ferritin as an indices of body iron stores and acute myocardeal infarction in diabetics

A152 CLINICAL CHEMISTRY, Vol. 55, No. 6, Supplement, 2009

Wednesday PM, July 22

Poster Session: 2:00 pm - 4:30 pmClinical Studies/Outcomes

The influence of nocturnal oxyhaemoglobin desaturation to fibrinolitic

dysfunction in hypertensive patients

D. Begovic, B. Pencic, V. Celic, M. Dekleva, Z. Caparevic, N. Kostic, R.Cvetkovic. Clinical Health Center Dr Dragisa Misovic, Belgrade, Serbia

Recent study confirmed that plasma levels of PAI-1 have positive correlation withblood pressure in both sexes. Nocturnal oxyhaemoglobin desaturation has been alsorecognized as potentional new risk factor for arterial hypertension. The aim of ourstudy was to evaluate the relation between PAI-1 and nocturnal oxyhaemoglobindesaturation in hypertensive patients before and after exercise test. Study included 40patients with essential hypertension, age< 65 without heart failure or pulmonarydiseases. Patients were divided according the oxyhaemoglobin desaturation in twogroups: group I - patients with oxyhaemoglobin saturation index (ODI)≥5/h and groupII - patients with oxyhaemoglobin saturation index (ODI)< 5/h. Groups were similarin demographic and clinical characteristics.Two days before exercise test allantihypertensive medica-mentations were withdrawn. In all patients we determinatedbody mass index, total cholesterol,LDL- cholesterol and HDL- cholesterol. PAI-1 (U/mL) was measured by spectrophotometric method in plasma one hour before exerciseand immidiately after the recovery phase. Patients in groupI had significantly higherPAI-1 levels before exercise( 3.28±1.00 U/mL ) than patients in group II ( 2.36± 0.71 U/mL). After exercise PAI-1levels were slightly, but not significantly more decreased in group II ( p= 0.41) than ingroup I ( p= 0.898).The final model of the stepewise multiple regression analysisshowed that nocturnal oxyhaemoglobin desaturation was independent risk factor forincreasing PAI-1 levels before ( B=2.806, Beta standardized coefficient = 0.227,p=0.000) and also affter exercising ( B= 0.122, Beta standardized cofficient= 0.727,p=0.030). Our results confirm that nocturnal oxyhaemoglobin is likely to bepredictive for increasing PAI-1 levels in patients with essential hypertension.

THE EFFECTS OF SOCIO DEMOGRAPHIC FACTORS ON

PLASMA ASCORBIC ACID AND ALPHA TOCOPHEROL ANTI

OXIDANTS DURING PREGNANCY IN NIGERIA WOMEN

S. E. Idogun1, P. Aikoriogie2. 1University of Benin Teach Hospital, Benin,Nigeria, 2University of Benin Teaching Hospital, Benin, Nigeria

Background: Plasma Ascorbic acid (vitamin C) and Alpha-tocopherol (vitamin E)are antioxidants known to alter during pregnancy.Objectives: Was to assess the plasma levels of vitamins C and E at the various stagesof pregnancy and to correlate the plasma levels of both vitamins with somesociodemographic factors of pregnant Nigerians.Setting: University of Benin Teaching Hospital, Nigeria.Study Design: A cross-sectional study.Patients and Methods: Patients were randomly recruited from the antenatal clinicaccording to the gestational ages. The controls were non-pregnant women of the sameage range, selected from the gynecology clinic. Blood specimens were collected fromboth the cases and controls for the assay of both vitamins. Ascorbic acid was analyzedusing the 2,4 Dinitrophenyl hydrazine photometric method and hexane extractionmethod was used for Alpha-tocopherol anlysis.Results: A total of 180 pregnant cases and 20 controls were studied. The mean plasmavitamins C and E in the pregnant cases and controls are shown in table 1.Thecorrelation of the mean plasma vitamins Cand E versus the parity of the cases was notsignificant; r=0.013, P>0.05; r=0.047, P>0.05, for vitamins C and E respectively.Themean plasma level of vitamin C was highest in the farmers and lowest in the housewives, although this was not significant; P>0.05. However, the correlation of vitaminC versus maternal age showed a significant negative relationship; r= -0.59, p<0.05;the mean plasma level of vitamin C declining with increase in maternal age.Conclusion: The mean plasma Ascorbic acid and Alpha-tocopherol are reducedduring pregnancy and the plasma vitamin E decline from first trimester and becamesignificantly low in the third trimester. Sociodemographic factors have mild effects on

the plasma levels of these vitamins but most significant is the effect of maternal ageon plasma level of vitamin C.

Investigation of Inflammatory Markers in Cerebrospinal Fluid from

Infectious and Non-Infectious Human Brain Disease

L. L. Wen1, C. Liang2, J. Wang3. 1En Chu Kong, Taipei Hsien, Taiwan,2Far Eastern Memorial Hospital, Taipei Hsien, Taiwan, 3National DefenseMedical Center, Taipei, Taiwan

Inflammation in the brain was associated not only with infections but also withneurodegeneration (Alzheimer’s disease, Parkinson’s disease, and stroke). Recentevidence suggested that protein concentration changed in cerebrospinal fluid (CSF)during central nervous system inflammation. In this study several markers wereexamined. The following were investigated: C-reactive protein (CRP): serum amyloidA (SAA); haptoglobin (HPT); tumor necrosis factor-alpha (TNF-α); interleukin-1b(IL-1β) and IL-6 due to inflammation in CSF of bacterial meningitis and stroke. CSFsamples were taken from three different groups of patients, classified as the following:(1) infection (bacterial meningitis; n=11) (2) non-infection (stroke; n=17) (3) normalcontrol group (n=18). The levels of change in concentration within the markers wereassessed. SAA, CRP and HPT were determined using BN II nephelometer® (DadeBehring Inc., Newark, DE, USA). The concentrations of TNF-α, IL-1 β and IL-6 weremeasured by chemiluminescence immunoassay with IMMULITE® automatedanalyzer (Diagnostic Products Corp., Los Angeles, CA, USA). The preliminary dataindicated that the relative change (folds of control) of IL-6 in CSF of meningitis wasthe highest compare to the other markers. Immunohistochemical staining studies alsorevealed elevated expression of IL-6 in activated of leukocytes of CSF were activatedduring cerebral infection by bacteria. Although the CRP was also increased inmeningitis and stroke patients, the CRP showed no significant difference betweenthese groups. These results suggested that TNF-α, IL-1 β and IL-6 in CSF could beimportant as useful diagnosis markers for infectious brain disease (bacterialmeningitis).

Association of Vitamin D receptor gene polymorphisms with Multiple

Sclerosis in an Indian Population

S. Vivekanandhan1, M. Murali1, N. Sonali1, V. Sneha1, M. Tripathi2.1Neurobiochemistry, All India Institute of Medical Sciences, New Delhi,India, 2Neurology, All India Institute of Medical Sciences, New Delhi, India

Purpose: Multiple Sclerosis is a chronic, immune-mediated inflammatory andneurodegenerative disease of the central nervous system (CNS), with an etiology thatis not yet fully understood. The prevalence of MS is highest where environmentalsupplies of vitamin D are lowest. The present study was undertaken to investigateVDR gene variation using three intragenic restriction fragment length polymorphisms(Apa I, Taq I and Fok I) in an Indian MS case-control population.Methods: 40 Multiple Sclerosis patients and 40 age, sex and ethnicity matchedcontrols were investigated for VDR variants (Taq I, Apa I and Fok I) genotypes usingpolmerase chain reaction -restriction fragment polymorphisms (PCR-RFLP). Allelefrequencies were derived from genotypic data. Case-control comparisons were madeusing Chi-square tests. Deviations from the Hardy-Weinberg equilibrium were alsotested.Results: The VDR Variants Taq I, Apa I and Fok I polymorphisms was seen to be inHardy-Weinberg equilibrium and showed significant genotypic and allelic associationbetween the case and control groups for the functional exon 9 VDR Taq Ipolymorphism. Genotypic: P = 0.05; OR = 0.39 (0.13-1.15), Allelic: P = 0.008; OR=2.79 (1.20-6.58). The Apa I and Fok I polymorphisms showed significant allelicassociation .Allelic : P = 0.0431,OR = 2.13(0.96-4.75), P= 0.05,OR= 2.15(0.93-5.04)

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Mean plasma vitamin C and E in the pregnant cases and controlsPregnant women (N=60)

Pregnant women (N=60)

Pregnant women (N=60)

Non-pregnant women (N=20)

P- value

Plasma levels of vitamins

1st Trimester

2nd Trimester

3rd Trimester

Controls

Vitamin C (microg/ml)

1.75±0.36 1.76±0.38 1.60±0.37 2.1±0.41 <0.0001

Vitamin E (mg/dl)

0.90±0.39 1.87±0.57 0.88±0.36 2.31±0.42 <0.0001

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CLINICAL CHEMISTRY, Vol. 55, No. 6, Supplement, 2009 A153

but no significant genotypic frequencies between MS and controls (P=0.174, OR =0.54).Conclusion: Our findings suggest that significant role of VDR gene polymorphismsin the risk of developing multiple sclerosis in an Indian population.

Atypical cells in the morning fresh urine, a and their possible

significance

S. Giju1, C. Flangea2, V. Dumitrascu3, L. Petrica4, D. Vlad1, S. Ursoniu5.1Counnty Emergency Hospital, Timisoara, Romania, 2Victor BabeUniversity of Medicine and Pharmacy, Biochemistry Department,Timisoara, Romania, 3Victor Babe University of Medicine and Pharmacy,Pharmacology Department, Timisoara, Romania, 4Victor Babe Universityof Medicine and Pharmacy, Department of Nephrology, Timisoara,Romania, 5Victor Babe University of Medicine and Pharmacy, Departmentof Public Health, Timisoara, Romania

Objective: The main goal of our study was to demonstrate the importance of theassessment of urinary atypical cells in the urinary sediment. Furthermore, weattempted to evaluate the significance of atypical cells according to their importance.In our study, we emphasize that atypical cells, even if they are 1/lpf, have diagnosticimportance.Relevance: When atypical cells are observed in the urinary sediment fromspontaneous urine, it is necessary to know the conditions of their appearance, as wellas their origin and significance. We proposed to us to emphasize in the urinarysediment the atypical cells, as well as the presence of other cells, which indicatevarious tumoral diseases (renal or non renal). To avoid any possible confusions, weused the parallelism of microscopic techniques and when necessary, the stained andunstained samples. Each photo belongs to one patient. In some circumstances we tookphotos (to eliminate any doubt) of the same atypical cells with different types ofmicroscopy. We can warn the patient and recommend a pathological examination. Thetechniques proposed by us are not expansive and they don’t last long. Anexperimented microscopist is required.Methodology: In these studies the urinary sediment was screened at a low power (x100) and thereafter the suspect structures were examined at a higher power (x 400).Initially we worked on a normal microscope but with reduced light with the condenserplaced at a lower position to increase the contrast. After that we used a phase contrastmicroscope, more suitable for the study because atypical cells are relatively difficultto identify using just the traditional bright field microscopy. The investigation wasperformed by using different microscopic techniques in the observation of thesediment elements in both unstained and stained samples (May-Grünwald-Giemsastain and Sternheimer-Malbin stain).Validation: The purpose of our study is to show the diagnostic importance of atypicalcells and also the high relevance of other types of cells. We studied a lot of 62 patientsadmitted to the hospital, most of them in different clinics of the County EmergencyHospital No.1 Timisoara. Due to the rarity of the cases, the study lasted almost threeyears (February 2005 - January 2008). For urinalysis we used either the first or thesecond morning urine specimen. The microscopic examination revealed the presenceof urinary atypical cells. To confirm our findings, we took photos of the same castwith different types of microscopy. The other types of cells are diagnosis elementstoo.Conclusions: To emphasis the atypical cells between the slide and cover slip (stainedand unstained), as well as in the smears, is a cytoprevention element, because a simplesuspicion of the presence of atypical cells determine us to recommend a pathologicalexamination. We say that because this department is the only one which can certainlyconfirm or not the tumoral cells presence. The microscopic study of the atypical cellsis a non - invasive and accurate method. It has the advantage of decreasing costs andeliminating the risks associated with invasive methods.

Evaluation of Chronic Liver Fibrosis Test, "Fibrometer" In Asian

Population

H. H. Liu1, C. Cheng2. 1Taiwan Specialty Reference Laboratory, Taipei,Taiwan, 2National Taiwan University Hospital, Taipei, Taiwan

Disease background: Chronic liver fibrosis is a chronic liver disease caused mainlyby Hepatitis B, Hepatitis C, fatty Liver, and chronic alcoholic consumptions. Over20% of chronic HBV patients and 85% of HCV patients eventually develop liver

cirrhosis. In Asia, 90% of liver cancer patients are due to chronic HBV infection. Over300 million HBV patients are in Asian and Number one cause of cancer death.Chronic liver fibrosis is a slow-developed liver injury. It represents a wound healingprocess responding to hepatocellular demages due to HBV, HCV infections or othercauses. It will develop into liver cirrhosis or hepatocarcinoma. The time to developthese severe liver conditions takes 20 to 30 years. There are many articles indicatedthe disease is treatable during the first 5-15 years of development. When livercirrhosis symptom is presented, it is often not treatable or curable any more.Therefore, early detection of chronic liver fibrosis is very significant clinically. HBVis a major infectious disease in Asia. Over 300 million HBV patients are residing inAsia. It is the number one cause in cancer death.Diagnosis of chronic liver fibrosis: Chronic liver fibrosis is traditionally diagnosedby liver biopsy then starting treatment. Due to the severe adverse effects of HCVtreatment (chronic flu-like symptom), it is recommended to treat patients with liverfibrosis Stages II or above. In United States, less than 10% of patients with chronicHCV accept liver biopsy. Therefore, many HCV patients are not treated.Synopsis for current study: In recent years, several blood testing methods aredeveloped. Fibrometer, Fibrosure (Fibrotest), APRI, Hepascore, and many more areused in US and Europe. Among them, Fibrometer has the best performance based onliterature reports. Fibrometer is developed using only Caucasian HCV, HBV patientsas study subjects. Our study is to evaluate results of Fibrometer performance amongAsian patient populations who has chronic hepatitis B or hepatitis C, compare them tothe results of Caucasian samples.Study methods: Fibrometer uses the following 9 assays- glucose, bilirubin, AST,ALT, GGT, Ferritin, Urea, Hyaluronic Acid (HA), and Alpha 2 Macroglobulin (A2M)plus Prothrombin index and platelets. Prothrombin time and platelets are done at siteof sample collection. The biochemical tests use Roche Hitachi and Elecsysinstruments. “Fibroscan”, an ultrasound based liver elasticity measuring instrumentwas also studied as a reference method to compare to the accuracy of “Fibrometer”.Results: The study results indicated that Fibrometer is an excellent test for hepatitis Binduced chronic liver fibrosis measurement. The AUROC of HCV and HBV arecomparable at 0.82 vs. 0.81. There are no significant differences between the twoapplications in diagnostic accuracy (0.87%). The diagnostic accuracy of Fibrometer isclosely correlated to Fibroscan but Fibrometer has a much superior accuracy (1.5x inIndex alpha ratio) for the clinically significant (Metavir Stages II and above).Conclusion: Based on this study, Fibrometer is an excellent tool to assess the stagingof Chronic Liver Fibrosis/Cirrhosis in Asian Patients who has chronic liver fibrosiscaused by either HBV or HCV.

Does Proteinuria Justify a Microscopic Examination of the Urine

Sediment (MEUS)?

C. Puza1, E. S. Pearlman2. 1South Boston Community Health Center,Boston, MA, 2Boston Medical Center, Boston, MA

A widely adhered protocol reflexes to a MEUS if urine dipstick chemistries (UDCs)are positive for hemoglobin (H), leukocyte esterase (LE), nitrite (N) or protein (P). Wequeried whether reflex MEUS was warranted when, of the above four analytes, only Pwas positive by UDC. We examined this issue by assessing data on 110 consecutivespecimens from ambulatory patients seen at two community health centers in Boston.UDCs were assessed using a Status urinalysis instrument (Siemens: Tarrytown NY)and were all negative for H, LE and N but were all =/> 1+ positive for P. Thepopulation consisted of 67 M and 43 F (z score=2.27 versus equal genderrepresentation; p<.03 [2-tailed]). The median (range) M age was 49 YO (2 mos.-91YO) and for Fs was 24 YO (7-85 YO) (p<.005; 2-tailed median test). Review of theelectronic medical record (EMR) indicated that proteinuria or renal insufficiency waspart of the patient problem list in 22 instances (20%). Associated conditions (numberof patients) possibly relevant to occurrence of proteinuria included hypertension (35),diabetes mellitus (21) hepatitis B/C (10), pregnancy (9) renal calculi (3) and gout (2).Relevant associated conditions were absent in 45 patients (41%). The magnitude ofthe proteinuria was 1+ (n=85), 2+ (n=23) and 3+ (n=2). Simultaneous urine culture(UC) was obtained in 41 patients but only one revealed =/> 10,000 CFU/mL of asingle organism. Repeat UDCs were obtained in 33 patients within 6 months of theinitial UDC and P reverted to negative/trace in 23 instances. A microalbumin/creatinine ratio (MACR) was obtained within 6 months in 33 patients and was withinthe reference interval (WRI) in 20. Serum creatinine concentration was determinedwithin 6 months in 64 patients and was WRI in 56 and was =/> 2 mg/dL in only 1patient. Potentially significant microscopic findings [Ringsrud & Linne, Urinalysisand Body Fluids (1995)] were not identified in 87 patients (79%). In the remainingpatients findings included 6-10 WBC/hpf (10), 6-10 RBC/hpf (5), 11-15 RBC/hpf (1),10-30 epithelial cells/hpf (11), 10-30 hyaline casts/lpf (2), 2-5 granular casts/lpf (1)

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and 3+ bacteria (1) [UC was negative in this case]. From review of the EMR over thesix months subsequent to the initial MEUS however, it was unclear how MEUSfindings specifically impacted patient management. Review of the EMR indicated thatwhen the finding of proteinuria was identified in provider notes the usual follow-upswere repeat of the UDC, MACR or serum creatinine. No specific reference to theMEUS in clinician/provider notes was identified. We concluded after review of theabove results that the yield in respect to data that would influence patient managementdid not justify continuation of the MEUS in P only specimens and a decision wasmade to reflex to an MACR in such cases.

Diagnostic Efficiency of Amylase and Type IV Collagen in Predicting

Chronic Pancreatitis

S. K. Das1, V. Balakrishnan2, D. M. Vasudevan3. 1Agartala Govt MedicalCollege, Agartala- 799006, India, 2Amrita Institute of Medical Sciences,Cochin 682 026, India, 3Amrita Institute of Medical Sciences, Cochin682026, India

BACKGROUND: Chronic pancreatitis, an irreversible inflammatory disease of thepancreas, is associated with the replacement of the destroyed parenchyma by extendeddevelopment of fibrosis. The biological cause of fibrosis is the accumulation ofexcessive amounts of extracellular matrix, which leads to tissue dysfunction andorgan failure. There is growing evidence that pancreatic fibrosis represents adysregulation of the normal repair processes after injury. The severity of pancreaticfibrosis can be assessed only by direct histologic analysis of pancreatic tissue. Theanatomic position and relationships of the pancreas make direct observation ofpancreatic pathology difficult. OBJECTIVE: Therefore, we examined the hematological and common biochemicalparameters in serum of chronic pancreatitis patients and analyzed their association, inparticular with serum amylase and type IV collagen levels.METHODS: A complete clinical history, including anthropometric measurements;haematological and biochemical investigations including renal function tests, liverfunction tests and lipid profile were performed among forty (40) chronic pancreatitispatients within 18 to 67 yrs. Type IV collagen level in serum was measured by latexturbidometric immunoassay.RESULTS: ESR level and alkaline phosphatase (ALP) activity elevated in 40% cases.Hyperglycemia was observed in 45% patients Though serum calcium level decreasedin 25% patients and serum amylase activity increased in 80% patients; it showedsignificant correlation with ALP (r=0.458, p=0.003), CA-19.9 (r=0.556, p<0.001),and calcium level (r= -0.472, p=0.002). Type IV collagen level in chronic pancreatitispatients also elevated (164.4 ± 55.5 ng/ ml) and showed significant negativecorrelation with calcium level (r= -0.505, p=0.001). However, no significantcorrelation was observed between amylase activity and type IV collagen (r=0.289, p=0.07).CONCLUSIONS: Pancreatic fibrosis is a key pathological feature of chronicpancreatitis and sometimes results in diabetes mellitus. The blood parameters such asamylase activity and type IV collagen level, and their correlations with hematocrit,calcium, alkaline phosphatase activity and CA-19.9 may be monitored in thesepatients. More study is necessary to identify the biomarker or combination ofbiomarkers for these patients.

Analysis of the influence of Etamsylate on creatinine using enzymatic

methods

Q. YANG. Chinese PLA general hospital, Beijing, China

Background: Etamsylate is widely used in clinical treatment as an antihemorrhagic.During clinical work, it has been found that patients administrated with Etamsylatehad poor results of creatinine(Cr)(using enzymatic methods), which later was found tobe discrepancy to clinical symptoms. The Cr results in dry chemistry isn't consistentwith results in wet chemistry(both are used enzymatic methods). So it might beEtamsylate that affects tests of Cr.Objective: This study is to investigate the influence of Etamsylate on enzymaticmethod assaying creatinine.Method: 20 healthy people's serum were collected and made into serum mixture, andit was divided into 10 groups , added with different doses of Etamsylate as 300, 150,75, 37.5, 18.75, 9.38, 4.69, 2.34, 1.17and 0mg/L according to the maximum bloodEtamsylate level. The same groups of saline were added the same levels of Etamsylate

as blank control. Dry and wet chemical methods were used to test each sample 4times.Result: The Cr result of the serum mixture that with 0mg/L Etamsylate was67.33±0.43µmol/L in dry chemistry and it was 66.23±0.13µmol/L in wet chemistry.When added 300mg/L Etamsylate(peak drug level), the Cr result was40.78±0.46µmol/L in dry chemistry and 17.35±0.39µmol/L in wet chemistry. Whenadded 150mg/L Etamsylate, the Cr result was 50.7±0.36µmol/L in dry chemistry and22.88±0.56µmol/L in wet chemistry. When the drug Etamsylate that added into theserum mixture was 9.38mg/L, the Cr result in dry chemistry was 66.2±1.12µmol/Land there was no difference between results of dry chemistry and 0mg/Lgroup(without Etamsylate, true value, p<0.01); when the level of Etamsylate is1.17mg/L, the Cr result in wet chemsitry was 66.13±0.59µmol/L and it had nodifference with 0mg/L group(without Etamsylate, true value, p<0.01).Discussion: Etamsylate in curable level does have inhibition effect on assayingcreatinine using enzymatic methods; when presented in blood, Etamsylate decreasesCr results. But the inhibition effect of Etamsylate fades with dose-dependent. To testCr in laboratory, it can be concluded from this study that at least 11 hours aftertreatment with Etamsylate, patient's blood is available for creatinine test and then itsresult is reliable.

The analysis of increasing Ca125 serum level in patients with CKD

J. Gao, Y. Tian. Dept. of Clinical Biochemistry, Chinese PLA GeneralHospital, Beijing, China

Objective: To find out the reason of elevated Ca125 serum level in patients withchronic kidney disease (CKD).Relevance: The elevated tumor marker’ serum level such as CA125 in patients withCKD always confused the nephrologists. Either there was tumor exited in the patientsor just an elevated level of tumor markers compared with chronic inflammation?Validation: 649 cases in our hospital were investigated retrospectively. The patientsless than 70 years old with the diagnosis of CKD according to DOQI guideline wereincluded in this study and the patients with tumor, rheumatic disease, tuberculosis,virus hepatitis or acute kidney disease were excluded.Methodology: The serum levels of 38 biochemical parameters including CA125,CA199, albumin (ALB), total cholesterol (TC), and triglyceride (TG) were detectedwith ROCHE modular analyzing system. Serum level of 35 U/ml was set as cutoffvalue of CA125, and all patients were divided into two groups. Data of two groupswere collected and analyzed. Univariate analysis was performed to compare datadifference of two groups. Binary logistic regression model (method was forward:conditional) was developed to assess factors affecting CA125 positive rate in CKD.CA125 serum levels were compared between IgA nephropathy and non-IgAnephropathy groups, between serousal effusion and no effusion groups and betweenhypoalbuminemia and no hypoalbuminemia groups.Results: The results showed there were significant differences in 24 parametersbetween CA125 positive group and negative group, including clinical diagnosis,pathological diagnosis, CA153, et al (P<0.01 or <0.05). Logistic regression analysisshowed serousal effusion, pathological diagnosis and hypertension were mainindependent factors affecting CA125 serum positive rate (P were 0.001, 0.025 and0.009; odds ratio were 21.141, 0.112 and 12.678; model log-likelihood values were -34.154, -29.562 and -31.105). CA125 serum level of IgAN was lower than non-IgAnephropathy ( 11.2 U/ml vs 26.0 U/ml,P<0.01); of hypoalbuminemia group washigher than no hypoalbuminemia group (37.0 U/ml vs 11.1 U/ml, P<0.01); of serousaleffusion group was much higher than no effusion group (154.4 U/ml vs 12.7 U/ml,P<0.01). Stratification analysis was used to calculate the correlation of CA125 serumlevel and ALB. The correlation coefficient of ALB with CA125 was -0.391 (P<0.01)when ALB serum level was less than 35 g/L. But it was only -0.085 (P>0.05) whenALB serum level was higher than 35 g/L.Conclusion: Patients with non-IgA nephropathy are at a high risk of getting elevatedCA125 serum level especially complicating hypertension or serousal effusion.However hypoalbuminemia also is an important factor on it.

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Association between chronic kidney disease and metabolic syndrome

M. Orisaka1, K. Kawamura1, M. Nakamura2. 1Iwate Health ServiceAssociation, Morioka, Japan, 2Osaka Medical Center for Health Scienceand Promotion, Osaka, Japan

Renal disorders showing a chronic course are generically termed chronic kidneydisease (CKD). Recently, its incidence has rapidly increased. The number of patientsundergoing dialysis has also increased. In the presence of insulin resistance,proteinuria is frequent, commonly leading to CKD via kidney hypofunction. Weinvestigated the relationship between CKD and metabolic syndrome (MS), which isclosely associated with insulin resistance.Subjects: The subjects were 18,556 patients, excluding those undergoing chronicnephritis treatment. They consisted of 11,244 males (mean age: 54.6±11.3 years,range: 19 to 90 years) and 7,312 females (mean age: 55.3±11.4 years, range: 20 to 92years). The estimated glomerular filtration rate (eGFR, mL/min/1.73m2) wascalculated using the Modification of Diet in Renal Disease (MDRD) formula: 0.741 x175 x Age-0.203 x creatinine-1.154 (females: x 0.742). Patients showing an eGFR below60 were regarded as having CKD. Furthermore, MS subjects were selected accordingto a revision of the NCEP-ATP III criteria (2005). Concerning abdominalcircumference, males with a circumference of 90 cm or more and females with that of80 cm or more were included.Results: MS subjects consisted of 2,713 males (24.1%) and 1,611 females (22.0%).We examined incidences of CKD with respect to gender, age, and the presence orabsence of MS. In males younger than 30 years, the incidences were 0.0 and 1.5% inthe MS and non-MS groups, respectively. Among those aged 30 to 39 years, thevalues were 6.2 and 2.9%, respectively. For those aged 40 to 49 years, the values were10.0 and 6.9%, respectively. Concerning those aged 50 to 59 years, the values were14.1 and 9.9%, respectively. For those aged 60 to 69 years, the values were 15.8 and12.9%, respectively. Among those aged over 70 years, they were 28.5 and 16.1%,respectively. In females younger than 30 years, the incidences were 0.0% in both theMS and non-MS groups. Among those aged 30 to 39 years, the values were 15.4 and3.8%, respectively. For those aged 40 to 49 years, the values were 4.6 and 7.6%,respectively. Concerning those aged 50 to 59 years, the values were 8.5 and 5.9%,respectively. For those aged 60 to 69 years, the values were 9.1 and 5.5%,respectively. Among those aged over 70 years, they were 14.7 and 1.7%, respectively;the incidence of CKD was higher in the MS group at all ages regardless of gender. Inmales aged over 40 years, there were significant differences between the MS and non-MS groups (p<0.01). In males overall, the incidences of CKD in the MS and non-MSgroups were 14.7 and 9.8%, respectively. In females overall, the values were 9.8 and6.6%, respectively. Among both males and females, the incidence of CKD in the MSgroup was 1.5 times higher than in the non-MS group, with a significant difference(p<0.01).Conclusion:The incidence of CKD was significantly higher in the MS group,indicating the association between CKD and MS. MS strategies must be established,considering CKD.

INCREASED PLASMA LEVELS OF NGAL AND IL-18

CORRELATE WITH REDUCED GFR PROVIDING EVIDENCE OF

TUBULOGLOMERULAL DEFECT IN PATIENTS WITH HbS/

ββββTHALASSEMIA

I. Papassotiriou1, A. Margeli1, E. Hantzi1, E. Terpos2, E. Voskaridou3.1Department of Clinical Biochemistry “Aghia Sophia” Children’sHospital, Athens, Greece, 2Department of Medical Research, 251 GeneralAirforce Hospital, Athens, Greece, 3Thalassemia Center, Laikon GeneralHospital, Athens, Greece

Promising novel biomarkers for acute kidney injury (AKI) or chronic kidney injury(CKI) include a plasma levels of cystatin C, Neutrophil Gelatinase-associatedLipocalin (NGAL) and Interleukin-18 (IL-18) and urinary levels of NGAL, IL-18 andKidney Injury Molecule-1 (KIM-1). These biochemical indices have been reported tobe useful for evaluating the duration and severity of kidney disease and for predictingprogression and adverse clinical outcomes. Furthermore, these biomarkers have givenpromising results in differentiating the various causes of AKI or CKI. Progressiverenal failure is one of the main complications in HbS/β-thalassemia (HbS/β-thal).Early identification of patients at high risk of developing renal failure is of greatimportance as it may allow specific measures to delay the progression of renal

damage and thus to reduce the incidence of end-stage renal failure and mortality.Continuing our previous studies (Voskaridou et al, Kidney Int, 69:2037-2042, 2006),we explored the activation of tubuloglomerular feedback in 58 adult patients (age42±17years, males/females 20/58) with HbS/β-thal by measuring the abovementioned parameters, along with the more classical ones such us β2-microglobulinand N-acetyl-β-D-glucosaminidase (NAG) in blood and urine using standardmethodology. GFR values were calculated according to the recently proposed cystatinC-based prediction equation using only each concentration in mg/L: GFR [mL/min/1.73m2] = 76.7 x Cystatin C-1.18. The main results of the study were: a) impairment ofGFR in 21/58 patients (36%); b) increased plasma concentrations of NGAL and IL-18in 34/58 (58%) and 58/58 (100%) patients, respectively; c) increased and/ordetectable urine concentrations of NGAL and IL-18 in 54/58 (93%) and 46/58 (79%)patients, respectively; d) significant negative correlations between GFR and plasmaNGAL and IL-18 (r=-0.735, p<0.0001 and r=-0.350, p<0.007, respectively) and e)significant positive correlations between cystatin C and urine NGAL and IL-18(binomial, p<0.005 and r=0.412, p<0.03, respectively). These findings suggest thattubuloglomerular feedback is activated in almost all studied patients with HbS/β-thalassemia. Measurements of plasma and urine NGAL and IL-18 contributesignificantly to the early detection and risk stratification of renal disease in thesepatients. However, prior to their clinical usefulness, these biomarkers must undergothrough rigorous validation in multiple cohorts.

Development of a System Check (Prozone Check) for Total Bilirubin

Test (BILTS) on Roche cobas c 311 and cobas c 501 Systems

H. Klima1, B. Loehr2, G. Sobczak1. 1Roche Diagnostics GmbH, Penzberg,Germany, 2Roche Diagnostics GmbH, Mannheim, Germany

Introduction: Interference of paraproteins in diagnostic tests is a well known problemin the routine clinical laboratory. Due to poor solubility of immunoglobulin proteinsthe samples become turbid during analysis. This turbidity interferes with the reactionkinetics of analyte determination. This interference seems to be independent from theconcentration and from the type of immunoglobulin in the patient sample.It is known from the literature and from internal investigations at Roche Diagnosticsthat in rare cases patient samples containing paraproteins give falsely elevated resultsin the Total Bilirubin liquid test (BILTS) on Roche cobas c 311 and cobas c 501analyzers.Objectives: In order to identify falsely elevated results in patient samples withuntypical reaction kinetics a system check, so called ‘prozone check’, wasimplemented in the applications of BILTS on Roche cobas c 311 and cobas c 501.Due to difference in the applications, two different system specific checks detectingabnormal reaction kinetics in patient samples were developed.The BILTS applications with and without prozone checks are currently evaluatedunder routine conditions using routine clinical laboratory samples in order to detectfalsely flagged samples.Study Design: In this study we analyzed whether applications for total bilirubin showfalsely elevated results for Total Bilirubin in gammopathic samples. Patient samples,n = 24, with known interference in the current BILTS test on cobas c 311 and cobas c501 systems were used for development and verification of the prozone check.Materials and Methods: The Serum I Index and the measurement with the RocheBILT2 liquid reagent were used as reference methods for verification of total bilirubinconcentration in discordant samples. Immunoglobulin concentration were measuredwith Roche IgA, IgG and IgM assays on cobas c 501 system.Results: The measurements of samples with known paraprotein interference hasproven that the prozone check on cobas c 311 and cobas c 501 analyzers allows todetect interference from gammopathic samples. A flag (>kin) occurred with all testresults for BILTS application having abnormal reaction kinetics. Discordant testresults in patient samples were confirmed by two independent methods for totalbilirubin in all paraprotein samples. The bias in the bilirubin value measured with theBILTS assay and the reference methods was found in the range of 17.6 µmol/l to 704.3 µmol/l in the patient samples.Conclusions: The reliability of BILTS applications was markedly improved withimplementation of the prozone check. The check can be used for detection of patientsamples with abnormal reaction kinetics. All samples tested were marked by theprozone check. Although it cannot be ruled out that these modified applications mightmiss patient samples with very special kinds of paraprotein.The prozone check for BILTS applications will be released in 2009 after the externalevaluation has been completed.

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A Comparative Evaluation of the Clinical Utility of the Premier

Enzyme Immunoassay (for IgG) and the Meridian Immunodiffusion

assay (for F antigen) in the Diagnosis of Coccidioidomycosis.

C. M. LaPolt1, E. I. Blackman2. 1University of California Berkeley,Berkeley, CA, 2Thousand Oaks Pathology Associates, Thousand Oaks, CA

Objective: The objective of this study is to compare the clinical utility of the PremierEnzyme Immunoassay for IgG (P-EIA) versus the Meridian Immunodiffusion assayfor F antigen (M-ID) for the diagnosis of Coccidioidomycosis in a communityhospital setting. Methods: We studied 472 consecutive specimens (from 447 patients)with orders for diagnostic serology for Coccidioidomycosis. P-EIA and M-ID wereperformed according to manufacturer’s specifications. Definitive testing at aspecialized referral lab (SRL), unrelated to our institution, was performed for 78 ofthese specimens (from 78 patients), including all specimens with a discordancebetween the P-EIA and M-ID results. Samples were assigned a reference diagnosisbased on the SRL results or on concordant P-EIA and M-ID results. SRL results wereconsidered positive if: the original specimen was positive by Complement Fixation(CF) or Immunodiffusion for IgG (ID-CF); or the original specimen was positive byImmunodiffusion for IgM (ID-TP) and a subsequent specimen was positive by CF orID-CF within 60 days of the original sample. Sensitivity, specificity, and positive andnegative predictive values were calculated for P-EIA and M-ID based on acomparison to the reference diagnosis. Statistical significance was determined by thez test, with a significance level of 0.05. Results: The reference diagnosis was positivefor 22 specimens (from 22 patients) and negative for 450 specimens (from 425patients). Sensitivity, specificity, and positive and negative predictive values were asfollows:

Conclusion: No statistically significant differences were observed in the clinicalperformance characteristics of the M-ID and P-EIA assays. P-EIA may be consideredas an acceptable alternative to M-ID for the diagnosis of Coccidioidomycosis.

Urine neutrophil gelatinase-associated lipocalin (NGAL) as a

biomarker for chronic cyclosporine nephrotoxicity after cardiac

transplantation

Y. Chen1, F. Gustafsson2, J. Sheedy2, H. Ross2, M. Liao3, Y. Wang3, P.Wong3. 1Department of Laboratory Medicine and Pathobiology, Universityof Toronto, Toronto, ON, Canada, 2Heart Failure and Transplant, TorontoGeneral Hospital, Toronto, ON, Canada, 3University Health Network,Toronto, ON, Canada

Background: Cyclosporine (CsA) has improved allograft survival and quality of lifeof for solid-organ recipients. However, after over two decades of CsA use its chronicnephrotoxicity remains a significant clinical problem, which is the most importantcause of renal dysfunction after cardiac transplantation. Urine neutrophil gelatinase-associated lipocalin (NGAL) is an emerging renal biomarker for a variety of clinicalsituations leading to acute kidney injury or to chronic kidney disease. The aim of thepresent study is to investigate the role of NGAL measurement in chronic CsA-inducednephrotoxicity following cardiac transplantation.Methods: Eight male and three female heart transplant recipients (mean age 54±8)were recruited in the study. Patients were treated with the combination of CsA(114±80 mg/day) with Everolimus (n=9, 1.58±0.38 mg/day) or Mycophenolatemofetil (n=2, 1500 mg/day or 3000 mg/day). Urinary NGAL were measuredconsecutively from 0 to 12 hours after CsA administration at 6 time points by anautomatic NGAL assay* on Architect® i2000 analyzer and a manual ELISA method(AntibodyShop, Denmark). On the day of study, serum creatinine was also measuredfor the estimated glomerular filtration rate (eGFR).Results: The urine NGAL levels peaked at 2 hr (C2) post CsA treatment in 3 patientswith a concentration above 70 ng/ml and then gradually decreased to baseline level atC12. This pattern correlates with the general pharmacokinetics of blood CsA thatpeaks 2 hr after oral administration and reach a through at 12 hr. Urine NGALconcentrations in other eight patients did not show such increase. C2 urine NGAL

levels of these two groups were 81.53±1.6 and 9.18±7.6 respectively (P<0.001).Urinary NGAL has shown an inverse correlation with eGFR (R=-0.607, P<0.05) anda correlation with time from transplantation (R=0.622, P<0.05). Analytically, theArchitect NGAL assay demonstrated a good precision with CVs of 4.35, 2.2%, 2% atlevels of 21, 393, and 1192 ng/ml. The Architect NGAL assay correlated very wellwith the ELISA method: Architect=1.0213xELISA-0.454 (R=0.992, P<0.001).Conclusion: NGAL is a novel marker for chronic CsA-induced nephrotoxicity aftercardiac transplantation. Monitoring urine NGAL may help adjusting dosage of CsA toprevent irreversible renal damage. The performance of the Architect NGAL assay wassatisfactory for routine laboratory practice.* in development

Serum TIMP-1 and HER-2/neu levels in metastatic breast cancer

(MBC) treated with either letrozole or tamoxifen

P. J. Hamer1, A. Lipton2, K. Leitzel2, L. Demers2, S. M. Ali3, D. B. Evans4,H. A. Chaudri-Ross5, S. Brown-Shimer1, K. Pierce1, W. P. Carney1, V.Gaur1. 1Oncogene Science/Siemens Healthcare Diagnostics, Cambridge,MA, 2Penn State University, Hershey Medical Center, Hershey, PA,3Lebanon VA Medical Center, Lebanon, PA, 4Oncology Research, NovartisPharma AG, Basel, Switzerland, 5Oncology Business Unit, NovartisPharma AG, Basel, Switzerland

Purpose: This was a combined analysis of serum TIMP-1and serum HER-2/neulevels in MBC patients treated with an aromatase inhibitor, Letrozole versustamoxifen to determine if levels of these biomarkers were associated with clinicaloutcome.Patients and Methods: Five hundred twenty-two patients estrogen receptor-positiveMBC were randomly assigned to receive first-line hormone therapy with letrozole ortamoxifen. Serum tissue inhibitor of metalloproteinases-1 (TIMP-1) and serum HER-2/neu pretreatment levels were measured using enzyme-linked immunosorbent assaysfor both biomarkers.Results: Among patients with available pretreatment serum, 298 patients had normalpretreatment serum levels of both TIMP-1 and HER-2/ neu (reference group), 72patients had elevated serum TIMP-1 and normal serum HER-2/neu, 104 patients hadnormal serum TIMP-1 and elevated serum HER-2/neu levels, and 48 patients hadelevated pretreatment serum levels of both TIMP-1 and HER-2/neu. Median time toprogression (TTP) for all three patient groups with either or both serum biomarkerselevated was significantly reduced compared with that of the reference group (normalpretreatment serum levels of both TIMP-1 and HER- 2/neu; P< .0001). In thisanalysis by treatment arm, patients with normal serum levels of both TIMP-1 andHER-2/neu, median TTP for letrozole was 14.4 months versus 9.2 months fortamoxifen (P< .01). In patients with elevated TIMP-1 and normal HER-2/neu, medianTTP for letrozole was 9.2 months versus 3.7 months for tamoxifen (P< .02). Inpatients with normal TIMP-1 and elevated HER-2/neu, median TTP was 7.4 monthsfor letrozole versus 4.6 months for tamoxifen (P< .13). Finally, in patients withelevation of both TIMP-1 and HER-2/neu, median TTP was 3.2 months for bothletrozole and tamoxifen (P<.45). Elevated versus normal serum TIMP-1 wasassociated with a significantly increased risk of death, both within the normal serumHER-2 subgroup (median survival, 26 v 42 months; P < .0006), and within theelevated serum HER-2 subgroup (median survival, 15 v 25 months; P<.01). Patientswith elevated serum levels of both TIMP-1 and HER-2 had the worst prognosis,which was significantly shorter compared with all other subgroups (median survival,15 months; P < .0001 compared to the reference group. We next compared overallsurvival for the four biomarker subgroups. Elevated versus normal serum TIMP-1level was associated with a significantly increased risk of death, both within thenormal serum HER-2 subgroup (median survival, 26 v 42 months; P < .0006), andwithin the elevated serum HER-2 subgroup (median survival, 15 v 25 months; P<.01).Patients with elevated serum levels of both TIMP-1 and HER-2 had the worstprognosis, which was significantly shorter compared with all other subgroups (mediansurvival, 15 months; P < .0001 compared to the reference group and p = .01 for bothremaining subgroups. Conclusion: Combined analysis of both serum TIMP-1 and HER-2/neu conferredadditional ability to predict significantly different clinical outcomes compared tousing either biomarker alone.

P-EIA M-ID p-valueSensitivity 21/22 (95.5%) 18/22 (81.8%) nsSpecificity 442/450 (98.2%) 448/450 (99.6%) nsPositive Predictive Value 21/29 (72.4%) 18/20 (90.0%) nsNegative Predictive Value 442/443 (99.8%) 448/452 (99.1%) ns

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Serum HER-2/neu levels in early stage HER-2/neu positive breast

cancer (HER-2+BC). Results from the NCCTG adjuvant intergroup

trial N9831

W. P. Carney1, A. Moreno-Aspitia2, A. Dueck3, W. Lingle4, L. Kutteh5, K.Tenner6, N. Davidson6, E. Perez2. 1Oncogene Science/Siemens HealthcareDiagnostics, Cambridge, MA, 2Mayo Clinic, Jacksonville, FL, 3MayoClinic, Scottsdale, AZ, 4Mayo Clinic, Rochester, MN, 5Oncology Associatesof Cedar Rapids, Cedar Rapids, IA, 6Johns Hopkins Oncology Center,Baltimore, MD

Background: Serum HER-2/neu is the circulating extracellular domain ( p97Kda-p115Kda) that is cleaved from full-length, membrane-bound p185 HER-2/neu protein.Previous studies support that increased serum HER-2/neu levels are stronglyassociated with poor prognosis and a predictor of poor response to chemotherapeuticand hormonal treatments in metastatic breast cancer patients. The role of serum HER-2/neu levels in early breast cancer patients in the adjuvant setting was evaluated instudy N9831. NCCTG N9831 is a randomized, phase III clinical trial comparing threedrug regimens: doxorubicin/cyclophosphamide followed by paclitaxel withtrastuzumab added concurrently, sequentially, or not at all as adjuvant therapy forwomen with HER2-positive resected breast cancer.Methods: The aims of the study was to analyze the association between baselineserum HER-2/neu and disease-free survival (DFS) in patients randomized to Arms A(standard chemotherapy) and C (standard chemotherapy with concurrent trastuzumab)of N9831. Baseline serum samples of 1423 patients from both arms were analyzed forserum HER-2/neu levels using the ADVIA Centaur® HER-2/neu test (SiemensHealthCare Diagnostics). Normal levels of serum HER-2/neu is less than 15 ng/ml. .Patients who cancelled prior to receiving therapy, ineligible patients, and patientswhose HER-2/neu status was not corroborated by central review were excluded fromanalysis. This study used Cox models stratified by hormone receptor status and nodalstatus to assess the association between serum HER-2/neu levels and DFS.Results: Baseline characteristics between patients with serum HER-2/neu levels <15ng/mL (N=1234) and patients with serum HER-2/neu levels ≥15 ng/mL (N=189)demonstrated differences between groups by age, menopausal status (higher serumHER-2/neu levels in pts ≥ 50 yrs/post-menopausal), and hormonal receptor status(lower serum HER-2/neu levels in hormone receptor + tumors). Hazard ratiosbetween serum HER-2/neu groups within Arm A and within Arm C showed that

pts with serum HER-2/neu ≥≥≥≥15 ng/mL had worse DFS than pts with serum HER-2/neu <15 ng/mL (A: HR=1.71, p =.004; C: HR=1.42, p =.22). Hazard ratios betweenarms within each serum HER-2/neu group demonstrated similar benefit fromtrastuzumab in each serum HER-2/neu group (<15: HR=.66, p=.006; ≥15: HR=.60,p=.10). Results remained consistent when including menopausal status and age in theCox models.Conclusions: 13% of HER2+BC pts of this study had high levels of serum HER-2/neu. In the standard chemotherapy arm, pts with high serum HER-2/neu hadsignificantly worse DFS than patients without. In the concurrent trastuzumab arm, asimilar trend was observed but did not reach statistical significance, potentially due toa smaller number of events

Decrease in serum HER-2/neu levels at 4 to 16 weeks is associated with

prolonged progression-free survival in metastatic breast cancer

patients treated with lapatinib monotherapy

W. P. Carney1, A. Lipton2, K. Leitzel2, S. M. Ali3, G. Platek4, L. O'Rourke4,A. Martin4, J. Maltzman4. 1Oncogene Science/Siemens HealthcareDiagnostics, Cambridge, MA, 2Penn State University, Hershey MedicalCenter, Hershey, PA, 3Lebanon VA Medical Center, Lebanon, PA,4Medicine Development Center, GlaxoSmithKline, Collegeville, PA

Background: The extracellular domain of the HER-2/neu oncoprotein is shed intoserum and can be quantitated using a standardized immunoassay. This studyinvestigated if changes in serum HER-2/neu levels at weeks 4, 8, 12 and 16 after startof lapatinib monotherapy was associated with prolonged progression free survival(PFS).Methods: Study EGF20009 investigated lapatinib monotherapy in 138 HER2-positive patients with metastatic breast cancer who had not previously receivedchemotherapy or trastuzumab. Serum was collected at baseline and every 4 weeksafter initiation of oral lapatinib (1,500 mg daily or 500 mg twice daily). Disease

assessment was performed at week 8, 12 and every 12 weeks thereafter. Disease statuswas assessed by an independent radiology committee, and response was measuredusing RECIST. Serum HER-2/neu levels were measured using the test from SiemensHealthCare DX. A serum HER-2/neu decrease or increase of 20% from baseline wasdefined as a significant change.Results: In this study baseline serum HER-2/neu levels were measured for all 138MBC patients. Of the 138 patients, 109 (79%) % had serum HER-2/neu baselinelevels > above the normal cutoff of 15ng/ml. During the first 16 weeks post therapy,serum HER-2/neu levels were measured every 4 weeks ( 4, 8,1 2 and 16) and thechanges from baseline measured. Baseline serum HER-2/neu levels did not predictpatient response not were baseline serum HER-2/neu levels associated with PFS,however, changes in serum HER-2/neu levels from baseline did have clinicalsignificance. A 20% decrease from baseline of serum HER-2/neu was associated withprolonged PFS, while a 20% increase from baseline was associated with shorter PFS.Patients who experienced a 20% decrease from baseline in serum HER-2/neu levels atweek 4 had a median PFS of 199 days (95% confidence interval [CI] = 145, 253)compared with 135 days (95% CI = 104, 171) for patients who did not (p = 0.016).Similarly, patients who experienced a 20% increase from baseline of serum HER-2/neu at week 4 had a median PFS of 113 days (95% CI = 59, 135) compared with 199days (95% CI = 142, 253) for patients who did not (p < 0.001). Similar associationswere observed at weeks-8,12 and 16.Conclusions: A 20% or greater decrease from baseline of serum HER-2/neu level atweeks 4, 8, 12 and 16 was strongly associated with prolonged median PFS, whereas a20% or greater increase from baseline was strongly associated with shorter medianPFS. When adjusted for changes in tumor burden, serum HER-2/neu was anindependent predictor of median PFS..

Disparity in estimated average glucose due to different hemoglobin

A1c assays and variant hemoglobins

Y. Zhu, L. Williams, B. Horne, M. Jenkins. Medical University of SouthCarolina, Charleston, SC

Background: It has been recommended that estimated average glucose (eAG) valuescalculated using hemoglobin A1c (A1C) results be reported in addition to A1c.However, many A1c assays based on various principles are used in clinicallaboratories, and therefore, different eAG results may be obtained when the sameequation is used. Also, different A1c methods may have variable susceptibility tovariant hemoglobin interference, which may further increase the difference in eAGvalues. The objective of this study is to compare eAG results calculated based on A1cresults measured with BioRad variant II TURBO (BioRad) and Beckman Coulter(Beckman) A1c assays in patients with normal and variant hemoglobins.Methods: BioRad variant II TURBO is an HPLC A1c assay, while Beckman Coultermethod is an immunoturbidimetric assay. Both methods are certified by the NationalGlycohemoglobin Standardization Program (NGSP). Freshly collected blood samplesin EDTA tubes from 129 patients with normal hemoglobin and 42 patients withheterozygous hemoglobin S were measured for A1c with both BioRad and Beckmanassays within 24 hours after collection. eAG values were calculated using thefollowing recently published equation: eAG (mg/dL) = 28.7 × A1c - 46.7. T-test wasused for the statistical analysis.Results: For 129 patients with normal hemoglobin, the mean eAG was 142.5 ± 62.8mg/dL and 133.1 ± 57.8 mg/dL (mean ± SD) based on BioRad and Beckman methodsrespectively. The eAG bias between these two methods was statistically significant (P= 0.0000) with a mean bias of 9.5 ± 10.2 mg/dL. The mean bias is larger for patientswith A1c ≥ 7.0% (17.7 ± 13.5 mg/dL), while it is smaller for patients with A1c < 7%(7.6 ± 8.4 mg/dL) (P = 0.0016). For 42 patients with heterozygous hemoglobin S, theaverage eAGs were 187.1 ± 76.3 mg/dL and 157.8 ± 68.5 mg/dL respectivelydetermined with BioRad and Beckman methods. The mean bias was 29.3 ± 15.4 mg/dL, and the difference was statistically significant (P = 0.000). Similar to patients withnormal hemoglobin, for patients with heterozygous hemoglobin S, the mean bias waslarger for patients with A1c ≥ 7.0% (37.3 ± 16.9 mg/dL, n = 17), while smaller forpatients with A1c < 7% (23.9 ± 11.7 mg/dL, n = 25) (P = 0.009). Further analysisshowed that the mean bias between patients with normal and variant hemoglobinswere statistically significant (29.3 ± 15.4 vs 9.5 ± 10.2 mg/dL).Conclusions: eAG results derived from A1c determined with BioRad and Beckmanassays are not interchangeable when the same equation is applied, although bothassays are certified by NGSP. The bias is more significant in patients with elevatedA1c and in patients with hemoglobins S. Since NGSP allows ± 1% A1c deviationfrom NGSP reference method, the bias in eAG is most likely due to different bias ofA1c assays to NGSP reference method. This problem can be solved by furtherstandardizing A1c assays.

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Infections with Nontyphoidal Salmonella Species Producing CTX-M-

38 and CMY-2 ββββ-Lactamases, in China

C. Jian, Z. Sun, J. Chen. Laboratory Medicine of Tongji Hospital affiliatedto Huazhong University of Science and Technology, Wuhan, China

A total of 102 (0.97%,102/10,464) nontyphoidal Salmonella (NTS) were isolatedfrom 10,464 stool cultures between 2003 and 2005 in Wuhan, China. S. entericaservoar Typhimurium (Stm), serovar Enteritidis (Sen), serovar Derby, serovarNewport, serovar Stanley and serovar Thompson accounted for 51.0% (52/102),28.4% (29/102), 9.8% (10/102), 3.9% (4/102), 2.9% (3/102) and 1.0% (1/102),respectively. Another 2.9% (3/102) could not been typed. The percentage of NTSfrom <1 year of age, 1~4year of age, 4~14 year of age and >14 year of age were61.7%, 25.4%, 2.9% and 10.0%. The susceptibility of NTS to 16 antibiotics showedthat the resistance of extended-spectrum cephalosporins (ESC) was between 2.9% and6.9% (7/102). In 7 isolates, CTX-M-38 ESBLs, CMY-2 AmpC and TEM-1 β-lactamase were all detected in plasmids. 4 were detected above two types of β-lactamases. None of β-lactamase was detected in 2 isolates. In conclusion, NTSinfection was mainly due to Stm (51.0%), following by Sen (28.4%). CTX-M-38ESBLs was firstly detected in NTS, and NTS producing CMY-2 AmpC β-lactamasehas not been reported in China other than Taiwan.

A study on the relationship between apolipoprotein E gene

polymorphism and hepatitis B infection

Z. Yin1, S. Yan2, J. Wang1. 1Medical Laboratory Center, BeijingChuiyangliu Hospital, Beijing, China, 2Department of LaboratoryMedicine, China-Japan Friendship Hospital, Beijing, China

Objective The rate of hepatitis B virus (hepatitis B virus, HBV) infection is currentlythe highest among hepatitis viruses in Han Chinese from northern China. Thedevelopment of different diseases after infection is related to individual on hepatitis Binfection occurred in the immune response and the virus itself factor as well. Researchhas shown that certain genetic polymorphisms can lead to difference on body immunefunction, thus affecting different clinical rehabilitation for HBV patients.Apolipoprotein E (apolipoprotein E, ApoE) gene polymorphism has multiplebiological functions. This study explored the relationship between the HBV infectionand ApoE gene polymorphism so as to provide evidence for the pathogenesis ofhepatitis B from genetic angle.Methods Multiplex Amplification Refractiry Mutation System (Multi-ARMS) wasused to detect apolipoprotein E genotype of 270 cases of hepatitis B patients withdifferent infection models and 112 normal healthy cases, and chemiluminescence wasused to detect serological indicators of hepatitis B for the samples mentioned aboveand HBV DNA qualitative was carried out.Results The frequency of ε2 allele of patients with different models of hepatitis Bvirus infection and HBV DNA infection positive rate were higher than that of thenormal control group, and with statistical significance (P <0.01). Compared to ε3 +ε4, the incidence of risk of hepatitis B in ε2 was more than 1.0 with the positivecorrelation. And also HBV DNA infection positive rate is also higher than normalpopulation (P <0.01)Conclusion There is correlation between Apolipoprotein E genotype and patientswith hepatitis B virus infection. This shows that Han Chinese from northern Chinawho carry ε2 allele are more susceptible to hepatitis B virus.

Bone biomarkers significantly enhances the predictability of

preclinical study outcomes and translation to first in man studies

targeted towards osteoporosis

K. K. Maddali, C. I. Starks, C. McDonough, J. Dharmadhikari, B. A.Litzenberger. Huntingdon Life Sciences, East millstone, NJ

Objective: Translational bone biomarkers applicable to the various preclinical speciescan provide important tools for studying the bone remodeling process in thepreclinical studies in response to Osteoporosis drugs. Circulating osteocalcin (OC) isassociated with changes in the rate of bone turnover and regarded as a specific markerfor bone formation. Recent studies have indicated that serum OC may provide a

useful, non-invasive indicator of skeletal health in preclinical species and humans.The rat is a common preclinical species utilized for assessing the responses of drugtoxicities in preclinical drug development.Method: In this study, the investigational drug is intended for osteoporosis therapyand the osteocalcin biomarker is an indicator of a drug’s efficacy at various doses inhealthy 6-7 week old normal rats. We describe the evaluation and application of anOsteocalcin (OC) enzyme-linked immunosorbent assay (ELISA) for measuringresponses to bone formation in rat serum. The Rat Osteocalcin ELISA (IDS) is anenzyme-linked immunosorbent assay for the quantitative determination of osteocalcinin rat serum and plasma. The Rat-MID™ Osteocalcin EIA is based upon thecompetitive binding of a monoclonal antibody to soluble osteocalcin or toimmobilized osteocalcin. Briefly, the monoclonal antibody is raised against humanosteocalcin and recognizes the mid-molecular part (amino acids 21-29) of themolecule.Results: The sensitivity of the OC ELISA was 50 ng/ml. The averaged intra- andinterassay variation was 4.36% and 7.43%. Averaged spiked recovery was 108 ng/ml.In vivo validation of osteocalcin ELISA was performed in 6-7 weeks old Sprague-Dawley (SD) rat models treated with PTH (Para Thyroid Hormone) analog; 200 ug/kg. In treated rats, the osteocalcin concentrations were approximately 35%, higherthan the sham group and exhibited acceptable sensitivity, accuracy, and selectivitynecessary to evaluate bone formation in a normal rat model.Conclusion: In conclusion, the inclusion of osteocalcin, a bone biomarker forassessing bone formation mechanisms in preclinical and clinical drug developmentprograms for osteoporosis adds value by providing additional information about themechanism of action of the investigational drug.

The Ordering Patterns of Serological Tests in the Diagnosis of Celiac

Disease

Y. Huang, A. C. Don-Wauchope, M. Mansour, V. L. Grey. Department ofPathology and Molecular Medicine, McMaster University, Hamilton, ON,Canada

Objectives: Current guidelines for the diagnosis of celiac disease in children includerecommendations to measure tissue transglutaminase antibodies (tTG). In adults thereare similar recommendations. We designed a laboratory audit to review the orderingpatterns of serological tests in celiac disease.Methods: The specimen number, age of patient, serological test result, and orderingphysician were extracted from the Laboratory Information System over three months atMcMaster University Medical Centre. The specimen number was used as the primaryidentifier to determine the ordering patterns. Intestinal biopsy results were retrieved forthose with a positive serology test and an age matched control group with negativeserology. Frequency data was analyzed by Fisher Exact Test or Chi-Square Test.Results: 1152 serological tests were ordered for 666 patients in three months (47.6%<=18y). The majority of pediatric sub-specialists ordered tTG alone , significantlydifferent (p <0.001) from the other groups. In adult practice the ordering pattern wassimilar across groups with the majority ordering celiac panel (Table 1). The overallpositive rate of tTG and celiac panel were 8.3% and 20.9% (tTG-Gliadin+ 15.7%). Thebiopsy reports in the following 6 months revealed that 75.0% tTG positive patients hadan intestinal biopsy which is similar to the celiac panel group (83.3%), when all 3 testswere positive. Fewer patients had a biopsy (37.5%) with a positive tTG and a negativegliadin (IgA or IgG). A negative tTG result with a positive gliadin (IgA or IgG) reducedthe biopsy rate to 24.6%, while with a negative gliadin to 16.0%.

Conclusions: The pediatric sub-specialists preferentially ordered tTG for children. Inadult practice, celiac panel was most common. Celiac panel appeared to influencebiopsy rate when the tTG and Gliadin results did not agree. These findings need to beconfirmed by a chart review.

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The ordering patterns of serological tests in celiac diseaseGastroenterologist and immunologist sub-specialization

PediatricianOther

hospital physician

Non-hospital

physicianNumber (%) of physicians 24(11.3) 23(10.9) 50(23.7) 114(54.0)

Number (%) of tests ordered 422(36.6) 231(20.1) 161(14.0) 338(29.3)

Test ordering patterns%

Children: Anti-tTG IgA (p<0.001) 71.1 40.3 15.8 10.2

Celiac panel * 21.0 59.0 63.2 77.3Adults: Anti-tTG IgA 8.9 --- 8.1 3.7

Anti-Endomysial IgA

16.1 --- 5.4 1.9

Celiac panel 69.6 --- 78.4 80.4* Celiac panel: Anti-tTG IgA and Anti-Gliadin IgA, IgG

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The clinical application of fully automated urine cell analyzer in rapid

diagnosis of urinary tract infections in children

Q. Wu, Y. Li, M. Wang, Z. Xia, H. Hu. Renmin Hospital of WuhanUniversity, Wuhan, China

Objectives: To evaluate the clinical application of fully automated urine cell analyzer inthe diagnosis of urinary tract infections (UTI) in children. Methods: We considered 322subjects, aged between 0 and 12, 139 males and 183 females. The samples were strictlycollected by using the midstream technique. Each sample was subjected tomicrobiological evaluation bacteria culture, dipstick tests and fully automated urine cellanalyzer examination. The results of the urine cell analyzer and urine bacteria culturewere compared to evaluate the value of the application of urine cell analyzer indiagnosis of UTI in children. Results: Out of the total 322 subjects 105 cases wasconsidered of UTI (account for 32.61%). The urine cell analyzer-based screeningmethod had a sensitivity of 0.89, a specificity of 0.87, a positive predictive value of0.72, a negative predictive value of 0.95 and a correctly classified incidence of 0.93.Conclusion: In this study, we considered that the bacteria counting cut-off value of UTIwas 3000/µl. The results of the urine cell analyzer-based screening method show a verygood correlation with the diagnosis of UTI in children,and have a better sensitivitycompared with the results (sensitivity 0.69) of dipstick tests in the same conditions.

Establishment of the accurate cutoff index and 95% interval for the

HBsAg qualitative test in Chinese population

Y. Li, H. Zhang, L. Song. Renmin Hospital of Wuhan University, Wuhan,China

Objectives: The Hepatitis B surface antigen kit (electrochemiluminescenceimmunoassay assay, ECLIA) has stated the borderline as 0.9-1.0 (COI) for the HBsAgqualitative test, but whether this borderline is suitable for Chinese people is unknown.Our purpose was to verify and establish the accurate cutoff index (COI) and 95%interval for the HBsAg qualitative test (ECLIA) in Chinese population. Methods:Following the NCCLS global consensus guideline, a series dilutions was made for thisstudy such as 0.32, 0.64, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.4,2.5, 5.0, 10.0, 16.0, 32.0 (COI). Every dilution were tested for 20 times, then determinedthe percentage of positive /negative results for each sample. It should be the accuratecutoff index at which concentration we can yield 50% positive results and 50% negativeresults. Test the concentration above / below the cutoff index, if we can yield 95%positive / negative results, the 95% interval can be determined. According to this cutoffpoint and 95% interval, we analyzed 100 clinical samples. All reactive results orborderline samples were investigated using an independent neutralization test (HBsAgConfirmatory Test). Negative predictive value (NPV) and positive predictive value(PPV) were calculated at last. Results: Not at 1.0 COI but at 1.4 COI, the percentages ofthe positive results and negative results were both 50%; and not at 0.8 and 1.2 COI, butat 0.8 and 2.0 COI, the percentages of the negative results and positive results were both95%. NPV and PPV of the 100 clinical samples were both 100%. Conclusion: It wassuggested that the accurate cutoff index for the HBsAg qualitative test (ECLIA) maybe1.4 COI in Chinese population; the proper 95% interval maybe 0.8-2.0 COI.

THE PREVALENCE OF GESTATIONAL DIABETES AT THE

UNIVERSITY OF PORT HARCOURT TEACHING HOSPITAL -

INTERIM RESULTS OF A PROSPECTIVE STUDY

A. A. Ejilemele1, S. A. Uzoigwe2, O. N. Okike2. 1Department of ChemicalPathology, University of Port Harcourt, Port Harcourt, Nigeria,2Department of Obstetrics and Gynaecology, University of Port Harcourt,Port Harcourt, Nigeria

Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance withonset or first recognition during pregnancy. It affects up to 14% of pregnant womenand is commoner in those who are obese, older than 25 years, have a previous historyof abnormal glucose metabolism or poor obstetric outcome, have first-degree relativeswith diabetes, or are members of ethnic groups with high prevalence of diabetes. Inthis environment, there is no available data on the prevalence of GDM or therelationship between GDM, one abnormal glucose value (OAV) and adverse

pregnancy outcomes. We therefore conducted this study to determine this relationshipif any.Women presenting at the antenatal clinic from July 1 - December 31 2008 wereevaluated for risk factors for development of GDM. All women who had more thanone risk factor were recruited into the study group. All patients underwent the 75g - 2hour oral glucose tolerance test. GDM was diagnosed according to the criteria set bythe American Diabetes Association. Glucose was measured by the glucose oxidasemethod in the hospital laboratory.After a six month period, a total of three hundred and sixty four patients wererecruited into the study. OGTT was more likely to be requested if the woman wasobese, had a family history of diabetes mellitus, previous history of GDM or had ahistory of poor fetal outcomes. The mean fasting, 1 hour and two hour blood glucosefor the study population was 4.05 ± 1.16, 6.4 ± 1.66, 5.64 ± 1.86 respectively. Theprevalence of GDM was 6.6% while that of one abnormal value was 4.7%. The resultsof the study of the adverse effects of GDM on pregnancy are ongoing.

SERIAL MEASUREMENTS OF B-TYPE NATRIURETIC PEPTIDE

(BNP) IN PATIENTS UNDERGOING CARDIAC SURGERY

H. Turner, J. Reeve, J. McNeilly, W. Mutch, R. Peake, D. Rae, P. Gibson,G. Hillis, B. Cuthbertson, B. Croal. Aberdeen Royal Infirmary, Aberdeen,United Kingdom

Background. Patients undergoing cardiac surgery are ultimately at risk of significantmorbidity and mortality in the post-operative period. While various scoringmechanisms exist to assist in the prediction of such adverse outcome, these arecomplex and not always reliable. We have looked at the significance of serialmeasurements of BNP taken before and after cardiac surgery.Methods. 331 consecutive patients undergoing cardiac surgery had samples assayedfor BNP (Siemens ADVIA Centaur) prior to surgery and at 6 and 24 hours followingsurgery. Patients were followed up in the immediate post-operative period and for oneyear following surgery.Results. BNP levels (pg/L) increased from baseline values through to 6 and 24 hours(median 62.1 v 135.2 v 305.8 respectively; p <0.001). In patients who subsequentlydied (n=14), BNP levels were higher at baseline (108.1 v 61.4; p=0.062), 6hrs (239.9v 134.0; p=0.002) and 24hrs (642.9 v 294.0; p<0.001) following surgery. ROCanalysis for the prediction of mortality using BNP levels taken 24hrs post surgerydemonstrated high diagnostic accuracy with an area under the curve of 0.841 (p <0.001). In addition a multivariate logistic regression model, indicated, that BNPmeasured at 24hrs remained the best predictor of mortality (p=0.014) even whenadjusted for age, sex and established risk scoring systems (Parsonnet and Euroscore).In a similar adjusted multivariate regression model, patients with a BNP level in thehighest quartile at 24 hours were more than 6 times likely to die (OR 6.33; p=0.003).

Conclusion. BNP levels measured post-operatively provide unique prognosticinformation regarding the short and long term mortality that is independent of alreadyestablished clinical risk scoring systems. Such information may allow direction ofmore aggressive treatment and follow up.

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Association between tumor necrosis factor-ββββ polymorphism andcoronary heart disease in a Chinese population

M. Wang1, W. Li2, Y. Li1. 1Renmin Hospital of Wuhan University, Wuhan,China, 2Department of Hematology, Renmin Hospital of Wuhan University,Wuhan, China

Objectives: To investigate the association between tumor necrosis factor-β (TNF-β)gene polymorphisms as well as its serum levels and coronary heart disease (CHD) in aChinese population. Methods: Polymerase chain reaction- sequence specific primers(PCR-SSP) was used for the detection of TNF-β C804A genotype in 210 patients withCHD and 186 controls. The serum TNF-β levels were measured by enzyme-linkedimmunosorbent assay (ELISA). Results: The frequencies of CC, CA and AAGenotypes of C804A were 25.7% and 37.1%, 49.5% and 45.7%, 24.8% and 17.2%,patients and controls respectively. There were statistically significant differences inthe distributions of the genotypes (P<0.05) and the allele frequencies (P<0.05)between two groups. The relative risk suffered from CHD of AA and CA genotypeswas 1.704 times of the CC genotype (OR=1.704, 95%CI: 1.109~2.617). The serumTNF-β and high sensitive C-reactive protein (hsCRP) levels of the patients weresignificantly different from those of the controls (both P<0.05), however, amongdifferent TNF-β genotypes of patients and controls respectively, there were nosignificant differences. Conclusion: The single nucleotide polymorphism at position804 in the exon 3 of TNF-β gene is associated with CHD and the allele A may be arisk factor for CHD in Chinese.

THE SIGNIFICANCE OF CYSTATIN C AND ITS DERIVED eGFR

IN PATIENTS UNDERGOING CARDIAC SURGERY

J. D. McNeilly1, J. L. Reeve2, H. Turner2, R. Peake2, W. J. Mutch2, D.Rae3, P. H. Gibson3, G. S. Hillis3, B. H. Cuthbertson3, B. L. Croal2.1Department of Clinical Biochemistry, Royal Hospital for Sick Children,,Glasgow, United Kingdom, 2Department of Clinical Biochemistry,Aberdeen Royal Infirmary, Aberdeen, United Kingdom, 3Department ofCardiology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom

Background: Renal dysfunction, as assessed by a reduced estimated glomerularfiltration rate (eGFR) is a potentially useful predictor of outcome in patients undergoingcardiac surgery. Cystatin C, a cysteine protease inhibitor, and its derived eGFR, arepotential alternatives which have the added advantage of improved sensitivity,specificity and less inter/intra-individual variability. We assessed the significance of pre-operative cystatin C in comparison to creatinine-based risk assessments in predictingsubsequent mortality in patients undergoing cardiac surgery.Method: 973 patients undergoing cardiac surgery had serum cystatin C (Siemens,Prospec) and creatinine (Siemens, Advia 2400) measured pre-operatively. GFR wasestimated by the adjusted 4-variable MDRD equation using creatinine (eGFR-Cr) andan equivalent equation using cystatin C (eGFR-CC). Patients were followed up for 1year.Results: Pre-operative creatinine and cystatin C levels were higher in those patientswho subsequently died in the first year following surgery (creatinine, 111 v 102 µmol/L;p=0.004) (cystatin C, 1.13 v 0.93 mg/L; p<0.001). Similarly, both eGFR-Cr (58.9 v69.9;p<0.001) eGFR-CC (72.6 v 100.6;p<0.001) levels were lower in patients whosubsequently died. Using Kaplan-Meier survival curve analysis, patients in the higherquartile for creatinine (p<0.001) and cystatin C (Figure 1) demonstrated highermortality Both eGFR-Cr and eGFR-CC remained independent predictors of mortality ina multivariate regression model adjusted for EuroScore, an established objectiveassessment of risk (eGFR-CC p=0.029, eGFR-Cr p=0.020)

Conclusion: This study demonstrates the potential usefulness of pre-operativemeasurements of creatinine, cystatin C and their derived estimates of GFR as riskprediction tools in patients undergoing cardiac surgery.

Renal function markers in the prognosis of patients presenting with

chest pain

J. L. Reeve1, H. Turner1, W. J. Mutch1, V. Mills2, J. D. McNeilly3, R.Peake1, J. Allison1, G. Hillis2, R. Soiza2, B. Croal1. 1Department of ClinicalBiochemistry, Aberdeen Royal Infirmary, Aberdeen, United Kingdom,2Department of Cardiology, Aberdeen Royal Infirmary, Aberdeen, UnitedKingdom, 3Department of Clinical Biochemistry, Royal Hospital for SickChildren, Glasgow, United Kingdom

Background: We have assessed the value of several markers of renal dysfunction(cystatin C, creatinine, eGFR and Neutrophil Gelatinase-Associated Lipocalin(NGAL), a novel marker of acute kidney injury) in predicting mortality in patientspresentating with chest pain.Methods: Admission levels of serum creatinine (umol/L; Siemens ADVIA 2400),cystatin C (mg/L; Siemens ProSpec) and their derived estimates of GlomerularFiltration Rate (eGFR-Cr and eGFR-CC, respectively) were measured in 210 patientspresenting with chest pain to a Coronary Care Unit. Serum NGAL (ng/mL; ELISA,AntibodyShop) was measured at 12 hours, while creatinine was monitored daily untildischarge. Patients were followed up for 6 months for subsequent mortality.Results: Levels of cystatin C (1.53 v 0.93; p=0.001) and peak creatinine (189 v 103;p=0.001) were higher in patients who subsequently died (n=16). Similarly, levels ofeGFR-Cr (48.8 v 77.8; p=0.010) and eGFR-CC (43.8 v 102.3; p=0.001) were lower inpatients who subsequently died. Serum NGAL levels were also higher in patients whodied, however this was not significant (250.1 v 207.1; p=0.159). Kaplan Meiersurvival curve analysis demonstrated higher mortality for those patients within thehigher quartiles for peak creatinine (p=0.002) and cystatin C (p=0.001), and the lowerquartiles for eGFR-CR (p=0.017) and eGFR-CC (p=0.001). In a regression model, acreatinine or a cystatin C level in the upper quartile equated to a much higher risk ofdeath by 6 months (OR 5.94, p=0.001; OR 11.79, p<0.001, respectively). Cystatin Cin the upper quartile remained an independent predictor of mortality even whenadjusted for the GRACE score prediction of mortality at 6 months (OR 10.35;p=0.006). Serum NGAL quartile status was significantly related to subsequent peakcreatinine levels (p<0.001) (see Figure).

Conclusions: Serum NGAL levels may predict future renal function deterioration;however, creatinine and cystatin C serve as more useful predictors of mortality.

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THE ROLE OF URINARY NGAL TO URINARY CREATININE

RATIO IN THE EARLY DETECTION OF CONTRAST AGENT

INDUCED ACUTE KIDNEY INJURY AFTER CORONARY

ARTERY ANGIOGRAPHY

K. Makris1, C. Demponeras2, F. Zoubouloglou1, S. Potamitis1, N. Kafkas2,I. Drakopoulos1, D. Rizos3, A. Nikolaou2, D. Babalis2, A. Haliassos4.1Clinical Biochemistry Department, KAT General Hospital, Athens,Greece, 2Cardiology Department, KAT General Hospital, Athens, Greece,3University of Athens, Aretaieio Hospital, Hormones Laboratory, Athens,Greece, 4National External Quality Assessment Scheme in ClinicalChemistry (ESEAP), Athens, Greece

Acute kidney injury (AKI), usually defined by an increase in the serum creatinine>0.5mg/dL or >25% from baseline, is a common complication of contrast agent usedduring percutaneous coronary interventions (PCI). This increase typically occurs 3-5days after contrast administration when patients are already discharged.Neutrophil gelatinase-associated lipocalin (NGAL) is highly accumulated in thehuman kidney cortical tubules, blood and urine after nephrotoxic and ischaemicinjuries, has been proposed as an early, sensitive biomarker for AKI.The aim of our study was to evaluate the use of the urinary-NGAL/urinary-creatinineratio in order to detect contrast-induced AKI. This ratio was used in order tonormalize the urine NGAL concentrations due to the different levels of diuresisamong our patients. Sixty patients undergoing either coronary angiography alone(n=21) or angioplasty with stenting (n=39) were included. According to admissionserum creatrinine (>1.2mg/dL), saline and/or n-acetylcysteine (NAC) administrationwas decided by the cardiologist. Low-osmolar contrast agent (iodixanol) was used inall patients. Serum and urine samples obtained just before PCI (baseline), 6-hoursafter contrast administration and 24 and 48 hours thereafter. NGAL was measuredwith ELISA (Bioporto,Gentofte,Denmark). Urine and serum-creatinine weremeasured with Jaffe method, and cystatin-C with immunoturbidometric assay onArchitect-16200 analyzer (Abbott Diagnostics, Abbott Park,Il).Table summarises our results.

Normal renal function observed in 42 patients at baseline (group-I) while renalimpairment (cystatine>1.0mg/L) in 18(group-II). Ten patients from group-I developedAKI (mean serum-creatinine increase 28.9%), while deterioration of kidney function(mean serum-creatinine increase 27.4%) was observed in 8 patients from group-II. InAKI-patients urinary-NGAL/urinary-creatinine ratio at 6hours was significantlyincreased from baseline as well as compared with non-AKI patients at the same time-point. Similar increase observed among the patients who did not receive NAC.We conclude that urinary-NGAL/urinary-creatinine ratio can be used to early identifypatients that develop AKI due to contrast administration.

24-Hour Bence-Jones Protein Determinations: Can We Insure

Accuracy?

J. Kaplan, G. Horowitz. Beth Israel Deaconess Medical Center, Boston, MA

Current guidelines recommend that Bence-Jones proteinuria be monitored with 24-hour urine collections. Implicit in this recommendation is that the collections beaccurate, but this is frequently not the case. We hypothesized that, in practice, Bence-Jones quantitation by this technique is often misleading and that, instead, one can userandom urine protein/creatinine ratios to provide better information.We reviewed data (urine creatinine (UCR), protein (UTP), total volume, andproportion of Bence-Jones protein (%BJP)) from 2003 through 2008 on all BIDMCpatients with Bence-Jones proteinuria who had 24-hour urine collections for proteinand creatinine. In addition, for these same patients, we found all other random urinesamples on which UCR, UTP, and %BJP were reported.We focused first on those patients who had more than four 24-hour collections. Therewere a total of 14 patients to evaluate, with 135 24-hour urine protein collections. Wefound that the urine creatinine excretion values for each patient, which should bereasonably constant, varied considerably (CV range 12%-39%). We then calculatedthe BJP protein excretion based on the 24-hour protein (i.e., assuming an accuratecollection) and based on a corrected value determined from that patient’s mean 24-hour creatinine excretion. The differences in these values ranged from -1377 to 2374mg/day.We looked at differences in serial 24-hour Bence-Jones excretion (mg/day) by eachmethod. We initially defined clinically significant as values with opposite signs (i.e.,an increase by one measure but a decrease by the other) or values whose magnitudewas at least 2-fold different and >100 mg/day. With these criteria, of 121 events, 44were clinically significant. We lowered this number to 37 by inspecting the data in thecontext of the patients’ overall level of BJP. Three patients had no clinicallysignificant differences; among the other eleven patients, the proportion of sampleswith clinically significant differences ranged from 17% to 80%. For all 14 patients,the median was 30%.We then evaluated the reliability of random urine samples to estimate 24-hour Bence-Jones excretion. From our original database, there were 23 random urine samples,from 11 different patients, on which BJP was measured within 10 days of a 24-hoururine collection. In 18 of 23 cases, the random sample gave results that wereconsistent with the clinical scenario and the normalized BJP on the corresponding 24-hour sample. In 4 of the remaining 5 cases, the random sample was probablyconsistent, but we did not have access to enough clinical data to be certain.We conclude that 24-hour urine collections for Bence-Jones proteinuria can, inpractice, often be misleading. At a minimum, one should verify that the 24-hourcreatinine excretion is accurate. In addition, it may be possible to use the protein/creatinine ratio from random urine samples to determine 24-hour Bence-Jonesexcretion.

Pulmonary Hypertension in Patients with Graves' Disease

T. Sugiura, H. Kataoka, N. Morimoto, T. Hisahara, S. Yamanaka, H.Takeuchi, Y. Kumon. Kochi Medical School, Nankoku, Japan

Back ground: Thyroid hormone affects almost all organs of the body includingcardiovascular system. The aim of this study was to determine the frequency andclinical correlates of pulmonary hypertension in patients with Graves' disease.Methods and Results: Fifty consecutive patients with Graves' disease were studiedby echocardiography and laboratory findings. Among 50 patients with Graves'disease, 14 patients (28%) had pulmonary hypertension (pulmonary artery systolicpressure ≥40mmHg calculated as the sum of the trans-tricuspid pressure gradient plus10mmHg). Although there was no significant difference in cardiac output between thetwo groups, patients with pulmonary hypertension had significantly higher free T3(12.68±7.45 vs 8.72±4.85pg/ml) and thyroid stimulating hormone receptor antibody(82.7±98.9 vs 18.9±22.9lu/l) compared to those without (p=0.031 and p<0.001,respectively). Pulmonary artery systolic pressure had a weak correlations with free T3(r=0.36, p=0.010), and a fair relation with thyroid stimulating hormone receptorantibody (r=0.52, p<0.001).Conclusion: Pulmonary hypertension was a relatively common complication inpatients with Graves' disease, and was associated with more severe hyperthyroid state.Moreover, autoimmune process of the pulmonary artery seems to be anotherimportant factor causing pulmonary hypertension in Graves' disease.

Group I Group II

* mean (range)Baseline cystatine

<1mg/LBaseline cystatine

>1mg/LNo AKI AKI NAC No NAC p value

n 32 10 10 8Age* 58.7(39-81) 62.2(49-74) 68.2(52-82) 68.6(48-80)contrast volume (mL)*

356(90-800)

426(130-1100)

290(80-740) 295(140-750)

stable angina/UA+AMI

10/20 4/6 2/8 2/6

uNGAL/uCREA baseline (ug/mg)*

0.43(0.04-2.23)

1.83(0.10-4.14)

3,75(0.02-8.93)

9,48(1.10-25.08)

p<0.001

uNGAL/uCREA at 6hrs*

2.48(0.18-7.20)

9.89(1.13-15.40

5.69(0.27-17.44)

17.07(4.83-29.87)

p<0.001


1.15(0.06-4.54)

9.69(1.09-24.24)

4.87(0.43-12.02)

12.60(4.67-19.26)

p<0.001


0.77(0.07-3.76)

7.83(0.35-18.01)

4.64(1.10-11.32

8.83(1.76-15.08)

p<0.001

p-value p<0.001 p<0.001 p=NS p<0.001sCreatinine at baseline (mg/dL)

0.86 0.83 1.24 0,95 p<0.001

sCreatinine change (%)

0.0% 28.9% 5.7% 27.4%

sCystatine at baseline (mg/L)

0.82 0.80 1.23 1.11 p<0.001

Serum cystatine change (%)

-2.4% 25.0% 0.0% 21.62%

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The relationship between the levels of serum testosterone and bone

metabolic biochemical markers in elder males

Z. Zhao1, F. Wang1, X. Yang1, Z. Jiang1, Y. Li2. 1Department of Geriatric,Renmin Hospital of Wuhan University, Wuhan, China, 2Department ofClinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, China

Objectives: Bone metabolic biochemical markers were associated with osteoporosis,and the levels of serum androgen also are associated with primary osteoporosis inelder males. However, a few studies have specifically addressed the relationshipbetween the levels of androgen and bone metabolic biochemical markers in eldermales. We assessed the relationship between the levels of serum testosterone and bonemetabolic biochemical markers in elder males. Methods: 186 elder males weredivided to three groups according to stage of age, 64 with Group A (60-69 years old),68 with Group B (70-79 years old), 54 with Group C (≥80 years old), respectively.The levels of serum testosterone (T), bone glaprotein (BGP), procollagen I C-terminalpeptides (PICP) were determined with ELISA method. Alkaline phosphatase (ALP)and urinary deoxypyridinoline (DPD) were determined with chemical luminescenttechnique, respectively. Results: As increasing age in elder males, the levels of serumT, BGP, PICP and ALP decreased, but the levels of urinary DPD increased (P<0.05 orP<0.01).The correlation analysis showed that the levels of serum T were positivecorrelation with the levels of serum BGP (r=0.57, P<0.05) and that were negativecorrelation with the levels of urinary DPD (r= -0.49, P<0.05). Conclusion: Our studysuggests that the levels of androgen are closely related to bone metabolism in eldermales. The change of levels of androgen plays an important role in primaryosteoporosis in elder males.

Personalized Medicine in the Treatment of Myasthenia Gravis

K. Nakatani1, Y. Sakamoto2, J. Nishioka2, N. Kawaguchi3, T. Nobori1. 1MieUniversity Graduate School of Medicine, Tsu, Mie, Japan, 2Mie UniversityHospital, Tsu, Mie, Japan, 3Chiba University Graduate School ofMedicine, Chiba, Chiba, Japan

[Background] Myasthenia gravis (MG) is a prototypic antibody-mediatedneurological autoimmune disorder. Thus, a wide array of immunosuppressivetreatments has been established. In addition to the most popular drug, glucocorticoid,the calcineurin inhibitors (CNI), tacrolimus and ciclosporin, became also usefulimmunosuppressive drugs for the treatment of MG. CNI are widely used after organtransplantation but has a narrow therapeutic range and its pharmacokinetic variabilitycomplicates its daily dose assessment. CNI are metabolized by cytochrome-P4503A(CYP3A) enzymes and CYP3A5 gene polymorphism has been shown to influencetacrolimus blood concentration. In this study, it was investigated whether appropriateadministration of tacrolimus for each subject was determined using CYP3A5genotype.[Subjects] 74 Japanese subjects with MG were recruited in Chiba University Hospital.This study was approved by the ethics committee of both Mie and Chiba Universities,and written informed consent was obtained from each subject.[Methods] Tacrolimus administration was started at 3 mg/day and the maintenancedosage was adjusted to target trough levels. DNAs were extracted from pieces of nailobtained from each subject, and CYP3A4 (A-392G, *1B) and 3A5 (A6986G, *3) weregenotyped by single primer extension method. To evaluate the effects of CNI and thepossibility of substitution of CNI for predonisolone (PSL) with strong side effects,MG activities of daily living (MGADL) scale and the dose of PSL were assessed atbaseline and 4 months later.[Results] Among 74 recipients, CYP3A4 *1B allele was not observed. The genotypefrequencies of CYP3A5 were 10.8% for *1/*1, 29.7% for *1/*3 and 59.5% for *3/*3.As 3 allele is known to possess no metabolic activity for tacrolimus, recipientswithout *1 allele showed significantly higher plasma concentration of tacrolimus thanthose having CYP3A5 with 1 at trough level. Moreover, recipient with CYP3A5 *1allele needed the higher maintenance dose of tacrolimus than those without *3 allele.Therefore, the maintenance dose of tacrolimus was strongly influenced by CYP3A5genotype but ciclosporin showed no infuence of CYP3A5 genotype.PSL dose could be decreased at 4 months after administration of both tacrolimus andciclosporin. Moreover, MGADL scores were similarly improved by both CNI. Thus,two CNI showed similar effects on MG treatment.[Conclusion] The great influence of CYP3A5 on the pharmacokinetics andpharmacodynamics of tacrolimus in recipients with MG suggests the need for pre-treatment screening of this polymorphism to improve tacrolimus therapy. Two CNI

with different pathway demonstrated the similar effects on the treatment of MG. Todate, use of the CYP3A5 genotype to guide the drug choice of CNI and predictappropriate dose of tacrolimus is the most promising option for individualization ofdrug therapy in MG.

Evaluation of clinical application of HBsAg confirmatory test for

samples with poor reactive HBsAg

J. Shi, Y. Li, J. Yang. Renmin Hospital of Wuhan University, Wuhan, China

Objectives: To Evaluate the clinical application of the HBsAg confirmatory test forsamples with poor reactive HBsAg. Methods: Out of 24281 serum samples routinelytested by electrochemiluminescence immunoassay (ECLIA) method there were 200samples of poor reactive HBsAg. The results of these samples were from 0.9 to 10.0COI. Both HBsAg Confirmatory Test and Enzyme Linked Immunosorbent Assay(ELISA) were performed on these 200 samples, and the results of both werecompared. Results: Total reactive result samples by HBsAg confirmatory test have197, the coincidence rate was 98.5%(197/200); The COI value in 0.9~1.5 sample have19, the confirmatory coincidence rate was 89.5%(17/19); The COI value in 1.5~2.0sample have 33, the confirmatory coincidence rate was 97%(32/33); The COI> 2.0sample confirmatory coincidence rate was 100%; The reactive results samples by theELISA test have 86 only because the sensitivity limit, the coincidence rate is43.7%(86/197)and the undetected rate was 56.3%(111/197). Conclusion: For thesamples with poor reactive HBsAg, undetected rate of ELISA test is very high, andthe sensitivity is not good. Therefore, patient and blood donor HBsAg screeningshould be performed using an automated immunoassay. Moreover, it was suggestedthat HBsAg confirmatory test should be performed for poor reactive HBsAg samplesthat COI rang from 0.9 to 1.5.

Hyperadiponectinemia predicts mortality in hemodialysis (HD)

patients

J. Racek1, M. Vostry1, D. Rajdl1, J. Eiselt1, L. Malanova2, T. Ladislav1.1Charles University, Faculty of Medicine, Pilsen, Czech Republic, 2B.Braun Avitum, Pilsen, Czech Republic

Introduction and aims. Low adiponectin (ADPN) level is associated with adversecardiovascular outcome and increased mortality in a variety of clinical conditions.Nevertheless, studies in HD patients addressing the ADPN value for survival andoutcome prediction give inconsistent results.Methods. We performed a prospective cohort study with 204 HD patients (median[IQR] of age = 68 [59.8-74] years, dialysis duration = 16 [5-38] months, BMI = 26.5[23.9-30.1] kg/m2, 76 males). Fasting serum total ADPN levels were measured usingcommercial ELISA kit.Results. During the follow-up period (28 [10-36] months) 92 patients died (45%).Non-survivors had significantly higher ADPN levels than patients who survived(22.76 [15.87-35.03] vs. 16.65 [23.9-30.1] mg/l, 95% CI for difference 2.68-9.00 mg/l, p<0.001). Survival analysis was performed by Kaplan-Meier plot and by a Coxmodel. In a multivariate Cox regression model (adjusted for age, sex, BMI, HDduration, albumin and CRP level) ADPN emerged as significant independentpredictive factor for all-cause mortality (relative risk per tertile 1.39, 95% CI 1.04-1.85, p = 0.025).Conclusions. Our results indicate that higher ADPN independently predicts all-causemortality in HD patients. This study is not the first to show such association and theresults support the hypothesis that in high-risk conditions such as uremia ADPN maynot mediate its protective effects and rather reflects the disease severity. In thiscontext, ADPN could promote and also mirror the progressive wasting of HDpatients.The study was supported by research project MSM 0021620819.

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Effects of heart operations under mild and profound hypothermal

cardiopulmonary bypass on red-cell immunity.

J. HU, Y. TIAN. Department of Clinical Biochemistry, PLA GeneralHospital, Beijing, China

Siegel raised the theory of red-cell immune system according to many research resultsof predecessors and those of themselves in 1981. It is suggested from the theory thatred cells have an immune function as well as a respiratory one. There are manymaterials on surface or in cytoplasm of erythrocytes associated with red-cellimmunity, among which complement receptor type 1 (CR1, also named CD35),membrane attack complex inhibition factor (MACIF, also named CD59) and Duffyantigen receptor for chemokines (DARC, also named ECKR) are especiallyimportant. CR1 mediates the binding of immune complexes(IC) to erythrocytes,which is the passage of ridding IC from the circulation. DARC can regulate plasmalevels of chemokines. While the binding of CD59 on erythrocytes to CD2 on Tlymphocytes can activate and modulate T lymphocyte immunity. As cardiopulmonarybypass (CPB) is not a physiological circulation, many complications happen in singleor multiple organs, which are mainly due to the damage of hematological system. Thefact that RBCs are severely damaged in morphology and respiratory function afteroperation under CPB provokes our interests to know how about red-cell immunity inthat condition. Eighteen patients with heart operation under CPB, 10 with mildhypothermia and 8 with profound hypothermia, were included in this study. Venousblood was drew to tube with EDTA at pre-CPB, minimum body temperature, end ofCPB, and 1 day, 3 days, 7 days after operation. The level of CD35 and CD59 weremeasured with flow cytometry by fluorescence-activated cell sorter, and the resultswere showed as geometric mean fluorescence intensity ratio (GMFIR). The resultsshowed that there were the same tendencies in mild hypothermal CPB (MHCPB)group and profound hypothermal CPB (PHCPB) group that CD35-GMFIR andCD59-GMFIR decreased at time of minimum body temperature and end of ECCcompared to that at pre-CPB, and then turned back on 1 day, 3 days, 7 days afteroperation. But the descend extents of CD35-GMFIR and CD59-GMFIR in MHCPBgroup were bigger that those in PHCPB group, while the recovery period were longer.We conclude that the immune function of RBCs is damaged by heart operations underCPB, and the extent of red-cell immunity damage in MHCPB group is more seriousthan that in PHCPB group.

Clinical evaluation of a new Cystatin C Assay on the Abbott Architect

cSystems and on AEROSET Clinical Chemistry Systems:

Commutability and eGFR.

F. Rota1, G. Borsani1, V. Maffulli1, R. Lucini1, J. Bjoerk2, M. Fritz3, J.Herzog3, H. Troonen3. 1Sentinel CH., Milan, Italy, 2Competence Centre forClinical Research, Lund University Hospital, Lund, Sweden, 3AbbottGmbH & Co. KG, Diagnostics, Delkenheim, Germany

Objective: To introduce the new Cystatin C assay on the ABBOTT ClinicalChemistry Systems. This is primarily based on robust assay standardisation. We havegenerated clinical data documenting commutability of results and equivalency ofreference range with the nephelometric immunoassay (PENIA). New eGFR equationswere derived and compared with those published.Materials/Instruments: The new Cystatin C assay, SENTINEL CH., is a particleenhanced turbidimetric immunoassay (PETIA). Abbott’s ARCHITECT cSystems andAEROSET Systems are random-access analyzers.Assay commutability: Review of published references ranges (13 PENIA, 4 PETIA,4 not specified) supported standardization of the new PETIA. Using PENIA as basistarget values were assigned to a panel of 8 human sera pools (HSP), ranging from 0.7to 5.8 mg/L in three external laboratories, which is used to assign target values oncommercial calibrator lots.Reference range study: 259 plasma samples from healthy blood donors were assayedon the Architect cSystem in duplicate. The reference range was defined at the 95%interval.eGFR derivation [GFR in ml/min per 1.73 m2]: 522 plasma samples from referredpatients (445 adults, 77 children <18 yrs) were assayed on the Architect cSystem induplicate. New and existing eGFR-prediction equations were derived and accuracywithin ±30% (P30%) and absolute % error (APE%) vs. reference iohexol assaymeasured GFR compared.

Results

Reference range [mg/L]Overall analysis: (n=259) 0.45 to 0.85Gender subgroups: Male (136) 0.45 to 0.93; Female (123) 0.44 to 0.88Age subgroups: <50 years (137) 0.44 to 0.76; >50 years (122) 0.44 to 0.93

eGFR can further be enhanced by adding creatinine. More complex equations offeredno significant improvement in accuracy.Conclusion: Reference range established with the new PETIA on the AbbottArchitect cSystem confirmed published PENIA data (0.53 to 0.93 mg/L). Predictionof eGFR by compact PETIA and Rule1 was essentially equivalent.

Comparison of Urinary Catecholamins Results for Specimens

Collected with Preservative Added at the Beginning or at the End of

the Collection

P. P. Chou1, W. Price2, K. Sisco1, N. Sherman1. 1Quest Diagnostics NicholsInstitute, Chantilly, VA, 2Carilion Labs, Roanoke, VA

Introduction: Catecholamines in urine are very labile thus need to be preserved withacid while the 24-hour urine is being collected. Although acetic acid and/or boric acidcan be used as the preservative, most laboratories prefer to use 25 mL 6N HCl as thepreservative. The use of 6N HCl as preservative presents a challenge because patientsmay inadvertently be exposed to the strong acid.Method: In this study, five (5) volunteers were recruited to collect 24-hour urinespecimens. Special urine containers (P-splitter) were used to collect the specimens.This container has two compartments (A and B). This container divides all urinefractions evenly into both compartments. Before the collection, 12.5 mL 6N HCl wasadded to compartment A. At the end of the collection, another 12.5 mL 6N HCl wasadded to the compartment B. Both aliquots were analyzed by HPLC withelectrochemical detector. Results:

Conclusions: With a few exceptions, the variations are within the inter-assayvariations for each analyte. Therefore, using limited number of specimens, we havedemonstrated that the preservative used for urinary catecholamines can be addedeither at the beginning or at the end of the collection. More specimens need to becollected and analyzed to confirm this finding. Once confirmed, the potential risk forpatients being exposed to caustic acid can be avoided.

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Best x-validated eGFR equations among adults (n=445)

P30%(95%CI)

APE%

New equations

Compact PETIA: 71.0 x CysC- 1.28 82(78 - 85)

15

PETIA: 848.8 x CysC- 0.796 x Crea- 0.468 x Age-0.089

x 0.857 (if female)92

(89 - 94)11

Existing equations

MDRD-IDMS78

(74 - 82)15

Rule1: 66.8 x Cys C- 1.30 83(79 - 86)

16

Mean of MDRD-IMDS and Rule 189

(86 - 92)11

ID Analyte Before After Diff CV(µg/24h) (µg/24h) % %

1 Norepinephrine 11 10 -9 6.7Epinephrine 36 32 -11 8.3Dopamine 214 210 -2 1.3

2 Norepinephrine 4 5 25 15.7Epinephrine 42 41 -2 1.7Dopamine 217 198 -9 6.5

3 Norepinephrine 13 12 -8 5.7Epinephrine 53 56 6 3.9Dopamine 264 309 17 11.1

4 Norepinephrine 14 13 -7 5.2Epinephrine 59 46 -28 17.5Dopamine 242 156 -35 30.6

5 Norepinephrine 22 22 0 0Epinephrine 57 66 16 10.4Dopamine 257 294 14 9.5

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UVA Clinical Evaluation of Abbott Next Generation Calcium on the

ARCHITECT® ci8200®

S. Colberg1, L. Legendre2, M. Edwards2, D. Armbruster1, Y. Lemma1.1Abbott Laboratories, Irving, TX, 2University of Virginia MedicalLaboratory, Charlottesville, VA

Objective: To compare assay performance of Abbott Clinical Chemistry NextGeneration Calcium (Next Gen Ca), on-market Abbott Clinical Chemistry Calcium(Ca), and Randox Ca (R-Ca) assays for Imprecision and Method Correlation.Background: The Next Gen Ca is a liquid, ready-to-use, concentrated reagent that isautomatically diluted on-board the instrument allowing for less operator interventionand instrument down time. The Next Gen Ca is available in multiple kit sizes to suitthe testing needs of varying size laboratories. The University of Virginia MedicalLaboratories (UVA) evaluated the Next Gen Ca assay for use on the ARCHITECTci8200 analyzer during one week in which a large population of renal patients wasscheduled for dialysis. Sporadic falsely increased Ca results for dialysis patientsprompted specific evaluation of factors that might affect Ca values in this patientpopulation.Method: A 5-day (N=20) precision study was conducted to evaluate the totalimprecision of the Next Gen Ca using a normal and abnormal control material assamples. Method correlation was performed to compare the Next Gen Ca, Ca, and R-Ca reagents. A total of 199 serum and plasma samples were collected. 103 of thesesamples were from renal patients (pre and post dialysis) including 25 geriatricsamples. The remainder of the samples consisted of 25 pediatric, 32 geriatric, 22samples with additions of high concentrations of magnesium (a common potentialinterferent), and 17 normal patient samples. All samples were run on the threereagents simultaneously.Results

Conclusion: The Abbott Next Generation Calcium Assay was very precise, deliversgood correlation on serum and plasma samples, including samples from dialysis,pediatric and geriatric patients and also lacks interference from high magnesium.Lastly, the kit size for Next Gen Ca meets the workflow needs of large volumelaboratories such as UVA, allowing for less operator intervention.

CAN THE RESULTS OF THE URISED AUTOMATED ANALYZER

BE USED IN EARLY PREDICTION OF URINARY TRACT

INFECTIONS

Y. U. Budak1, K. Huysal2. 1Sevket Yilmaz Devlet Hospital, Bursa, Turkey,2Yuksek Ihtisas Training Hospital, Bursa, Turkey

Aim: The aim of this study is to evaluate if its possible to predict the positive culturesby analyzing the Urised (77 electronica, Budapest, Hungary) automated urineanalyzer results.Methods:Leucocyte and bacteria counts were estimated by the analyzer. Urinecultures were performed by using conventional methods based on colony countingtechnique.Results: 386 patients attended at the laboratory for urinalysis and culture at the sameday were included in the study. 68 % (263/386) of the patients were female, 32%(123/386) were male. 317 of the cultures did not give bacterial growth; culturepositivity was found in 69 of the patients. Among the individual measures analyzedwith Urised analyzer to discriminate culture positivity, we found a specificity 79.8%,

sensitivity 56.5%, PPV 37.3%, NPV 89.3%, accuracy 75.6% of urine leukocyteparameter. Accuracy value was 80.8 % when using bacteriuria and leukocyteuriaparameters together. The highest specificity (99 %) and accuracy (82.3 %) level wasestablished with urine leucocyte and bacteria and LE and, NO2 combination. Thesensitivitity of this combination was 2.8%.Conclusion: Although the outcome of the Urised analyzer results’ specificity was inaccaptable limits we observed the sensitivity value as low. Thus, Urised urinalysisresults do not accurately predict the outcome of cultures.

Comparison of Aqueous sCD44 Level in Patients with Degenerative

Myopia and Primary Open-Angle Glaucoma

Y. U. Budak1, M. Akdogan1, K. Huysal2. 1Sevket Yilmaz Devlet Hospital,Bursa, Turkey, 2Yüksek htisas Training Hospital, Bursa, Turkey

The transmembrane glycoprotein CD44 is a major hyaluronic acid (HA) cell surfacereceptor widely distributed in eye tissues and fluids. The shed ectodomain of CD44 isknown as soluble sCD44 and is toxic to human TM cells and retinal ganglion cells incell culture. sCD44 is released from cell surface by metalloproteases in response tometabolic stress or hypoxia.Glaucoma, one of the world's leading causes of visual impairment and blindness, ischaracterized by excavation of the optic nerve head and selective apoptotic loss ofretinal ganglion cells (RGCs), resulting in a progressive decline in visual function.There are several types of glaucoma (e.g., primary open-angle, normal tension, andearly onset-to name a few). Patients with degenerative myopia (spherical equivalent atleast -6.0 D) are more susceptible to glaucoma .The purpose of this study was to investigate the concentration of soluble sCD44 in theaqueous humor of normal subjects and patients with primary open-angle glaucoma(POAG) and degenerative myopia without glaucoma to understand if the presence ofthis molecule may represent a protein marker of POAG.For this case control study, aqueous samples were collected from normal patients(n=16), patients with POAG (n=8) and patients with degenerative myopia (n=8) whounderwent phacoemulsification surgery for mature or immature cataract. Aqueousspecimens from anterior chambers were obtained during phacoemulsification surgeryin all patients. The aqueous concentration of sCD44 was measured using acommercial ELISA kit (Bender MedSystems, Wien, Austria).In normal aqueous samples the sCD44 concentration was 5.40 ±1.21 ng/ ml, indegenerative myopia patients the sCD44 concentration was 5.76 ±1.15 ng/ ml. Therewas no statistical significant difference between these two groups (p >0.05). Theaqueous sCD44 concentration of cases with POAG (12.2 ±10.1 ng/ ml) was higherthan control group (p< 0.05).sCD44 concentration in aqueous is a possible protein biomarker of visual field loss inPOAG.

COMPARISON OF THREE AUTOMATED SYSTEMS FOR URINE

CHEMISTRY AND URINE SEDIMENT ANALYSIS

Y. U. Budak1, K. Huysal2. 1Sevket Yilmaz Devlet Hospital, Bursa, Turkey,2Yüksek htisas Training Hospital, Bursa, Turkey

Urine analysis is an essential component of patient assessment, which is used forscreening, diagnosing and planning care. Its assessment includes evaluation ofphysical characteristics, biochemical parameters (urine pH, blood, glucose, ketones,bilirubin, urobilinogen, and protein) and microscopic sediment evaluation (RBC,WBC, organisms, epithelial cells, crystals, and casts). Automated urinalysis systemscan save labor and time and is more feaseble for high-volume laboratory workload.Aim of this study is to compare the diagnostic performance of 3 automated urinalysissystems (and the chemical units of these systems); namely the Iris iQ200 (AX-4280)(Iris Diagnostics, Chatsworth, CA), Sysmex UF-1000i (Urisys 2400) (SysmexCorporation, Kobe, Japan) and Urised (LabuMAt) (77 electronica, Budapest,Hungary) and determine the convenience of use in the routine hospital laboratory.A total of 412 consecutive fresh urine samples, which were sent to the laboratory forurinalysis were analyzed by three automated urinalysis systems. The performance ofthe systems was evaluated and the differences between the instruments wereconverted to same scalas before data analysis. The pairwise concordance rate betweenthe 3 instruments was defined by the percent of results within +/-1 grading from thebest fit line.Correlation between the 3 instruments represented as within 1 grading difference was

Study Results

5-Day Precision

Next Gen Ca

Mean: 8.7 mg/dL

Mean 11.3 mg/dL

Total % CV 1.0 Total % CV 1.4

CaMean: 8.6 mg/dL

Mean 11.3 mg/dL


R-CaMean: 8.2 mg/dL

Mean 10.8 mg/dL


Method Correlation

Next Gen Ca vs. CaR = 0.994, Slope = 0.97, Int = 0.4

mg/dL

Next Gen Ca vs. R-CaR = 0.998, Slope = 1.02, Int = 0.3

mg/dL

R-Ca vs. CaR = 0.995, Slope = 1.00, Int = 0.6

mg/dL

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better between the LabUMat and AX-4280 chemistry systems for pH, blood,bilirubin, glucose and nitrite (>90 %). For protein, LE and nitrite, better correlationwas observed between the Urisys 2400 and LabUMat (> 87 %), while the AX-4280and Urisys 2400 conveyed better corralation for blood, glucose, urobilinogen andketone (>90 %).When we compared the microscopic units; correlation between the 3 instruments wasbetter between the iQ200 and Urised (92 %) for red blood cell data. For white bloodcell data (86 %) and epithelial cell data(90 %) UF1000i and iQ200 conveyed bettercorrelation. Bacteria counts showed the greatest differences by the 3 systems. Castswere difficultly differantiated by all of the systems.The automated urinalysis systems demonstrated acceptable correlations with eachother in urine chemistries. On the other hand, automated microscopy units could beused as a screening procedure but some manual microscopy was still necessary. Wesuggest that measurements by urinalyzers may be reliably employed as a screeningmethod to discard negative urine samples,while sediment examination may beemployed as confirmation tests.This approach would increase the throughput andstandardisation of laboratories.

Uniparental Disomy Analysis using Linkage Mapping Set Markers

J. Lee, A. Chhibber, B. Johnson, C. Davidson, D. Rodriguez, A. Pradhan,R. Padilla, R. Fish, S. Berosik, S. Hung, L. Joe, A. Felton, R. Petraroli. LifeTechnologies, Foster City, CA

In a departure from classical Mendelian genetics, there are a few imprinted genes thatare only expressed from the allele inherited from one parent. Uniparental disomy(UPD) occurs when an individual inherits both copies of a chromosome pair fromonly one parent and no copies from the other parent. Individuals with UPD that have adeficiency in the expression of imprinted genes on Chromosome 15 are subject to twogenetic disorders, Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Onemethod used to determine the presence or absence of the maternal or paternalchromosome is to interrogate the DNA from the individual and both parents bymicrosatellite or STR (Short Tandem Repeat) analysis with fluorescently labeledprimers. We demonstrate new methods to compare the DNA via fragment analysis bycapillary electrophoresis (CE). Such methods could help in producing consistentresults across a wide range of laboratory environments. We also present a softwareanalysis procedure to rapidly generate results and draw conclusions.

Ferritin an indices of body iron stores and acute myocardial infarction

in diabetics

A. M. Jarari, M. M. Abdelmoneim Farag, H. Hyder, J. R. Peela, R. Pathak.Department of Biochemistry,Al-Arab Medical University, Benghazi, LibyanArab Jamahiriya

Body keeps most of the iron in the bound form by sequestering it in transport andstorage proteins as a defence against oxidants. It is the catalytic form of iron which isresponsible for the generation of free reactive oxygen radicals. Speroxide and otheroxidants have also been involved in the release of ionic form of iron from ferritin. Ironis a transition metal that can catalyze toxic redox reactions, and it has been suggestedto be involved in many harmful biological processes and diseases in the human bodysuch as diabetes and cardiovascular diseases. The role of tissue iron and elevated bodyiron stores play in causing type 2 diabetes and or the pathogenesis of its importantcomplications, particularly diabetic nephropathy and cardiovascular disease is latelyunder extensive investigations. A causative link with iron overload is suggested of theimprovement in insulin sensitivity and insulin secretion with frequent blood donationand decreased iron stores. Viewing the participation of the free radicals in potentiatingthe pathogenesis of diabetes and it’s complications such as cardiovascular disease, itwas fascinating to investigate the body iron status and lipid peroxidation activity intype 2 diabetics at the onset of acute myocardial infarction.The present study investigated 30 acute myocardial infarction patients with or withoutdiabetes for the serum levels of ferritin, total iron, total iron binding capacity anderythrocyte lipid peroxidation activity by employing authentic analytical methods andcompared with age and sex matched 30 healthy controls.A statistically significant elevated levels of serum iron (P<0.02), ferritin (P<0.001)and a decrease in total iron binding capacity (P<0.03) were observed in diabetics andnon-diabetics also showed similar trends at the onset of acute myocardial infarction ascompared with healthy controls. The erythrocyte lipid peroxidation activity was

significantly raised in study subjects (P<0.03) in comparison to controls. Serum iron,ferritin and erythrocyte lipid peroxidation statistically correlated positively whilenegatively with total iron binding capacity. However, there were no significantchanges in these parameters between diabetic and non-diabetic acute myocardialinfarction patients.The interplay of serum ferritin, total iron, total iron binding capacity and erythrocytelipid peroxidation activity in potentiating and progression of diabetes and of it’scomplication, an acute myocardial infarction has been discussed in the light of theobservation that iron-catalyzed oxidative stress mediate apoptosis of pancreatic isletswith a resultant decrease in insulin secretory capacity and the vascular endothelialdysfunction and ischemic myocardial damage.

Correlation between homocysteine and biochemical markers of renal

function in the general population.

G. Lippi1, G. Targher2, M. Montagnana1, G. L. Salvagno1, G. C. Guidi1.1Laboratorio di Biochimica Clinica, University of Verona, Italy, 2Sezione diEndocrinologia e Malattie del Metabolismo, University of Verona, Italy

Context. Despite the well-known influence of renal function on homocysteinemetabolism, there is contradictory information on the biological interrelationshipsbetween homocysteine and traditional or innovative markers of renal function.Material and method. Results of homocysteine, creatinine, cystatin C and folate,which were performed on consecutive outpatients referred by general practitioners toour laboratory for routine testing over the previous 12 months. Results. Cumulativeresults were retrieved for 140 adults >35 years old. After stratifying plasmahomocysteine values according to the thresholds of creatinine, estimated glomerularfiltration rate (e-GFR), and cystatin C, significant differences were observed insubjects with values of these biochemical markers suggestive for reduced renalfunction. Accordingly, the prevalence of homocysteine values ≥15 µmol/L wassignificantly higher in subjects with biochemical markers suggestive for reducedglomerular filtration rate. Although plasma homocysteine was strongly associatedwith serum cystatin C, creatinine and e-GFR (all p<0.001) in multiple linearregression analysis, the only biological interrelationship remaining statisticallysignificant after adjustment for age, sex and folate status was that with cystatin C(p<0.001) (table 1, Data are expressed as means and 95%CI or percentages.).

Conclusions. These results suggest that the measurement of cystatin C might bewarranted to improve the clinical usefulness of homocysteine testing for assessing thethrombotic risk in the general population

Simple,rapid and sensitive detection of Toxoplasma gondii by loop-

mediated isothermal amplification

J. Zhang1, M. Jiang2. 1Department of Biochemistry,Wuhan UniversitySchool of Medicine, Wuhan, China, 2Department of EtiologicalBiology,Wuhan University School of Medicine, wuhan, China

Objective: The traditional clinical diagnosis of Toxoplasma gondii (T. gondii )infection requires time-consuming cell culturing,mouse inoculation,which are notvery sensitive and specific.Polymerase chain reaction(PCR)-based detection methodsincluding nested PCR and real-time PCR need sophisticated equipments foramplification.In this study,a sensitive,specific, rapid and simple loop-mediatedisothermal amplification(LAMP) method was developed for the detection of T. gondiiDNA in blood and amniotic fluid from patients with suspected PCR diagnosedpositive. The sensitivity and specificity of the LAMP were compared with thoseconventional and nested PCR.

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Methods: DNA from clinical samples was extracted with conventional phenol/chloroform extraction.4 primers specific for 6 distinct regions of the T. gondii B1gene(GeneBank AF179871) were designed.After amplifying DNA under isothermalconditions(65°C) within 1 h by Bst DNA polymerase with strand-displacementactivity. The LAMP reaction products can be simply judged with eye inspection by awhite turbidity of magnesium phrophosphate or a colour change of a mixture withSYBR Green 1. In addition,LAMP and PCR products can be confirmed by 1.5%agarose gel electrophoresis and SYBR Green 1 staining. T. gondii tachyzoites andsome parasites were used as positive,negative controls,respectively.Results: The LAMP assay showed higher sensitive than the conventional PCR and assensitive and specific as nested PCR with a detection limit of 3 T. gondii tachyzoitesper reaction.All the nested PCR positive samples were positive by LAMP.There wasno cross-reaction with DNA of other parasite controls.Conclusions:The results demonstrated that the LAMP assay is asensitive,specific,simple and rapid diagnostic method for T. gondii infection.One ofthe most important advantages of LAMP may be its simplicity and conveniencerequiring only a heating block or water bath for the reaction and can satisfy clinicaland field applicability.

The clinical value of fluorescent in situ hybridization and SYBR Green

I real-time quantification PCR in rapidly detection and identification

of Enterococcus faecalis in urine samples

Y. Tang, Y. Li, M. Wang, Z. Xia, J. Gu, Q. Wu, H. Hu. Renmin Hospital ofWuhan University, Wuhan, China

Objectives: To evaluate the clinical value of tow rapid methods to detect and identifyEnterococcus faecalis in urine samples. Methods: Using fluorescent in situhybridization (FISH) with species specific oligonucleotide probe, fluorescentlylabeled with fluorescein Cy3, targeting the 16S rRNA of Enterococcus faecalis washybridized for the urine samples. Specific primers were synthesized according to thespecific gene sequence of heat shock proteins' groES of Enterococcus faecalis. Toselect 180 urine samples were simultaneously tested by cultivation, FISH, FQ-PCR.Results: The positive was detected by Cultivation, FISH, FQ-PCR each other in 9, 11,11 of 180 samples, the negative was in 165 of 180 samples in common. The sensitivityof cultivation, FISH, FQ-PCR was 75% (9/12), 91.6% (11/12), 91.6% (11/12).Conclusion: Fluorescent in situ hybridization (FISH) and SYBR Green I real-timequantification PCR (FQ-PCR) are rapid, specific, highly sensitive technique for theearly detection of Enterococcus faecalis. They represent a considerable advancementand a great potential for diagnosis urinary tract infection.

Estimation of renal function in hospitalized patients in Spain:

Concordance between MDRD 4 and MDRD 6.

E. Fernandez1, A. de Francisco2, J. Cruz3, M. Casas4, J. Gomez-Gerique2,A. Leon5, F. Cava6, J. Bedini7, A. Enguix8, E. Ripoll9, L. Borque10, A.Fernandez11, M. Arias12. 1Hospital Cabuenes, Gijon, Spain, 2HospitalMarques de Valdecilla, Santander, Spain, 3Departamento Estadistica.Universidad Autonoma, Madrid, Spain, 4Hospital Virgen de la Arrixaca,Murcia, Spain, 5Hospital Virgen del Rocio, Sevilla, Spain, 6FundacionHospital Alcorcon, Madrid, Spain, 7Hospital Clinic, Barcelona, Spain,8Hospital Virgen de laVictoria, Malaga, Spain, 9Hospital Ramon y Cajal,Madrid, Spain, 10Hospital San Pedro, Logroño, Spain, 11HospitalMexoeiro, Vigo, Spain, 12Hospital Marque de Valdecilla, Santander, Spain

Background: More than 500 million people worldwide have some form of chronickidney damage (CKD). Despite this high prevalence, there has been relatively littleattention focused on the prevalence of CKD among hospitalized patients, a populationwho generally is receiving potentially nephrotoxic drugs, exposed to major surgery orto radio contrast agents. It has been suggested that improved estimation of GlomerularFiltration Rate (eGFR) may be possible in sick by incorporating albumin and BUNlevels. Objective: The aim of this study was to assess the agreement between MDRD4 and MDRD 6 formulae in hospitalized patients. Materials and methods: Patients(n = 14 658) were recruited into this study from a population of adults (≥18 years)hospitalized at 10 centers in Spain since May to June 2007. Patients belonging toObstetrical and Nephrology Departments were excluded. Serum samples were takenfor the analysis of hemoglobin, creatinine, albumin and urea nitrogen upon at the time

of hospital admittance. eGFR was estimated using the full MDRD equation containingsix variables (MDRD 6) (n=8611) and the MDRD 4 (4 variables) equation (n=14658).Laboratories using a creatinine method calibrated to be traceable to IDMS used theIDMS-Traceable MDRD Study equation. Agreement between MDRD 4 and MDRD 6classification in the present study was evaluated utilizing a Kappa statistic. Results: Acorrelation coefficient r of 0.9557(CI 95% 0.9559-0.9594) (P < 0.0001 ) (n = 8611)was found between eGFR MDRD 4 and eGFR MDRD 6. The strength of agreementbetween MDRD 4 and MDRD 6 CKD NKF/KDOQI stages 3-5 was very good, with aKappa statistic 0.868 (CI 95%: 0.857-0.879).In a group of patients (n = 3032) withhypoalbuminemia (serum albumin <3.5 g/l) there was also a good MDRD 4 to MDRD6 correlation: r = 0.9675 (CI 95%: 0.9652-0.9657); P < 0.0001. Conclusions: To ourknowledge, this is the first study demonstrating a very good strength of agreementbetween MDRD 4 and MDRD 6 for the distribution of CKD NKF/KDOQI stages 3-5in a large population of hospitalized patients belonging to different medical andsurgical departments. Although a previous study states that MDRD equations are notreliable measures of actual level of renal function and may be unsuitable for clinicalapplication in this population, their value is limited due to the fact that inpatientselection was based on the individual nephrologists’ perception of laboratory valueswhich is not reflective of actual GFR and nearly half of the patients included in theinvestigation were at intensive care units. In our study only 4.6% of the studypopulation was in the intensive care unit. The strength of agreement was similar evenwhen the study was performed only in malnourished patients with decreased serumalbumin concentration. However, limitations of this study include the fact that theMDRD equations have not been thoroughly validated in a large number of illhospitalized patients, for whom the laboratory variables may be distorted.

Multiparametric classification of fibrogenic liver diseases as

exemplified by non-invasive Fibrotest/Actitest: Whom can you trust?

O. A. Gressner, N. Beer, S. Stanzel, A. M. Gressner. RWTH-UniversityHospital, Aachen, Germany

Introduction: Non-invasive, i.e. serum-based assessment of liver fibrosis andfibrogenesis is still an important challenge although multiple single tests andmultiparametric panels of biomarkers have been proposed (1).Aim: Assessment of the diagnostic validity of non-invasive biomarkers of liverfibrosis.Methods: Two approaches were used to assess the reliability of the tests: (i) Practicalapproach: Haptoglobin, ALT, gammaGT, alpha 2-macroglobulin, apolipoprotein A1and bilirubin were measured in sera of 4 patients with histological proven fibrosis(staging F1-F4, grading A1-A3) were determined in 6 different quality-controlledlaboratories. Inter-laboratory variations of the calculated Fibrotest Scores for stagingand Actitest Scores for grading (both by BioPredictiveTM, France), and their errorratios compared to the results obtained by biopsy were calculated. (ii) Theoreticalapproach: The variability of obtained Fibrotest/Actitest Scores depending on 64differential combinations of the allowed analyte-specific maximum/minimumpermissible values for the parameters listed above as determined by the externalquality control of the German Association of Clinical Chemistry and LaboratoryMedicine (DGKL) was determined and the frequency distribution of the resultscalculated.Results: (i): Fibrotest and Actitest Scores were largely reproducible among thedifferent laboratories. However, the error ratio was 77% for all Fibrotest- and 73% forall Actitest results when compared to the histological findings. (ii): Calculated scoresvaried among F2 (9%), F3 (31%), F3-F4 (6%), and F4 (53%) (Fibrotest), as well asA1/A2 (48%), A2 (9%), A2-A3 (5%), and A3 (38%) (Actitest).Conclusion: Despite reproducibility of Fibro- and Actitest results among the sixtested laboratories, large scale investigation (n=64) displayed increasing variability ofthe results depending on interlaboratory variation of measured analyte concentrationsthat were still within the quality controlled, analytically acceptable range.Furthermore, calculated scores coincided with histological findings only in less than25% of all cases. Thus, the diagnostic accuracy of these tests must be considered aslow, if histology is accepted as gold standard.(1) Gressner, O. A. et al. Clin Chim Acta 381 (2007) 107-113

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Fecal Calprotectin Levels in Patients with Colonic Polyposis

A. Barassi1, R. Pezzilli2, A. M. Morselli-Labate2, S. Finazzi3, L. Fantini2,G. Gizzi2, M. Lotzniker3, V. Villani2, R. Corinaldesi2, G. Melzi d'Eril1.1University of Milan, Milan, Italy, 2Sant’Orsola-Malpighi Hospital,University of Bologna, Bologna, Italy, 3Central Laboratory, LegnanoHospital, Legnano, Italy

Introduction. Calprotectin is a cytoplasmic antimicrobial compound prominent ingranulocytes, monocytes, and macrophages. It accounts for approximately 60% of thetotal protein of the cytosol. Calprotectin can inhibit bacterial proliferation as acomponent of the innate immune response and through its iron-binding capacity (1).Theusefulness of stool calprotectin determination in diagnosis of inflammatory disease ofthe colon has been reported; information about its usefulness for patientswith polyposisare scarce, however. Aim. To evaluate the significance of stool calprotectinconcentrations for patients affected by colonic polyposis. Material and methods.Sixty-three consecutive patients (35 males, 28 females, mean age 60.3 years, range 39-78 years) were enrolled: 26 patients (41.3%) with polyps, 17 patients (27.0%) withasymptomatic diverticular disease, and 20 subjects (31.7%) with normal endoscopicappearance of the colon. Calprotectin concentrations were measured by use of acommercial ELISA kit (CalprotectinELISA Kit, Immundiagnostik, Bensheim,Germany) based on a two-site sandwich technique with two selected monoclonalantibodies which bind to human calprotectin. Two 100-mg samples of faeces from asingle stool sample from each participant were assayed, and the mean of the twomeasurements was recorded. According to the manufacturer, the reference range was<15 µg/g of faeces. The within run %CV (n = 20) was 9.1 at 10.7 µg/g and 4.2 at 22.2µg/g and the between-run %CV (n = 12) was 12.3 at 9.9 µg/g and 6.1 at 22.5 µg/g; thedetection limit of the test was 2.9 µg/g. Results. Stool calprotectin concentrations were17.4 ± 24.5 µg/g for patients with colonic polyposis, significantly higher thanconcentrations for patients with diverticulosis (7.1 ± 5.7 µg/g; p = 0.026) or for patientswith normal appearance of the colon (6.0 ± 5.8 µg/g; p = 0.003). For patients with asingle polyp, stool calprotectin concentrations were similar to those for patients withmultiple polyps. Calprotectin fecal concentrations for patients with sessile polyps andthose with flat polyps were not significantly different (p < 0.05). Calprotectinconcentrations were not significantly related to the size of the polyps. Conclusion. Thisstudy demonstrated that the presence of colonic polyps may increase stool calprotectinconcentrations, which suggests that these colonic lesions should be suspected in cases ofelevated stool calprotectin concentrations. Further studies are needed to confirm thisinitial promising observation. Reference. 1) D’Inca R, et al. Calprotectin and lactoferrinin the assessment of intestinal inflammation and organic disease. Int J Colorectal Dis2007 ;22:429-37.

Serum adhesion molecules in acute pancreatitis: time course and early

assessment of disease severity

A. Barassi1, R. Pezzilli2, M. M. Corsi3, A. M. Morselli-Labate2, A.D'Alessandro4, G. Dogliotti3, L. Fantini2, A. Malesci5, R. Corinaldesi2, G.Melzi d'Eril1. 1University of Milan, Milan, Italy, 2Sant’Orsola-MalpighiHospital, University of Bologna, Bologna, Italy, 3Institute of GeneralPathology, University of Milan, Milan, Italy, 4Department ofGastroenterology, San Bortolo Hospital, Vicenza, Italy, 5HumanitasHospital, Milan, Italy

Introduction. Inflammatory mediators play an important role in acute pancreatitis andin the development of distant organ complications of the disease. However, in acutepancreatitis, there is also microcirculatory derangement; the substances capable ofdetermining these alterations and released during the inflammation have beencharacterized and they are called adhesion molecules (VCAM-1, ICAM-1, E-selectin,P-selectin, and L-selectin). Aim. We designed the present study to evaluate the behaviorof adhesion molecules in the early phases of acute pancreatitis and to explore thepossibility that these proteins are also useful in assessing the severity of the disease.Material and methods. Fifteen consecutive patients with acute pancreatitis (7 males, 8females; average age 63.1±14.5 years) were studied. The patients were admitted to theemergency room within 6 hours after the onset of pain. Diagnosis was based on a historyof upper abdominal pain associated with at least a 2-fold increase in serum lipase andwas confirmed by contrast-enhanced computed tomography. The pancreatitis was ofbiliary origin in 6 patients, and of unknown origin in the remaining 3. According to theAtlanta criteria, the patients were classified into 2 subgroups: 10 had the mild form and

5 had the severe form of the disease. In the patients with acute pancreatitis, bloodsamples were taken on admission to the emergency room and afterwards daily, at thesame time, for the following 2 days. All serum samples were frozen immediately aftercollection and stored at -20 °C until analysis. VCAM-1, ICAM-1, E-selectin, P-selectin,and L-selectin were quantified by a biochip array analyzer (Randox Laboratories). Thebiochip used to measure all 5 molecules simultaneously consists of a 9.9-mm substrateon which discrete test regions have been constructed. After a simple enzyme-linkedimmunosorbent assay procedure, each spot is imaged to capture the chemiluminescentsignals generated at each spot on the array. The light signal is captured by a charge-coupled device camera as part of an imaging station and converted by image-processingsoftware to provide results compared with calibration curves for each location on thebiochip. Intraassay imprecision was 8.2% to 10.0% and interassay imprecision was8.6% to 11.0%. Results. Acute pancreatitis patients had vascular cell adhesion molecule1 and P-selectin concentrations significantly lower and L-selectin concentrationssignificantly higher than the healthy subjects. Only E-selectin was significantly higherin severe than in mild disease (P = 0.029); a value of E-selectin ranging from 3.83 to3.92 ng/mL was the best cutoff value for differentiating severe from mild acutepancreatitis (sensitivity: 60.0%, specificity: 90.0%, cases correctly classified: 80%). E-selectin and P-selectin entered the multivariate logistic regression analysis, and a scorewas calculated showing a sensitivity of 93.3% and a specificity of 86.7% in identifyingthe patients with severe pancreatitis. Conclusions. This score seems to be useful for theearly assessment of the severity of acute pancreatitis.

Genetic Variations in CYP2B6 and Risk of Cyclophosphamide Toxicity

in Patients Undergoing Myeloablative Hematopoietic Stem Cell

Transplantation

S. E. Melanson1, K. Stevenson2, H. Kim2, J. H. Antin2, M. H. Court3, V. T.Ho2, J. Ritz2, F. C. Kuo1, J. Longtine1, P. Jarolim1. 1Brigham and WomensHospital, Boston, MA, 2Dana Farber Cancer Institute, Boston, MA, 3TuftsUniversity School of Medicine, Boston, MA

Introduction: Genetic variations in cytochrome P450 2B6 (CYP2B6) may predicttoxicity and survival in patients receiving high dose cyclophosphamide (CPA) prior toallogeneic stem cell transplantation (SCT). CPA requires biological activation to 4-OHCPA, primarily by cytochrome P450 2B6 (CYP2B6), to exert its effects. Patients withhigh enzyme activity, as reported for the *4 allelic variants of CYP2B6, may have anincreased risk for CPA-associated toxicity, while patients with low enzyme activity, asreported for the*6 allelic variants of CYP2B6, may have a decreased risk of toxicity andimproved survival.Methods: A total of 392 DNA samples were obtained from patients with hematologicmalignancies who received CPA as part of a myeloablative SCT. Geneticpolymorphisms in CYP2B6 were analyzed by a pyrosequencing method, whichdetected three SNPs, 516A>G, 785A>G and 1459C>T, associated with four allelicvariants: *4 (785A>G), *5 (1459C>T), *6 (785A>G, 516G>T) and *7 (785A>G,516G>T, 1459C>T). Outcomes were veno-occlusive disease (VOD) and CPA-relatedcardiac toxicity from 0 to 100 days post-transplant and overall and progression-freesurvival.Results: Fifty-six (14%) patients experienced toxicity (cardiac = 8; VOD = 48). Theprevalence of the *4, *5, *6 and *7 alleles was 5%, 12%, 36% and 4%. Patients withallelic variations in CYP2B6 had similar rates of toxicity with those patients withoutallelic variation of CYP2B6 (i.e. *1/*1). When grouped by phenotype, patients with lowprotein activity (i.e. slow metabolizers), *6/*6, *6/*7, did not have a lower rate oftoxicity compared to those with intermediate or high protein activity (i.e. intermediateand high metabolizers), *1/*1, *1/*4, *1/*5, *1/*6, *1/*7 and *5/*5, (p=0.72).However, slow metabolizers had a trend towards improved overall and progression freesurvival (p values of 0.22 and 0.16) (Figure).

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Conclusion: Patients with allelic variants of CYP2B6 that underlie the slow metabolizerphenotype may have improved survival.

Validation of a Sex Hormone-Binding Globulin (SHBG) Immunoassay

on the ADVIA Centaur® Immunoassay System from Siemens

Healthcare Diagnostics

C. Ahnadi1, J. Pomerleau1, M. Leonard2, R. Ludewig3, S. Tan3, J. Fan3, W.Canfield3, J. Martel3, A. Grant1. 1Collaborative Reseach for EffectiveDiagnostics, CHUS, Sherbrooke, QC, Canada, 2White Plains Hospital,White Plains, NY, 3Siemens Healthcare Diagnostics, Tarrytown, NY

Sex hormone-binding globulin (SHBG) is a glycoprotein, produced by the liver, witha high binding affinity for steroid hormones such as estradiol, dihydrotestosterone,and testosterone. SHBG measurement is a useful indicator of androgen disorders,including hirsutism, when it is combined with testosterone measurement to calculate afree testosterone index (FTI). The FTI is determined by calculating the ratio oftestosterone to SHBG.Siemens is developing a fully automated SHBG immunoassay* for testing plasma orserum samples on the ADVIA Centaur platform. The objective of this study was toevaluate the analytical performance of the SHBG assay on this platform.The ADVIA Centaur SHBG assay is a quantitative, two-site sandwich immunoassay.Relative light units (RLUs) detected by the system are directly proportional to theamount of SHBG present in the test sample. The assay range is 0-180 nmol/L. TheFTI is automatically calculated and reported by the ADVIA Centaur system.Imprecision was assessed at two clinical sites (US and Canada) on repeatedmeasurements using calibrators, control material and prepared pools. The within-runCVs ranged from 2.7% to 4.0% and total CVs from 3.2% to 4.3% on samples withSHBG concentrations of 9.62-52.15 nmol/L. The distribution of within-run CVs for250 patient samples showed that more than 90% of samples had a CV of ≤5%.A method comparison against the Roche Elecsys 2010 was performed at one site on250 samples, including 50 pediatric samples, covering the range of the assay. Two lotsof SHBG reagent were tested; their correlation coefficients were 0.992 and 0.993.In conclusion, preliminary assessment of the results of this clinical evaluation indicatethat the ADVIA Centaur SHBG immunoassay is a precise method for measuringSHBG in serum across a wide range of clinically relevant concentrations and showsequivalent performance to the Roche Elecsys 2010 SHBG assay.* In development. The performance characteristics of this product have not beenestablished. Not available for sale.

Gestation Age Specific Reference Intervals for Coagulation Tests

During Uncomplicated Pregnancy Delivery/Caesarians and Early

Postpartum Period

P. B. Szecsi, M. Jørgensen, A. Klajnbard, N. P. Colov, M. R. Andersen, A.Barfoed, K. Haahr, B. Bjørngaard, S. Stender. Gentofte Hospital, Hellerup,Denmark

The physiological changes occurring during pregnancy may affect biochemicalparameters. Most reference values are based upon samples from non-pregnant womennot necessarily useful for clinical decision in pregnant women.Eight hundred and one women were recruited among 2147 women attending firsttrimester screening. Among these, 391 women with a totally uncomplicatedpregnancy, delivery/caesarians and puerperium were identified.Plasma were obtained at gestational week 13-20, 21-28, 29-34, 35-42, at active laborand one and two days postpartum.All tests were assayed on the STA-R Evolution coagulation analyzer with reagentsfrom Stago.Reference ranges (2.5th and 97.5th percentiles) were calculated for each test andgestational period using RefVal ver. 4.11 as recommended by IFCC.Free protein S decreased slightly (≈20%) while protein S activity decreasedsubstantially (≈50%). In contrast, Factor VII (≈50%), VIII (≈100%), IX (≈50%), D-dimer (≈400%) and fibrinogen (≈50%) increased during pregnancy. Quickprothrombin time, aPTT, antithrombin, protein C, coagulation factors II, V, X, XI, &XII did not change significantly.Protein S deficiency during pregnancy is not easily revealed, however measurementof free protein S antigen might be indicative. If protein S activity is measured,gestational specific reference intervals is mandatory, however, the lower reference

limit is as low as 20%. Gestational age specific reference values are alsorecommended to be use for fibrinogen, factor VII, VIII & IX, while the usefulness ofmeasuring D-dimer during pregnancy is doubtful. Nearly all women from gestationweek 20 had D-dimer values above the conventional cut-off point of 0.5 mg/l withincreasing levels peaking at delivery. We had too few cases with thromboembolism,but it is unlikely that D-dimer levels can differentiate between normal andpathological cases.Most values fluctuated somewhat more around the delivery, andwere similar independent of delivery method.

Analytical Performance of the Enhanced Liver Fibrosis (ELF™)

Assays on the ADVIA Centaur® Immunoassay Systems from Siemens.

Comparison of ADVIA Centaur and Immuno1™ ELF Scores Using

Patient Samples.

P. W. Dillon1, R. Payne1, H. Leipold1, R. Cross2, B. Bossen1, C.DiPasquale1, L. Halik1, C. Krumm1, L. Oppenheimer1, A. Yarovoy1.1Siemens Healthcare Diagnostics, Tarrytown, NY, 2iQur, Ltd, Southampton,United Kingdom

Background: The Enhanced Liver Fibrosis (ELF) score is calculated from thenoninvasive test results of three direct markers of liver fibrosis: hyaluronic acid (HA),N-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of matrixmetalloproteinase 1 (TIMP-1). The ELF score has performed well clinically (see, e.g,Rosenberg et al. Gastroenterology. 2004;127:1704-1713). It is CE marked andavailable in Europe on the Immuno1™ Immunoassay System. The assays and thescore function are under development for use on the ADVIA Centaur ImmunoassaySystems.*Objectives: Determine if the individual assays have acceptable precision on theADVIA Centaur systems. Determine the correlation of the ADVIA Centaur ELFassays and score with the Immuno1 ELF assays and score.Methods and Materials: Remnants of routine serum samples from a referencelaboratory, iQur, Ltd., were assayed in duplicate on 1 day at Siemens’ Tarrytown, NY,laboratory with the ELF assays on an ADVIA Centaur XP Immunoassay System. Thesamples had been assayed at iQur over a period of 13 months (with recalibrations atleast every 2 months) on the Immuno1 system. Patient sample correlations werecalculated for each assay (and their logarithms) and ADVIA Centaur ELF scorecoefficients estimated from the Immuno1 coefficients and the correlation parameters.The resultant ADVIA Centaur ELF score was correlated with the Immuno1. Passing-Bablok regressions (Analyse-it software) were used exclusively. The ADVIA CentaurELF score within-run precision profile was estimated from the sample duplicates.Precisions of the individual assays were estimated from assays of single-analytecontrols, calibrators and standards in CLSI EP-5A-like protocols run on ADVIACentaur XP and, for PIIINP and TIMP-1, on ADVIA Centaur CP systems.Results: A total of 136 patient samples were included in the correlations. TheImmuno1 score equation includes a term proportional to the logarithm of eachmarker’s concentration and a constant. It is straightforward to adjust its coefficients toADVIA Centaur using the parameters from the fitted correlations of the threeindividual methods. The resultant ADVIA Centaur score correlated well with theImmuno1 score: (Centaur)=0.991(Immuno1) + 0.0811, r=0.96, range=6.4-13.8The slope and intercept were not significantly (95% confidence) different from 1 or 0.The samples ranged from negligible fibrosis to cirrhosis. Discounting one outlier,patient sample within-run score SDs ranged from 0.00 to 0.14 (CVs are inappropriatefor ELF scores). For the individual assays, within-run precisions with controls, etc.were less than 11% (HA), 6% (PIIINP) and 5% (TIMP-1). The corresponding totalprecisions were less than 15% (HA), 10% (PIIINP) and 6% (TIMP-1).Conclusions: The ADVIA Centaur and Immuno1 ELF scores have excellentcorrelation. The ADVIA Centaur ELF scores and the individual assay concentrationshave acceptable precisions.* The ADVIA Centaur assays are not commercially available, and the Immuno1assays are not available in the US.

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The Clinical Performance of the Immuno1™ Enhanced Liver Fibrosis

(ELF™) Score Is Robust to the Biopsy Staging System and

Dichotomization Level Used to Fit Its Coefficients

P. W. Dillon1, J. Parkes2, S. Harris2, R. Cross3, A. Burt4, W. Rosenberg5.1Siemens Healthcare Diagnostics, Tarrytown, NY, 2The University ofSouthampton, Southampton, United Kingdom, 3iQur, Ltd., Southampton,United Kingdom, 4School of Clinical and Laboratory Sciences, Universityof Newcastle Upon Tyne, Newcastle Upon Tyne, United Kingdom, 5Centrefor Hepatology, University College London & iQur, Ltd., London &Southampton, United Kingdom

Background: The “gold standard” for assessment of liver fibrosis is liver biopsy, aprocedure with many drawbacks. IVD markers, as well as other noninvasivesubstitutes, are the subject of great interest. IVDs may include both direct and indirectmarkers of fibrosis. Indirect markers reflect liver function, whereas direct markers aredirectly involved with fibrogenesis and/or fibrolysis. The ELF score is calculatedfrom three direct markers: hyaluronic acid (HA), N-terminal propeptide of type IIIcollagen (PIIINP), and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1).* Thescore coefficients have been fitted using logistic regression of Ishak stage 4-6 vs. 0-3(one of many biopsy staging systems).Objectives: To determine the score performance over the entire range of fibrosis aswell as in differentiating the categories of fibrosis used to set its coefficients.Determine if performance depends on the biopsy staging system used to set itscoefficients.Methods and Materials: Sera were collected at 13 European sites from 921 patientsundergoing liver biopsies. Each was evaluated against the Ishak (0-6) and Scheuer (0-4) systems. Patients had different etiologies. Fibrosis ranged from none to cirrhosis.Immuno1 HA, PIIINP and TIMP-1 assays were run on each sample. Scorecoefficients were defined by a logistic regression of the probability that the Ishakscore was greater than I (for I=0 to 5) vs. the sum of ln(Concentration) terms:Logit[Prob(Ishak > I)]=AI + Σ3M=1 BI,Mln(ConcM)The commercial ELF score was evaluated with I=3. An ordinal regression thatsimultaneously fits all six dichotomizations with fixed BMs but variable AIs was done(as a score, the six AIs were averaged). Similar calculations were done vs. Scheuerstaging. The regressions were done using SAS PROC LOGISTIC.The clinical performance of each score equation is estimated by the area under anROC curve (AUROC). A curve for discriminating between, say, Ishak 0-1 and 2-6may be calculated using any score (e.g., that fitted vs. Scheuer 0-3 vs. 4). Is bestperformance achieved using the same dichotomization for both fitting anddiscriminating? As seen below, the AUROC for any discrimination is effectivelyindependent of the score chosen. ROC curves were calculated, using Analyse-Itsoftware, for all combinations of score equations and discriminations. The data usedto set scores was also used to calculate AUROCs.Results: The AUROC depends on the discrimination chosen, increasing from milderto more severe fibrosis, but is effectively independent of score equation chosen. TheAUROCs for discriminating Ishak stages:0/1-6: 0.714-0.722,0-1/2-6: 0.764-0.768,0-2/3-6: 0.784-0.787,0-3/4-6: 0.828-0.832,0-4/5-6: 0.855-0.860,0-5/6: 0.874-0.876.For Scheuer:0/1-4: 0.714-0.722,0-1/2-4: 0.781-0.7840-2/3-4: 0.829-0.833,0-3/4: 0.869-0.872.Conclusions: The AUROC of the Immuno1 ELF score to differentiate any particularlevel of fibrosis is independent of the level (or staging method) of fibrosis used toestimate its coefficients. A single score equation may be used over the entire range offibrosis.* The Immuno1 ELF markers are CE marked and available in the UK through iQur,Ltd. They are not available for sale in the US.

Erythropoietin therapy leads to a reduction in hepcidin in chronic

renal disease patients

M. Busbridge1, D. Ashby1, D. P. Gale2, R. Chapman1, M. Patrick1, S.Bloom1. 1Imperial College Healthcare NHS Trust, London, UnitedKingdom, 2University College London, London, United Kingdom

Background: The hepatic hormone hepcidin prevents iron overload by inhibiting ironabsorption and transport, but its increase in inflammatory states restricts iron availablefor erythropoiesis contributing to the anaemia of chronic disease. Levels are also highin renal failure, preventing the absorption of dietary iron, and possibly underlyingerythropoietin resistance. However, using a recently developed immunoassay tomeasure circulating hepcidin in haemodialysis patients, we found that higherythropoietin requirements were associated with low, rather than high, hepcidinlevels.Methods: Using a competitive radioimmunoassay, plasma hepcidin was measured instable chronic kidney disease patients with moderate anaemia, before and afterstarting treatment with intravenous iron or erythropoietin.Results: Following a single intravenous dose of 200mg iron sucrose in 4 patients,hepcidin was markedly increased (59.3+/-18.6 vs 18.1+/-9.6ng/ml, p=0.047). In 7patients hepcidin levels were significantly reduced after a single dose of 20mcgdarbepoitein alfa (60.7+/-6.0 vs 71.0+/-4.7ng/ml, p=0.045) remaining at the lowerlevel after several weeks of continued therapy (60.0+/-7.2ng/ml). There were noconcurrent changes in iron indices, or in circulating levels of the putative hepcidinregulator GDF15 (growth differentiation factor 15).Conclusion: Erythropoietin therapy leads to a reduction in circulating hepcidin. Theresulting improvement in iron transport may be an important component of itsefficacy in renal anaemia. Studies characterising this effect in healthy controls areplanned.

Hepcidin levels in inflammatory bowel disease compared with healthy

controls

M. Busbridge1, J. Arnold2, A. Sangwaiya2, R. Chapman1. 1ImperialCollege Healthcare NHS Trust, Clinical Biochemistry, London, UnitedKingdom, 2Ealing Hospital NHS Trust, Gastroenterology Department,London, United Kingdom

Introduction: Hepcidin is the principle regulator of iron homeostasis and has beenconsidered as a key player in refractory anaemia in chronic inflammatory diseases,such as inflammatory bowel disease (IBD). We report the concentrations of hepcidinin IBD, measured using a competitive radioimmunoassay (RIA)Aims & Methods: Hepcidin, prohepcidin and ferritin along with haemoglobin (Hb),mean corpuscular volume (MCV) and iron were measured in 61 patients with IBD.Iron Deficiency Anaemia (IDA) was defined as Hb <12.5g/dL, MCV <80fL and iron<10.6µ mol/L. Patients with IDA (n=18), IBD-IDA group. Patients with normal Hb,normal or high ferritin, normal or high iron and normal MCV (n=41), IBD-nonanaemic group. Mean age of patients with IBD was 44.2yrs (18-88). Compared withhealthy controls (n=25), mean age 36.04yrs (20-62)Results: Mean hepcidin in IBD-IDA was 4.14± 3.06ng/mL. The mean prohepcidin267.1± 457.59ng/mL. The mean ferritin 15.28± 19.35µ g/L. Hepcidin correlation withferritin (r=0.693, p=<0.001). The mean hepcidin level in IBD-non anaemic group was6.81± 7.68ng/mL, mean prohepcidin 104.56± 76.8ng/mL and mean ferritin 110.62±138.34µ g/L. The mean hepcidin level in healthy control group was 15.3± 15.71ng/mL, mean prohepcidin 236.88± 83.68ng/mL and mean ferritin 109.8± 128.08µ g/L.Conclusion: The mean hepcidin level in IBD-IDA group and IBD-non anaemic groupwere significantly lower (p<0.05) when compared with healthy controls. There wasalso a clear positive correlation between ferritin and hepcidin (p=<0.001). The lowhepcidin in patients with IBD maybe due to loss of hepcidin from the intestinalmucosa and may perpetuate inflammation by reduction in the natural protective effectas part of innate immunity.

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The use of an automated 12-part dipstix for routine urinalysis

T. C. Aw, K. Teo, W. R. Lim, P. Q. Leow, Q. Liu, S. P. Tan. Changi GeneralHospital, Singapore, Singapore

Background: Microscopic urinalysis is labor-intensive and operator-dependent.Some laboratories advocate the use of negative dipstix via chemical screening toobviate microscopy. However, manual dipstix evaluation is affected by wide observervariability and subjectivity. As such a manual 10-parameter dipstix is included as partof our routine urinalysis.Objective: We evaluated the use of a newly available automated 12-part dipstixanalyzer (Clinitek Atlas, Siemens) for urinalysis.Methods: The Clinitek assesses 12 parameters: color, clarity, glucose, Bilirubin,ketone, specific gravity, hemoglobin (blood), pH, protein, urobilinogen, nitrite (N),and leukocyte esterase (LE) in 16 seconds (225 samples per hour) from as little as 2mL of urine. A roll of 490 test strips can be loaded at one time. The Clinitek canaccommodate 20 sample racks containing 10 samples each. Freshly collected urinesamples without preservatives submitted to our laboratory for diagnostic urinalysiswere studied; 605 dipstix normal samples were independently examined bymicroscopy and urine culture.Results: Microscopy and dipstix concurred in 81.1% (491/605) of the cases; 71 ofthese 491 urines (14.5%) had positive culture results; 114 of the dipstix negative cases(18.9%) had abnormal microscopy reports including 30 culture positive cases. It isdisconcerting that 48/101 (47.5%) culture positive samples contained mixed bacteriaflora i.e. contaminated collections, underscoring the need for proper sampleprocurement. In this cohort of 605 samples microscopy as a diagnostic test had asensitivity of 29.7% (30/101), false positive rate of 73.7% (30/114), specificity of83.3% (420/504) and negative predictive value (NPV) of 85.5% (420/491); NPV fordipstix was 83.3% (504/605). In another 1601 samples submitted for urine culture theperformance of the Clinitek LE, N were evaluated. When LE, N were both negative83.4% of 1371 samples were also culture negative; 14.1% of the samples werecontaminated. When LE, N were both positive 79.5% of 230 samples were culturepositive; 23% of the samples were contaminated.Conclusion: The improved precision, elimination of personnel variability, ease of use,automation, and high throughput of the Clinitek has encouraged us to use it to triageour urinalysis samples. When all 12 Clinitek parameters are normal microscopy is notperformed except for patients from the nephrology service. Our average turnaroundtime for urinalysis from sample receipt to result release is now 15 minutes.

Analytical Reproducibility of the BD Viper™ System with XTR

Technology (extracted mode) for the Detection of Chlamydia

trachomatis and Neisseria gonorrhoeae

C. Welborn1, W. LeBar2, S. Taylor3, E. W. Hook4. 1Becton DickinsonDiagnostic Systems, Sparks, MD, 2Hospital Consolidated Laboratories-Providence Hospital, Southfield, MI, 3LSU Health Sciences Center, NewOrleans, LA, 4University of Alabama at Birmingham, Birmingham, AL

Objective: The objective of this study was to determine the reproducibility ofqualitative test results obtained with a sixteen panel member panel of simulatedChlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) positive and negativeurine and swab specimens using the BD Viper™ System with XTR Technology (BDViper™ System with XTR) and BD ProbeTec™ CTQ and GCQ Amplified DNAAssays. The study included analysis of within run and between run reproducibility inaddition to instrument to instrument reproducibility across three viper instruments.The target levels tested were designed to span above and below the analytical limits ofdetection (LODs) of the CTQ and GCQ assays.Methods: Three clinical sites were each provided five identical reproducibilitypanels, each consisting of 90 simulated urine and swab samples. The 90 panelmembers were prepared using BD ProbeTec CT/GC Qx Swab Diluent (Qx SwabDiluent) and were either left unspiked (0x) or spiked with known quantities of CTserovar H and/or GC strain ATCC 19424 at 2x or 5x the specified analytical LOD foreach analyte. Samples that were not spiked with one or both organisms wereconsidered to be negative for the respective analyte(s). Additionally, simulated swabpanels contained a clean endocervical swab. Panels were run once a day at eachfacility over five consecutive days. A second series of 90 panel members wasprepared in Qx Swab Diluent at target levels of 1:10 (0.1x) and 1:100 (0.01x) of thespecified analytical LOD for each organism. The levels for these panel members wereselected to fall within the dynamic range of the analytical LOD curves of the assays.

Samples in each panel were randomized and blinded to the user prior to testing.Validation: The study generated a total of 2250 results for each assay. For the CTQassay, the percentage correct for the panel members at 0x, 2x, and 5x the analyticalLOD is 99.6% (538/540), 100.0% (540/540), and 100.0% (270/270) respectively,across days and sites. For the second panel members, the percent positive at 0.1x theLOD was 67.7% (303/450) across days and sites, and the percent positive for thepanel members at 0.01x the LOD was 10.4% (47/450) across days and sites. For theGCQ assay, the percentage correct for the panel members at 0x, 2x, and 5x the LOD is99.2% (536/540), 100.0% (540/540), and 100.0% (270/270) respectively, across daysand sites. For the second panel members, the percent positive for the panel membersat 0.1x the LOD was 91.8% (413/450) across days and sites, and the percent positivefor panel members at 0.01x the LOD was 26.7% (120/450) across days and sites.There was no statistical difference in performance between sites.Conclusions: For the CT/GC negative simulated urine and swab samples and thosespiked with organisms at either 2x or 5x the specified analytical LODs of the CTQand GCQ assays, there was >99% agreement with the expected results. As expected,lower levels of agreement were obtained with samples spiked below the analyticalLODs of the two assays.

Report on detecting Cystatin C concentration in serum and pleural

effusions

W. Chen, J. Tu*, H. Hu, M. Lv. Department of Laboratory Medicine,Center for Genetic diagnosis, Zhongnan Hospital,Wuhan University,Wuhan,Hubei, China

Objective: To study the correlation between Cystatin and traditional renal biomarkerCreatinine and BUN, we analyzed Serum BUN, Creatinine, also measured Systatin Cconcentration with samples from pleural effusions.Materials and methods: 695 (male 352, female 342) serum samples fromhospitalized patients had been collected and used to detect concntration of BUN,Creatinine and Cystatin C. 48 pleural effusion sampls were used to determin CysClevel on on automatic biochemical analyzer (Aeroset, Abbott).Results: Out of 353 male cases, mean age: 45±11, BUN: 5.91±6.52mmol/L (40 casesoff range, 11.33%), Cr: 103±107µmol/L (31 cases off range, 8.78), Cysc:1.08±0.74mg/L (63 cases off range, 17.85%), 342 felmale cases, mean age: 43±11,BUN: 3.96±3.11mmol/L (8 off range, 2.34%), Cr: 67.9±18.3µmol/L (2 off range,0.58%), Cysc: 0.86±0.33mg/L (36 off range, 10.53%),Among 48 pleural effusion cases, mean age: 61±17, BUN: 6.73±5.30mmol/L (14cases off range, 29.1%), Cr: 103±87µmol/L (6 off range, 12.5%), serum Cysc:1.33±0.92mg/L (18 off range, 37.5%), pleural effusion CysC: 2.16±1.05 (44 casesover serum reference range, 37.5%). Average pleural effusion CysC conc. is about 1.6folds of serum Cysc conc.Conclusions: Pleural effusion has higher CysC level than serum.less than 20%patients has abnormal CysC level in male and less than 10% in female patients. Whenuse Cr as cut off standard, less than 10% patients found Cr off range in both male andfemale which indicate CysC is more sensitive than Cr.1=Department of Laboratory Medicine, Center for Genetic diagnosis, ZhongnanHospital, Wuhan University* To Whom correspondence should be addressed [email protected]

Validation of the Brahms Kryptor Procalcitonin Assay in Surgical ICU

and Trauma ICU Patients

E. K. O'Keeffe, C. K. Lee, H. G. MacNew, J. M. Jenkins, A. K. May, A.Woodworth. Vanderbilt University Medical Center, Nashville, TN

Background: Sepsis is a major cause of death among critically ill patients. Earlydiagnosis is essential for patient management and outcome. Measurement of serumprocalcitonin (PCT) in Intensive care unit (ICU) patients helps identify patients withsepsis earlier than bacterial cultures. However, PCT is elevated after surgery ortrauma in the absence of sepsis. The diagnostic utility of PCT to identify early sepsismay be different among patients with recent surgery or trauma. Objective: To validatethe BRAHMS Kryptor®PCT assay for use in trauma and surgical ICU patients.Methods: All patient samples were left-over serum or plasma specimens collectedfrom Surgical or Trauma ICU physician ordered CRP or prealbumin analysisperformed in the Vanderbilt University Core Laboratory. Sepsis was determined basedupon criteria defined by the Society of Critical Care Medicine. Procalcitonin assays

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were performed on the BRAHMS Kryptor, a Time Resolved Amplified CryptateEmission assay. For stability studies, known concentrations of pooled patient sampleswere incubated at 25°C, 4°C and -20°C for up to 72 hours. 44 corresponding plasmaand serum samples were analyzed for specimen type comparisons. The utility of PCTto predict sepsis was determined by ROC analysis. Results: Precision, linearity andrecovery for the Kryptor®PCT assay were previously validated. PCT was stable inserum samples stored at 4 and -20°C for up to 72 hours. PCT concentrationsdecreased by 12% after 24 hours in samples stored at 25°C. PCT was significantlyelevated in patient samples with high calcitonin. Patients with calcitoninconcentrations of ~300 and 6200 pg/mL yielded PCT results of 7.5 and 274.4 ng/mLrespectively. PCT concentrations were consistently higher in plasma compared withserum from matched patient samples, where plasma=1.24(serum)-0.2 (r2=0.99). 113samples from 56 patients were collected from the TICU (n=42 samples, 18 patients)and SICU (71 samples, 38 patients). Of these, 34 patients (61%) developed sepsis; 10(56%) were from the TICU and 24 (63%) were from the SICU. The areas under theROC curves were 0.7 (95% CI, 0.59-0.8), 0.84 (0.71-0.96) and 0.62 (0.48-0.76) forthe combined population, TICU and SICU respectively. Using ROC curves, anoptimal cutoff with maximum sensitivity (74%) and specificity (65%) was 0.5ng/mLfor the total population, 0.5ng/mL for the TICU (sensitivity= 56%, specificity=94%)and 15 ng/mL for the SICU (sensitivity=27%, specificity= 6%). Odds ratios fordeveloping sepsis were 0.2 (p<0.0001), 20.3 (p=0.001), and 3.3 (p=0.044) for the totalpopulation, TICU and SICU respectively. Conclusions: The BRAHMS Kryptor®PCTassay is a precise, rapid method for detecting PCT in serum and plasma of patientsfrom the SICU or TICU. The difference in measured PCT between serum and plasmasuggests that different specimen types should not be used interchangeably. PCT isunstable in serum at 25°C after 24 hours. Different optimal PCT cut-offs forevaluating sepsis in TICU and SICU patients were observed, likely due to non-septicelevation of PCT after surgery or trauma. Individual reference ranges for PCT must bedetermined for unique patient populations in the context of clinical picture.

Clinical Utility of Procalcitonin in ICU Patients after Major Trauma

or Surgical Event

E. O'Keeffe, C. K. Lee, H. G. MacNew, J. M. Jenkins, A. K. May, A.Woodworth. Vanderbilt University Medical Center, Nashville, TN

Background: Detection of Procalcitonin (PCT) in the serum of critically ill patientsaids in the diagnosis and monitoring of sepsis. PCT concentrations are associated withdisease severity and can be used to guide antibiotic therapy and predict prognosis.Non-septic elevations of procalcitonin occur post trauma and surgery, therefore it isnot regularly used as a sepsis marker in surgical or trauma intensive care units (ICU).PCT concentrations in serum rise within 4 hours of a bacterial insult, trauma orsurgery. PCT stays elevated in patients with infection and/or sepsis while the infectionpersists. PCT concentrations decrease within 48 hours of a trauma in the absence ofinfection. Monitoring the change in PCT over time may allow for its routine use as asepsis marker in the trauma and surgical ICU patients. Objective: To evaluate thediagnostic and prognostic utility of PCT in trauma and surgical ICU (SICU andTICU) patients. Methods: All patient samples were left-over plasma specimenscollected from SICU or TICU physician ordered basic metabolic panels performed inthe Vanderbilt University Core Laboratory. Samples were collected daily from 100patients (51 SICU, 49 TICU) for 7 consecutive days (572 samples total). Infectionwas determined based upon the results of blood, urine, or bronchial cultures.Procalcitonin assays were performed on the BRAHMS Kryptor®, a Time ResolvedAmplified Cryptate Emission assay. Significances were determined using student t-tests. Results: Out of the 100 patients, 19 died during or shortly after their ICUadmission. The maximum change and total change in PCT over the 7 day collectionperiod was calculated for each patient, no significant correlation with overall survivalwas observed (p=0.22 and 0.051 for max and total PCT change/7 days respectively).There was also no correlation between the highest PCT value and patient outcome(p=0.47). The mean, median and maximum PCT values over the 7 day collectionperiod were determined for each patient and none was a significant predictor of death(p=0.88, 0.85, and 0.46 for mean, median and maximum respectively). PCT was alsoa poor predictor of bacterial culture positivity in the SICU and TICU (RelativeRisk=1.0, p = 1.0). Patients’ PCT values were separated according to number of dayspost surgery or trauma when collected. A significant difference was observed in meanPCT concentrations between samples collected less than (mean ± SD = 4.5±0.6) orgreater than (mean ± SD = 1.2±0.4) 48 hours of a trauma or surgery (p=0.0007).Conclusion: PCT is a poor predictor of infection and prognosis when evaluated singlyor over 7 consecutive days in both Trauma and Surgical ICU patients. Procalcitoninvalues were good predictors of number of days post trauma or surgery. Thus, single orconsecutive measurements of PCT should not be used alone to predict infection or

prognosis in SICU and TICU patients. PCT should be used as a tool in combinationwith clinical parameters (including days post trauma or surgery) and other laboratoryvalues in order to evaluate infection and sepsis in these patients.

Validation of the IMMULITE® 2000 Syphilis Screen Assay from

Siemens Healthcare Diagnostics

N. M. Ureda1, A. Wu1, Z. Bostanian2, M. Edwards3, L. Silverman3, J.Martel4, C. M. Conarpe4, W. Gentzer4, Y. Luna4, A. Zhao4, M. Ghadessi4,M. Mahue4, R. Streefkerk5, M. van Westreenen5. 1University of California,San Francisco General Hospital, San Francisco, CA, 2NorthridgeDiagnostic Laboratory Inc, Northridge, CA, 3University of Virginia,University Hospital, Charlottesville, VA, 4Siemens Healthcare Diagnostics,Tarrytown, NY, 5Erasmus University MC, Rotterdam, Netherlands

Syphilis is caused by the spirochete Treponema pallidum. The disease is transmittedprimarily via sexual contact but can also be transmitted from mother to fetus orthrough transfusion of blood and blood components. Syphilis that is untreated andprogresses to the tertiary stage may cause irreversible and potentially lethal damage toneurologic, cardiovascular, hepatic and bone tissues. Therefore, early diagnosis ofsyphilis is important.Diagnosis of syphilis is primarily made through a combination of eithernontreponemal and/or treponemal diagnostic techniques. Siemens is developing anassay* on the IMMULITE family of instruments for the detection of T. pallidumantibodies in human serum and heparinized plasma.In-house precision results (CVs) were 5.1%-7.9% (within-run) and 6.3%-12.6%(total) for lot A; CVs for lot B were 5.0%-6.9% (within-run) and 6.7%-8.7% (total).External clinical trials were conducted at two US sites and one European site tovalidate the performance of the IMMULITE 2000 Syphilis Screen* compared to theperformance of the DiaSorin LIAISON Treponema Assay. Clinical enrollmentincluded representation from high-risk populations which included HIV-positivepatients. These results demonstrated good sensitivity and specificity.Lots A and B Combined

Results of the preliminary evaluations indicate that the Siemens IMMULITE 2000Syphilis Screen is a precise immunoassay for the detection of antibody to T. pallidumin human sera.* CE marked. In the US, for investigational use only. The performance characteristicsof this product have not been established.

Analytical Performance of the VITROS® 5600 Integrated System

Y. Qiu, K. A. Madden, J. Petersen, A. Mohammad, A. O. Okorodudu.University of Texas Medical Branch at Galveston, Galveston, TX

Background: The VITROS® 5600 System integrates clinical chemistry andimmunoassay testing to increase laboratory productivity for more than 100 differentchemistry, immunoassay and infectious disease assays on a single system. The systemuses a sample-centered processing approach that allows individual samples to beaccessed independently and in parallel for chemistry and immunoassay testing.Objective: The purpose of this study was to evaluate the overall system performance,ease-of-use, and analytical performance of the MicroSlide, MicroTip, and MicroWellassays on the new VITROS 5600 Integrated System. Results from the VITROS 5600System were compared to those obtained from VITROS ECi ImmunodiagnosticsSystem and the VITROS 5,1 FS Chemistry System.

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Site nSensitivity

(95% CI)Specificity(95% CI)

Total Agreement

Positive Predictive

Value

Negative Predictive

ValueNorthridge Diagnostic Laboratory Inc.

38299%

(97%-100%)99%

(97%-100%)99% 96% 100%

University of Virginia

431100%(n/a)

99%(98%-100%)

99% 97% 100%

Erasmus Medical Center

33098%

(96%-100%)99%

(98%-100%)99% 98% 100%

All Sites Combined

114399%

(98%-100%)99%

(98%-100%)99% 97% 100%

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Methods: Forty two assays (50 samples per assay) were evaluated using samplesselected to cover the entire analytical range. CLSI EP9-A2 and EP12-A protocolswere followed for the method comparison evaluations for quantitative (EP9) andqualitative (EP12) assays respectively vs VITROS 5,1 FS Chemistry System andVITROS ECi/ECiQ Immunodiagnostic System. Each sample was tested in triplicatefrom one cup with the exception of %hemoglobin A1c samples, where the testingincluded one replicate per cup. The means of the triplicate results were compared tothe results with either the VITROS 5,1 FS Chemistry System or the VITROS ECI/ECiQ Immunodiagnostic System. The evaluation also included within run and day today precision testing, using an abbreviated CLSI EP5-A2 design. Two replicates oftwo precision runs (AM and PM) were performed per day using appropriate precisioncontrol fluid on the VITROS 5600 System. The means, standard deviations and %CVwere calculated for each assay.Results: The regression analyses for the entire menu of 42 assays had slopes that arewithin 1.00 + 0.10 (except for PSA with y = 0.84x + 0.185) with most slopes within1.00 + 0.05. All the assays tested had clinically negligible intercepts. The overallwithin and between run precisions were less than 5%.Conclusion: The analytical performance of the VITROS 5600 Integrated System isequivalent to that of the VITROS 5,1 FS Chemistry System and the VITROS ECi/ECiQ Immunodiagnostic System. The VITROS 5600 Integrated System provides anadditional feature of workstation consolidation for testing done on routine clinicalchemistry and immunoassay systems.

VITROS® 5600 Integrated System Testing: Throughput/Turnaround

Time Study

K. A. Madden, Y. Qiu, J. Peterson, A. Mohammad, A. O. Okorodudu.University of Texas Medical Branch, Galveston, TX

Background: Sample through-put as indicated by turn around time (TAT) for patientsamples that require both immunoassay and routine chemistry is often prolongedbecause of multiple sample handling requirements. Different laboratories havehandled this problem by protocols that include pre-analytical aliquotting requiringcreation of daughter tubes to be run simultaneously on different analyzers. Thisapproach may improve the TAT intermittently. The VITROS® 5600 Integrated Systemis designed to optimize TAT and enhance productivity by intelligently accounting forvariable sample and test mixes, eliminating the need to split the sample or movesample trays between modules. The System also offers an increased menu capacitywith 150 reagent positions that allow over 100 assays to be on-board at once.Objective: The purpose of this study was to assess the efficiency of handling ourlaboratory’s workload on the new VITROS 5600 Integrated system. This wasevaluated by comparing a sample workflow between the VITROS 5,1 FS ChemistrySystem, VITROS ECi Immunodiagnostic System and VITROS 5600 IntegratedSystem. The results from the VITROS 5600 System were analyzed and compared tothe results from the VITROS 5,1 FS System and VITROS ECi System combined.Methodology: A workflow test design was created and preprogrammed exactly thesame on each of the three analyzers: VITROS ECi System, VITROS 5600 System,and VITROS 5,1 FS System. The test design consisted of 9 trays, each of which wastimed and recorded according to when the tray was placed on the specific analyzer,when the tray was first sampled from, when all tests were completed for that tray,when the tray was removed from that analyzer and when all results were completedfor all 9 trays. A side-by-side comparison of the 9 individual tray completion times,across the 3 analyzers, was used to determine the efficiency of the VITROS 5600System, as well as the total tray completion time. The tray completion time wasdetermined by measuring the time that the analyzer first started sampling tocompletion of all results for that particular tray.Results: The VITROS 5600 System proved to be much more efficient, with theaverage difference for each tray time (delta time), between the VITROS 5600 Systemand VITROS 5,1 FS/VITROS ECi Systems, being 17.57+4.31 minutes. Whenassessing the total time of completion for all 9 trays, based on the time the analyzerstarted the first tray to when the last tray was completed, the VITROS® 5600 had a 56minutes TAT advantage relative to the comparative analyzers, thus proving muchmore efficient than the comparative analyzers combined.Conclusion: Based on these customized workflow studies, the VITROS 5600Systemwas found to be more efficient than the combination of the VITROS 5,1 FS and theVITROS ECi Systems running in parallel, thus making the VITROS 5600 System avaluable asset to laboratories that have substantial sample workloads. The VITROS5600 System should make the laboratory TAT more reliable in terms of percentage ofsamples meeting defined TAT goals.

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Poster Session: 2:00 pm - 4:30 pmEndocrinology/Hormones

Reclassifications of thyrotropin (TSH) results using free thyroxine

(FT4)-dependent TSH reference ranges for paired TSH-FT4

measurements with normal FT4

K. D. Perry, J. D. Landmark, D. F. Stickle. University of Nebraska MedicalCenter, Omaha, NE

Background: Results combining abnormal TSH (reference range: 0.4-5.0 mIU/L) withnormal FT4 (reference range: 0.8-2.0 ng/dL) are the subject of frequent inquiries.Despite the known linear relationship of log TSH to FT4, by convention a FT4-independent reference range for TSH is used even in the context of paired TSH-FT4measurements. Objective: We evaluated the extent to which TSH measurements inabnormal TSH-normal FT4 pairs would be reclassified when using a FT4-dependentreference range for TSH. Methods: Primary data were paired TSH-FT4 patientmeasurements made during a 6-month interval for which FT4 was normal (n = 6426).Median values for TSH were determined as a function of FT4. A conservativeestimate of the FT4-dependent reference interval was calculated by assuming a log-normal distribution and width (1.1 log units) of the standard TSH reference interval,which width was then centered on the median TSH for each value of FT4. Theclassifications of TSH (low, normal, high) with and without FT4-dependent TSHreference intervals were compared. Results: 24.5% of TSH concentrations wereabnormal using the conventional TSH reference range (12.5% low, 12.0% high).Median TSH concentrations were log-linear with respect to FT4: log TSH (mIU/L) =-0.4783 FT4 (ng/dL) + 0.8463 (r2 = 0.963). Designating this correlation as f1(FT4),FT4-dependent TSH reference ranges were estimated as f2(FT4) = f1(FT4)±0.55,where 0.55 = one-half of 1.1 log units. Using the TSH reference range f2(FT4), 23.5%of TSH measurements were abnormal (13.3% low, 10.2% high), with reclassification(from either low, normal or high) of 7.8% of TSH measurements. Areas ofreclassification (a) for TSH are shown in Figure.

Conclusions: Approximately 8% of TSH measurements are reclassified when usingestimated FT4-dependent reference ranges for TSH. In practice, the calculations andFigure have proven useful for discussion with clinicians regarding association ofabnormal TSH with normal FT4.

Determination of kinetic model parameters for glycated hemoglobin

formation consistent with the relationship of estimated average glucose

(eAG) vs. %hemoglobin A1c

D. F. Stickle, J. D. Landmark. University of Nebraska Medical Center,Omaha, NE

Background: In the standard model for hemoglobin glycation, formation ofirreversibly glycated hemoglobin occurs according to the following scheme:A↔B→C, where A represents nonglycated hemoglobin, B represents reversibly

glycated hemoglobin, and C represents irreversibly glycated hemoglobin. Ourobjective was to determine parameters for the kinetics of A1c formation according tothe standard model that were most consistent with the recently established linearrelationship between % hemoglobin A1c (%A1c) and estimated average glucose(eAG) (Nathan et al., Diabetes Care 2008;31:1473-8). Methods: Species A, B and Cwere designated as fractions of total hemoglobin (A+B+C=1). The model assumed areversible equilibrium relationship of B to A as a function of glucose concentration, G,according to a single dissociation constant, K: B/(A+B) = G/(G+K). The rate offormation of C was assumed to be proportional to B: dC/dt = kB. This model has asimple solution C(t) over the lifetime of a red cell under conditions of constant G:C(t)=C(0)+(1-C(0))(1-exp(-αt)) (Eqn. 1), where α = kG/(G+K). Values of α(G) weredetermined such that average C(t) for the entire red cell population (0-120 days age)for a constant value of G was equal to A1c(G) given by the eAG equation: eAG (mM)= 1.59 A1c (%) - 2.59 (Eqn. 2). The model differs from previous models in that 1. it isnot assumed that dB/dt=0; 2. it is not assumed that C(0)=0; and 3. A1c(eAG) per Eqn.2 is targeted. Results: Values for α(G) were linear with G, as calculated based on%A1c(eAG) in the range of A1c = 4-14 % (G = approximately 4-20 mM): α (1/day) =0.000119 G (mM), r2 > 0.999 (Eqn. 3). A model constraint was that α(G=0) should beequal to 0. This constraint determined a finite value for C(0)=0.0126 (A1c=1.26%). Anon-zero value for C(0) was consistent with literature data (nascent red blood cells donot have %A1c = 0), and with the non-zero x-intercept for the eAG(%A1c) correlation(A1c(G=0) = 1.63%, Eqn. 2). The observed linear function α(G)=k'G, in comparisonto the nonlinear function α(G)=kG/(G+K) predicted by the glycation formationmodel, means simply that K is a large number relative to the operative range of G (K>> 20 mM), such that k' ≈ k/K. Determination of the function α(G) (Eqn. 3) consistentwith the eAG vs. A1c correlation (Eqn. 2) is useful in particular because %A1c(t) maybe predicted by step-wise model simulation (using Eqn. 1), accounting for cellturnover, for any arbitrary input function G(t). For instance, model calculations forA1c(t) after step changes in G predicted boundaries for the maximum possible delta%A1c(t) over a 120-day interval that were highly consistent with maximum observeddelta %A1c's observed among patient data for a given initial %A1c. Conclusions:Using the standard kinetics model for formation of irreversibly glycated hemoglobin,model parameters consistent with the known relationship of %A1c to average glucosewere successfully determined. Model simulations are useful in evaluating predicted%A1c(t) for varying inputs G(t).

High molecular weight / total adiponectin ratios are decreased in HIV-

infected women receiving protease inhibitors

F. Omar1, J. A. Dave2, J. A. King1, N. S. Levitt2, T. S. Pillay1. 1Division ofChemical Pathology, University of Cape Town & National HealthLaboratory Service, Cape Town, South Africa, 2Division of Endocrinology,University of Cape Town, Cape Town, South Africa

Relevance: We use two Highly Active Anti-Retroviral Therapy (HAART) regimensfor the treatment of HIV. Regimen 1 contains 2 nucleotide reverse transcriptaseinhibitors (NRTIs) (stavudine and lamivudine) and a non-NRTI (efavirenz), whileregimen 2 contains a protease inhibitor (PI) (liponavir/ritonavir) and 2 NRTIs(zidovudine and didanosine). HAART (including PIs and NRTIs) is associated withthe lipodystrophy syndrome, with insulin resistance as an important feature.Furthermore, total adiponectin (TA), an insulin sensitizing hormone is also decreasedin these patients. Recently, the high molecular weight (HMW) form of adiponectinhas been shown to be the active form and the HMW:TA ratio is known to be a bettermarker of insulin resistance than either form individually.Objectives: To establish whether HMW:TA ratios are lower in HIV patients receivingPIs, compared with those not on PIs and to establish whether the HMW:TA correlateswith other markers of insulin resistance in these patients.Methodology: 66 HIV-infected African females were recruited: 22 on regimen 2 (PIgroup) for at least 6 months, 22 on regimen 1 (non-PI group) for the same period and22 treatment naïve (TN). All the groups were matched for BMI and age. Patients withovert diabetes, renal failure or cardiovascular disease were excluded. Waist hip ratios(WHR) were measured. Adiponectin levels (TA and HMW) were analysed on fastedserum samples using the Alpco DiagnosticsTM Adiponectin (Multimeric) enzymeimmunoassay. Glucose (fasting and 2h post 75g oral glucose) and fasting insulin weremeasured and mathematical models of insulin resistance (HOMA-IR, HOMA-β% andQUICKI) calculated. Data were analysed non-parametrically.Results: The PI and non-PI groups had significantly lower HMW adiponectin levelsthan the TN group (median 2.23 and 3.49, respectively, vs 5.29mg/l; p<0.005), but didnot differ significantly from each other. Similarly, TA was significantly lower in bothtreatment groups compared with the TN group (median 5.64 and 7.3, respectively, vs9.03mg/L; p<0.05), but did not differ significantly between the PI and non-PI groups.

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Endocrinology/Hormones Wednesday, July 22, 2:00 pm - 4:30 pm



In contrast, the HMW:TA ratio was significantly lower in the PI group than in both theNPI (p<0.05) and TN (p<0.0001) groups, and was also lower in the non-PI than in theTN group (p<0.05). Insulin, glucose, HOMA-IR, HOMA-β% and QUICKI did notdiffer significantly amongst the groups. The HMW:TA ratio correlated negativelywith WHR (p<0.005) and fasting insulin (p<0.005). A negative, though notstatistically significant, correlation was also found between HMW:TA ratio andfasting and 2h glucose levels, as well as age and BMI.Conclusion: These data demonstrate that both PI- and non-PI containing HAARTregimens significantly lower the HMW:TA ratio in HIV patients, with the ratio moresignificantly decreased in the PI-containing regimen, implying that PIs and NRTIshave an additive effect on the HMW:TA ratio. Although the HMW:TA ratio correlatednegatively with indirect markers of insulin resistance, no overt insulin resistance wasdemonstrated. HMW:TA ratio may therefore be a more sensitive marker of insulinresistance in these patients. (Funding was provided by the NHLS, UCT and NRF.)

The effect of hypothyroidism, hyperthyroidism, and their treatment on

parameters of oxidative stress and antioxidant status.

H. Erdamar1, H. Demirci2, H. Yaman3, K. Erbil3, T. Yakar4, B. Sancak2, S.Elbeg2, G. Biberoglu2, I. Yetkin2. 1Tunceli Government Hospital, Tunceli,Turkey, 2GAZI University Faculty of Medicine, Ankara, Turkey, 3GulhaneMilitary Medical School, Ankara, Turkey, 4Beytepe Military Hospital,Ankara, Turkey

BACKGROUND: Free radical-mediated oxidative stress has been implicated in theetiopathogenesis of several autoimmune disorders. Also, there is growing evidencesupporting the role of reactive oxygen species in the pathogenesis of thyroiddisorders. The aim of this study was to investigate the influence of hypothyroidism,hyperthyroidism, and their treatments on the metabolic state of oxidative stress, andantioxidant status markers. METHODS: A total of 20 newly diagnosed patients withovert hypothyroidism due to Hashimoto's thyroiditis, 20 patients with overthyperthyroidism due to Graves' disease, and 20 healthy subjects as the control groupwere enrolled in the study. Fasting blood samples (12 h), taken at the initiation, afterthe 30th and 60th day of therapy were analyzed for malondialdehyde, nitrite, vitaminE, vitamin A, beta-carotene, ascorbate, and myeloperoxidase and superoxidedismutase activity. No patient presented additional risk factors for increased reactiveoxygen species levels. RESULTS: Malondialdehyde, nitrite, vitamin E, andmyeloperoxidase activity increased in patients with hypothyroidism. After 2 months,the levels of nitrite and vitamin E were reduced to control levels by treatment. Thepatients with hyperthyroidism had increased levels of malondialdehyde andmyeloperoxidase activity in comparison with the controls. Treatment withpropylthiouracil attenuated these increments after 1 month. CONCLUSIONS: Ourresults reveal an increased generation of reactive oxygen species and impairment ofthe antioxidant system in patients with hyperthyroidism, and particularly in patientswith hypothyroidism. These findings indicate that thyroid hormones have a strongimpact on oxidative stress and the antioxidant system.

Plasma advanced glycation endproduct, methylglyoxal-derived

hydroimidazolone, is elevated in young, complication-free patients

with Type 1 diabetes

Y. Han1, E. Randell1, S. Vasdev1, V. Gill1, M. Curran1, L. Newhook2, M.Grant2, D. Hagerty2, C. Schneider1. 1Memorial University ofNewfoundland, St. John's, NL, Canada, 2Eastern Health, St. John's, NL,Canada

Objectives: Elevated advanced glycation end-products (AGEs) are implicated incomplications of diabetes. Methylglyoxal-derived hydroimidazolone (MG-H) is oneof the most abundant AGEs in vivo in body fluids and cellular proteins. The purposeof this work was to develop a novel time-saving and specific method for measurementof free MG-H in human plasma and to determine the levels in complication-freeyoung individuals with Type 1 diabetes (T1DM) compared to young individualswithout T1DM. The relationship of plasma free MG-H to hemoglobin A1C (A1C) andplasma methylglyoxal levels was also determined.Design and methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to measure plasma MG-H levels. The method involvedsolid phase extraction of 100µl of acidified plasma using an Oasis® MCX mixedmode ion exchange column. The bound extract was eluted using 5% ammonium

hydroxide in acetonitrile, dried under nitrogen and reconstituted in water. An aliquotof the reconstituted extract was chromatographed on an Atlantis® HILIC Silicacolumn using a water gradient (10% to 55%) against acetonitrile and with formic acidconcentration maintained at 10mM. Free plasma MG-H levels were measured in 40complication-free T1DM patients (DM group), ages 6 to 21 years, and 11 individualswithout diabetes (ND group), ages 6 to 22 years. Methylglyoxal was measured using aLC-MS/MS method previously developed by our laboratory. A1C was measured by aTosoh G7 automated high-performance liquid chromatograph.Results: The developed method showed high recovery (101 ± 0.8%), sensitivity (69nmol/L) and a short run-time (6 minutes) for the measurement of free MG-H inplasma. Plasma free MG-H (nmol/L) was significantly higher (p < 0.001) in DMgroup (1318 ± 569; mean ± standard deviation) as compared to ND group (583 ± 419).Within the DM group, free MG-H in plasma did not correlate with plasmamethylglyoxal (r = -0.046, p = 0.779) or A1C (r = 0.06, p = 0.720).Conclusions: We have developed a novel LC-MS/MS method for measurement offree MG-H in human plasma. This method may be useful for future clinicalapplication. Plasma MG-H levels are elevated prior to the appearance ofcomplications of diabetes. The increased levels of free MG-H observed in individualswith diabetes, are not merely the result of short term changes in glucose ormethylglyoxal, but may reflect long-term alterations to tissue proteins.

HbA1c and variant haemoglobins

L. Owen, S. J. Lockhart, B. G. Keevil. University Hospital of SouthManchester, Manchester, United Kingdom

Many laboratories report HbA1c in patients with variant haemoglobin and append acomment stating that variant haemoglobin was detected and to treat the results withcaution. However, reporting the HbA1c value may give misleading low results. Somelaboratories prefer to obtain a total glycated haemoglobin (GHb) result using affinitychromatography as this will give an indication of overall glycation status. Weanalysed data from patients with variant haemoglobin obtained by our HbA1c assayand compared this to a DCCT aligned total glycated haemoglobin affinitychromatography assay to assess if the HbA1c assay can be used to give a morereliable estimate of glycation when a GHb assay is not available for routine use.Data was collected over a 29 month period. During this time 52, 442 samples weresent to the laboratory for HbA1c analysis. Analysis was performed on an in-houseMono-S ion exchange method calibrated to give results that are aligned to theDiabetes Control and Complications Trial. Of these samples any with S, C or F carrierstatus were selected for data analysis. Also any new or rare variants that did notinterfere chromatographically with HbA1c or HbAo measurement were included.Exclusions included homozygotes, compound heterozygotes and carriers of D, E andany rare haemoglobins that can not be resolved chromatographically. HbA1c valuesobtained in the variants were compared to GHb result obtained from an affinitychromatography method (Primus). An estimate of glycated variant was calculatedusing %HbA1c, %HbAo and %variant. This was then added to HbA1c to give anestimate of GHb and this was compared to measured GHb.Of the 52,442 samples sent, 564 data sets were analysed. Of these 121 were A/C, 387were A/S, 20 were A/F and 36 were rare variants. Approximately 50 other data setswere excluded as the peaks could not be resolved on the chromatogram. A Passing-Bablock regression analysis of HbA1c and measured GHb gave the equation,measured GHb = 1.49 x HbA1c - 0.54 with a R value of 0.87. The Passing-Bablock ofestimated GHb against measured GHb was, measured GHb = 0.97 x HbA1c - 0.87with a R value of 0.90. There were no significant differences when each of thevariants were compared individually.Reporting the HbA1c results in patients with variant carrier status can give misleadingresults for the patient and the clinician, especially when used to compare to DCCTtargets. We have shown that estimating the total glycated haemoglobin can giveresults much closer to DCCT aligned results obtained using an affinitychromatography method for the vast majority of patients. Furthermore, the equationabove can be applied to the data to give a derived DCCT aligned value. The smallpercentage of patients with variant haemoglobin that cannot be resolvedchromatographically may be monitored using an alternative marker of glycation statussuch as fructosamine or glucose profiles when a GHb assay is not available.

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Wednesday, July 22, 2:00 pm - 4:30 pm Endocrinology/Hormones



Comparison of clinical measurements with Elecsys PTH STAT and

PTH

Y. Gao. Shanghai Sixth People's Hospital, Shanghai Jiao Tong University,Shanghai, China

Objective: To study if there are some differences in clinical measurements of intactparathyroid hormone (iPTH) with Elecsys PTH STAT assay and PTH assay.Method: Plasma samples from 113 patients with or without parathyroid glanddisorders were collected and determined simultaneously with 9-minute PTH STATassay and 18-minute PTH assay on Elecsys 2010 immunoassay analyzer (ECLIA,Roche Diagnostics GmbH, Mannheim, Germany). Wilcoxon and McNemar test forpossible differences in the two iPTH assays were performed and their correlation wasanalyzed with SPSS 13.0 soft ware (SPSS, Inc., Chicago, Ill.,USA).Results: The median (P5~P95) values of iPTH were 51.7 (9.9~477.0) pg/mL and 54.6(9.8~497.0) pg/mL for PTH STAT assay and PTH assay, respectively. Though a slightdifference in the measurements was seen statistically (Z=-6.440, P<0.001), but a highcorrelation (Spearman r=0.993, P<0.001) and a total of 94.7% (107/113) agreement ofsample classification were achieved (Kappa=0.906, P<0.001) with the two assays(Figure 1).

Conclusion: The Elecsys PTH STAT has almost identical detectivity with the PTHassay on clinical plasma samples, and is more suitable for intraoperative use toconfirm the completeness of parathyroid surgery with short assay duration as well asshort biological half-life of iPTH in the blood stream.

Evaluation of an Improved Bio-Rad VARIANT™ II TURBO HbA1cKit

C. B. Tapply1, F. H. Yu2, H. K. Lee3. 1Dartmouth-Hitchcock MedicalCenter, Lebanon, NH, 2Dartmouth College, Hanover, NH, 3DartmouthMedical School & Dartmouth-Hitchcock Medical Center, Lebanon, NH

Background: The Bio-Rad VARIANT II TURBO HbA1c kit - 2.0, an improvedversion of the current Bio-Rad VARIANT II TURBO HbA1c kit, was evaluated. Thenew kit utilizes a reduced particle size cartridge, new prefilters and enhanced buffersto improve the separation of HbA1c from potentially interfering forms of hemoglobinsuch as Carbamylated Hb (CHb) and Labile A1c (LA1c). The improved chemistryalso presents several workflow advantages, such as reduced priming and calibrationfrequency, and eliminating the need to optimize temperature. The current VARIANTII TURBO HbA1c kit uses an analytical and guard cartridge format for analyzingHbA1c. Calibration and priming needs to be performed after every new analytical orguard cartridge change, and temperature optimization is required to assure that theHbA1c retention time is within limits. The interference threshold for Hemoglobin F(HbF) and CHb concentrations are also lower on the current kit, resulting in someHbA1c results not being reportable. Objectives: This study aimed to validate theimproved VARIANT II TURBO HbA1c kit - 2.0 (Test method) using the current kit asthe Reference method. Methods: Correlation of patient samples between the twomethods was performed using 200 samples with HbA1c values between 4-13%. Forty

samples with HbA1c values determined by an NGSP Secondary Reference Laboratory(SRL) were also analyzed on the Test method. Intra-day precision was determined onthe Test method by running the high and low controls 20 times in one day. Inter-dayprecision was determined by running the controls in duplicate for two runs per dayover 10 days. Interference of HbA1c analysis by CHb and HbF was investigated usingnon-diabetic and diabetic blood samples with 5 levels of CHb and HbF. Thesesamples were analyzed in quadruplicate. Results: Regression analysis for thecorrelation of patient samples between Test (y) and Reference (x) methods showed aslope of 1.1038, intercept of -0.7677, and correlation coefficient of 0.9932. The BlandAltman Bias plot for the NGSP samples comparison study showed that the Testmethod met the 0.75% acceptance criteria for level 1 laboratory certification with alower 95% confidence limit of -0.55 and an upper 95% confidence limit of 0.20. Intra-day precision study showed a CV of 0.87% and 0.31% for the low and high controlsrespectively. Inter-day precision study showed a CV of 1.07% and 0.81% for the Testmethod, while the Reference method showed a CV of 1.31% and 1.1% for the twocontrols. No interference of HbA1c analysis by CHb and HbF were detected insamples with CHb levels up to 6.2% and HbF levels up to 28.5%. Conclusions: Thetest method exhibits better precision and the increased ability to report HbA1c in thepresence of higher levels of CHb and HbF. The reference and test methods correlatedwell in the HbA1c range of 4-13%. Recalibration and priming frequency was reducedand the need to optimize temperature was eliminated.

Development of a Second Generation Anti-Mullerian Hormone

(AMH) ELISA*

A. Kumar, B. Kalra, A. S. Patel, L. McDavid. DSL, Inc., a BeckmanCoulter Company, Webster, TX

Objective: Development of AMH Gen II ELISA, standardized to the Immunotech-Beckman Coulter (IOT) AMH assay for the quantitative measurement of AMH inserum.Relevance: AMH is a glycoprotein dimer composed of two 72kDa monomers linkedby disulfide bridges. It belongs to the transforming growth factor-β family. AMHperforms various physiological functions. In males, AMH is secreted by the Sertolicells. During embryonic development, AMH is responsible for Mullerian ductregression. AMH continues to be produced by the testicles until puberty and thendecreases slowly to residual post-puberty values. In females, AMH is produced insmall amounts by ovarian granulosa cells after birth until menopause, and thenbecomes undetectable.Methodology: A two-step, sandwich-type enzymatic microplate assay has beendeveloped to measure AMH levels in 20 µL of sample in less than 3 hours. The assaymeasures mammalian AMH (i.e. human, bovine, mouse or rat). AMH calibratorsrange from 0.2-28 ng/mL. The antibodies used in the assay bind to the mature regionof AMH, which is more stable against proteolysis compared to pro-hormone region.This highly characterized dual monoclonal antibody pair is specific to AMH and doesnot detect inhibin A, activin A, FSH and LH at 2 times physiological concentrations.Validation: The AMH Gen II assay was standardized to the IOT AMH assay. AMHGen II, when compared to IOT using 38 serum samples in the range of 1.5-33 ng/mLyielded a correlation coefficient of >0.97 and a slope of 1.0 with an intercept of -0.29ng/mL. AMH Gen II when compared to the current Diagnostic Systems Laboratories(DSL) assay, using 180 male and female serum samples, in the range of 0.07-18 ng/mL yielded a correlation coefficient of >0.99 and a slope of 1.34 with an intercept of -0.01 ng/mL. The current DSL AMH patient values could be normalized to the AMHGen II assay by multiplying the patient concentrations by 1.34. Total imprecision,calculated on 4 samples over 40 runs, 4 replicates per run, between two lots usingCLSI EP5-A guidelines, was 5.7% at 3.8 ng/mL, 7.7% at 4.4 ng/mL, 5.3% at 14 ng/mL and 5.8% at 16.4 ng/mL. The average analytical sensitivity calculated by theinterpolation of the mean plus two standard deviations of 16 replicates of the zerocalibrator on two independent lots was 0.75 ng/mL. Dilution and spiking studiesshowed an average recovery of 91-110%. Lot-to-lot comparison of two independentlots testing 38 serum samples (1.5-33 ng/mL range) yielded a slope of 1.01, interceptof -0.08 ng/mL and r2 of 0.98. When potential interferents (hemoglobin, triglycerides,bilirubin) were added at 2 times physiological concentrations, AMH concentrationswere within ±10% of the control.Conclusions: A highly specific and reproducible microplate AMH Gen II assay hasbeen developed to standardize the measurement of AMH between methods. Theperformance of the AMH Gen II assay is ideal for investigation into the physiologicroles of AMH in men and women.*For Research Use Only. Not for use in diagnostic procedures

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Development of a Simplified Second Generation Inhibin B ELISA

B. Kalra, A. Kumar, K. Patel, A. S. Patel, L. McDavid, M. J. Khosravi.DSL, Inc., a Beckman Coulter Company, Webster, TX

Objective: Development of a simplified and sensitive Inhibin B Gen II ELISA for thequantitative measurement of inhibin B in serum and plasma*.Relevance: Inhibins are heterodimeric protein hormones secreted by granulosa cellsof the ovary in the female and Sertoli cells of the testis in the male. The currentcommercially available Oxford Brooks Innovation (OBI) and Diagnostics SystemsLaboratories (DSL) inhibin B assays require pre-treatment of samples with hydrogenperoxide to oxidize two methionines in the epitope to the sulfoxide for fullimmunoreactivity. Both the assays require overnight incubation of the sample withcumbersome steps. Publications have assessed inhibin B levels in Sertoli cellfunction, ovarian reserve and granulosa cell tumors.Methodology: We have developed a two-step, sandwich-type enzymatic microplateassay to measure inhibin B levels in less than 3.5 hours. Sample pre-treatment is notrequired. The assay measures inhibin B in 50 µL of serum or Li-Hep plasma samples.Inhibin B calibrators range from 10-1000 pg/mL. The highly characterized dualmonoclonal antibody pair is specific to inhibin B and does not detect inhibin A,activin A, activin B, activin AB, AMH, FSH, LH and follistatin 315 at 2 times theirphysiological concentrations.Validation: The Inhibin B Gen II assay was compared against two commerciallyavailable assays using 60 male and 60 female samples, ranging in age from 20-50years. The assay showed significant positive linear correlations to OBI and DSLassays (r=0.98; P<0.0001; & r=0.94; P<0.0001, respectively). Method comparison toOBI and DSL resulted in the following slope and intercept (Gen II =1.03OBI - 6.77pg/mL & Gen II = 1.57DSL + 11.29 pg/mL), respectively. Matched serum and Li-Hepplasma samples (n=120) showed a correlation coefficient of >0.99 and a slope of 0.97with zero intercept. Total imprecision calculated on 3 samples and 2 controls over 40runs, 3 replicates per run, between two lots using CLSI-EP5-A guidelines was 6.8% at19.3 pg/mL, 4.4% at 76.0 pg/mL, 4.3% at 275.3 pg/mL, 5.4% at 99.9 pg/mL and 5.7%at 363.9 pg/mL. The average analytical sensitivity calculated by the interpolation ofthe mean plus two standard deviations of 16 replicates of the zero calibrator on twoindependent lots was 1.6 pg/mL. The functional sensitivity of the assay at 10% and 15% CV were 9.3 and 5.6 pg/mL, respectively. Dilution and spiking studies showed anaverage recovery of 90-110%. Lot-to-lot comparison of two independent lots testing120 serum samples (5-600 pg/mL range) yielded a slope of 0.98, intercept of 0.13 pg/mL and r2 of 0.99. When potential interferents (hemoglobin, triglycerides, bilirubinand human serum albumin), were added at 2 times their physiological concentration,inhibin B concentrations were within ±10% of the control.Conclusions: A highly specific, sensitive, simplified and reproducible microplateInhibin B assay has been developed to measure inhibin B in serum and plasma. Theperformance of the assay is ideal for investigation into the physiologic roles of inhibinB in men and women.* For Research Use Only. Not for use in diagnostic procedures

Development of an Intact PTH* assay for the VITROS® ECi/ECiQ

Immunodiagnostic Systems, the VITROS 3600 Immunodiagnostic

System and the VITROS 5600 Integrated System

E. Lindhout1, M. Sieswerda1, M. Martens1, A. Vacca Smith2, D.Montague3, S. Edwards2. 1Future Diagnostics, Wijchen, Netherlands,2Ortho-Clinical Diagnostics, Rochester, NY, 3Ortho-Clinical Diagnostics,High Wycombe, United Kingdom

Parathyroid hormone (PTH) is involved in the maintenance of calcium homeostasis,and its measurement plays an important role in the diagnosis and management ofcalcium metabolism disorders.The Intact PTH assay is performed using the VITROS ECi/ECiQ ImmunodiagnosticSystems, VITROS 3600 Immunodiagnostic System and VITROS 5600 IntegratedSystem using Intellicheck® Technology. An immunometric immunoassay technique isused, which involves the reaction of Intact PTH present in the sample with abiotinylated antibody (goat polyclonal anti-Intact PTH) and a horseradish peroxidase(HRP)-labeled antibody conjugate (goat polyclonal anti-Intact PTH). The antigen-antibody complex is captured by streptavidin on the wells. Unbound materials areremoved by a washing step. The bound HRP conjugate is measured by a luminescentreaction. A reagent containing luminogenic substrates (a luminol derivative and aperacid salt) and an electron transfer agent, is added to the wells. The HRP in the

bound conjugate catalyzes the oxidation of the luminol derivative, producing light.The electron transfer agent (a substituted acetanilide) increases the level of lightproduced and prolongs its emission. The light signals are read by the system. Thebound HRP conjugate is directly proportional to the concentration of Intact PTHpresent.The VITROS Intact PTH assay under development has a reportable range of up to5000 pg/mL. Limit of Blank (LoB) was measured in line with CLSI EP17-A andshown to be <0.7 pg/mL. A precision study based on CSLI EP05-A2 was conductedover 5 days. Between run imprecision at 16, 200 and 825 pg/mL was <7.5%. Withinrun imprecision was <2.0%. Method comparison, carried out based on CLSI EP09-A2, versus a commercially available assay using 88 patient samples covering a rangeof 0.15 to 1308 pg/mL shows a good correlation (r = 0.980; intercept = 12.3 pg/mL,standard error = 3.94 pg/mL; Slope = 0.89, standard error 0.014). No high does hookeffect was observed with PTH concentrations up to 1,000,000 pg/mL. Cross-reactivityversus PTH fragments 1-34, 39-68, 53-84, 44-68 and 39-84 was assessed based onCLSI EP07-A2 as < 0.01%. Linearity across the assay range was assessed based onCLSI EP06-A with Intact PTH spiked into normal human serum. Due to the widerange linearity was assessed in 4 experiments using overlapping concentration rangesand these studies show that the assay is linear from 7.04 to >3000 pg/mL. The time tofirst result is 18 minutes.In conclusion, the VITROS Intact PTH assay combines good analytical performancewith the advantages of rapid automated continuous random access immunoassaysystems.* Under Development

Unravelling The Diagnostic Challenges Of Polycystic Ovary Syndrome

(PCOS): Should We Do More And Can We Do Better?

O. A. Mojiminiyi, F. Safar, H. Al Rumaih, T. Al Rammah, M. Diejomaoh.Faculty of Medicine, Kuwait University, Kuwait, Kuwait

PCOS, the most common endocrine disorder in women of reproductive age, arisesduring puberty and results in infertility and increased risk of type 2 diabetes andcardiovascular disease. However, despite the recognition of a major role for metabolicdisorders in the pathogenesis of PCOS, no metabolic feature is included in thedefinition of the syndrome. In this study we explore the hypothesis that PCOS is apredominantly metabolic disorder and evaluate if we could use metabolic indicatorsfor screening and early diagnosis. We measured fasting follicular phase LH, FSH,estradiol, androstenedione, DHEA-S, testosterone, SHBG, free androgen index (FAI)(calculated from testosterone and SHBG levels), fasting lipid profile, adiponectin,leptin, leptin receptor, glucose, insulin and insulin resistance (homeostasis modelassessment (HOMA-IR) and Quantitative Insulin-Sensitivity Check Index (QUICKI))in 136 women diagnosed a posteriori as PCOS (n= 92) and normal controls (n = 44)using the Rotterdam criteria. We used univariate and multivariate regression analysesto find the associations of these variables with each other and PCOS and used receiveroperating characteristic (ROC) analysis to determine the diagnostic performance ofthe biochemical parameters. Insulin resistant (22.8%), overweight/obese (82.6%) andhyperandrogenic (clinical and/or biochemical) phenotypes (96.7%) are thepredominant phenotypes of PCOS in our population. The best diagnostic tests with thehighest area under the ROC curve (AUC) were: FAI (AUC = 0.868), SHBG (0.765),HDL-cholesterol (0.715), HOMA-IR (0.670), QUICKI (0.658), adiponectin (0.644),leptin:adiponectin ratio (0.658). Oligomenorrhea (53.3%) and polycystic ovaries(68.2%) were not found in all PCOS and high serum androgens showed variablecorrelations with clinical indicators (acne (68.5%), hirsutism (77.2%), and alopecia(38%)) of hyperandrogenism. Binary logistic regression analysis showed that oddsratio (OR) of the significant associations with PCOS were: age (0.89), testosterone(3.44), FAI (2.70), HDL-Cholesterol (0.10), SHBG (0.98), adiponectin (0.89),HOMA-R (2.56) androstenedione (1.62) and DHEA-S (1.42). Whereas the clinicalpresentation of Oligomenorrhea, chronic anovulation and hyperandrogenism arestressed as the major diagnostic criteria for PCOS, our data show that some simplemetabolic parameters have comparable diagnostic performance and should be usefuladjuncts for screening. As early diagnosis and treatment may improve thereproductive sequelae and reduce cardio-metabolic risks, should we do more and canwe do better to screen for PCOS? Yes, we can.

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Serum Aminotransferase Estimation Should Be An Integral Part Of

Cardio-Metabolic Risk Assessment

O. A. Mojiminiyi, T. Al Rammah, N. Abdella. Faculty of Medicine, KuwaitUniversity, Kuwait, Kuwait

Increasing prevalence of obesity has made non-alcoholic fatty liver disease (NAFLD)a major health problem that is found in association with increased cardio-metabolicrisk. Aminotransferase (aspartate aminotransferase (AST), alanine aminotransferase(ALT), concentrations have been shown to be associated with indices of obesity andmay reflect NAFLD. This study explores the associations of ALT and AST withindices of obesity, adipokines, insulin resistance and components of the metabolicsyndrome in an alcohol free normoglycemic population with negative medicationhistory and hepatitis screen. Anthropometric measurements and fasting adiponectin,leptin, leptin receptor, insulin, glucose, high-sensitivity C-reactive protein (hs-CRP)and lipid profile were measured in 429 (155M, 274F) subjects. Univariate regressionand multivariate logistic regression analyses were used to relate the aminotransferaseswith indices of obesity, adipokines as well as with the degree of adiposity (BMI≤25,25.1 - 29.9 or ≥30), insulin resistance (homeostasis model assessment (HOMA-IR ≤2or ≥2)) and the number of the criteria of the metabolic syndrome (MS) (InternationalDiabetes Federation (IDF) criteria). 89 (21%) of subjects were classified as MSpositive. All the study subjects had normal ALT and AST but gender dimorphism wasnoted with males having higher mean ALT (27.7 IU/L) and AST (23.5 IU/L) thanfemales (17.4 and 20.0 IU/L respectively). ALT and AST showed stepwise increasewith increasing categories of obesity, insulin resistance and the number of criteria ofthe metabolic syndrome. ALT showed significant correlations with age (r = 0.13),indices of obesity (r = 0.32 for BMI; r= 0.36 for waist circumference), beta cellfunction (%B) (r= 0.14) insulin (r= 0.18), HOMA-IR (r= 0.27), triglycerides (r= 0.24),hs-CRP (r= 0.25) and inverse correlations with adiponectin (r= -0.29), insulinsensitivity (%S) (r= -0.27) and HDL-cholesterol (r= -0.25). AST showed similarcorrelations. Adiponectin, HOMA-IR, %B and %S retained their significance afterpartial correlation analysis correcting for indices of obesity. In logistic regressionmodels adjusting for age and sex, subjects in the 4th quartiles of ALT (odds ratio (OR)3.8 (95% CI 1.3-10.9) and AST (OR 2.5 (95% CI 1.1-5.7) had significant risk of MScompared with those in the 1st quartile. We conclude that aminotransferases aresignificantly associated with cardiometabolic risk factors and should be included inroutine laboratory risk assessment. As concern is growing about NAFLD, not onlybecause it is a common liver disorder, but also because it is one of the leading causesof chronic liver disease, laboratories may need to lower the upper normal referencelimits to facilitate earlier detection of obesity-associated increases inaminostransferases.

Effect Of Single Nucleotide Polymorphisms In The Adiponectin Gene

On Circulating Adiponectin And Cardio-Metabolic Risk Factors In

Patients With Polycystic Ovary Syndrome.

F. H. Safar1, O. A. Mojiminiyi1, H. Al Rumaih2, F. Al Mulla1, T. AlRammah1, M. Diejomaoh1. 1Faculty of Medicine, Kuwait, Kuwait, 2IVFUnit, Maternity Hospital, Kuwait, Kuwait

The genetic mechanisms underlying the hyperandrogenism and metabolic disorders inpolycystic ovary syndrome (PCOS) are complex. We postulate that polymorphisms inthe gene for adiponectin, an adipose tissue-derived hormone, determine theadiponectin level and play a significant role. In a study on 92 patients with PCOS and108 controls, we used real time PCR to determine the 45T>G (rs2241766) and276G>T (rs1501299) polymorphisms in the adiponectin gene and measured follicularphase LH, FSH, estradiol, testosterone, androstenedione, DHEA-S, SHBG, fastinglipid profile, adiponectin, glucose and estimated insulin resistance using thehomeostasis model assessment (HOMA-IR). Body fat% was measured byBioimpedance. Univariate and multivariate regression analyses were used to find theassociations of these variables with each other, adiponectin gene polymorphism andPCOS. Adiponectin showed significant inverse correlations with waist circumference,fat %, HOMA-IR, free androgen index (FAI) and triglycerides but showed positivecorrelations with HDL-cholesterol and SHBG. Multivariate regression analysisshowed that adiponectin is a significant determinant of insulin, HOMA-IR, SHBGand FAI. The distributions of the genotypes of both polymorphisms were notsignificantly different in PCOS patients and controls but TT and GT genotypes of rs2241766 and TT and TG genotypes of rs 1501299 were associated with significantlylower adiponectin and HDL-cholesterol and significantly higher insulin, HOMA-IR,

triglycerides and FAI despite similar obesity indices. Binary logistic regressionanalysis showed that the TT (OR = 0.89) and GT (OR = 0.83) genotypes of rs2241766 and TT (OR = 0.90) and TG (OR = 0.87) genotypes of rs 1501299 weresignificant determinants of PCOS risk. We conclude that polymorphisms in theadiponectin gene are associated with lower adiponectin that may contribute to thepathogenesis of insulin resistance and hyperandrogenism in the patients.

Leptin, Leptin Receptor Free Leptin Index and Metabolic Phenotypes

of PCOS in the Kuwaiti Population.

F. H. Safar1, O. A. Mojiminiyi1, H. Al Rumaih2, T. Al Rammah1, M.Diejomaoh1. 1Faculty of Medicine, Kuwait, Kuwait, 2IVF unit, MaternityHospital, Kuwait, Kuwait

Polycystic ovaries syndrome (PCOS) is a common complex endocrine disorderaffecting women in their reproductive age with heterogeneous presentation anddifferent phenotypes. The aim of the study was to find the association, if any, betweencirculating leptin, leptin receptor, free leptin index (FLI) with the metabolicphenotypes of PCOS.We measured follicular phase LH, FSH, estradiol, testosterone (T), androstenedione(D4), DHEA-S, SHBG, fasting lipid profile, leptin, leptin receptor, FLI, glucose,insulin and estimated beta-cell function (%B), insulin sensitivity (%S) and resistanceusing the homeostasis model assessment (HOMA-IR) and quantitative insulin checkindex (QUICKI). IDF criteria were used to define the metabolic syndrome (MS)status. Spearman Rank correlations, univariate and logistic regression analyses wereused to find the associations of these variables with each other and with metabolicphenotypes of PCOS. PCOS patients had significantly higher leptin (p=0.031), FLI(p=0.022) and lower leptin receptor (p=0.032) than controls.Among PCOS patients34.1% were metabolic syndrome positive compared with 24.3% controls. Leptin (p=0.007) and FLI (p=0.009) were significantly higher in MS+ve PCOS patients whileleptin receptor was significantly lower (p=0.031).The levels of leptin (p=0.004) andFLI (p=0.002) were also significantly higher while leptin receptor levels weresignificantly lower (p=0.008) among PCOS women with HOMAI-IR ≥2. However,no significant differences were detected in leptin, leptin receptor and FLI betweenhyperandrogenic and normoandrogenic PCOS. Spearman correlation analysis showedthat leptin and leptin receptor had significant direct correlations with waist, hipcircumference,BMI, TG, insulin, HOMA-IR, %B, FAI and inverse correlations withSHBG, %S, QUICKI and HDL-cholesterol. Similar significant correlations werefound with leptin receptor but in the inverse direction. Logistic regression analysisshow that leptin (O.R 1.040, 95% C.I 1.014-1.068, p=0.003), leptin receptor (O.R.0.875, 95% C.I 0.779-0.984, p=0.025) and FLI (O.R. 1.318, 95% C.I. 1.072-1.619,p=0.009) were significantly associated with MS. Logistic regression analysis alsoshow that leptin (O.R 1.048, 95% C.I 1.019-1.078, p=0.001), leptin receptor (O.R.0.823, 95% C.I 0.708-0.956, p=0.011) and FLI (O.R. 1.459, 95% C.I. 1.149-1.852,p=0.002) were significantly associated with insulin resistance phenotypes. However,no significant associations were found with androgens. We conclude that leptin, leptinreceptor and FLI are significantly associated with metabolic phenotypes and insulinresistance phenotype of PCOS but leptin does not appear to contribute to thehyperandrogenic status in PCOS patients.

Evaluation of the Roche Testosterone II Assay on the MODULAR

E170 Analyzer

W. E. Owen1, M. L. Rawlins1, W. L. Roberts2. 1ARUP Laboratories Inc.,Salt Lake City, UT, 2Department of Pathology, University of Utah HealthSciences Center, Salt Lake City, UT

Background: The androgen testosterone is produced in Leydig cells of the testes inmales. In females testosterone is produced principally in the ovaries. Normalconcentrations of testosterone in females are approximately 10-fold less than in males.In males decreased testosterone may indicate hypogonadism, hypopituitarism,hyperprolactinemia, renal failure, hepatic cirrhosis, or Kleinfelter’s syndrome, whileelevated testosterone can be caused by adrenal and testicular tumors. In femalesincreased testosterone can be caused by polycystic ovary syndrome, stromalhyperthecosis, ovarian and adrenal tumors, or congenital adrenal hyperplasia. Thepurpose of our study was to evaluate a new generation Testosterone II assay (Testo II)from Roche Diagnostics. Methods: Testo II performed on a MODULAR ANALYTICS E 170 module is an

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automated random access electro-chemiluminometric assay. It was evaluated forimprecision over 21 days using 2 levels of control material and 5 patient pools withtestosterone concentrations from 35 to 1321 ng/dL and method comparison with highperformance liquid chromatography-tandem mass spectrometry assay (LC-MS/MS)used in our facility. Method comparison samples were tested using the currentgeneration Roche Testosterone assay (Testo I) and the Beckman Coulter Testosteronechemilumimometric assay (Access) and compared to LC-MS/MS. Serum samplesfrom 100 men, 100 women, 50 boys (ages 0 to 17 years), and 50 girls (ages 0 to 17years) were used for the comparison studies. Samples outside the analyticmeasurement range were eliminated from data analysis. Passing-Bablok regressionwas used for method comparison and included statistics for slope, intercept, r, andSMAD (scaled median absolute deviation), a non-parametric error estimate.Results: Repeatability (within-run) and within laboratory imprecision ranged from1.6 to 2.6 and 2.3 to 5.1 %CV respectively. Passing-Bablok regression statistics aresummarized in the table below.

Conclusions: The Roche Testo II assay demonstrated excellent precision andimproved correlation with LC-MS/MS for samples from men, women, and girls.

Direct measurement of serum free testosterone by ultrafiltration and

liquid chromatography-isotope dilution tandem mass spectrometry

Y. Chen1, M. Yazdanpanah2, Y. Wang2, B. Hoffman1, E. Diamandis1, P.Wong1. 1Department of Laboratory Medicine and Pathobiology, Universityof Toronto, Toronto, ON, Canada, 2University Health Network, Toronto,ON, Canada

Background: Currently there is no reliable method for serum free testosterone (FT)measurement in routine clinical laboratory practice. The objective of this study was todevelop a liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for the direct detection and quantification of serum freetestosterone.Methods: After ultrafiltration (UF) with 0.5 ml of patient serum, an Agilent 1200Series HPLC system coupled to an API 5000 mass spectrometer equipped with anatmospheric pressure chemical ionization ion source was used to separate, detect andquantify serum testosterone by using the method we previous developed (ClinBiochem, Epub Dec. 3, 2008, in press). Ion-transitions of m/z 289.2 → 109.1 and294.2 → 113.2 were used to monitor testosterone and testosterone-2,2,4,6,6-d5 ,respectively. This direct UF-LC-MS/MS method was validated by use of split-samplecomparisons with calculated free testosterone (cFT) and equilibrium dialysis (ED)followed by LC-MS/MS.Results: Within-run and total imprecision of the method demonstrated coefficient ofvariations (CV) of 3.6%, 2.9% and 5.5%, 4%, at levels of 158 pmol/L and 67 pmol/Lrespectively (n=20 each). The method demonstrates a dynamic linear response up to atleast 2500 pmol/L and the functional sensitivity was 8.7 pmol/L at CV 20%. Themethod comparison showed the correlation of FT by UF-LC-MS/MS with cFT basedon total testosterone values measured by Architect® i2000 (R=0.816) and by LC-MS/MS (R=0.8996). UF-LC-MS/MS method correlated very well with ED-LC-MS/MS(R=0.9873). Average bias of UF-LC-MS/MS were -13.29%, -3.33% and 8.1%compared with cFT by Architect®, LC-MS/MS and ED-LC-MS/MS. The accuracy ofthe UF-LS-MS/MS for FT was validated by spiking standard material at three levelsand the recoveries were at 92-109%.Conclusion: We set up a new simple and definitive method for measuring serum freetestosterone with UF-LC-MS/MS. This method is reliable and rapid as a referencemethod and can be easily used in routine clinical laboratories.

Hemoglonin Yamagata[HBB: c.399A>T; p.Lys133Asn]: Hemoglobin

variant detected by HbA1c test

K. Hong1, C. Jung1, M. Lee1, W. Chung1, Y. Sung1, S. Lee2, C. Ki2. 1EwhaWomans University MokDong Hospital, Seoul, Republic of Korea,2Samsung Medical center, Sungkyunkwan University School of Medicine,Seoul, Republic of Korea

Hemoglobin (Hb) Yamagata is a rare Hb variant which has been reported only twotimes in Japan and Korea, respectively. It arise from Lys→Asn substitution due to achange of AAA to AAC or AAT at codon 133 of beta-globin gene. This study reportsthe third case of Hb Yamagata [HBB: c.399A>T; p.Lys133Asn] and its impact onHbA1c measurement. This variant was detected during routine follow-up of a 70-year-old Korean man with diabetes mellitus (DM). The result of HbA1c by Variant llTurbo (Bio-Rad, USA) was 47.9%, abnormally high level. It was impossible tomeasure HbA1c correctly by Variant ll Thalassemia Mode (Bio-Rad, USA) because ofinterference between variant and A1c peaks. On the contrary, the HbA1c wasmeasured as low as 5.0% by HLC-723 G7 (Tosoh Corp., Japan) while those by CobasIntegra (Roche diagnostics, Switzerland) and NycoCard (Axis-Shield, Norway) were8.0% and 7.9%, respectively. At the time of this evaluation, the patient’s fasting bloodglucose value was 177 mg/dL which is in the diabetic range. His peripheral bloodindices showed a Hb level of 12.9 g/dL, Hct 37.2%, MCV 87.3 fL, MCH 30.3 pg, andRDW 12.7%. The peripheral blood smear also showed normocytic normochromic redblood cells. The DNA sequence analysis revealed the presence of Hb Yamagata inwhich the Lys→Asn substitution was due to a change of AAA to AAT at codon 133 ofbeta-globin gene. In Capillary EP (Sebia, France), fractions of Hb Yamagata, HbA,and HbA2 were 40.9%, 54.4%, and 3.0%, respectively. This study demonstrates thatHb Yamagata can interfere accurate measurement of HbA1c in a Korean patient withDM. Considering that HPLC-based Variant ll Turbo and Tosoh G7 can be interferedby Hb Yamagata, an immunoassay or an affinity-chromatography can be the methodof choice for accurate measurement of HbA1c with this variant. In conclusion, a Hbvariant can be suspected when the concentration of HbA1c shows abnormally high orlow level or discrepancy between the result and clinical status. Then the HbA1cvalues should be determined with a method based on different principles, which canimprove the management of DM patients.

Establishment of a reference range for the Bone Alkaline Phosphatase

(BAP) with DiaSorin Liaison in a biologically well-designed

population.

E. Cavalier, P. Delanaye, A. Bekaert, I. Carlisi, J. Chapelle. UniversityHospital of Liège, University of Liège, Liège, Belgium

Introduction: Defining a reference range is not an easy task. As requested by ISO15189, laboratories must check the reference range published by the manufacturers ofthe kits, but generally, the way these values have been obtained is unclear. Theconcept of “what is a reference range” is also controversial. From our point of view, a“healthy” population, like young blood donors cannot be considered as a “reference”population. We prefer to use biological or clinical variables to establish our referencepopulation. Indeed, by using these variables as a reference, any lab will have more orless the same values and, more important, we will know exactly on which populationwe will compare our patients.Materials and Methods: We determined the BAP on Liaison (BAP OSTASE,DiaSorin, Stillwater, MN) in duplicate. We selected our population using thesevariables: age >18 yo, sex, menstrual status, levels of 25-OH vitamin D (25VTD) >30ng/mL, levels of intact parathormone (PTH) <58 pg/mL, calcium and phosphorous inthe laboratory reference range and eGFR> 60 mL/min/1.73m² (estimated by theMDRD equation). For each subclass (men/non-menopause women/post-menopausewomen), we included 120 individuals. We used the Kolmogornov-Smirnov test tocheck if the population was Gaussian. If it wasn’t the case, we used a non-parametricmethod. We took the 95% right-sided result as the cut-off.Results: Our selected population presented a mean 25VTD level of 37 ng/mL(Lowest: 32; Highest: 58), a mean PTH of 38 pg/mL (L:18 - H: 57) and a mean age of58 yo (L:22; H: 85). In men, the BAP cut-off was found to be 20.3 µg/L (90% CI: 16.1- 25.2 µg/L), 23.6 µg/L in non-menopause women (90% CI: 16.9 - 26.6 µg/L) and20.4 µg/L in menopause women (90% CI: 17.8 - 25.0 µg/L). We did not find anystatistical difference between the three populations. We thus combined all the data.The cut-off observed in the whole population (n=360) was then 21.3 µg/L (90% CI:18.3 - 24.2 µg/L).

Method Comparison StatisticsSlope Intercept SMAD r n LC-MS/MS Range (ng/dL)

Men - Testo I 1.085 - 4 42 0.972 100 60 - 1440Men - Testo II 1.165 - 19 35 0.984 98 60 - 1250Men - Access 0.971 7 41 0.955 100 60 - 1440Women - Testo I 1.231 0 12 0.848 96 4 - 671Women - Testo II 1.040 - 3 10 0.989 93 4 - 671Women - Access 1.061 6 13 0.920 88 10 - 671Boys - Testo I 1.021 1 9 0.982 47 4 - 1260Boys - Testo II 1.038 - 4 10 0.979 38 5 - 1260Boys - Access 0.948 8 10 0.992 39 5 - 1260Girls - Testo I 1.848 - 2 16 0.673 49 1 - 65Girls - Testo II 1.422 - 6 12 0.739 46 4 - 65Girls - Access 1.600 3 10 0.768 48 1 - 65

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Discussion: We have evaluated the reference range for the BAP OSTASE in a wellbiologically designed population. We did not find any difference between men, non-menopause and menopause women. This is quite new, as the reference ranges forBAP generally distinguish these populations. However, one should notice that in thestudies showing that differences exist between these subclasses, 25VTD levels havenever been taken into consideration. This is of importance as 25VTD deficientpatients can suffer from secondary hyperparathyroidism, leading to an increase boneturnover and increased BAP values.Conclusions: We present here the reference range for BAP OSTASE (Liaison) in abiologically well designed cohort. Our population is representative of the “normal”population for the studied parameter. Our approach could be generalized so thatlaboratories and manufacturers could establish the reference ranges on biologicallyand clinically well designed populations, leading to a better harmonization.

Translating Sex Hormone Binding Globulin (SHBG) Measurements

into Indices: Reference Intervals for Apparent Free Testosterone

Concentration (AFTC), Bioavailable Testosterone and Free Androgen

Index (FAI)

D. Young, J. M. Mattke, C. Hodges-Savola, J. Xu, P. Marquet, A. Morel-Montero. Beckman Coulter, Inc., Chaska, MN

Objective: The objective of this study was to establish readily available referenceintervals, to be used as examples, for SHBG, Free Androgen Index (FAI) calculatedfree testosterone and bioavailable testosterone.Relevance: Sex hormone-binding globulin (SHBG) is responsible for transport oftestosterone and estradiol. Less than 2% of biologically active sex hormones are freein circulation with the remainder being bound with high affinity to SHBG and lowaffinity to albumin. Free sex hormones and those weakly bound to albumin areavailable to the tissues. The sum of these fractions represents “bioavailable” hormone.Androgen status assessment in various conditions requires more information thantotal testosterone (TT) measurements alone. Albumin, SHBG and TT can bemeasured and used to calculate “free” and “bioavailable” testosterone to provide abetter evaluation of androgen status. These calculated indices are recognized as theFAI, Apparent Free Testosterone Concentration (AFTC) and BioavailableTestosterone. These calculated levels offer the convenience of automatedimmunoassays with results equivalent to technically difficult reference methods.Methodology: Reference intervals were established using a determination of themedians and 95% non-parametric reference intervals, with a bootstrap estimation ofthe 95% confidence interval of each of the intervals for 3 populations. Subjectsincluded were: a.) 160 healthy males aged 20-50 years; b.) 149 healthy, cyclingfemales aged 20-46 years; and c.) 141 healthy, postmenopausal females not onhormone replacement therapy, aged 47-91 years. Access® Testosterone and AccessSHBG assays from Beckman Coulter were performed on samples from all subjects. Aconstant of 4.3 g/dL was utilized for albumin. “Free” and “bioavailable” testosteroneresults employed the Vermeulen calculations available at http://www.issam.ch/freetesto.htm. FAI was calculated by: FAI = (TT/SHBG)*100.Validation: Nonparametric reference intervals for:SHBG:13.2-89.5 nmol/L with a median of 36.9 nmol/L for males, 18.2-135.7 nmol/Lwith a median of 48.3 nmol/L for cycling females and 16.8-106.9 nmol/L with amedian of 49.6 nmol/L for postmenopausal females.FAI : 22.2-110.2% with a median of 51.5% for males, 0.65-10.93% with a median of2.64% for cycling females and 0.23-5.40% with a median of 1.70% forpostmenopausal females.AFTC: 0.11-0.66 nmol/L with a median of 0.38 nmol/L for males, 0.006-0.055 nmol/L with a median of 0.018 nmol/L for cycling females and 0.002-0.033 nmol/L with amedian of 0.011 nmol/l for postmenopausal females.Bioavailable Testosterone: 2.8-15.5 nmol/L (25.7-70.5%) with a median of 8.8 nmol/L (47.3%) for males, 0.13-1.30 nmol/L (14.8-57.4%) with a median of 0.43 nmol/L(33.2%) for cycling females and 0.049-0.762 nmol/L (15.3-59.0%) with a median of0.26 nmol/L (31%) for postmenopausal females.Conclusions: As the FAI and calculated values for free testosterone and bioavailabletestosterone become more accepted by the endocrinologist community, it is importantto have reference intervals readily available. These reference intervals are useful asexamples; however each laboratory should verify these intervals or establish theirown intervals to assure proper representation of specific populations.Disclaimer: Product is currently not available in the US.

Comparison of Three Methods for Sex Hormone Binding Globulin

(SHBG)

D. Young1, J. M. Mattke1, A. Artus1, P. Marquet1, A. Morel-Montero1, J.Guéchot2, H. J. Roth3. 1Beckman Coulter, Inc., Chaska, MN, 2Hôpital SaintAntoine, Laboratoire de Biochimie, Paris, France, 3Limbach Laboratory,Heidelberg, Germany

Objective: The objective of this study was to compare performance of the newAccess® SHBG immunoassay from Beckman Coulter with three other commerciallyavailable SHBG assays.Relevance: Sex hormone-binding globulin (SHBG) is a glycoprotein responsible forblood transport of testosterone and estradiol. SHBG is synthesized in the liver and hasa high binding affinity for 17-hydroxysteroid hormones. Less than 2% of biologicallyactive steroids are free in circulation, with the majority being bound with high affinityto SHBG and low affinity to albumin. The measurement of SHBG can be an importantindicator of chronic or excessive androgenic activity where clinical symptomsindicate androgen excess, but upon measurement the androgen levels are found to benormal. Elevated SHBG levels can be seen in persons with androgen insensitivities,hyperthyroidism, cirrhosis and in patients on oral contraceptives or antiepilepticdrugs. Decreased concentrations of SHBG are often seen in men with hypothyroidismand androgen replacement therapy; where women with hirsutism, virilism, polycysticovarian syndrome, elevated androgen levels, obesity and acromegaly will alsodemonstrate decreased SHBG levels. Methodology: Serum samples were collected from residual samples for analysisacross the range of the Access SHBG assay. We assessed the performance of the newAccess SHBG immunoassay from Beckman Coulter with the Siemens Immulite®SHBG immunoassay. A smaller sample set was employed for another methodcomparison between the Access SHBG assay, the Immulite SHBG assay and theImmunotech SHBG radioimmunoassay (RIA). An additional evaluation with anotherresidual sample set was performed between the Access SHBG assay and the RocheElecsys® SHBGValidation: The results generated between the Access, Immulite and RIA methodsdemonstrated good agreement, as did the results between the Access and Elecsysmethods. The Method Comparisons yielded the following Deming regressionequations:Access = 1.12 (RIA) - 3.45 nmol/L, R = 0.96 (n = 41; range = 6 - 186 nmol/L)Access = 1.08 (Immulite) + 2.90 nmol/L, R = 0.97 (n=158; range = 6 - 190 nmol/L)Immulite = 0.93 (RIA) - 0.37 nmol/L, R = 0.97 (n = 41; range = 6 - 186 nmol/L)Access = 1.07 (Elecsys) -1.60 nmol/L, R = 0.99 (n = 123; range = 9 - 160 nmol/L)Conclusions: This study effectively demonstrates that the results from the automatedAccess SHBG assay correlate well to the Immulite SHBG, Immunotech SHBG RIA,and Elecsys SHBG assays. Laboratories transitioning between methods should expectvery good analytical agreement.Disclaimer: Product is currently not available in the US.

Comparison of a Reference Method and Three Immunoassay Methods

for Estradiol (E2)

D. Young, J. M. Mattke, A. Artus, S. Villeneuve, E. Romeu, W.Roudebush. Beckman Coulter, Inc., Chaska, MN

Objective: The objective of this study was to determine the correlation between theAccess® Estradiol assay and the International Federation of Clinical Chemistry(IFCC) ID-GCMS estradiol reference method. Two other automated immunoassayswith ID-GCMS standardization were also selected for correlation.Relevance: Assay standardization is essential for superior patient care and ensuringhigh quality, reproducible results. The IFCC recognizes reference methods and/orreference standards for use to ensure commutability between methods. For estradiol,the IFCC recognized reference method is isotope dilution-gas chromatography massspectrometry (ID-GCMS). In women, estradiol levels are used to monitor ovulatorystatus. Since estradiol levels reflect follicular maturation in women, they are avaluable tool in the assessment and treatment of sexual development, etiology ofamenorrhea, causes of infertility and menopause. In men, abnormally high levels ofestradiol are indicative of feminizing syndromes such as gynecomastia.Methodology: A set of 63 serum samples were collected for analysis across the rangeof the Access Estradiol assay for comparison and standardization to the IFCCrecognized ID-GCMS method. Samples had Access Estradiol values ranging from 23-4576 pg/mL. Additionally, a set of 247 serum samples was obtained from residual

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samples for comparison to the Access Estradiol assay and the Elecsys® Estradiol IIelectrochemiluminescence assay (Roche Diagnostics) and the Architect® Estradiolassay (Abbott Laboratories Diagnostics Division). Samples had Access Estradiolvalues ranging from 21-4572 pg/mL.Validation: The Access Estradiol assay and the ID-GCMS method demonstratedgood agreement. The Deming Method Comparison yielded the following regressionequation: Access = 1.02 (ID-GCMS) -6.18 pg/mL, R = 1.00Correlations between the three immunoassay methods demonstrated good agreement,but there is bias of approximately 28% between the Architect and both the Elecsysand Access assays. The Deming Method Comparison yielded regression equations of:Access = 1.07 (Elecsys) - 41.49 pg/mL, R = 0.99Access = 1.28 (Architect) - 38.03 pg/mL, R = 0.99Elecsys = 1.29 (Architect) - 56.19 pg/mL, R = 0.99Conclusions: This study effectively demonstrates that the results from the automatedAccess Estradiol method correlate well with the ID-GCMS and Elecsys methods.Interestingly, the Access Estradiol and Elecsys Estradiol II assays both demonstrate asimilar bias when correlated with the Architect Estradiol assay. Laboratoriestransitioning between Access and Elecsys should expect similar patient values.Transitioning between Architect and either Access or Elecsys may translate todifferent patient values and reference intervals, thus requiring discussions withphysicians.

Comparison of a newly developed ultrasensitive estradiol

immunoassay with the ID-GCMS reference method

D. Young, J. M. Mattke, S. Bord, J. Fieschi. Beckman Coulter, Inc.,Chaska, MN

Objective: To aid in the diagnosis of conditions where low concentrations of estradiol(E2) must be measured with precision, an ultrasensitive E2 immunoassay (usE2) wasdeveloped for the family of Access® Immunoassay Systems from Beckman Coulter.Relevance: Estradiol-17β (estradiol, E2) is the most potent natural estrogen inhumans and plays a major role during the sexual development and regulation ofreproductive function. Most commercially available immunoassays accuratelymeasure E2 concentrations during the ovulatory peak, luteal phase and pregnancy.However, due to assay sensitivity, most assays fail to accurately measure lowconcentrations of E2 which is necessary for several conditions such as precocious ordelayed puberty, onset of menopause, investigation of the menstrual cycle(amenorrhea, infertility, Polycystic Ovarian Syndrome [PCOS]) or gynecomastia. Inaddition to the clinical need for an ultrasensitive estradiol assay, the InternationalFederation of Clinical Chemistry (IFCC) recognizes the international referencemethod as isotope dilution-gas chromatography mass spectrometry (ID-GCMS) forestradiol assays.Methodology: The Access usE2 is a sequential, two-step competitive immunoassaywith calibrators traceable to the ID-GCMS reference method. Assay performance,including limit of detection, imprecision and functional sensitivity were evaluated, inaddition to a method comparison to the ID-GCMS reference method.Validation: Assay performance: The Access usE2 assay has a reportable range of <1to 200 pg/mL, defined by the limit of detection and the highest calibrator. Within-run,between-run and total imprecision coefficients of variation (CV) were < 10% between10 - 200 pg/mL. The functional sensitivity of the assay is <4 pg/mL. Time to firstresult is approximately 50 minutes.Comparison to ID-GC/MS: The Access usE2 calibrators were assigned values basedon a reference curve established with 54 human serum samples values assigned by theID-GCMS reference method. Correlation of this initial sample set achieved a Demingregression equation of Access = 0.99 ID-GCMS - 1.04, R = 0.98 (n=54; range 2.6 -231pg/mL). The accuracy of the standardization was confirmed with a second set of31 samples also tested by the ID-GCMS reference method. This comparison resultedin a Deming regression equation of Access = 0.98 ID-GCMS - 6.06, R = 0.98 (n = 31;range 6.0 - 181 pg/mL).Conclusions: This study effectively demonstrates that the results from the AccessusE2 method correlate well to the ID-GCMS reference method. Analysisdemonstrated a high level of precision and accuracy for low concentrations ofestradiol which is preferable for use in patients with diminished estradiol levels, suchas postmenopausal women.Disclaimer: Product is currently not available in the US.

Determination of intact parathyroid hormone (iPTH) reference

intervals based on iPTH values from ambulatory patients

M. M. Desouki, J. E. Madory, Y. Zhu. MUSC, Charleston, SC

Background: Serum iPTH reference intervals can be determined according to iPTHvalues in normal populations, but this approach requires recruitment of apparentlyhealthy individuals. Since iPTH level is closely related to the levels of serum calcium(Ca) and vitamin D (25-OHD), and renal function, we may be able to define iPTHreference intervals based on its levels in ambulatory patients with normal serum Ca, 25-OHD, and creatinine (Cr). The objective of this study is to determine if we need all orpart of these three analytes to define iPTH reference intervals from ambulatory patients.Methods: We identified 2033 ambulatory patents who had PTH testing with ADVIACentaur two-site sandwich immunochemiluminescent assay from our laboratoryinformation system. From these individuals, we searched patients with normal levels ofserum Ca (8.9-10.3 mg/dL), Cr (0.4-1.3 mg/dL), and 25-OHD (25-80 ng/mL). Sincethese laboratory results were obtained from ambulatory patients instead of normalcontrol subjects, we employed Hoffmann statistical approach to define iPTH referenceintervals. We used Microsoft Office Excel 2007 to generate Hoffman plot. The lowestand the highest iPTH levels in the linear portion of the S-shaped Hoffman plot aredefined as reference intervals.Results: From 2033 subjects, we identified 1415 patients with normal Ca levels, 523with normal Ca and Cr, 369 with normal Ca and 25-OHD, and 147 with normal Ca, Cr,and 25-OHD. iPTH reference intervals were determined as 10-110, 10-80, 10-75, and10-70 pg/mL, respectively based on iPTH levels from ambulatory patients in the abovefour groups.Conclusions: iPTH reference intervals determined based on its levels in patients withnormal Ca plus Cr, normal Ca plus 25-OHD, and normal Ca, Cr, and 25-OHD are veryclose to the manufacturer provided iPTH reference interval (14 - 72 pg/mL) obtainedfrom 142 apparently healthy individuals with normal Ca levels. However, iPTHreference intervals determined from patients with normal Ca alone is higher. This studysuggests that iPTH reference intervals can be determined from ambulatory patients withnormal Ca plus normal Cr and/or 25-OHD levels. Serum calcium alone should not beused to determine iPTH reference intervals if the subjects are not from healthypopulations.

Evaluation of the Beckman Coulter Access® Testosterone Assay

A. M. Dnistrian, R. Gonzalez-Espinosa, F. Celucia, A. Mathew, M.Fleisher. Memorial Sloan-Kettering Cancer Center, New York, NY

Introduction: The Beckman Coulter Access® is recognized for the reliability of tumormarker assays for the management of prostate cancer patients (total PSA and freePSA). The availability of a sensitive and reliable testosterone assay on the sameinstrument as the tumor markers would be useful in monitoring prostate cancerpatients during the course of androgen deprivation therapy, the basic intervention forhormone dependent prostate cancer.Methods: We evaluated the performance characteristics of the Access testosteroneassay, a chemiluminescent immunoassay for the quantitative determination of totalserum testosterone using the fully automated Access Immunoassay System. Thesensitivity, accuracy, precision, linearity and reportable range for the Accesstestosterone assay were validated by standard statistical procedures for the ClinicalLaboratory. A method correlation with the manual Siemens Coat-A-Count® RIAprocedure was performed with male specimens (N = 100) with testosteroneconcentrations ranging from 10 to 1205 ng/dL.Results: The Access testosterone assay exhibited an excellent analytical sensitivity(10 ng/dL) and a reportable range up to 1600 ng/dL. The within run imprecision was1.8 - 3.2 % at three different levels of quality controls (140, 454 and 838 ng/dL,respectively), and the between run imprecision was 4.6 - 7.8 %. Regression analysisof correlation data for the automated procedure (y) with the manualradioimmunoassay method (x) yielded the equation y = 1.046x + 0.2 (R=0.9909)indicating the results were substantially equivalent. Discordant results between thetwo methods were observed for 7 of 27 specimens (26 %) with testosteroneconcentrations < 40 ng/dL signifying that accurate results at ultra low levels were notpossible with these methods.Conclusion: The Beckman Coulter Access testosterone assay is a fast, reliable andreproducible method for the evaluation of male endocrine function. The test correlatedwell with a sensitive radioimmunoassay procedure but accurate measurements forpatients with concentrations < 40 ng/dL would require an ultrasensitive method.

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The relevance of hormones ratio between cerebral spinal fluid

(CSF)and serum for patients with pituitary tumors

M. Purice1, M. Gheorghiu2, M. Coculescu2. 1"C.I.Parhon" NationalInstitute of Endocrinology, Bucharest, Romania, 2"Carol Davila“University of Medicine and Pharmacy, Bucharest, Romania

It was previously shown by our group and others that pituitary tumors (PT) alter thepermeability of blood-brain barrier (BBB) for the anterior pituitary hormones.Albumin CSF/serum ratio is generally considered an accurate index of the integrity ofBBB (AR, albumin index).The aim of the study was to assess if evaluation ofhormonal CSF/serum ratio (RC/S) has or not relevance for the damage of BBB and if itcan help for the diagnostic of the tumor.Methods and patients: We evaluated CSF/serum ratio for GH, PRL, LH, FSH andTSH and albumin for 52 patients with pituitary adenomas (17-79 years, 25 M/27F, 16before and 36 after pituitary surgery) comparative to a control group (n=10, 21-79years, 6M /4 F, before undergoing abdominal or peripheral surgery). The study had theapproval of the local Ethical Committee. Anterior pituitary hormones and albuminwere simultaneously measuerd in serum and CSF by rapid fluoroimmunoassay (FIA-DELFIA Wallac)) and nephelometry (ARRAY, Beckman Coulter), respectively. FIAmethod for serum was prior validated for CSF and compared with a standard IRMAmethod. The report RC/S > 1 was considered as an index of hormonal secretion directin CSF. An albumin index >0.007 was considered abnormal.Results: We found significant higher CSF/serum ratio comparative to control(p<0.001) for all pituitary tumor types but with normal concentrations LH,FSH andTSH in serum .For at least one pituitary hormone a RC/S > 1 was found in significantlymore patients with tumors in contact with BBB- either before pituitary surgery (47%)or after surgery (56%)- compared with only 6% in patients with PT without contactwith BBB before surgery (p = 0.001). Only patients with acromegaly andprolactinoma show significant high concentrations of GH and PRL, both in serum andCSF, comparative with control group (p<0.001) but with CSF/serum ratio <1.Albumin in CSF, in serum and the albumin index, were not statistically differentbetween contact and non-contact tumors or in patients with hormonal CSF/serum ratio>1, compared to those with hormonal CSF/serum ratio (RC/S) <1.Conclusions: Pituitary hormones CSF/serum ratio can be a good index for BBBalteration but only for those hormones that are in low or normal serumconcentrations (LH, FSH, or TSH). The alteration is correlated with a significanthigher CSF/serum ratio mainly for tumors with suprasellar extension. CSF/serumalbumin evaluation shows that there is no alteration of the CSF flow rate in patientswith pituitary adenomas and with increased CSF/serum ratio for the anterior pituitaryhormones, compared to controls.

A case of pseudonormocalcemia and catch 22

A. Viljoen1, F. Kaplan1, S. Pereira1, P. J. Twomey2. 1East and NorthHertfordshire NHS Trust, Stevenage, United Kingdom, 2Ipswich HospitalNHS Trust, Ipswich, United Kingdom

We present a case of dual pathology related to calcium homeostasis, assimilating to a‘normal’ calcium concentration. We coin the term, pseudonormocalcemia which hasnot been used before in the medical literature.Calcium homeostasis is a complex process which primarily involves: serum calciumand phosphate, 1,25-dihydroxyvitamin D and parathyroid hormone.Hypoparathyroidism is a relatively uncommon condition in which parathyroidhormone secretion is deficient or absent with subsequent hypocalcemia andhyperphosphatemia. It is most commonly seen following neck surgery.Hypoparathyroidism may also be due to developmental defects of the parathyroidglands as seen in the DiGeorge / Velocardiafacial syndrome (DGS/VCFS).Hyperthyroidism is associated with modesty raised calcium concentrations, with somereports of significant hypercalcemia. This association was noted more than a centuryago.We present a case of an 18 year old female patient who initially presented withthyrotoxicosis and became acutely and severely hypocalcemic following treatmentwhich yielded her to be clinically euthyroid. Subsequent investigations for thehypocalcemia revealed that the patient had hypoparathyroidism. The cause for thiswas shown to be DGS/VCFS, also known as CATCH22, and confirmed by FISHanalysis revealing a deletion at the 22q11.2 location.As depicted by figure 1, the initial calcium concentrations at the time of diagnosiswere near normal. The patient then became acutely hypocalcemic following

carbimazole treatment for the hyperthyroidism, which uncovered the diagnosis ofhypoparathyroidism. The hypoparathyroidism was managed with Calcichew andAlphacalcidol. The patient initially responded to treatment for both disorders but poorcompliance of all therapies lead to a rebound ‘pseudonormocalcemia’. Most recently,in light of good compliance, the patient is clinically euthyroid with truenormocalcemia.To our knowledge this is the 10th report of hyperthyroidism associated with DGS/VCFS. As this syndrome is associated with other auto-immune conditions weinvestigated the possible underlying immunological mechanisms betweenhyperthyroidism and DGS/VCFS.

A Case of Biotin Interference in the Roche Elecsys 2010 Intact

Parathyroid Hormone Assay

D. L. Meany, S. M. Jan de Beur, M. J. Bill, L. J. Sokoll. Johns HopkinsMedical Institutions, Baltimore, MD

Objective: The objective of this study was to investigate biotin as an interferingsubstance in the Roche Elecsys 2010 intact PTH assay in a patient with renalimpairment taking high doses of biotin.Background: A 64-year-old female with end-stage renal disease (ESRD) wasevaluated for management of renal osteodystrophy. Adynamic bone disease (ABD)was suspected because of low normal serum intact parathyroid hormone (PTH),intermittently elevated serum calcium, and severe osteoporosis. However, her mildlyelevated serum alkaline phosphatase (range: 149-196 U/L, reference range: 30-120 U/L) was inconsistent with the low bone turnover observed in ABD. This discrepantclinical profile prompted investigation into the PTH assay used at our institution.Simultaneous samples were analyzed for intact PTH on our Roche Elecsys 2010immunoassay analyzer and at a reference laboratory on the Siemens Immulite 2000immunoassay analyzer. Discrepant values of 48 ng/L and 786 ng/L were obtained,respectively. Dilution studies confirmed the presence of a negative interference sincePTH was higher in the diluted samples compared to the undiluted sample (undiluted =48 ng/L; diluted 1:20 = 567 ng/L). After heterophilic blocking reagent had revealedno effects, we reviewed the patient’s medical history which suggested that biotin maybe the cause of the interference since the Elecsys 2010 assay uses biotin-streptavidinmechanisms.Methods: We first determined the biotin concentration in the specimen to be 4.8 µg/L, approximately ten fold higher than the reference range upper limit (200-500 ng/L).Two different approaches were subsequently used to examine the interfering role ofbiotin: (i) studying the effect of added biotin and (ii) removing the effect of biotin, inthis case using streptavidin-coated microparticles. The first approach was carried outby adding various amounts of free biotin (0- 160 µg/L) into sera with normal andelevated intact PTH concentrations (33 and 487 ng/L, respectively). This secondapproach treated 50 µL of the patient’s specimen with the streptavidin microparticlesfor 1 hour at room temperature with shaking.Results: The first approach failed to mimic the interference, whereas the secondapproach clearly identified biotin as the interference. In this experiment, the intactPTH concentration in the patient’s specimen increased from 32 ng/L pre-treatment to419 ng/L post-treatment. The PTH recovery was >1,000% compared to recoveries ofapproximately 80% in 3 control specimens from patients not taking large doses ofbiotin. To further confirm the interfering role of biotin, serum intact PTH wasmeasured in both laboratories after the patient stopped taking biotin for two weeks.The Elecsys 2010 result, 158 ng/L, was consistent with the result measured on theImmulite 2000 (223 ng/L) using the same specimen.

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Changes in calcium concentration related to thyroid function

Mar 07 Aug 07 Sept 07 Apr 08 Jan 09Reference Interval

Treatment notes

Treatment started for

thyrotoxicosis 'Pseudo-

normocalcemic'

Treatment started for

hypo-calcemia

Clinically euthyroid

Non-compliance to treatment

'Pseudo-normocalcemia'

Good compliance. Clinically euthyroid.

True normo-

calcemia

Adjusted calcium concentration

8.04 (2.01) 6.24 (1.56 ) 8.24 (2.06 ) 9.56 (2.39 ) 9.16 (2.19)

8.8-10.4 mg/dL (2.2-2.6

mmol/L)Thyroid stimulating hormone

<0.03 <0.03 0.03 <0.03 <0.030.3-5.6 mIU/L

Free thyroxine (FT4)

43 19.2 11.8 58.4 13.510-21

pmol/L

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Conclusions: Using the streptavidin microparticle treatment, we determined theinterfering role of biotinylated molecules present in specimens from patients withrenal impairment who take high doses of biotin with immunoassays that use biotinstreptavidin interactions. An in vitro interference study using exogenous biotin failedto mimic the interference, which may be due to the discrepancy between exogenousbiotin and endogenous biotinylated molecules.

Thyroid Autoimmunity and Trimester Specific Reference Intervals for

Thyroid Function Tests in Pregnant Chinese Women

S. Lu, Z. Xie, M. Wang, B. Zhu, Y. Sun. Zhejiang University, Hangzhou,China

Background: Proper maternal thyroid function is important during pregnancy.However, thyroid disorders are common in women of reproductive age, and maternalthyroid dysfunction is associated with a number of adverse outcomes. Because ofphysiological changes associated with pregnancy, interpretation of thyroid functiontests (TFT) in pregnant women requires use of trimester specific reference intervals.In addition, ethnic and dietary differences are also known to influence thyroidfunction. For these reasons, reference intervals for TFT should ideally be establishedusing local patient samples. The objective of our study was to establish trimesterspecific reference intervals for thyroid function tests in pregnant Chinese women.Methods and results: Serum samples were collected from 1409 pregnant women aspart of routine antenatal care (n = 438, 400, and 571 for 1st, 2nd, and 3rd trimesters,respectively). All samples were tested for TPO-Ab, Tg-Ab, TSH, FT4, TT4, and TT3using the Abbott ARCHITECT analyzer. In the study population, 18.7% of womenwere positive for TPO-Ab and/or Tg-Ab. Reference intervals (2.5th - 97.5th percentile)were calculated using antibody negative samples, and are reported in the table below.

Conclusions: Laboratory evidence of thyroid autoimmunity was relatively in the studypopulation. Trimester specific reference intervals for TFT were established inpregnant Chinese women. These reference intervals were significantly different fromthose in non-pregnant women, and should prove useful as an aid in the interpretationof TFT during pregnancy.

Variability in Calculated Serum Free Testosterone Concentrations

J. Hackbarth, J. Hoyne, S. K. Grebe, R. J. Singh. Mayo Clinic, Rochester,MN

Background: Free testosterone (fT) levels are important in the assessment ofandrogen status. However, equilibrium dialysis (ED) and immunoassay, two commonmethods of measuring fT, are either expensive and laborious or inaccurate,respectively. One alternative is to calculate fT levels using mass action equationsbased on serum albumin, sex hormone binding globulin (SHBG), and totaltestosterone (TT) levels. Several different formulas are commonly used. The goal ofthis study was to compare fT calculated from an equation with fT measured by ED todetermine if variables including albumin, SHBG, association constants, andpopulation makeup affect goodness of fit.Method: Serum concentrations of albumin, SHBG, TT and fT were measured for twoseparate groups of patients who underwent fT testing at Mayo Clinic Rochester: A(n=191, 93M/98F) and B (n=209, 171M/38F). Albumin, SHBG, and TT levels were

determined by colorimetric assay, two-site chemiluminescence, or LC-MS/MS,respectively. fT concentration was determined either by ED or the mass actionequation described by Vermeulen, et al. Association constants for testosterone bindingto albumin and SHBG were previously published by Vermeulen, et al., Sodergard, etal. and Emadi-Konjin, et al. or individually derived for each patient group to yield thebest fit with the ED data (cA and cB). fT concentrations measured by ED or calculatedusing these association constants were compared by weighted linear regression usingAnalyse-It. Statistical significance between groups was determined using JMP.Results: Individually derived association constants gave the best fit for theirrespective patient groups compared to previously published constants (A/cA:y=1.01x-0.23, r=0.941 and B/cB: y=1.00x-0.02, r=0.947). However, these constantsyielded a poorer fit when applied to the other group (A/cB: y=1.29x-0.37, r=0.937 andB/cA: y=0.79x+0.03, r=0.952). Roughly 10-25% of each group had a poor fit (>15%CV) between calculated and ED fT results. SHBG levels in this subset weresignificantly higher than the subset with better fit (p<0.01). Albumin levels hadminimal impact on fit, as calculated fT results were statistically similar whetheralbumin was measured or assumed to be 4.3 g/dL (p=0.94).Conclusion: Use of a mass action equation to calculate fT is a cheaper and fasteralternative to ED. However, the choice of association constants has a significantimpact on the fit between calculated and ED-measured fT. Constants that wereindividually derived to specific patient groups yielded a better fit for their respectivegroups than previously published constants, but yielded poorer fits when used on theother dataset. Poor fit is associated with higher SHBG levels and differences in fitbetween the two groups are likely due to their different gender makeup. Thisdifference may be attributable to gender variability in the levels of other steroidhormones that bind SHBG. These results indicate that constants need to be verified ineither large population sets or between numerous smaller datasets, and that differentconstants may be required for males and females to ensure goodness of fit.

Evaluation of the automated DxI® direct Testosterone assay.

D. Gruson, J. Ketelslegers, O. Alexopoulou, D. Maiter, C. Fillée. CliniquesUniversitaires St Luc, bruxelles, Belgium

Background: Radioimmunoassay (RIA) and chemiluminescence immunoassays arestill the most widely used methods for measuring total testosterone (TT). RIA methodsincorporating extraction procedure and chromatography offer several advantages,including removal of interfering proteins and separation of cross-reacting steroids, butare labor intensive and use a large serum aliquots to increase the sensitivity. Therefore,high-throughput and low volume direct automated assays employing serum or plasmawithout any extraction are often preferred nowadays but their performances andaccuracy for measuring TT in male and female patients is still a matter of debate. Theaim of this preliminary study was to assess the analytical performances of the DxI®Testosterone assay and to compare it to our conventional RIA method.Methods: The linearity, precision and functional sensitivity were determined for theBeckman Coulter DxI® Testosterone assay. Additionally, 100 patient plasma samplessent to the Cliniques Universitaires St-Luc Laboratory for testosterone testing were usedto measure and compare the testosterone values obtained by the RIA method and theDxI® Testosterone assay, respectively. Statistical analysis was performed on log-transformed data with the Medcalc software. Results: Within-run precision testingindicated maximum coefficients of variation (CV) for the DxI® Testosterone assay of27% at 0.35 nM, 1.8% at 7.4 nM and 2.2% at 15.7 nM. The between-run precision CVsfor five levels of plasma pools were 24% at 0.43 nM, 9.9% at 1.0 nM, 8.7% at 1.5nM,5.7% at 7.7nM and 4.7% at 16.0 nM. The functional sensitivity of the DxI®Testosterone assay was estimated to be 0.65 nM. The average recoveries after dilution oftwo plasma samples titrated at 37.3 nM and 35.0 nM were 109% for a dilution factor of2 - 97% for a dilution factor of 4 and 126% for a dilution factor of 8, reflecting a goodlinearity for this assay. The comparative analysis of testosterone results yielded thefollowing regression equations: log[DxI] = 0.95 log[RIA] + 0.23 in males (r=0.953;p<0.001; n=74) and log[DxI]= 1.31 log[RIA] + 0.17 in females (r=0.757, p<0.001;n=26). Bland and Altman plots showed a systematic positive bias for the DxI® for bothmales and females with mean differences of 4.1nM and 0.51 nM, respectively.Conclusions: The results of this preliminary study show that the DxI Testosterone assayappears to be a reliable method for the routine determination of TT without previousextraction. The accuracy and sensitivity of this assay are suitable for the determinationof TT in both men and women. For both sex groups, the correlation between the DxI®assay and our reference RIA was excellent but with a systematic positive bias for DxI®.Additional studies should confirm the exact reference intervals using well-defined andcharacterized populations and the reliability of the DxI® assay for its use in paediatricpopulations.

Trimester, Median (2.5th-97.5th percentile)First Second Third

TSH (mIU/L)1.219

(0.155-3.781)1.314

(0.339-3.512)1.619

(0.336-4.316)N=365 N=346 N=480

FT4 (pmol/L)14.05

(10.925-17.741)12.0850

(9.288-15.240)10.66

(7.871-14.075)N=365 N=346 N=480

TT4 (nmol/L)102.59

(73.148-164.422)130.725

(89.656-174.913)118.31

(83.493-170.143)N=365 N=346 N=480

FT3 (pmol/L)3.82

(2.892-5.019)3.765

(2.914-4.603)3.65

(2.85-4.45)N=365 N=346 N=480

TT3 (nmol/L)1.67

(1.202-2.570)2.18

(1.464-3.01)2.055

(1.37-2.889)N=365 N=346 N=480

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Technical performance of the first fully automated assays for human

soluble fms-like tyrosine kinase 1 and human placental growth factor

E. Schneider, A. Gleixner, R. Haenel, W. Kraus, T. Ciesiolka, B. Denk, D.Gassner. Roche Diagnostics GmbH, Penzberg, Germany

Objective: An imbalance in circulating soluble fms-like tyrosine kinase 1 (sFlt-1), asplice variant of Flt1, and placental growth factor (PlGF) appears to be involved in thepathogenesis of preeclampsia (PE). Pregnant women who develop PE exhibit higherlevels of sFlt-1 and decreased levels of PlGF. sFlt-1, an antagonist of PlGF (andVEGF), contributes to PE by absorbing free VEGF and PlGF in the maternalcirculation and thereby preventing interaction with their respective membranereceptors. Automated immunoassays were established for sFlt-1 and PlGFmeasurements in human serum as an aid in diagnosis of PE in conjunction with otherclinical findings.Materials and Methods: Elecsys sFlt-1 and PlGF are sandwich immunoassays forthe quantitative determination of sFlt-1 and PlGF. Each assay applies a biotinylatedmouse monoclonal capture and a monoclonal detection antibody labeled with aruthenium complex. PlGF capture antibody is directed to an unique epitope on thePlGF molecule involved in receptor binding. sFlt-1 antibodies used do not interferewith the PlGF binding site of sflt-1.Basic assay characteristics were assessed. Molecular forms of sFlt-1 and PlGF innormal pregnant and PE samples were analyzed by Elecsys sFlt-1 and PlGF followinggel filtration over Superdex 200. sFlt-1/PlGF ratios in normal pregnancies andpregnancies diagnosed with PE were determined.Results: Elecsys PlGF in serum detects biologically active PlGF, i.e. free PlGF aswell as that part of sFlt-1-complexed PlGF which can still bind receptor. Elecsys sFlt-1 detects total sFlt-1 (free multimeric and ligand-complexed multimeric sFlt-1)independent on PlGF binding. Destabilization of receptor ligand interaction by heattreatment released receptor masked PlGF from multimeric sFlt-1. Elecsys sFlt-1covers a measuring range of 10 to 85000 pg/mL. The limit of detection (LOD) usingCLSI protocol EP17A was found <10 pg/mL . The limit of quantitation (LoQ) was<15 pg/mL. Assay standardization against sVEGFR-1 ELISA (R&D Systems)demonstrated good agreement (P/B: y = 1.025*x-7.1, r = 0.967, n= 120, range < 2000pg/mL). Total precision (CLSI protocol EP5-A2) resulted in CVs between 1.8 to 5.0%for concentrations from 59 to 72350 pg/mL. No high dose hook effect was found up to200000 pg/mL. Elecsys PlGF covers a measuring range from 3 to 10000 pg/mL. LoDwas <3 pg/mL. LOQ <10 pg/mL. Standardization against PlGF ELISA (R&DSystems) yielded comparable results (P/B: y = 1.013*x+0.58, r = 0.994, n= 119, range< 1000 pg/mL). Total precision CVs ranged from 1.0 to 2.6% for concentrationsbetween 17.8 to 5730 pg/mL. No high dose hook effect was observed up to 100000pg/mL. Both assays enable results within 18 minutes. In 75 normal pregnancies PlGF(sFlt-1) values ranged from 23 to 1818 pg/mL, mean 429 pg/mL (354 to 5282 pg/mL,mean 1883 pg/mL). The mean sFlt-1/PlGF ratio was 12.5 (range 1-114) in controls vs.590 (range 145 -1440) in 21 pathological cases.Conclusion: Elecsys sFlt-1 and PlGF are the first, fast and automated immunoassaysfor convenient and reliable measurement of PE markers in maternal serum.Availability of these assays will offer new possibilities in the diagnosis of PE.

Temperature Effects on Free Thyroid Hormone Measurements by

Ultrafiltration and Isotope-Dilution Liquid Chromatography-Tandem

Mass Spectrometry

T. Guo1, A. Xu1, S. J. Soldin1, S. J. Soldin2. 1NMS Labs, Willow Grove, PA,2Georgetown University, Washington, DC

BACKGROUND: The free thyroid hormones, including free thyroxine (FT4) and free3,5,3'-triiodothyronine (FT3), are the most physiologically active fractions of thyroidhormones. The two recommended laboratory tests to assess thyroid function aresensitive thyroid stimulating hormone (TSH, thyrotropin) and FT4 tests. Most clinicalchemistry laboratories measure FT4 by various immunoassays (IAs), which have beencriticized for their lack of specificity. Recently, isotope dilution (ID)-liquidchromatography tandem mass spectrometry (LC-MS/MS) assays have been reportedto measure FT4 and FT3 in serum, following a physical separation by eitherequilibrium dialysis (ED) or ultrafiltration (UF) of serum. We previously developedthe first UF and ID-LC-MS/MS assay to simultaneously measure FT4 and later FT3 inundiluted serum, with UF performed at 25°C. A small comparative study had shownresults were higher at 37°C. The results at 25°C were, however, identical to the thengold standard Nichols equilibrium dialysis method, which was performed at 37°C. In

a more detailed study, we have now further investigated the ultrafiltration temperatureeffects on free thyroid hormone measurements by UF ID-LC-MS/MS, and comparedits analytical performance with ED ID-LC-MS/MS at 37°C.METHODS: An API-5000 triple-quadrupole mass spectrometer (AB-Sciex) coupledwith electrospray ionization (ESI) source and Shimadzu HPLC system was usedemploying isotope dilution with 13C6-labeled internal standard (IS) for each analyte.400 µL of undiluted serum/plasma were placed in a Centrifree YM-30 ultrafiltrationdevice (30,000 MW cut-off, Millipore), and centrifuged at 2900 rpm for 30 min at25°C. A duplicate ultrafiltration was repeated at 37°C. 150 µL of ultrafiltrates werefurther deproteinized by adding 450 µL of methanol containing internal standards(IS). After centrifugation, 500 µL of supernatant were diluted with 400 µL ofdeionized water, and 650 µL aliquot was injected onto a C-18 column. The tandemmass spectrometer was operated in negative ion mode, and thyroid hormones werequantified in multi-reaction monitoring (MRM) mode (649.7/126.7 for T3; 655.8/127for T3-13C6; 775.7/127 for T4; 781.8/127 for T4-13C6).RESULTS and CONCLUSION: The results of ultrafiltration temperature effectdemonstrated a linear least squares regression equation of y = 1.515x - 0.097 (n = 28;r = 0.974; Syx = 5.59; range 1.14-85.2 pg/mL) for FT4, and y = 1.495x + 0.038 (n =24; r = 0.964; Syx = 1.414; range 0.55-23 pg/mL) for FT3. Both the serumconcentrations of FT4 and FT3 show an expected temperature-dependency: byswitching the ultrafiltration temperature from 25 to 37°C, the results for both free T4and free T3 increase by a factor of ~1.5. A comparison study between theultrafiltration tandem mass spectrometric method and an equilibrium dialysis tandemmass spectrometric method was also performed at 37°C, and results show goodagreement, with a linear least squares regression equation of y = 0.428x + 2.712 (n =37; r = 0.847; Syx = 0.803; range 5-21 pg/mL) for FT4.

Performance Characteristics of Six Intact Parathyroid Hormone

Assays

S. L. La'ulu1, W. L. Roberts2. 1ARUP Institute for Clinical andExperimental Pathology, Salt Lake City, UT, 2Department of Pathology,University of Utah Health Sciences Center, Salt Lake City, UT

The aim of this study was to evaluate the performance characteristics of six intactparathyroid hormone (PTH) assays: Beckman Coulter Access, Abbott ARCHITECTi2000SR, Siemens ADVIA Centaur, Roche Modular E170, Siemens IMMULITE2000, and DiaSorin LIAISON. Imprecision studies were performed using threeconcentrations of commercially available quality control materials. Two runs ofduplicate testing were conducted per day, for 5 days with a minimum of 2 hoursbetween runs. For method comparison, 203 serum samples were tested by all methodswith the E170 as the comparison method. A study comparing sample types and theirstabilities was conducted where 20 individuals had two types of BD Vacutainer tubesdrawn: red top and SST. An aliquot from each tube was tested after being subjected tothe following conditions: tested immediately, frozen immediately and tested afterthawing, after 24 hours at room temperature, and after 24 hours and 48 hoursrefrigerated. Total CV’s ranged from 1.6 to 10.9%, 1.5 to 8.1%, and 1.1 to 5.5% forlevels 1, 2, and 3, respectively. The E170 always had the lowest within laboratory CVand the LIAISON had the highest. Method comparison results by Passing-Bablokregression had slopes of 1.01-1.32 and correlation coefficients of 0.93-0.99. Theresults of the stability study are summarized (Table).

*Indicates a statistically significant difference (p<0.05) between fresh and otherstorage conditions. For red top tubes, one freeze-thaw cycle was not different from immediate analysis forall methods except the IMMULITE. Serum samples show statistical differences after

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Percent recovery compared to freshRed Top Frozen 24h RT 24h 4C 48h 4CAccess 87.9 82.4* 86.5* 92.4*ARCHITECT 102.4 86.4* 91.3* 96.6Centaur 99.6 82.0* 87.4* 86.4*E170 101.2 92.7* 96.6 100.5LIAISON 100.4 85.9* 94.0 97.1IMMULITE 81.0* 77.0* 84.6 86.0*SST Frozen 24h RT 24h 4C 48h 4CAccess 95.2* 79.1* 89.3* 84.0*ARCHITECT 99.9 80.8* 91.1* 89.0*Centaur 103.2* 76.7* 84.9* 80.5*E170 99.2 86.2* 97.3 92.4*LIAISON 96.0* 82.3* 98.4 98.3IMMULITE 92.0* 116.5 95.7 86.3

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24 hours refrigerated for three of the six methods. In summary, imprecision wasacceptable for all methods. All methods correlated well to the E170 (r = 0.92 to 0.99).The IMMULITE exhibited the highest positive bias. The types of collection tube andsample storage conditions are more important for some methods than others.

Comparison of RIA for the measurement of 25-OH Vitamin D with

automated Liaison CIA and with LC-MS/MS: A need for

standardization.

R. Kreller, M. K. Gupta, T. M. Daly. Cleveland Clinic Foundation,Cleveland, OH

With the recognition of high prevalence of Vitamin D insufficiency/deficiency ingeneral population there has been growing interest in the screening for 25-OHVitamin D levels. The increasing volume requires a simple reproducible method thatwill accurately estimate the total circulating 25-OHD independent of circulating25OHD2 and D3. There is a growing confusion and concern about the variabilityamong available Vitamin D assays i.e RIA vs. automated chemiluminescenceimmunoassay [CIA, Diasorin] and LC-MS/MS. To assess this variability we selected148 samples in different range [between <7-150 ng/mL by RIA, Diasorin] and froze 4aliquots and shipped one to a lab performing 25OHD analysis by CIA [Liaison,Diasorin] and another to a lab performing analysis by LC-MS/MS and compared theirresults with RIA.Both RIA and CIA utilize same antibody that reacts to both D3 and D2 forms equally.Data showed the median values [25-75 percentile range] of 23.65 (12-32.8 ng/mL) forRIA, 29 (10.2-32.8 ng/mL) for CIA and 32.0 [14.5-42.5]for LC-MS/MS. Linearregression analysis showed the following equations: CIA vs. RIA = 0.944 + 2.05 (r =0.899); LC-MS/MS vs. RIA = 0.74 + 0.94 (r=0.91) LC-MS/MS vs CIA.= 0.75 + 1.42(r=0.89). The paired T-test statistics showed significant differences in the mean levelsbetween LC-MS/MS and RIA or CIA [P (one tailed) = <0.0001]. Our results showthat although all three assays correlated well with each other the 25OHD levels weresignificantly higher by LC-MS/MS than immunoassays. Using a cutoff level of 30 ng/mL[defined by RIA] for optimum levels, about 18 % of patients had insufficientlevels by RIA and CIA had adequate levels by LC-MS-MS. This number is higher[28%] if we use 25 as an optimum cutoff value that is defined with this LC-MS/MSmethod.In conclusion, Vitamin D assays compared here show good correlation but differ indefining an individual as deficient vs. adequate. Our results indicate the need ofstandardization of these methods in terms of reference cutoff level.

LC-MS/MS methods for the measurement of androstenedione and

testosterone in serum.

E. Erdogan1, M. M. Kushnir2, B. Yue2, T. L. Blamires2, W. L. Roberts1, A.W. Meikle1, A. L. Rockwood1. 1Department of Pathology, University ofUtah, Salt Lake City, UT, 2ARUP Laboratories, Salt Lake City, UT

Introduction: Androgens, a group of 19-Carbon steroids derived from cholesterol, havedirect effects on diverse physiological and behavioral systems. Androstenedione andtestosterone are frequently measured for detection of androgen-secreting tumors ofovarian and adrenal origin, evaluation of inborn errors of sex-steroid metabolism anddisorders of puberty. Sensitivity, specificity and high throughput make LC-MS/MSmethods advantageous over immunoassays for androgen analysis. Here we describe anLC-MS/MS method for the measurement of androstenedione and a two dimensional(2D)-LC-MS/MS method for the simultaneous measurement of androstenedione andtestosterone using Agilent 6410 triple quadruple (QQQ) tandem mass spectrometer withHot Box Upgrade (Santa Clara, CA). Methods: LC/MS-MS method: samples werederivatized using aqueous hydroxylamine and extracted using methyl tert-butyl ether.Chromatographic separation was performed on a 1200 series HPLC system (Agilent);4.6 x 50 mm Rapid Resolution HT C18 column with 1.8-µm particles (Agilent), themobile phase (methanol with 10 mol/L aqueous formic acid). Analysis was performedusing Agilent 6410 QQQ in the multiple-reaction monitoring (MRM) mode using masstransitions 317 to 124 and 317 to 112 for androstenedione and 307 to 124 and 307 to 112for d3-testosterone (internal standard). 2D-LC-MS/MS method: the sample preparationwas performed as described above using d3-testosterone and d7-androstenedione. A two-dimensional chromatographic separation was performed using a Gemini Phenylcartridge with mobile phase (gradient, methanol with 10 mol/L aqueous formic acid) asthe first dimension and a Gemini C18 column with mobile phase (gradient, acetonitrile

with 10 mol/L aqueous formic acid) as the second dimension. Analysis was performedusing Agilent 6410 QQQ with Hot Box Upgrade. Mass spectrometry conditions were asdescribed above for the LC-MS/MS method with the addition of the MS/MS transitionsfor testosterone: 304 to 124 and 304 to 112. Results: LC-MS/MS method results: limitof quantification (LOQ) and upper limit of linearity (ULOL) were 0.33, and 92 ng/mL,respectively. Intraassay CVs were 15.9% (0.3 ng/mL) and 3.8% (2.3 ng/mL) andinterassay CVs were 5.0% and 3.6% at concentrations of 0.3 and 2.3 ng/mL,respectively. The LC-MS/MS method comparison with chemiluminescentimmunoassay (Siemens) resulted in a regression equation y= 0.59x + 2.02, r= 0.41, Sy/x= 0.80 (n=31 samples). Out of 22 structurally similar steroids, none seemed to interferewith the analysis. The 2D-LC-MS/MS method results: LOQ and ULOL were 0.05 and40 ng/mL, respectively. Total imprecision of the method, at concentrations 0.7-3.0 ng/mL, was less than 15% for androstenedione and testosterone. The method comparisonwith a chemiluninescent immunoassay for androstenedione resulted 2D-LC-MS/MSevaluated= 0.53*CEIA + 0.06, r=0.93, Sy/x= 0.17 (n=20 samples); and testosteronecomparison with an LC-MS/MS resulted 2D-LC-MS/MSevaluated = 1.00*LC-MS/MSreference + 0.04, r=0.98, Sy/x= 0.01 (n=20 samples). No interference was observed with19 structurally similar steroids. Conclusion: LC-MS/MS method is free of crossreactivity and has acceptable sensitivity for the measurement of androstenedione inserum. 2D-LC-MS/MS showed acceptable performance using Agilent 6410 QQQ withHot Box Upgrade and allowed the simultaneous measurement of androstenedione andtestosterone.

The Establishment of Reference Intervals of Thyroid Peroxide

Antibodies, Thyroglobulin Antibodies, and Thyroglobulin Assay by

Modular E170 in Korean Population Compared with Immulite 2000

W. Lee, S. Yoon, O. H. Kwon, J. Kim. Yonsei University College ofMedicine, Seoul, Republic of Korea

Measurement of thyroid peroxidase antibodies (TPOAb) is useful in diagnosingpatients with autoimmune thyroid disease (AITD). Measurement of thyroglobulinantibodies (TgAb) is used to detect potential interferences with thyroglobulin (Tg)immunoassays and in limited situations for the diagnosis of AITD. The primary use ofserum Tg measurements is as a tumor marker for patients carrying a diagnosis ofdifferentiated thyroid cancer.We determined TPOAb, TgAb, and Tg by Roche Modular E170 analyzer. Wedetermined the reference intervals for above items using 139 healthy subjects (Male/Female, 79/60; mean age, 39.2; range, 25~49). We used the following exclusioncriteria for reference subjects: blood glucose <50 mg/dL or >126 mg/dL; AST >36 U/L (Male), AST >30 U/L (Female); ALT >50 U/L; Hb <11.0 g/dL; platelet <100,000/mcL or >1,000,000/mcL; WBC <3,000/mcL or >15,000/mcL; and TSH <0.1 mIU/mLor >8 mIU/mL. Volunteers with known underlying diseases such as diabetes mellitus,hypertension, thyroid disease, gastric ulcer, cancer, and those taking medications wereexcluded. We also compared E170 with Immulite 2000 and calculated the kappaagreement ratio of each test to determine abnormal subjects.Nonparametric reference intervals (2.5 to 97.5 percentile) were 2.5 to 13.6 IU/mL(TPOAb), 5.0 to 124.2 IU/mL (TgAb), and 2.5 to 32.5 ng/mL (Tg). Passing-Bablokcomparison of each test items (y) with Immulite 2000 (x) were as follows: y=0.637x-0.872 (r=0.77, n=57) for TPOAb, y=5.913x-108.3 (r=0.84, n=123) for TgAb,y=1.281x-0.156 (r=0.94, n=130) for Tg. Kappa agreement ratios were 0.81 (TPOAb),0.64 (TgAb), and 0.76 (Tg).We could establish reference intervals of TPOAb, TgAb, and Tg in Koreanpopulation. Although there were some bias of those test between Modular E170 andImmulite 2000, the concordance rate were relatively good.

Prolactin Reference Intervals With Polyethylene Glycol Precipitation

on VITROS® 5600 Integrated System

S. SAW, C. Wang, S. Sethi. NATIONAL UNIVERSITY HOSPITAL,SINGAPORE, Singapore

Background: Recently we have seen publications providing reference intervals formonoprolactin samples treated with polyethylene glycol (PEG) precipitation ofdifferent immunoassay platforms. With the placement of the new VITROS® 5600Integrated System (Ortho Clinical Diagnostics, USA) in our laboratory we chose todetermine the reference intervals on this platform.Methods: We assayed anonymised blood donor samples from healthy males (n=113)

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and healthy females (n=109) for prolactin on VITROS 5600. From these samples 12male subjects and 21 female subjects were greater that the reference intervalpublished by the manufacturer (male: 78-380mIU/L; female: 64-395mIU/L) andomitted. 250uL of patient serum was mixed with equal volumes of 25% PEG 6000 inPBS (137mmol/L sodium chloride, 10mmol/L sodium phosphate), pH 7.4. Sampleswere mixed well and incubated for 10 minutes, then centrifuged at 10000g for 30minutes. The supernatant required a dilution 1:1 in normal saline prior to aspiration onVITROS 5600.Results: Post PEG treatment prolactin results for male: mean 137, minimum 29,maximum 224, median 139, SD 45; for female: mean 131, minimum 18, maximum219, median 132, SD 47. The PEG treated means recovery was 53%, range 19-66%(male) and 50%, range 18-79% (female). These histograms showed a central tendencyfor both sexes in the range of 45-65%.Conclusions: We compared the results obtained to our current method of reportingand interpretation: ”Recovery >60%: sample contains mostly monomeric prolactin.Recovery 40-60%: greyzone - in addition to monomeric prolactin, sample alsocontains macroprolactin and/or oligomeric prolactin. Results need to be reported assuch. Further assessment is necessary (e.g filtration chromatography). Recovery<40%: sample contains mostly macroprolactin results need to be correlated to clinicalfindings”. This shows that our current reporting format is not likely to be transferablebetween instruments. It is known that there are differences in immunoassay antibodyspecificity for the prolactin isoforms. With this in mind it may then mandatemanufacturers to include a reference interval for PEG treated samples to allowappropriate interpretation.

Determination of Trimester-specific Reference Intervals for Thyroid

Function Tests in a mixed South East Asian Population

T. Loh1, S. Sethi1, S. Saw1, B. Saw1, S. Ong1, P. Wong1, M. Thevarajah2, N.W. Tee3, N. Sabir2, J. Sickan4. 1National University Health System,Singapore, Singapore, 2University of Malaya Medical Centre, KualaLumpur, Malaysia, 3KK Women and Children's Hospital, Singapore,Singapore, 4Abbott Diagnostics South Asia, Singapore, Singapore

The prevalence of thyroid autoimmunity and possibility of relative iodine deficiencyduring pregnancy appear to influence and determine the occurrence of thyroid under-function. In addition, physiological changes in thyroid hormone status during thecourse of pregnancy complicate the assessment of maternal thyroid status. Weundertook this study to establish a trimester-specific reference interval for a SouthEast Asia population.Serum free-T4, free-T3, total T4, total T3, TSH and TPO-Ab were measured in 1024pregnant women of mixed South East Asian ethnic origin using Abbott AxSYManalyzers. Thyroid autoantibodies were detectable in 11.7% (n = 120).We found significant difference in mean values between the 1st and 2nd, 1st and 3rd aswell as 2nd and 3rd trimester using post-hoc ANOVA (p < 0.05) in our analysis of fT3,fT4, TSH and fT4:fT3 ratio. However, the mean values of fT3, fT4:TT4 ratio andfT4:fT3 ratio failed to show significance between the 2nd and 3rd trimester. Nosignificant difference was found among the different trimesters for TT4 and TT3.

Fine tuning of the management of subtle thyroid dysfunction during gestation requiresa strict definition of the reference intervals of functional thyroid hormones, since bothhypothyroidism and hyperthyroidism can lead to maternal and fetal complications.Data indicates significant differences among the mean values of the thyroid hormonesacross the three trimesters, supporting the utility of trimester-specific referenceintervals.

Glycated hemoglobin, insulin resistance and glucose tolerance in

individuals at high risk of developing type 2 diabetes

K. Santos-Rey, P. Fernández-Riejos, J. Mateo, V. Sánchez-Margalet, R.Goberna. HUVM, Sevilla, Spain

Objective To compare the glycated hemoglobin (HbA1c) levels with insulin resistanceand glucose tolerance in individuals at high risk of developing type 2 diabetes.Research design and Methods A total of 685 patients with at least two risk factorsfor the developement of type 2 diabetes (obesity, dyslipidemia, hypertension, previoushistory of impaired fasting glucose or family history of diabetes) underwent an oralglucose tolerance test (OGTT) with 75 g glucose, as well as an HbA1c and a basalinsulin measurement. Insulin resistance was assesed by Homeostasis ModelAssessment (HOMA). Our laboratory follows the NGSP recommendations for HbA1cmeasurement, with a Level I Certification. Results From the 685 patients, 234 wereeuglycemic (1st group), 185 had impaired fasting glucose (2nd group), 116 presentedimpaired glucose tolerance (3rd group) and 150 met the diagnostic criteria for type 2diabetes (4th group). First group presented a main HbA1c value of 5.02 % ± 0.36, and aHOMA value of 2.01± 1.6; in the second group, HbA1c was 5.35 % ± 0.35, HOMA3.14 ± 1.7; third group, HbA1c = 5.66 % ± 0.33, HOMA = 4 ± 2.4; fourth group,HbA1c = 6.13 % ± 0.61, HOMA = 5 ± 2.6. Statistically significant differences wereobserved for HbA1c values in each group. Differences for HOMA values did not reachstatistical significance. Conclusions HbA1c assay could be usefull to classify patientsat high risk of developing diabetes.

RELATIONSHIP BETWEEN GESTATIONAL DIABETES

MELLITUS AND PERIODONTAL DISEASES.

P. Fernández-Riejos1, G. Cisneros1, K. Santos1, J. Mateo1, R. Jaramillo2, R.Santos2, J. Rios2, P. Bullon2, V. Sánchez-Margalet1, R. Goberna1.1Department of Clinical Biochemistry, Virgen Macarena UniversityHospital, Seville, Spain, 2School of Medicine, Seville, Spain

Background: The association between periodontal diseases and diabetes mellitus hasbeen recognized in the dental literature for several decades. The recent accumulatingevidence of a relationship between periodontal infections and certain systemicdiseases has renewed interest in this association. However, there are no comparablepublished studies evaluating this relationship in subjects with gestational diabetesmellitus (GDM).Objective: To investigate the relationship between periodontaldiseases and GDM in pregnant women. Methods: We conducted a cross-sectionalprospective pilot study to investigate the relationship between periodontal diseasesand GDM in pregnant women from Seville, who came to University Hospital fromNovember 2007 to November 2008. The subjects completed a questionnaire andunderwent full-mouth periodontal examinations at enrollment (prior to 32 weekgestational age). MAIN OUTCOME MEASURES: Plaque and bleeding scores,pocket probing depth and loss of attachment. Hemoglobin A1C level and C-reactiveprotein (CRP) were collected prospectively. Hemoglobin A1C reflects the mean bloodglucose concentration over the preceding 1-3 months and CRP is a marker of systemicinflammation. Periodontitis was defined as the presence of at least four teeth with oneor more sites with probing depth ≥4 mm and clinical attachment loss ≥3 mm. Knownrisk factors like smoking, alcohol, drug consumption, socio-economic status and theperiodontal status were also recorded.Results: Data were collected from 165 subjects. A Mann-Whitney U test and aStudent’s t-test were carried out in which N.D.S. were found. Conclusion:There is agood correlation between biochemical markers and the risk of periodontal diseases inwomen with GDM.

Connective tissue growth factor: A marker for liver fibrogenesis?

E. Kovalenko1, O. Gressner1, R. Weiskirchen1, A. Janetzko2, C. E.Geacintov3, A. M. Gressner1. 1RWTH University Hospital, Aachen,Germany, 2DRG Instruments GmbH, Mountainside, NJ, 3DRGInternational, Inc., Mountainside, NJ

CTGF, a member of the CCN protein family, is relevant in many physiological andpathological processes (1). Both, clinical and experimental studies have demonstrated

Mean 95% CI

1st TrimesterfT4 (pmol/L)TSH (mIU/L)

13.571.26

12.97- 14.171.11 - 1.40

2nd TrimesterfT4 (pmol/L)TSH (mIU/L)

9.931.72

9.70 - 10.161.61 - 1.84

3rd TrimesterfT4 (pmol/L)TSH (mIU/L)

9.252.01

9.02 - 9.471.90 - 2.12

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that CTGF is overexpressed in fibrotic human liver and in experimental animalmodels of liver fibrogenesis (2). CTGF is detectable in various human fluids (serum,plasma, urine) and can potentially provide information about fibrotic remodellingprocesses (3). We established a novel method for the measurement of serum CTGFlevels and tested if this CCN protein is a suitable marker for staging liver fibrosis. Weexamined 138 patients with chronic Hepatitis C virus infection (median age 47 years,range 17-85 years; 76 males and 62 non-pregnant females) and healthy controls (n=80). CTGF levels were measured using a novel ELISA test that utilized capture anddetection antibodies directed against the C-terminal and N-terminal part of CTGF.The CTGF level was determined to 15.4±10.0 µg/L in healthy volunteers and53.8±34.68 µg/L in patients with chronic Hepatitis C. Moreover, the CTGFconcentration was elevated in all patients, irrespective of the severity of hepaticfibrosis that was scored after Desmet Scheuer (F1 47.5 (median 45; range 11-171) µg/L, F2 48.2 (median 42; range 14-189) µg/L, F3 52.9 (median 41; range 11-150) µg/L,F4 81.5 (median 76; range 21-148) µg/L). Therefore, we conclude that serum CTGFlevels determined by the new DRG ELISA are usable for classification of diseasesthat are associated with fibrogenesis in general.References cited

1.Brigstock DR. Endor Rev 1999;20:189-206.2.Paradis V, Dargere D, et al. Hepatology 1999;30:968-76.3.Moussas E, Brigstock DR. Mol Genet Metabolism 2000;71:276-92.

Understanding Diabetes Mellitus in Singapore

S. Ong1, X. Lin2, D. Poo2, S. Sethi1. 1National University Hospital,Singapore, Singapore, 2National University Singapore, Singapore,Singapore

Aim: To use data mining techniques in the understanding of diabetes mellitus inSingapore.Method: We extracted 65536 records in year 2003-2008 from the LIS database atNational University Hospital of diabetes mellitus patients diagnosed on fastingplasma glucose ≥7.1mmol/L criterion. Each data set was anonymised with a uniquenumber. Data field included patient’s demographic, fasting glucose, HbA1c andlipids(cholesterol, LDL-C, HDL-C, triglycerides). Descriptive statistics were done bycalculating the demographic-based breakup for diabetes related tests for ethnicChinese, Malay and Indian. Predictive modeling was done by regression analysis.Exploratory study was constructed using Microsoft Excel and SPSS 15.0 for cleaningand statistical analysis. Permission to use database was approved by our DomainSpecific Review Board Ethic CommitteeResults: Descriptive modeling indicated that diabetics fasting glucose mean±sd was9.7±3.3mmol/L (n=5186; male:female=1.1:1.0; age 16-86 years) withChinese:Malay:Indian ethnicity of 65%:20%:15%. The ethnic Malay subjects hadhighest mean fasting glucose of 10.3mmol/L. Diabetic HbA1c was 8.1±1.8%(n=1307), total cholesterol was 5.2±1.1mmol/L (n=3271), LDL-C was 3.2±1.0mmol/L (n=3273), HDL-C was 1.18±0.40mmol/L (n=3403) and triglycerides was1.9±1.7mmol/L (n=3453). 60% of patients had HbA1c ≥ 7.0% and 40% has LDL-C ≥2.6mmol/L with 75% diabetics had both HbA1c and LDL-C in these ranges. EthnicChinese diabetics had the lowest mean HbA1c, total cholesterol and LDL-C andhighest HDL-C. The Indian male had higher fasting glucose than female, Malayfemale has the highest HbA1c and females had higher LDL-C than males. Inpreliminary calculation of predictive modeling (n=869) of fasting glucose and LDL-C, we used Tenure = (collection date - collection date of first visit)/age 30 * 24 *3600. ANOVA analysis at p<0.05 change rates are: a) one year increase in ageincreased monthly change rate of fasting plasma glucose by 0.05 mmol/L, b) Fastingglucose of Chinese female had higher tendency to decrease over time compare toChinese male, c) 1 mmol/L increase in baseline fasting plasma glucose resulted inmonthly change rate of fasting glucose decrease by 0.79 mmol/L, d) 1 mmol/Lincrease in baseline LDL-C increases monthly change rate of fasting glucose by 0.46mmol/L (R-square=0.268).Discussion: Based on criterion of fasting glucose≥7.1mmol/L, status of subject’sduration of diabetes mellitus was not known. Further calculation of predictivemodeling would include the contribution of factors such HbA1c, smoking, familyhistory, body-mass-index measurements and blood pressure readings.Conclusion: Database mining tools appear to be able to define the theoreticalfoundation for the development of clinical decision support systems to assist in themanagement of diabetes mellitus.

Measurement of serum Testosterone: Waters Quattro Premier XE

Micromass® System versus Elecsys®Testosterone II and

Elecsys®Testosterone

F. Streit, N. von Ahsen, G. Brandhorst, M. Oellerich. University MedicineGoettingen, Goettingen, Germany

Immunoassays for quantification of testosterone can be affected by cross-reactivitywith structurally related compounds, in particular with specimens in the low assayrange (e.g. females) or from dialysis patients 1, 2. The purpose of the present study wasto compare the performance of the new Elecsys®Testosterone II assay (RocheDiagnostics) with the current Elecsys®Testosterone (I) assay. The accuracies of thetwo immunoassays were evaluated against an in house lc-ms/ms-method, which inturn had been validated with a reference id-gc-ms-method (x): y=1.05*x-0,032(r=0,998; n=38). Imprecision of the new Elecsys II assay was determined according tothe CLSI-Protocol. Within run imprecision was 3.2% and 1.2% while totalimprecision was 6.0% and 3.4% at testosterone concentrations of 0.22µg/L and13.00µg/L respectively. The measuring range for the Elecsys II was from 0.02 µg/L(LLOQ) to 15 µg/L. A method comparison (Passing Bablock) with the specific lc-ms/ms method is shown in the following Table.

The new Elecsys Testosterone II immunoassay showed good precision in the lowassay range as well as markedly improved accuracy in female patients andhemodialysis patients as compared with the first generation assay.(1) Fitzgerald R, Herold DA. Clin. Chem. 1996, 42, 749-755. (2) Taieb J, Benattar C,Birr AS, Lindenbaum A. Clin. Chem. 2002, 48, 583-585.

Development of a Cortisol Assay using LOCI® technology on the

Dimension® Vista Intelligent Lab System

S. A. Lewisch, C. Clark, V. Desai, M. Drinan, L. Schiavoni, M. Sharma, Z.Teng. Siemens Healthcare Diagnositics, Newark, DE

Cortisol is a glucocorticoid hormone secreted in a diurnal pattern by the adrenalglands in response to adrenocorticotropic hormone (ACTH) which is released by thepituitary. Additionally, cortisol levels are increased due to stress. Measurements ofcortisol aid the evaluation of adrenal insufficiency and excess. Low levels of cortisolimply hypocortisolism; high levels of cortisol indicate hypercortisolism. We describethe development and initial analytical performance of a fully automated, highsensitivity immunoassay for cortisol* employing LOCI® technology on theDimension Vista® System (Siemens Healthcare Diagnostics, Newark, DE).LOCI® technology is a homogenous, chemiluminescent immunoassay approachutilizing singlet oxygen transmission between sensibead and chemibead particles,incorporating two bead reagents and a biotinylated species. A generic bead reagent(sensibead) is coated with streptavidin and contains a photosensitive dye. A secondbead reagent (chemibead) is coated with a cortisol analog. The chemibead contains achemiluminescent dye as the signal generating component; it competes with analytefor limited amount of biotinylated antibody specific for cortisol. During the progressof the assay, the three reactants combine to form a bead-aggregated immunocomplex.Illumination of the complex by light at 680 nm generates singlet oxygen fromsensibeads, which diffuses into chemibeads to trigger a chemiluminescent reactionthat is measured at 612 nm. The resulting signal is inversely proportional to theconcentration of analyte in the sample.The analytical range is 0.1 to 60 µg/dL cortisol (spanning the interval from the limit ofdetection, mean plus 2 SDs of an analyte-free sample, to the upper calibrationstandard). The assay has a 16 minute turn around time and demonstrates excellentprecision (testing was conducted over a ten day interval, using commercial qualitycontrol materials). Repeatability was 2.6 % CV and within lab precision was 4.8 %CV at 4.3 µg/dL; at 23.0 µg/dL repeatability was 2.0 % CV and within lab precision

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(µg/L)slope intercept r md95

Male120 Elecsys I 0.063-12.55 1.055 0.043 0.976 0.817120 Elecsys II 0.04 - 12.86 1.056 0.016 0.982 0.702

Female151 Elecsys I 0.02 - 5.09 1.336 -0.031 0902 0.388144 Elecsys II 0.02 - 4.15 1.074 -0.035 0.959 0.206

Dialysis male20 Elecsys I 1.61 - 7.71 1.431 -0.150 0.765 1.01120 Elecsys II 1.07 - 5.76 1.196 -0.332 0.993 0.19

Dialysis female20 Elecsys I 0.08 - 1.4 5.098 -0.255 0.668 0.07317 Elecsys II 0.07 - 0.61 1.098 -0.029 0.661 0.211

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was 3.7 % CV; at 39.9 µg/dL repeatability was 2.3 % CV and within lab precision was4.1 % CV. Good agreement was observed between the comparative system, SiemensADVIA Centaur®, and the Dimension Vista® system: Dimension Vista® cortisol =(1.02 X Siemens ADVIA Centaur®) + 0.9 µg/dL (r = 0.97, n = 71). No significantinterference (<10 % bias) was seen from lipemia (3000 mg/dL triglycerides),hemolysis (1000 mg/dL hemoglobin) or icterus (40 mg/dL conjugated bilirubin or 80mg/dL unconjugated bilirubin). Minimal cross reactivity is observed.We conclude that use of LOCI® technology provides excellent sensitivity, precision,turnaround time, and dynamic range suitable for measurement of cortisol.*product under development - not available for sale

Commutability of Serum-based Reference and Proficiency Testing

Materials for Total Testosterone with Selected Immunoassays

J. C. Botelho, C. Shacklady, R. Razdan, A. Smith, H. W. Vesper. Centersfor Disease Control and Prevention, Atlanta, GA

Testosterone is important in the diagnosis of androgen diseases and disorders such asandrogen deficiency in men or polycystic ovary syndrome in women. Problems in theperformance of testosterone tests have been described and the need to improvetestosterone testing has been stated by many researchers and organizations. CDC isaddressing this with its steroid hormone standardization project. One tool to helpaddress current problems in testosterone testing is the use of reference materials toassess assay trueness and allow for traceable calibration. One material characteristicthat describes the suitability of a reference material for use with certain assays is itscommutability. Currently, no information about the commutability of referencematerials (RM) and proficiency testing (PT) materials for testosterone testing exists.The aim of this study was the assessment of commutability of serum-based referenceand PT materials.Serum-based RM from NIST (SRM 971) at two levels: 27.6 ng/dL (F) and 641 (M)ng/dL, dilution of these RM with assay specific diluents, and 6 materials intended forPT programs were assessed for commutability. 14 immunoassays from 8manufacturers using 40 fresh-frozen patient samples (3.66-788 ng/dL) over 3 days(n=7) were used to make the evaluation. Measurement results obtained by theimmunoassays were compared against those obtained by a mass spectrometry-basedcertified reference method. Initial assessment of commutability was made followingCLSI protocol EP14-A2 “Evaluation of Matrix Effects”. Results are summarized inthe table below. This initial assessment found the materials and dilutions beingcommutable for most immunoassays. For some assays SRM materials werecommutable while PT materials were not commutable, which may lead to incorrectdetection of bias in the PT study.

Prolactin Isoforms in Healthy Subjects Versus Individuals with

Increased Thyroid Autoantibodies

R. T. Daher, R. A. Nassar, D. S. Sarriedine, N. K. Cortas. Pathology andLaboratory Medicine, American University of Beirut, Beirut, Lebanon

Hyperprolactinemia described in autoimmune diseases, supports animmunomodulatory role for prolactin (PRL) through activation and enhancement ofthe immune process by binding to PRL receptors. The biological significance of PRL

molecular variants is not fully understood. Macroprolactin (macroPRL) has beenlisted as a diagnostic pitfall, and it is recommended to screen all patients for it in theroutine investigation of hyperprolactinemia. Many studies have shown the associationof macroPRL with certain autoimmune diseases particularly those with increased IgGtype (e.g. SLE), hence our interest in individuals with increased thyroidautoantibodies (tAb+). We compared different PRL isoforms in healthy individualswith those having tAb+: anti-thyroglobulin greater than 105 IU/ml and/or antithyroidperoxidase greater than 30 IU/ml (both assayed by Abbott Axsym). PRL was assayedusing Roche Modular analytics. Serum macroPRL was precipitated after treatmentwith equal volume of 25% polyethylene glycol (PEG) solution. MonoPRL was thendetermined in the supernatant; a recovery greater than 60% was negative formacroPRL. The method precision was 5.4% and 3.9% (between-run) for PRL &monoPRL respectively, and 0.94% (within-run). The analytical measuring range is0.05-470 ng/ml. Serial dilutions on a sample with high PRL gave a linear responsebetween 2.4-161.7 ng/ml and 1.1-69.3 ng/ml for PRL and monoPRL respectively.

The ranges for PRL and monoPRL obtained for the healthy individuals in this studywere used as cutoffs for normal. As described in the table, 3 out of 55 individuals withtAb+ had increased PRL. One additional male had increased monoPRL only. Arecovery of 53.5%, highly suspicious for macroPRL, was found in one of the twofemales. In comparison with the control group, both PRL and monoPRL levels werehigher in the group with tAb+, but this increase was not considerably associated withthe presence of macroPRL.

The analysis of 25-hydroxyvitamin D in serum using semi-automated

solid phase extraction and LC/MS/MS

L. J. Calton1, B. J. Molloy1, B. G. Keevil2, D. P. Cooper1. 1WatersCorporation, Manchester, United Kingdom, 2University Hospital SouthManchester (UHSM), Manchester, United Kingdom

Introduction: In recent years the demand for serum 25-hydroxyvitamin D (25OHD)analysis has increased significantly. In addition to the role vitamin D plays in bonemetabolism, several clinical studies now link vitamin D deficiency with increased riskfor certain cancers, multiple sclerosis and heart disease. 25OHD concentration isaccepted as the clinical indicator for determining vitamin D status and is alsoimportant for monitoring supplementation therapy, which is available in two forms,vitamin D2 and vitamin D3. Many clinical laboratories have now adopted LC/MS/MSbased methods for measuring 25OHD to enable the simultaneous and reliablemeasurement of 25OHD2 and 25OHD3. Some immunoassays may under report thetotal 25OHD level for patients on Vitamin D2 supplementation due to the cross-reactivity of the antibody for 25OHD2 being less than 100%. The analysis of 25OHDby LC/MS/MS requires a number of sample pre-treatment steps to release 25OHDfrom Vitamin D binding protein and to minimise matrix effects. This methoddescribes a semi-automated sample pre-treatment protocol in combination withUPLC/MS/MS for the simultaneous analysis of 25OHD2 and 25OHD3.Methodology: For the initial study, twenty samples were analysed using anestablished manual liquid/liquid solvent extraction LC/MS/MS method at UHSM. Allserum samples were anonymised and stored at -20°C. Calibrators were prepared inhorse serum (Sigma) over the concentration range 2.5-100ng/mL and QC sampleswere prepared independently. Commercially available calibrator and QC materialfrom Chromsystems were also analysed. Primary serum samples and calibrators wereplaced on a robotic liquid-handling system and identified by bar code to be trackedthroughout the extraction procedure. Hexa-deuterated internal standard was added tothe dispensed samples prior to protein precipitation. Following centrifugation (off-line), the supernatant was transferred to a conditioned Oasis® HLB® µElution SPEplate and washed. The retained analytes were eluted by the liquid-handling systemand the eluant was chromatographed using a Waters ACQUITY UPLC™ with anACQUITY BEH Phenyl column (2.1x50mm) with a water/methanol/ammonium

Assay %CV Male %CV Female Material Percent Commutable1 6.21 13.2 SRM-M Undiluted 71.4%2 3.28 9.19 SRM-M Dilution 1 71.0%3 3.00 7.52 SRM-M Dilution 2 92.9%

4 11.5 24.3SRM-M

Dilution 392.9%

5 10.8 21.8 SRM-F Undiluted 92.9%

6 10.9 29.5SRM-F

Dilution 192.9%

7 2.68 5.17SRM-F

Dilution 285.7%

8 2.00 5.19 PT 1 71.4%9 1.67 3.75 PT 2 100%10 2.38 10.4 PT 3 71.4%11 2.07 8.36 PT 4 71.4%12 1.27 6.21 PT 5 100%13 2.81 11.8 PT 6 100%14 6.67 7.71

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Healthy tAb+Males Females Males Females

Number (N) 34 26 11 44Prolactin (ng/ml)(ranges)

3.6-19.7 4.1-23.0 6.1-24.6 4.1-65.1

Monomeric (ng/ml)(ranges)

2.8-16.9 3.8-19.5 5.2-20.2 3.6-55.4

Recovery (%)(ranges)

60.0-95.6 75.5-99.0 82.0-100.0 53.5-96.3

Increased PRL (N)Increased MonoPRL (N)Macroprolactin (N)

000

000

120

221

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acetate gradient. A Waters TQD mass spectrometer was used to quantify 25OHD2 and25OHD3, monitoring two transitions for each analyte.Results: The assay was linear over the range 2.5-100ng/mL with a coefficient ofdetermination (R2) of > 0.997. The inter- and intra-assay imprecision at three levelswas less than 10% CV (n=5, over 5 days). The calculated 25OHD concentrations ofthe twenty patient samples were in good agreement with the LC/MS/MSmeasurements from UHSM, using Passing-Bablok analysis Waters=1.01(UHSM)-1.45. Ion suppression studies were conducted and demonstrated minimal ionsuppression for 25OHD3.Conclusion: A routine semi-automated solid phase extraction UPLC/MS/MS methodfor the analysis of 25OHD2 and 25OHD3 in serum has been developed. The assay hasacceptable performance characteristics (linearity, sensitivity, precision and accuracy)and allows the analysis of up to 196 samples per working day. The semi-automatedprocedure significantly reduces the manual operation steps, eliminates solventevaporation, reconstitution and operator variability to ensure more consistent andreproducible results.

ANALYTICAL AND CLINICAL EVALUATION OF THE

AUTOMATED LIAISON® INSULIN-LIKE GROWTH FACTOR

(IGF-I) ASSAY

H. Mekouar, D. Gruson, D. Maiter, M. Maes, J. Cumps, J. Ketelslegers.Clinique Universitaire Saint-luc, UCL, brussels, Belgium

Background. IGF-I assay is essential for management of Growth HormoneDeficiency (GHD) in children and adults as well as diagnosis and follow-up ofacromegaly. The Liaison® from Diasorin allows automation of IGF-I assay and isbased upon a sandwich chemiluminescence immunoassay, after neutralization of IGF-I binding proteins by excess of IGF-II. The performances of this automated assay arecurrently poorly documented. The aim of this study was to determine the analyticaland clinical performances of the IGF-I Liaison® assay.Methods. Precision, linearity and functional sensitivity were determined for the IGF-ILiaison® assay using different serum pools covering a broad spectrum of IGF-Iconcentrations. IGF-I Liaison® assay was also compared with 490 samples previouslymeasured with the former Advantage® Nichols Institute Diagnostics (NID). Inaddition, normal values over the life span were derived from the correlation withAdvantage® assay. Finally, to assess the concordance with our reference values, wedetermined IGF-I in patients samples with clinically established GHD (children andadults) or active acromegaly.Results. Intra-assay coefficient of variation (CV) obtained with 20 replicates of serumpools: were 6.4% at 19ng/mL, 6.5% at 57ng/mL, 2.0% at 251ng/mL and 4.0% at575ng/mL. Inter-assay variation were assessed with four levels IGF-I serum pools (9,20, 21 and 31 ng/mL) run as singles (n = 20) for 2 weeks with 4 calibrations, CVsobtained were 33.2%, 15.3%, 12.7% and 8.1% respectively, consistent with afunctional sensitivity of 15 ng/mL . After serial dilutions of serum pools, the linearitywas supported. After log transformation of the data, the Liaison® IGF-I assaycorrelated closely with the Advantage® IGF-I assay: Liaison® = 0.315 + 0.875 xAdvantage® (r = 0.99; n = 490; range: 15 - 1246 ng/mL). Passing and Bablockregression analysis (distribution of Studentized residuals) showed no significantdeviation of linearity. Estimated reference values for Liaison® were derived using theregression equation, from 1656 normal samples previously published. Liaison®

baseline values (0 - 5 years) rose from 71 (30 - 170) ng/mL to a peak of 372 (211-655)ng/mL (13 - 16 years); values declined to 240 (130 - 440) ng/mL (20-25 years), andfurther decreased to 145 (93 - 225) ng/mL at 60 - 70 years (geometrical means; 95%CF). To evaluate the clinical concordance of our reference values, IGF-I was tested in11 GHD children (3 - 11 years), before treatment, IGF-1 was < 15 ng/mL (n = 4) orlow (17 to 32 ng/mL); eleven adult patients with GHD were also evaluated; 4 subjectswith childhood onset GHD (19 - 48 years) had IGF-I levels of <15 - 28 ng/mL; in 7patients with adulthood GHD (47 - 84), IGF-I ranged from <15 ng/mL to 34 ng/mL.In 14 active acromegal patient, IGF-I range was: 530ng/mL to 903 ng/mL.Conclusion. Liaison® IGF-I assay offers analytical performances suitable for routinedetermination of IGF-I and excellent correlation with the used reference assay. Afterestablishment of the assay reference values, we also demonstrated the reliability ofthis assay through different patient’s samples.

LC-MS/MS Reference Intervals for Androgens in Serum of Children

M. M. Kushnir1, T. Blamires1, W. L. Roberts2, B. Yue1, A. L. Rockwood2,A. M. Bunker1, E. Erdogan2, A. W. Meikle3. 1ARUP Institute for Clinicaland Experimental Pathology, Salt Lake City, UT, 2Department ofPathology, University of Utah, Salt Lake City, UT, 3Departments ofMedicine and Pathology, University of Utah, Salt Lake City, UT

Dehydroepiandrosterone (DHEA) and androstenedione (A4) are androgen precursorsof male and female sex hormones. Measurement of DHEA and A4 is useful indiagnosing sex hormones-related disorders because abnormal production of thesesteroids may cause pathological changes in masculine and feminine characteristics.Gender and age-specific reference intervals (RI) for androgens are required forinterpreting results of diagnostic testing. Available RI of DHEA and A4 establishedwith automated analyzers are reagent- and method-specific and not transferablebetween instruments of different manufacturers. As a general rule validated liquidchromatography tandem mass spectrometry (LC-MS/MS) methods provide accuratemeasurement of analytes, independent of the instrument type and vendor of thereagents and are transferrable between laboratories. We developed a high sensitivity/high specificity LC-MS/MS method for DHEA and A4 and established age andTanner Stage specific RI in children 7 - 17 years of age. The method utilizes solventextraction and derivatization of the androgens to form oxime derivatives to enhancesensitivity. Instrumental analysis was performed using two-dimensionalchromatographic separation and MRM acquisition on an API 4000 (AppliedBiosystems/ MDS Sciex) LC-MS/MS. The assay was linear up to 9 ng/mL for DHEAand 40 ng/mL for A4. Total imprecision of the method at concentrations of 0.05, 0.3,0.7, and 2.5 ng/mL was less than 9.3% and 11.2% for DHEA and A4, respectively.The lower limit of quantitation was 0.05 ng/mL for DHEA, 0.01 ng/mL for A4.Method comparison with another LC-MS/MS yielded linear regression equations of Y=0.89 * X - 0.04, r =1.00, Sy,x = 0.05; and Y =0.95 * X - 0.50, r =1.00, Sy,x = 0.27 forDHEA and A4, respectively. Nonparametric reference intervals for children aresummarized in the table. The method requires small sample volume (0.2 mL) and issufficiently sensitive to measure DHEA and A4 in all population groups.

Performance evaluation of an automated immunoassay for the

determination of Cortisol on the Olympus AU3000i™ Immunoassay

System

M. Evans, A. O'Sullivan, S. Hennessey, J. Hanley, S. Gaston. Olympus LifeScience Research Europa (GmbH), Co.Clare, Ireland

Cortisol, also known as hydrocortisone, is the major glucocorticoid produced andsecreted by the adrenal cortex and has a number of functions including the regulationof carbohydrate, lipid and protein metabolism together with immunosuppressive andanti-inflammatory activity. Cortisol circulates either bound to transport proteins(90%) or as free hormone. Only the free, unbound cortisol fraction can interact withits receptor and is thus physiologically active. In individuals with normal renalfunction, about 1% of the plasma cortisol is excreted unchanged into the urine andlevels can be related to the concentration of biologically active plasma free cortisol.Plasma cortisol levels normally follow a diurnal rhythm, with maximumconcentrations (around 750nmol/L) usually reached early in the morning andfollowed by a gradual decrease during the day. The Olympus Cortisol assay is a one step paramagnetic particle enzymeimmunoassay. It is based on the competitive principle and is used to quantitate cortisolin serum, plasma and urine.Reported here are results from the development and evaluation of the automated assayfor cortisol on the Olympus AU3000i™ Immunoassay System. This assay is

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Gender and age N Androstenedione, ng/mL DHEA, ng/mLBoys 7 to 9 206 0.031-0.31 0.092-2.46Boys 10 to 11 140 0.072-0.41 0.3-3.81Boys 12 to 13 143 0.11-0.64 0.58-4.11Boys 14 to 15 141 0.18-1.01 0.87-6.64Boys 16 to 17 136 0.31-1.14 1.21-7.63Girls 7 to 9 206 0.038-0.49 0.12-2.70Girls 10 to 11 148 0.04-1.27 0.55-3.92Girls 12 to 13 142 0.19-2.09 0.81-6.34Girls 14 to 15 143 0.43-2.09 1.23-7.63Girls 16 to 17 138 0.39-2.15 1.46-9.51

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referenced to ID-GC-MS with a limit of detection and limit of quantification of5nmol/L and 8nmol/L, respectively.Assay imprecision was characterized over 5 days according to CLSI protocol EP15-A.Total coefficients of variation (CV) for low (<230nmol/L), medium (600-950nmol/L)and high (>1500nmol/L) human serum and urine pools were determined to be 6.5%,4.0% and 3.2 % respectively for serum and less than 7% for all urine pools tested.A method comparison was performed against the Roche Cobas® e411Cortisol assayusing serum samples over a concentration range of 10-2000nmol/L, producing a slopeof 1.05, an intercept of -1.09nmol/L and a correlation coefficient of 0.95.Dilution linearity was demonstrated over the entire range of the assay from 10-3000nmol/L. No significant interference was detected from bilirubin, hemoglobintriglycerides, Intralipid, protein and urea. Reagents were optimized to negate RF andHAMA interference. No significant cross-reactivity was observed with any of thecompounds and drugs normally associated with automated cortisol immunoassay;these include all other major steroid hormones and compounds known to causeinterference in cortisol assays, e.g. cortisone, 11-deoxycortisol, dexamethasone,methylprednisolone, prednisolone and progesterone.Based on our evaluation, we conclude that the Olympus Cortisol assay is a sensitive,precise, and specific method that meets the requirements of measuring cortisol inhuman serum, plasma and urine.

Performance evaluation of an automated immunoassay for the

determination of estradiol on the Olympus AU3000i™ Immunoassay

System

P. O'Dea, P. Curley, B. de Grimaudet de Rochebouët, A. O'Sullivan, S.Gaston. Olympus Life Science Research Europa (GmbH), Co.Clare,Ireland

Quantitation of estradiol (E2) in human serum and plasma has been utilized as ameans for monitoring ovulatory status and is used extensively in in-vitro fertilizationmonitoring. Distinguishing low levels of estradiol in pre- and postmenopausal womenalso enables risk assessment of common fertility related diseases in older women.Abnormally high levels of estradiol in males are indicative of gynecomastia and otherfeminizing syndromes.The Olympus Estradiol assay is a paramagnetic particle, chemiluminescentimmunoassay for the quantitative determination of estradiol levels in human serumand plasma using the Olympus AU3000i™ Immunoassay System.Reported herein are results from the development and evaluation of the automatedassay for estradiol on the Olympus AU3000i Immunoassay System.This assay is referenced to ID-GC-MS assigned sera. Limit of detection andfunctional sensitivity (at a CV of 20%) were determined to be 15pmol/L and 70pmol/L respectively. Assay imprecision was characterized according to CLSI protocolEP15-A. Within run coefficients of variation (CV) for low (<184pmol/L), medium(184pmol/L -1835pmol/L), and high (>1835pmol/L), human serum pools weredetermined to be 4%, 3%, and 1% respectively. Total precision was determined to be5%, 3%, and 2% for the same serum pools. Method comparisons performed againstthe Roche Cobas® e 411 Estradiol assay using 126 serum samples over aconcentration range of 50pmol/L to 6000pmol/L yielded a slope of 0.9874, y-interceptof -69.2pmol/L and correlation coefficient of 0.984. No significant interference wasdetected from bilirubin, hemolysate, triglycerides and Intralipid. Reagents wereoptimized to negate RF and HAMA interference. No significant cross-reactivity wasobserved with estrone, estriol and other potentially cross-reactive analogues.Based on our evaluation, we conclude that the Olympus Estradiol assay is anexceptionally sensitive, precise, and specific method that meets the requirements ofmeasuring estradiol in human serum and plasma.

Correlation of Factors affecting the AMH Normal Range for Women

in the Reproductive Years

G. A. Agramonte1, J. M. Cortes2, K. Gelman3, N. Goodman4, C. Ferguson1,A. Menocal1. 1Unilab, Fort Lauderdale, FL, 2Unilab of Dade, FortLauderdale, FL, 3Infertility and Reproductive Medicine of South Broward,Pembroke Pines, FL, 4Neil Goodman, MD, Miami, FL

Background: AMH has important functions for both male and female reproductiveorgan development. AMH/HIS begins circulating after puberty, when menstrualcycling begins and slowly decreases throughout life until it becomes undetectable at

menopause. AMH is produced in the primordial follicle. In conjunction with FSH andAge, it is the most used marker for estimating ovarian reserve.Unilab of Dade is a private laboratory dedicated to reproductive medicine. Proceduresused for patient diagnosis are first validated and then verified from time to time forlinearity, normal range, specimen requirement and additional parameters that may bedeemed necessary by our Director or clinicians.This study evaluated 900 orders received for AMH levels during 2008. The followingconditions were analyzed:1. Specimen Stability2. Validity of the calculated normal range3. AMH levels vs. Age4. AMH levels vs. FSH5. AMH level and incidence of pregnancyMethod: Subjects were screened for AMH levels using the Diagnostics SystemsLaboratories (DSL) ACTIVE® MIS/AMH ELISA procedure. This is anenzymatically amplified two-site immunoassay.All orders received were used for the calculated range. Patients that were followed intreatment towards pregnancy were used in the physiological range. Three age groupswere evaluated: 18-30 years, 31-36 years and 37-50 years. These samples wereevaluated for Normal Range, Age, FSH and Pregnancy correlation.Results:

1. Sample Stability according to the insert range is 24 hours at 2-8°C. Stability wasexpanded to one week at 2-8°C with less than <1% decay.2. The normal estimated range was first calculated for each age group by determininga Mean and 2 Standard Deviations for results obtained between <0.05-5.0 ng/mL.3. Physiological range was obtained by determining the AMH level detected for eachgroup with patients resulting in an elevated HCG:4. Additional relationships:A. AMH vs. AGEB. AMH vs. FSHC. None detectable levels of AMHConclusions:

1. Normal levels of AMH correlate with age and FSH.2. Age Group 18-30 years: AMH levels of 0.5-5.0 ng/mL correlate well with FSHvalues of 0.0-8.0 miu/ml3. Age Group 31-36 years: Incidence of higher FSH values begins to increase withlower AMG counterparts.4. Age group 37-50 years: Shift of FSH to higher levels with large percent inabnormal range. AMG values continue to decrease with a high percent of Non-Detectable noted.5. AMH Non-Detectable may be inducible towards pregnancy as occurred in twocases of this study.6. An AMH normal level is not sufficient to determine the successful rate ofpregnancy.

Increased leptin expression in placenta from pathological pregnancies

A. Perez-Perez1, Y. Gambino2, J. Maymo2, J. L. Dueñas1, R. Goberna1, C.Varone2, V. Sanchez-Margalet1. 1Virgen Macarena University Hospital,Seville, Spain, 2University of Buenos Aires, Buenos Aires, Argentina

Objectives: Leptin is a regulatory hormone in many cellular systems, includingtrophoblastic cells, where it is produced and may function as a trophic factor. In thepresent work we aimed to study the expression level of leptin in pathologicalpregnancy (gestational diabetes and pre-eclampsia) compared with control placentafrom normal pregnancies.Materials and Methods: We have studied trophoblastic samples from placentaobtained from donors after full-term delivery. 8 control placenta from normalpregnancies, 6 placenta from gestational diabetes, and 5 placenta from pre-eclampsiawere studied. Leptin expression level was determined by real time PCR and theprotein level by immunoblot. Student´s t test was employed to assess statisticaldifferences.Results: We have found that every placenta from pathological pregnancy haveincreased expression level of leptin, compared with control placenta from normalpregnancy. Both placenta from gestational diabetes and preeclampsia expressed inaverage twice as much leptin levels than control placenta, significant differences(p<0.001) between placenta from pathological pregnancy and control placenta werefound.Conclusions: Pathological pregnancy (gestational diabetes and pre-eclampsia)produces an increase in the leptin expression in trophoblast, and leptin may be a usefulmarker in the diagnosis and follow-up of these pathological pregnancies.

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Free Cortisol Measurement in Human Serum by Combined

Equilibrium Dialysis and LC-MS/MS

J. Twentyman, K. Cradic, S. Grebe, R. Singh. Mayo Clinic, Rochester, MN

Background: Free serum cortisol is perceived to be superior to total serum cortisol forassessment of adrenal function in severely ill patients. Historically, free serum cortisolhas been calculated from the concentrations of total serum cortisol and cortisolbinding globulin (CBG). Questions have been raised as to the validity of thesemethods.1 Equilibrium dialysis combined with LC-MS/MS offers an effective meansof measuring free cortisol directly. We offer a method for directly quantifying freecortisol in serum using equilibrium dialysis and LC-MS/MS.Methods: Serum samples were dialyzed in phosphate dialysis buffer, pH 7.4, in asingle-use Pierce rapid equilibrium dialysis (RED) plate. A pipetting station was usedto transfer all components of the assay which greatly improved speed and precision ofthe assay. 250 µL of serum was added to the sample chamber and 500 µL of dialysisbuffer was added to the buffer chamber of the RED plate. The plate was sealed andincubated for 16-18 hours at 37°C on an orbital shaker. After equilibrium wasreached, 150 µL of the dialysate was removed and transferred directly into a 96 deep-well plate. Deuterated cortisol (d3-cortisol) was added as an internal standard. Theresulting dialysate mixtures underwent on-line high-throughput liquidchromatography (HTLC) followed by conventional liquid chromatography on anXDB-C18 analytical column. Cortisol and d3-cortisol were analyzed using a heatednebulizer ion source (APCI) and tandem mass spectrometry.Results: Intra-assay imprecision (CV) was found to be 3.4-8.9% (n=20) for threedifferent levels (0.29-6.84 µg/dL). Linearity of the dialysate (4 patient dialysates)ranged from 78-107% with an average of 95% for samples ranging from 0.238-7.99µg/dL. Free cortisol results by equilibrium dialysis showed significant systematic biasto calculated free cortisol, with a slope of 0.3637 and intercept of 0.3639 (n=5, onesample removed as it exceeded the highest calibrator for our method).Conclusions: The dialysis method provides a simple and reproducible means ofdirectly quantitating free cortisol. There is a systematic difference when compared tocalculated free cortisol. Assuming that equilibrium dialysis is closer to the true in vivofree cortisol levels, this suggests that calculated free cortisol measurements result ininaccurate estimation of free cortisol levels.1. Vogeser et.al. Clin Chem Lab Med. 2007;45(4):521-5.

Performance Evaluation of the VITROS® 3600 Immunodiagnostic

System Estradiol Assay

D. Giacherio, E. VasBinder, K. Forbing. University of Michigan Hospital,Ann Arbor, MI

Background: Estradiol measurement is a key component in the assessment ofreproductive function in women, including infertility, oligo-amenorrhea, andmenopausal status. It is also commonly used by in vitro fertilization programs formonitoring ovulation. Immunoassays for estradiol have historically had accuracy andreproducibility issues at low estradiol concentrations. This study evaluated theperformance of the estradiol immunoassay on the new Ortho Clinical Diagnostics'VITROS® 3600 Immunodiagnostic System.Methods: The VITROS 3600 estradiol assay is a competitive immunoassaytechnique. Estradiol in the sample competes with a horseradish peroxidase labeledestradiol conjugate for a limited number of biotinylated anti-estradiol antibodybinding sites. The antibody complex is captured on streptavidin coated well.Following washing to remove unbound material, the HRP catalyzes the oxidation of aluminogenic substrate producing light. The VITROS 3600 estradiol assay wascompared to the Siemens ADVIA Centaur® estradiol assay which is routinely used inour clinical laboratory. A subset of patients also had estradiol determinations by anLC/MS methodology.Results: To assess day-to-day assay imprecision, BioRad® LiquichekTM

Immunoassay Plus control material was analyzed for 10 consecutive days. The CV’sfor Levels 1, 2, and 3 were 7.25 %, 7.20 %, and 6.70 % respectively on the VITROS3600 System, and 28.7 %, 6.03%, and 7.14 % respectively on the ADVIA Centaur.Three serum pools with low estradiol concentrations were analyzed on the Vitros3600 System with 5 replicate determinations of each. These gave the following meanand CV data: Pool 1 = 9.66 pg/mL and 9.9 %, Pool 2 = 21.62 pg/mL and 6.3 %, Pool3 = 33.75 pg/mL and 2.9 %. A method comparison with the Centaur estradiol assayusing 177 serum samples with Centaur values ranging from 12 to 1074 pg/mL yieldeda regression equation of VITROS = 0.59 Centaur + 2.3, r = 0.9536. The two

calibrators for the Centaur assay with assigned estradiol concentrations 97.9 pg/mLand 726 pg/mL gave values of 67.3 pg/mL and 599 pg/mL when analyzed on theVITROS 3600. Analysis of 18 samples by LC-MS yielded regression data as follows:VITROS = 0.99 LCMS - 6.4, r = 0.9168, and Centaur = 1.06 LCMS + 1.3, r = 0.9450.Conclusion: Based on this limited evaluation, the new VITROS 3600 Systems’estradiol assay is a sensitive and precise assay that readily meets the routine needs ofthe clinical laboratory. The assay demonstrated excellent precision at low estradiolconcentrations. The VITROS 3600 assay correlates well with the ADVIA Centaurassay, and it appears that a significant amount of the difference between the assayresults can be attributed to assignment of calibrator values. The VITROS 3600 assaygave an acceptable correlation with a LC-MS estradiol assay performed at a referencelaboratory.

Performance characteristics of the Tosoh G8 for the determination of

Hemoglobin A1c.

R. Khoury, A. H. Gandhi, B. P. Salmon, A. D. Patel, P. Gudaitis, D.Gudaitis, S. S. Gibbs. Aculabs, Inc., East Brunswick, NJ

Background: Diabetes is a chronic illness marked by high levels of blood glucose dueto either an improper insulin production, insulin action, or both; more than 10 millionor 20 % of people age 60 and older in the Unites States have diabetes. The first step indiabetes care is the glycemic control to prevent and reduce the risk of thecomplications. Techniques to assess the effectiveness of a management plan onglycemic control include: Self-Monitoring of Blood Glucose and the measurement ofhemoglobin A1c. The objective of this study is to validate the Tosoh G8 and comparethe results to the Tosoh G7 and Roche Integra 800 results.Methodology: The Tosoh G8 Glycohemoglobin analyzer uses non-porous ionexchange HPLC and microcomputer technology. There is no interference from Schiffbase and no pretreatment is required. The assay is NGSP certified, and there is aminor difference between G7 and G8 which helped reducing the assay time to 1.6minutes from 2.2 minute for G7, although the assay time is shorter but high resolutionwas maintained. We evaluated the assay accuracy, linearity, precision, reportablerange, and samples correlation with Tosoh G7 and Roche Integra 800.Results: The assay %CV was 0.9 % for a mean A1c of 5.9%, the assay linearity wasconfirmed with an average recovery of 100.9%. Reportable range was 3.0 to 18.1%;the following is the summary of the correlation:

Conclusion: the Tosoh G8 offers the gold standard technology for the measurement ofA1c which eliminate interferences, it is precise, fully automated, user friendly, offersup to 290 samples loader; in addition it offers the fastest throughput of the 3instruments tested.

Relationship between Extremely Elevated Serum Human Chorionic

Gonadotropin (hCG) and the Thyroid Hormones Thyroid Stimulating

Hormone (TSH) and Free Thyroxine (fT4)

C. M. Lockwood1, D. G. Grenache2, A. M. Gronowski1. 1WashingtonUniversity School of Medicine, St. Louis, MO, 2University of Utah HealthSciences Center, Salt Lake City, UT

Background: During pregnancy, when hCG concentrations are highest, there is atransient suppression of serum thyroid stimulating hormone (TSH). Numerous studieshave established that hCG can stimulate release of the thyroid hormones, whichinhibit the pituitary release of TSH through negative inhibition. In normal pregnancy,TSH suppression is a transient phenomenon and TSH concentrations generally remainwithin non-pregnant reference intervals; however, in some patients TSH is loweredbelow the non-pregnant reference interval. It has been reported that suppressed TSHconcentrations are usually only observed in cases when the hCG concentrationexceeds 50,000 IU/L. In a pilot study, we reported that if there is an hCGconcentration above which all serum TSH or free thyroxine (fT4) concentrations canbe expected to be abnormal, it is >330,000 IU/L. However, the pilot study was limitedbecause it contained very few specimens with hCG concentrations >200,000 IU/L(n=18).Objectives: To determine: 1) if there is an hCG concentration, above which, TSH

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D-124 Instrument Correlation Coefficient Slope InterceptG8 vs. G7 0.9993 1.03 -0.19G8 vs. Integra 800 0.9945 1.00 -0.14

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concentrations consistently fall outside the normal reference interval; 2) how quicklythyroid hormone concentrations change in response to changes in hCGconcentrations; and 3) the clinical symptoms in patients with such extremely elevatedhCG concentrations.Methods: Over a twenty-six month study period, 15,597 physician-ordered hCG testswere performed. 69 specimens from 63 women with hCG concentrations >200,000IU/L were identified with sufficient sample remaining to measure TSH and fT4concentrations. Medical records were reviewed for clinical symptoms and pregnancydiagnosis. Approval for this study was provided by each institution’s IRB.Results: 37% (23/63) of subjects had hyperemesis gravidarum, 19% (12/63) hadgestational trophoblastic disease (GTD), and 10% (6/63) had a twin pregnancy (5 ofwhich also had hyperemesis gravidarum). TSH was suppressed below the referenceinterval in 74% of the study specimens and 100% of specimens with an hCGconcentration >400,000 IU/L. fT4 concentrations were elevated in 32% of specimensand in 80% of specimens with an hCG concentration >400,000 IU/L. Interestingly,only 6% (4/63) of subjects had documented signs of hyperthyroidism. During thestudy period, multiple hCG concentrations >200,000 IU/L were collected in 8% (5/63) of serially monitored subjects, which allowed an assessment of how rapidly thethyroid hormones changed relative to hCG concentration. In all cases, as hCG varied,fT4 and TSH also changed in a predictable manner.Conclusions: These data confirm the relationship between very high hCGconcentrations and the thyroid hormones TSH and fT4. TSH is consistentlysuppressed at an hCG concentration >400,000 IU/L, a threshold which is substantiallyhigher than previously published estimates. This study also illustrates that varyinghCG concentrations can rapidly influence serum fT4 and TSH. Finally, despite thepresence of biochemical hyperthyroidism in 32% of study subjects, very few (6%)exhibited overt hyperthyroid symptoms.

Sensitive and Specific Quantification of 1-84 PTH in Serum and

Plasma by Immunocapture-in situ digestion LC-MS/MS

V. Kumar, D. R. Barnidge, J. M. Twentyman, K. W. Cradic, S. K. Grebe, R.J. Singh. Mayo Clinic College of Medicine, Rochester, MN

Background: Specific 1-84 PTH assays have been reported to be reflective of thebioactivity of the PTH hormone. Most existing immunoassays reflect the sum of 1-84PTH and other cross-reacting PTH fragments (predominantly 7-84 PTH) thataccumulate in patients with Chronic Kidney Disease (CKD). In addition, regardless ofthe degree of crossreactivity, consistent calibration over time remains an issue. Aphysico-chemical reference methodology may help address these problems.Methods: We have developed an accurate and robust method for measuring 1-84 PTHin serum and plasma. 1-84 PTH and 15N-PTH (internal standard) were recovered fromserum and plasma by immunocapture using anti-44 - 84 PTH antibody coupled toquarter -inch polystyrene beads. Briefly, after washing the beads with PBS, the boundPTH molecules were digested in 50mM Ammonium Bicarbonate buffer, pH 8.0,containing 1µg trypsin to generate the unique proteotypic N-terminal tryptic peptide1-13 PTH (SVSEIQLMHNLGK) that was analyzed using LC-MS/MS method. Thetryptic peptides were resolved by high performance liquid chromatography carried outon a C18 column with a gradient flow rate of 0.25mL/min either with acetonitrile ormethanol organic solvent. The PTH results obtained from 111 patient samples byimmunoassays were compared with their corresponding LC-MS/MS values. Thepatient samples were subdivided into three groups: i) 29 patients in whom th PTHlevels were between 150 and 299 pg/mL, the desired range for PTH in patients withstage 5 CKD according to KDOQI guidelines, ii) 54 CKD patients undergoingdialysis at Mayo clinic (PTH: 20 - 3,638pg/mL), and (iii) 28 patients undergoingparathyroidectomy (IOPTH) at Mayo clinic (PTH: 10 - 1,951pg/mL).Results: Standard curves were made in charcoal stripped human serum usingrecombinant 1-84 PTH in concentration of 20-5,000 pg/mL (r2= 0.996). The intra-assay CV’s for replicate extracts of three levels of pooled controls-sera (27.2, 83.1 and269.3 pg/mL) were 14%, 9.1% and 12.8%, respectively. The LC-MS/MS methodshowed poor correlation (R2 = 0.68, slope = 0.925) with the Roche Cobas®immunoassay in group 1 patients. Furthermore, 10 of 29 immunoassay values showeda significant (>2SD) positive bias compared to the LC-MS/MS method. Comparisonin Dialysis patients showed a good correlation (R2 = 0.93, slope = 0.87) and only 3 of54 samples showed a significant (>2SD) positive bias compared to LC-MS/MSmethod. Finally, comparison for IOPTH patients showed a poor correlation (R2 =0.59, slope = 0.57), but only 1/28 samples showed a significant (>2SD) positive biascompared to LC-MS/MS method.Conclusions: The LC-MS/MS method we have developed quantifies specifically theunique proteotypic N-terminal tryptic fragment 1-13 derived from immunocaptured 1-84 PTH. This novel PTH method is robust and reproducible over an analytical range

of 20-5,000 pg/mL. The significant postive bias shown in certain patient cohorts bythe Roche immunoassays compared to LC-MS/MS, suggests interference by variousnon-1-84 PTH fragments in these immunoassays. The LC-MS/MS method ofquantification presented here is not plagued by interference by non-1-84 PTHfragments and therefore has clear advantages over ICMA and IRMA immunoassayscurrently used to quantify 1-84 PTH.

Plasma Concentrations of Adiponectin Hormone in Type 1 and Type 2

Diabetes

S. L. Barnes, E. P. Womack, M. Bon Homme, W. Tucker, R. Valdes, S. A.Jortani. University of Louisville, Louisville, KY

Background: It has been previously shown that patients with metabolic syndrome andtype 2 diabetes have significantly lower plasma concentrations of adiponectincompared with non-diabetic controls. However, it is not clear as to whether non-obesepatients such as type 1 diabetics would also have reduced plasma concentrations ofadiponectin. This hormone is released by the adipose tissue to slow the production ofglucose by the liver and to initiate the oxidation of fatty acids and glucose in themuscle. Ironically, obese individuals have reduced expression of adiponectin and itsreceptors. Furthermore, patients with reduced concentrations of adiponectin havedifficultly controlling insulin and obesity. Hypothesis: Since plasma concentration ofadiponectin is inversely proportional to the degree of obesity, we hypothesized thattype 2 diabetics generally known to be obese would have lower expression ofadiponectin than do type 1 diabetics. Methods: Plasma specimens from patients >18years of age with blood glucose values greater than 200 mg/dL (n = 59) and those withblood glucose values less than 120 mg/dL (n = 60) were collected. Plasmaconcentrations of adiponectin were measured using a singleplex adiponectin kit on theLuminex 100 analyzer. Plasma concentrations of C-peptides were also measured.Various parameters such as clinical diagnosis of type 1 versus type 2 diabetes, height,weight and gender were recorded and body mass indices (BMI) were calculated.Results: As expected, patients with glucose >200 mg/dL had significantly higher C-peptide concentrations compared with patients with normal glucose values (p < 0.05).Clinically diagnosed type 2 diabetics had significantly lower plasma adiponectinconcentrations compared with clinically diagnosed type 1 diabetics (10.26 ± 5.85 vs.16.47 ± 9.86 µg/mL, p < 0.05). In this cohort, we also noted that adiponectin in theleaner patients (i.e. normal BMI) who had elevated glucose (>200 mg/dL) did notdiffer from those with elevated BMIs regardless of their diabetes status. In type 1diabetics, plasma concentration of adiponectin was not reduced and was comparableto non-diabetic controls. Conclusions: We conclude that plasma concentrations ofadiponectin are lowered in patients diagnosed with type 2 diabetes. The possibilityexists that this phenomenon is related to the degree of obesity in such patients.

Use of Serial Patient Differences of HPLC HbA1c to Determine Long

Term Instrument Performance

C. L. Wiley1, D. V. Tran2, T. N. Higgins3, L. Vandergouwe4, G. S.Cembrowski2. 1Marshfield Clinic, Marshfield, WI, 2University of Alberta,Edmonton, AB, Canada, 3DynaLIFE Dx, Edmonton, AB, Canada,4Chinook Health Region, Lethbridge, AB, Canada

Background: The usefulness of measuring HbA1c to assess glycemic control indiabetic patients is well established. The quality of the HbA1c assay is inverselyproportional to the variation of the assay. Often estimates of assay imprecision arebased on the repeated analysis of quality control specimens. These estimates aredependent on the matrix of the control specimen which may differ from actual patientspecimens. We have developed a unique approach to determining the imprecision ofHbA1c methods.Methods: HbA1c measurements of outpatient blood sample pairs drawn between 1and 30 days were made on two Tosoh high performance liquid chromatographyassays: Tosoh 2.2+ (total pairs, n=598) and Tosoh G7 (total pairs, n=626). The datacollections period was two years. The standard deviation of duplicates was calculatedfor three day time intervals up to 30 days (1 to 3 days, 4 to 6 days, etc.). These intra-individual variations were then plotted. Extrapolation to zero time yields acombination of biologic and analytic variation (Figure). Since HbA1c is slow-formingand its biological variation is low, we assumed that the majority of this variation attime zero is due to analytic imprecision.Results: At the mean HbA1cs of 7.51 and 6.96% for populations tested on the Tosoh

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2.2+, and the Tosoh G7, respectively, the total analytic imprecisions (coefficient ofvariation, CV) are 1.66% and 1.14%, respectively.

Conclusion: Using our approach, the total analytic variation is attributed to thevariation in multiple reagent lots, multiple calibrators and multiple calibrations duringthe study period as well as long and short-term environmental differences includingvariations in ambient temperature. Our analysis takes into consideration the variationobserved in specimens that are separated over periods as long as 30 days. Thevariation of the patient duplicates is thus a measure of the average variation over thisextended period.





Poster Session: 2:00 pm - 4:30 pmProteins/Enzymes

Establishment of the biomarker signature of age-related skeletal

muscle weakness

K. Ohlendieck, P. Donoghue, K. O'Connell, J. Gannon. National Universityof Ireland, Maynooth, County Kildare, Ireland

Frailty and fragility among sedentary elderly patients is related to an impaired structureand function of contractile fibres. Proteomic research into the mechanisms that underlieage-related skeletal muscle wasting investigates the scientific basis of evidence fordesigning new diagnostic and therapeutic strategies for treating sarcopenia of old age.Once new treatment options such as cell therapy or pharmacological intervention havebeen developed, it will be essential to also establish more reliable diagnostic markers.With respect to preventive medical approaches to sarcopenia, the identification of areliable set of disease-specific biomarkers would be crucial for the proper diagnosis ofage-dependent muscle degeneration long before the onset of serious clinical symptoms.To identify novel sarcopenia markers, we have conducted a mass spectrometry-basedproteomic profiling exercise to investigate muscle specimens from young, adult andsenescent rat and human fibres. We are currently comparing the soluble proteincomplement from muscle versus distinct subcellular fractions representing microsomalmembranes, mitochondria and the contractile apparatus. Using fluorescently taggedadult versus old muscle proteomes and high-resolution two-dimensional gelelectrophoresis (1st dimension: pH 3 to pH 10; 2nd dimension: 10 kDa to 220 kDa), thedifferential expression pattern of several thousand muscle proteins could be determined.For statistical purposes, 4 biological repeats and 3 technical repeats were performed peranalysis using differential fluorescent labeling with the dyes Cy5, Cy2 and Cy3. Both,peptide mass fingerprinting and peptide fragmentation technology was used for theidentification of tryptic digests of proteins that exhibited a marked change in abundance.Immunoblotting surveys and confocal microscopy was used to confirm these findings.Major age-related alterations in the muscle proteome included proteins involved in fibrecontraction (myosin heavy chains, myosin light chains, tropomyosin, troponins, actin),excitation-contraction coupling (dihydropyridine receptor), ion homeostasis (SERCA-type calcium pumps, sarcalumenin, sodium-calcium exchanger, plasma membranecalcium pump), metabolite transportation (fatty acid binding protein, myoglobin),cellular stress response (small heat shock prteins), and metabolism (glycolytic enzymes,mitochondrial enzymes). Based on these proteomic findings, we are currently screeningdifferent sub-types of muscle fibers to establish a comprehensive biomarker signature ofsarcopenia. Hopefully muscle proteomics will lead to a better molecular understandingof age-related muscle wasting and thereby help in the future design of superiordiagnostic procedures to evaluate muscle biopsies.

Human ribosomal protein S17 inhibits splicing of its own pre-mRNA

K. A. Cheremisina, A. A. Malygin, G. G. Karpova. Chemical biology andfundamental medicine, Novosibirsk, Russian Federation

The computer and statistical analysis of the number of EST sequences correspondingto human ribosomal protein genes showed that large variations in the expression ofthese genes take place in different normal adult human tissues. Moreover, a number ofstudies have detected changes in the expression of ribosomal protein genes associatedwith the oncogenic state, including increase of amount of specific ribosomal proteinmRNAs in tumors. Since eukaryotic ribosomal protein gene expression iscoordinately regulated at the transcription and translations steps, it is possible toassume that differences in quantities of ribosomal protein mRNAs in the cell areconcerned with regulation of their genes expression at the pre-mRNA splicing stage.Our investigation was aimed at studying regulation of splicing of human ribosomalprotein S17 (rpS17) pre-mRNA. Interestingly, overexpression of this protein wasobserved in tumorous lymph tissues.The cDNA of rpS17 was cloned into the expression vector pET-15b. Large-scaleproduction of the recombinant protein was carried out in E. coli cells and highlypurified protein was isolated. A method for refolding the protein from inclusionbodies was optimized. The secondary structure content of the refolded protein wasanalyzed by CD spectroscopy. In the present work, we showed that rpS17 binds

specifically with its pre-mRNA and suppresses the formation of mature mRNA duringsplicing in vitro. Human ribosomal protein S17 possesses high affinity to fragment ofits own pre-mRNA, containing the first intron. Weak binding of the pre-mRNAfragment to other ribosomal proteins was an evidence for the specific nature of therpS17 binding to its own pre-mRNA.Results of the present work together with the data obtained earlier for humanribosomal proteins S13 and S26 make it possible to draw a conclusion on existence ofthe pathway of feedback auto regulation general for all eukaryotes which provides theexact control of ribosomal proteins biosynthesis in the cell in the course of splicing oftheir pre-mRNAs. Probably, transgressions in this regulation result in uncontrolledprotein synthesis typical for tumor cells.

LIVER PROTEIN PROFILING OF MICE WITH HIGH FAT DIET-

INDUCED NONALCOHOLIC FATTY LIVER DISEASE

I. Kirpich, M. Bon-Homme, I. Deaciuc, C. McClain. University ofLouisville, Louisville, KY

Introduction. Non-alcoholic fatty liver disease (NAFLD) is present in up to one-third ofthe general population and in the majority of patients with metabolic risk factors such asobesity and diabetes. NAFLD refers to a wide spectrum of liver damage, includingsimple steatosis, steatohepatitis, fibrosis and cirrhosis. Insulin resistance and oxidativestress play an important role in NAFLD development and progression to non-alcoholicsteatohepatitis (NASH). In spite of a large number of clinical and experimental studies,the intimate molecular mechanism of NAFLD and progression to the more complicatedform, NASH, remains to be elucidated. The goal of the present study was to achieve amore detailed understanding of the molecular changes in response to high fat diet-induced liver steatosis through the identification of differentially expressed liver proteins.Materials and Methods. C57/BL6 mice were fed high fat diet (HFD, 60% lard) orcontrol diet (CD) for eight weeks. Liver injury was evaluated by histopathology andplasma alanine aminotransferase (ALT) activity; hepatic and plasma triglyceride (TAG)and free fatty acid (FFA) were measured. Hepatic proteome was analyzed by two-dimensional difference gel electrophoresis with mass spectrometry. Results. At the end of the experiment, HFD-fed mice developed hepatic steatosis (fataccumulation in the shape of macrovacuoles) and showed increased plasma ALT activity(58.3 + 7.4 vs 26.2 + 1.8 U/L, p > 0.001). Hepatic TAG (148.0 + 6.8 vs 42.0 + 2.9 mg/g ofliver, p > 0.05) and FFA (11.9 + 0.8 vs 8.5 + 0.29 µmoles/g of liver, p > 0.001) weresignificantly higher compare to their CD-fed cohorts, as well as plasma TAG (80.4 + 4.4vs 56.4 + 4.3 mg/dL, p > 0.05) and plasma FFA (0.37 + 0.02 vs 0.29 + 0.02 mEq/L, p >0.05). Proteomic analysis identified 9 proteins whose expression levels changed bygreater than 1.5 fold with significant changes (p > 0.05) in the HFD group compared tothe CD group. Of these, five proteins were up-regulated and seven were down-regulated.The identified proteins can be divided into six different categories according to theirfunctional properties: (i) proteins involved in cholesterol and triglyceride metabolism,such as apolipoprotein E and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase, bothup-regulated; (ii) enzymes involved in carbohydrate metabolism, such as ketohexokinase,up-regulated; (iii) proteins involved in stress response and xenobiotic metabolism, suchas selenium-binding protein 2, glutathione S-transferases mu1 and pi, all down-regulated;(iv) proteins involved in methionine metabolism, such as methionine adenosyltransferaseI alpha, up-regulated; (v) molecular chaperons, such as chaperonin subunit 5 epsilon,down-regulated; (vi) intracellular inhibitor of the lysosomal cysteine proteinases, such asstefin A, up-regulated.Conclusion. In summary, the current study has identified a set of proteins linking HFD-induced metabolic shift to the development of hepatic steatosis.

Multicenter evaluation OF a NEW Roche CRP (LATEX) ASSAY ON

HITACHI and COBAS SYSTEMS

R. Roeddiger1, F. Ceriotti2, A. Bezzu2, C. Müller3, F. Enzmann3, G.Sarfati4, M. Castro-Castro5, M. Widmann1. 1Roche Diagnostics GmbH,Mannheim, Germany, 2Diagnostica e Ricerca San Raffaele S.p.A., Milano,Italy, 3Virchow Klinikum, Berlin, Germany, 4Hôpital Cochin, Paris,France, 5Hospital Universitario de Bellvitge, L'Hospitalet de Llobregat,Catalonia, Spain

Background/Objective: The present abstract describes the analytical performance ofa new microparticle-enhanced turbidimetric assay for C-reactive Protein (CRP Gen. 3,Roche Diagnostics GmbH, Mannheim, Germany) with an extended primary

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measuring range for serum and plasma on RD/Hitachi and cobas c systems.Methods: The new method uses a reagent which was prepared by coupling twomonoclonal antibodies independently to latex particles of two different sizes. The twomicroparticle reagents are mixed in an appropriate ratio. On both systems, the sampleis first mixed with reaction buffer and thereafter with latex reagent. Theimmunoreaction is monitored at 800/570 (sub/main) nm after the addition of the latexreagent. The CRP concentration in patient samples is determined automatically from asix-point calibration curve.Results: The primary measuring range, which was defined by linearity testing, wasconfirmed from 0.3 to 350 mg/L (recovery rate: 0.3 - 5 mg/L ± 0.5 mg/L; 5 - 350 mg/L 100 ± 10 %), with an extended measuring range of up to 700 mg/L achieved by anadditional dilution step that is automatically performed by the analyzer. Limit ofQuantitation (CV 10 %) was found within a range of 0.44 - 0.61 mg/L. Within-labprecision (%CV) ranged from 1.4 - 4.9 for concentrations between 1.4 - 350 mg/L.Statistical Passing/Bablok analysis of method comparison against Tina-quant® [a]CRP and Tina-quant® [a] CRP HS yielded correlation coefficients > 0.98, slopesbetween 0.91 - 1.05 (Median 0.98) and intercepts ranged from - 0.38 to - 0.05 mg/L(Median - 0.15 mg/L). 10 calibrations at each site were performed to verify thecalibration reproducibility. CV ranged from 0.4 to 1.4 % for standards 2 to 6.Conclusions: The new, sensitive CRP assay provides highly precise and accuratemeasurements with a broad primary measuring range from 0.3 to 350 mg/L.Disclaimer: The Roche CRP Gen. 3 assay is not cleared for use in the U.S.

Cystatin C estimated Glomerular Filtration Rate (eGFR) in the

Elderly.

R. Bäck1, P. Thylén2, I. Klarin2, U. Bergman3, I. Odar-Cederlöf4, A.Helldén4. 1Clinical Chemistry, Karolinska University Hospital Huddinge,Stockholm, Sweden, 2Dept. of Geriatrics, Karolinska University HospitalHuddinge, Stockholm, Sweden, 3Clinical Pharmacology, KarolinskaUniveristy Hospital Huddinge, Stockholm, Sweden, 4ClinicalPharmacology, Karolinska University Hospital Huddinge, Stockholm,Sweden

Introduction: Elderly patients have decreased kidney function caused by ageing anddiseases. Information on renal function is important for clinical assessment of thepatient and for drug selection and dosing. Determination of plasma creatinine is easilyavailable but may present unreliable results in elderly patients, due to influence ofmuscular mass. Plasma cystatin C has been considered a better marker of renalfunction than creatinine and allows calculation of estimated glomerular filtration rate(eGFR).Its reliability in very old patients (>75 years) has not been studied.Patients and methods: Ninety-seven inpatients, 64 women and 33 men, aged >75years were examined. P-cystatin C was analysed with Dade-Behring’s assay on aBNProspec nefelometer. The routine equation for adults (>20years) used atKarolinska University Laboratory was used for calculation of cystatin C estimatedGFR (eGFR) and compared with GFR determined by iohexol clearance. Wecalculated the percent of estimates falling within 30% and 50% above or below themeasured GFR (i.e. iohexol clearance), which is considered a measure of accuracy.Results and discussion: When we used the routine equation for cystatin C eGFR, wefound that only 62% of the estimates were within 30% deviation from iohexolclearance (gold standard). We therefore derived a new equation based on thecorrelation between cystatin C and iohexol clearance in these elderly patients:y=60.173*CystC-1.0721 With this new equation 84% of the estimates fell within 30%deviation from the gold standard. GFR below 30 mL/minute is frequentlyrecommended as “a cut off point”. When we calculated eGFR with our routineequation for adults 18% had a eGFR below 30 mL/min/1.73 sq.m. body surface area(BSA), compared to 31% when we used our new equation. Our data suggest thatcystatin C based eGFR may overestimate GFR in old patients unless the routineequation is adapted to this group of patients.It should be taken into consideration that analysis of Cystatin C is not standardizedbetween laboratories so algoritms cannot be used generalized without adjustment forthe methods used.Conclusion: eGFR based on Cystatin C adds information on renal function, but theequation needs to be adjusted for elderly patients.

Re-Optimisation of Angiotensin Converting Enzyme Activity on

Dimension RXL MAX using reagent from Trinity Biotech plc

O. A. Joshi, I. Halsall. Cambridge University Hospital NHS Trust,Cambridge, United Kingdom

Introduction: Kinetic measurement of Angiotensin Converting Enzyme (ACE; EC3.4.15.1) was performed on Dimension RXL Max (Siemens Healthcare DiagnosticsInc) using kit from Trinity Biotech plc (Catalogue No. 305-10).Concerns following poor performance (positive bias) in external quality controlscheme (Biorad Immunoassay Programme 2) and imprecision at low activity,prompted re-optimisation of the instrument method parameters.Method: Concentrated substrate (N-[3-(2-furylacryloyl)-L-phenylalanylglycylglycine(FAPGG), was loaded on the analyser and automatically diluted at the time ofanalysis. Dimension RXL Max method kinetic programme was utilised to optimisethe following method parameters: Reaction times, Reagent and sample mix rate andlimit of detection. Calibration stability was also determined. Patient samples wereanalysed in duplicate to determine within batch imprecision. Total imprecision wasassessed using quality control material.Results: See Results Camparison Table

Discussion: Following re-optimisation, performance in the external quality controlscheme and precision at low activity has improved. Method kinetics has demonstratedthat previously recommended measurements at 5 and 10 minutes leads to imprecisiondue to, sample specific, varied rate of decrease in absorbance. If the readings are takenafter 7 minutes then precision improves, particularly at low activities.Conclusion:This method allows the users of Dimension RXL Max (SiemensDiagnostics Inc) to add ACE activity measurement to their routine chemistryrepertoire, improving turn around time whilst reducing sample handling. Singletonanalysis and improved on-board reagent stability has reduced reagent cost per test.

A Multicenter Prospective Evaluation of the Access® Soluble

Transferrin Receptor (sTfR) Assay and the sTfR / log Ferritin Index

(sTfR Index) for Differential Diagnosis of Iron Deficiency Anemia and

Anemia of Chronic Disease

B. S. Skikne1, K. Punnonen2, P. H. Caldron3, M. T. Bennett4, M. Ervasti2,G. H. Gasior5, J. S. Chamberlin5, L. A. Sullivan5, K. R. Bray5, P. C.Southwick5. 1University of Kansas Medical Center, Kansas City, KS,2Kuopio University Hospital, Kuopio, Finland, 3Arizona Arthritis andRheumatology Associates, Paradise Valley, AZ, 4Medical AssociatesResearch Group, San Diego, CA, 5Beckman Coulter, Inc., San Diego, CA

Background: Iron deficiency anemia (IDA) and anemia of chronic disease (ACD) arethe most prevalent forms of anemia. Standard tests of iron status used in differentialdiagnosis are affected by inflammation, making clinical interpretation difficult. Incontrast, soluble transferrin receptor (sTfR) is a valuable indicator of iron deficiencyand is unaffected by inflammation.Objectives and Methods: Our study assessed the clinical utility of the newautomated Access® sTfR assay (Beckman Coulter) and sTfR/log ferritin index (sTfRIndex) in a large prospective multicenter clinical trial consisting of patients withcommon disorders associated with anemia, and included a sufficient number ofsubjects to determine definitive cutoffs. Four sites consecutively enrolled 145patients.Results: Subjects with IDA or ACD+IDA showed significantly higher sTfR and sTfRIndex values than subjects with ACD (p<0.0001). The following cutoffs were derived

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Result Comparison Table

ParameterBefore Re-

optimisationAfter Re-

optimisationReaction times 5:00-10:00 minutes 07:30-11:30 minutesMix rate Moderate ModerateLimit of detection 5 U/L 10 U/LCalibration stability Daily 3 monthsTotal imprecision: Mean (%CV, n)

Mean: 17 U/L (30.5, 5) Mean:15 U/L, (8.1, 73)

Total imprecision: Mean (%CV, n)

Mean: 45 U/L, (14.8, 58) Mean:44 U/L, (3.3, 28)

Total imprecision: Mean (%CV, n)

Mean: 64 U/L, (6.8, 15) Mean: 64 U/L,(1.7, 19)

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using ROC curves: sTfR 21 nmol/L (1.55 mg/L), sTfR Index 14 (using nmol/L incalculations) or 1.03 (using mg/L in calculations). The figure below illustrates thedistribution of sTfR Index values by diagnostic group, with the double y-axis showingvalues in either nmol/L or mg/L in the Index calculation; note the clear separation ofACD subjects from those with ACD+IDA or IDA. The sTfR Index is superior to sTfR(AUC 0.87 vs 0.74, p<0.0001). Use of all three parameters in combination increasedsensitivity from 41% (ferritin alone) to 92% (ferritin, sTfR, sTfR Index).

Conclusions: The Access sTfR assay and sTfR Index improve detection of IDA,particularly in situations where routine markers provide equivocal results. Ourfindings demonstrate a significant advantage in the simultaneous determination offerritin, sTfR and the sTfR Index in anemic patients requiring a differential diagnosisof IDA and ACD. Obtaining a ferritin level alone may delay diagnosis or recognitionof the presence of combined IDA and ACD.

ANALYTICAL PERFORMANCE OF THE NEW ACCESS®

SOLUBLE TRANSFERRIN RECEPTOR (sTfR) ASSAY AND THE

sTfR / LOG FERRITIN INDEX (sTfR INDEX) ON THE ACCESS

IMMUNOASSAY SYSTEM

J. S. Chamberlin, G. H. Gasior, L. A. Sullivan, P. C. Southwick, K. R. Bray.Beckman Coulter, Inc., San Diego, CA

Background: Anemia of chronic disease (ACD) and iron deficiency anemia (IDA),the most common forms of anemia, are difficult to differentiate by standard testswhich are affected by chronic inflammatory diseases or infections. sTfR is unaffectedby inflammation and therefore provides an advantage over standard measures. ThesTfR assay may also be used in conjunction with an Access ferritin measurement toprovide a calculated sTfR / log ferritin Index. sTfR and the sTfR Index are intended asaids in the diagnosis of IDA and for the differential diagnosis of IDA and ACD.Objectives and Methods: This study was performed at Specialty Laboratories todetermine assay performance of the new sTfR assay and Index on the Access® 2Immunoassay System, a random access automated analyzer. The sTfR assay is a two-site immunoenzymatic (“sandwich") immunoassay. A serum or heparinized plasmasample is added to a reaction vessel along with paramagnetic particles coated withanti-sTfR antibody and an anti-sTfR alkaline phosphatase conjugate. After incubationin a reaction vessel, materials bound to the solid phase are held in a magnetic fieldwhile unbound materials are washed away. The chemiluminescent substrate Lumi-PhosTM 530, is added to the vessel and light generated by the reaction is measuredwith a luminometer. The light production is directly proportional to the concentrationof sTfR antigen in the sample. The amount of analyte in the sample is determinedfrom a stored, multi-point calibration curve. The time to first result is 35 minutes. TheIndex is determined by dividing sTfR by the log of ferritin.Results: The analytical range of the sTfR assay is 3-150 nmol/L. The followingresults were obtained in our studies. The analytical sensitivity was 0.03 nmol/L when

averaging 6 runs performed over 3 days. Using a Deming regression analysis, 271values ranging from 9.16 to 87.94 nmol/L were compared using the Access sTfRassay and the R&D Systems Inc. Quantikine® sTfR ELISA. The slope was 0.90(0.87-0.93 [95% CI]), the intercept was 0.55 (-0.59-1.68 [95% CI]) and the correlationcoefficient was 0.96 (0.95-0.97 [95% CI]). The sTfR assay exhibited within-runimprecision of ≤1.74 %CV and total imprecision of ≤5.4 %CV using 4 controls (9.91-94.28 nmol/L) analyzed 2 times per day over 20 days for a total of 80 individualresults. Imprecision of the sTfR / log of ferritin index was ≤6 % when analyzing thevariability of duplicate CV% on 334 samples. Using a Deming regression analysis,263 values ranging from 10.02-40.33 nmol/L were compared in the Access sTfRassay using matched serum (SST) and plasma (Li-heparin PST) samples. The slopewas 0.95 (0.92-0.97 [95% CI]), the intercept was 0.16 (-0.37- 0.68 [95% CI]) and thecorrelation coefficient was 0.98 (0.97- 0.98 [95% CI]). Conclusions: The AccesssTfR assay and sTfR Index demonstrated acceptable assay performance on the Accessautomated analyzer sufficient to support accurate detection of IDA and thedifferentiation between IDA and ACD in the presence of chronic inflammatorydiseases and infections.

The establishment of the reference range of the ratio between the urine

amylase and urine creatinine

Y. L. Hou, H. Jiang, Y. P. Tian. Chinese PLA General Hospital, BeiJing,China

Objective: To establish the reference ratio of urinous amylase and urinous creatininein local laboratory.Methods: The samples were derived from 11 acute pancreatitis in-patient (acutepancreatitis group), 326 urine specimens of normal local adults (man 169,woman 157)(control group) and 126 urine specimens of 21 nomal adults (6 specimens in eachperson are separately taken before and after three meals in a day). Analyticalinstrument: Roche Modular P was used in the test.Result: The reference of ratio of urinous amylase and urinous creatinine of 326 urinespecimens of nomal local adults is 9.36~19.48. The reference of urinous amylase of11 acute pancreatitis in-patient is 918.9±868.2 (97.7-2784.9), the reference ratio ofurinous amylase and urinous creatinine of 11 acute pancreatitis in-patient is58.60±26.70 (33.12-129.95), The reference of ratio of urinous amylase and urinouscreatinine of 21 urine speciments of nomal adults are 12.29±12.91, 12.57±1.98,12.19±1.94, 12.24±1.84, 12.19±1.74 and 12.26±1.80 separately. The reference ofurinous amylase of 326 urine speciments of nomal local adults is 128.4±98.4 (7.8-626.4), The reference of urinous amylase and urinous creatinine of 326 urinespeciments of nomal local adults is 14.42±5.06 (4.11-29.26).Conclusion:1. When the reference value of urinous amylase of in acute pancreatitisin-patient may be normal, the ratio of urinous amylase and urinous creatinine may farvarid from the normal level. 2. The ratio of urinous amylase and urinous creatininemay be stable while the urinous amylase and the urinous creatinine of the nomaladults alter greatly. 3. Using the ratio between the urine amylase and urine creatinineis better than urine amylase singly on acute pancreatitis diagnosis.Discussion:There was no significant difference between each reference value of ratioof urinous amylase/creatinine in nomal adults (P>0.05). It is showed that individualratio of urinous amylase/creatinine may change little in a day and the ratio fromrandom urine could reflect the normal level of whole day. The urinous amylase inrandom urine of acute pancreatitis often increased greatly, but it was not sometime.And it could be easily detected with the help of the ratio of urinous amylase/creatinine, which was more sensitive than the urinous amylase in acute pancreatitis.With the ratio, it is easy to differentiate the acute pancreatitis from other kinds of acuteabdominal pain in clinic.

Comparison of the mass and activity of serum isoenzyme of creatine

phosphokinase(CK-MB) in children

R. Zheng, H. Jiang, Y. P. Tian. Chinese PLA General Hospital, BeiJing,China

Background: CK-MB is specific enzyme in myocardial cells and is becoming animportant biochemistry marker to diagnose myocarditis and myocardial injury inhospitals. Nowadays, there are two main methods, one is the enzyme inhibitionimmunoassay, and another is the electrochemiluminescence immunoassay, but thereference intervals are mainly defined for adults, which may not always be

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appropriate for children.Objective: In order to know the children's CK-MB value by comparing the mass andactivity of CK-MB in children with two methods.Materials and methods: 165 children were divided into 5 groups from 1 to 15 yearsold and 19 adults were in group 6 as control. The activity and mass of CK-MB weredetected by the method of enzyme inhibition immunoassay andelectrochemiluminescence immunoassay, with the reference interval of 0-16U/L and0-6.5ng/ml.Results: CK-MB activity and mass analysis were linear correlated (P<0.01). CK-MBactivity mean value from group 1 to group 6 were 23.903U/L, 20.872U/L, 15.310U/L,13.530U/L, 11.688U/L, 8.916U/L; and their 5th-95th percentile confidence intervalswere 22.05~25.76U/L, 17.84~23.90U/L, 11.75~18.87U/L, 11.03~16.04U/L,10.66~12.72U/L, 6.51~11.32U/L. CK-MB mass mean value from group 1 to group 6were 4.899ng/ml, 4.999ng/ml, 3.010ng/ml, 2.862ng/ml, 2.725ng/ml, 2.926ng/ml; andtheir 5th-95th percentile confidence intervals were 4.57~5.23ng/ml, 3.61~6.39ng/ml,2.46~3.56ng/ml, 2.56~3.17ng/ml, 2.43~3.02ng/ml, 2.51~3.34ng/ml. It showed acommon tendency that the age of children were younger, the value of CK-MB activityand mass were higher. And CK-MB activity test results in group 1 and group 2 agebelow 5 years old were beyond its reference interval, meanwhile, all CK-MB masstest results and other groups of CK-MB activity test results were in their referenceintervals. Thus it is found that there were no significant correlation with CK-MBactivity and CK-MB mass analytical methods between group 1 and group 2(P>0.05).Conclusion: Children CK-MB activity and CK-MB mass value are higher thanadults. It is advisable to define young children's CK-MB reference value based ontheir physiological feature. At present, CK-MB mass analysis is better for clinicaldiagnosis of young children.

Rapid Identification of Amyloid Proteins from Formalin-Fixed

Paraffin-Embedded Sections Using Laser Microdissection and Triple

Quad MS/MS

J. D. Gamez. Mayo Clinic, Rochester, MN

Amyloidosis is a condition in which one of 25 known proteins form non-solublefibrils in various organs and tissues throughout the body. Amyloid deposits areidentified by staining sections of formalin fixed paraffin embedded (FFPE) tissuebiopsies with Congo Red. When amyloidosis is confirmed immunohistochemistry isperformed in order to determine the subtype. In a number of cases the results areambiguous, and a subtype is not defined. In amyloidosis definitive identification ofthe amyloid protein is essential for proper patient treatment. We have developed a newmethod that determines amyloid subtypes using laser microdissection, isotopicallylabeled synthetic peptides (acting as internal standards), and triple quad massspectrometry. We have tested 40 samples with this method consisting of 15transthyretin, 5 serum amyloid-associated protein 10 immunoglobulin light chainlambda, and 10 immunoglobulin light chain kappa. These samples incorporate a widevariety of tissue samples: (heart, lung, GI, kidney, and bone marrow). These sampleswere previously characterized using the LTQ orbitrap MS/MS method andimmunohistochemistry. In this study we used these results to gauge the level ofsensitivity with the triple quad mass spectrometer. Laser microdissection was used tocollect amyloid plaques from a 10 micron thick FFPE section exhibiting positiveCongo Red staining. Proteins were extracted, digested with trypsin, spiked withisotopically labeled peptides to the 4 amyloid subtypes, and MS/MS performed.Analyst software was used to interpret the data. A majority of the time we were ableto identify the 4 amyloid subtypes using this method. In clinical testing the ambiguoussamples would be reflexed to testing on the LTQ orbitrap mass spectrometer, thecurrent clinical testing method. With this new Triple Quad method we are able toidentify amyloid proteins in a high through put manner with accuracy and sensitivity.

The Correlation of Peroxiredoxin Levels and Pain in Patients with

Varicella Zoster Reactivation: A Novel Biomarker for Postherpatic

Neuralgia

M. P. Thompson, M. Kuro-o, J. Pastor, D. A. Payne. UT-Southwestern,Dallas, TX

Postherpetic neuralgia (PHN) affects approximately 30% of shingles patients. Theaim of this study was to identify predictive biomarkers of shingles-associated PHN.Thirty patients presenting with newly diagnosed shingles had their blood collected on

the day of presentation (day 0), eight days after presentation (day 8), and twenty-eightdays after presentation (day 28) for analysis. On day 28, the patients were clinicallyexamined for post-herpetic neuralgia, with nine patients exhibiting PHN and fourteenpatients without PHN (six patients that were immunocompromised were excludedfrom this study and one patient was lost to follow-up, n = 23). All of the whole bloodsamples of the nine patients with PHN and the fourteen without PHN on day 28 werethen divided into PHN-positive and PHN-negative groups and pooled by time ofcollection to yield four sample groups: PHN-positive, day 0; PHN-negative, day 0;PHN-positive, day 8; PHN-negative, day 8. The pooled serum of the patients withPHN were compared to those without PHN using 2-dimensional gel electrophoresisfollowed by Progenesis Discovery v.2005 software analysis. All protein spots with a ±2 fold change between the PHN-positive and PHN-negative groups on either days 0 or8, were considered significant and eligible for mass spectrometric identification. Ofthese protein spots, spot 636 was the most significant, exhibiting a greater than 3 foldincrease on both day 0 and day 8 in the group that ultimately developed PHN whencompared to the group that did not develop PHN. Mass spectrometry followed bytandem mass spectrometry were utlilized to identify spot 636 as peroxiredoxin, athioredoxin peroxidase. Western blot analysis of the pooled whole blood samples witha peroxiredoxin-specific antibody followed by band density analysis verified andquantified the significant upregulation of peroxiredoxin in the patients with PHNversus the patients without PHN on day 0.This is the first description of increased serum peroxiredoxin in association with painand the first association peroxiredoxin levels as a predictor of pain The peroxiredoxinfamily of proteins function as reducers of reactive oxygen species and are associatedwith oxidative stress. In vivo, peroxiredoxin levels have been shown to correlate withincreases in reactive oxygen species. Furthermore, reactive oxygen species have beenshown to play a vital role in both neuralgic and inflammatory nociception. Our futureresearch into the novel association of peroxiredoxin and nociception may provebeneficial in understanding the pathophysiology of nociception, the role of theperoxiredoxin family in nociceptive signaling, and may ultimately lead to therecognition of the peroxiredoxin family as sensitive, objective biomarkers of reactiveoxygen species-associated pain.

The transferrin soluble receptor (sTfR): an application protocol for

the Roche/Hitachi Modular P800 Chemistry System

M. E. Mendes, P. Romano, V. R. Morsoleto Batista, P. A. Rezende Ebner,M. N. Burattini, N. M. Sumita. Central Laboratory Division &Laboratories of Medical Investigation (LIM-03) of Hospital das Clínicasda Faculdade de Medicina da Universidade de São Paulo (HCFMUSP),São Paulo, Brazil

Introduction: Some studies in hematology therapy suggest that blood concentrationof transferring soluble receptor (sTfR) should be monitored to maximize efficacycontrol to reposition of iron. The transferrin receptor is an essential component ofcellular uptake of iron, and it binds to serum transferrin. Recently, 2 different types oftransferrin receptors have been recognized: transferrin receptor (TfR or transferrinreceptor 1) and transferrin receptor 2. Most cells possess an ubiquitous systemcontrolling the biosynthesis of TfR at the posttranscriptional level to avoid excess ironinflux into the cells through TfR. During the process of recycling of transferrinreceptors, some are shed and appear as soluble or serum transferrin receptors.Measurement of serum transferrin receptor is a new marker of iron metabolism thatreflects body iron storage and total erythropoiesis. It has been shown that serumtransferrin receptor and ferritin ratio has significant predictive value for differentiatingiron deficiency anemia from non-iron deficiency anemia, such as anemia of chronicdisorders.Objective: The aim of this study was to evaluate the performance of sTfRimunoturbidimetric Tina-Quant assay from Roche ((Roche Diagnostics, Mannheim,Germany).Materials and Methods: The reagent contains antibody anti-sTfR linked to a latexreagent in a bovine serum albumin (BSA) matrix. The complex antigen-antibodyagglutination is measured by turbidimetric method. The assay has been optimized tomeasure total sTfR from 0.50 to 40.0 mg/L. The assay was performed on Roche/Hitachi Modular P800 (Roche Diagnostics, Mannheim, Germany), using low and highcontrol samples. The detection limit was defined evaluating a sample of serumwithout sTfR for 20 times. The recovery test was assessed using a saline solutionadding a sample containing sTfR. Two samples with different concentrations wereobtained: 1.05 mg/L and 3.60 mg/L.Results: The assay was found to be linear up to 20.7 mg/L. The detection limit was0.01 mg/L in 20 dosages. The functional sensitivity was 0.22 ± 0.014 µg/mL. The

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within-run precision ranged from 3.44± 0.07% to control sample with a concentrationof 2.11 mg/L and 0.7±0.06% for concentration of 7.2 mg/L. The between-runprecision ranged from 4.25±0.09% to control sample with 2.11 mg/L and 4.66±0,39%for concentration of 7.20 mg/L. The recovery test ranged from 92.8% to 2.11 mg/Land 109.3% to 7.20 mg/L.Conclusion: The Tina-Quant sTfR assay is a simple fast and convenient alternative toevaluate sTfR in blood serum sample. This parameter is useful in the diagnosis of irondeficiency, especially for patients with concurrent chronic disease, where routine testsof iron status are compromised by the inflammatory condition.

Meagerment of Beta-2 microglobulin Intermediate in serum by

Capillary Electrophoresis

Y. Uji1, M. Miura2, Y. Motomiya3, Y. Ando4, C. Yoshida1, I. Kitajima1.1Clinical Laboratory Center,Toyama University Hospital, Toyama, Japan,2Department of Clinical Chemistry,Hokuriku University, Kanazwa, Japan,3Suiyukai Clinic, Kashihara, Japan, 4Department of LaboratoryMedicine,Kumamoto University Graduate School, Kumamoto, Japan

Background: The beta-2 microglobulin (B2 m) conformer has been proven to be anamyloidogenic intermediate using capillary electrophoresis(CE) in several studies[Biol Chem 2002,277,11184-9 (1)and Electrophoresis 2002,23-918-25(2)] . However,this technique has never been used to determine the presence of the B2 m conformerin clinical specimens, such as hemodialysis(HD) patients serum. We establishedmeasurement of amyloidgenic intermediate B2m(I-B2m) and native B2m(N-B2m) inserum by CE. Methods and Materials: All experiments performed on a P/ACE 2100 CE system(Beckman Coulter Instruments, Inc., Fullerton, Calif., USA). Fused-silicacapillaries(50 micro meter of ID, 57 cm of length) were also purchased from BeckmanCoulter Instruments. Before the CE analysis, the serum samples were centrifuged for30 min at 2,500 g using a Sartorius Ultrafilter Centrisart I (Sartorius AG, GoettingenGermany) to exclude proteins of more than 20,000 kDa in the sample. Using apressure injection of 0.5 psi (5 s) applied to the capillary inlet for the sample(approximately 12.5 nL) in each cycle, capillaries were pretreated with 1 M NaOH for1 min, followed by 3 min of washing with water and 5 min with 100 m M phosphatebuffer solution. A positive polarity of 15 kV was applied at the inlets from the anodeside to the cathode side, and through all experiments the currents varied from 89 to 90micro A. The capillary was maintained at 24 ° C, and the wavelength was fixed at 200nm. All serum samples were obtained at Suiyukai Clinic (Nara,Japan) after receivinginformed consent for this study. The purified B2m from human urine was purchasedfrom Sigma(St. Louis, MO.,USA) for a standard B2m. Polyclonal antibody of B2mwas purchased from Sanyo Kasei (Kyoto,Japan).Results: Standard B2m and all serum samples including healthy subjects showed twopeaks, major and minor, with migration time of 9.8 and 10.2 min, respectively. Wejudged the major peak to be the N-B2m and the minor peak to be the I-B2m usingaffinity column of B2m polyclonal antibody and according to (1) and (2). Linearity upto 100mg/L for N-B2m and 50 mg/L for I-B2m. Detection limits was 0.38 mg/L. Thewithin-assay and between assay precision (CVs) were less than 6.5% (N=10).Recovery studies were performed 96-104% was achieved. The concentrations of N-B2 m and I-B2 m were: 29.4± 6.8 (17.5-48.6) and 2.7 8±1.4 (0.8-6.2) mg/L in HDpatients (N=31) ; 4.4 ± 1.9 (2.2-6.4) and 0.6 ± 0.6 (0.1-1.6) mg/L in non-dialysischronic renal failure patients (N=5), and 1.2 8± 0.1 (1.0-1.3) and 0.2 ± 0.1 (0.1-0.3)mg/L in healthy subjects (N=5). In addition, dialysis related amyloidosis (DRA)patients (N=13) of I-B2m concentration (N=13) were significantly lower than non-DRA patients (N=13, p<0.004).Conclusion: The proposed method suggested that can be new tool for diagnosis ofDRA.

Oxidized urinary albumin directly effects on the electrophoretic

mobility

A. Nakayama1, M. Sakatsume2, T. Kasama1, T. Kawara1, F. Gejyo2, M.Isobe1, K. Shiba3, K. Sato1. 1Tokyo Medical and Dental Univeresity, Tokyo,Japan, 2Niigata University, Niigata, Japan, 3Bunkyo Gakuin University,Tokyo, Japan

In patients with glomerulonephritis (GN), thirteen different mobility of urinaryalbumin bands were identified in the molecular mass range of 55 to 172 kD [1]. We

found that high molecular mass forms of albumin were aggregation andintermolecular link of albumin and other protein by disulfide bond. We undertook thepresent study to clarify the characterization of the low molecular mass forms ofalbumin and its possible relation to oxidative damage. H2O2-treated and -untreatedhuman serum albumin (HSA), representative urine sample with GN and healthy urinesample were analyzed by non-reducing SDS PAGE, Western Blot using anti albuminantibody. These samples were also incubated with D-biotin-hydrazide (BHZ) to detectprotein carbonyl using Western Blot based method [2]. As a result, among otherproteins, the low molecular mass form of albumin (approximately 60 kD) was clearlycarbonylated in GN subject (band no. 2 in the figure) similarly to the H2O2-treatedHSA as a reference. The results from in-gel digestion and matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry showed that the peptideincluding N-terminal of the sequence was preserved in the 60 kD of albumin, and thedetected peptides pattern was well-correlated with un-carbonylated albumin (bandno.1 in the figure). These findings suggest that the electrophoretically low molecularmass form of albumin is not a simple fragment and strongly relevant for the mobilityshift to the cathode due to oxidation, and carbonylated urinary albumin provides anew aspect for further understanding of posttranslational modification.[1] Nakayama A et al. Molecular heterogeneity of urinary albumin inglomerulonephritis: Comparison of cardiovascular disease with albuminuria. ClinChim Acta 2009 in press.[2] Oh-Ishi M et al. Proteomic method detects oxdatively induced protein carbonyls inmuscles of a diabetes model Otsuka Long-Evans Tokushima Fatty (OLETOF) rat.Free Radic Biol Med. 2003;34(1):11-22.

Evaluation of a new Cystatin C assay on the ABBOTT ARCHITECT

cSystems and AEROSET Clinical Chemistry Systems

F. Rota1, R. Dioli1, L. De Angelis1, R. Lucini1, J. Herzog2, H. Troonen2.1Sentinel CH., Milan, Italy, 2Abbott GmbH & Co. KG, Diagnostics,Delkenheim, Germany

Objective: A new Cystatin C assay was evaluated on the ABBOTT ARCHITECTcSystems and AEROSET Clinical Chemistry Systems. The scope of the study was toverify analytical performance and agreement with NACB’s guidelines for EmergingBiomarkers of Cardiovascular Disease and Stroke (allowed total analytical error15%).Materials/instruments: New Cystatin C reagent, manufactured by SENTINEL CH.,is a latex enhanced immunoturbidimetric (PETIA) assay based on microparticlescoated with anti-hu CysC (rabbit). The assay was optimized to enhance datatransferability and comparability with the nephelometric (PENIA) Cystatin C. TheAbbott ARCHITECT c8000 and AEROSET Systems are random-access clinicalchemistry analyzers. ARCHITECT c8000 can be integrated with ARCHITECTi2000SR, an immunoassay module, to form the ci8200.Study Design: Assay performance was investigated using modified CLSI protocols.Acceptance criteria were set at ≤5.0% CV for total imprecision and at ≤5.0% foraccuracy throughout the study (allowed Total analytical error ≤13.2%). LOQ wasdefined as the analyte concentration at which imprecision was less than 20% CV.Analytical measurement range was defined through linearity. Method comparisonconsisted in comparing ARCHITECT and/or AEROSET results for patient serumsamples with those generated by PENIA on the BNA II system (n=61, 0.58 to 6.91

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mg/L). Interference due to endogenous substances and drugs was investigatedfollowing CLSI EP7-A2.

Results:

Interfering substances (max allowable bias: 0.1 mg/L): Bilirubin (conjugated 66 mg/dL, unconjugated 60 mg/dL), Hemoglobin (1000 mg/dL), Intralipid (1000 mg/dL),and RF (500 IU/L) did not interfere.19 drugs and 9 drug combinations were tested and found to give a bias < 3%.Conclusion: Performance of the new Cystatin C assay fully meets NACB’sguidelines for Emerging Biomarkers of Cardiovascular Disease and Stroke on bothABBOTT systems.

A New Method for Cystatin C Evaluated on the ADVIA® Chemistry

Systems from Siemens Healthcare Diagnostics

P. Datta, M. Losapio, J. Dai. Siemens Healthcare Diagnostics, Tarrytown,NY

Objective: Quantitative determination of cystatin C in human serum or plasma isuseful in the diagnosis and treatment of renal insufficiency. Serum concentrations ofcystatin C are almost totally dependent on the glomerular filtration rate (GFR). Areduction in GFR causes a rise in cystatin C concentration. Cystatin C has not beenshown to be affected by factors such as muscle mass and nutrition, factors which havebeen demonstrated to affect creatinine values. In addition, a rise in serum creatininedoes not become evident until the GFR has fallen approximately by 50%. Recently,Siemens released a new cystatin C assay (CYSC) to measure cystatin C on theautomated, random access ADVIA 1650, ADVIA 1800, ADVIA 2400, and ADVIA1200 Chemistry Systems. Our evaluation of this method included precision, linearity,correlation, and interference performance studies.Methods: All ADVIA Chemistry Systems use the same CYSC reagent packs,calibrators, and commercial controls. In this assay, a specimen containing humancystatin C is diluted and then reacted with antibody (rabbit) coupled to latexmicroparticles. The increased turbidity is measured at 571 nm. By constructing a six-level standard curve (water is used as reagent blank) from the absorbances ofstandards, the analyte concentration of the sample is determined.Results: The within-run and total CVs (40 replicates per sample) of the new methodwith a two-level commercial control and three serum pools (~0.6, 3.0, 0.6, 2.0, and 5.0mg/L cystatin C) on all ADVIA Chemistry Systems were <1.8% and <3.1%,respectively. The analytical range/linearity of the method (all ADVIA systems) wasfrom 0.1 mg/L to the cystatin C concentration in the highest level of calibrator (7.7-8.3 mg/L). The new method (CYSC) on the ADVIA 1650/1800 (y) correlated wellwith the BN™ II System (Siemens) Cystatin C method (x): y = 1.00x + 0.02 (r = 0.99,n = 55, range: 0.13-7.75 mg/L). The ADVIA 2400 and 1200 CYSC methods, in turn,agreed with the ADVIA 1650/1800 CYSC method: ADVIA 2400 CYSC = 0.99(ADVIA 1650/1800 CYSC) + 0.05 (r = 0.99, n = 102, range: 0.18-7.57 mg/L); andADVIA 1200 CYSC = 1.03 (ADVIA 1650/1800 CYSC) - 0.02 (r = 0.99, n = 104,range: 0.18-7.57 mg/L). The new method showed <10% interference with bilirubin(conjugated or free) up to 60 mg/dL, hemoglobin up to 1000 mg/dL, lipids(INTRALIPID®, Fresenius Kabi AB Corporation) up to 1000 mg/dL, and rheumatoidfactors up to 1200 IU/mL. The method has a minimum of 60 days’ on-system andcalibration stability on all ADVIA Chemistry Systems. No prozone was observed withthe method on any platform up to the highest cystatin C concentration tested in asample (56 mg/L).Conclusion: We conclude that the new Cystatin C method, when used on any ADVIAChemistry System, can measure serum or plasma cystatin C concentrations preciselyand accurately over a broad range in routine laboratory use.

Evaluation and Method Comparison of Three ELISAs for Quantifying

S-100B in Serum

J. A. Erickson1, D. G. Grenache2. 1ARUP Institute for Clinical andExperimental Pathology, Salt Lake City, UT, 2University of Utah HealthSciences Center, Department of Pathology, Salt Lake City, UT

Background: S-100B is a calcium binding protein with a molecular weight ofapproximately 22 kD found in astroglial cells, Schwann cells and melanocytes.Several studies have demonstrated that serum concentrations of S-100B can be usedto monitor ongoing brain damage and predict outcome in patients with acquired braininjury. Other studies have demonstrated that S-100B is a useful tumor marker formonitoring the treatment of malignant melanoma.Objectives: The purpose of this study was to evaluate the performance characteristicsand perform method comparison studies for three commercially available S-100BELISAs. Assays included in the study were the Sangtec® 100 ELISA (DiaSorin,Stillwater, MN), CanAg® S100 EIA (Fujirebio Diagnostics, Malvern, PA) and YK150Human S-100B ELISA (Yanaihara Institute, Inc, Shizuoka, Japan).Methods: Residual serum samples sent to ARUP Laboratories for routine testing wereutilized in this study. S-100B protein was measured according to each kitmanufacturer’s testing protocol. Performance characteristics and split-samplecomparison studies were determined for all assays with the exception of between-runprecision, sample stability and reference interval determination for the YK150 assay.Reference intervals (97.5th percentile) were established by non-parametric analysis ofserum S-100B results generated from 150 healthy adult volunteers (75 males, 75females). This study was approved by the University of Utah’s Institutional ReviewBoard.Results: The results are given in the table below.

Conclusions: The performance characteristics of the Sangtec and CanAg S-100BELISAs are comparable and both assays have lower limits of detection, betterlinearity and are more precise than the YK150 assay. Although agreement is pooramong all assay combinations, the Sangtec and CanAg ELISAs demonstrate bettercorrelation versus comparison with the YK150 ELISA.

PerformancesARCHITECT

c8000ARCHITECT

c16000AEROSET

Assay range 0.05 to 8.0 mg/LPrecision(10-days)Units: mg/L

2.1% at 0.62.0% at 0.81.9% at 1.1

1.5% at 0.81.1% at 1.71.2% at 4.1

4.3% at 0.62.9% at 0.91.9% at 1.1

LOD 0.03 mg/L 0.05 mg/L 0.04 mg/LLOQ 0.04 mg/L NA 0.06 mg/LComparison vs. Penia on BNASlopeInterceptR

0.9320.1100.998

0.9290.1260.998

0.9180.1230.998

Calibration stability 45 days

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Sangtec CanAg YK150CanAg vs.

Sangtec(N = 39)

YK150 vs.

Sangtec(N = 40)

YK150 vs.

CanAg(N = 39)

Upper limit of AMR 5000 ng/L 3500 ng/L 6300

ng/L NA NA NA

Limit of Detection 15 ng/L 12 ng/L 39 ng/L NA NA NA

LinearityLinear

Regression 1.014x+65.1 1.038x+31.1 1.123x-105.4 NA NA NA

R2 0.999 0.999 0.997 NA NA NA

Precision

Within-Run

(CV, Mean)

5.7%(222 ng/L)

1.6%(1182 ng/L)

2.1%(3217 ng/L)

6.3%(47 ng/L)

2.9%(680 ng/L)

1.9%(1837 ng/L)

10.8%(335 ng/

L)4.7%

(704 ng/L)

4.6%(2677 ng/L)

NA NA NA

Between-Run

(CV, Mean)

11.3%(228 ng/L)

7.6%(1162 ng/L)

5.4%(2912 ng/L)

6.2%(80 ng/L)

4.1%(509 ng/L)

3.5%(1604 ng/L)

ND NA NA NA

Analyte Stability

Ambient 24 h 24 h ND NA NA NA4 °C 7 d 7 d ND NA NA NA

Reference Interval ≤ 141 ng/L ≤ 96 ng/L ND NA NA NA

Comparison of Methods

Deming Regression NA NA NA 0.339x+24.1 0.226x-

140.01.376x-

13.1

R2 NA NA NA 0.932 0.690 0.860

NA = Not ApplicableND = Not Done

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Determination of asymmetric dimethylarginine (ADMA) and

symmetric dimethylarginine (SDMA) in human plasma: analytical

and clinical validation

M. Plebani1, C. Artusi1, M. Ivanova1, G. Boffa2, M. Zaninotto1.1Department of Laboratory Medicine, University-Hospital, Padova, Italy,2Department of Cardiology, University-Hospital, Padova, Italy

Background: Due to the growing number of reports in the literature on ADMA as apossible marker of endothelial dysfunction, its use in the clinical practic is ofincreasing interest. In contrast there are a less studies concerning the role of SDMA,the structure isomer of ADMA, as an endogeneous marker of renal function.Objectives: The purposes of this study were: 1) Determination of reference values ofADMA and SDMA plasma concentrations after validation of a chromatographicHPLC method; 2) Evaluation of plasma concentrations of ADMA (p-ADMA) inpatients with various disorders characterized by endothelial dysfunction; 3)Estimation of the relationship between plasma concentrations of SDMA (p-SDMA)and estimated GFR (eGFR) as well as plasmatic creatinine in patients with chronickidney disease (CKD). Materials and methods: Blood samples were taken from 140blood subjects (107 males, 33 females) aged from 18 to 65 years for determination ofreference values. We selected 4 groups of patients with a high risk of cardiovascularcomplications for evaluation of p-ADMA and p-SDMA. A total of 168 patients wereanalyzed: 66 with chronic heart failure, 43 with type II diabetes (30 patients of thisgroup were complicated with renal insufficiency), 42 with CKD, 17 with pulmonaryarterial hypertension. Sample cleanup was performed by solid-phase extraction onpolymeric cation-exchange columns. After derivatization with orthophthaldialdehydereagent, analytes were separated by isocratic reversed-phase HPLC with fluorescencedetection. Results: Calibration curve is linear throughout the selected ranges (0.5-10.0 µmol/L); the absolute and relative recoveries (96-106%) were assessed by sixdeterminations at 3 concentrations; the within- and between- day imprecision (CV<5%) were evaluated by performing al least 5 replicate analyses of quality controlsamples at 3 different concentrations; the accuracy (-3.57/+6.00%, bias -0.03/+0.06µmol/L) was obtained by measurement of recovery because certified referencestandards in the matrix is not available. Reference values (2.5-97.5 percentiles) for p-ADMA and p-SDMA are 0.34-0,63 µmol/L and 0.26-0.57 µmol/L respectively.ADMA levels were significantly elevated in all groups compared with controls(p<0.001). In contrast there isn’t a statistically significant difference of p-ADMAbetween the group of patients with type II diabetes and controls. This can be explainedby the fact that these patients are under tight glycemic control. SDMA levels weresignificantly elevated (p<0.0001) both in the patients with CKD and in patients withtype II diabetes complicated with renal insufficiency. We found that SDMA levelscorrelated highly with both eGFR (R=0.740) and plasmatic creatinine (R=0.700).Conclusions: Our study, showing elevated p-ADMA levels in all groups of patientsstudied, suffering from diseases characterized by endothelial disfunction, suggests thepeculiar usefulness of this measurement in the biochemical evaluation of endothelialfunction. Furthermore, the preliminary results suggest that SDMA could be a reliablemarker of renal function, but further studies are in progress in order to investigatewhether SDMA is indeed a stable and clinical reliable marker of renal function,especially in pediatric populations in which the use of eGFR is not recommended.

Development and Validation of an Immunochemiluminometric Assay

for 14-3-3 Protein

C. M. Preissner, A. J. Aksamit, J. E. Parisi, S. K. Grebe. Mayo Foundation,Rochester, MN

Background: Elevation of 14-3-3 protein in cerebrospinal fluid (CSF) is one of theWorld Health Organization criteria supporting an antemortem diagnosis of sporadicCreutzfeldt-Jakob Disease (CJD). However, 14-3-3 testing is performed essentiallyexclusively by Western blot, relying on subjective visual interpretation. Elevatedneuron specific enolase (NSE) levels in spinal fluid have also been found in CJDpatients.Objective: To develop and validate an immunochemiluminometric (ICMA)microtiter plate assay for 14-3-3 protein.Assay Methodology: Two mouse monoclonal antibodies are used in the 14-3-3 assay.White high-binding 96-well plates are coated overnight with 0.5µg antibody per well.The wells are then washed and blocked with Superblock® for 1 hour at roomtemperature. Duplicate 200µL aliquots of standards, controls and patient samples areadded and incubated overnight at 4°C. After washing, 200µL of acridinium ester-

labeled antibody are added for 6 hours at 4°C. Following a further wash the plate iscounted in a luminometer, with the measured relative light units (RLUs) beingproportional to the 14-3-3 concentration in the sample.Until 11/20/2007 NSE was measured with an in-house ICMA. Since then, the samplesare analyzed on the BRAHMS Kryptor® Compact using time resolved amplifiedcryptate emission technology. For data reduction purposes of this study, the resultsobtained after 11/20/2007 were recalculated using the regression equation derivedfrom a method comparsion between the old ICMA and the Kryptor NSE assay.Results: The 14-3-3 standard curve ranges from 2 to 250ng/mL with a net increase ofover 150,000 RLUs. Assay imprecision was <20% at concentrations from 3.8 to40.6ng/mL. Linearity of serial dilution of 4 CSF samples with elevated 14-3-3 levelsshowed a mean recovery of 110%, range 93-132%.For clinical validation, CSF samples submitted from 2002-2008 (N=949) for NSEtesting and then frozen, were thawed and analyzed for 14-3-3 protein in 2008.Thirteen samples came from autopsy-proven CJD cases and 936 came from patientswith other neurological disorders. Using ROC analysis, the optimal cutoffs weredetermined to be 43ng/mL for NSE (76.9% sensitivity; 94.0% specificity) and 9.6ng/mL for 14-3-3 (76.9% sensitivity; 96.7% specificity). In another group of samplesfrom 33 clinically highly probable CJD cases without histological confirmation NSEwas elevated in 28 (84.8%) cases, while14-3-3 was elevated in 24 (72.7%). Thespecificity of the 9.6 ng/mL cut-off for 14-3-3 was further validated in 235 CSFsamples submitted for red and white blood cell counting (CJD was not suspected). Ofthese, 222 (94.5%) had 14-3-3 values ≤9.6ng/mL. Some of the elevated results (9.8-34.8ng/mL) came from patients with disorders known to elevate CSF 14-3-3, such asGuillain-Barre syndrome and viral encephalitis.Since erythrocytes contain NSE and 14-3-3, interference by blood-contamination wasassessed. The 14-3-3 concentrations in 82 visibly blood-tinged CSF samples were 0-281ng/mL, mean 47.8ng/mL, with 74 samples (90.2%) showing levels >9.6ng/mL.Conclusions: The ICMA performs well with adequate precision and linearity, but isaffected by hemolysis. When measured along with NSE it provides additionaldiagnostic specificity in patients in whom CJD is being considered.

EVALUATION OF NEUTROPHIL GELATINASE-ASSOCIATED

LIPOCALIN AS A MARKER OF OUTCOME IN PATIENTS

FOLLOWING CARDIAC SURGERY

H. Turner, J. Reeve, J. McNeilly, W. Mutch, R. Peake, D. Rae, P. Gibson,G. Hillis, B. Cuthbertson, B. Croal. Aberdeen Royal Infirmary, Aberdeen,United Kingdom

Background: Serum neutrophil gelatinase-associated lipocalin (NGAL) has beensuggested as an early marker of acute kidney injury in patients following cardiacsurgery. We have evaluated NGAL along with, creatinine, cystatin C and their derivedestimated glomerular filtration rates (eGFR-Cr; eGFR-CC respectively), at predictingmortality in patients following cardiac surgery.Methods: Serum NGAL (ng/mL, ELISA, AntibodyShop) was measured in earlypost-operative samples (6hr) in patients undergoing cardiac surgery (n=284). Pre-operative levels of cystatin C (mg/L, Siemens Prospec) and creatinine (umol/L,Siemens ADVIA 2400) were measured prior to surgery and their respective eGFRscalculated. Creatinine continued to be measured until discharge. Patients werefollowed up for one year post surgery.Results: Early post-operative NGAL levels were not predictive of mortality(p=0.645) at 1 year. However, there were significant differences in the pre-operativecystatin C (1.13 v 0.94; p<0.001), eGFR-CC (72.6 v 99.8; p<0.001) and eGFR-Cr(65.9 v 82.4; p<0.001) levels in patients that subsequently died. Quartiles of NGALwere not useful in predicting survival (p=0.52), however cystatin C quartiles did showsignificant differences (p=0.001). Interestingly, post-operative renal function, asreflected by the highest measured creatinine or the lowest eGFR-CR, was related tothe quartile status of early post-operative measurement of NGAL (p<0.001; p<0.001respectively). In a linear regression model, quartile status for NGAL was predictive ofthe subsequent highest creatinine level measured during the 10 days following surgery(p<0.001). However, this was not predictive of the maximum rise in creatinine frombaseline during this period (p=0.062)

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Conclusion: NGAL measured during the early post-operative period would appear tobe a potentially useful predictor of subsequent deterioration in renal function,however it does not appear to be related to subsequent mortality.

S100B PROTEIN AS A SERUM MARKER OF SURVIVAL AFTER

TRAUMATIC BRAIN INJURY (TBI)

M. C. GONZALEZ MAO, A. REPARAZ ANDRADE, E. ALVAREZGARCIA, P. POSADA GONZALEZ, C. VARA PEREZ, A. ANDRADEOLIVIE. COMPLEJO HOSPITALARIO VIGO, Vigo -pontevedra, Spain

BACKGROUND: In the last years, there has been an increasing need of a newperipheral biochemical marker specific for the activity of damaged brain tissue thatallows assessing the severity of traumatic processes. The early identification of severepatients after TBI is essential to assist them and crucial to establish prognosis.OBJECTIVE: To establish the relationship between S100B protein levels and thesurvival of patients with TBI.METHODOLOGY: N (137) patients with TBI (110 males and 27 females), admittedto hospital Intensive Care Unit (ICU) during the last year and with an average age of42.7 years (15-84), were selected. Inclusion criteria: arrival at hospital in the 6 hoursfollowing the injury, TBI as the main diagnosis, previous neurological assessment.Clinical variables assessed: survival/exitus. Analytical criteria: S100B levels in bloodin the first 6, 24, 48 and 72 hours after the injury. Negative urine drug screening atadmission. S100B concentration was determined with a quantitative automatedluminometric inmunoassay (LIASON Sangtec 100 Diasorin). Detection limit 0.02 µg/L Inter-assay variation (10.7% at 0.11 µg/L; 2.2% at 2.6; 3.2% at 18.4)We used SPSS 16.0 statistical package (SPSS, Chicago, IL, USA). p<0,05 wasconsidered statistically significant. Odds ratio (OR) and 95% confidence ratio arecalculated.RESULTS:

CONCLUSIONS: S100B protein levels after 6, 24 and 48 hours are a good survivalpredictive marker after TBI. Significantly higher S100B levels are observed in thosepatients with fatal outcome. Each increase in one unit of the S100B level at 6, 24 and48 hours of the injury rises the risk of death 1.2, 3.1 and 2.7 times respectively. Thevalue with the highest predictive significance is taken 24 hours after TBI.

TRAUMATIC BRAIN INJURY (TBI): S100B PROTEIN VERSUS

GLASGOW SCALES

M. C. GONZALEZ MAO, E. ALVAREZ GARCIA, A. REPARAZANDRADE, C. VARA PEREZ, P. POSADA GONZALEZ, A. ANDRADEOLIVIE. COMPLEJO HOSPITALARIO UNIVERSITARIO VIGO, Vigo -pontevedra, Spain

BACKGROUND: Being able to predict the presence and severity of Nervous CentralSystem (NCS) injury is vital at Neurological Intensive Care Units (NICUS). Theavailability of a marker before clinical symptoms allows modifying treatment criteria.Until now we have lacked a really specific marker to assess the severity of traumaticprocesses and to early detect adverse neurological effects.OBJECTIVES: To study S100B predictive capacity as a brain injury markercomparing this protein levels with patients’ clinical assessments using Glasgow ComaScale (GCS), and S100B usefulness as a predictor of adverse neurological effects withpatients’ neurological assessments at hospital discharge measured with GlasgowOutcome Scale (GOS).METHODOLOGY: 137 patients with TBI (110 males and 27 females), admitted tohospital ICU during the last year, with an average age of 42.69 (15-84) were selected.Inclusion criteria: arrival at hospital in the 6 hours following the injury, TBI as a maindiagnosis, previous neurological assessment. Clinical variables assessed: GCS, GOS.Analytical criteria: S100B levels in blood in the first 6, 24, 48 and 72 hours after theinjury. Negative urine drug screening. S100B concentration was determined with aquantitative automated luminometric inmunoassay (LIASON Sangtec 100 ).Thedetection limit is 0.02 µg/L. Inter-assay variation (10.7% at 0.11 µg/L; 2.2% at 2.6;3.2% at 18.4). We used SPSS 16.0 . p<0,05 is considered statistically significant.Pearson’s correlation among S100B levels and GSC and GOS scales is calculated.RESULTS:

CONCLUSIONS: S100B measured in the initial 24, 48 and 72 hours correlates withGCS scale as a marker of brain injury severity and with GOS scale as a predictor ofadverse neurological effects in moderate and severe TBI. S100B 6h only correlates insevere TBI. No correlation is observed in mild TBI, except with GOS scale at 72hours.

Development of a Cystatin C assay using monoclonal antibodies for

automated clinical chemistry analyzers

S. Nakayama, Y. Nakamura, H. Takahashi, H. Yago, K. Ushizawa. TsukubaResearch Institute, Ryugasaki, Japan

Estimation of the glomerular filtration rate (GFR) is essential for the evaluation ofpatients with chronic kidney disease (CKD). Cystatin C has been suggested as a new

S100B values (µg/L) in TBI (n= 137)S100B

6hS100B

24hS100B

48hS100B

72hMean(Confidence ratio 95%)

2.18(1.6-2.7)

1.34(1.0-1.7)

1.17(0.6-1.8)

0.60(0.4-0.8)

Median 1.11 0.68 0.45 0.38Range (0.04-26) (0.05-14) (0.04-28) (0.07-5.5)S100B marker as mortality predictorMean S100BExitus (Y/N)

3.6 / 1.7 2.9 /0.7 3.5 / 0.6 1.3 / 0.5

p 0.002 0.000 0.04 0.05(n.s)NExitus (Y/N)

36/89 31/89 21/83 14/65

Predictive capacity regarding: Survival/lethality. Logistic regressionOdds Ratio“OR”

1.2(1.1-1.4)

3.1(1.8-5.2)

2.7(1.4-5.2)

p 0.01 <0.001 0.002

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r Pearsonpn

S100B6h

S100B24h

S100B48h

S100B72h

GCS

Mild TBI-0.2140.204

37

-0.1560.395

32

-0.0440.826

28

-0.2210.323

22

Moderate TBI-0.0850.626

35

-0.44**0.012

33

-0.43**0.021

29

-0. 36**0 .034

23

Severe TBI-0.32**0.011

63

-0.37**0.006

55

-0.21**0.015

47

-0.44**0.010

34

Total TBI-0.41**0.000121

-0.25 **0.009104

GOS

Mild TBI-0.2940.078

37

-0.2580.154

32

-0.2790.150

28

-0.50**0.017

22

Moderate TBI-0.250.152

35

-0.62**0.0033

-0.54**0.003

29

-0.63**0.001

23

Severe TBI-0.31**0.013

63

-0.47**0.000

55

-0.36**0.012

47

-0.40**0.018

34

Total TBI-0.53**0.000120

-0.380.000104

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marker of the GFR. We have developed a specific and sensitive reagent for themeasurement of Cystatin C levels using various automated clinical chemistry analyzers.The assay is based on latex-enhanced immunoturbidimetry with a combination of anti-human Cystatin C mouse monoclonal antibodies. The Cystatin C concentration of asample is determined by measuring the change in absorbance that results fromagglutination of latex particles. For standard analysis ueing the Hitachi 917 autoanalyzer, 2.4 µL of human serum or plasma was mixed with 120 µL of the first buffersolution and incubated for 5 min. at 37ºC. Then 120 µL of the second reagent, whichcontains the monoclonal antibody-coated latex particles, was added and the absorbancewas monitored at 570 nm /800 nm (main/sub wavelengths) for 5 min. The detectionlimit for Cystatin C was 0.05 mg/L. The measurable range was defined 0.5 mg/L to 10mg/L. The within-run CV (n=10) at 0.5, 1, 2, 4, and 10 mg/L was 0.8%, 0.5%, 0.4%,0.5%, and 0.9%, respectively. The between-run CV (n=10) at 0.6, 2.1, and 6.8 mg/L was1.3%, 0.5%, and 1.7%, respectively. Interference studies showed no effect of bilirubin,hemoglobin, and rheumatoid factor (RF) at concentrations of 20 mg/dL, 500 mg/dL, and500 IU/dL, respectively. Comparison with the N-Latex Cystain C kit using a BNprospec (Dade-Behring) yielded a correlation coefficient of 0.99 and an equation of Y(present method) = 0.965 X (nephelometric method) + 0.117 (n = 55). We conclude thatthis ready-to-use assay is an accurate, precise, and simple method for measuringCystatin C concentrations in serum or plasma samples from patients with CKD.

Improved enzymatic cycling method for determination of ethanol

concentration in Biological sample.

T. Wang, Y. Tang, P. He. Yujiang Medical College for Nationalities,Baiso,Guangxi, China

INTRODUCTION: Gas chromatography is considered to be the reference method forethanol determination. However, enzymatic ethanol assays have been selected as routinescreening method. The most frequently used enzymatic assay utilizes the oxidation ofethanol to acetaldehyde by alcohol dehydrogenase with concurrent reduction ofnicotinamide adenine dinucleotide (NAD) to NADH while monitoring the increase inabsorbance at 340 nm. Previously, several authors reported that increased concentrationsof lactate and lactate dehydrogenase (LDH) can cause false-positive results inhaemolytic sample, because of the NADH produced by LDH. The another disadvantageof the NAD-ADH system is insensitive in low alcohol sample. In this paper, we presentan enzymatic cycling method to overcome the weakness of the NAD-ADH system,which includes two steps, in first step, ethanol is converted to acetaldehyde by ADH,and the potential lactate is depleted. In second step, acetaldehyde formed issubsequently oxided to acetic acid by aldehyde dehydrogenase, while thio nicotinamideadenine dinucleotide(thio-NAD) is reduced to reduced thio nicotinamide adeninedinucleotide(thio-NADH), in the presence of NADH, acetic acid is reversely catalyzedto acetaldehyde,. Thio NADH signal is amplified during the cycling reaction and can bemonitored at 405nm.METHODS: The method involves use of thio-NAD(+), NADH , alcoholdehydrogenase(EC 1.1.1.1) and aldehyde dehydrogenase (EC. 1.2.1.5) , andmeasurement of the increase in absorbance at 405 nm of thio-NADH at 37 degrees C.RESULTS: The calibration curve for ethanol was linear (r=0.99) between 0.5 and 120mmol/l. Analytical recoveries of exogenous ethanol added to serum and urine were 100-105% and 98-103%, respectively. Within-run and between-run coefficient of variation(CV) were 2.8%(1 mmol/l), 1.6%(30 mmol/l) and 0.8%(80mmol/l) in blood sample,and 3.4%(1 mmol/l), 2.5%(30 mmol/l) and 1.2%(80mmol/l) in urine sample,respectively. There were no significant difference(p>0.05) in the results of thecongenerous samples with or without lactate and LDH added(lactate 3.0 mmol/l, LDH400 U/l).CONCLUSIONS: An improved enzymatic cycling assay for ethanol determinationwas successfully developed, which was more sensitive and able to eliminate theinterference of lactate and LDH. This method is especially suit for the low ethanolsample and haemolytic sample.

Development of a novel IRI immunoturbidimetry assay for clinical

chemistry analyzers

J. Kondou, M. Yamamoto, H. Yago, K. Ushizawa. Tsukuba ResearchInstitute, Ryugasaki, Japan

Insulin resistance is defined as a clinical state in which a normal or elevated insulinlevel produces an attenuated biologic response. Insulin resistance is observed patients

with Type 2 diabetes, obesity, essential hypertension, and coronary heart disease. Thusdiagnosis of insulin resistance is very important and immunoreactive insulin (IRI) hasbeen used as a marker of insulin resistance.We have developed a method based onlatex-enhanced immunoturbidimetry for measurement of the serum and plasma IRIconcentrations. The IRI concentration of a sample is determined by measuring thechange in absorbance that results from agglutination of latex particles. Our assay hastwo reagents and shows excellent performance. For standard analysis using a Hitachi917 autoanalyzer, 10 µL of human serum or plasma sample was mixed with 150 µL ofthe first reagent and incubated for 5 min. at 37ºC. Then 50 µL of the second reagent,which contains antibody-coated latex particles, was added and the absorbance wasmonitored at 570 nm /800 nm (main/sub wavelengths) for 5 min. The detection limitfor IRI was 1 µU/mL. The measurable range was defined as 1 µU/mL to 200 µU/mL.The within-run CV (n=10) at 4.2, 51.4, and 206.2 µU/mL was 2.8%, 0.9%, and 1.2%,respectively. Comparison with data obtained using LUMIPULSE INSULIN N(FUJIREBIO) yielded a correlation coefficient of 0.96 and a regression formula of Y(present method) = 0.989 X (nephelometric method) + 0.263 (n = 142). We concludethat this ready-to-use assay is an accurate, precise, and simple method formeasurement of IRI in serum or plasma samples from patients with insulin resistance.

Simultaneous measurement of interleukins, chemokines and growth

factors in a single sample with an Evidence biochip array.

P. Lowry, L. Farry, J. Porter, F. M. McPhillips, V. Toner, R. I. McConnell,S. P. Fitzgerald. Randox Laboratories, Crumlin, United Kingdom

Background: Cytokines are investigated in multiple fields of research as they playcentral roles in many biological processes. Cytokines are often pleiotropic andnormally function as part of a complex network. Evidence Biochip Array Technologyenables simultaneous measurement of multiple analytes with a single sample and thisis beneficial as a patient profile is generated in real time. This multi-analyte profilingapproach provides more information on the cytokine network than single-analytedeterminations and leads to reduced consumption of sample/reagents and better cost-effectiveness of the tests.Relevance: We report multiplexed immunoassays on biochip array for simultaneousquantitation of interleukins, chemokines and growth factors per sample using a singleanalytical device. The biochip array enables determination of eotaxin, IL-1Ra, IL-12p40, IGF-I, IP-10, PDGF-AA, PDGF-BB, RANTES.Methodology: Simultaneous sandwich chemiluminescent immunoassays areemployed. Antibodies for all the analytes are bound to a solid substrate (the biochip)in discrete test regions defining arrays. Assay specific reagents and sample are appliedto the biochip and incubated under controlled conditions. Signal detection, dataprocessing and storage were carried out using the semi-automated bench top analyserEvidence Investigator.Results: Initial evaluation of the biochip array shows that the immunoassays aretarget specific. All analytes are measured in picogram quantities with sensitivityranging from ≤ 5pg/ml for PDGF-BB (calibration range 0 to 3,000pg/ml) to ≤140pg/ml for PDGF-AA(calibration range 0 to 2,000pg/ml). For all the immunoassays, theintra-assay precision expressed as %CV is typically less than 12%. Conclusion: Biochip array technology is applicable to the real time measurement ofinterleukins, chemokines and growth factors. The use of a multi-analytical approachcan provide a better understanding of the inter-relationship of cytokines in diseasestates with a view to aiding earlier diagnosis and potentially identifying new routes fortreatment

Diagnostic Values of NMDA Receptor Peptide for Acute Ischemic

Stroke

S. A. Dambinova. Emory University, Atlanta, GA

Introduction. NR2 peptide, an N-methyl-D-aspartate (NMDA) receptor fragment,has been proposed as a biomarker for TIA and ischemic stroke providing real-timeevidence of neurotoxicity. Early microembolic/thrombotic processes activate NMDAreceptor cleavage by thrombin-activated serine proteases, resulting in peptidefragments entering the bloodstream. Ability of the NR2 peptide to signal acutecerebrovascular events and diagnostic utility of this biomarker in different clinicalsettings were studied.Methods. An NR2 peptide assay based on magnetic-particle (MP) ELISA wasdeveloped and assay performance characteristics were established. Plasma specimenswere collected at 3 clinical settings, where feasibility studies of the NR2 peptide assay

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for assessment of transient ischemic attack (TIA) and ischemic acute stroke wereperformed. Of 232 patients recruited between January 2006 and June 2008 accordingto local Institutional Review Board approvals, 141 had TIA/acute stroke. To definetissue-based evidence of injury all patients had DWI/MRI. Blood samples were drawn1, 3, 6, 12, and 24 h after TIA/acute stroke onset. Plasma samples were also drawnfrom apparently healthy persons (n=154).Results. The reference interval calculated from the samples (central 94th percentile)was found to be 0.1-0.5 ng/mL for both genders. Positive likelihood ratio of the NR2peptide assay to detect cortical ischemic events varied depending on time from onset:

A significant correlation of NR2 peptide to stroke volume from 2 to 250 cc wasobserved. In patients with white matter strokes and lacunar lesions, NR2 peptideshowed low sensitivity (about 63%), while specificity remained significant (95%).Conclusions. NR2 peptide is brain-borne microvascular biomarker that is releasedinto the bloodstream as tissue-based evidence of cerebrovascular injury. Thelikelihood ratios, when used in conjunction with clinical findings, have the potentialto decrease time to referral to a stroke specialist for patients suspected of having acerebral ischemic event.

Evaluation of Enzyme Assays on the Abbott ARCHITECT® c4000

Clinical Chemistry System

S. Shaw, C. Kasal, J. Lucio, P. Bathory, J. Salazar, D. Bozimowski. AbbottLaboratories, Irving, TX

Objective: Verification studies established performance of enzyme assays on a clinicalchemistry analyzer under evaluation, the Abbott ARCHITECT® c4000* ClinicalChemistry System.Methodology: The ARCHITECT c4000 Clinical Chemistry System uses the reagentconfiguration, calibration processes, reaction modes, photometric technology andIntegrated Chip Technology® (ICT) that are available on the ARCHITECT® c8000System and the ARCHITECT® c16000 System and the Abbott AEROSET® System.The ARCHITECT c4000 is a fully automated analyzer that can be run as a stand-alone instrument or integrated with the ARCHITECT® i1000SR (the immunoassaymodule) to form the ARCHITECT ci4100. The ARCHITECT ci4100 is the newestmember of the ARCHITECT family of instruments.Results: Assay performance (Precision, Sensitivity, Linearity and MethodComparison was characterized using CLSI-derived protocols. Precision (5 days) wasdetermined using a minimum of two levels of controls. LOQ was demonstrated tomeet or exceed values previously determined for the ARCHITECT cSystem. Theanalytical range represents the observed linear low and high levels verifying theLinearity claim. Method Comparison was evaluated by assaying serum patientsamples across the entire range of the assay, comparing the ARCHITECT c4000 to theARCHITECT c16000. Bias was determined at the medical decision level(s). SigmaMetrics ([TEa - bias] / CV) were calculated and ranged from 6.2 (Acid Phosphatase)to 22.7 (Amylase).

Conclusion: The ARCHITECT c4000 Clinical Chemistry System demonstratedacceptable performance characteristics that achieved or exceeded all pre-established

analytical goals. The method correlation data support the equivalence of theARCHITECT c4000, ARCHITECT c8000, ARCHITECT c16000 and AEROSETSystems. The common reagent and commodity requirements across the four systemsallow the laboratory the flexibility to use either instrument depending on the userneeds. The ability to integrate the c4000 with an i1000SR immunoassay moduleprovides the capacity to analyze most routine clinical requests on one system.* = In-development

Evaluation of Diazyme Total Bile Acids Assay on the Olympus AU680

Chemistry-Immuno Analyzer.

M. Lin, C. Hoke, R. Dlott, T. Lorey. TPMG Regional Laboratories,Berkeley, CA

Total bile acids are metabolized in the liver, and hence, serve as a marker for normalliver function. Serum total bile acids are increased in patients with acute hepatitis,chronic hepatitis, liver sclerosis, and liver cancer. Total bile acid testing is also asensitive test for obstetric cholestasis in pregnancy, where the flow through the bileducts is reduced.The Diazyme Total Bile Acid reagents are in a stable liquid formulation that allowsfor ease of use. In the presence of Thio-NAD, the enzyme 3-α-hydroxysteroiddehydrogenase (3-α-HSD) converts bile acids to 3-keto steroids and Thio-NADH.The reaction is reversible and 3-α-HSD can convert 3-keto steroids and Thio-NADHto bile acids and Thio-NAD. In the presence of excess NADH, the enzyme cyclingoccurs efficiently and the rate of formation of Thio-NADH is determined bymeasuring specific change of absorbance at 405 nm.Deming’s linear regression was used to compare the Diazyme Total Bile Acid assay tothe LC/MS/MS method with the following results (n = 40): r = 0.9852, y = 1.04x -1.53, Syx = 2.00, average bias = -1.15 umol/L, range 0.8-57.4 umol/L.

Precision was determined following CLSI Protocol EP5. The analytical measurementrange (1 umol/L to 180 umol/L) and reference range from 66 males and 68 females (<10 umol/L, mean 3.2 umol/L) were determined.The Diazyme Total Bile Acid test was easily implemented on the “open channel”Olympus AU680 analyzer. The assay demonstrated good performance and can beconveniently integrated into a high volume laboratory setting.

Method Comparison of Quantitative Serum Free Light Chain

Immunoassays Performed on Nephelometric Versus Turbidimetric

Platforms

S. LeSourd, K. Sweat, M. Johnston, J. Marshall, J. Bornhorst. University ofArkansas for Medical Sciences, Little Rock, AR

Background: The serum free light chain assay, Freelite™ (The Binding Site, Inc),is aquantitative serum-based immunoassay for free circulating light chains, a surrogatemarker for many plasma cell dyscrasias. The immune complexes formed usingFreelite™ antibody reagents assay can be quantified by both nephelometry (lightscatter) and turbidimetry (light absorption). The objectives of this study were tocompare performance characteristics and analytical results of serum free light chaintesting on a turbidimeter (The Binding Site SPAPLUS™) versus the nephelometer(Dade Berhing BNII®). Freelite™ reagents have been validated for both platforms andshould provide comparable results.Methods: Intra-assay (ten repeats of control and pooled patient samples) and inter-assay (control samples run seven days three time daily) imprecision were analyzed forkappa and lambda free light chains on a nephelometer and a turbidimeter usingabnormal and normal control samples provided by the manufacturer and a highabnormal patient pool provided by UAMS. Patient and control serum sample resultswere compared between both platforms using Freelite™ kit reagents and standards.

Clinical settingCut off,

µg/LHours from

onsetSensitivity

%Specificity

%

Positive likelihood

ratioSurgery 0.5 1-6 88 95 18.5Neurology Clinic 0.9 2-12 99 90 9.7Emergency Department

1.5 12-24 83 84 5.2

Assay

Control Imprecision(Total %CV)

Level 1 Level 2

Limit of Quantitation(Sensitivity)

(U/L)

Analytical Range(U/L)

Method Comparison(vs. c16000)

Slope R % BiasAcid Phosphatase 1.52 1.33 0.4565 0 - 88 1.05 0.9994 -1Activated Alanine Aminotransferase 2.92 1.52 4.1825 6 - 5066 0.99 0.9997 3

Activated Aspartate Aminotransferase 1.14 0.30 3.5490 6 - 5450 0.99 1.0000 1

Alanine Aminotransferase 2.22 1.02 2.6579 6 - 4200 1.00 1.0000 0

Alkaline Phosphatase 2.13 0.61 5.0424 4 - 4712 1.00 1.0000 -2Amylase 0.62 0.35 2.2977 3 - 6720 1.00 1.0000 1Aspartate Aminotransferase 0.98 1.50 2.4844 3 - 4531 0.98 1.0000 -2

Creatine Kinase 1.33 0.91 4.9207 6 - 4419 1.01 1.0000 2GGT 0.86 0.71 2.2674 4 - 9396 1.01 1.0000 2Lactate Dehydrogenase 1.45 1.14 0.7363 3 - 5057 1.03 0.9999 2Lipase 2.60 1.78 3.8589 3 - 1256 0.99 0.9999 1

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Precision Within-run (N=20)Mean (umol/L) SD %CV

11.3 0.4 3.9109.8 0.6 0.5Total Precision (N=20)Mean (umol/L) SD %CV11.0 0.5 4.731.5 1.1 3.6109.0 5.7 5.3

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Deming regression analyses were performed for method comparison.Results: The precision for kappa and lambda reagents on the SPAPLUS wasdetermined for both intra-assay (CV range 1.31 - 3.22%) and inter-assay (CV range3.4 - 8.3%) analysis. Historically, precision of the BNII for the assay has beencomparable (average CV range - 2.75 - 6.94%). Normal serum range for free kappaconcentrations is 0.33-1.94 mg/dL and for free lambda concentrations is 0.571-2.63mg/dL. Free kappa results from normal and myeloma subjects ranged from 0.036 to982.0 mg/dL on the SPAPLUS and 0.030 to 1110.0 mg/dL on the BNII (n=129);analyses between instruments for kappa reagents demonstrated a correlationcoefficient of 0.9774 P<0.0001. The Deming regression equation for the kappamethod comparison is SPAPLUS= 0.9790BNII + -1.3286. Free lambda results fromnormal and myeloma subjects ranged from 0.060 to 1153.0 mg/dL on the SPAPLUS and0.040 to 1010.0 mg/dL on the BNII (n=129); analyses between instruments forlambda reagents demonstrated a correlation coefficient of 0.9933 P<0.0001. TheDeming regression equation for the lambda method comparison is SPAPLUS=1.1289BNII + -5.3222. General agreement between methods for measured free kappa,free lambda and Free Kappa/Lambda ratios values were observed in the normalpatient samples.Conclusion: Method comparison of the serum free light assay, Freelite™, bynephelometry and turbidimetry demonstrated that either platform produce comparableand precise results. Both platforms represent viable options for clinical serum lightchain analysis.

Method Comparison for the Gentian Immunoturbidimetric Cystatin

C Assay for Beckman Coulter’s Synchron System Versus a Siemens

Nephelometric Cystatin C Assay on the Siemens BNII System

S. LeSourd, K. Sweat, R. C. Faught, K. Sanderson, J. Marshall, J.Bornhorst. University of Arkansas for Medical Sciences, Little Rock, AR

Background: Cystatin C is a non-glycosylated peptide produced by all nucleated cellsand is present in all body fluids. It is removed from circulation by glomerular filtrationand is often considered a good indicator of glomerular filtration rate (GFR). Serumcystatin C concentrations are relatively unaffected by age, gender, muscle mass orinflammatory disease. Recently an user defined reagent immunoturbidimetric assayfor Cystatin C by Gentian for the Beckman Coulter automated Synchron and UniCelsystems was released for human serum or plasma. A method comparison wasperformed on the Beckman Coulter platform versus a nephlometric cystatin C assayon the Siemens BN II System. Precision studies and a patient linearity were alsoperformed to validate the immunoassay on the Beckman Coulter UNICEL® DXC 800System.Methods: Sixty five serum specimen Cystatin C results were compared between theGentian assay on the Beckman Coulter UNICEL® DxC 800 System and the SiemensCystatin C assay on the Siemens BNII System. Data from two calibrations for eachassay were included in this data set. Samples were obtained from patients with low ornormal estimated glomerular filtration rates (eGFR). Imprecision was analyzed on theBeckman Coulter DxC using low and high patient samples and 2 different levels ofcontrol material. Linearity was confirmed by diluting a high patient sample. Thestated package insert analytical measurement range is 0.4-8.0 mg/L for the Cystatin Cassay by Gentian and 0.2-6.8 mg/L for the Siemens Cystatin C assay.Results: The method comparison was performed on 65 patient serum samples rangingfrom 0.51 to 6.53 mg/L for the BNII and 0.7 to 7.99 mg/L for the DXC. The Demingregression equation for the method comparison is DxC=1.127(BNII assay) + 0.137and with a correlation coefficient (r-value) of 0.9970. Intra-assay precision analysisfor the Beckman Coulter DxC system had a CV range of 0.6-2.8%. Inter-assayprecision studies are ongoing. Cystatin C precision studies performed on the SiemensBN II instrument showed comparable imprecision with an intra-assay CV range of1.5-3.5%. Linearity was demonstrated on the Beckman Coulter system using theGentian cystatin C assay over a measured range of 0.52 to 7.97 mg/L using a highpatient serum sample (slope was 0.96 ± 0.4 and the intercept was 0.00 ± 0.19.)Conclusion: While the these two assays showed comparable precision, the GentianBeckman Coulter cystatin C immunoassay as tested on the UNICEL® DxC 800system demonstated a ~13% positive bias when compared to the Siemens BNIIsystem Dade-Behring nephelometric cystatin C assay. The stated package insertreference interval for the Siemens BNII assay is 0.53-0.95mg/L, and the statedreference interval Gentian Cystatin C assay is 0.62-1.15mg/L, roughly reflecting theobserved positive method comparison bias. The Gentian cystatin C turbidimetricassay appears to be a potential alternative to the Siemens nephlometric cystatin Cassay.

A New Enzymatic Method to Measure Adenosine Deaminase in

Human Body Fluids

P. P. Chou, M. Lara, K. Sisco, N. Sherman. Quest Diagnostics NicholsInstitute, Chantilly, VA

Introduction: Adenosine deaminase (ADA) is an enzyme catalyzing the deaminationreaction from adenosine to inosine. This enzyme is widely distributed in humantissues, especially high in T lymphocytes. Elevated serum ADA activity has beenobserved in patients with acute hepatitis, alcoholic hepatic fibrosis, chronic activehepatitis, liver cirrhosis, viral hepatitis, and hepatoma. Increased ADA activity is alsoobserved in patients with tuberculous effusions.Method: The ADA assay is based on the enzymatic deamination of adenosine toinosine, which is converted to hypoxanthine by purine nucleoside phosphorylase.Hypoxanthine is then converted to uric acid and hydrogen peroxide by xanthineoxidase. Hydrogen peroxide is further reacted with N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline and 4-aminoantipyrine in the presence of peroxidase togenerate quinone dye which is monitored kinetically.

Conclusions: We have developed and validated the ADA assay for human cerebralspinal, pleural and peritoneal fluids.

Characterization of Abnormal Lactate Dehydrogenase Isoenzyme with

the Semi-Automated Hydrasys Electrophoresis System

W. Lee, Y. Park, S. Yoon, J. H. Lee, O. H. Kwon. Yonsei University Collegeof Medicine, Seoul, Republic of Korea

Macroenzymes are complexes of serum enzymes with a plasma protein, having highermolecular weight with prolonged half-life. Their presence can cause an elevation inthe serum enzyme levels, possibly leading to misdiagnosis.Herein we present a 30-year-old man found with persistent elevation of LDH levelmeasuring 885-1083 IU/L in a routine health check-up. Other laboratory findingsincluding complete blood counts, routine chemistries, liver function tests andserologic tests for hepatitis virus were not remarkable. Radiologic studies includingabdominal ultrasonography also showed no significant finding. The electrophoresis ofLDH isoenzymes was performed with the Hydragel Iso-LDH kit used in conjunctionwith the semi-automated Hydrasys gel electrophoresis system, and the electrophoreticseparation showed abnormal migration patterns of LDH2 and 3 isoenzymes withdensely stained LDH3. The immunoelctrophoresis of LDH isoenzyme with anti-human IgG, IgA, IgM, kappa and lambda light chain antibodies was performed toidentify the antibodies bound to LDH. The patient's LDH was presented to be boundto IgA and kappa light chain.This modified immunoelectrophoresis with semi-automated system could be useful tocharacterize these kinds of abnormal LDH isoenzyme pattern.

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Results

CSFPleural Fluid

Peritoneal Fluid

Intra-assay Mean, U/L CV, % Mean, U/L CV, % Mean, U/L CV, %Level 1 0.35 20.20 3.55 1.99 4.23 2.73Level 2 25.63 1.42 22.97 0.75 27.72 0.15Level 3 39.92 0.23 134.67 1.01 46.35 0.20Inter-assay Mean, U/L CV, % Mean, U/L CV, % Mean, U/L CV, %Level 1 0.35 15.65 3.55 2.35 4.23 15.50Level 2 25.63 1.41 22.97 1.09 27.72 1.88Level 3 39.92 0.96 134.67 0.99 46.35 0.38Recovery, % 95.2-101.2 103.1-114.6 94.6-97.5Limit of Qn, U/L 0.35 2.70 0.70Ref Range, U/L <7.0 <9.2 <7.6

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Comparison of Cystatin C to various markers in chronic kidney

disease patients

Y. Minakawa1, Y. Meguro1, Y. Sato1, T. Asaumi2. 1Denka Seiken Co., Ltd.,Tokyo, Japan, 2Tokyo Medical University Hachioji Medical Center, Tokyo,Japan

Objective: We compared clinical utility of serum Cystatin C in estimating GFR tovarious serum and urine markers that have been traditionally used for kidney functionmonitoring and estimation. We also address potential discordance in GFR estimationby the use of different methods for Cystatin C determination with the lack of thestandardization. Methods: Serum Cystatin C, serum and urine beta2-microglobulin, serum and urinealpha1-microglobulin, creatinine and microalbumin were determined in 100 subjects.Each biochemical marker was evaluated for correlation with creatinine clearanceestimation of GFR. The correlation with creatinine clearance was also evaluated insubgroups of subjects from each stage of chronic kindly disease. Statisticalsignificance of each biochemical marker among different stages of chronic kindlydisease was also evaluated. A latex-enhanced TIA (Denka Seiken, Japan) was usedfor serum Cystatin C measurement.Results: Serum 1/Cystatin C showed the best correlation with creatinine clearanceamong the various biochemical markers tested (r = 0.821). The following equationwas derived to estimate GFR by serum Cystatin C: eGFR (mL/min) = 61.58 xCystatin C (mg/L) -1.3691. Even when the subjects were subdivided into 5 groupsaccording to the GFR levels, serum Cystatin C showed the good correlation withcreatinine clearance in all the subgroups. We previously reported that there is asubstantial difference in test results among Cystatin C assays from differentmanufactures (the slope of correlation curves varies from 0.88 - 1.11). The currentstudy indicates in conjunction with such previous findings that there can be 53 - 73mL/min discordance in estimating GFR by the use of different Cystatin Cassays.Conclusions: Cystatin C was demonstrated as the most sensitive and specificbiochemical marker estimating GFR in this study. Once an international referencepreparation becomes available and a common equation for eGFR is established,Cystatin C will be further valuable tool for diagnosing and monitoring kidney status.

Multiplexing combined with immuno-mass spectrometry: An

approach to simultaneous quantification of multiple analytes in

biological fluids

V. Kulasingam1, I. Batruch2, C. R. Smith2, T. Martz3, D. A. Jeffery3, A.Buckler3, T. Rezai4, M. F. Lopez4, E. P. Diamandis5. 1University of Toronto,Toronto, ON, Canada, 2Mount Sinai Hospital, Toronto, ON, Canada,3Novartis Institutes for BioMedical Research, Inc., Cambridge, MA,4Thermo Fisher Scientific, Cambridge, MA, 5University of Toronto; MountSinai Hospital, Toronto, ON, Canada

Currently, a major bottleneck in the verification phase of putative biomarkers is the lackof methods/reagents to quantify low levels of analytes in biological fluids. The aim ofthis study was to establish a high-throughput multiple reaction monitoring (MRM) assaycapable of quantifying 4 analytes simultaneously. HER-2/neu, IGFBP2, VEGF and IL-8were selected to demonstrate the feasibility of this approach since these molecules arepresent in low pg/mL to ng/mL range in serum. Digested human recombinant proteinsfor the 4 analytes were used to select proteotypic peptides as well as MRM transitionson the TSQ Quantum Ultra mass spectrometer (triple quadrupole, Thermo). PeptidesASPLTIEGR (m/z 472), LIQGAPTIR (m/z 484), FMDVYQR (m/z 479) andENWVQR (m/z 416) corresponding to HER-2/neu, IGFBP2, VEGF and IL-8,respectively, were used for quantification. An additional 1-2 peptides per protein wereused for verifying the identity of the protein. The experimental procedure consisted ofan immunoextraction step on a 96-well microtiter plate of the 4 analytes from a complexmixture. Specifically, each well on the plate was coated with 100 ng/well of each of fourmonoclonal antibodies (400ng/well in total), followed by immunocapture of theanalytes in the sample, trypsin digestion in-well, with subsequent MRM on the triplequadrupole mass spectrometer. In-house ELISA assays were developed for all 4analytes using commercially available reagents and used to validate the findings of theimmuno-MS approach. The amount of spiked "heavy" peptide for each analyte was keptconstant across the samples so that quantification was performed by generating acalibration curve using a ratio of the light:heavy peptide. This method demonstrated alimit of detection of 0.1 - 1 ng/mL with coefficient of variation < 20%. We conclude that

this methodology could be adapted to other candidates, thus providing a bridge betweenthe discovery and validation platforms for biomarkers. Furthermore, with thismethodology, there is the potential to multiplex commonly co-requested analytes inroutine clinical chemistry laboratories.

Liquid stable immunoturbidimetric assay for the measurement of

cystatin C on RX series analysers.

P. Armstrong, E. Donnelly, P. McGivern, J. Campbell, S. P. Fitzgerald.Randox Laboratories, Crumlin, United Kingdom

Background: Cystatin C is a small cysteine proteinase inhibitor that is steadilyproduced by all nucleated cells. The small molecular weight of cystatin C allows it tobe freely filtered by the glomerular membrane and therefore cystatin C levels in theblood are indicative of a normal or impaired glomerular filtration rate(GFR). TheGFR gives a good indication of renal function. Due to the strong link between kidneydysfunction and later cardiovascular problems, cystatin C has also been proposed forthe early detection of coronary heart disease, stroke and death in the elderly. CystatinC is broken down in renal tubular cells and is not secreted nor excreted by thekidneys. Therefore, levels of cystatin C in serum/plasma are almost entirelydependant on GFR. The use of methods enabling accurate determination of thiscompound is advantageous for the estimation of renal function in clinical andtherapeutic applications.Relevance: We report the development of a liquid stable immunoturbidimetric assayfor the measurement of cystatin C with an analytical range that allows determinationsin human serum/plasma samples without additional dilutions. The assay is applied tothe fully automated RX series analysers. This is of value as an accurate, stable andconvenient tool for determination of cystatin C using these automated systems.Methodology: The principle of the assay is immunoturbidimetric. A latexagglutination complex (read at 570nm) is found between cystatin C and latexparticles. The assay is applicable to the fully automated RX series analysers, RXDaytona and RX imola. These systems require 2.1µl of neat sample, generate the firstresult after 14 minutes and include dedicated software for data management. On-board and calibration stabilities were tested by storing two lots of reagent uncappedon the RX Series analysers for a period of 28 days. Stressing studies were also carriedout for the reagents, calibrators and controls. All were stored at 37oC for 3 weeks andthen performance was compared to fresh material. Within-run and total precision wereassessed by testing serum samples at defined medical decision levels, 2 replicatestwice a day for 11 days. A correlation was conducted with 40 serum samples usingcommercially available cystatin C assay.Results: The liquid assay reagent presents an on-board stability of 28 days atapproximately 80C and a calibration frequency of every 7 days. The liquid calibratorsand controls are stable to expiry at 2 - 80C.Evaluation of the performance parametersshows an assay sensitivity of 0.40mg/L and linearity up to 10mg/L. The within-runprecision and total precision for three different concentration levels (n=44) andexpressed as %C.V. were typically<6.5. Correlation with other commerciallyavailable assay generated the following linear regression equation: Y= 0.90x + 0.07; r=1.0. Conclusion: Data shows optimal analytical performance of the assay for themeasurement of cystatin C on the fully automated RX series analysers with the addedadvantage of the stability of the liquid reagent, calibrators and controls. This is ofvalue in the accurate determination of this analyte in human serum/plasma for clinicaland therapeutic applications.

Effectiveness of a predictive algorithm combining total Tau, p-Tau181,

Aββββ42 in CSF/plasma to discrimate early Alzheimer Disease from age-associated MCI and secondary dementias

A. SCOGNAMIGLIO1, M. Tondelli2, P. Nichelli3, T. Trenti1. 1ClinicalPathology Department - Nuovo Ospedale S.Agostino Estense,Baggiovara,Modena, Italy, 2Neuroscience Department- Nuovo OspedaleS.Agostino Estense, Baggiovara,Modena, Italy, 3Neuroscience Department- Nuovo Ospedale S.Agostino Estense, Baggiovara,Modena, Italy

Diagnostic biomarkers for Alzheimer Disease (AD) would be especially valuable asaids in the early diagnosis in the disease course, when correct diagnosis is difficult andwhen therapeutic drugs have the greatest effective potential. It is well known that totalTau protein, p-Tau protein (p-Tau181) and β-amyloid peptides (Aβ40 and Aβ42) inCSF/plasma are laboratory tests of moderate sensitivities and specificities when

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performed alone to diagnose AD. The strategy of combining the biomarkers for ADdiagnosis is appealing as the combination might result in increased sensitivity andspecificity. These markers may be useful to discriminate early or incipient AD fromage-associated memory impairment, depression, and some secondary dementias.The aim of the present study was to evaluate the role of total Tau, p-Tau (p-Tau181) β-amyloid peptides (Aβ40 and Aβ42) in CSF and β-amyloid peptides (Aβ40 and Aβ42)in plasma as combined biomarkers able to distinguish AD in patients when the clinicalpresentation required to differentiate AD from some problematic differentialdiagnoses, especially age-associated memory impairment, depressive pseudo-dementia, Parkinson’s disease, alcoholic dementia, etc.Analysis of CSF t-tau, p-tau and CSF/plasma b-amyloid (Aβ1-42) was performedusing quantitative enzyme-linked-immunoassay (ELISA) (InnotestInnogenetics,Ghent Belgium) applied on Biomeck 3000 (Beckman Coulter USA).Microsoft Excel Macro was set up to visualize the results obtained with theINNOTEST β-amyloid (1-42), INNOTEST hTau Ag, and INNOTEST PHOSPHO-Tau in cerebrospinal fluid. These Macro program, based on up to date literature,combine biomarker results to increase sensitivity and specificity for discriminatingAD from healthy controls and other neurologic disorders. The study protocol willfollow The STARD Iniziative-Towards Complete and Accurate Reporting of Studieson Diagnostic Accuracy Checklist.Results: CSF t-tau, p-tau and CSF/plasma b-amyloid (Aβ1-42) were determined on allconsecutive patients admitted to our neurological department for investigation ofcognitive disturbances when it was not possible a definitive diagnosis nevertheless theuse of clinical information and brain-imaging techniques i.e. SPECT and MRI. Thestudied patients were reviewed after approximately 1 year period and the revieweddiagnosis was the “diagnostic golden standard” used in this research, also in this case,when necessary were used brain-imaging techniques together clinical information to adefinitive diagnosis. 33 patients were evaluated, 15 female end 18 male aged 43- 84. 8out of 33 patients showed a full concordance between the laboratory biomarker indexand the AD ultimate diagnosis, 22 out of 33 patients showed a full agreement in theexclusion of AD between laboratory data and clinical final evaluation (the mostcommon diagnosis performed were: Parkinson’s atypical disease, depression,frontotemporal dementia, normal pressure hydrocephalus). 3 out of 33 patientsshowed laboratory data not in agreement with the clinical finding, however up to nowit was not possible a definitive diagnosis. In conclusion, in this study, nevertheless thelimited but selected patients, the biomarkers for Alzheimer Disease diagnostic use,when combined by the means of a predictive algorithm, exhibit effectiveness in thediagnosis of early AD and they have the potential to help to differentiate AD fromsome problematic differential diagnoses.

Evaluation of the VITROS® 5600 Integrated System for IgA, IgG and

IgM determination in serum

J. Teixeira, A. Denooz, J. P. Chapelle. University of Liège, Liège, Belgium

Background : The VITROS® 5600 Integrated System (Ortho Clinical Diagnostics)merges five technologies (MicroSlide, MicroTip, MicroWell, MicroSensorTM andIntellicheck®) into a single system, integrating a menu of chemistry,immunodiagnostic and infectious disease assays for both routine and STAT testing.Objective : We evaluated the performance of the system for measuringimmunoglobulin (Ig) concentrations in serum by immunoturbidimetry.Methods : IgA, IgG and IgM concentrations were determined according to the assayprotocols on the VITROS 5600 System. The comparison method wasimmunonephelometry on the BN II analyser (Siemens Diagnostics). The evaluationwas performed using CLSI protocols.Results : The new immunoglobulin assays showed excellent precision at allconcentration levels on serum pools and OCD controls. Intra-assay CVs ranged from1.30 to 2.13 %. Inter-assay CVs were 1.67 - 3.55 % for IgA at concentrations from 97to 687 mg/dL, 1.80 - 3.83 % for IgG (384 - 1850 mg/dL) and 1.79 - 3.92 % for IgM(40 - 227 mg/dL). Determination of successive dilutions of high concentrationsamples (n = 10) in kit buffer showed excellent agreement between expected andmeasured values (r² > 0.99 for the three Ig). The correlation with the comparisontechnique (r² = 0.99 for IgA, 0.98 for IgG, 0.99 for IgM) was assessed from dataobtained on 90 patient samples covering the whole concentration range (up to 948 mg/dL for IgA, 2210 mg/dL for IgG, 292 mg/L for IgM). Serum concentrations > 800 mg/dL (IgA), 2700 mg/dL (IgG), 400 mg/dL (IgM) are automatically diluted by theVITROS 5600. We verified that values exceeding these limits (up to 1050 mg/dL forIgA, up to 3200 mg/dL for IgG and up to 575 mg/dL for IgM) were correctlyprocessed by the system.Conclusions : These results indicated that the VITROS 5600 has excellent analyticalperformance for immunoglobulin determinations in serum.

Urinary KIM-1: a Novel Sensitive Marker of Renal Tubular Damage

for Confidence in Safety

A. McGuinty, P. Cleall, R. Allavena, E. Horsley, D. Brees, A. Rossi, R.Walley, S. Sultana. Pfizer, Sandwich, United Kingdom

INTRODUCTION: There is a need for early and more sensitive markers of kidneydamage. Traditional markers, serum urea and creatinine lack sensitivity to mildkidney injury leading to a delay in diagnosis and treatment. Kidney Injury Molecule-1(KIM-1) is elevated in urine in response to proximal tubular damage due tonephrotoxicity and ischaemia. In order to assess the utility of Kim-1 as a translatablebiomarker of kidney injury we validated a pre-clinical assay using a rat model ofnephrotoxicity and tested a novel assay for human KIM-1 in healthy male volunteers.METHODS: Pre-clinical study -A novel therapeutic known to cause tubulardegeneration and necrosis in rats was administered at doses of 0, 50 and 250 mg/kg/day for 2 weeks (n=10/group) followed by a 2 week and 4 week (n=5/group) recovery.Urine samples from various time points were analyzed for Kim-1 levels with acommercially available ELISA assay using Mesoscale Discovery (MSD) technology.Clinical stud y- 28 healthy males [21-54 yrs] volunteered in a non-drug study eachproviding 4 urine samples over 4 days. Urinary KIM-1 levels were measured with aprototype ELISA assay using MSD technology, and urinary creatinine measured onthe Advia 1650 analyzer.RESULTS: Pre-clinical study - Prior to study start the rat Kim-1 assay was validatedwith results as follows: Linear Range 0.006 - 2.59 ng/ml, Precision (Intra = 5.0%,Inter = 9.5%), Recovery = 88.5 - 94.2%. A dose related increased incidence of renaltubular degeneration/necrosis was observed after 14 days of dosing. The microscopicfindings were associated with statistically significant (p<0.05) dose related increasesin urinary Kim-1 of up to 4.5 fold (day 4) in 250 mg/kg group, and to 2.5 fold (day 1)in 50 mg/kg group. In contrast, no changes were noted in the traditional biomarkers,serum creatinine and urea over the same time course. During the recovery phase, nomicroscopic findings were observed at 2 or 4 weeks; indicating recovery. Recoverycould also be monitored with Kim-1, as levels returned to baseline shortly aftercessation of treatment (day 18).Clinical study - Prior to study start the human KIM-1 assay was validated with resultsas follows: Linear Range 0.0365 - 10.0 ng/ml, Precision (Intra = 5.4%, Inter = 15.2%),Recovery = 104.3 - 121.8%. In the healthy male volunteer study, the geometric meancreatinine-corrected urinary KIM-1 was 0.426 ng/mg, a range of 0.113 ng/mg -1.182ng/mg. Based on published data, a cutoff of 1.6 ng/mg was established, giving 100%specificity for these data.CONCLUSION: In the rat study Kim-1 correlated well with histopathology, provinga predictive and sensitive marker of renal proximal tubular damage and recovery. Thenovel assay for human KIM-1 performed well in healthy volunteers. Further work isneeded to establish assay performance in patients with acute renal injury and chronickidney disease but these data along with published literature suggest utility in clinicalmonitoring for acute proximal tubular damage. The findings give greater confidencein renal safety monitoring, allowing the clinical assessment of compounds known tocause preclinical nephrotoxicity.

Quantification of C-reactive protein in human serum with affinity

purification and isotope-dilution tandem mass spectrometry

E. L. Kilpatrick1, W. Liao2, I. V. Turko2, J. E. Camara2, N. G. Dodder2, D.M. Bunk2. 1National Institute of Standards and Technology, Charleston,SC, 2National Institute of Standards and Technology, Gaithersburg, MD

Objective: Characterize the performance of affinity purification coupled with isotope-dilution tandem mass spectrometry in the quantification of low abundance proteins inserum.Relevance: Certified reference materials are presently not available for C-reactiveprotein (CRP) at normal and moderately elevated levels primarily due to the lack ofdefinitive methods. This study advances the development of a higher order methodnecessary to generate future reference materials with levels better matched to clinicaldiagnostic cut-off points, thereby reducing issues related to commutability andincreasing interlaboratory harmonization.Methodology: The coding sequence for mature recombinant C-reactive protein (rCRP)was inserted into a vector with a hexahistidine leader sequence and expressed inArcticExpress (DE3) strain of E. coli with media containing 15N-ammonium chlorideas the sole nitrogen source. 15N-labeled rCRP (15N-rCRP) was purified sequentially byimmobilized metal affinity and phosphatidylcholine binding with a yield of

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approximately 60 micrograms. Tosyl-activated superparamagnetic polystyrene beadswere conjugated with monoclonal antibody against CRP and incubated with humanserum spiked with either 15N-rCRP alone or with 15N-rCRP and a 5 fold spike ofpurified human CRP. Following elution, the CRP was trypsin-digested and the proteinquantified by analyzing five tryptic peptides by isotope-dilution tandem massspectrometry with both external and standard addition calibration . The serum sampleanalyzed was a commercially available stock commonly used for control purposes with~1.6 mg/L concentration ( n = 3) as determined by the manufacturer byimmunoturbidimetric assay.Validation: Protein purity of 15N-rCRP was greater than 99% as assessed by sodiumdodecyl sulfate - polyacrylamide gel electrophoresis. Analysis by matrix-assisted laserdesorption ionization mass spectrometry indicates that the principle product has a massconsistent with CRP with 15N incorporation along with the hexahistidine leadersequence. Incorporation as a percent of total nitrogen was estimated to be greater than99% as determined by isotopic profile analysis. CRP binding was linear with r2regression coefficient > 0.99. Mixtures of CRP and 15N-rCRP in specific ratiossubjected to affinity purification, digestion and LC-MS/MS analysis were linear (r2 >0.99) demonstrating similar binding and digestion characteristics. External calibrationcurves for quantification of CRP in serum were linear (r2 > 0.99) and a produced valueof 1.3 mg/L (%CV = 19%). Standard addition calibration produced a similar value (1.3mg/L (%CV = 18%)).Conclusions: The incorporation of isotope-dilution mass spectrometric quantificationinto the affinity purification of CRP from human serum requires the generation of astable-isotope labeled internal standard. The present study demonstrated the generationof 15N-rCRP intact protein of sufficient purity, label incorporation and quantity toperform small scale analysis of CRP in human serum utilizing affinity purification incombination with isotope-dilution tandem mass spectrometry. This technique will play asignificant role in the future development of higher order methods for the measurementof CRP and other low abundance proteins in serum. Present work is focused on thedevelopment of a yeast protein expression system in order to increase yields andgenerate recombinant protein structurally identical to native forms.

Evaluation of a New Olympus CRP Assay on the Olympus AU400TM,

AU640 TM, AU2700 TM and AU680TM Immunochemistry

Analyzers.

M. Burkhard1, M. D. McCusker1, L. O'Gorman1, P. Turley2, S. Mellen2, T.Bertsch3, C. Aschenneller3. 1Olympus Life Science Europa GmbH (IrishBranch), O'Callaghan's Mills, Ireland, 2York Hospital, York, UnitedKingdom, 3Klinikum Nurnberg, Nurnberg, Germany

CRP is one of the most sensitive acute-phase reactants providing information aboutacute and chronic inflammatory processes. CRP levels in serum can be elevated toorders of magnitude above the normal level (<5mg/L) following infection, myocardialinfarction, trauma, inflammation or surgery.The new CRP Latex assay, OSR61157, provides a dynamic assay range from 4-480mg/L.This assay principle utilizes sample mixed with R1 buffer and R2 reagent containinglatex material coated with anti-human CRP antiserum. Turbidimetric absorbance[600(660)nm] of complexes formed during the antigen-antibody reaction is measuredat 37oC for approximately 5 minutes. The reaction is a calibrated spline method,utilizing 6 liquid stable calibrators (ODC00032) traceable to ERM®-DA470.Prozone hook effect does not occur until >750mg/L. Imprecision CV values are <5%(4-10 mg//L) and <3% (10-480 mg/L) for within run and <5% total CV for allanalysers tested using NCCLS EP5-T2. Interferences measured at app10 mg/L are asfollows: lipemic and haemolytic <5% (1000mg/L Intralipid, 5g/L hemoglobin), icteric<10% (684 µmol/L bilirubin) (NCCLS EP7-P). Reagent displays 90 day on boardstability with open claim recalibration. Two comparisons with current Olympus CRPreagents were performed: OSR6147 in York District Hospital, UK and OSR6199 inKlinikum Nürnberg, Germany. The following table shows the results of thecomparison of the assay on AU2700.

Tissue-based Quality Control Materials for Tests of the Intestinal

Disaccharidases

J. Lu1, D. G. Grenache2. 1ARUP Institute for Clinical and ExperimentalPathology, Salt Lake City, UT, 2Department of Pathology, University ofUtah Health Sciences Center, Salt Lake City, UT

Background: Carbohydrate malabsorption is due to a deficiency of one or moreintestinal disaccharidases. Disaccharidase activity is determined by measuring theglucose liberated from a disaccharide substrate after incubation of the substrate with ahomogenate made from a biopsy of duodenal mucosa. The lack of tissue-based qualitycontrol material is a significant limitation for clinical laboratories that performintestinal disaccharidase tests.Objective: To develop tissue-based quality control materials for intestinaldisaccharidase tests.Methods: Duodenal mucosa from freshly slaughtered adult pigs was utilized.Samples (~5 mg) were homogenized in 500 µL of saline for 2 minutes using a BulletBlenderTM (Next Advance, Inc., Averill Park, NY). Individual substrate solutions (56mM lactose, maltose, palatinose, and sucrose) were prepared in 0.1 M sodium maleatebuffer, pH 6.0. Equal volumes (30 µL) of homogenate and each substrate werecombined and incubated at 37°C for 60 minutes, with the exception of maltose, whichwas incubated for 30 minutes. Following incubation, the enzyme reactions werestopped by heating at 100°C for 5 minutes. The Roche Cobas c501 analyzer (RocheDiagnostics, Indianapolis, IN) was used to measure both glucose (hexokinase) andtotal protein (benzethonium chloride). Enzyme activities were expressed in units ofµmol/min/g protein. Unheated tissue samples (N=15) were used as baseline controls.Test samples were incubated at variable temperatures for 5-10 minutes prior tohomogenization. The percent of remaining activity in the test samples was calculatedrelative to that of baseline controls.Results: Shown in the table below.

Conclusions: Sucrase is the most heat-labile and palatinase is the most heat-stable ofthe 4 enzymes. Porcine mucosal samples can be heated prior to homogenization tocreate a low-activity quality control material while unheated tissue can serve as a“normal” activity control.

Validation of the SEBIA Mini-Capillary System (MINICAP) for

Serum Protein Electrophoresis and Hemoglobinopathy Analysis

R. L. Fitzgerald1, C. B. Alday2, E. A. Coker2, D. A. Herold1. 1VAMC/UCSD, San Diego, CA, 2VAMC, San Diego, CA

Objective: SEBIA has recently developed a mini-capillary system that uses twocapillary electrophoresis (CE) columns which is designed for the analysis of serumproteins and hemoglobin variants in mid sized laboratories. The objective of this studywas to compare the CE system for the analysis of serum proteins and hemoglobinvariants with gel electrophoresis and high performance liquid chromatography,respectively. Relevance: Analysis of serum proteins is performed to confirm a varietyof disease states but is primarily used as a screen for monoclonal populations ofimmunoglobulins. Hemoglobinopathy analysis is performed to identify thalassemiasas well as structural variants. Methodology: For CE protein analysis, serum wasloaded onto the SEBIA Minicap, diluted with buffer, and injected into the capillaries.Migration occurs for 4 minutes. Proteins are detected at 200 nm. The data systemcalculates each fraction; albumin, alpha 1, alpha 2, beta 1, beta 2 and gamma. Beta 1and beta 2 were combined as one fraction using a manual overlay with the referencepattern. For proteins, the comparison method was Hydrasys gel electrophoresis. ForCE hemoglobinopathy analysis, red blood cells from EDTA anticoagulated blood areloaded into the mini-capillary system, hemolyzed, and injected into the capillaries.Migration takes place for 8 minutes and hemoglobin is detected at 415 nm.Hemoglobin variants are tentatively identified based on component migration patterns

Method Comparison OSR6147 OSR6199 NA

Test methodOSR61157

(new assay)OSR61157

(new assay)Range (mg/L) 5-300 4 - 480Slope 0.990 1.014Intercept 2.1 mg/L 6.4 mg/LCorrelation coefficient 0.996 0.998Specimen type Serum LiHe plasmaNo. of samples 959 997

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at various temperaturesMean Activity of Unheated Tissue

(µmol/min/g protein)

50°C10 min

65°C10 min

70°C10 min

80°C10 min

100°C5 min

Lactase 47.2 146% 108% 31% 0% 0%Maltase 432.8 143% 63% 72% 20% 0%Palatinase 14.1 151% 96% 105% 17% 0%Sucrase 36.7 141% 20% 17% 0% 0%

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relative to reference windows. The comparative method for hemoglobinopathyanalysis was the Bio-Rad Variant. Validation: For proteins, the CE method was linearfor the 5 fractions (albumin, alpha 1, alpha 2, beta, and gamma) at concentrationstypically encountered in a clinical laboratory. For a patient with a monoclonalgammopathy, within and between run coefficient of variations of all fractions wereless than 5.5%. A paired t-test demonstrated that the CE and gel electrophoresissystem were not significantly different for quantifying alpha 2, beta or gammaregions, while albumin and alpha 1 were significantly different for 55 patient samples.On the CE system, albumin was lower and alpha 1 was higher as compared with gelelectrophoresis. Most importantly, all monoclonal bands detected by CE wereconfirmed by gel electrophoresis. For CE hemoglobinopathy analysis, the method waslinear for A2, S and C for concentrations typically encountered in a clinicallaboratory. Within run coefficient of variations of Hb A, F, S and C was < 4 % for CE.The run-to-run precision of Hb A2 was 2.9 %. A paired t-test on 10 AS traitspecimens demonstrated that CE reported significantly higher values for hemoglobinS. One hemoglobinopathy case had elevated hemoglobin F (6.9%) by HPLC and nodetectable hemoglobin F by CE. This specimen appeared to be a rare hemoglobinvariant, otherwise specimens from 42 patients run on the CE hemoglobinopathyprogram generally agreed well with HPLC. Conclusions: This is the first clinicalevaluation of the Sebia mini-capillary system for proteins and hemoglobin variantanalysis. Our results demonstrated comparable performance of CE with gelelectrophoresis and HPLC. For hemoglobinopathy analysis, the Sebia mini-capillarysystem provided complementary information for rare hemoglobin variants.

Evaluation of analytical performance of a novel immunoassay system

for determination of serum tartrate-resistant acid phosphatase type 5b

T. Miura1, Y. Igarashi2, Y. Mochizuki3, W. Kikuchi1, K. Ito1, K. Sasagawa1,R. Kojima1, K. Katayama1, F. Nomura4. 1Biochemical Laboratory, NittoBoseki Co., Ltd., Fukushima, Japan, 2Tsuchiya Children's Hospital,Saitama, Japan, 3Department of Obstetrics and Gynecology, DokkyoMedical University, Tochigi, Japan, 4Department of Molecular Diagnosis,Graduate School of Medicine, Chiba University., Chiba, Japan

Tartrate-resistant acid phosphatase type 5b (TRACP 5b) is a reliable bone resorptionmarker derived specifically from osteoclasts and its serum levels are not affected byfeeding and renal dysfunction. The development of TRACP 5b specific assays hasbeen complicated by the presence of the highly similar isoform TRACP 5a which isderived from macrophages and an abundant inactive TRACP 5b fragments in serum.Recently we have developed a new ELISA which detects the enzyme activity ofTRACP 5b specifically based on a novel assay system called fragments absorbedimmunocapture enzymatic assay (FAICEA, OsteolinksTRAP-5b®: Nitto Boseki Co.,Ltd, Fukushima, Japan). The assay uses two different monoclonal antibodies, Trk49and Trk62, generated with immunization of TRACP 5b from human bone. Theantibody Trk49 is highly specific to inactive TRACP 5b fragments. In contrast theTrk62 is highly specific to intact active TRACP 5b. The microwell plate coated withboth antibodies is used for the assay. At the immunoreaction, the Trk49 binds inactiveTRACP 5b fragments firstly, thereby making Trk62 more available to bind activeTRACP 5b in reaction mixture. After the immunoreaction, binding TRACP 5bactivity is measured by 2-chloro-4-nitrophenyl phosphate (CNPP) as a substrate. TheCNPP is more specific substrate for TRACP 5b than traditional substrate, 4-nitrophenyl phosphate (PNPP). In this study, we examined analytical performance ofthe FAICEA system. Then the influences of TRACP 5a and inactive TRACP 5bfragments for FAICEA were compared with that for another commercially availableimmunoassay kit for TRACP 5b (BoneTrap®: IDS, Boldon, UK). Sensitivity: Theminimum detection limit of the FAICEA is 0.1 U/L, determined by the upper 3 SDlimit in a zero standard precision study. Recovery: Spike recovery was determined byadding a known quantity of purified TRACP 5b to serum samples with different levelsof endogenous TRACP 5b. The recovery was 92-103%. Precision: Intra-assay wasdetermined for 8 replicate of 2 sera. Inter-assay was determined for 8 replicate for7days for 2 sera. The CVs of Intra-assay were 2.0% and 2.2%. The CVs of inter-assaywere 1.6% and 1.9%. Assay Range: The assay range of FAICEA is 0.1-15 U/L. In thespike test of TRACP 5a into serum, increasing amounts of purified TRACP 5a wasadded to normal human serum. The FAICEA measurements were not affected byincreasing TRACP 5a levels. In contrast, TRACP 5b levels measured by BoneTrap asincreased by 67%. To investigate possible inactive fragment interference, serumsamples (three TRACP levels) were diluted serially with saline. The FAICEAdemonstrated excellent linearity (93-102% recovery for highest dilutions), whereasBoneTrap measured highly increased values after dilution (127-164% recovery forhighest dilutions), suggesting fragment interference in the undiluted samples. In

conclusion, the FAICEA for detection of TRACP 5b demonstrates good analyticalperformance and less influences of TRACP 5a and inactive TRACP 5b fragmentscompare to the other immunoassay kit.

Phenotyping and Quantitation of αααα-1-Antitrypsin by LC-MS/MS: ANovel Methodology for the Laboratory Evaluation of αααα-1-AntitrypsinDeficiency

M. Snyder, Y. Chen, L. M. Benson, J. A. Katzmann, H. R. Bergen. MayoClinic, Rochester, MN

Background: α-1-antitrypsin (A1AT) is the primary inhibitor of neutrophil elastase inthe lungs, functioning to prevent tissue damage that would result from uncontrolledprotease activity. Mutations in the A1AT gene lead to decreased concentrations of theprotease inhibitor, resulting in A1AT deficiency disease. The two most commonalleles associated with A1AT deficiency are the S and Z alleles. Diagnosis of thisdisorder requires quantitation of A1AT in conjunction with identification of thespecific deficiency alleles. The current algorithm of laboratory testing for A1ATdeficiency uses a combination of quantitation, genotyping, and/or phenotyping. Here,we describe a novel multiplex liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the quantitation of A1AT and identification of the specificdeficiency alleles.Method: Peptides containing the common S and Z deficiency alleles, as well as aninvariant peptide, were identified by LC-MS/MS of a purified A1AT tryptic digest.Each of the identified peptides was synthesized in unlabeled and 13C/15N-labeledforms. Serum samples (n=18) were denatured and digested with trypsin. Afteraddition of labeled internal standard peptides, the digested plasma samples wereanalyzed by LC-MS/MS on a Finnigan TSQ Quantum Ultra mass spectrometer. TheS, Z, and wild-type alleles were identified based on detection of the specific peptidesand comparison to the internal standard. The results were compared to the currentphenotyping assay performed by isoelectric focusing (IEF) electrophoresis using theHydragel 18 A1AT Isofocusing system (Sebia) according to the manufacturer’sinstructions. For quantitation, varying concentrations of unlabeled invariant peptidewere used to construct a standard curve. For each sample, the ratio of the invariantpeptide peak area to the internal standard peak area was calculated, which was thencompared to the standard curve. The quantitation results obtained by LC-MS/MSwere compared to those determined by immunonephelometry as performed on a DadeBehring II nephelometer according to manufacturer’s instructions.Results: For the A1AT allele identification, in 17 out of the 18 samples, the LC-MS/MS results were identical to those obtained by IEF gel electrophoresis, resulting in aconcordance of 94%. The single discrepant result was due to administration ofreplacement therapy to a patient with known A1AT deficiency. A1AT quantitationwas assessed for precision and accuracy. The inter- and intra-assay CVs were <5% for2 peptide standards and <10% and <5%, respectively, for 3 serum samples. To assessaccuracy, quantitation of the serum samples by LC-MS/MS was compared to thecurrent nephelometric assay. This comparison demonstrated a linear relationship, witha correlation coefficient of 0.93 and a slope near unity.Conclusion: We have developed a novel multiplex LC-MS/MS method for A1ATquantitation and allele identification. This method demonstrates excellent correlationwith current phenotyping and nephelometric assays and is analytically robust. It is apromising method that offers numerous improvements over the current testingmethodologies used for the diagnosis of genetic A1AT deficiency.

Changes in serum free light chain values do not precede changes in M-

spike in a series of patients with relapsed/refractory intact

immunoglobulin multiple myeloma

S. N. Uljon1, P. G. Richardson2, C. Grudzien1, P. H. Schur1, M. J.Tanasijevic1, N. I. Lindeman1. 1Brigham and Women's Hospital, HarvardMedical School, Boston, MA, 2Dana-Farber Cancer Institute, HarvardMedical School, Boston, MA

BACKGROUND: Serum free light chains (SFLC) are used to diagnose and followmultiple myeloma, particularly for patients with light chain or hyposecretory disease.SFLC may also, however, be useful in monitoring patients with intactimmunoglobulin multiple myeloma (IMM), where the short circulating half-life offree light chains relative to intact immunoglobulin may make the SFLC test valuableas an earlier indicator of treatment response than quantification of M-spikes from

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scanned serum protein electrophoresis (SPEP) gels or heavy chain nephelometrymeasurements. To test the hypothesis that the SFLC test detects changes in IMMdisease status before M-spike, we reviewed SFLC, M-spike, and heavy chain resultsobtained from individual patients at multiple time points during the course of theirdisease. Further, we tested the specificity of the SFLC assay for free light chains in thepresence of abundant intact monoclonal immunoglobulin, as is seen in patients withIMM.METHODS: 630 samples from 17 patients with relapsed/refractory myelomaparticipating in a phase I clinical trial were reviewed; mean follow-up was 24 mos(range, 8-36 mos). Each patient’s SFLC, M-spike, and heavy chain results werereviewed along with bone marrow biopsy, medication records, and clinical notes.SFLC assay specificity was assessed by direct measurement of intact IgG-kappaimmunoglobulin and by spike-and-recovery studies with normal serum; intact IgG-kappa was purified from pooled sera from 12 patients with IMM. SFLC and heavychains were measured on a Siemens BN2 analyzer; Helena SPIFE 3000 was used forSPEP.RESULTS: 15/17 (88%) cases showed M-spike, total heavy chain, and SFLCmeasurements that rose and fell in near-synchrony throughout the patient’s course. Inone patient, changes in serum free light chain preceded changes in M-spike and heavychain by an average of 3 mos, while in another patient, SFLC results lagged behindthe M-spike and heavy chain measurements by 1 mo. In eight patients, the SFLCconcentrations showed short-term fluctuations in the absence of correspondingchanges in M-spike, heavy chain, or clinical treatment or status. Specificity studiesdemonstrated minimal (<0.5%) cross-reactivity of the free light chain assay for theintact IgG-kappa immunoglobulin.DISCUSSION: In 16 of 17 refractory myeloma patients tested over ~2 yrs, SFLCmeasurements did not show changes earlier than M-spike measurements other thanshort-term fluctuations that reversed on subsequent measurement and that did notcorrelate with other laboratory values or changes in treatment or clinical condition.The synchrony between the SFLC and M-spike results was not attributable to cross-reactivity in the SFLC assay, which showed >99% specificity for free light chains inthe presence of abundant intact immunoglobulin. For practical clinical management inthis population of patients with refractory/relapsed IMM, SFLC measurements did notadd value as an adjunct to conventional SPEP with M-spike quantification.

CYSTATIN C AND MDRD ESTIMATED GLOMERULAR

FILTRATION RATE DIFFER DURING NORMAL PREGNANCY

A. Larsson1, L. Hansson2, M. Palm3, O. Axelsson3. 1Department ofMedical Sciences, Clinical Chemistry, Uppsala, Sweden, 2LaboratoryMedicine, Karolinska Institute, Sweden, 3Department of Women’s andChildren’s Health, Obstetrics and Gynecology, Uppsala, Sweden

Background. Estimation of the glomerular filtration rate (eGFR) is used to monitorpatients with suspected kidney disease and to optimize the dosage of drugs that areeliminated by the kidneys. Plasma creatinine and cystatin C are the two most widelyused GFR markers, Both markers are recommended to be reported as estimated GFRbut there is a lack of reference intervals for pregnant females.Methods. We have studied creatinine (eGFRMDRD) and cystatin C (eGFRcystc)estimated GFR during 52 normal pregnancies from pregnancy week ten to deliveryand postpartum. Each woman was sampled repeatedly and the samples were groupedaccording to gestational age into the following periods: week 7 - 16; week 18 - 24;week 24 - 28; week 28 - 31; week 31 - 34; week 34 - 38; -2 - 0 weeks prior to deliveryand postpartum (>6 weeks after delivery). The 2.5 and 97.5 percentiles for thesemarkers were calculated according to the recommendations of the InternationalFederation of Clinical Chemistry on the statistical treatment of reference values.Results. In healthy pregnant females eGFRcystc was higher in the first two trimestersand lower prior to delivery in comparison with eGFRMDRD. eGFRcystc and eGFRMDRDgive different results. No significant correlations between the two estimates werefound in any of the time groups.Conclusions. It is important to distinguish between the two GFR estimates and useseparate gestational specific reference intervals for pregnant females.

SIGNIFICANT DIFFERENCES WHEN USING CREATININE,

MDRD OR CYSTATIN C FOR ESTIMATING GLOMERULAR

FILTRATION RATE IN ICU PATIENTS

M. Lipcsey1, M. Furebring2, L. A. Hansson3, S. Rubertsson4, A. Larsson5.1Section of Anaesthesiology & Critical Care, Department of SurgicalSciences Uppsala University Hospital, Uppsala, Sweden, 2Sections ofInfectious Diseases, Department of Medical Sciences, Uppsala UniversityHospital, Uppsala, Sweden, 3Laboratory Medicine, Karolinska Institute,Stockholm, Sweden, 4Section of Anaesthesiology & Critical Care,Department of Surgical Sciences, Uppsala University Hospital, Uppsala,Sweden, 5Clinical Chemistry, Department of Medical Sciences, UppsalaUniversity Hospital, Uppsala, Sweden

Background. Renal dysfunction is associated with increased morbidity and mortalityin intensive care patients. In most cases the glomerular filtration rate (GFR) isestimated based on serum creatinine and the Modification of Diet in Renal Disease(MDRD) formula but cystatin C estimated GFR is being used increasingly. The aim ofthis study was to compare creatinine and MDRD and cystatin C estimated GFR inintensive care patients (ICU).Methods. Retrospective observational study was performed, on patients treatedwithin the general ICU during 2004-2006, in a Swedish university hospital.Results. GFR markers are frequently ordered in the ICU. Ninety-two percent of thepatient test results had Cystatin C estimated GFR (eGFRCystatin C) ≤ 80 mL/min/1.73m2, 75 % had eGFR ≤ 50 mL/min/1.73 m2 and 30 % had eGFR ≤ 20 mL/min/1.73 m2.In contrast, only 46% of the patients had reduced renal function assessed by plasmacreatinine alone and only 47% had eGFRMDRD ≤ 80mL/min/1.73 m2. The meandifference between eGFRMDRD and eGFRCystatin C was 39 mL mL/min/1.73 m2 foreGFRCystatin C values ≤ 60 mL/min/1.73 m2.Conclusions. GFR is commonly assessed in the ICU. Cystatin C estimated GFRyields markedly lower GFR results than plasma creatinine and eGFRMDRD. Manydrugs are eliminated by the kidneys and their dosage is adjusted for kidney function.Thus, the differences in GFR estimates by the methods used indicate that the GFRmethod used in the intensive care unit may influence the treatment.

The effect of the acute phase response on ceruloplasmin concentrations

P. J. Twomey, C. A. Hockings. The Ipswich Hospital, Ipswich, UnitedKingdom

Introduction: Wilson’s Disease (WD), also known as hepatolenticular degeneration,is an inborn error of copper metabolism affecting approximately 1 in 30,000 livebirths. A significant reduction in the ATP7B P-type ATPase activity results indecreased excretion of copper into bile by hepatocytes leading to hepatic injury andcopper deposition in the brain, cornea and kidneys. An additional consequence is thefailure to incorporate copper into ceruloplasmin, reducing its half-life in plasma.Accordingly, serum ceruloplasmin concentrations are often employed to screen forWD. However, ceruloplasmin is also an acute phase response protein. The objectiveof this study was to use routine clinical data to determine the relationship betweenceruloplasmin and C-reactive protein (CRP).Methods: Using the laboratory information system, all serum requests that had bothceruloplasmin and CRP analysed were retrospectively obtained since this laboratoryintroduced in-house ceruloplasmin. Requests for patients <18 years of age wereexcluded as were subsequent requests for the same individual patients. Bothceruloplasmin and CRP were analysed on an AU2700 (Olympus Diagnostica, CountyClare, Ireland) using Olympus reagents in line with the manufacturer’s instructions.Least squares linear regression analysis was carried out using MS Excel to determinethe exact relationship (slope, intercept and correlation co-efficient) betweenceruloplasmin and CRP. The t-distribution was employed to calculate the p-value forthe correlation co-efficient.Results: There were 312 result sets that met the criteria. The linear regressionequation was ceruloplasmin = 0.4706 x CRP + 260.56 with r2 = 0.0743. The p-valuefor r was <<0.001.Discussion: The main desirable attribute of a screening test is a high clinicalsensitivity, that is, not to miss a diseased patient. An acute phase response is seenmuch more frequently in routine clinical practice than a WD case. Patients withabnormal liver function tests are often tested for ceruloplasmin to exclude WD, manyof whom will have an inflammatory response. The relevant clinical question is - what

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is the chance that an acute phase response will result in a patient’s ceruloplasmingoing from below the lower reference interval to above it? A CRP of 22 mg/L wouldbe associated on average with a ceruloplasmin about 10 mg/L higher than if the CRPwas 1 mg/L. Therefore, many patients with concurrent viral infections or minor injurywill have their serum ceruloplasmin concentrations increased by approximately 10 -20 mg/L. A CRP of 150 mg/L would be associated on average with a ceruloplasminabout 70 mg/L higher. While patients with such CRP values are not common, they aremore common the patients with WD. Thus, the acute phase response may result insome WD patients whose “healthy” level is just below the lower reference limithaving a normal serum ceruloplasmin concentration due to an acute phase response.Conclusion: Laboratories should consider measuring CRP with serum ceruloplasminrequests to increase the clinical sensitivity of ceruloplasmin for the detection of WD.



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