Innovation & Information

1. Issue 2012

Immundiagnostik AG • Stubenwald-Allee 8a • 64625 Bensheim • Germany • Tel.: +49 (0) 62 51/7 01 90-0 • Fax: +49 (0) 62 51/84 94 30 • info@Immundiagnostik.com • www.Immundiagnostik.com

PhiCal® Calprotectin –

no test is better!Test result „BRAVO“ in the latest inter-laboratory trial of Instand e.V. (March 2012)

(page 1)

MRSA-PCR Now with corresponding lysis buff er – convenient and quick analysis (page 2)

PSEUDOMONAS AERUGINOSAPCR for the determination of intestinal bacteria (page 2)

HISTAMINE INTOLERANCE New ELISAs analyze the concentration of the neurotransmitter (page 2)

INSULIN RESISTANCE We off er a comprehensive biomarker-package for routine & research (page 3)

New: Med-Science team in BerlinImmundiagnostik establishes offi ce in Berlin)(page 7)

Lab-Tip: Multiple dilutions ofstool samples (page 7)

Faecal Calprotectin

Leading Test!

One step ahead in the standardization of 25(OH) vitamin Dstatus determination (page 4)

New avenues in LC-MS/MS laboratory diagnosticsCooperation with instrument manufacturers (page 6)

PhiCal® Calprotectin ELISA in Abbott‘s FIRE-study (page 6)

ID-products in use25(OH) vitamin D from dried blood spots as well as calprotectin and myostatin (pages 3-5)

NEW ON THE MARKET

i2

Break-through: oxPTH analysis Determination of OxPTH enables for the first time adequate PTH monitoring in dialysis patients (page 6)

i2 Innovation & Information

Faecal Calprotectin

Leading Test!

„For the same sample, the Bühlmann assay reports up to 3,8 times higher faecal calprotectin concentrations than the Immundiagnostik and Eurospital assays.“

(Whitehead SJ et al. (2012) Annal Clin Biochem 49(1):106-107)

While other commercial assays produce incorrect data due to standardization problems, the PhiCal® Calprotectin ELI-SA delivers precise, clinically correct values in an english interlaboratory trial (UK NEQAS) (Whitehead et al. 2012).

Precise analysis in international comparison

Superior in the discrimination of IBD and IBS

I m m u n d i a g n o s t i k ‘ s PhiCal® Calprotectin assay (K 6927) is the predominant assay in

the determination of faecal calprotectin for diagnostics and therapy monitoring of inflamm-atory bowel diseases. The excellent reliability of the test was rated „BRAVO“ in a recent German interlaboratory trial of Instand e.V.

New data from the Focus 2012 meeting confi rm the predomi-nance of our PhiCal® Calprotectin test in comparison to com-peting assays in the discrimination of infl ammatory bowel diseases (IBD) and irritable bowel syndrome (IBS) in clinical routine (Tomkins et al., 2012).

„Overall, PhiCal performed better than EK-CA in distinguishing IBS from active IBD using 50 µg/g cut-off “.

(Tomkins CR et al. (2012) Annal Clin Biochem 49(1):102-103)

Stabilization buff er enables faultless calprotectin determination

A crucial factor in the measurement of faecal calprotectin is the stability of the parameter. The calprotectin concentration falls signifi cantly in faecal samples after only one day, inde-pendent of storage conditions (e.g. room temperature, 4°C, or -20°C). An accurate determination of calprotectin is especially diffi cult in samples with high concentrations. The PhiCal® Cal-protectin assay employs a specifi c buff er which keeps calpro-tectin stable for several weeks at room temperature, thereby enabling a precise determination and an accurate clinical di-agnosis.

Stabilization of Calprotectin in Stool Samples at Room Temperature for more than 4 weeks

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Stablization successful over 4 weeks using our propriotory Immundiagnostik buffer ! ELISA better than automated rapid test

The clinical accuracy of the ELISA is superior to that of an automated rapid test, which produces more false data in direct com-parison (>10%, in a measurement range of 30-300 µg calprotectin/g stool), especially more false positive data.

(Wassell et al., 2012; Ann Clin Biochem 49:55-58)

Test result „Bravo“ in German interlaboratory trial

PhiCal® Calprotectin ELISA – no test is better!

1

„We will adopt the monoclonal assay from Immun-diagnostik as it is automated.”

(Srinivas et al., 2012, DDW abstract)

A British study assessed the signifi cance of calprotectin in the primary diagnosis of newly admitted patients in a clinic setting and in therapy monitoring of patients with chronic infl ammatory bowel diseases. The analytical precision and cli-nical relevance of four commercial calprotectin ELISAs were compared. The PhiCal® Calprotectin ELISA achieved an excel-lent correlation with intestinal infl ammation and came out on top as automated test (Srinivas et al., 2012).

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MRSA-PCRNow with correspon-ding lysis buff er for a convenient and quick analysis

MutaCLEAN® PLUS (KG1036)MutaREX® MRSA (KG290396)MutaPLEX® MRSA (KG190396)

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for many diseases that are difficult to treat. The bacterium is resistant against beta-lac-tam antibiotics (e.g. Penicillin) as well as against cephalosporins and is therefore a serious threat to hospitals and nursery homes.

Our MutaPLEX® & MutaREX® MRSA real time PCR kits have specifically been de-signed for the direct MRSA-identification

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NEW ON THE MARKET

PSEUDOMONAS AERUGINOSA-PCRfor the analysis of in-testinal bacteria groups

MutaPLATE® Pseudomonas aeruginosa (KE19006)

The PCR test enables the quantitative determination of the eubacteria group Pseudomonas aeruginosa in faecal samples for the discrimination of irritable bowel syndrome (IBS) and inflammatory bowel disease. Pseudomonas aeruginosa is indicative for IBS since it proliferates in a peak-like manner in IBS-patients in contrast to healthy subjects. Furthermo-re, the role of Pseudomonas aeruginosa in the development of the leaky-gut-syndrome is a matter of current research.

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HISTAMINEINTOLERANCENovel test panel ana-lyses the status of the signalling molecule

Histamine (Stool) (K 8213)Histamine (EDTA-Plasma, Urine) (K 8212)DAO ELISA (Serum, Stool) (K 8500)DAO REA 3H (Serum) (K 8220)

Many people suff er from histamine indu-ced food intolerance, with corresponding symptoms such as migraine headache, digestive problems, irritation of the nasal mucosa or other allergy-like symptoms. The enzyme diamine oxidase (DAO) de-grades histamine and is often impaired in these patients. Our new histamine ELISAs and our DAO-tests now enable a comprehensive analysis of histamine me-tabolism in diff erent matrices. This tool box has been designed for routine dia-gnostics of histamine intolerance as well as for research on the function of hista-mine as a neurotransmitter.

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Innovation & Information i2

OSTEO-PROTEGERINNew ELISA determines the bone metabolism marker in just one day

OPG (Serum & Plasma) (K 1011)

Osteoprotegerin (OPG) is a bone prote-in, that is produced by a number of dif-ferent tissues and cell types including osteoblasts. The glycoprotein represents a negative regulator of bone resorption by acting as a decoy receptor for RANKL, thereby neutralising its function in osteo-clastogenesis.In addition to its specific protective role in bone formation, OPG appears to be involved in the development of vascu-lar calcification during the inflammatory events of atherosclerosis.The determination of OPG in serum is therefore useful in the research of vari-ous illnesses, such as osteoporosis, di-seases with locally induced bone resorp-

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tion activity, arthritis or cardiovascular pathologies. Furthermore, OPG has been identified together with other bone me-tabolism parameters as predicitive mar-ker for a progressive course of Morbus Bechterew. In this context, OPG is a po-tential candidate for therapy monitoring and optimization as well as for pharma-ceutical development.

Our new, CE-certified OPG-ELISA fea-tures a high sensitivity, is suited for small sample sizes (20 µl) and can conveniently be performed in just one day.

in clinical samples which contain coagu-lase-negative staphylococci as well as methicillin-resistant Staphylococcus aure-us. A preceding cultivation of the patho-gen is not necessary and the determina-tion takes less than two hours.

The MRSA analysis with the PCR kits MutaREX® MRSA & MutaPLEX® MRSA is now facilitated by the corresponding lysis buffer MutaCLEAN® PLUS.

The buffer enables a quick lysis of gram positive bacteria and is therefore ideal for MRSA sample preparation. An elaborate DNA extraction is not necessary anymo-re: The sample simply incubates in the ly-sis buffer for 15 min. before PCR analysis.

Find more products on our website

www.immundiagnostik.com/en/home/products/product-portfolio.html

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ID-PRODUCTS IN USE

DRIED BLOOD SPOT VITAMIN D ANALYSIS

25(OH) Vitamin D analysis from dried blood spots now clinically validated

To facilitate collection and shipping of blood samples for vitamin D status ana-lysis, Immundiagnostik has developed the D-Vital ID® test set for dried blood spots from capillary blood. This material can be mailed to a laboratory for ana-lysis via conventional couriers without cooling. Vitamin D determination of the D-Vital ID® samples is exclusively cou-pled to Immundiagnostik‘s 25-OH vita-min D ELISA. This market-proven assay delivers reliable vitamin D data which are comparable in their diagnostic accuracy to reference methods such as HPLC or LC-MS/MS.

The D-Vital ID® test set supplements Immundiagnostik‘s large product range in vitamin D analysis with a product, that signifi cantly simplifi es the preparation and shipping of blood samples, thereby enabling vitamin D status monitoring on a larger scale with little eff ort.

INSULIN RESISTANCE

We off er a unique biomarker panel for diabetes-II prevention

RBP4 (K 6110)

RBP4 (1-point calibration) (K 6120)

Proinsulin intact (K 7821)

Adiponectin total (K 6250)

Leptin (KD2395)

Resistin (K 8029)

Visfatin (KA44VISFTH05)

Myostatin (K 1012)

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The metabolic syndrome is characterized by adipositas, hypertension, disorders of the lipid metabolism and a resulting eleva-ted risk for type-II diabetes, often already recognizable in a reduced glucose tole-rance (pre-diabetes).

Immundiagnostik offers a comprehen-sive ELISA routine diagnostic panel for diabetes-II prevention: Risk patients can be tested for proinsulin as a reliable indi-cator of an imminent insulin resistance or for RBP-4 as a marker for undetected in-sulin resistance, when other clinical sym-ptoms of diabetes-II (fasting glycaemia & HbA1C normal) are still missing.

Furthermore, we offer a broad test panel

fasting glucose waist circumference blood pressure triglycerides HDL-cholesterol

pathological values?fasting glucose waist circumference

blood pressure

fasting glucose waist circumference

blood pressure

Metabolic syndrome is confirmed when patho-logical levels are reached in 3 out of 5 criteria

D-Vital ID®

Apply 50 µl capillary blood here

Please read the manual !Prevention

Sampling device

Day of sampling

Patient name

Praxis stamp or sender‘s address

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Test set D-Vital ID® for dried blood spots

D-Vital ID® (DZ9002)

i2 Innovation & Information

Immundiagnostik‘s dried blood spot vita-min D analysis from capillary blood using the D-Vital ID® test set has recently been tested at the University of Potsdam and the University of Mainz in a clinical setting:

Samples from capillary and venous blood were acquired in parallel from 96 sub-jects. The concentration of 25(OH) vi-tamin D was subsequently determined with Immundiagnostik‘s ELISA using the dried blood spot and venous blood samples. The data comparison showed a very good agreement of both methods (r2 = 0,63). The results of this study con-fi rm that the vitamin D analysis from capillary blood with the D-Vital ID® test set is comparable to the classical deter-mination from venous blood. The novel test set is therefore an ideal alternative to conventional vitamin D screening.

Hocher B et al. (2012) „Easy determination of the 25-OH Vitamin D Status from Dried Blood Samples on a Filter Paper .“

Abtract für Eur Soc Nutrition, 2012, Prag

including unique tests for research on the causal events leading from cardiovas-cular diseases to diabetes-II.

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CONGESTIVE HEART FAILURE

Myostatin ELISA validated for clinical research on congestive heart failure

Myostatin is a negative regulator of mus-cle growth and presumably plays a regu-latory role in the cardiovascular system. The fi rst ELISA for the determination of human myostatin, developed by Immun-diagnostik, has now been used by the University of Cologne together with the University hospital of Mainz in the clinical monitoring of patients with decompen-sated congestive heart failure (CHF).The parameter was fi rst signifi cantly ele-vated in the patient group and dropped in the course of an ongoing therapy with standard CHF-medication. The authors observed a myostatin-lowering eff ect during CHF-therapy in parallel to the expected drop in NT-proBNP concentrati-on (s. Fig. 3A)

LEADING THE WAY

in the standardization of 25(OH) vitamin D status analysis

The determination of 25(OH) vitamin D is a routine analysis in laboratory diagnostics. However, there is an urgent need to stan-dardize the various methods that are currently used in order to establish a uni-versal reference method for correct and clinically valid data interpretation.

For example, the processing of higher sample numbers by fully automated im-munoassays is error-proned due to the incomplete separation of the vitamin D binding protein (VDBP) (Heijboer et al. 2012): In samples with high VDBP con-centrations (e.g. from pregnant women), this incomplete purifi cation of vitamin D leads to false-low results and in samples with low VDBP concentrations (e.g. from intensive care patients) to false-elevated results.

In direct comparison to the 25(OH) vita-min D Immundiagnostik-ELISA the other commercially available automated im-

munoassays deliver variable data, the results are to a large extent dependent on the used method (Farrell et al.; Carter; Moon et al.; all 2012).

To be on the safe side rather use the gold standard: Immundiagnostik‘s 25(OH) vi-tamin D ELISA features an excellent cor-relation to chromatographic methods (r= 0,92 with LC-MS/MS) and meanwhile has been established as benchmark re-ference assay in the standardization of 25(OH) vitamin D analysis (Scharla & Lem-pert, 2012).

The test is automatable to process larger sample volumes and the Xpress-version requires only 4 hours.

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......................................................25(OH) Vitamin D Xpress (K 2107)

Goldstandard in25-OH Vitamin D analysis*

www.Immundiagnostik.com

25-OH Vitamin D ELISA

Simply the best!

Simply accurate

Simply monoclonal

Simply automatable

Simply different versions for your needs

*S. Scharla & U. Lempert; P60, Osteologie 2012

The data confi rm that Immundiagnostik‘s Myostatin ELISA reliably refl ects the cli-nical status and therapeutical eff ect in CHF-patients. The validated ELISA is the-refore ready for a broad range of applica-tions in cardiovascular research, e.g. for research on cardial cachexia.

Wintgens KF et al. (2012) „Plasma myostatin measured by a compe-titive ELISA using a hig specifi c antiserum.“

Clin Chim Acta 413(15-16):1288-94

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......................................................Myostatin ELISA (K 1012)

Innovation & Information i2

Myo immunoreactivity in CHF patients of NYHA classes II–IV comparedwith control patients [8]. However, another study employing the sameassay [17] yielded the opposite results although it was somewhathampered by the fact that the control group consisted of only 11patients whowere compared with 70 CHF patients. In our CHF patients,plasma levels at baseline were increased by ~50% over those measuredin controls, which corroborates not only the principal findings but alsothe extent of elevation reported by George et al. and Gruson et al. [7,8].

According to Hill et al. [20], most if not all plasma Myo in micecirculates in the latent complex; hence, if their results are fullyapplicable to man in general and human CHF in particular we willlikely have measured Myo latent complex which we call here “Myoimmunoreactivity”. This may pose a relevant problem since it isevident that “immunoreactivity” does not translate into “bioactivi-ty” at all under these particular circumstances. Hence, further

studies, e. g.,mass spectrometry followingMyo-specific immunoaffinityenrichment, are needed to elucidate the precise composition of “Myoimmunoreactivity” in human plasma. Besides, attention has to befocused on the activation mechanism(s) and levels of activators invivo: according to Lee [21], the metalloprotease bone morphogeneticprotein-1 activates latent Myo in mice; and in human heart failure [7],increased circulating levels of this enzyme have already been reported.This topic, Myo activity versusMyo concentration in plasma, remains tobe investigated in further studies.

In contrast to increased tissue levels of the N-terminal pro-peptide[7] indicating primarily enhanced local activation, the rise of circulatingMyo immunoreactivity may also reflect elevated gene expression.Relevant parts of the signaling pathways stimulating myocardialMyo expression have already been defined: insulin-like growthfactor-1, which itself is up-regulated by increased cyclic stress;

Fig. 3. Myo immunoreactivity is increased in CHF patients and diminished in response to therapy. A: Myo (left) and NT-proBNP (right) immunoreactivities, depicted as box plotswith median and interquartile range, in plasma samples of control and CHF patients (n=20 each). CHF patients admitted in NYHA stadium III (CHF pre) were recompensated usingiv. diuretics and vasodilators (CHF post). P≤0.05: *, vs. control; #, CHF post vs. CHF pre. B and C: Correlation between NT-proBNP and Myo values in control (B) and CHF (C) pa-tients. For CHF patients, all values measured before and after therapy were included. The Spearman rank correlation coefficient was non-significant (NS) in both cases.

1292 K.F. Wintgens et al. / Clinica Chimica Acta 413 (2012) 1288–1294

Myo

stat

in

Heijboer AC et al. (2012), Clin Chem 58(3): 543-548

Farrell CJL et al. (2012), Clin Chem 58(3): 531-542

Carter GD et al. (2012), Clin Chem 58(3): 486-488

Scharla S & Lempert U (2012), P60, Osteologie 2012

Moon HW et al. (2012), Clin Biochem 45: 326-330

Fig. 3A.: Myostatin as therapy indicator. Box-plot diagram, CHF pre = patients before, CHF post = after treatment

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i2 Innovation & Information

ACUTE KIDNEY INJURY

PhiCal® Calprotectin ELISA identifies intrinsic acute kidney damage

The prognosis of patients suff ering from acute kidney injury depends to a large extent on an early diagnosis and the im-mediate start of an appropriate therapy. The quicker the underlying cause of kid-ney malfunction has been identifi ed the better are the chances for a successful treatment.

Especially the diff erentiation of prere-nal and intrinsic acute kidney injury is signifi cant since both diseases require fundamentally diff erent therapies: Pre-renal damage represents a problem that is located at the front-end of the kidney, e.g. massive fl uid loss and/or hypotensi-on. Since the nephrons itself are intact, a simple volume adjustment can already improve the patient‘s condition. An intrinsic kidney injury involves dama-ge of the organ itself, caused for example

ID-PRODUCTS IN USE

Heller F et al. (2011) „Urinary calprotectin and the distinction between prerenal and intrinsic acute kidney injury“.

Clin J Am Soc Nephrol 6(10):2347-55

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PhiCal® Calprotectin (K 6935)(Serum, Plasma, Urine)

PROJECTS & COOPERATIONS

OXIDATIVE STRESS MARKER IN DIABETES PATIENTS

Joint project with Boehringer Ingel-heim and Bayer Healthcare

Together with Boehringer Ingelheim Pharma GmbH & Co. KG and Bayer Healthcare AG Immundiagnostik investi-gates the role of oxidative stress in the development and progression of chronic kidney diseases (e.g. diabetic nephropa-thy). The project focuses on the eff ect of novel pharmaceuticals, such as DPP-4-inhibitors as well as on activators of the guanylate cyclase. The intention is to fol-low up on initial data from animal experi-ments in phase II/III clinical studies.

Berthold Hocher and colleagues presen-ted fi rst results of this cooperation on this year‘s ADA (American Diabetes Associati-on) congress in Philadelphia/USA.

RELAXIN-2 AS VASCULAR MODULATOR

Cooperation with Charité Berlin and the University of Potsdam

Relaxin-2 infl uences the cardiovascular system via G-protein-coupled receptors and exhibits a compensatory function in patients with heart failure. Thomas Dschietzig and colleagues have emplo-yed Immundiagnostik‘s relaxin assay in animal experiments and demonstrated a protective role of relaxin in TNF-α-induced endothelial dysfunction. (Dschietzig et al., Cardiovascular research 04/2012; DOI: 10.1093/cvr/cvs149)

LABORATORY DIAGNOSTICS OF OXIDATIVE STRESS

Advancement of Immundiagnostik‘s test panel

Oxidative stress is involved in the patho-genesis and progression of a variety of diseases, e.g. atherosclerosis, diabetes, neurodegenerative disorders, rheumatoid arthritis and even cancer. The University of Graz identifi es and va-lidates in cooperation with Immundia-gnostik analytic methods and diagnostic parameters of oxidative stress, such as carbonylated proteins, oxidized LDL or ni-trotyrosin. The goal of these research pro-jects is to enable and optimize therapeu-tic strategies to fi ght oxidative stress. Our Med-Science team will feature a seminar „Diagnostics of oxidative stress“ in 2013.

More information: marion.kronabel@immundiagnostik.com

protein, urinary creatinine, urinary sodium, and plasmasodium were not significantly different in the two groups(P � 0.05 each). FENa tended to be higher in intrinsic AKI,but the difference did not reach any significance (P � 0.10).There were no urinary tract infections in the group of prer-enal AKI and 23 cases in the intrinsic group (P � 0.001).

Urinary Calprotectin and Calprotectin/Creatinine Ratios inAKI

Assessment of urinary calprotectin concentration wassuccessful in the whole study population. Figure 1A pres-ents the individual results of calprotectin measurements ineach group. The values ranged from 0 to 20,400 ng/ml. Totake the current concentration status of the urine into ac-count, urinary creatinine was assessed, and calprotectin/creatinine ratios were calculated ranging from 0 to 112,710(ng/ml)/(g/L). The data are presented as medians andinterquartile ranges. Calprotectin concentrations were notsignificantly different in healthy controls (45 ng/ml, 19 to139) and prerenal AKI (28 ng/ml, 13 to 73; P � 0.25).Median urinary calprotectin was 60.7 times higher, how-ever, in intrinsic AKI (1692 ng/ml, 765 to 4735) than inprerenal AKI (P � 0.01). Calprotectin/creatinine ratios didnot differ significantly between healthy controls (70 (ng/ml)/(g/L), 14 to 162) and prerenal AKI (52 (ng/ml)/(g/L),

23 to 123; P � 0.97). The calprotectin/creatinine ratio inintrinsic AKI, however, was 71.1 times higher than inprerenal AKI (P � 0.01). The calprotectin and calprotectin/creatinine ratios in intrinsic AKI were significantly higherthan those of healthy controls (P � 0.01 each). Becauseurinary tract infections go along with leukocyturia, poten-tially leading to high urinary calprotectin concentrations, weexcluded all cases of urinary tract infection in a second anal-ysis. Figure 1B illustrates the results of this analysis, showingthat exclusion of urinary tract infection did not lead to aqualitative change of the findings in the overall study popu-lation. There were no cases of urinary tract infection in theprerenal AKI group; healthy controls were not tested forurinary tract infection. Accordingly, calprotectin and calpro-tectin/creatinine ratios did not change in these groups. Uri-nary calprotectin concentration was 36.1 times higher in in-trinsic AKI (1007 ng/ml, 465 to 2,675) than in prerenal AKI(P � 0.01). The calprotectin/creatinine ratio was 38.2 timeshigher in intrinsic AKI (1987 (ng/ml)/(g/L), 1009 to 4744)when compared with prerenal AKI (P � 0.01).

Diagnostic Accuracy of Urinary Calprotectin in PredictingIntrinsic AKI

The accuracy of urinary calprotectin in the detection ofintrinsic AKI was assessed by ROC curve analysis as pre-

Figure 1. | Individual measurement results of urinary calprotectin and calprotectin/creatinine ratio of healthy controls, prerenal acute kidneyinjury (AKI), and intrinsic AKI of the complete study population (A) and all subjects without urinary tract infection (UTI) (B). The data arepresented as scatter plots (logarithmic y axis; medians are indicated by horizontal lines). Calprotectin concentrations and calprotectin/creatinineratios did not significantly differ between healthy controls and prerenal AKI but were significantly higher in intrinsic AKI than in prerenal AKI andcontrols (P � 0.001 each). The difference remains highly significant after exclusion of subjects with urinary tract infection.

Clin J Am Soc Nephrol 6: 2347–2355, October, 2011 Calprotectin in AKI, Heller et al. 2351

by acute tubular necrosis, interstitial ne-phritis, glomerulonephritis or vasculitis – a complex, costly and intensive therapy is required.

In their search for a reliable routine para-meter for the discrimination of prerenal and intrinsic kidney injury, clinical resear-chers of the Charité in Berlin have deter-mined the inflammation marker calpro-tectin in the urine of patients with acute kidney injury:

Subjects with intrinsic kidney injury had a signifi cantly higher calprotectin con-centration than patients with prerenal damage (s. Fig. 1A). The authors conclude from these data that calprotectin in urine is an adequate diagnostic marker for the diff erentiation of prerenal (calprotec-tin low) and intrinsic (calprotectin high) acute kidney injury.

In addition, with a sensitivity of 92,3% and a specifi city of 97,1% the PhiCal® Calprotectin ELISA has been validated as a reliable test for clinical routine.

Fig. 1A from Heller et al.: High calprotectin levels indicate intrinsic acute kidney injury

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Innovation & Information i2

NEW AVENUES IN LC-MS/MS-DIAGNOSTICSCooperation with instrument manu-facturers to advance product portfolio

For the further development of the chro-matography product pipeline Immundi-agnostik counts on a close cooperation with leading manufacturers of diagnostic instruments. The new biomarker LC-MS/MS product line will focus on indications in the areas of cardiovascular diseases and bone metabolism.

One successful example is the develop-ment of the innovative and highly pre-cise 1,25(OH)

2 Vitamin D ImmunoTube®

LC-MS/MS kit: Immundiagnostik valida-ted the test protocol and proprietory Im-munoTube® affi nity purifaction sample preparation step in close collaboration with ThermoScientifi c, AB Sciex and Wa-ters, utilizing their powerful instruments.

This fruitful industrial cooperation will be continued in a joint development of mass spectrometry tests for the quali-tative and quantitative analysis of dia-

gnostic marker proteins for use in routine and research.

Immundiagnostik organizes already today individual demos together with leading manufacturers of LC-MS/MS instruments, e.g. for the determination of 1,25(OH)

2

vitamin D.

BREAK-THROUGH FOR KIDNEY THERAPY CONTROL

Determination of OxPTH enables for the first time adequate PTH monitoring

Secondary hyperparathyreoidism is a fre-quent result of progressive kidney mal-function in patients suff ering from chro-nic renal failure. The level of parathyroid hormone (PTH) rises in the course of the disease and can lead to bone loss, frac-tures, and vascular calcifi cation with an increased risk of cardiovascular mortality. Monitoring of the PTH level to identify and treat secondary hyperparathyreoi-dism in time is therefore an essential part of therapy control in patients suff ering from chronic renal failure.

However, a precise PTH analysis in clini-cal routine is diffi cult: Some assays deter-mine the total PTH concentration, inclu-ding degradation products, which leads to false-elevated data. Other tests avoid

this problem but still lack clinical accuracy in the diagnosis of hyperparathyreoidism.

Immundiagnostik and the Institute for Nutritional Sciences of the University Potsdam have jointly developed a com-pletely novel PTH test, that can exactly determine the concentration of the bio-active hormone: Given that an elevated oxidative stress burden in patients with chronic renal failure leads to PTH oxidation and a re-sulting loss of PTH receptor binding, the immunoassay is based on the discrimina-

tion of oxidized PTH (oxPTH, biologically inactive) and non-oxidized PTH (biologi-cally active). An initial sample preparati-on step employs an affi nity chromatogra-phy to remove all oxPTH from the patient plasma. Subsequently, non-oxidized, biologically active PTH is detected in this pre-treated sample with a conventional Sandwich-ELISA.

Using this novel assay, researchers could demonstrate that in dialysis patients a considerable, yet individually variable part of the PTH is oxidized and therefo-re biologically inactive. The test will now be used in clinical studies on the corre-lation of oxPTH and bone disorders as well as cardiovascular diseases in dialysis patients. First results of the cooperation have been published (s. below) and will be presented at the ISN Nexus 2012 con-ference in Kopenhagen (20.-23.09.).

(„Measuring PTH in patients with oxidative stress – do we need a fourth generation parathyroid hormone assay?“, Hocher et al., PLoS One).

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Fig.: Human PTH (amino acids 1-84)Oxidation of two methionines (labelled red) lead to the biological inactivation of the hormone.

CALPROTECTIN MONITORING IN CROHN‘S DISEASEIn Service: PhiCal® Calprotectin ELISA in Abbott‘s FIRE-Study

The determination of calprotectin in stool is an established diagnostic marker for chronic intestinal bowel diseases. The si-gnifi cance of the calprotectin concentrati-on in predicting relapse in Morbus Crohn patients is currently under investigation in the multicentric FIRE-study by Abbott (FIRE = Faecal Marker of Intestinal Infl am-

mation for RElapse prediction in routine monitoring of Morbus Crohn patients). The goal of the FIRE-study is, among other aspects, to identify appropriate basal le-vels of calprotectin during remission as well as to determine threshold concentra-tions for the prediction of a relapse risk. The time span between a relevant rise in calprotectin level and the onset of clinical relapse is of particular signifi cance for the-rapeutic intervention.

Using the PhiCal® Calprotectin ELISA the faecal calprotectin concentration will be

assessed in approximately 500 Morbus Crohn patients in 50 participating German clinical institutions over a period of two years. The collected data will provide in-formation on the predicitive potential of calprotectin with respect to the aff ection pattern, the duration and the course of the Crohn‘s disease, thereby establishing the value of the parameter for a persona-lized therapy.

LAB-TIP

Multiple determinations of serial stool sample dilutions deliver correct data

For a precise determination of calprotectin concentrations it is helpful to dilute stool samples several times, e.g. 1:2500, 1:5000 and 1:10000. These multiple determinations of diff erent dilutions validate the single data points, which is especially useful in samples with high calprotectin concentrations. This procedure is recommended in par-ticular for those labs that participate in interlaboratory trials. Sample dilution with Immundiagnostik‘s stabilization buff er included in the PhiCal® Calprotectin ELISA kit guarantees for genuine data and correct clinical results.

7

Immundiagnostik AGStubenwald-Allee 8aD-64625 BensheimTel.: +49 (0) 62 51-70 19 00Fax: +49 (0) 62 51-84 94 30info@immundiagnostik.comwww.immundiagnostik.com

i2 Innovation & Information / 1. Ausgabe 2012

i2 Innovation & Information

COMPANY NEWS

NEW:„MED-SCIENCE“ TEAM IN BERLIN

Immundiagnostik AGUhlandstr. 4-510623 BerlinTel.: +49(0)30 / 367 435 44 and 030/ 288 635 41Fax: +49(0)30/ 516 426 87 marion.kronabel@immundiagnostik.com

As of now, Immundiagnostik operates a Med-Science office in Berlin that is in char-ge of customer liaison and support as well as of training und continued education in the areas of routine diagnostics and re-search. The bureau will advance the publi-

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cation of novel research with established markers and the introduction of innova-tive parameters in clinical routine. Other tasks of the team include scientific marketing, the identification of new busi-ness areas, networking, and the coordina-tion of research cooperations.

The team:Marion Kronabel has previously been di-rector of european affairs at BIO Deutsch-land, director of the european working group for pharma biotechnology (EAPB) and more than 10 years deputy director of Heidelberg‘s technology park. Thomas Dschietzig, Professor at the Charité Berlin, is a specialist for cardiovascular diseases,

Contact of the med-science office in Berlin isDr. Marion Kronabel

esp. heart failure. Berthold Hocher is Pro-fessor at the University of Potsdam. His re-search focuses on nephrological diseases and their impact on the cardiovascular sy-stem (www.uni-potsdam.de/eem/index/prof-hocher.html).

• Biomedical Laboratory Science 18. – 22. August, Berlin

• INDC (12th International Nutrition & Diagnostics Conference)27. – 30. August, Prague, Czech Republic

• ADMA 30. – 31. August, London, UK

• ASBMR (American Society of Bone & Mineral Research)

EVENTS

Visit us at our booth!(Our team at the AACC 2011)

12. – 15. October, Minneapolis, USA Booth #425

• UEGW (United European Gastro- enterology Week) 20. – 24. October, Amsterdam, The Netherlands Booth #35

CONTACT