accurate results in the clinical laboratory || issues with immunology and serology testing

C H A P T E R

18

Issues with Immunology and Serology TestingAmer Wahed, Semyon Risin

University of Texas Health Sciences Center at Houston, Houston, Texas

INTRODUCTION

Clinicians depend on clinical laboratories for obtain-ing accurate results in immunology and serology test-ing, and inaccurate results may have tremendousimpact not only on the diagnosis but also on thepatient. For example, a false-positive HIV test resultmay have a devastating psychological impact on thepatient. Various methods are used in immunology andserology testing, including electrophoresis, immunoas-says, and various other analytical techniques. Even fora single analyte, multiple technologies may be avail-able. As expected, no method is free from analyticalerrors. In this chapter, various sources of errors inimmunology and serology testings are addressed. Theemphasis is on how to minimize errors as well as elim-inate them when possible.

CHALLENGES IN HEMOGLOBINOPATHYDETECTION

Multiple methodologies exist for testing for hemo-globinopathies in the clinical laboratory (Table 18.1).The most common ones employed are conventionalelectrophoresis, capillary electrophoresis, and high-performance liquid chromatography (HPLC). In hemo-globin electrophoresis, red cell lysates are run inelectric fields under alkaline (alkaline gel) and acidic(acid gel) pH. This can be carried out on filter paper, acellulose acetate membrane, a starch gel, a citrate agargel, or an agarose gel. Separation of different hemoglo-bins is largely but not solely dependent on the chargeof the hemoglobin molecule. Change in the amino acidcomposition of the globin chains alters the charge ofthe hemoglobin molecule, resulting in a change in thespeed of migration.

HPLC utilizes a weak cation exchange column sys-tem. A sample of a red blood cell (RBC) lysate inbuffer is injected into the system. Hemoglobin mole-cules are adsorbed onto the column as they arecharged molecules in the buffer system. An elutingbuffer is then injected into the system. The hemoglobinfraction then elutes off the column. The time requiredfor different hemoglobin molecules to elute is referredto as retention time. The eluted hemoglobin moleculesare detected by light absorbance. HPLC permits theprovisional identification of many more variant hemo-globins than can be distinguished by conventional gelelectrophoresis.

In capillary electrophoresis, a thin capillary tubemade of fused silica is used. When an electric field isapplied, the buffer solution within the capillary gener-ates an electroendosmotic flow that moves toward thecathode. Separation of individual hemoglobins takesplace due to differences in overall charges. Other lesscommonly used methodologies include isoelectricfocusing, DNA analysis, and mass spectrometry.

Any one of the previously discussed methods canbe used for screening purposes. Detection of abnormalhemoglobin requires validation by a second method.In addition, relevant clinical history, review of thecomplete blood count (CBC), and peripheral smearprovide important correlation in the pursuit of anaccurate diagnosis.

Hemoglobinopathy Diagnosis Errors

Blood specimens for hemoglobinopathy diagnosismay be sent to outside clinical laboratories requiringspecimens to be transported by couriers. Sometimesthe specimens are mailed. Unduly long transittimes without refrigeration may result in artifactualbands on gel electrophoresis causing confusion in

295Accurate Results in the Clinical Laboratory.

DOI: http://dx.doi.org/10.1016/B978-0-12-415783-5.00018-9 © 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-415783-5.00018-9

interpretation and delay in final diagnosis [1]. Themethod of preparing the red cell lysate can sometimesbe important. One study demonstrated that for accuratequantification of hemoglobin H from electrophoresis onagarose gel at alkaline pH, the lysate carbon tetrachlo-ride is required. If other lysates are used, much lesshemoglobin H is detected, which could result in miss-ing its (Hb H) detection [2]. When HPLC is used, arecognized problem is carryover of sample from oneto the next [3]. For example, if the first sample belongsto a patient with sickle cell disease (Hb SS), then asmall peak may be seen at the “S” window in thenext sample. This can lead to diagnostic confusionas well as the sample needing to be re-run. Relevantto the final diagnosis is the transfusion history. Bloodtransfusion-acquired hemoglobinopathy is an estab-lished phenomenon [4]. This potential for transfusion-acquired hemoglobinopathy exists because heterozygousindividuals show no significant abnormalities during theblood donor screening process [5]. The abnormal hemo-globin in recipients accounts for between 0.8 and 14% ofthe total hemoglobin [4]. Transfusion histories thusremain vital in explaining such findings.

Common hemoglobin disorders are Hb S, Hb E, HbC, Hb D, Hb G, and Hb O (Table 18.2). Hb S is themost common hemoglobinopathy in the United States,whereas Hb E is the most common hemoglobinopathy

in Southeast Asia. These two hemoglobinopathiesaccount for the majority of all hemoglobinopathiesdetected worldwide. Although only a handful ofhemoglobinopathies are dealt with in the clinical labo-ratory, more than 1000 hemoglobinopathies have beendescribed, the vast majority of which are rare or clini-cally insignificant.

Hemoglobin A2

In the diagnosis of β-thalassemia trait, the propor-tion of Hb A2 relative to the other hemoglobins is clin-ically important [6]. In certain cases, Hb A2 variantsmay also be present. In such cases, the total Hb A2(Hb A2 and Hb A2 variant) needs to be considered forthe diagnosis of β-thalassemia [6]. Hb A20 is the mostcommon of the known Hb A2 variants and has beenreported in 1 or 2% of African Americans [7]. Hb A20

has been detected in heterozygous and homozygousstates and in combination with other Hb variants andthalassemia [8�11]. The major clinical significance ofHb A20 is that for the diagnosis or exclusion of β-thal-assemia minor, the sum of Hb A2 and Hb A20 must beconsidered. When present, Hb A20 accounts for a smallpercentage (1 or 2%) in heterozygotes and is difficultto detect by gel electrophoresis [12]. However, it is eas-ily picked up by capillary electrophoresis and HPLC.By HPLC, Hb A20 elutes in the “S” window. In Hb AS

TABLE 18.1 Common Methodologies for Detection of Variant Hemoglobins

Methodology Advantages Disadvantages

Agarose gelelectrophoresis

Cheaper to perform Labor-intensive and time-consuming; common variant hemoglobinsdistributed in the four major lanes

HPLC Faster turnaround time; greater resolution ofcommon variant hemoglobins

Expensive

Capillaryelectrophoresis

Faster turnaround time; common varianthemoglobins distributed in 15 zones

Isoelectricfocusing

Greater resolution More difficult to interpret; particularly suitable for small samplesincluding dried blood spots and is thus often used for neonatal screening

TABLE 18.2 Common Hemoglobin Disorders

Disorder Defective

Chain

Comments

Hb S β chain defect Most common variant hemoglobin seen in the United States; mostly seen in African Americans

Hb C β chain defect Implies ancestry is from Western Africa. HB SC is a sickling disorder

Hb E β chain defect Most common variant hemoglobin seen in Asia

Hb D β chain defect Seen in African, Indian, Pakistani, as well as English individuals. Hb SD is a sickling disorder

Hb G α chain defect Most common α chain variant. Found in African Americans and African Caribbeans. It is of no clinicalsignificance

Hb A20 δ chain defect Most common δ chain defect. Seen in 1�2% of the African American population

Hb O β chain defect Wide geographic distribution—from Africa to Middle East and eastern Europe. HB SO is a sickling disorder

296 18. ISSUES WITH IMMUNOLOGY AND SEROLOGY TESTING

ACCURATE RESULTS IN THE CLINICAL LABORATORY

trait and HB SS disease, Hb A20 will be masked by thepresence of Hb S. In Hb AC trait and Hb CC disease,glycosylated Hb C will also elute in the “S” window.In these conditions, Hb A20 will remain undetected.Conversely, sickle cell patients on chronic transfusionprotocol or recent, efficient RBC exchange may have avery small percentage of Hb S, which the pathologistmay misinterpret as Hb A20.

It has been documented that the Hb A2 concentra-tion may be raised in HIV during treatment [13]. Othercauses of a raised Hb A2 level are thought to be veryunusual [14]. Severe iron deficiency anemia can reduceHb A2 levels [15,16], and in some reports this has beenshown to interfere with the diagnosis of β-thalassemiatrait.

Hemoglobin F

An increase in the percentage of hemoglobin F isassociated with multiple pathologic states, includingβ-thalassemia, δβ-thalassemia, and hereditary persis-tence of fetal hemoglobin (HPFH). The former is asso-ciated with high Hb A2, and the latter two states areassociated with normal Hb A2 values. Hematologicmalignancies are associated with increased hemoglo-bin F and include acute erythroid leukemia (M6) andjuvenile myelomonocytic leukemia. Aplastic anemia isalso associated with an increase in Hb F %. When elu-cidating the actual cause of high Hb F, it is importantto consider the actual percentage of Hb F, Hb A2values, as well as correlation with CBC and peripheralsmear findings.

It is also important to note that drugs (hydroxyurea,sodium valproate, and erythropoietin) and stresserythropoiesis may also result in high Hb F.Hydroxyurea is used in sickle cell disease patients toincrease the amount of Hb F, the presence of whichmay help to reduce the clinical effects of the disease.Measuring the level of Hb F may be useful for deter-mining the appropriate dose of hydroxyurea. In15�20% of pregnancies, Hb F may be raised as muchas 5%.

Hb F quantification may be an issue when HPLCis used. Fast variants (e.g., Hb H or Hb Bart’s) maynot be quantified because they may elute off the col-umn before the instrument begins to integrate inmany systems designed for adult samples. This willaffect the quantity of Hb F. If an α-globin variantseparates from Hb A, often an Hb F variant separatesfrom normal Hb F but may not separate from otherhemoglobin adducts present so that the total Hb Fwill not be adequately quantified. Hb F variants mayalso be due to mutation of the γ-globin chain, andagain this may result in a separate peak and incorrectquantification. Some β chain variants and/or theiradducts may not separate from Hb F, leading to

incorrect quantification [17]. Capillary zone electro-phoresis has an advantage over HPLC in that hemo-globin adducts (glycated hemoglobins and the agingadduct Hb X1d) do not separate from the mainhemoglobin peak so that interpretation is easier thanwith HPLC. If Hb F appears to be greater than 10%on HPLC, its nature should be confirmed by an alter-native method to exclude misidentification of Hb Nor Hb J as Hb F [18].

Hemoglobinopathy S

Hemoglobin S hemoglobinopathy is the most com-mon hemoglobinopathy detected in the United States.Possible diagnoses of patients with Hb S hemoglobin-opathy include sickle cell trait (Hb AS), sickle cell dis-ease (Hb SS), and sickle cell disease status post RBCtransfusion/exchange. Patients with sickle cell traitmay also have concomitant α-thalassemia, and thediagnosis of Hb S/β-thalassemia (0/1/11) is alsooccasionally made. Double heterozygous states of HbSC, Hb SD, and Hb SO are important sickling statesthat should not be missed.

Patients with Hb SS disease may have increased HbF. The distribution of Hb F among the haplotypes ofHb SS is as follows: Hb F, 5�7% in Bantu, Benin, orCameroon; 7�10% in Senegal; and 10�25% in Arab/Indian types [19]. Hydroxyurea also causes an increasein Hb F. This is usually accompanied by macrocytosis.Hb F can also be increased in Hb S/HPFH.

Hb A2 values are typically increased in sickle celldisease and more so by HPLC. This is because thepost-translational modification form of Hb S, Hb S1d,produces a peak in the A2 window. This elevatedvalue of Hb A2 may produce diagnostic confusionwith Hb SS disease and Hb S/β-thalassemia. It isimportant to remember that microcytosis is not a fea-ture of Hb SS disease, and patients with Hb S/β-thalas-semia typically exhibit microcytosis.

Hb SS patients and Hb S/β-0-thalassemia patientsdo not have any Hb A, unless the patient has beentransfused or has undergone red cell exchange.Glycated Hb S has the same retention time (approxi-mately 2.5 min) as Hb A in HPLC [19]. This will pro-duce a small peak in the A window and raise thepossibility of Hb S/β1 -thalassemia.

Hb S/α-thalassemia is considered when the percent-age of Hb S is lower than expected. Classical cases ofsickle cell trait are 60% of Hb A and approximately35�40% of Hb S. Cases of Hb S/α-thalassemia willhave lower values of Hb S, typically below 30% withmicrocytosis. A similar picture will also be present inpatients with sickle cell trait and iron deficiency.Common challenges in hemoglobinopathy detectionare summarized in Table 18.3.

297CHALLENGES IN HEMOGLOBINOPATHY DETECTION


DETECTION OF MONOCLONALPROTEINS

A monoclonal protein (paraprotein or M protein) is amonoclonal immunoglobulin that is secreted by anabnormal clone of plasma cells [20]. The M protein can bean intact immunoglobulin, only light chains (light chainmyeloma, light chain deposition disease, or AL amyloid-osis), or rarely only heavy chains (heavy chain disease).

Serum protein electrophoresis (SPEP) is an inexpen-sive, easy-to-perform screening procedure for detectionof monoclonal proteins [21]. It is usually done by theagarose gel method or by the capillary zone electro-phoresis method. Monoclonal bands are usually seenin the γ zone but may be seen in proximity of the βband or rarely in the α2 area. Urine protein electropho-resis is analogous to SPEP and is used to detect mono-clonal proteins in the urine. Ideally, it should beperformed on a 24-hr urine sample. Common pro-blems encountered in SPEP are listed in Table 18.4.

When a monoclonal band is identified on SPEP,serum immunofixation and 24-hr urine immunofixa-tion is typically recommended. There are certain situa-tions in which a band may be apparent that in realityis not a monoclonal band. Examples include thefollowing:

1. Fibrinogen is seen as a discrete band whenelectrophoresis is performed on plasma. Thisfibrinogen band is seen between the β and γregions. If the electrophoresis is repeated after theaddition of thrombin, this band will disappear orserum immunofixation study will be negative.

2. With intravascular hemolysis, the free hemoglobinbinds to haptoglobin. The hemoglobin�haptoglobincomplex may appear as a large band in the α2 area.Serum immunofixation studies should be negativein such cases.

3. In patients with iron deficiency anemia,concentrations of transferrin may be high. This mayresult in a band in the β region.

4. If electrophoresis is performed on a nephroticsyndrome patient, total protein and serumalbumin is typically low. The condition alsoproduces increases in α2 and β fractions. Bands ineither of these regions may mimic a monoclonalband.

5. When performing gel electrophoresis, a band maybe visible at the point of application. Typically, thisband is present in all samples performed at thesame time.

If the quantity of the M protein is low, this may notbe detected by serum electrophoresis. There are alsocertain situations in which a false-negative interpreta-tion may be made on serum electrophoresis:

1. A clear band is not seen in cases of α heavy chaindisease (HCD). This is presumably due to thetendency of these chains to polymerize or due totheir high carbohydrate content [22�25].

2. In μ-HCD, a localized band is found in only 40% ofcases [26]. Panhypogammaglobulinemia is aprominent feature of such patients.

3. In occasional cases of γ-HCD, again a localizedband may not be seen [24,27,28].

4. When an M protein forms dimers, pentamers,polymers, or aggregates with each other or whenthey form complexes with other plasmacomponents, this may result in a broad smear ratherthan a discrete band.

TABLE 18.3 Common Challenges in HemoglobinopathyDetection

Scenario Cause of Challenge

HPLC Carryover of sample from one to the next

Transfusion Transfusion from donors who are, for example, Hb AS(S trait) or Hb AC (C trait)

Medication Hydroxyurea raises Hb F levels

Irondeficiency

Lowers Hb A2 levels, thus masking diagnosis ofβ-thalassemia trait

HIV Falsely increases Hb A2 levels

Agarose gel Small percentages of abnormal hemoglobins (e.g., HbA20) may be undetected

HPLC Hb A20 elutes in the S window. Thus, in sickle celldisease or trait, Hb A20 will be undetected

HPLC Fast hemoglobin variants (e.g., Hb H and Hb Bart’s)may not be quantified effectively. Hb F quantificationwill also be affected

TABLE 18.4 Common Problems Encountered in Serum ProteinElectrophoresis

1. SPE performed on plasma will result in a band due to fibrinogen.Subsequent immunofixation will be negative

2. A band may be seen at the point of application. Typically, thisband is present in all samples performed at the same time

3. If the concentration of transferrin is high (e.g., due to irondeficiency), this may result in a band in the β region

4. In nephrotic syndrome, prominent bands may be seen in α2 and βregions that are not due to monoclonal proteins

5. Hemoglobin�haptoglobin complexes (seen in intravascularhemolysis) may produce a band in the α2 region

6. M proteins may form dimers, pentamers, polymers, or aggregateswith each other, resulting in a broad smear rather than a distinctband

7. In light chain myeloma, the light chains are rapidly excreted inthe urine and thus SPE may fail to show a band



5. Some patients produce only light chains. These lightchains are rapidly excreted in the urine [29]. Serumelectrophoresis fails to show any band. Urinestudies are typically more fruitful. When the lightchains cause nephropathy and result in renalinsufficiency, excretion of the light chains ishampered. It is at this point that a band may beseen in serum electrophoresis.

6. In some patients with IgD myeloma, the M proteinspike may be small enough to be disregarded.

Hypogammaglobulinemia

Hypogammaglobulinemia may be congenitalor acquired. Among the acquired causes aremultiple myeloma and primary amyloidosis.Panhypogammaglobulinemia can occur in approxi-mately 10% of cases of multiple myeloma. Most ofthese patients have a Bence�Jones protein in the urinebut lack intact immunoglobulins in the serum [30,31].Bence�Jones proteins are monoclonal-free κ or λ lightchains in the urine. Panhypogammaglobulinemia canalso be seen in 20% of cases of primary amyloidosis. Itis important to recommend urine immunofixationstudies when panhypogammaglobulinemia is presentin serum protein electrophoresis.

Immunofixation Studies

An apparent monoclonal protein on serum proteinelectrophoresis may or may not be a true monoclonalprotein. Also, M proteins may not be apparent on elec-trophoresis. To confirm the former and because of thelatter, if there is a high index of clinical suspicion, immu-nofixation studies are required. Immunofixation can beperformed on both serum and urine specimens. It ispreferable to perform urine immunofixation on a 24-hrurine sample. Immunofixation studies are more sensitivethan regular electrophoresis and also determine theparticular isotype of the monoclonal protein. However,they cannot estimate the quantity of the M protein(which the electrophoresis can do). One source of possi-ble error in urine immunofixation study is the “step lad-der” pattern. Here, multiple bands are seen in the κ(more often) or λ lanes and are indicative of polyclonalspillage rather than monoclonal spillage into the urine.

Capillary Zone Electrophoresis

This is an alternative method of performing serumprotein electrophoresis. Protein stains are not required.A point of application is not seen. It is considered tobe faster and more sensitive compared to agarose gelelectrophoresis. Classical cases of monoclonal

gammopathy produce a peak, typically in the γ zone.However, subtle changes in the γ zones may also rep-resent underlying monoclonal gammopathy.Interpretation can be subjective. Pathologists with ahigh index of suspicion will refer a high percentage ofcases for ancillary studies such as immunofixation.Others, disregarding the subtle changes, may poten-tially miss positive cases.

Free Light Chain Immunoassay

Patients with monoclonal gammopathy may havenegative serum protein electrophoresis as well asserum immunofixation studies. Reasons include verylow level of M proteins and light chain gammopathy,where the light chains are very rapidly cleared fromthe serum by the kidneys. Because of the latter, urineelectrophoresis and urine immunofixation are part ofthe workup for cases in which monoclonal gammopa-thy is a clinical consideration. Urine electrophoresisand urine immunofixation studies are also performedto document the amount (if any) of potentially nephro-toxic light chains being excreted in the urine in thecase of monoclonal gammopathy.

Quantitative serum assays for κ and λ free lightchain (FLC) have increased the sensitivity of serumtesting strategies for identifying monoclonal gammo-pathies, especially the light chain diseases [32�35].Cases that appear as nonsecretory myeloma canactually be cases of light chain myeloma. FLC assaysallow disease monitoring as well as provide prognosticinformation for monoclonal gammopathy of undeter-mined significance (MGUS) and smoldering myeloma.

The rapid clearance of light chains by the kidney isreduced in renal failure. Levels may be 20�30 timeshigher than normal in end-stage renal disease. In addi-tion, the κ:λ ratio may be as high as 3:1 in renal failure(normal, 0.26�1.65). Therefore, patients with renal fail-ure may be misdiagnosed as having κ light chainmonoclonal gammopathy. If a patient has λ light chainmonoclonal gammopathy, with the relative increase inκ light chain in renal failure, the ratio may becomenormal. Thus, a case of λ light chain monoclonal gam-mopathy may be missed. It is also important to beaware that the presence of circulating M proteins mayinterfere with other laboratory tests. The most commonerrors that occur are falsely low high-density lipopro-tein cholesterol, falsely high bilirubin, as well asaltered values of inorganic phosphate. Other tests inwhich altered results may occur include low-densitylipoprotein cholesterol, C-reactive protein, creatinine,glucose, urea nitrogen, and inorganic calcium.

There is a potential for inappropriate clinical deci-sions based on altered lab values.

299DETECTION OF MONOCLONAL PROTEINS


Cerebrospinal Fluid Electrophoresis

Qualitative assessment of cerebrospinal fluid (CSF)for oligoclonal bands is the most important diagnosticCSF study when determining a diagnosis of multiplesclerosis (MS). In MS, elevation of the CSF immuno-globulin level relative to other protein componentsoccurs, suggesting intrathecal synthesis. The immuno-globulin increase is predominantly IgG, although thesynthesis of IgM and IgA may also be increased.

Oligoclonal bands are defined as at least two bandsseen in the CSF lane with noncorresponding band pres-ent in the serum lane. Thus, it is crucial to perform CSFand serum electrophoresis simultaneously. Oligoclonalbands may be found in 95% or more of patients withclinically definite MS [36]. However, they may also beseen in central nervous system infections (e.g., Lymedisease), autoimmune diseases, brain tumors, and lym-phoproliferative disorders. Thus, it is important to real-ize that an oilgoclonal band is not equivalent to MS.

The first step when interpreting CSF electrophoresisis to establish that the sample is indeed a CSF sample.The presence of the prealbumin band and the band inthe β2 regions due to desialated transferin establishesthat the sample is indeed a CSF sample. Both of thesebands are not present in the serum lane. The presenceof oligoclonal bands may then be confirmed by per-forming immunofixation studies.

An abnormality of CSF IgG production can be dem-onstrated in 90% of clinically definite MS patients [37].There are various ways to document this. The CSF IgGlevel may be expressed as a percentage of the totalprotein, as a percentage of albumin, or by the use ofthe IgG index. It is important to correlate the findingsof the electrophoresis with the IgG index, CSF study,as well as any pertinent magnetic resonance imagingand clinical findings.

In MS, CSF is grossly normal, and CSF pressure is nor-mal. The total leukocyte count is normal in the majorityof patients. If the CSF white blood cell count is elevated,it rarely exceeds 50 cells/μL [38]. Lymphocytes are thepredominant cell type. CSF protein is also usually nor-mal. If there is a systemic immune reaction or a monoclo-nal gammopathy, then bands will be seen in both theserum and the CSF lanes. These bands will correspondwith each other, and they are not oligoclonal bands.

CHALLENGES IN HIV TESTING

Testing for HIV can be broadly divided into screen-ing tests and confirmatory tests. Screening testsinclude standard testing, rapid HIV testing, and com-bination HIV antibody and antigen testing.Confirmatory testing is performed by Western blot.

Standard screen is performed by enzyme immunoas-say (EIA). The test is based on the detection of IgG anti-body against HIV-1 antigens in the serum. These HIVantigens include p24, gp120, and gp41. Antibodies togp41 and p24 are the first detectable serologic markersfollowing HIV infection [39]. IgG antibodies appear6�12 weeks following HIV infection in the majority ofpatients and generally persist for life. Assays for IgMantibodies are not used because they are relativelyinsensitive.

HIV viruses are categorized into the followinggroups: M, N, O, and P (Table 18.5). M is consideredto be the pandemic strain and accounts for the vastmajority of strains of HIV. Group O strains are fromcertain areas of Africa (Cameroon, Gabon, andEquatorial Guinea). Groups N and P are fromCameroon. Group M viruses are divided into 10 sub-types, A�J. Subtype B is the most common one foundin the United States and Europe.

The two important issues regarding HIV screen testsare the ability of the test to detect non-M strain andthe timing of the test post exposure. If the patient hasnot been seroconverted, as expected, the antibody isabsent and the individual may have a false-negativetest. There are also rare patients with HIV infectionwho become seronegative even though they showedseropositive results after exposure to HIV virus [40,41].Other causes of false-negative results include thefollowing:

• Fulminant HIV infection• Immunosuppression or immune dysfunction• Delay in seroconversion following early initiation of

antiretroviral therapy.

False-positive serologic test for HIV is extremelyrare. However, false-positive test results for HIV infec-tion have been documented in individuals who have

TABLE 18.5 HIV Types and Groups

HIV

Type

Group Description

I Related to viruses found in chimpanzees andgorillas

I M M denotes “major.” Most common type of HIV;responsible for the AIDS pandemic

I N N denotes “non-M, non-O”; seen in Cameroon

I O O denotes “outlier”; most common in Cameroon;not usually seen outside west-central Africa

I P P denotes “pending the identification of furtherhuman cases”; the virus was isolated from aCameroonian woman residing in France

II Related to viruses found in sooty mangabeys



received HIV vaccines in vaccine trials [42]. Some ofthese individuals who became HIV positive on screentests also had a positive Western blot. HIV RNA test-ing is an approach that should be used to resolve suchissues. HIV testing methods are listed in Table 18.6.

Rapid HIVAntibody Tests

Rapid HIV antibody testing is available in theUnited States. Results are available within minutesbecause the tests can be done on-site and can be readby the provider. Rapid tests may not be as sensitive assecond-generation EIAs. In one study of more than14,000 specimens, rapid testing failed to detect 16 sam-ples, which were positive by EIA and subsequentlyconfirmed by Western blot [43]. A positive rapid testshould be considered as “preliminary” and requiresconfirmation by Western blot. Reports of false posi-tives do exist in the literature. A negative test may bereported as such and does not require further workup.However, early testing prior to seroconversion willnaturally result in false-negative results.

Combined Antibody Antigen Tests

Fourth-generation tests have the ability to detectboth HIV antibody and the p24 antigen. Sensitivityand specificity of such tests are generally excellent.The primary advantage of these assays is the ability todetect HIV infection prior to seroconversion.

HEPATITIS TESTING

Hepatitis infection is a worldwide problem.Hepatitis A is a problem in many developing countriesbecause infection may spread from contaminatedwater (fecal�oral route). However, hepatitis A testingis straightforward; IgM anti-hepatitis A virus (HAV)denotes recent infection, and IgG anti-HAV appears inthe convalescent phase of acute hepatitis. Hepatitis Evirus (HEV) is also an enterically transmitted virus.HEV can also be transmitted by blood transfusion, par-ticularly in endemic areas. Chronic hepatitis does notdevelop after acute HEV infection, except in the

transplant setting and possibly in other settings ofimmunosuppression. Fulminant hepatitis can occur,resulting in an overall case fatality rate of 0.5�3%. Forreasons as yet unclear, the mortality rate in pregnantwomen can be as high as 15�25%, especially in thethird trimester. The diagnosis of HEV is based on thedetection of HEV in serum or stool by polymerasechain reaction (PCR) or on the detection of IgM antibo-dies to HEV. Antibody tests against HEV alone are lessthan ideal because they have been associated with fre-quent false-positive and -negative results. The hepatitisD virus (HDV; also called the delta virus) is a defectivepathogen that requires the presence of the hepatitis Bvirus (HBV) for infection. HDAg can elicit a specificimmune response in the infected host, consisting ofantibodies of the IgM and IgG class (anti-HDV).Hepatitis B and C infection serology, which is the mostimportant component of hepatitis testing, is discussedin detail in the following sections.

Serology for Hepatitis B

Serologic markers available for hepatitis B infectionare HBsAg (hepatitis B surface antigen), HBeAg (hepati-tis B e antigen), anti-HBc (antibody against hepatitis Bcore antigen; both IgG and IgM), anti-HBs (antibodyagainst hepatitis B surface antigen), anti-HBe (antibodyagainst hepatitis B e antigen), and HBV DNA(Table 18.7). HBsAg is the first marker to be positive afterexposure to HBV. It can be detected even before theonset of symptoms. Most patients may clear the virusand HBsAg typically becomes undetectable within 4�6months. Persistence of HBsAg for more than 6 monthsimplies chronic infection. The disappearance of HBsAgis followed by the presence of anti-HBs. During the win-dow period (after the disappearance of HBsAg andbefore the appearance of anti-HBs), evidence of infectionis documented by the presence of anti-HBc (IgM).

TABLE 18.6 HIV Testing Methods

Screening Confirmatory

Standard testing (EIA) Western blot

Rapid HIV testing HIV RNA by PCR

Combination HIV antigen and antibody test

EIA, enzyme immunoassay; PCR, polymerase chain reaction.

TABLE 18.7 Hepatitis B Serology

Antigen

HBsAg First detectable agent in acute infection

HBcAg Not tested because it is not detectable in blood

HBeAg Indicates virus is replicating and patient is highlyinfectious

Antibody

Anti-HBc First antibody to appear; should be positive when othertests for hepatitis B are negative during the windowperiod (HBsAg is negative and anti-HBs is not yetdetectable)

Anti-HBe Virus is not replicating

Anti-HBs Patient is immune

301HEPATITIS TESTING


Coexistence of HBsAg and anti-HBs has been documen-ted in approximately 24% of HBsAg-positive indivi-duals. It is thought that the antibodies fail to neutralizethe virus particles. These individuals should be consid-ered as carriers of HBV. A subset of patients haveundetectable HBsAg and are positive for HBV DNA.Most of these patients have very low viral load withundetectable levels of HBsAg. Uncommon situations areinfection with HBV variants that decrease HBsAg pro-duction or mutant strains that have altered epitopesnormally used for detection of HBsAg.

Individuals with recent infection will develop anti-HBc, IgM antibodies. Individuals with chronic infec-tion and individuals who have recovered from aninfective episode will develop anti-HBc, IgG antibo-dies. However, anti-HBc, IgM antibodies may remainpositive for up to 2 years after an acute infection.Levels may also increase and be detected duringexacerbations of chronic hepatitis B. This may lead todiagnostic confusion.

Some individuals have isolated anti-HBc antibodypositivity. The clinical significance of this finding isunclear. Some of these individuals have been found tohave HBV DNA by PCR. This is true for samples fromserum or liver. Transmission of hepatitis B has beenreported from blood and organ donors who have hadisolated anti-HBc antibody positivity. On the otherhand, a certain percentage of individuals who haveanti-HBc are false positive. The presence of HBeAgusually indicates that the HBV is replicating and thepatient is infectious. Seroconversion to anti-HBe typi-cally means the virus is no longer replicating. This isassociated with a decrease in serum HBV DNA andclinical remission. In some patients, seroconversion isstill associated with active liver disease. This may bedue to low levels of wild-type HBV or HBV variantsthat prevent or decrease the production of HBeAg.

Serology for Hepatitis C

The tests for hepatitis C virus (HCV) include sero-logic assays and molecular assays for HCV RNA. Thecommonly utilized screening assay to detect anti-HCVantibody is an EIA. The latest version is EIA-3, whichhas better sensitivity and specificity compared to EIA-1 or EIA-2. In addition, the mean time to detection ofseroconversion has been reduced by 2 or 3 weeks.Rapid tests for detection of anti-HCV also exist. If anindividual is positive for anti-HCV, then the logicalnext step is to test for HCV RNA. If there is true infec-tion, both anti-HCV antibody and tests for HCV RNAshould be positive. If the HCV RNA is negative, thenthe possibilities include the following:

1. A false-positive anti-HCV antibody test.

2. If the individual is a newborn, the anti-HCV may bethat of the mother with transfer of antibodies acrossthe placenta.

3. Intermittent viremia.4. Past infection.

For establishing the diagnosis of past infection,another test, recombinant immunoblot assay (RIBA),may be undertaken. If the anti-HCV is a false-positivetest, then RIBA will be negative. If it is a case of pastinfection, RIBA will be positive.

There may be individuals who have HCV RNA butanti-HCV antibody testing is negative. This can beseen in immunocompromised individuals or in the set-ting of early acute infection. HCV RNA tests are posi-tive earlier than anti-HCV antibody tests.

ANTI-NUCLEAR ANTIBODIES

Anti-nuclear antibodies (ANA) are serologic hall-marks of systemic autoimmune diseases [44]. A posi-tive ANA may be seen in individuals with systemic ororgan-specific autoimmune diseases and a variety ofinfections. They can also be found in otherwise normalindividuals. Examples of organs implicated in organ-specific autoimmune diseases are thyroid, liver, andlung. Examples of infections in which ANA may befound to be positive include infectious mononucleosis,hepatitis C infection, subacute bacterial endocarditis,tuberculosis, and HIV infections [45�47]. If they arepositive in otherwise normal individuals, these indivi-duals are most likely women and elderly.

The ANA assay is performed by incubating humanepithelial cell tumor line (Hep2) cells fixed with meth-anol and/or acetone with the patient’s serum.Fluorochrome-labeled antihuman globulin is added,which binds to the antigen�antibody complex. Theslide is then viewed through a fluorescent microscope.Antibodies present in the patient’s serum will bind tothe nuclear antigen and should also produce a patternthat is noted. The dilution of the serum at which thereaction pattern disappears is also noted.

The accurate interpretation of patterns requires con-siderable experience. Also, the pattern type has beenrecognized to have a relatively low sensitivity andspecificity for different autoimmune disorders. Asmentioned previously, false positives may be seen innormal individuals. The majority of these are presentin low titer.

False negatives are also an established phenomenondue to technical and physical issues that includemethod of substrate fixation, solubility of the antigenin question, and localization of the antigen outside thenucleus. Once the ANA is positive, further testing is



required. These tests include antibodies to double-stranded DNA, anti-histone antibodies, antibodies tochromatin, and antibodies to other nuclear proteinsand RNA�protein complexes.

CONCLUSIONS

Clinicians rely on accurate results from clinical labo-ratories for diagnosis of hemoglobinopathy, HIV, andhepatitis. A false-positive or false-negative test resulthas serious consequences for patient management.Moreover, a monoclonal band, if missed, can also be aserious patient safety concern. In this chapter, the pit-falls of immunology and serology testing wereaddresses, with emphasis on both false-positive andfalse-negative test results and how to eliminate someof these errors in the clinical laboratory.

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