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SOURCES OF ERROR IN SEROLOGIC AND IMMUNOLOGIC LAB Lecture by: Dr. Forouzan Karimi; PharmD, PhD [email protected]

SOURCES OF RROR IN SEROLOGIC AND IMMUNOLOGIC …iacld.ir/DL/co/14/immunology/sourcesoferrorinserologyandimmunology... · SOURCES OF ERROR IN SEROLOGIC AND IMMUNOLOGIC LAB Lecture

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Page 1: SOURCES OF RROR IN SEROLOGIC AND IMMUNOLOGIC …iacld.ir/DL/co/14/immunology/sourcesoferrorinserologyandimmunology... · SOURCES OF ERROR IN SEROLOGIC AND IMMUNOLOGIC LAB Lecture

SOURCES OF ERROR INSEROLOGIC AND

IMMUNOLOGIC LABLecture by:Dr. Forouzan Karimi; PharmD, [email protected]

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C-REACTIVE PROTEINRAPID LATEX AGGLUTINATION TESTRAPID LATEX AGGLUTINATION TEST

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SOURCES OF ERROR

False-positive results may be observed if:False positive results may be observed if:Serum specimens are lipemic, hemolyzed, or heavilycontaminated with bacteria.If the reaction time is longer than 2 minutes, a false-positive result may also be produced from a dryingeffecteffect.

False-negative results may be observed in:Undiluted serum specimens because of high levels ofp gCRP (antigen excess). A 1:5 dilution of serum is alsotested for this reason.

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TRADITIONAL SCREENING TESTFOR INFECTIOUS MONONUCLEOSISOR C OUS O O UC OS S

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False-positive reactions have beenFalse-positive reactions have beenobserved:In conditions such as hepatitis infectionIn conditions such as hepatitis infectionand Hodgkin’s disease.A i l i ti t d illAn improperly inactivated serum willproduce hemolysis (When using RBCs).

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PREGNANCY LATEX SLIDEAGGLUTINATION

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TECHNICAL SOURCES OF ERROR

Reagents should never beReagents should never beexpired; latex reagent must be wellshaken and agglutination should beshaken and agglutination should beread within 3 minutes to avoiderroneous results caused byerroneous results caused byevaporation.

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FALSE-POSITIVE RESULTS

If a patient has been given an hCG injection (e.g.,p g j ( g ,Pregnyl) to trigger ovulation or lengthen the luteal phaseof the menstrual cycle, trace amounts can remain in thepatient’s system for as long as 10 days after the lastpatient s system for as long as 10 days after the lastinjection. This will produce a false positive result. Twoconsecutive quantitative hCG blood assays cancircumvent this problem. If the hCG level increases bythe second test, the patient is probably pregnant.Chorioepithelioma hydatidiform mole or excessiveChorioepithelioma, hydatidiform mole, or excessiveingestion of aspirin may give false-positive results.

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FALSE-POSITIVE RESULTS (CONT’D)

In men, a test identical to that used for pregnancy may be, p g y yperformed to detect the presence of a testicular tumor. IfMab against the β subunit is not used, other hormoneswith the same α unit may cross react and cause a falsewith the same α unit may cross-react and cause a false-positive reaction.

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FALSE-NEGATIVE RESULTS

Testing before reaching detectable levels of hCGTesting before reaching detectable levels of hCGwill yield false-negative results.

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ABO BLOOD GROUPING(REVERSE GROUPING)( )

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If a patient has been recently transfused withp ynon–group specific blood, mixed-fieldagglutination may be observed. If largequantities of non group specific blood have beenquantities of non–group-specific blood have beentransfused, determination of the correct ABOgrouping may be impossible.Discrepancies in forward typing can result fromconditions such as weak antigens, alteredexpression of antigens caused by diseaseexpression of antigens caused by disease,chimerism, or excessive blood group substances.

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Excess amounts of blood group–specific solubleg p psubstances present in the plasma in certaindisorders (e.g., carcinoma of stomach or pancreas)neutralize the reagent anti A or anti B leavingneutralize the reagent anti-A or anti-B, leavingno unbound antibody to react with the patient’serythrocytes.This excess of blood group–specific substanceproduces a false-negative or weak reaction in theforward grouping If the patient’s erythrocytesforward grouping. If the patient s erythrocytesare washed with saline, the substance should beremoved and a correct grouping can be observed.

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Incorrect typing can also result from additionalIncorrect typing can also result from additionalantigens, caused by the following:

Polyagglutinable RBCsy ggAcquired B-like antigen; acquired A-like antigenComplexes attached to RBCspAgents causing nonspecific erythrocyte

agglutinationAntibody-sensitized RBCs: effect of colloids and

antiantibodies (e.g., hemolytic disease of theb i tibl t f i t inewborn, incompatible transfusion, autoimmune

process)

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Discrepancies in serum (reverse) grouping can result p ( ) g p gfrom additional or missing antibodies caused by the following:

P i l i d i l ti i• Passively acquired isoagglutinins• Alloantibodies• Rouleaux formation• Rouleaux formation• Auto–anti-I; iso–anti-I• Anti-A1 in Ax, A2, and A2B blood, ,• Anti-H in A1B, A1, B, and Bombay blood• Anti-IA and IA

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CAUSES OF WEAK OR MISSING ANTIBODIESINCLUDE THE FOLLOWING:

• Deteriorated reagent erythrocytesg y y• Hypogammaglobulinemic• Elderly patients• Newborn infants• Chimerism• Rare variants of A or B

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TECHNICAL SOURCES OF ERROR

Each manufacturer provides with eachEach manufacturer provides, with eachpackage of antiserum, detailed instructionsfor the use of anti-A and anti-B Becausefor the use of anti-A and anti-B. Becausethe details vary, it is important to followthe directions for the specific antiserum inthe directions for the specific antiserum inuse.

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PROCEDURES THAT APPLY TO ALL TESTS FORABO GROUPING INCLUDE THE FOLLOWING:

1. Do not rely on the color of dyes to identifyy y yreagent antisera. All tubes must be properlylabeled.

2 D t f t t t t t hi h2. Do not perform tests at temperatures higherthan room temperature (20° C to 24° C [68° F to75° F]).

3. Perform observations of agglutination with awell-lit background.

4 R d l i di l f b i4. Record results immediately after observation.5. Remember that contaminated blood specimens,

reagents or supplies may interfere with the testreagents, or supplies may interfere with the testresults.

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LIMITATIONS

Antisera prepared from human sources arep pcapable of detecting A1 and A2 groups. Except inthe case of newborn and very young infants, areverse cell typing should also be performed toreverse cell typing should also be performed toverify the results of forward typing.

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ELECTROPHORESIS TECHNIQUES

Sources of ErrorThe prozone phenomenon is an incompleteprecipitin reaction caused by antigen excess( ti t tib d ti t hi h) P i(antigen-to-antibody ratio too high). Prozoningshould be suspected if a precipitin arc appears torun into a trough, if an L chain appears fuzzywhen an H chain is increased, or if an arcappears to be incomplete.

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CLASSIC VDRL PROCEDURE: VDRL QUALITATIVE SLIDE TESTS

Sources of Error:False-negative reactions can occur in a variety of situations.

These include the following:1. Technical error (e.g., unsatisfactory antigen or technique)2. Low antibody titersPatients may have syphilis, but the reagin concentration is too

low to produce a reactive test result. A low concentration ofreagin may be caused by several factors such as an infectionreagin may be caused by several factors, such as an infectionthat is too recent to have produced antibodies, the effects oftreatment, latent or inactive disease, and patients who have notproduced protective antibodies because of immunologictolerance.

These seronegative patients may demonstrate apositive reaction with more sensitive treponemalp ptests such as the FTA-ABS.

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3. Presence of inhibitors in the patient’s serump4. Reduced ambient temperature (<23° C to 29° C

[<73° F to 84° F])5. Prozone reaction

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A prozone reaction is encountered occasionally.p yThis type of reaction is demonstrated whencomplete or partial inhibition of reactivity occurswith undiluted serum and minimal reactivity isyobtained only with diluted serum. The prozonephenomenon may be so pronounced that only aweakly reactive or rough nonreactive resulty goccurs in the qualitative test by a serum that willbe strongly reactive when diluted. It isrecommended that all sera producing a weakp greaction or rough nonreactive results inqualitative testing be retested with aquantitative procedure before a final report of theq p pVDRL slide test is issued.

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Weakly reactive results can be caused by they yfollowing:

1. Very early infection2. Lessening of the activity of the disease after

treatment3 Improper technique or questionable reagents3. Improper technique or questionable reagentsFalse-positive reactions can also occur. Of allpositive serologic tests for syphilis, 10% to 30%may be false-positive biologic reactions.

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Nonsyphilitic positive VDRL reactions have been reported withthe cardiolipin type of antigen in the following:1. Lupus erythematosus2. Rheumatic fever2. Rheumatic fever3. Vaccinia and viral pneumonia4. Pneumococcal pneumonia5 I f ti l i5. Infectious mononucleosis6. Infectious hepatitis7. Leprosy8. Malaria9. Rheumatoid arthritis10 Pregnancy10. Pregnancy11. Older individuals

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Contaminated or hemolyzed specimens can alsoy pproduce false-positive results

Limitations:The VDRL procedure is not specific for syphilisand may demonstrate positive reactions in otherand may demonstrate positive reactions in otherreagin-producing disorders, autoimmunedisorders, infectious diseases, and in pregnancy or

i i l h i laging in normal physiology.

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RAPID PLASMA REAGIN CARD TESTSources of Error

E b i t d d i t t t lt b f f t h t i tiError can be introduced into test results because of factors such as contamination of rubber bulbs or an improperly prepared antigen suspension.False-positive biological reactions have been reported with cardiolipin type of antigens in the following conditions:

L h• Lupus erythematosus• Rheumatic fever• Vaccinia and viral pneumonia• Pneumococcal pneumonia Pneumococcal pneumonia• Infectious mononucleosis• Infectious hepatitis• Leprosy• Malaria• Rheumatoid arthritis• Pregnancy• Aging individuals Aging individuals

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False-negative reactions can result from thegfollowing:• Poor technique• Ineffective reagents• Ineffective reagents• Improper rotationAgain, if mechanical rotation is below or abovegthe 95- to 110–rpm acceptable range, theclumping of the antigen tends to be less intensein procedures with undiluted specimen; thus,p p ; ,some minimal reactions may be missed. Inquantitative tests, rotation above 110 rpm tendsto produce a decrease in titer, approximately onep , pp ydilution lower.

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LIMITATIONS

A diagnosis of syphilis cannot be made based on ag ypsingle reactive result without clinical signs andsymptoms or history.Pl i h ld t b d t t bli hPlasma specimens should not be used to establisha quantitative baseline from which changes intiter can be determined, particularly forevaluating treatment. The RPR cards should notbe used for testing CSF. Little reliance should beplaced on cord blood serologic testing for syphilisplaced on cord blood serologic testing for syphilis.The RPR procedure has adequate sensitivity andspecificity in relation to the clinical diagnosis.

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PASSIVE LATEX AGGLUTINATION FOR DETECTION OFANTIBODIES TO CYTOMEGALOVIRUS

Sources of ErrorIncorrect test results may be caused by a variety offactors:Specimens that are incorrectly collected or stored canproduce errors in the test results. The use of componentsor procedures other than those previously described mayor procedures other than those previously described mayalso lead to erroneous results.

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LIMITATIONS

Several limitations are inherent in CMV antibody detection,as follows:1. Patients with acute infection may not have detectableantibody.2. Seroconversion may indicate recent infection, but anincrease in antibody titer by this method does not differentiatebetween a primary and secondary antibody response.3. The timing of antibody responses during a primary infectionmay differ slightly. The pattern of antibody response during aprimary CMV infection has not been demonstrated.4. Test results from neonates should be interpreted withcaution because the presence of CMV antibody is usually theresult of passive transfer from the mother to the fetus.

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LIMITATIONS

5. Although the CMV latex procedure will detectIgM and IgG antibodies, detection of IgA and IgE

tib di h t t b d t t dantibodies has not yet been demonstrated.A negative CMV test result may be useful in

excluding possible infection, but the diagnosis ofexcluding possible infection, but the diagnosis ofan actual CMV infection should be documentedby demonstrating the presence of the virusdirectly or by viral culturedirectly or by viral culture.

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PAUL-BUNNELL SCREENING TEST

Sources of ErrorFalse-positive reactions have been observed inconditions such as hepatitis infection and Hodgkin’sdidisease.An improperly inactivated serum will producehemolysis.hemolysis.

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MONOSLIDE TEST

Sources of ErrorFor accurate results, only clear, particle-free serum or

plasma specimens should be used.False-positive results can be caused by the following:

1. Observing agglutination after the observation time2 Mi i t ti l ti ti2. Misinterpreting agglutination3. Simultaneous occurrence of infectious mononucleosisand hepatitis has been reported.p pA result interpreted as a false-positive may be caused byresidual heterophile antibody present after clinical

t h S b id dsymptoms have Subsided.

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WESTERN BLOT

The WB technique is time-consuming and expensive. It is alsoq g popen to considerable interpretation and has many sources oferror. Variables in the test include the following:• The technical skill and experience of the technologist• The technical skill and experience of the technologistperforming the procedure• Characteristics of the technical methodology• General sensitivity of the WB in detecting antibodiesspecific for various HIV-1 antigens (especially during thewindow period of seronegativity)• Frequent lack of specificity because of contamination of theviral reference preparation by histocompatibility and otherantigens that electrophoretically migrate with p24 and gp41antigens that electrophoretically migrate with p24 and gp41.

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Variation in band reactivity patterns in sera fromi di id l th f HIV 1 i f tian individual over the course of HIV-1 infection

Indeterminate test results account for 4% to 20% ofWB assays with positive bands for HIV-1 proteins.Indeterminate WB results can be caused by theyfollowing:

Serologic tests in the process of seroconversion;anti-p24 is usually the first antibody to appear.

End-stage HIV infection, usually with loss of coreantibody

Cross-reacting nonspecific antibodies, as seen withll l di t i dicollagen vascular disease, autoimmune diseases,

lymphoma, liver disease, injection drug use, multiplesclerosis, parity, or recent immunization

Infection with O strain or HIV 2Infection with O strain or HIV-2Recipients of HIV vaccine

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Perinatally exposed infants who arey pseroconverting (losing maternal antibody)Technical or clerical errorIn addition, nonspecific reactions producingindeterminate results in uninfected persons haveoccurred more frequently in pregnant women oroccurred more frequently in pregnant women ormothers than in persons in other groupscharacterized by low HIV seroprevalence. Theincidence of indeterminate WB results isincidence of indeterminate WB results isrelatively low. The immunofluorescence assaycan be used to resolve an EIA-positive, Wb-indeterminate sample.

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The most important factor in evaluatingi d t i t lt i i k t P ti t iindeterminate results is risk assessment. Patients inlow-risk categories with indeterminate test resultsare almost never infected with HIV-1 or HIV-2.Repeat testing usually continues to showp g yindeterminate esults, and the cause of this pattern isseldom established.Follow-up serology testing at 3 months is

d d t if th i lt P ti trecommended to verify the previous results. Patientswith indeterminate tests who are in the process ofseroconversion usually have positive WB test resultswithin 1 month; repeat tests at 1, 2, and 6 months are; p , ,generally advocated, with appropriate precautions toprevent viral transmission in the interim.False-positive WB results, especially those with a

j it f b d t lmajority of bands, are extremely uncommon.

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DIRECT ANTIGLOBULIN TEST

Sources of Error:False-positive and false-negative results can occur.

False-positive results in the DAT can be caused by thefollowing:

• Contamination of AHG antisera or supplies• Overcentrifugation• Bacterial contamination of specimen or reagentsp g• Fibrin clot in cell suspensionFalse-negative results usually occur because of technicalerror Common causes of false-negative reactions include theerror. Common causes of false negative reactions include thefollowing:• Failure to add AHG reagent• I d t hi g f RBC• Inadequate washing of RBCs• Weak or inactive AHG

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RAPID SLIDE TEST FORANTINUCLEOPROTEIN

Sources of ErrorSources of ErrorFailure to observe the test mixture at theappropriate time can yield false results.pp p y

LimitationsNo one test has been shown to beNo one test has been shown to becompletely reliable for the diagnosis ofSLE because many of the ANAsaccompanying this disease are alsodemonstrated in other SRDs, such ash t id th itirheumatoid arthritis.

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ANTINUCLEAR ANTIBODY VISIBLEMETHOD

Sources of ErrorFalse-negative results can occur if the ANAhappens to be specific for an antigen other thanth d i th dthe one used in the procedure.False-negative results may also occur if thesubstrate is fixed in acetone and is inadequatelysubstrate is fixed in acetone and is inadequatelywashed. Without fixation, however, some solublenuclear antigen may be lost.F l i l l b l d hFalse-negative results may also be related to thebinding of antinuclear factor to circulatingimmune complexes and to a low antibody titer.p y

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ANTINUCLEAR ANTIBODY VISIBLEMETHOD (CONT’D)

False-positive interpretations may occur becausep p yof nonspecific staining, which may resemble aspeckled pattern of reactivity.Th t i i ti h thThese staining reactions occur whenever theconjugate or serum contains antibodies to othertissue antigens. Careful rinsing and removal ofexcess fluoresceinated conjugate minimize therisk of some nonspecific staining reactions.

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Although IIF is considered to be the goldg gstandard, it suffers from being a nonstandardizedmanual test, has subjective interpretation ofresults and has low reproducibility. Recently,p y y,manufacturers have automated thepreevaluation and evaluation phases of IIF ANAtesting, including using automatic fluorescentg, g gimage analysis to provide a virtual titer, whicheliminates the process of staining a series ofdiluted samples manually.p yEIAs and solid-phase methods (e.g., microarraysand bead-based assays) are popular. However,IIF currently remains the gold standard ofIIF currently remains the gold standard oftesting.

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LimitationsNo diagnosis should be based solely on theresults of laboratory testing. Clinical data,

tib d tit d th l b t fi diantibody titers, and other laboratory findingsshould all be reviewed before a definitivediagnosis is established.

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REFERENCE:

Turgeon ML. Immunology & Serology ing gy gyLaboratory Medicine. 5th Edition. Elsevier; 2014.