update in the evaluation of antiphospholipid antibodies silvia s. pierangeli, phd louisville apl...

UPDATE IN THE EVALUATION OF UPDATE IN THE EVALUATION OF ANTIPHOSPHOLIPID ANTIBODIESANTIPHOSPHOLIPID ANTIBODIES

SILVIA S. PIERANGELI, PhDSILVIA S. PIERANGELI, PhD

LOUISVILLE APL DIAGNOSTICS, INCLOUISVILLE APL DIAGNOSTICS, INC

DORAVILLE, GA USADORAVILLE, GA USA

Louisville APL Diagnostics, Inc

Louisville APL Diagnostics

1984-19851984-1985

Problems with false positive resultsProblems with false positive results

aCL positive in a wide variety of infectious diseases aCL positive in a wide variety of infectious diseases and in non-APS related autoimmune diseases. and in non-APS related autoimmune diseases.


ANTIPHOSPHOLIPID SYNDROMEANTIPHOSPHOLIPID SYNDROMEDiagnostic TestingDiagnostic Testing

1.) Laboratory confirmation is vital for diagnosis of APS.1.) Laboratory confirmation is vital for diagnosis of APS.

2.) Anticardiolipin antibody and lupus anticoagulant tests together 2.) Anticardiolipin antibody and lupus anticoagulant tests together diagnose vast majority of APS patients-these tests are relatively diagnose vast majority of APS patients-these tests are relatively standard and well understood.standard and well understood.

3.) More specific test for APS should be developed and broadly tested 3.) More specific test for APS should be developed and broadly tested (include anti-cardiolipin positive/APS negative sera in testing).(include anti-cardiolipin positive/APS negative sera in testing).

4.) More specific tests may enable confirmation of diagnosis in 4.) More specific tests may enable confirmation of diagnosis in equivocal and unusual clinical presentations to APS.equivocal and unusual clinical presentations to APS.


ANTIPHOSPHOLIPID SYNDROMEANTIPHOSPHOLIPID SYNDROMELaboratory TestsLaboratory Tests

Tests should be positive in most patients with disorder Tests should be positive in most patients with disorder (sensitivity).(sensitivity).

Tests should be largely confined to patients with disorder (specificity)Tests should be largely confined to patients with disorder (specificity)

Tests should be performed reproducibly in most laboratoriesTests should be performed reproducibly in most laboratories. .


APhLAPhL®®ELISA KitELISA Kit - Principle - Principle

Based on observation that antiphospholipid Based on observation that antiphospholipid antibodies crossreact with negatively charged antibodies crossreact with negatively charged phospholipids but syphilis and other infectious phospholipids but syphilis and other infectious diseases sera largely limited to cardiolipin binding diseases sera largely limited to cardiolipin binding (no crossreactivity)(no crossreactivity)

Construction of a kit with negatively charged Construction of a kit with negatively charged phospholipids might eliminate non-specific binding. phospholipids might eliminate non-specific binding.


ANTI-CARDIOLIPIN ANTIBODIESANTI-CARDIOLIPIN ANTIBODIES

BINDSBINDS

CardiolipinCardiolipin

PhosphatidylserinePhosphatidylserine

PhosphatidylglycerolPhosphatidylglycerol

Phosphatidic AcidPhosphatidic Acid

DOES NOT BINDDOES NOT BIND

PhosphatidylcholinePhosphatidylcholine

?phosphatidylethanolamine?phosphatidylethanolamine


Sensitivity of the assaysSensitivity of the assays

Relative Sensitivity/SpecificityRelative Sensitivity/Specificity

Assay # of APS samples tested

# of APS samples that tested positive

Sensitivity (%)

ACL ELISA 54 54 100

APhL®ELISA

54 53 98

anti- 2GP1 54 40 74


Prevalence of aCL, APL and anti-Prevalence of aCL, APL and anti-22GPI in various GPI in various

infectious diseasesinfectious diseases

Diagnosis ACL ELISA APhL®ELISA anti- 2GP1

Kala-azar 3/30(10) 0/30(0%) 16/30(53%)

Syphilis (69)

23/69(32%) 2/70(2.9%) 8/70(11.4%)

Leptospirosis (33)

13/33(33%) 3/33(9%) 6/34(17%)

Specificity 73% 96.3% 70%


Evaluation of different assays to Evaluation of different assays to diagnose APS: Multicenter study.diagnose APS: Multicenter study.

Samples testedSamples tested: :

APS=56APS=56

Healthy controls=145Healthy controls=145

Non-APS including infectious diseases (syphilis, HIV, other Non-APS including infectious diseases (syphilis, HIV, other autoimmune diseases)= 206.autoimmune diseases)= 206.


Evaluation of different assays to diagnose APS.Evaluation of different assays to diagnose APS.

*Centers involved and assays utilized:*Centers involved and assays utilized:

Univ Texas San Antonio: Univ Texas San Antonio: APhLAPhL®® ELISA ELISA test. test.

Morehouse School of Medicine: aCL (in-house) and anti-Morehouse School of Medicine: aCL (in-house) and anti-22GPI (INOVA)GPI (INOVA)

University of Milan: in-house anti- University of Milan: in-house anti- 22GPI and anti-prothrombin.GPI and anti-prothrombin.

* participating centers were blind to the identity of the samples* participating centers were blind to the identity of the samples

Univ of UtahUniv of Utah: : Coordinating centerCoordinating center. .


Sensitivity and Specificity of the Sensitivity and Specificity of the assaysassays

AssayAssay APS samplesAPS samples

# positives# positives

(sensitivity)(sensitivity)

Healthy Healthy controlscontrols


Other Other diseasesdiseases


Non-APS Non-APS combinedcombined

# # positivespositives

SpecificitySpecificity Positive Positive Predictive Predictive

valuevalue

aCL aCL

(in-house)(in-house)

42/56 (75%) 42/56 (75%) 1/150 (0.6%)1/150 (0.6%) 27/206 (13%)27/206 (13%) 28/356 28/356 (7.8%)(7.8%)

91.8%91.8% 59.1%59.1%

aaGPI GPI (INOVA)(INOVA)

44/56 44/56 (78.5%)(78.5%)

15/143 15/143 (10.5%)(10.5%)

58/197 58/197 (29.4%)(29.4%)

73/340 73/340 (21.4%)(21.4%)

78.6%78.6% 37.6%37.6%

aPT (in-aPT (in-house)house)

13/54 (24%)13/54 (24%) 15/132 15/132 (11.3%)(11.3%)

19/178 19/178 (10.7%)(10.7%)

34/310 34/310 (10.9%)(10.9%)

88.4%88.4% 25.4%25.4%

aaGP(in-GP(in-house)house)

43/5443/54

(79.6%)(79.6%)

13/14513/145

(8.9%)(8.9%)

9/192 (4.6%)9/192 (4.6%) 22/337 22/337 (6.5%)(6.5%)

93.2%93.2% 65.1%65.1%

APhL ELISA APhL ELISA kitkit

45/56 45/56 (80.3%)(80.3%)

4/147 (2.7%)4/147 (2.7%) 14/204 14/204 (6.8%)(6.8%)

18/351 18/351 (5.1%)(5.1%)

94.0%94.0% 68.1%68.1%


Evaluation of CAP survey samples in Evaluation of CAP survey samples in FDA aCL approved assaysFDA aCL approved assays

SampleSample Kit 1Kit 1 Kit 2Kit 2 Kit 3Kit 3 APhL APhL ResultResult

99-499-4 negneg negneg negneg negneg negneg

99-699-6 94.294.2 95.695.6 >200>200 >200>200 pospos



00-300-3 93.393.3 9191 66.766.7 69.869.8 pospos

01-401-4 50.150.1 56.156.1 50.050.0 50.950.9 pospos


01-601-6 24.424.4 15.415.4 26.526.5 26.426.4 pospos



02-302-3 negneg negneg negneg negneg NegNeg


CAP survey results: IgG APhL ELISACAP survey results: IgG APhL ELISA

Sample CAP median low high APhL ELISA Apls result interpretation ACL-01 56 42 88 76.8 positive OK ACL-02 60 28 91 56.6 positive OK

ACL-03* 14 10 18 <10 negative *


CAP survey: IgM APhLCAP survey: IgM APhL

Sample CAP median Low High APhL ELISA IgM APhL ELISA IgM Interpretation

ACL-01* 10 1 27 3.1 negative * ACL-02* 21 4 51 20.3 positive * ACL-03 2 0 4 0 negative OK


APhLAPhL®®ELISA kitELISA kit

Six pre-diluted calibrators (ready to use)Six pre-diluted calibrators (ready to use)

3 x 30 minutes incubation steps3 x 30 minutes incubation steps

Peroxidase and alkaline phosphatase systems availablePeroxidase and alkaline phosphatase systems available

All other reagents in “ready-to-use” formAll other reagents in “ready-to-use” form

Determination of IgG and IgM aPL antibodiesDetermination of IgG and IgM aPL antibodies

12 month expiration date.12 month expiration date.

Good to be used in automated systems.Good to be used in automated systems.


APhLAPhL®®ELISA kitELISA kit

Antigen composed fo mixture of phospholipids - Antigen composed fo mixture of phospholipids - ßß22GP1GP1

Sensitivity of APS (greater than 90%)Sensitivity of APS (greater than 90%)

More specific than anticardiolipin test and at least as More specific than anticardiolipin test and at least as specific (or more) compared to anti-ßspecific (or more) compared to anti-ß22GP1GP1

Incorporation of an in-house positive controlIncorporation of an in-house positive control

Can be utilized for first line testing, and certainly Can be utilized for first line testing, and certainly important in confirmation of APSimportant in confirmation of APS


Conclusion Conclusion

Since sensitivity ofSince sensitivity of APhLAPhL®®ELISA kit ELISA kit is is comparable to anticardiolipin test, it can comparable to anticardiolipin test, it can be used for first line testing in place of the be used for first line testing in place of the anticardiolipin test. The anticardiolipin test. The APhLAPhL®®ELISA kit ELISA kit will enable greater specificity - APS will enable greater specificity - APS diagnosisdiagnosis

Clinical Features of APS

Anticardiolipin (IgG, IgM) and Lupus anticoagulant tests

(APhL ELISA® test)

Negative Anticardiolipinand

Negative Lupus anticoagulantbut

Highly Suggestive Clinical Features

Low Positive Anticardiolipin High/Medium PositiveAnticardiolipin

orPositive

Lupus anticoagulant

anti-2GP1or

APhL ELISA® Test Negative Positive aCL

AntiphospholipidSyndrome


Confirmation with anti-2GP1or

APhL ELISA® test

Both Negative Either Positive

Consider IgAAnticardiolipin


Repeat test

Negative Positive



Anticardiolipin ELISA publicationsAnticardiolipin ELISA publications

Harris EN. Annotation: antiphospholipid antibodies. Br J Haematology. 1990; 74:1-9Harris EN. Annotation: antiphospholipid antibodies. Br J Haematology. 1990; 74:1-9

Harris EN, Pierangeli SS. Anticardiolipin and lupus anticoagulant testing and significance. J Clin Immunol Harris EN, Pierangeli SS. Anticardiolipin and lupus anticoagulant testing and significance. J Clin Immunol 1994; 17:1-8.1994; 17:1-8.

Harris EN, Pierangeli SS, Birch D. Anticardiolipin wet workshop report: Vth International Symposium on Harris EN, Pierangeli SS, Birch D. Anticardiolipin wet workshop report: Vth International Symposium on Antiphospholipid antibdies. AM J Clin Pathol 1994: 101: 616-624.Antiphospholipid antibdies. AM J Clin Pathol 1994: 101: 616-624.

Harris EN, Pierangeli SS. “Equivocal” Antiphospholipid Syndrome. J Autoimm. 2000; 15:81-85.Harris EN, Pierangeli SS. “Equivocal” Antiphospholipid Syndrome. J Autoimm. 2000; 15:81-85.

Pierangeli SS, Gharavi AE, Harris EN. Testing for antiphospholipid antibodies: problems and solutions. Pierangeli SS, Gharavi AE, Harris EN. Testing for antiphospholipid antibodies: problems and solutions. Clin Obstet Gynecol 2001:44:48-57.Clin Obstet Gynecol 2001:44:48-57.

Harris EN, Pierangeli SS. Revisiting the anticardiolipin test and its standardization. Lupus 2002; 11:269-Harris EN, Pierangeli SS. Revisiting the anticardiolipin test and its standardization. Lupus 2002; 11:269-275.275.

Pierangeli SS, Harris EN. Anticardiolipin testing. Clin Chim Acta. 2005; Pierangeli SS, Harris EN. Anticardiolipin testing. Clin Chim Acta. 2005;


ELISAs for IgG and IgM aPLELISAs for IgG and IgM aPL

For InVitro Diagnostic Use

3988 FLowers Rd Ste 620Doraville, GA 30360.USA

APhLAPhL ®® ELISA Kit ELISA Kit(IgG and IgM)

FOR THE MEASUREMENT OF ANTIPHOSPHOLIPID ANTIBODIES

1-800-624-3192CUSTOMER SERVICE

email: [email protected]: [email protected]

website: www.louisvilleapl.com

Louisville APL Diagnostics, Inc.

770-455-7129PHONE

770-455-6499FAX


ELISAs for IgG or IgM aPLELISAs for IgG or IgM aPL


ELISAs for IgA aPLELISAs for IgA aPL


Louisville APL Diagnostics, IncLouisville APL Diagnostics, Incweb site: web site: www.louisvilleapl.comwww.louisvilleapl.comemail:[email protected]:[email protected]


Silvia S. Pierangeli, Ph.D.Silvia S. Pierangeli, Ph.D.

update in the evaluation of antiphospholipid antibodies silvia s. pierangeli, phd louisville apl...

Documents

n phosphatidylcholine

n samples

n problems

n nonaps

n healthy controls

disorder specificity

crossreactivity n construction

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