UPDATE IN THE EVALUATION OF ANTIPHOSPHOLIPID ANTIBODIES

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UPDATE IN THE EVALUATION OF ANTIPHOSPHOLIPID
ANTIBODIES

• SILVIA S. PIERANGELI, PhD
• LOUISVILLE APL DIAGNOSTICS, INC
• DORAVILLE, GA USA

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1984-1985

• Problems with false positive results
• aCL positive in a wide variety of infectious
  diseases and in non-APS related autoimmune
  diseases.

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ANTIPHOSPHOLIPID SYNDROMEDiagnostic Testing

• 1.) Laboratory confirmation is vital for
  diagnosis of APS.
• 2.) Anticardiolipin antibody and lupus
  anticoagulant tests together diagnose vast
  majority of APS patients-these tests are
  relatively standard and well understood.
• 3.) More specific test for APS should be
  developed and broadly tested (include
  anti-cardiolipin positive/APS negative sera in
  testing).
• 4.) More specific tests may enable confirmation
  of diagnosis in equivocal and unusual clinical
  presentations to APS.

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ANTIPHOSPHOLIPID SYNDROMELaboratory Tests

• Tests should be positive in most patients with
  disorder (sensitivity).
• Tests should be largely confined to patients with
  disorder (specificity)
• Tests should be performed reproducibly in most
  laboratories.

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APhLELISA Kit - Principle

• Based on observation that antiphospholipid
  antibodies crossreact with negatively charged
  phospholipids but syphilis and other infectious
  diseases sera largely limited to cardiolipin
  binding (no crossreactivity)
• Construction of a kit with negatively charged
  phospholipids might eliminate non-specific
  binding.

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ANTI-CARDIOLIPIN ANTIBODIES

• BINDS
• Cardiolipin
• Phosphatidylserine
• Phosphatidylglycerol
• Phosphatidic Acid

• DOES NOT BIND
• Phosphatidylcholine
• ?phosphatidylethanolamine

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Relative Sensitivity/Specificity
Sensitivity of the assays
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Prevalence of aCL, APL and anti-?2GPI in various
infectious diseases
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Evaluation of different assays to diagnose
APS Multicenter study.

• Samples tested
• APS56
• Healthy controls145
• Non-APS including infectious diseases (syphilis,
  HIV, other autoimmune diseases) 206.

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Evaluation of different assays to diagnose APS.

• Centers involved and assays utilized
• Univ Texas San Antonio APhL ELISA test.
• Morehouse School of Medicine aCL (in-house) and
  anti-?2GPI (INOVA)
• University of Milan in-house anti- ?2GPI and
  anti-prothrombin.
• participating centers were blind to the
  identity of the samples
• Univ of Utah Coordinating center.

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Sensitivity and Specificity of the assays
Assay APS samples positives (sensitivity) Healthy controls positives Other diseases positives Non-APS combined positives Specificity Positive Predictive value
aCL (in-house) 42/56 (75) 1/150 (0.6) 27/206 (13) 28/356 (7.8) 91.8 59.1
a??GPI (INOVA) 44/56 (78.5) 15/143 (10.5) 58/197 (29.4) 73/340 (21.4) 78.6 37.6
aPT (in-house) 13/54 (24) 15/132 (11.3) 19/178 (10.7) 34/310 (10.9) 88.4 25.4
a??GP(in-house) 43/54 (79.6) 13/145 (8.9) 9/192 (4.6) 22/337 (6.5) 93.2 65.1
APhL ELISA kit 45/56 (80.3) 4/147 (2.7) 14/204 (6.8) 18/351 (5.1) 94.0 68.1
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Evaluation of CAP survey samples in FDA aCL
approved assays
Sample Kit 1 Kit 2 Kit 3 APhL Result
99-4 neg neg neg neg neg
99-6 94.2 95.6 gt200 gt200 pos
00-1 neg neg neg neg neg
00-2 neg neg neg neg neg
00-3 93.3 91 66.7 69.8 pos
01-4 50.1 56.1 50.0 50.9 pos
01-5 neg neg neg neg neg
01-6 24.4 15.4 26.5 26.4 pos
02-1 neg neg neg neg neg
02-2 neg neg neg neg neg
02-3 neg neg neg neg Neg
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CAP survey results IgG APhL ELISA
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CAP survey IgM APhL
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APhLELISA kit

• Six pre-diluted calibrators (ready to use)
• 3 x 30 minutes incubation steps
• Peroxidase and alkaline phosphatase systems
  available
• All other reagents in ready-to-use form
• Determination of IgG and IgM aPL antibodies
• 12 month expiration date.
• Good to be used in automated systems.

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APhLELISA kit

• Antigen composed fo mixture of phospholipids -
  ß2GP1
• Sensitivity of APS (greater than 90)
• More specific than anticardiolipin test and at
  least as specific (or more) compared to
  anti-ß2GP1
• Incorporation of an in-house positive control
• Can be utilized for first line testing, and
  certainly important in confirmation of APS

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Conclusion

• Since sensitivity of APhLELISA kit is
  comparable to anticardiolipin test, it can be
  used for first line testing in place of the
  anticardiolipin test. The APhLELISA kit will
  enable greater specificity - APS diagnosis

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Clinical Features of APS
Anticardiolipin (IgG, IgM) and Lupus
anticoagulant tests (APhL ELISA test)
High/Medium PositiveAnticardiolipinorPositiveL
upus anticoagulant
Negative AnticardiolipinandNegative Lupus
anticoagulantbutHighly Suggestive Clinical
Features
Low Positive Anticardiolipin
Repeat test
anti-?2GP1orAPhL ELISA Test
AntiphospholipidSyndrome
Negative
Positive aCL
AntiphospholipidSyndrome
Both Negative
Either Positive
Consider IgAAnticardiolipin
AntiphospholipidSyndrome
Confirmation with anti-?2GP1orAPhL ELISA test
Negative
Positive
AntiphospholipidSyndrome
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Anticardiolipin ELISA publications

• Harris EN. Annotation antiphospholipid
  antibodies. Br J Haematology. 1990 741-9
• Harris EN, Pierangeli SS. Anticardiolipin and
  lupus anticoagulant testing and significance. J
  Clin Immunol 1994 171-8.
• Harris EN, Pierangeli SS, Birch D.
  Anticardiolipin wet workshop report Vth
  International Symposium on Antiphospholipid
  antibdies. AM J Clin Pathol 1994 101 616-624.
• Harris EN, Pierangeli SS. Equivocal
  Antiphospholipid Syndrome. J Autoimm. 2000
  1581-85.
• Pierangeli SS, Gharavi AE, Harris EN. Testing for
  antiphospholipid antibodies problems and
  solutions. Clin Obstet Gynecol 20014448-57.
• Harris EN, Pierangeli SS. Revisiting the
  anticardiolipin test and its standardization.
  Lupus 2002 11269-275.
• Pierangeli SS, Harris EN. Anticardiolipin
  testing. Clin Chim Acta. 2005

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ELISAs for IgG and IgM aPL
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ELISAs for IgG or IgM aPL
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ELISAs for IgA aPL
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Louisville APL Diagnostics, Incweb site
www.louisvilleapl.comemailsupport_at_louisvilleapl.
com
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Silvia S. Pierangeli, Ph.D.