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© 2006 Diagnostica Stago - All rights reservedDIAGNOSTICASTAGO
Laboratory Diagnosisof the
Anti-Phospholipid Syndrome (APS)
Olivier Morboeuf, Ph.D
Diagnostica StagoAsnières / Seine, France
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APS
Clinical criteria
Sapporo criteria 1999(Wilson et al. Arthritis Rheum. 1999)
Laboratory criteria
auto-antibodies
ACALA
vascular thrombosis
pregnancy morbidity
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Specificity of antiphospholipid antibodies(Galli et al. Lancet 1990; Mc Neil et al. 1990)
APS is a syndrome with a wrong name !
auto-antibody
anionic phospholipids
target protein
auto-antibody
anionic phospholipids
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Possible target proteins for antiphospholipid antibodies binding to
anionic phospholipids
2-glycoprotein I prothrombin • protein S• protein C• EPCR• HMWK
• factor XII• Annexin V• TFPI• complement factor H• phospholipases• tPA …
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2-glycoprotein I
2-glycoprotein I
• structure : 5 "sushi" domains
• synthesis : liver
• function : "in vitro" anticoagulant
• congenital deficiency : asymptomatic
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Generally accepted as the clinical relevant antibodies
Antibodies against 2-glycoprotein I
2-glycoprotein I
binding site foranionic phospholipids
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Antibodies against 2-glycoprotein I(de Laat et al. Blood 2006; 107 : 1916-1924)
Auto-antibodies increase the affinity of -glycoprotein I
for anionic phospholipids
I
II
III
IVV
auto-antibody2GPI
I
II
III
IVV
conformationalchange
affinity(surface-dependent) I
II
III
IVV
Kd 2GPI170 nM
I
II
III
IVV
I
II
III
IV
stabilisation Kd dimer 2GPI5 nM
V
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LA, ACA and anti-2GPI antibodies are related antibodies with overlapping specificity but they are not
identical antibodies
ACALA
anti-2GPI
2GP1
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0,1 1 10 100 1000
LA/ACA/a2GPI Any thrombosis
ACA/a2GPI Abortions
LA Any thrombosis
Antibody positivity Clinical scenario
ACA/a2GPI Any thrombosis
ACA Any thrombosis
a2GPI Any thrombosis
(Pengo et al. Thromb. Haemost. 2005; 93 : 1147-1152)
Antiphospholipid Profile & APS: Multivariate Analysis of 100 Patients
OR, 95% CI
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Revised Sapporo criteria 2006(Miyakis et al. J. Thromb. Haemost. 2006)
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APS
Clinical criteria Laboratory criteria
ACALAvascular thrombosis
pregnancy morbidity
anti-2GPI
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Revised Sapporo criteria 2006Laboratory Criteria
(Miyakis et al. J. Thromb. Haemost. 2006)
LA : - present in plasma- 2 or more occasions, 12 weeks apart- detected according ISTH guidelines
ACA : - IgG and/or IgM- titer > 40 GPL (or MPL) or > 99th percentile- present in plasma or serum- 2 or more occasions, 12 weeks apart- measured by standardized ELISA
anti-2GP1 : - IgG and/or IgM- titer > 99th percentile- present in plasma or serum- 2 or more occasions, 12 weeks apart- measured by standardized ELISA
and/or
and/or
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APS patient classificationbased on laboratory
findings(Miyakis et al. J. Thromb. Haemost. 2006)
2 main categories:
• Category I:
→ More than one laboratory criteria present (LA, ACA , anti-2GP1)
• Category II :
→ IIa : LA present alone→ IIb : ACA present alone→ IIc : anti-2GP1 present alone
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Proposed pathogenic mechanisms of antiphospholipid antibodies
(Franchini Clin. Lab. 2006)
• Interference with the function of the coagulation cascade- inhibition of 2-glycoprotein 1- decreased activation of PC- inhibition of AT activity- inhibition of fibrinolysis- inhibition of annexin V binding- upregulation of tissue factor activity
• Activation of endothelial cells- increased expression of adhesion molecules- increased expression of tissue factor- secretion of proinflammatory cytokines
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Proposed pathogenic mechanisms of antiphospholipid antibodies
(Franchini Clin. Lab. 2006)
• Activation of platelets and stimulation of platelet aggregation- increased synthesis of thromboxane A2
• "Heparin-induced thrombocytopenia"-like mechanism- vascular damage with exposure to negatively charged phospholipids- formation of immune complexes between APA & 2-glycoprotein 1 bound to membrane phospholipids with further vascular damage and platelet activation
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Lupus
Anticoagulant
(LA)
Detected by phospholipid-
-dependent clotting tests
AntiphospholipidAntibodies
(ACA, anti-2GPI)
Detected by ELISA
Laboratory diagnosis of APS
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Lupus
Anticoagulant
(LA)
Detected by phospholipid-
-dependent clotting tests
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Laboratory diagnosis of LA
• No screening reagent is 100% sensitive and 100% specific
• International recommendations by the Scientific Subcommittee (SSC) on LA
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LA testing flow chart following international recommendations
normal plasma +patient plasma
Mixing study
Incubated mixing study
Corrects
Abnormal
Factor assays
factor deficiency
Corrects
Specific inhibitor
Test 1
Test 2
Confirmatory
tests
LA confirmed
Failure to correct
Positive
Negative
Phospholipiddependance
Stop evaluation for LA
- Sensitive to LA- 2 different principles- Low PL concentration
Test 1
Screening
tests
Test 2
Normal
Normal
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LA DiagnosisScreening tests
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(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)
" PTT-LA is the most sensitive APTT reagent "
APTT reagents sensitivitytowards LA
0
20
40
60
80
100
PTT-LA V W X Y Z
APTT
PTT-LA Silimat PTT-A Platelin APTT ActinFSL
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Screening test :DRVV Screen Reagent
- Insensitive to FVII, FXII, FXI, FIX and FVIII deficiencies
- Insensitive to FVIII inhibitors- Insensitive to anticoagulants within therapeutic
ranges
- High specificity :
- Screen reagent : - RVV = FX activator
- phospholipid- calcium- heparin inhibitor
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" PTT-LA (87% positive) and DRVVT (59% positive) may be discordant, but the combination of these two tests provide a very sensitive procedure, and immediately available."
0
20
40
60
80
100
PTT-LA V W X Y Z
DRVVT
APTT
Combination of APTT and DRVV tests for LA screening
PTT-LA Silimat PTT-A Platelin APTT ActinFSL
(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)
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0
20
40
60
80
100
PTT-LA Silimat PTT-A Platelin APTT ActinFSL
KCT
DRVVT
APTT
P. Cauchie. High sensitivity of a new APTT reagent in Lupus anticoagulant detection. XIVth congres of the ISTH, Thromb Haemost, 1993, 69,N°6.
Combination of APTT, DRVVt and KCT for LA screening
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LA DiagnosisConfirmatory tests
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Confirmatory test : Staclot® LASensitivity and specificity close to 100%
• Hexagonal phase phospholipids• Sensitive and specific to LA
– Diagnoses LA at low titer– Eliminates false positive
• Insensitive to heparin– Allows direct diagnosis of LA in heparinized
patients
• Insensitive to factor deficiencies– Allows LA testing in warfarin patients, patients with
specific factor deficiency or inhibitor
(Triplett et al. Thromb. Haemost. 1993; 70, 787-793)
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Confirmatory test :DRVV Confirm reagent
Confirm reagent :
- RVV = FX activator- phospholipid +++- calcium- heparin inhibitor
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AntiphospholipidAntibodies
(ACA, anti-2GPI)
Detected by ELISA
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Minimal requirements for APA ELISAs(Tincani et al. Thromb. Res. 2004; 114 : 553-558)
• to run the samples in duplicate
• to determine the cut-off level in each laboratory with at least 50 samples from normal subjects
• to calculate the cut-off level in percentiles
• to use humanized monoclonal antibodies as calibrators and controls (proposal : HCAL for IgG and EY2C9 for IgM)
• to use arbitrary units (eg. MAU/GAU; MPL/GPL)
standardization = decrease the inter-laboratory variability
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Screening for Antiphospholipid antibodies :Asserachrom® APA Screen
Qualitative determination of APA (IgG, IgM & IgA)
Screening for ACA/APA : Asserachrom® APA IgG,M
Quantitative determination of APA (IgG and/or IgM)
Asserachrom® APA line
cardiolipinphosphatidylserinephosphatidic acid
2GP1
1/ TMB+ wait for5 min.
2/ H2SO4sampledil. 1:101
30 min.room temp.+ wash (5X)
anti-human IgG,M,(A)coupled withperoxidase
30 min.room temp.+ wash (5X)
read at450 nm
APA
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Asserachrom® APA line
Screening for anti-2GP1 antibodiesAsserachrom® Anti-2GP1 IgG
Quantitative determination of anti-2GP1 antibodies (IgG)
Asserachrom® Anti-2GP1 IgM Quantitative determination of anti-2GP1 antibodies (IgM)
human 2GP1
1/ TMB+ wait for5 min.
2/ H2SO4sampledil. 1:101
1 hourroom temp.+ wash (5X)
anti-human IgG or IgMcoupled withperoxidase
1 hourroom temp.+ wash (5X)
read at450 nm
anti-2GP1
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LA correlates best with thrombotic complications
2Glycoprotein 1 is the relevant antigen in the diagnostic assays (LA,
ACA, anti-2GP1)
Anti-2GP1 assay is more specific than ACA assay for APS diagnosis
Revised Sapporo laboratory criteria should demonstrate an improvement of
APS diagnosis
Conclusion
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Thank youfor
your attention !