1. Anti-Phospholipid Syndrome (APS)
Laboratory Diagnosis
of the
Anti-Phospholipid Syndrome (APS)
Olivier Morboeuf, Ph.D
Diagnostica Stago
Asnières / Seine, France

2. (Wilson et al. Arthritis Rheum. 1999)
Sapporo criteria 1999
(Wilson et al. Arthritis Rheum. 1999)
Laboratory criteria
Clinical criteria
auto-antibodies
vascular thrombosis + LA
ACA
pregnancy morbidity
APS

3. APS is a syndrome with a wrong name !
Specificity of antiphospholipid antibodies (Galli et al. Lancet 1990; Mc Neil et al. 1990)
auto-antibody
auto-antibody
target protein
anionic phospholipids
anionic phospholipids
APS is a syndrome with a wrong name !

4. Possible target proteins for antiphospholipid antibodies binding to anionic phospholipids
b2-glycoprotein I
prothrombin
protein S
protein C
EPCR
HMWK
factor XII
Annexin V
TFPI
complement factor H
phospholipases
tPA …

5. b2-glycoprotein I structure : 5 "sushi" domains synthesis : liver
function : "in vitro" anticoagulant
congenital deficiency : asymptomatic
b2-glycoprotein I

6. Generally accepted as the clinical relevant antibodies
Antibodies against b2-glycoprotein I
binding site for
anionic phospholipids
b2-glycoprotein I
Generally accepted as the clinical relevant antibodies

7. Antibodies against b2-glycoprotein I (de Laat et al
Antibodies against b2-glycoprotein I (de Laat et al. Blood 2006; 107 : ) I II
III IV V
auto-antibody
b2GPI I II
III IV V
stabilisation
Kd dimer b2GPI
5 nM
affinity
(surface-dependent) I II
III IV V
Kd b2GPI
170 nM I II
III IV V
conformational
change
Auto-antibodies increase the affinity of b2-glycoprotein I for anionic phospholipids

8. LA, ACA and anti-b2GPI antibodies are related antibodies with overlapping specificity but they are not identical antibodies
b2GP1 LA
ACA
anti-b2GPI

9. Antiphospholipid Profile & APS: Multivariate Analysis of 100 Patients
0,1 1 10
100
1000
LA/ACA/ab2GPI Any thrombosis
ACA/ab2GPI Abortions
LA Any thrombosis
Antibody positivity Clinical scenario
ACA/ab2GPI Any thrombosis
ACA Any thrombosis
ab2GPI Any thrombosis
OR, 95% CI
(Pengo et al. Thromb. Haemost. 2005; 93 : )

10. + Revised Sapporo criteria 2006 APS Laboratory criteria
(Miyakis et al. J. Thromb. Haemost. 2006)
Laboratory criteria
Clinical criteria
vascular thrombosis + LA
ACA
pregnancy morbidity
anti-b2GPI
APS

11. Revised Sapporo criteria 2006
Laboratory Criteria
(Miyakis et al. J. Thromb. Haemost. 2006)
LA : - present in plasma
- 2 or more occasions, 12 weeks apart
- detected according ISTH guidelines
and/or
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
and/or
anti-b2GP1 : - IgG and/or IgM
- titer > 99th percentile
- present in plasma or serum
- 2 or more occasions, 12 weeks apart
- measured by standardized ELISA

12. APS patient classification based on laboratory findings
(Miyakis et al. J. Thromb. Haemost. 2006)
2 main categories:
Category I:
→ More than one laboratory criteria present
(LA, ACA , anti-b2GP1)
Category II :
→ IIa : LA present alone
→ IIb : ACA present alone
→ IIc : anti-b2GP1 present alone

13. Proposed pathogenic mechanisms of antiphospholipid antibodies
(Franchini Clin. Lab. 2006)
Interference with the function of the coagulation cascade
- inhibition of b2-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

14. 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 &
b2-glycoprotein 1 bound to membrane phospholipids
with further vascular damage and platelet activation

15. Laboratory diagnosis of APS
Lupus
Anticoagulant
(LA)
Detected by phospholipid-
-dependent clotting tests
Antiphospholipid
Antibodies
(ACA, anti-b2GPI)
Detected by ELISA

16. Detected by phospholipid- -dependent clotting tests
Lupus
Anticoagulant
(LA)
Detected by phospholipid-
-dependent clotting tests

17. Laboratory diagnosis of LA
No screening reagent is 100% sensitive and
100% specific
International recommendations by the Scientific
Subcommittee (SSC) on LA

18. Incubated mixing study
Stop evaluation for LA
- Sensitive to LA
- 2 different principles
- Low PL concentration
Test 1 
Screening tests
Test 2
Normal
LA testing flow chart following
international recommendations
normal plasma +
patient plasma 
Mixing study
Incubated mixing study
Corrects
Abnormal
Factor assays
factor deficiency
Specific
inhibitor
Test 1
Test 2 
Confirmatory tests
LA confirmed
Failure to correct
Positive
Negative
Phospholipid
dependance

19. LA-sensitive APTT reagent
Screening test : PTT-LA
LA-sensitive APTT reagent
Ph. Cauchie :
" PTT-LA is the most sensitive APTT reagent "
(Cauchie et al., Thromb. Haemost. 1993; 69,N°6)
J. Arnout :
“ PTT-LA clearly showed the highest responsiveness among
the APTT reagents. It is clear that the LA sensitivity of the
APTT largely depends on the reagents used”
(Arnout at al., Thromb. Haemost. 1999; 81: 929)

20. LA Diagnosis Screening tests

21. APTT reagents sensitivity
towards LA
PTT-LA Silimat PTT-A Platelin APTT ActinFSL
" PTT-LA is the most sensitive APTT reagent "
(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

22. Screening test : DRVV Screen Reagent
- RVV = FX activator
- phospholipid
- calcium
- heparin inhibitor
- High specificity :
- Insensitive to FVII, FXII, FXI, FIX and FVIII deficiencies
- Insensitive to FVIII inhibitors
- Insensitive to anticoagulants within therapeutic ranges

23. Combination of APTT and DRVV tests for LA screening
PTT-LA Silimat PTT-A Platelin APTT ActinFSL
" 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."
(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

24. Combination of APTT, DRVVt and KCT for LA screening
P. Cauchie. High sensitivity of a new APTT reagent in Lupus anticoagulant detection. XIVth congres of the ISTH, Thromb Haemost, 1993, 69,N°6.

25. LA Diagnosis Confirmatory tests

26. (Triplett et al. Thromb. Haemost. 1993; 70, 787-793)
Confirmatory test : Staclot® LA Sensitivity 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 ; 70, )

27. Confirmatory test : DRVV Confirm reagent
- RVV = FX activator
- phospholipid +++
- calcium
- heparin inhibitor

28. Incubated mixing study
Test 1:
Screening tests
Test 2:
Stop evaluation for LA
Normal
LA testing flow chart following
international recommendations
PTT-LA
Abnormal
normal plasma +
patient plasma
Mixing study
100% specificity
SSC on LA, 1995
STA Staclot
DRVV Screen
Failure to correct
Test 1:
Test 2:
Confirmatory tests
Incubated mixing study
Corrects
Staclot LA
Factor assays
factor deficiency
Corrects
Positive
LA confirmed
100% sensitivity
Cauchie P.
Thromb. Haemost, 1993.
STA Staclot
DRVV Confirm
Negative
Specific
inhibitor

29. Antiphospholipid
Antibodies
(ACA, anti-b2GPI)
Detected by ELISA

30. Minimal requirements for APA ELISAs
(Tincani et al. Thromb. Res. 2004; 114 : )
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

31. Asserachrom® APA line 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)
cardiolipin
phosphatidylserine
phosphatidic acid
b2GP1
1/ TMB
+ wait for
5 min.
2/ H2SO4
sample
dil. 1:101
30 min.
room temp.
+ wash (5X)
anti-human IgG,M,(A)
coupled with
peroxidase
read at
450 nm
APA

32. Asserachrom® APA line Screening for anti-b2GP1 antibodies
Asserachrom® Anti-b2GP1 IgG
Quantitative determination of anti-b2GP1 antibodies (IgG)
Asserachrom® Anti-b2GP1 IgM
Quantitative determination of anti-b2GP1 antibodies (IgM)
human b2GP1
1/ TMB
+ wait for
5 min.
2/ H2SO4
sample
dil. 1:101
1 hour
room temp.
+ wash (5X)
anti-human IgG or IgM
coupled with
peroxidase
read at
450 nm
anti-b2GP1

33. Correlation between Stago calibrators and HCAL / EY2C9
(Woodhams et al. Thromb. Res. 2004; 114 : 669)

34. Conclusion LA correlates best with thrombotic complications
b2Glycoprotein 1 is the relevant antigen in the diagnostic assays (LA, ACA, anti-b2GP1)
Anti-b2GP1 assay is more specific than ACA assay for APS diagnosis
Revised Sapporo laboratory criteria should demonstrate an improvement of APS diagnosis

35. Thank you
for
your attention !