Laboratory Diagnosis of The Anti-Phospholipid Syndrome (APS)

Laboratory Diagnosis
of the
Anti-Phospholipid Syndrome (APS)
Olivier Morboeuf, Ph.D
Diagnostica Stago
Asnières / Seine, France
DIAGNOSTICA
STAGO © 2006 Diagnostica Stago - All rights reserved
Sapporo criteria 1999
(Wilson et al. Arthritis Rheum. 1999)
Clinical criteria Laboratory criteria

auto-antibodies
vascular thrombosis
+ LA ACA
pregnancy morbidity
APS
DIAGNOSTICA
Specificity of antiphospholipid antibodies
(Galli et al. Lancet 1990; Mc Neil et al. 1990)
auto-antibody
auto-antibody
target protein
anionic phospholipids
APS is a syndrome with a wrong name !
DIAGNOSTICA
Possible target proteins for
antiphospholipid antibodies binding to
 2-glycoprotein I • factor XII

 prothrombin • Annexin V
• protein S • TFPI
• protein C • complement factor H
• EPCR • phospholipases
• HMWK • tPA …
DIAGNOSTICA
2-glycoprotein I
• structure : 5 "sushi" domains
• synthesis : liver
• function : "in vitro" anticoagulant
• congenital deficiency : asymptomatic
2-glycoprotein I
DIAGNOSTICA
Antibodies against 2-glycoprotein I
binding site for

2-glycoprotein I
Generally accepted as the clinical relevant antibodies

DIAGNOSTICA
Antibodies against 2-glycoprotein I
(de Laat et al. Blood 2006; 107 : 1916-1924)
2GPI II auto-antibody
III
IV
V
affinity I
(surface-dependent) I I I
II conformational II stabilisation II
II
Kd dimer 2GPI
Kd 2GPI change 5 nM
170 nM III III III
III
IV
IV IV IV
V V V V
Auto-antibodies increase the affinity of -glycoprotein I

DIAGNOSTICA for anionic phospholipids
LA, ACA and anti-2GPI antibodies are related
antibodies with overlapping specificity but they are not
identical antibodies
2GP1
LA ACA
anti-2GPI
DIAGNOSTICA
Antiphospholipid Profile & APS:
Multivariate Analysis of 100 Patients
Antibody positivity Clinical scenario
LA/ACA/a2GPI Any thrombosis
ACA/a2GPI Abortions
LA Any thrombosis
a2GPI Any thrombosis
ACA/a2GPI Any thrombosis
ACA Any thrombosis
0,1 1 10 OR, 95% CI 100 1000
DIAGNOSTICA (Pengo et al. Thromb. Haemost. 2005; 93 : 1147-1152)

Revised Sapporo criteria 2006
(Miyakis et al. J. Thromb. Haemost. 2006)
Clinical criteria Laboratory criteria
vascular thrombosis
LA ACA
+
pregnancy morbidity
anti-2GPI
APS
DIAGNOSTICA
Revised Sapporo criteria 2006
Laboratory Criteria
LA : - present in plasma
- 2 or more occasions, 12 weeks apart
and/or - detected according ISTH guidelines
ACA : - IgG and/or IgM

- titer > 40 GPL (or MPL) or > 99th percentile
- present in plasma or serum
and/or - measured by standardized ELISA
anti-2GP1 : - IgG and/or IgM

- titer > 99th percentile
- present in plasma or serum
- measured by standardized ELISA
DIAGNOSTICA
APS patient classification
based on laboratory
findings
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
DIAGNOSTICA
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
DIAGNOSTICA
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
DIAGNOSTICA
Laboratory diagnosis of APS
Lupus Antiphospholipid
Anticoagulant Antibodies
(LA) (ACA, anti-2GPI)
Detected by phospholipid-
Detected by ELISA
-dependent clotting tests
DIAGNOSTICA
Lupus
Anticoagulant
(LA)
Detected by phospholipid-
-dependent clotting tests
DIAGNOSTICA
Laboratory diagnosis of LA
• No screening reagent is 100% sensitive and

100% specific
• International recommendations by the Scientific

Subcommittee (SSC) on LA
DIAGNOSTICA

Screening LA testing flow chart following
tests international recommendations
Test 1

Abnormal Mixing
Normal
study
Test 2 normal plasma +
patient plasma

Normal Failure to Confirmatory Phospholipid
Corrects correct tests dependance
Stop evaluation
for LA Incubated Test 1
mixing study
- Sensitive to LA Positive LA
- 2 different principles confirmed
- Low PL concentration Test 2
Negative
Corrects
Factor assays
factor Specific
DIAGNOSTICA deficiency inhibitor
LA Diagnosis
Screening tests
DIAGNOSTICA
APTT reagents sensitivity
towards LA
100
80
60
APTT
40
20
0
PTT-LA V
PTT-LA Silimat W
PTT-A X
Platelin Y
APTT Z
ActinFSL
" PTT-LA is the most sensitive APTT reagent "
(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

DIAGNOSTICA
Screening test :
DRVV Screen Reagent
- Screen reagent :
- RVV = FX activator
- phospholipid
- calcium
- heparin inhibitor
- High- specificity : FVII, FXII, FXI, FIX and FVIII

Insensitive to
deficiencies
- Insensitive to FVIII inhibitors
- Insensitive to anticoagulants within therapeutic
ranges
DIAGNOSTICA
Combination of APTT and DRVV tests
for LA screening
100
80
60
DRVVT
40 APTT
20
0
PTT-LA V
PTT-LA Silimat W X
PTT-A Platelin Y
APTT Z
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)
DIAGNOSTICA
Combination of APTT, DRVVt and
KCT for LA screening
100
80
60 KCT
DRVVT
40
APTT
20
0
PTT-LA Silimat PTT-A Platelin APTT ActinFSL
P. Cauchie. High sensitivity of a new APTT reagent in Lupus anticoagulant detection.

XIVth congres of the ISTH, Thromb Haemost, 1993, 69,N°6.
DIAGNOSTICA
LA Diagnosis
Confirmatory tests
DIAGNOSTICA
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
DIAGNOSTICA (Triplett et al. Thromb. Haemost. 1993; 70, 787-793)

Confirmatory test :
DRVV Confirm reagent
Confirm reagent :
- RVV = FX activator
- phospholipid +++
- calcium
- heparin inhibitor
DIAGNOSTICA
Antiphospholipid
Antibodies
(ACA, anti-2GPI)
Detected by ELISA
DIAGNOSTICA
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
DIAGNOSTICA
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)
anti-human IgG,M,(A)
sample coupled with
dil. 1:101 peroxidase 2/ H2SO4
1/ TMB
+ wait for
5 min.
2GP1
APA
30 min. 30 min.
cardiolipin room temp. room temp. read at
phosphatidylserine + wash (5X) + wash (5X) 450 nm
phosphatidic acid
DIAGNOSTICA
Asserachrom® APA line
 Screening for anti-2GP1 antibodies
Asserachrom® Anti-2GP1 IgG
Quantitative determination of anti-2GP1 antibodies (IgG)
Asserachrom® Anti-2GP1 IgM
Quantitative determination of anti-2GP1 antibodies (IgM)
anti-human IgG or IgM

sample coupled with
dil. 1:101 peroxidase 2/ H2SO4
1/ TMB
+ wait for
5 min.
anti-2GP1
1 hour 1 hour
human room temp. room temp. read at
2GP1 + wash (5X) + wash (5X) 450 nm
DIAGNOSTICA
Conclusion
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
DIAGNOSTICA
Thank you
for
your attention !
DIAGNOSTICA

Laboratory Diagnosis of The Anti-Phospholipid Syndrome (APS)

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Laboratory Diagnosis of The Anti-Phospholipid Syndrome (APS)

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Laboratory Diagnosis

Olivier Morboeuf, Ph.D

Clinical criteria Laboratory criteria

APS is a syndrome with a wrong name !

 2-glycoprotein I • factor XII

• structure : 5 "sushi" domains

• function : "in vitro" anticoagulant

• congenital deficiency : asymptomatic

binding site for

Generally accepted as the clinical relevant antibodies

Auto-antibodies increase the affinity of -glycoprotein I

Antibody positivity Clinical scenario

LA/ACA/a2GPI Any thrombosis

a2GPI Any thrombosis

ACA/a2GPI Any thrombosis

ACA Any thrombosis

0,1 1 10 OR, 95% CI 100 1000

DIAGNOSTICA (Pengo et al. Thromb. Haemost. 2005; 93 : 1147-1152)

Clinical criteria Laboratory criteria

ACA : - IgG and/or IgM

anti-2GP1 : - IgG and/or IgM

• Interference with the function of the coagulation cascade

• Activation of endothelial cells

• Activation of platelets and stimulation of platelet aggregation

• "Heparin-induced thrombocytopenia"-like mechanism

• No screening reagent is 100% sensitive and

• International recommendations by the Scientific

" PTT-LA is the most sensitive APTT reagent "

(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

- High- specificity : FVII, FXII, FXI, FIX and FVIII

(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

P. Cauchie. High sensitivity of a new APTT reagent in Lupus anticoagulant detection.

• Hexagonal phase phospholipids

DIAGNOSTICA (Triplett et al. Thromb. Haemost. 1993; 70, 787-793)

• to run the samples in duplicate

• to determine the cut-off level in each laboratory

• to calculate the cut-off level in percentiles

• to use humanized monoclonal antibodies

• to use arbitrary units (eg. MAU/GAU; MPL/GPL)

 Screening for ACA/APA :

anti-human IgG or IgM

2Glycoprotein 1 is the relevant

Anti-2GP1 assay is more specific

Revised Sapporo laboratory criteria

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