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Pengo Vittorio Torino 13° Convegno Patologia Immune E Malattie Orfane 21 23 Gennaio 2010
1. Torino, 23 Gennaio 2010
Revisione dei criteri diagnostico-
laboratoristici dell’APS
Vittorio Pengo M.D.
Clinical Cardiology, Thrombosis Center
University of Padova, Italy
Tiziano Vecellio: presentazione di Maria al Tempio. Venezia, Gallerie dell’Accademia
2. Laboratory criteria
1. Lupus anticoagulant present in plasma detected according to the guidelines
of the International Society on Thrombosis and Hemostasis.
2. Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma,
present in medium or high titer (i.e. >40 GPL or MPL, or >the 99th percentile) ,
measured by a standardized enzyme-linked immunosorbent assay.
β
3. Anti-β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or
plasma (in titer >the 99th percentile), measured by a standardized enzyme-linked
immunosorbent assay, according to recommended procedures.
Positive tests must be confirmed on 2 or more occasions at least 12 weeks apart
Miyakis S, JTH 2006
3. Investigators are strongly advised to classify APS patients
in studies into one of the following categories:
I: More than one Laboratory criteria present (any combination)
IIa: Lupus Anticoagulant present alone
IIb: Anti-cardiolipin antibody present alone
IIc: Anti-β2 glycoprotein-I antibody present alone
Miyakis JTH 2006
4. APS?
• Male 55 years of age
• IgG aCL antibodies = 30 GPL (vn<21*)
• IgG aß2GPI antibodies = 8 U (vn<16*)
• LAC negative
• IgM aCL and IgM aß2GPI: negative
• Acute myocardial infarction
*99th percentile
Yes: classification category IIb
8. Padua Thrombosis Centre
20 PATIENTS
( aCL + medium-high titer/
LA -; a human β2-GPI -)
8 previous thrombosis APS?
5 +ve in bovine β2-GPI ELISA
2 +ve in other CL-binding proteins ELISA
V.Pengo & A. Biasiolo, Thromb Haemost 2001
9. Antiphospholipid antibody profiles as risk factors
of thrombosis
Lupus anticoagulant/ Thrombosi No Odds Ratio
Anti-cardiolipin antibodies/ s thrombosis
Anti-β2-glycoprotein I (N=340) (N=278)
antibodies no.(%) no.(%)
univariat 95% CI Multivariate 95% CI
e °
LA+/aCL+/ab2+ 34 (10) 2 (1) 14.9 3.5-62.7 33.3 7.0-157.6
LA+/aCL-/ab2- 0 (0) 5 (2) NA - NA -
LA-/aCL+/ab2+ 18 (5) 13 (5) 1.2 0.6-2.5 2.2 1.0-5.2
LA-/aCL+*/ab2- 7 (2) 13 (5) 0.5 0.2-1.2 0.8 0.3-2.1
LA-/aCL-/ab2+ 4 (1) 4 (1) 0.9 0.2-3.5 1.3 0.3-5.7
* > 40 GPL/MPL
V Pengo et al, 2005
10. Anticardiolipin and Thrombosis
The detection of lupus anticoagulants
and, possibly, of immunoglobulin
G (IgG) anticardiolipin antibodies at
medium or high titers helps to identify
patients at risk for thrombosis.
Galli M, Blood 2003
11. Invitation to a debate on the serological criteria
that define the antiphospholipid syndrome
Proposals for the next update of the criteria:
• implementation of strict guidelines for the
performance of LAC assay,
• exclusion of aCL measurements in their
current application from the criteria
• limitation of the measurement of aß2-GPI
antibodies to IgG.
Galli M, Reber G, de Moerloose P & Philip G. de Groot JTH 2008
12. Sydney consensus: ACL >40 GPL
or > the 99th percentile
• In our lab:
99th percentile is 17 GPL
Therefore a patients with 30GPL and
myocardial infarction is classified as a
syndrome only when using the 99th
percentile as a cut-off level.
13. IgG aCL titre and laboratory profile
Ruffatti A. JTH 2008
14. IgG aCL titre and clinical features
Ruffatti A. JTH 2008
15. Patients with thromboembolic
events
Consider single positivity for aCL antibodies
as non APS patients. More information
from clinical studies on homogeneous
cohort of patients with single positivity are
needed.
16. APS?
• Male 60 years of age
• IgM aCL antibodies = 50MPL (vn<10)
• IgM aß2GPI antibodies =8 U (vn<8)
• IgG aCL and IgG aß2GPI: negative
• LAC negative
• TIA
Yes: classification category IIb
17. IgM aCL
• Associations with thrombosis in ELISA tests
were only found for IgG and not for IgM isotype.
Galli M, Reber G, de Moerloose P & Philip G. de Groot JTH 2008
22. IgG antibodies that recognize epitope Gly40-Arg43
in domain I of 2–glycoprotein I cause LAC,
and their presence correlates strongly with thrombosis
Bas de Laat, Ronald H. W. M. Derksen,
Rolf T. Urbanus, and Philip G. de Groot
Blood 2005
31. APS?
• Male 52 years of age
• Lupus Anticoagulant: dRVVT mixing test
(ratio) = 1,22 (v.n <1.2)
• SCT or KCT mixing test = normal
• TIA
32. LAC potency
p=0.02
2.5
dRVVT ratio
2.0
1.5
1.0
LAC/thrombosis LAC/no thrombosis
Pengo V. ATVB 2007
33. 5
4
Ratios
3
2
1
group1 group 2 group1 group 3
dRVVT KCT
Ratios of coagulation time in mixing studies divided by that of normal plasma for
dRVVT and KCT in patients positive in both tests (group 1) are compared with ratios
in patients positive for the sole dRVVT (group 2) or the sole KCT (group 3).
Pengo V et al., JTH 2007
34. LAC positive patients
Consider False positive Consider LAC potency Consider aCL/aβ2GPI
•Heparin •Ratio in mixing studies
•Patient’s age •Positivity in more
•First diagnosis than one test
•Mild in potency
•Oral anticoagulant treatment
35. Rate of Antiphospholipid syndrome in unselected
LAC positive patients
LAC+/APS LAC+/no APS p
N=152 N=79
Multiple positivity (IgG)* —no. (%) 96 (63) 24 (30) <0.001
LAC positive only—no. (%) 42 (28) 40 (51) <0.001
Multiple Positivity = Positive LAC and IgG aCL and/or IgG aβ2GPI
Pengo V et al., ATVB 2007
36. RATIO
(Risk of Arterial Thrombosis In relation to Oral contraceptives)
• Large multicentre population-based
case-control study
• Enrolled women aged under 50 years
• Included 175 patients with first ischaemic stroke,
203 patients with myocardial infarction, and 628
healthy controls
Urbanus RT, Lancet Neurology 2009
38. RATIO
(Risk of Arterial Thrombosis In relation to Oral contraceptives)
The presence of lupus anticoagulant and
any additional antiphospholipid antibody
subpopulation did not affect the risk of
myocardial infarction or ischaemic stroke,
compared with the risk in patients with
only lupus anticoagulant.
Urbanus RT, Lancet Neurology 2009
39. LAC specific assays to detect
pathogenic anti β2 Glycoptrotein I
antibodies?
40. LA + antiβ2GPI + LA + antiβ2GPI -
2.5 2.5
2.0 2.0
Ratio
Ratio
1.5 1.5
1.0 1.0
0.5 0.5
10 5 10 5
Final CaCl 2 concentration [mM] Final CaCl 2 concentration [mM]
V Pengo et al 2004
48. Protein concentration and activity in ELISA of affinity-
purified anti human ß2-GPI autoantibodies
Purified anti ß-2-GPI
Control IgG 1 2 3 4 5
Protein concentration µg/ml 75 61 39 49 68 34
0.015 2.378 2.658 2.297 2.242 2.255
Anti ß-2-GPI IgG
ELISA
0.005 2.081 2.037 1.645 1.806 1.648
aCL IgG ELISA
V. Pengo et al. Thromb Haemost 1999
50. 2,5
American
Diagnostic
2 s (n=23)
dRVVT Ratio
LAC
1,5 Screen IL
(n=27)
1 LA1 Dade
(n=17)
0,5
0 100 200 300 400
IgG Anticardiolipin (GPL)
Pengo V et al. 2006
51. Antiphospholipid antibody profiles as risk factors
of thrombosis
Lupus anticoagulant/ Thrombosi No Odds Ratio
Anti-cardiolipin antibodies/ s thrombosis
Anti-β2-glycoprotein I (N=340) (N=278)
antibodies no.(%) no.(%)
univariat 95% CI Multivariate 95% CI
e °
LA+/aCL+/ab2+ 34 (10) 2 (1) 14.9 3.5-62.7 33.3 7.0-157.6
LA+/aCL-/ab2- 0 (0) 5 (2) NA - NA -
LA-/aCL+/ab2+ 18 (5) 13 (5) 1.2 0.6-2.5 2.2 1.0-5.2
LA-/aCL+*/ab2- 7 (2) 13 (5) 0.5 0.2-1.2 0.8 0.3-2.1
LA-/aCL-/ab2+ 4 (1) 4 (1) 0.9 0.2-3.5 1.3 0.3-5.7
* > 40 GPL/MPL
V Pengo et al, 2005
52. Antiphospholipid profile and subsequent TE in obstetric APS
100,0%
aCL and aβ2GPI positive
80,0%
Cumulative proportion of TE events
60,0%
40,0%
20,0%
0,0% LA positive, aCL and aβ2GPI positive
0 2 4 6 8 10 12 14 16 18
Time (years)
Ruffatti et al. 2006
53. Personal Considerations
Patients with thromboembolic events
• Consider triple positive patients (LA positive, IgG or IgM
aCL> 40GPL, IgG or IgM ab2GPI> 99th percentile) as a
high risk population related to a single pathogenic
autoantibody (definite APS).
• Consider the presence of double positivity (LA negative
and aCL> 40GPL, ab2GPI> 99th percentile, same
isotype) as a scenario in which APS is highly probable in
low risk patients.
• Consider single positivity patients for LA, aCL or ab2GPI
antibodies, as non APS patients. However, further
studies on homogeneous cohorts of patients with single
positivity are required.
54. Diagnosis of APS is more likely if:
• The patient is less than 50 years of age
• Thromboembolic events are idiopathic
(other causes are excluded)
• The predominant immunoglobulin isotype
is IgG
• anti DmI ELISA is positive
55. Appropriateness of request for aPL
antibodies detection in pts with thrombosis
(Generalized searches are highly discouraged)
Low: venous (VTE) or arterial thromboembolism in elderly
patients;
Moderate: accidentally found prolonged aPTT in
asymptomatic subjects, VTE or ATE in young patients;
High: unprovoked VTE and (unexplained) arterial thrombosis
in young patients (< 50 years of age), thrombosis at unusual
sites, any thrombosis in patients with autoimmune diseases
• Perform LAC, IgG aCL and IGg aβ2GPI and analyze antiphospholipid profile.
• Confirm positivity on a second occasion >12 weeks after the initial testing.
