laboratory assessment of the antiphospholipid syndrome · 2016. 4. 1. · elisa a elisa b 9.4 1.8...

Laboratory assessment of the Antiphospholipid Syndrome

Katrien Devreese, MD, PhD

Coagulation Laboratory

Ghent University Hospital, Ghent, Belgium

20th Annual Meeting BSTH Antwerp 2012

The antiphospholipid syndrome (APS)

§  autoimmune disease

§  antiphospholipid antibodies (aPL)

§  thrombosis

§  pregancy morbidity

§  How to measure the aPL

§  How to use these tests in risk stratification

Introduction

(adapted from Giannakopoulos B et al. Blood 2009;113:985-994) ©2009 by American Society of Hematology

The antiphospholipid syndrome (APS)

Introduction

One clinical and one laboratory criterion

Laboratory criteria Sapporo (1999) Sydney (2006)

LAC Screening-, mixing and confirmation test

Screening-, mixing- and confirmation test

Interval 6 weeks Interval 12 weeks

aCL ELISA (ß2GPI), IgG and IgM

ELISA, IgG and IgM

high or medium titer > 40 GPL/MPL of > 99th perc.

Interval 6 weeks Interval 12 weeks

aß2GPI ELISA, IgG and IgM, > 99th perc. Interval 12 weeks

Wilson et al, Arthritis Rheum 1999; 42: 1309-11 Miyakis et al, J Thromb Haemost, 2006; 4:295-306

cofactor requirement

(Mc Neill et al 1990, Galli et al 1990, Matsuura et al, 1990)

PL-BP

aPL

ß2 glycoproteïne I (ß2GPI)

Antiphospholipid antibodies

Introduction


Lupus anticoagulants (LAC) Anticardiolipin antibodies (aCL) Beta-2-glycoprotein I antibodies (ß2-GPI)

LAC

aCL

Introduction

Coagulation assays

Solid phase assays (ELISA)

Lupus anticoagulants (LAC) Anticardiolipin antibodies (aCL) Beta-2-glycoprotein I antibodies(ß2-GPI)

LAC

aCL


Introduction

Diagnosis of the APS

Introduction

Incidence of clinical features is high and often determined by other underlying factors.

Diagnosis of the APS relies predominantly on the laboratory results.

Assays with optimal diagnostic power: Sensitivity and specificity

•  Importance of sensitivity: –  Prevent false negatives –  Patients with APS need long-term anticoagulation to

prevent recurrence

•  Importance of specificity –  Prevent false positives –  Patients without APS getting anticoagulation (the

bleeding risk) without the benefit

Laboratory diagnosis

Laboratory diagnosis of the APS

•  Lack of standardization in the assays –  Overdiagnosis/misdiagnosis –  Inter-laboratory variation

•  Diagnosis of APS: lack of a “golden standard” –  LAC (ISTH SCC Pengo et al, 2009)

–  Solid phase assays •  aCL antibodies •  aβ2GPI antibodies



assays Dependent on antibodies against

LAC “all” aPL: ß2GPI antibodies, prothrombin antibodies, other?

aCL ELISA aCL (ß2GPI-dependent) antibodies

aß2GPI ELISA ß2GPI antibodies


Strenghts and weaknesses


§  Laboratory criteria

§ LAC § aCL § aß2GPI



•  Phospholipid dependent coagulation tests

•  functional activity, qualitative test •  Multiple steps (Brandt et al 1995, Pengo et al 2009)

Laboratory diagnosis of the APS LAC

Screening Mixing Confirmation

Presence of aPL Presence of an inhibitor; exclusion of

factor deficiency

Demonstrate the PL-dependent character

of the aPL

Low concentration of PL

Mix PP and NPP 1:1, no incubation; Home made or

commercial (PPP, 100% clotting

factors)

Excess of PL Hexagonal or bilayer

•  Preanalytical conditions –  Screening assays: low concentration of PL –  Blood collection

•  Platelet poor plasma (<107/mL) residual platelets: give false negative results

•  double centrifugation:

1) 2000g 15 min RT; 2) 2500g 10 min

–  avoid repeated freeze/thawing cycles


LAC: methodology

Choice of assays Any PL-dependent assay?

Pengo et al, , J Thromb Haemost, 2009 two phospholipid-dependent coagulation tests different assay principle

assay of choice? -reduce the inter-laboratory variation

Commercially available and quality controlled -robust and highly reproducible

dRVVT and aPTT (low conc. PL and silica activator)


LAC: methodology

One of the two assays positive

APA

•  Calculation cut-off values


LAC: methodology

Screening Mixing Confirmation

-Express results as normalized ratios -Use local cut-off values -> 99th percentile of a normal population

> normalized ratio > Rosner index ((APTT 1:1mix – aPTT NPP)/ aPTT PP)) x 100 > clotting time of a normal distribution in sec

> % correction: (screen-confirmation) / screen x100

Integrated test systems -Screening+confirmation in one assay:

dRVVT or aPTT: low (screen) PL high (confirmation) PL

on mixture of PP and NPP (Staclot-LA) neat plasma (dRVVT)

-Interpretation:

-Lupus ratio : screen/confirm -% correction: (screen-cfr)/screen x100

LAC: methodology


traditional test integrated test

screening test + confirmation test

(+)

LA screen/confirm ratioorpercentage correction

(-) (+)(+)

confirmation test confirmation test within reference interval

(-) (+) (+)(-)

LAC negative LAC positive LAC positive

screen and confirm on mix

(-) (+)

other inhibitor LAC positive

screening test

screening test on mix

LAC positive

Two step method Three step method

LAC: methodology


Yes Mixing test No

•  To mix or not to mix? –  False positive results by no mixing

•  Screen/Confirm positive on neat plasma with mixing test on screen negative (Devreese JTH 2010)

majority aCL and/or aβ2GPI negative

–  False negative results by no mixing •  Strong LAC (Favaloro et al, JTH 2010)

•  “lupus cofactor” (Tripodi and Pengo, JTH 2011)

–  False negative results by mixing •  Dilution of weak LAC: clinical relevance?

(Aboud et al, Clin Chem 2007; Thom et al, JTH 2003; Moore et al, Throm Res 2008, Reber and Meijer Lupus 2012)

LAC: methodology


AVK Heparin New OAC check INR Thrombin time, anti Xa Specific assays Analysis of LAC After AVK, INR<1.5 ? Mix 1:1 PPP/NPP ? No analysis INR >3 No analysis No analysis Interference aPTT and dRVVT

FP and FN

aPTT and dRVVT FP

aPTT and dRVVT FP

Interference with anticoagulant therapy

LAC: pitfalls

Samples spiked with CRP: PTT-LA & LA-Screen normalised ratio

PTT-LA and LA-Screen Normalised Ratio

0,90

1,00

1,10

1,20

1,30

0 5 10 15 20 25 30

CRP (mg/dL)

Nor

mal

ised

Rat

io LA Screen

PTT-LA

Analytical imprecision PTT-LAand LA Screen

2.4 mg/dL

8.6 mg/dL

LAC: pitfalls

ECAT exercise in 2010: NPP enriched with CRP

“positive for LAC”

94% screening (PTT-LA)

72% positive mixing

86% positive confirm (Staclot)

(Schouwers and Devreese, Thromb Res 2010)

LAC: pitfalls

•  Analytical interference of CRP: imitates aPL Binding of CRP to negatively charged PL can influence test results for LAC, causing false positive results

•  Type of PL in the reagent is important –  PTT-LA and Staclot-LA (aPTT) are sensitive –  No interference observed for dRVVT reagents

used •  Interference increases with increasing CRP

•  False positive results –  Cut-off 99th percentile –  Two PL-dependent assays –  Three steps: screen, mix and confirm –  Anticoagulation: heparin contamination, new

anticoagulants, AVK INR >3 –  Specific coagulation factor inhibitors –  CRP –  Repeat testing after 12 weeks

•  False negative results –  Improper plasma preparation –  Diluting effect of mixing studies ? Laboratory diagnosis

LAC: pitfalls

•  Clinical characteristics –  Low:

•  VTE or AT in elderly patients –  Moderate:

•  accidentally prolonged aPTT without symptoms •  Recurrent spontaneous early abortion •  Provoked VTE in young patients

–  High: •  Unprovoked VTE and AT in young patients (<50y) •  Thrombosis at unusual sites •  Late pregancy loss •  TEC or pregancy complications in AID

No generalized searches in asymptomatic patients (increase of FP)

LAC: patient selection


(Pengo et al, 2009)

aCL and ß2GPI antibodies

ß2GPI

Adapted from Giannakopoulos B et al. Blood 2009;113:985-994 Laboratory diagnosis

aCL aβ2GPI

•  Guidelines (Miyakis et al, 2006)

ELISA, IgG and IgM > 40 GPL/MPL (aCL) of > 99th perc. Interval 12 weeks

•  Minimum requirements

-Calculate cut-off values in percentiles (99th)

-aCL in GPL/MPL units and range and aβ2GPI in universal units -Inter-assay variation should be less than 20% (CV) (10%)

-Samples should be run in duplicate (automated systems?)

-reliable standard with traceability for calibrators

Methodological problems Interassay and interlaboratory variation


Laboratory diagnosis (Tincani et al, 2004; Wong et al, 2004; Reber et al 2005; Pierangeli et al 2008 ; Lakos et al 2012)

Comparison of titers between systems •  IgG Sapporo standard (HCAL) 10.7 µg/ml

ELISA vs new automated systems

aCL IgG aβ2GPI IgG


Laboratory diagnosis (Van Hoecke and Devreese, Int J Lab Hematol, 2012)

•  Commercial ELISAs •  Same samples •  Tested in different labs


(Pengo et al, 2007) Laboratory diagnosis

Interassay variation •  Lack of uniformity in calibration resulting in large titer variation between

assays

–  Harris standards (polyclonal IgG and IgM) –  Secundary standards (heterogenous) –  Monoclonal antibodies HCAL (IgG) and EY2C9 (IgM) –  internal standard

•  No universal units –  aCL expressed as GPL/ MPL, U/ml –  ß2GPI expressed as AU, IU/ml, U/ml, µg/ml,…

•  differences in production of the kits (type of microtiter plate, PL, blocking agents, source of β2GPI, …)

Interlaboratory variation Ø  in numeric results Ø  cut-off values



(Favaloro et al , 2007; Pengo et al, 2007, Reber et al 2002, Devreese 2012)



§ Clinical relevance?


APS diagnosis

The lupus anticoagulant (LAC) correlates better with thrombosis than aCL.

Giannakopoulos B et al. Blood 2009;113:985-994 Galli et al, Blood 2003; 101: 1827-1832

©2009 by American Society of Hematology

LAC and thrombosis

APS diagnosis

LAC and thrombosis

•  Discrepancies in reported risk: Odds Ratio

–  VTE: 3.6-9.4 (de Groot et al, JTH 2005; Ginsberg et al, Blood 1995)

–  Ishemic stroke: 1.8-43.1 (Brey et al Stroke 2002; Urbanus et al, Lancet Neur 2009)

Causes: -Study population (age, sex, control population) -Methodology for LAC (choice of assays, cut-off values, two

separate analyses...)

APS diagnosis

aCL §  Isotype:

§  IgG (Pengo et al, JTH 2010) (Galli, JTH 2010) §  Only IgM: no firm association in clinical studies

§  high titer aCL (99th percentile)

•  Odds Ratio aCL –  not consistent (Galli et al, Blood 2003; Urbanus et al, Lancet Neur 2009; Ahmed et al

Stroke 2000, Brey et Stroke 2000; Naess et al JTH 2006, …)

–  VTE: 4.7-5.5 (Sanmarco et al, 2007; Ginsburg et al,1992)

–  Arterial thrombosis: 1.4-15 (Wu et al, 1992; Saidi et al, 2009)

•  Discrepancies: study design (small studies, retrospective studies, control population), different assays, single sample measurement,...

aCL and aß2GPI: clinical relevance

APS diagnosis

ß2GPI antibodies •  No or modest risk:

VTE: 1.6-2.4 (Petri et al 2010; de Groot et al, 2005) MI: 2.5 (Meroni et al, 2007) stroke: 2.3 (Urbanus et al, 2009)

§  current available ELISA for IgG ß2GPI antibodies OR - 4 -15.4 (Devreese et al, Blood 2010; Van Hoecke and

Devreese , Int J Lab Hematol, 2012) - 7.6-11.7 (de Moerloose et al, JTH 2010 )

aCL and aß2GPI: clinical relevance

APS diagnosis

Study population and type of assay

aCL and aß2GPI: clinical relevance ß2GPI antibodies Diagnostic weakness: §  more specific, easy to standardise? §  Heterogeneous group of antibodies

§  subpopulation of ß2GPI antibodies (domain I)

OR - 18.9 (de Laat et al, Blood 2005)

- 3.5 (de Laat et al, JTH 2009) (n=442)

APS diagnosis

Giannakopoulos B et al 2009

§  Laboratory criteria (Miyakis et al, 2006)

§  Relation with thrombosis:

§ LAC: + § aCL: ? § aß2GPI: ±

APS diagnosis


Isolated LAC Isolated aCL Isolated aß2GPI No association with thrombosis (Pengo et al 2005, de Groot et al 2005)

No association with thrombosis (Pengo et al 2005, Ruffati et al 2008, Runchey et al 2002, Proven et al 2004)

Except in SLE (Les et al, Semin Thromb Hemost 2009)

No association with thrombosis (Pengo et al 2005, Urbanus et al 2009)

not ß2GPI-dependent

not ß2GPI-dependent

Non pathogenic antibodies

Diagnostic value of LAC, aCL, ß2GPI antibodies

0 50 100 150 200

90

80

70

60

50

40

30

20

10

0

B2GPI_IgG__Bio_Rad_(GAU/ml))

aCL_

IgG

__B

io_R

ad_

(GP

L/m

l)

0 5 10 15 20 25

25

20

15

10

5

0

-5

b2GPI IgG TBS

aCL

IgG

TB

S

aß2GPI (GAU or µg/ml)

aC

L (G

PL o

r µg

/ml)

r=0.954 r=0.825

aCL and ß2GPI antibodies ELISA A ELISA B

9.4

1.8

0.91

0.59

54 LAC positive patients

Laboratory tests

(Devreese et al, Thromb Haemost 2011)

0 5000 10000 15000 20000 25000 30000-1000

0

1000

2000

3000

40005000

6000

7000

8000

9000

B2GPIIgG

aCLIgG

Automated System

34 APS patients (Van Hoecke and Devreese IJLH 2012)

r= 0.783


•  Antibody profiles (Pengo et al, 2005)

–  LAC, aCL, ß2GPI antibodies –  LAC+ aCL and ß2GPI antibodies same isotype (IgG) = high risk

for thrombosis

(Giannakopoulos B et al. Blood 2009;113:985-994; Ruffatti et al, Thromb Haemost 2006; 96: 337-341)

Positivity on multiple assays (LAC/CL-ELISA/direct β2GPI-ELISA) is associated with an increased risk of thrombosis and pregnancy complications.

©2009 by American Society of Hematology APS diagnosis


APS diagnosis

Miyakis et al 2006: Classification into subcategories:

I: more than one laboratory criterion present

IIa: LAC present alone

IIb: aCL present alone

IIc: aß2GPI present alone

Pengo et al 2010: type and number of positive assays I: triple positivity (LAC, aCL and aß2GPI)

II: double positivity (aCL and aß2GPI)

III: single positivity

(Pengo et al, Lupus 2010)

Pengo et al, Lupus 2012; Pengo et al. JTH 2005; Pengo et al JTH 2010; Ruffatti et al.Thromb Haemost 2006; Ruffatti et al JTH 2008; Lee et al Throm Res 2003; Pengo et al Blood 2011;


APS diagnosis

Triple positivity -Cumulative increasing incidence of thrombosis (recurrence)

-Carriers are at risk for a first event

Triple positivity (LAC, aCL, aß2GPI)

Marked association with thrombosis Clear definite APS

Double positivity (aCL, aß2GPI)

Lower risk? Classification as APS?

Single positivity


•  Results depend on quality and standardization of assays!

•  Interassay variability •  Methodological shortcomings

–  LAC: progress++: ISTH SSC updated guidelines –  aCL and aß2GPI : more guidelines and standardization needed

•  Relate lab results to clinical symptoms –  Integration and interaction of laboratory and clinician –  Clinical probability for APS/ laboratory probability for

APS

APS diagnosis

LAC

aCL


Conclusions

§  Perform all three assays: LAC, aCL, ß2GPI AB

§  Antibody profiles

–  LAC, aCL, ß2GPI antibodies

§  Medium/ high titers §  IgG > IgM > IgA

§  Persistent antibodies (> 12 weeks)

Thank you for your attention

“What is the opposite of ‘Eureka !’?”

laboratory assessment of the antiphospholipid syndrome · 2016. 4. 1. · elisa a elisa b 9.4 1.8...

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