Antiphospholipid syndrome

Prakriti Gupta

Sir Gangaram Hospital

New Delhi

Definition

The antiphospholipid syndrome (APS) is an

autoimmune disease defined by presence of

thromboembolic complications and/or

pregnancy morbidity with persistently

increased titers of antiphospholipid antibodies

(APA).

Epidemiology

• APA antibodies- 1-5% young, healthy

• SLE – 12-34%

• F > M

History

• 1907- Wasser a s o ple e t fi atio test for s philis usi g reagin (phospholipid)

• 1941- reagin = anionic phospholipid= cardiolipin

• 1950s- false positive syphilis test

- nonspecific coagulation inhibitor

- presence of a factor in some plasmas that

prolonged the clotting time, even when

the plasma was diluted with normal

pooled plasma

• 1963 - association of this anticoagulant with

thrombosis instead of bleeding

• 1972-Feinstein and Rapaport introduced the

ter lupus a ticoagula ts (LACs) for an

inhibitor directed against coagulation cascade

PLs

• 1980s – radioimmunoassay and ELISA for aCL

Clinical spectrum

PAPS- Primary :

Patients without clinical evidence of another autoimmune diseases

secondary causes.

SAPS- Secondary :

– Autoimmune or connective tissue diseases e.g. SLE

– Infection

– Drugs- proacinamide, hydralazine, quinidine, phenothiazines,

penicillin

APS classification.

CAPS - Catastrophic Antiphospholipid Syndrome :

aggressive form of APS; widespread thrombosis, multiple

organ sites; significant morbidity and/or mortality

SNAPS - Seronegative Antiphospholipid Syndrome

patients with typical clinical manifestations of APS but

negative for a range of aPL tests/assays

Antibodies against...

• β2 GPI

• Prothrombin

• Annexin V

• Factor V

• Protein C

• Annexin II

• HMWK

• Protein S

• Plasmin and tPA

Antiphospholipid antibody

• Misnomer

• Antibodies are directed against phospholipid

binding proteins

β2 glycoprotein I

• β2 glycoprotein I (Apolipoprotien H)

• 50kd glycoprotein found in plasma (150-300µg/ml)

• 5 domains (Domain I to V).

• Physiological function unclear

– Lipid metabolism

– Clearance of apoptotic bodies from circulation

– Protection against atherosclerosis

Procoagulant/ proinflammatory phenotype

Prothrombotic

•Platelets: Activation via Apo ER2 receptor

•Potentiates TxA2 production

•Interacts with GPIbα subunit of GPIb/IX/V

•Endothelial cells: Procoagulant and

proinflammatory phenotype via Annexin A2 and

TLR4

•Monocytes: Increased Tissue factor expression

Antifibrinolytic • Annexin A2 – Colocalizes tPA and

and Plasminogen

• Β2- anti β2 complex binds to

Annexin A2 and inhibits its

fibrinolytic function

Antithrombin

•Activated Protein C Pathway

•Competes for limited PL binding sites

•Acquired protein S deficiency

•Disturbs the inhibition of activated

factor Xa

•Annexin V

•Disturbs the anticoagulant sheild on

trophoblast surface

THROMBOSIS

Intrinsic (F XII,XI,IX, VIII)

Endothelial &

platelet

activation;

TF expression

FDP

Plasmin PAI

Ca2+

Extrinsic (FVII)

Fibrin Fibrinogen

FX Xa

Va Ca2

II IIa Ca2

ATIII

Pr.C,S,APCR

Anti-Annexin A2

& anti-β2 GPI

anti-plasmin

Anti-tPA

Anti-ProteinC, anti-

ProteinS, anti-

Thrombomodulin,

anti-annexin A5

Others:

Complement

activation; augmented

atherosclerosis

Clinical

types

of APA

LA( LUPUS

ANTICOAGULANT???)

Against a complex of synthetic anionic phospholipid bound prothrombin used in aPTT

Anti β2

glycoprotein I

aCL

Serum IgG against anionic

phospholipid protein

complexes

In ELISA

Reduces access of

coagulation factors to

anionic PL

Prolongation of

clotting times of PL

dependent tests

Mechanism of in vitro anticoagulant effect

of LA

Asymptomatic

• Persistently raised clotting time

• Unexplained

• High index of suspicion

CLINICAL MANIFESTATIONS

• Venous thrombosis

• Arterial thrombosis

• Microangiopathy – CAPS

• Pregnancy related complications

• Asymptomatic

Venous thrombosis

• DVT

• Ascites (Budd-Chiari

syndrome)

• Tachypnea (pulmonary

embolism)

• Peripheral edema (renal

vein thrombosis)

• Abnormal funduscopic

examination (retinal vein

thrombosis)

Arterial thrombosis

•CVA

•Digital ulcers, Gangrene of

distal extremities

•myocardial infarction

•Heart murmur, [aortic or

mitral insufficiency (Libman-

Sacks endocarditis]

•Abnormal fundoscopic

examination (retinal artery

occlusion)

Neurological

• Cerebrovascular accident

• Transient ischaemic attack

• Transverse myelitis

• Dementia

• Optic neuropathy

Renal

• Renal vein thrombosis

• Assosiated nephropathy

Cardiac valve disease

Livedo reticularis

Thrombocytopenia

Skin manifestions

• Ulcerations

• Pseudo-vasculitic lesions

• Digital gangrene

• Superficial phlebitis

• Malignant atrophic papulosis-like lesions

• Subungual splinter hemorrhages

• Anetoderma (a circumscribed area of loss of dermal elastic tissue)

Other neurological manifestations

• Cognitive dysfunction

• Chorea

• headache or migraine

• Multiple Sclerosis

Pregnancy morbidity

Anti β2-GPI ab on trophoblast

Early fetal loss

Disruption of annexin V

anticoagulant shield

Exaggerated complement activation on the trophoblast

surface resulting in thrombosis

Thrombosis of placental vessels

resulting in infarction

Clinical criteria (Sydney Criteria, 2006)

for diagnosis

• Vascular thrombosis:

– ≥ episode of e ous, arterial or s all essel thrombosis confirmed by imaging/ Doppler USG/

Histopathology

• Pregnancy morbidity:

– ≥1 pregnancy loss after 10 weeks of gestation

– ≥ preg a losses efore eeks of gestatio

– ≥1 preterm birth <34 weeks of gestation due to

pre-eclampsia or placental insufficiency

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).

J Thromb Haemost 2006;4:295–306.

Laboratory criteria

• Positive lupus anticoagulant test: – Prolongation of at least 1 PL dependent test with PPP

– Failure to correct by mixing patient & normal plasma

– Confirmation of lupus anticoagulant by correction with excess PL

– Exclusion of alternative coagulopathies using specific assays

or

• Positive aCL test (mod- titer / high titer IgG or IgM antibodies) or

• Positive β2GPI antibody test (>99th percentile IgG/IgM antibodies) and

• Laboratory anomaly should persist for 2 or more occasions atleast 12 weeks apart

CAPS

• Asherson Syndrome

• Rare < 1% of APS patients,

• rapid onset

• high mortality (50%)

• In about 25% of cases it may be complicated

by DIC

Asherson RA. CAPS:Lupus 2003;12:530–4.

• Multiple acute microangiopathy of small-vessel leading to

multi organ failure

• Clinical features -manifestations of organ and tissue ischemia-

– renal failure due to renal thrombotic microangiopathy, -

– acute respiratory failure due to adult respiratory distress

syndrome,

– cerebral injury due to microthrombi and microinfarctions,

– and myocardial failure due to microthrombi

Diagnostic criteria for CAPS

1. Vascular Thrombosis in three or more organs

2. Development of manifestations simultaneously or in

< 1 wk

1. Histological evidence of small vessel thrombosis in at

least one organ or tissue

2. Laboratory confirmation of the presence of APA

Interpretation:

• Definite Catastrophic APS: All 4 criteria

• Probable Catastrophic APS:

All 4 criteria but with involvement of only 2 organs,

systems, and/or tissues

All 4 criteria but with the absence of repeat detection of APA

at least 12 weeks apart due to the early death of a patient

who had never been tested for APA before the CAPS event

1, 2, and 4

1, 3, and 4

• Positive APA test can be associated with infections or anticoagulation

• False- negative APA may occur during acute APS

• Sepsis and CAPS share many similarities

• Acute thrombotic microangiopathic conditions

(TTP, HUS, HELLP) mimic CAPS

Mechanisms of CAPS

Thrombosis augments further thrombosis (Thrombosis Storm)

Decreased PAI-1

Consumption of ProteinC and AT

Widespread thrombosis

Two hit hypothesis

Infection + Cytokines from dead cells

Systemic inflammatory response syndrome

Multiorgan failure

Antiphospholipid

antibodies Uremia –

Platelet function

defect

Factor VIII

inhibitor

Immune

thrombocytopenia

Antiprothrombin

Abs

CAPS Consumptive

coagulopathy

Hypoprothromb

inemia

Bleeding

Pathogenesis of Haemorrhagic Lupus

Antiphospholipid

antibodies

Thrombosis

Arterial: Stroke,

MI

Venous: DVT

Capillary: CAPS

Pregnancy

morbidity

Prothrombotic:

late pregnancy

loss, premature

births

Non-

thrombotic:

Early

pregnancy loss

Bleeding

Thrombocytopenia

Reduced

Prothrombin

Anti-factor VIII

antibodies

Hemolysis

AIHA

MAHA

Categorising patients

Low risk

Venous or arterial thromboembolism in elderly patients

Moderate risk

• Accidentally found prolonged activated partial thromboplastin time in

asymptomatic subjects

• Recurrent spontaneous early pregnancy loss

• Provoked venous thromboembolism in young patients

High risk

• Unprovoked venous thromboembolism

• Unexplained arterial thrombosis in young patients (<50 years)

• Thrombosis at unusual sites

• Late pregnancy loss

• Any thrombosis or pregnancy morbidity

• In patients with autoimmune diseases (SLE, rheumatoid arthritis,

autoimmune thrombocytopenia, autoimmune hemolytic anemia)

Blood collection

1. Blood collection before the start of any anticoagulant drug or a

sufficient period after its discontinuation

2. Fresh venous blood in 0.109 M sodium citrate 9:1

3. Double centrifugation for platelet poor palsma( < 10,000/ul)

4. Quickly frozen plasma is required if LA detection is postponed

5. Frozen plasma must be thawed at 37 C

6. No filtration

1. Prolongation of a phospholipid-dependent clotting time

1. DRVVT

2. APTT

3. Two or more tests with different assay principles should

be performed for screening

2. Inhibition of the prolongation must be demonstrated via a

mixing test

1. Mixing of patient plasma with normal plasma does not

correct the prolonged clotting time

3. Phospholipid dependence of the prolongation and inhibition

must be demonstrated

1. DRVVT confirmatory test

2. APTT-based hexagonal phase phospholipid neutralization

test

3. APTT-based platelet neutralization procedure

3. Exclude/ evaluate for confounding coagulopathies

1. Factor deficiencies and inhibitors

2. Heparin

3. Warfarin

4. Direct oral anticoagulants

4. Recommend testing for serum antiphospholipid (aPL)

antibodies (IgG and IgM isotypes)

1. Cardiolipin antibodies

2. Beta-2-glycoprotein I antibodies

Tests used to detect LA

• Prothrombin time based assays

– Dilute PT

• Activated partial thromboplastin time dependent assays

– APTT with a lupus sensitive agent

– Kaolin clotting time (KCT)

• Snake venom based assays

– Dilute Russell viper venom time

– Textarin ecarin ratio

Choice of the test

1. Two tests based on different principles – false

positive rates increase if > 2

2. DRVVT should be the first test considered

3. The second test should be a sensitive aPTT

(low phospholipids and silica as activator)

4. LA should be considered as positive if one of

the two tests gives a positive result

DRVVT (β 2 GP Ab)

APTT KCT

FDP

Plasmin PAI

Intrinsic (F XII,XI,IX, VIII)

Ca2+

Extrinsic (FVII)

Fibrin Fibrinogen

FX Xa

Va Ca2 + PL

II IIa AT

Pr.C,S,APCR

Ca2 + PL

LA-Screening tests

Prolongation of a phospholipid-dependent

clotting test - SCREENING

Demonstration of the presence of an inhibitor

by mixing tests- MIXING

Demonstration of the phospholipid dependence

of the inhibitor- CONFIRMATION

Integrated tests

• Include SCREENING and CONFIRMATION in a single test

• DRVVT and aPTT done with 2 reagents

– LA1 screen: low conc of PL

– LA2 confirm: High conc of PL

• Calculated as

– LA ratio [screen/confirm]

– [screen-confirm/screen X 100]

• may benefit from normalizationof results against a PNP run in

parallel with the test plasmas-

[(screen/confirm) patient ÷ (screen/confirm) PNP]

• Eliminates mixing

DRVVT integrated

Suspected LA

LA1 screen

LA2 confirm

LA ratio:

LA1/LA2

LA not

detected

>1.2: LA

positive

Normal LA2 CT

Normal

LA1 CT

• Quantification

LA ratio

1.2-1.5: weak positive

1.5-2: moderate positive

>2: strongly positive

McGlasson & Fritsma. STH 2013; 39:315-319

Mixing tests

• Mixing assays must show evidence of

inhibitory activity by the effect of patient

plasma on an equal volume of normal pooled

plas a i i g

• Mixing normal plasma with test plasma

replenishes any factors deficient in the test

plasma.

• Recommended 1:1

Rosner index

(ICA – index of circulating anticoagulant)

• ICA = [(b-c)/a] x 100

(where a, b, c = clotting times of patient,

mixture, normal, respectively)

• For Rosner index/ICA - local determination of

value (generally between 10-20%)

• Improves specificity

• Introduces a dilution factor and may make weak LA samples appear negative.

• If ratio < 1.2 and LAC Screen and LAC Confirm clotting times are prolonged, then mixing studies should be performed ????

Not mixing can lead to false negative LA in some

cases of strong LA

• Very strong LA, dispatched to 93 RCPA QAP participants

• Clinical information provided:

76 year old female patient, B-cell lymphoma diagnosed 4

years ago on 3-month follow-up with no specific therapy.

During recent follow-up visit identified to have increasing

abdominal distension, night sweats, weight loss and a

significant drop in haemoglobin.

Routine coagulation tests performed at satellite facility

showed PT and APTT. Mixing tests did not show any

substantial correction.

Favaloro et al. JTH 2010; 8: 2828–31

• 38.1% participants concluded -LA negative

• 91.7% of these performing dRVVT testing without

mixing LA ratio- ~1.0 due to prolonged DRVVT screen and DRVVT confirm times

The normal LA ratio obtained using neat (non-mixed) plasma - inability of the

phospholipid level present in the confirm reagents to neutralize LA activity

within this sample

sample mixing

level of LA and phospholipids sufficiently match to enable neutralization of the

LA, correction or shortening of the LA confirm assay, and a raised LA ratio.

47.6% participants identified a strong LA

96.7% of these having performed mixing studies.

•Recommend the use of the DRVVT plus 1 additional LA test

(APTT or dilute prothrombin time)

•Suggest that mixing study may obscure weak LA

•State that confirmatory test is essential to demonstrate

phospholipid dependence

•Caution about LA test specificity in the presence of vitamin K or

heparin anticoagulant therapy

•Suggest antiphospholipid immunoassay tests

•Recommend stratifying selection of patients for testing

•Recommend repeat testing to demonstrate persistent positivity

≥ eeks

ISTH SSC 2009 CLSI H60 2014

NPP as denominator RI as denominator

99th percentile 97.5th percentile

ISTH 2009 CLSI 2014

Testing patients on

VKA

Undiluted plasma if

INR < 1.5; Mix with

NPP if INR > 1.5 < 3.0

Screen and confirm

on 1:1 mixture with

NPP; TSVT þ ET or

PNP

Testing patients on

UFH

Interpret with

caution

Can detect LA in

some cases where

heparin neutralizer

is effective

Anticardiolipin antibody detection • ELISA based assays

• Most anticardiolipin antibodies responsible for APS are β2 glycoprotein I dependent

• Positive assay requires repeat testing as many other infections can lead to transient false positive results

• Both IgG and IgM subtypes, IgG subtype more relevant

Anti-β2 glycoprotein I antibody detection

• ELISA developed with microtiter plates coated

with β2 glycoprotein I to detect anti- β2

glycoprotein I antibodies

• More specific than anticardiolipin

• Removes the problem with false positive aCL

results due to infections

Categories of APS based on lab tests

• Type I: combination of lab criteria

– Strongly associated with TE; recurrent events

• Type IIa: LAC alone

– Insensitive; may be positive in elderly

• Type IIb: aCL antibodies alone

– Obstetric complications

• Type IIc: anti-β2GPI antibodies alone

– Obstetric complications

Management

• Asymptomatic individuals who have positive

laboratory tests but do not meet APS criteria - do

not require specific treatment

• Correction of underlying risk factor

• Significant thrombotic events or obstetric

complications- anticoagulant therapy

• Even if the venous or arterial occlusion occurred

many years previously- long-term treatment with

oral anticoagulant therapy

• Ischaemic stroke – aspirin

or

low intensity warfarin (INR2.0 to 3.0)

• Non cerebral arterial thrombosis – aspirin

( 81mg)

low intensity warfarin (INR2.0 to 3.0)

Crowther MA . Thromb Res. 2005

Standard intensity warfarin ( INR 2-3)

vs

High intensity warfarin (3.1 to 4 or 4.5)

(Thrombotic risk and minor bleeding more with high intensity

anticoagulation)

Finazzi G J Thromb Haemost. WAPS 2005;3(5):848-853

Pregnancy

LMWH ( enoxaparin 40mg OD) + low dose

aspirin ( LDA- 75-100mg)

Or

unfractionated heparin( UFH 5000U s/c BD)

+ LDA

LMWH vs UFH

Int J Gynaecol Obstet. 2011Mar;112(3):211-5 Chest 2012; 141( supp 2)

.

CAPS

Intensive treatment with

• Corticosteroids

• Immunosuppression

• Intravenous Ig and/or

• Plasma exchange