antiphospholipid syndrome
TRANSCRIPT
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