Definition

The antiphospholipid antibody syndrome (APS) is autoimmune or alloimmune disorder characterized by production of Ab that interact with phospholipid or phospholipid-protein complex. Called syndrome as it is associated with a group of clinical manifestations.

Classification of APS ;
1.autoimmune (persistant and associated with clinical complication ) Primary : dont fulfill criteria for SLE Secondary : SLE Other connective tissue disease Drug induced: procainamide , penecillin. phenothiazine. 2. allo immune
(transient not associated with clinical complication)

Infection ; viral, bacterial, fungal, protozoal. Malignancy ; LPD, hairy cell lukaemia

The APS is characterized by antibodies against either phospholipids or plasma proteins that are bound to phospholipids. they do not bind directly to anionic phospholipids .

APL, which are directed against plasma proteins bound to anionic phospholipids,may be detected as:

Lupus anticoagulants (LA) Anticardiolipin antibodies(aCL) Antibodies to 2 glycoprotein-I Other antibodies, including those to prothrombin, annexin V, phosphatidylserine, phosphatidylinositol, and others.

auto-antibody
auto-antibody target protein

anionic phospholipids anionic phospholipids

APS is a syndrome with a wrong name !

The most commonly accepted explanation for the development of aPL is that they occur in susceptible individuals following incidental exposure to infectious agents (first-hit). A "second-hit" is required for the development of the fullblown syndrome .

Potential candidates for the delivery of such a second hit are: smoking, prolonged immobilization, pregnancy and the postpartum period ,oral contraceptive use, hormone replacement therapy, malignancy, nephrotic syndrome, hypertension, and hyperlipidemia

Thrombo resistant properties of normal endothelium

Anti platelet properties
Produce prostacyclin and NO Express CD 39 (modulate th effect of ADP AMP

Anti coagulant properties

Heparan sulphate like molecule on endothelial surface bind AT III Thrombo modulin +thrombin APC

Fibrinolytic properties
Produce TPA (tissue plasminogen activator)

APA interfere with these thrombo resistant properties as follow: 1. prostacyclin (PG I2) 2.interfere with protein c pathway by : Protein s deficiency Antibodies to thrombomodulin Antibodies to factor Va,VIII 3.Inhibition of heparan sulphate ATIII complex 4.increase plasminogen activator inhibitor 1 5.injury to endothelium by interlukin 1 TNF & endotoxin expresion of phospholipid anti APA production 6.endothelin 1 and endothelial derived platelet activating 7.disruption of annexin V (placental anticoagulant protien 1 ) 8.cross reactivity to oxidised LDL 9. cell adhesion molecules receptors on endothelium .

aPL cause obstetrical complications primarily by inducing thrombosis in the uteroplacental circulation. Thrombosis could be induced by a variety of pathways ; as an example: aPL can reduce the levels of trophoblast-associated annexin V (placental anticoagulant protein-1).
complement activation is a critical component of APS-related adverse pregnancy outcomes .

aPL have a direct effect on human placental trophoblast, inhibiting the expression of hCG, inducing apoptosis, decreasing trophoblast fusion, altering expression of adhesion molecules, limiting trophoblast invasiveness, and inhibiting decidualization of cultured endometrial stromal cells

venous or arterial thromboses. pregnancy morbidity . aPL-related clinical manifestations that are not part of the APS Classification Criteria, such as livido reticularis, thrombocytopenia, cardiac valve disease, or aPL-nephropathy .

History

Thromboembolic events. Obestatric complications in female patients, Other thrombosis risk factors. Medications known to be associated with aPL production include the phenothiazines, hydralazine , procainamide , and phenytoin. Exclude systemic lupus erythematosus.

Physical examination
There are no pathognomonic physical findings of APS.

Abnormal features may be related to ischemia or infarction of the skin, viscera, or nervous system.
Physical examination findings may include the presence of livedo reticularis, digital ischemia, gangrene, deep venous thrombosis, or neurological lesions consistent with a stroke.

Clinical
Vascular thrombosis
Pregnancy morbidity

Laboratory

Clinical Criteria:

Vasculer thrombosis. Arterial, venous or microvasculer thrombosis occuring in any tissue or organ.

Pregnancy morbidity.
One or more unexplained deaths of a morphologically normal fetus 10 weeks gestation.

One or more premature births of a morphologically normal neonate <34 weeks gestation due to eclampsia,severe preeclampsia or placental insufficiency.
Three or more unexplained consecutive spontaneous abortions <10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded.

Sapporo criteria (1999)

Sydney criteria (2006)

Screening, mixing, and confirmation tests (ISTH guidelines) Two or more occasions at least 12 weeks apart Detected by standardized ELISA IgG and/or IgM Medium or high titer (>40 units IgG or IgM phospholipid antibody titer or >99th percentile) Two or more occasions at least 12 weeks apart IgG and/or IgM Titer >99th percentile Two or more occasions at least 12 weeks apart

LACs

Screening, mixing, and confirmation tests (ISTH guidelines) Two or more occasions at least 6 weeks apart

aCL antibodies

Detected by standardized 2GPIdependent ELISA IgG and/or IgM Medium or high titer

Two or more occasions at least 6 weeks apart

Anti-2GPI antibodies

Sapporo criteria 1999

(Wilson et al. Arthritis Rheum. 1999)

Clinical criteria

Laboratory criteria
auto-antibodies

vascular thrombosis

pregnancy morbidity

LA

ACA

APS

Revised Sapporo criteria 2006

(Miyakis et al. J. Thromb. Haemost. 2006)

Clinical criteria

Laboratory criteria

vascular thrombosis

LA

ACA

pregnancy morbidity anti-b2GPI

APS

APS is diagnosed if at least one clinical and one laboratory criteria are met on at least 2 occasions 12 weeks apart (transient Ab may occur, e.g infection)

Screening test: demonstration of the prolongation of a phospholipid-dependent clotting time beyond the upper limit of the reference interval. Mixing test: confirmation of the presence of an inhibitor and the exclusion of a coagulation factor deficiency. Confirmation: that the inhibitor is phospholipid-dependent and not directed against a specific coagulation factor. Rule out other coagulopathies.This step is critical in identifying other situations which may yeild false positive results if only the first three steps are employed.

Pre-analytical varibles in the diagnosis:

If Heparin is initiated before the studies of thrombophilic work up is done compromises the potential diagnosis of LA and also other functional tests for physiological inhibitors. Patents who present with thrombotic events or during pregnancy will have evidence of acute phase reaction resulting in increase in various plasma proteins including Fibrinogen,Factor 8,Fibronectin shorten the baseline of coagulation studies.

The most important aspect is the preparation of platlet poor plasma (PPP).
Ideally the PPP should contain <5000 platlets/ml. Failure to prepare apprpriate PPP missing the diagnosis of LA. If the PPP is to be frozen and tested ,the plasma is filtered through a 0.22m filter. Residual platlets in improperly prepared plasma may neutralise LA upon thawing,with rupture of platlets and exposure of PL.

aPTT
Some manufacturers offer aPTT reagent which contains a low amount of phospholipid, therefore it is more sensitive for lupus anticoagulant

Prothrombin Time:
patients with LA will have a normal PT unless they are receiving oral anticoagulants or they develop an inhibitor to prothrombin (PT reagents contain more phospholipids than PTT reagents)

DRVVT (screening)
Activates factor X which in the presence of PL, calcium, and factor V activates prothrombin, leading to the formation of a fibrin clot Inhibition by LA leads to prolongation After positive screen, perform the mixing study- if does not correct then:

DRVVT (confirm)
Adds a higher amount of phospholipids to neutralize the lupus anticoagulant Ratio is derived from the screen clotting time divided by the confirmatory clotting time If ratio exceeds the established cutoff, then lupus anticoagulant is in the specimen

o Modified PT assay o Thromboplastin, which is rich in phospholipid, can be diluted so that its concentration becomes the rate limiting step o Inhibition of prothrombinase by a LA will cause prolongation of the PT assay o Due to the various PL and its concentration in the reagent, the test varies in its sensitivity and specificity

o o

o o o

Two part aPTT screening assay for LA Patients plasma is mixed with buffer (screening test) or hexagonal phase phosphatidyl ethanolamine (confirmatory test) to neutralize any lupus anticoagulant present Mixtures are incubated with normal plasma to correct any coagulation factor deficiency Measure aPTT in both mixtures If specimen contains LA, the aPTT of the confirmatory test will be significantly shorter than that of the screening test

Phospholipid antibody positive= difference in the clotting times between the two tubes is greater than + 8.0 seconds. The aPTT reagent in this assay contains a heparin inhibitor which makes the test system insensitive to heparin levels up to 2.0 U/mL.

Kaolin clotting time

Sensitive for LA when no additional PL is used LA is identified when the KCT fails to correct after the addition of even large amounts of plasma Problems with the KCT, owing to the particular nature of kaolin, is that it is unsuitable for some photo-optical devices, which makes full automation difficult

Presence of aPL
Various aPL may be present in some people who are otherwise healthy, have autoimmune or rheumatic disease, have been exposed to certain drugs or infectious agents.

Recurrent pregnancy loss

Venous thrombosis
Inherited and acquired coagulation and anticoagulation factor disorders (eg, protein C and protein S deficiency, factor V Leiden deficiency) II. Defective clot lysis III. Cancer and myeloproliferative disorders IV. Nephrotic syndrome V. Others.
I.

Arterial thrombosis

Atherosclerosis Embolic disease Atrial fibrillation or, much less common, atrial myxoma Marked left ventricular dysfunction Endocarditis Cholesterol emboli Paradoxical embolism Decompression sickness (Caisson's disease) TTP/HUS systemic vasculitis

Arterial and Venoous thrombosis

Heparin-induced thrombocytopenia,Defective clot lysis due to dysfibrinogenemia or plasminogen activator deficiency II. Homocysteinemia III. Myeloproliferative disorders, polycythemia vera (P vera), or paroxysmal nocturnal IV. hemoglobinuria V. Hyperviscosity due to P vera, Waldenstrom's macroglobulinemia, sickle cell disease VI. Systemic vasculitis, such as those associated with antineutrophil cytoplasmic antibodies VII. Paradoxical embolism
I.

It is an accelerated form of APS characterized by multiple small vessel thrombosis that can lead to multiorgan failure. It is also called Ashersons syndrome. CAPS represents less than 1% of the APS cases. In contrast to classic APS, single venous or arterial medium to large blood vessel occlusions are uncommon.

60% of patients appear to have a triggering factor, especially infections,

Development of SIRS which is common link between sepsis and CAPS.

Role of ADAMTS13 deficiency in the development of CAPS.

ANA, Lupus anticoaguloant and or anti cardiolipin ab

THROMBOCYTOPENIA ,hemolytic anemia and schisticytes
Finding consistent with DIC :prolonged PT and PTT , increased FDP and hypofibroginaemia

Thank you for your attention !