ANTIPHOSPHOLIPID ANTIBODIES

History
In 1952- Conley and Hartmann published the first description of phospholipid dependent coagulation inhibitor in two patients with SLE. In 1957- Laurell and Nelsson described an association between a chronic false +ve serologic test for syphillis,a circulating anticoagulant and recurrent pregnancy loss. In 1963- Bowie et al recognized the association between phospholipid dependent coagulation inhibitors and thrombosis rather than bleeding. In 1972- Feinstein and Rapaport proposed the term LA.

In 1980- An immunoglobulin monoclonal antibody displaying LA characteristics was demonstrated to react with anionic phospholipids other than cardiolipin. In 1990- It was recognized that a subset of these antibodies actually recognized a plasma protein B2GP1 in complex with phospholipids

LUPUS ANTICOAGULANT
The Lupus Anticoagulant is an acquired autoantibody found in a variety of autoimmune disorders and sometimes even in otherwise healthy individuals  Antibody detected against protein bound phospholipid that delays in vitro coagulation without a bleeding tendency but associated with thrombotic risk

Nature of antibodies
LA are acquired antibodies of heterogenous nature directed against anionic phospholipids thereby inhibiting in vitro generation of prothrombin activator complex with consequent prolongation of clotting time in most of phospholipid dependent coagulation tests

LA - Actually a misnomer
Associated with clotting, not anticoagulation in vivo. Recurrent venous thromboembolism  Recurrent abortions Cerebrovascular Accidents

Anticoagulant Effect of LA in Vitro

Inhibits activation of factor IX Inhibits activation of factor X Inhibits activation of prothrombin to thrombin

LA ACA
APLS

B2GP1

APLS
AntiPhospholipid Antibody Syndrome
APLS is an acquired disorder in which patients have thrombotic manifestation and/or recurrent spontaneous pregnancy loss with lab evidence of autoantibodies that recognize anionic phospholipid protein complexes

APLS

Primary APLS

Secondary APLS

Catastrophic APLS

Catastrophic APLS
Characterized by severe widespread vascular occlusions sometimes leading to death. Patients present with evidence for severe multi organ ischemia/infarction usually a concurrent microvascular thrombosis. Few patients experience large vessel occlusion and can present with massive thromboembolism along with respiratory failure and stroke.

Clinical Diagnosis associated with APLA

Primary APLA Secondary APLA  SLE  Other autoimmune and connective tissue disorders  Drug Induced Malignancies  Lymphoproliferative disorders Infections

Neurologic Disorders Liver Disease Valvular Heart Disease Peripheral Heart Disease Chronic Renal Failure

Arterial and Venous Thromboembolic Disease

Most common clinical presentation of patients with LA and ACL antibodies. Affecting about 70% of patients. Most common site is extremity deep vein thrombosis,but occasionally unusual sites are also involved such as axillary,retinal and hepatic veins and cerebral venous thrombosis.

Obstetric Aspects of APLS

APA are associated with obstetric complications including - Intrauterine growth restriction - Preeclampsia - Recurrent spontaneous fetal loss

Obstetric criteria for diagnosis of APA

1. One or more unexplained death of morphologically normal fetus at or beyond 10th week of gestation 2. One or more premature births of morphologically normal neonate before 34th week of gestation 3. Three or more unexplained consecutive spontaneous abortion before 10th week of pregnancy

Mechanisms involved in APA associated fetal loss

The pregnancy loss in the APL syndrome may be a consequence of marked reduction in the placental anticoagulant protein, annexin A5. on the apical membrane of placental syncetiotrophoblasts. Reduction of this protein which normally coats the interface between the maternal and fetal circulation may disrupt a consecutive antithrombotic mechanism within the intervillus blood circulation.

There is also an evidence to suggest that Annexin A5 plays an antithrombotic role in physiologic conditions . Phosphotidyl serine is present on the apical membranes of syncytialized trophoblasts where it is covered by a binding layer of Annexin A5 . Dissociation of Annexin A5 from surface of human placental trophoblasts and human umblical vein endothelial cells, accelerates the coagulation of plasma exposed to those cells so Annexin A5 plays a thrombomodulary role on the surface of cells lining placental vasculature.

Thrombocytopenia
Moderate immune thrombocytopenia is observed in approx 50% of patients with lupus inhibitors. Patients with thrombocytopenia of obscure etiology, including those with ITP like syndromes, probably should be screened for the LA and ACL antibodies

 When bleeding occurs in patients with APLS, this is generally attributed to coexistent thrombocytopenia, platelet dysfunction, prothrombin deficiency or other underlying coagulopathies.

Dermatologic Disorders
Livedo reticularis Acrocynosis Widespread cutaneous necrosis More common in patients with SLE and in females

Cardiac Disorders
 A high incidence of APA is seen in patients with peripheral arterial disease who experience an associated increased risk of early graft thrombosis Coronary artery disease also fulfils the APS thrombosis criterion.

Pulmonary Disease
Ischemic and Thrombotic pulmonary disease is linked to APA including Pulmonary embolism Pulmonary hypertension Intra alveolar pulmonary hemorrhage Adult respiratory distress syndrome

Proposed Pathogenic mechanism of Thrombosis in APLS

1. APLA mediated disruption of Annexin a5 anticoagulant shield. 2. Effects of APLA on vascular endothelium cells. 3. Interference with components of protein C pathway. 4. Induction of tissue factor activity by leucocytes. 5. Inhibition of the anti thrombin pathway. 6. Effects of APLA on platelets.

Effects of APLA on vascular endothelium

APL antibodies have been found to recognize, injure and activate the endothelial cells. APL antibodies leads to tissue factor expression and promote thrombosis.

Interference with components of protein C pathway

APL antibodies can interfere with protein C system by

 Decreasing activation of protein C by thrombomodulin thrombin complex.  Inhibiting assembly of protein C complex.  Inhibiting activity of protein C directly or via its cofactor protein S.  Binding to factor Va and VIIIa in a manner that protects them from proteolysis by APC.

Inhibition of the antithrombin pathway

Antithrombin is a member of serine protease inhibitor family. Individuals with deficiency of antithrombin are at increased risk of deep vein thrombosis.The antithrombotic activity of this protein is markedly accelerated by the presence of heparin or in vivo by heparan sulfate proteoglycans. Some APL antibodies cross react with heparan sulfate molecules and inhibit their effect on acceleration of antithrombin activity.

Effects of APLA on platelets

APL antibodies can stimulate platelet aggregation. Dimers of B2-GP1 which mimic effects of B2-GP1-anti B2-GP1 antibody complexes, increase platelet adhesion to collagen and thrombus formation in a flow system.

Indications for LA
Tests for the presence of LA should be carried out in  All young individuals with unexplained thrombosis , especially if there is no family history  In women with recurrent second trimester fetal loss  When LA is present the first line tests usually show a normal TT and a prolonged PTTK with a normal/ abnormal PT

Criteria for diagnosis of LA

LA presence in plasma is detected according to the guidelines of ISTH in following steps: 1) Prolonged phospholipid dependent coagulation demonstrated with a screening tests (APTT,KCT,Drvvt) 2) Failure to correct the prolonged coagulation time on the screening test by mixing with normal PPP.

Cont
3) Shortening or correction of prolonged coagulation time on the screening test by addition of excess PL. 4) Exclusion of other coagulopathies

Approach to the diagnosis of LA

Heat Stability Test

Principle Immunoglobulins are not adsorbed to aluminium hydroxide and are stable at 56C whereas most clotting factors are removed from plasma or inactivated by this treatment.When the adsorbed and heated plasma is mixed with fresh normal plasma and PTTK performed,any immunoglobulin inhibitor will still be present and will prolong the clotting time.

Reagent
Aluminium hydroxide moist gel:4g of moist gel/100ml water 2. Kaolin0.05% in 0.2mol/l tris buffer pH 7.4 3. Phospholipid 4. CaCl2 0.025mol/L 5. Fresh control plasma 6. PPP from patient and control Materials required 56C water bath 1.

Procedure
1. Place 0.5 ml of control plasma and 0.5ml of patient plasma in separate plastic tubes. 2. Add 50ul of AlOH suspension and incubate the tubes at 37C for 3 mins. 3. Centrifuge the tubes at 2000g for 15 mins and carefully pipette out the supernatant in fresh tubes which are capped and placed at 56C for 30mins. 4. Then centrifuge the tubes for 10mins at 2000g and pipette supernatant in plastic tubes. 5. Mix 0.1ml of treated patient or control plasma with 0.1ml of fresh normal plasma in a glass tube at 37C and carry out PTTK.

Interpretation
If an immunoglobulin inhibitor is present, the clotting time of the mixture of treated patient plasma and fresh control plasma will be at least 2sec longer than that of the treated and fresh control plasma.

Dilute Thromboplastin Inhibition Test

Principle: When the thromboplastin used for
PT is diluted, the PT becomes prolonged. At a certain point the conc. of phospholipid is low enough for the test to become sensitive to phospholipid binding antibodies and when an LA is present the ratio of test plasma to normal plasma clotting time increases.

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

Textarin / Ecarin Ratio

Principle
This ratio is a sensitive and relatively specific test for LA based on fractions of two snake venoms. Textarin is a protein fraction of Pseudonaja Textilis venom(Australian Eastern Brown snake) that activates prothrombin in the presence of phospholipid,factor V and Ca++.Ecarin is a protein fraction of Echis carinatus(Indian Saw scaled viper)venom that activates prothrombin

Cont.
in absence of cofactors.The activation of prothrombin by Textarin yeilds thrombin, whereas Ecarin yeilds meizothrombin(an intermediate of prothrombin activation).In presence of LA , the Textarin time is prolonged whereas the Ecarin time is unaffected

Results: The results are reported as a ratio of Textarin / Ecarin times. A positive result is indicated by a ratio greater than 1.3  This test has been reported to be useful in identifying LA in samples from patients who have received Warfarin because the action of Ecarin is not affected by lack of vit K

Kaolin Clotting Time

Principle When the PTTK is performed in absence of platelet substitute reagent,it is particularly sensitive to the LA.If the test is performed on a range of mixtures of normal and patient plasma, different patterns of response are obtained indicating the presence of LA ,deficiency of one or more coagulation factors or the Lupus cofactor effect.

dRVVT
Intended use : LA1 (screening reagent)- To screen the presence of LA LA2 (confirmation reagent)Phospholipid rich dRVV reagent for specific correction of LAs.

Principle
The RVV is present in dRVV screen and dRVV confirm reagents acts in the presence of calcium as an activator of factor X and so triggers the coagulation cascade downstream from factor X, thus eliminating the influence of coagulation factors acting upstream. dRVV screen is performed with a low conc.of phospholipid .If LA is present the clotting time will be lengthened. dRVV confirm contains a higher conc.of phospholipid which neutralizes LA present in plasma thus the clotting time obtained will be shortened.

Reagent
1. LA-1 2. LA-2 Contain RVV,phospholipids,antiheparin agents, calcium,stabilizers,sodium azide and dyes.

Materials required
1. 10x75mm glass test tubes 2. Water bath, Stop watch 3. Pipettes

1. Pre warm a slight excess of LA1 and LA2 at 37C in a reagent reservoir allowing 200ul per test. 2. Dispense 200ul of test plasma into glass tube and warm for 1 min at 37C. 3. Add 200ul of prewarmed reagent to plasma and note time from moment of reagent addition to clotting end point. 4. Repeat for duplicate test values and report the average of these as result.

Procedur e

LA-1
Patient Plasma Normal + Patient

LA-2
Patient Plasma Normal + Patient

Diagnosis

N Abn Abn Abn Abn

N Abn N Abn Abn

N N Abn Abn Abn

N N N N Abn

LA not detected

LA present Factor deficiency LA+Factor deficient Other inhibitor

Hexagonal Phase Array Test

Anti phospholipid antibodies can recognize phosphotidyl ethanolamine in the hexagonal phase array configuration but not in the lamellar phase. Principle Incubation of plasma with the hexagonal phase phosphatidyl ethanolamine should absorb the LA antibodies, if present and so normalizes a prolonged APTT because of LA.

Contt.
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

Platelet Neutralization Test

Principle
When platelets are used instead of phospholipid reagents in clotting tests,the test become insensitive to the LA.This may be partly due to the high concentration of phospholipids active in coagulation but it may also be due to presence of other functional sites and receptors at the platelet membrane that favours the coagulation process rather than binding of the anti phospholipid immunoglobulins.To utilize this property of platelets they are washed to remove contaminating plasma proteins and activated to expose their coagulation factor binding sites.

REAGENTS
Commercial platelet extract reagent or washed normal platelets. ACD anticoagulant solution, pH 5.4.For use, one part of this anticoagulant is added to6 parts of blood. Na2EDTA. 0.1 mol/l in saline. Calcium-free Tyrodes buffer. To prepare dissolve 8 g NaCl, 0.2 g KCL, 0.625 g Na2HPO4, 0.415 g MgCl2 and 1.0 g NaHCO2 in 1L of water. AdjustpH if necessrary to 6.5 with 1 mol/l HCL>

PROCEDURE
Collect normal blood into ACD and centrifuge at 270 g for 10min. Pipette the supernatant plateletrich plasma (PRP) into a plastic container, and centrifuge for a second time to obtain more PRP, which is added to the firat lot. Dilute the PRP with an equal volume of the calcium-free buffer, and add sufficient EDTA to give a final concentration of 0.01 mol/l. Centrifuge the mixture in a conical or round-bottom tube at 2000g for 10 min,and discard the supernatant. Gently resuspend the platelet pellet in buffer and 0.01 mol/l EDTA,and centrifuge again.

 Again discard the supernatant, and resuspend the pellet in buffer alone. Then centrifuge the platelets a third time, and finally resuspend the pellet in buffer without EDTA to give a platelet count of atleast 400*10 /l. The washed platelets may be stored below -20C in volumes of 1-2 ml. Before use,they must be activated by repeatedly thawing and refreezing 3-4 times. Use the washed platelets or the commercial reagent in the dilute RVV test or in the PTTK in place of the usual phospholipid reagent. First, determine a suitable dilution by testing a range of doubling dilution in the test system with control plasma. A suitable dilution will give a similar clotting time to thatobtained using control plasma and the phospholipid reagent.

Anti Cardiolipin Antibody Screening

Anti Cardiolipin is an indirect solid phase enzyme immunoassay for the simultaneous quantitative measurement of IgG,IgM and IgA class of autoantibodies against cardiolipin in human serum / plasma.

Principle
Highly purified cardiolipin is bound to microwells saturated with B2 GP1. Antibodies against these antigens if present in diluted serum / plasma ,bind to the respective antigens.  Washing of the microwells removes unspesific serum/plasma components  HRP conjugated anti human IgG,IgM and IgA immunologically detects the bound patient abs forming a conjugate/Ag/Ab complex.

Contt.
 Washing of the microwells removes unbound conjugate.  An enzyme substrate in the presence of bound conjugate hydrolyzes to form a blue color.  The addition of an acid stops the reaction forming a yellow end product  The intensity of the yellow color is measured photometrically at 450 nm

Materials Required: -Microplate consisting of 12 modules of 8 wells each, coated with highly purified bovine cardiolipin and saturated with B2 GP1. -Wash solution(PBS,NaN3 0.1%) -Microplate reader capable of endpoint measurement at 450nm -Vortex mixer -Timing device

Reagents Required 1. Sample Buffer 2. Enzyme Conjugate soln. 3. TMB Substrate soln. 4. Stop soln.(1M Hcl) Sample Preparation Dilute patient sample 1:100 with sample buffer before assay by taking 10ul of sample + 990ul of sample buffer and mix well.

Procedure
1. Prepare a sufficient no.of microplates to accommodate controls and prediluted patient samples. Pipette 100ul of controls and patient sample in duplicate in wells. Incubate for 30 mins at RT. Discard the contents and wash the wells 3 times with 300ul of wash solution. Dispense 100ul of Enzyme Conjugate into each well. Incubate for 15 mins at RT.

2. 3. 4. 5. 6.

Contt..
7. Discard the contents of microwells and wash 3 times with 300ul wash solution. 8. Dispense 100ul of TMB substrate into each well. 9. Incubate for 15 mins at RT. 10. Add 100ul of stop soln. to each well and incubate for 5 mins at RT. 11. Read OD at 450nm and calculate the results.

Interpretation of Results
Evaluation of ACA is carried out by direct comparison of O.D of each sample with O.D of controls.

-ve +ve

O.D of patient< O.D cut off ctrl O.D of patient>O.D cut off ctrl

The cut off has been set for ACA in a normal study with serum sample from healthy donors by performing ACA test on them.

Cut Off

10U/ml

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