Lupus Anticoagulant
M. D. Creagh, M. Greaves S UMMA R Y. Acquired antibodies to phospholipids form a heterogeneous group, which may be detected in vitro by the inhibition of phospholipid dependent tests of coagulation (Lupusanticoagulant) and also by immunological assays, such that a combined approach is required for their reliable detection. While initially described in sufferers from systemic lupus erythematosus, these antibodies are increasingly recognised in a broad spectrum of disease, most importantly in relation to thromboembolism and recurrent fetal loss; occasionally they may also be found in otherwise healthy individuals. The mechanisms underlying the prethrombotic state associated with these antibodies have not been defined, although interference with the natural anticoagulant systems seems possible. Identification of antiphospholipid in subjects with spontaneous thromboembolism may influence therapeutic decisions, while their presence in women with recurrent fetal loss has lead to attempts to alter the outcome of further pregnancies with anticoagulant and immunosuppressive regimens, however the optimum management has not yet been determined. The recognition of these antibodies and their clinical associations is therefore highly relevant to clinical and laboratory haematology.
It is nearly 40 years since Conley and Hartmann’ described inhibitors of coagulation in 2 patients with systemic lupus erythematosus (SLE). Subsequently these ‘lupus-type’ inhibitors were detected in other subjects with SLE and the associated tendency to thrombosis recognised.’ Paradoxically the term ‘lupus anticoagulant’ (LA) was introduced to describe such inhibitors.3 Over the last decade interest in the phenomenon has grown, LA and related antibodies to negatively charged phospholipids, such as cardiolipin, having been found in a variety of disease states as well as in otherwise healthy individuals, such that this area is clinically important to physicians across many specialist fields. The observation that the presence of these antibodies is associated with adverse pregnancy outcome in young women has also generM. D. Creagh, Lecturer, M. Greaves, Reader and Consultant in Haematology, Royal Hallamshire Hospital, Glossop Road, Sheffield SlO 2JF, UK. Correspondence to Dr M. Greaves. Bhd Reviews (1991) 5, 162-167 0 1991 Longman Group UK Ltd
ated considerable interest. The recognition of the diversity of clinical and laboratory findings has led to the use of the term the ‘anti-phospholipid syndrome’ (APS) to describe the association of antibody to phosphohpid with the clinical occurrence of thrombosis, thrombocytopenia or recurrent miscarriage. Lupus anticoagulants are therefore part of a heterogenous group of antibodies with differing specificities for negatively charged phospholipid. The presence of these antibodies may be determined by their ability to inhibit phospholipid dependent coagulation tests (lupus anticoagulant) or to react with negatively charged phospholipid in a solid phase immunoassay (for example antibody to cardiolipin ACL).4 The biological false positive test for syphilis is also a feature. These antibodies are usually polyclonal and they exhibit considerable laboratory heterogeneity, hence a comprehensive methodological approach is required for their determination.
BLOOD REVIEWS
The Laboratory Identification of Antibodies to Phospbolipid Coagulation-based Assays for LA LA act by inhibition of the prothrombinase complex, prolonging clotting times in the intrinsic pathway and less frequently the extrinsic pathway. The determination of the presence of LA thus requires the finding of prolongation of a phospholipid dependent coagulation test, due to an inhibitor directed at phospholipid and not at a specific coagulation protein. Pre-analytical Variables. Several tests have been promoted as being sensitive and specific for LA determination. Common to all tests is the importance of preanalytical variables, as sample collection and handling may influence the results. Disrupted platelets expose phospholipid to which antibodies to phospholipid (APL) may bind, thus quenching the LA effect. Blood should be processed without delay, with separation of the plasma ideally within an hour. Coagulation activation which may occur with the use of evacuated blood collection systems does not seem to influence results and samples collected in this way can be tested.5 Use of a microfilter or double centrifugation ensures maximal removal of residual platelet fragments and such manoeuvres are strongly recommended.6 Choice of Test. Of the screening tests for LA the APTT is used most frequently. Although more sensitive when a reagent with a low phospholipid content is used, nevertheless this test may be inadequate for detection of weak inhibitors. Furthermore, previous recommendations for the diagnosis of the presence of an inhibitor7-that a prolonged APTT should remain so by greater than 4 s over the control in a 1 : 1 mix with normal plasma-are too stringent; often samples will correct with 50% plasma, but not with 20%, yet LA is diagnosed by a more specific test. The modification of the APTT using aluminium hydroxide absorption and heating’ is unreliable. The dilute thromboplastin time’ is sensitive, however its specificity is limited and it may be affected by Factor VIII or Factor IX antibodies and heparin therapy, although the latter should be suspected from prolongation of the thrombin time. Recent recommendations for the detection of LA9 support the use of the APTT with either the kaolin clotting time (KCT)” or the dilute Russell’s viper venom time (DRWT). ” The KCT is a sensitive test, l2 however it utilises PPP with no added phospholipid and being sensitive to residual platelet debris, requires a filtration procedure. It is a manual procedure, in full entailing a series of dilutions of patient to normal plasma, although for screening purposes the ratio of a 1 : 4 mix of patient to normal plasma compared to a control time is suitable. The DRWT is a sensitive test comparable to the KCT,9*‘3 that
163
may be automated. The result is expressed as a ratio of the patient time to that of normal plasma. Confirmation that the inhibitor is directed at phospholipid rather than coagulation factors may be obtained using a platelet neutralisation procedure (PNP) 14,1Jin either the APTT or the DRVVT. Freeze thawed platelets may be used as a source of dilute phospholipid in place of the platelet substitute in the test: a significant shortening of the test plasma relative to normal plasma, also tested using freeze thawed platelets, confirms the presence of an inhibitor. The confirmation of an inhibitor by a neutralisation procedure helps exclude the likelihood of a significant factor deficiency, nevertheless if doubt remains then coagulation factor assays should be performed to exclude a specific inhibitor. Often the apparent concentration of several coagulation factors will be decreased, with non parallel assays. Testing During Anticoagulant Therapy. Assays for LA should be performed after discontinuation of anticoagulant therapy. Where this is not possible, in the subject taking warfarin a 1 : 1 mix with normal plasma may be used to restore the Vitamin K dependent factors to a level such that the DRVVT should be normal, while a strong LA will still be detectable. No acceptable method for testing in the presence of heparin has been devised.
Assays for Cardiolipin Antibody Introduction of the solid phase cardiolipin antibody assay4 has enabled the screening of large numbers of samples. In this test bovine cardiolipin is dissolved in ethanol, applied to microtitre plates and allowed to evaporate overnight. Test serum is added to the plates, incubated and an anti-human globulin is used to determine the titre of the cardiolipin antibody either, as originally described, by radioimmunoassay or more commonly by ELISA.i6 The assay can be adapted for the assessment of cross-reactivity by substitution of other phospholipids for cardiolipin. Positive results are commonly obtained with a range of negatively charged phospholipids only. The clinical relevance of the particular patterns of reactivity is not yet known. It may be explained by the recent important observation, by two independent groups, that antiphospholipid activity is primarily directed against a 50-kDa plasma glycoprotein which itself binds to negatively charged phospholipids,17*** although the importance of this ‘cofactor’ has been disputed. While estimation of cardiolipin antibodies has become widely practised, the interpretation of results has been limited by a lack of standardisation. This is particularly relevant to weakly positive tests. However an international standardisation exercise has been undertaken to improve the correlation between the results of participating laboratories and to enable multinational studies using these standardised tests.
164
LUPUS ANTICOAGULANT
It has also been recognised that the cardiolipin antibody assay is susceptible to non-specific binding. lg The inclusion of appropriate control wells for each sample is essential in order to avoid false positive results. In the presence of high titre rheumatoid factor estimation of IgM cardiolipin antibodies is unreliable. Also, cardiolipin antibody titres on samples from healthy subjects are not normally distributed. In many laboratories the normal range is therefore constructed from the mean value plus three or more standard deviations. In our own clinical practice to determine the presence of antiphospholipid we use an APTT method with a low phospholipid content and the DRWT to screen for LA, confirmed by a platelet neutralisation procedure. Serum is tested for IgG and IgM cardiolipin antibodies using a standardised ELISA. Positive results lead to repeat testing with an interval of at least two months between the tests, to avoid transient positives of doubtful significance. Our indications for screening are listed in Table 1. Clinical Associations of Antibodies to Phospholipid APL have been reported with variable frequency in a wide range of clinical states (Table 2) as well as, very occasionally, as an incidental finding in the apparently healthy. Whilst the detection of LA and/ or ACL may be predictive for arterial or venous thrombotic disease or for poor pregnancy outcome, in many cases the finding appears not to be associated with such risk. Antibody development during acute infections or in response to drug therapy may thus not carry the same significance as that arising as part of a lupus-like illness, although the thrombotic risk
Table 1 Indications for screening for antiphospholipid 1. Spontaneous venous thromboembolism presenting below 45 years of age 2. Arterial thrombosis presenting below 45 years of age in the absence of risk factors 3. Recurrent idiopathic first and second trimester fetal loss, early severe pre-eclampsia 4. For the assessment of thrombotic risk in cases of SLE 5. To investigate disorders with a possible thrombotic component, e.g. ischaemic optic neuropathy, chorea and livid0 reticularis
Table 2 Clinical conditions associated with, or in which APL have been found
1. SLE, rheumatoid arthritis, systemic sclerosis, temporal arteritis, psoriatic arthropathy and Sjogren’s syndrome 2. Acute infection, syphilis, malaria and HIV infection 3. Thrombotic stroke, transient cerebral ischaemia, retinal vein occlusion, ischaemic optic neuropathy and chorea 4. Myocardial infarction and ischaemic heart disease, peripheral arterial occlusion, venous thromboembolism, pulmonary hypertension and valvular disease 5. Recurrent first and second trimester fetal loss, intrauterine growth retardation and early severe eclampsia 6. Medications particularly phenothiazines and also hydralazine, procainamide, phenytoin and quinidine
in each clinical situation has not yet been adequately quantitated. Thrombosis Most frequently phospholipid antibodies have been identified in association with thromboembolic disease. This was first noted by Bowie et al2 in subjects with SLE; here the correlation with LA appears stronger than that with ACL and it is reported that the presence of LA confers between a 2- and 5-fold increase in the incidence of thrombosis compared to SLE sufferers who are negative.20v2’ Paradoxically the presence of thrombocytopenia may increase the risk of thrombosis,22 although APL also commonly occur in patients with immune thrombocytopenia in the absence of overt thrombosis. In those who do not have SLE, but do have APL, a 25% incidence of thrombotic events has been reported 23 although selection bias may contribute to this apparently high incidence. About 70% of thrombosis occurs in the venous system, most commonly in the deep veins of the leg;23 arterial sites include peripheral, coronary and cerebral vessels. In relation to myocardial infarction and after coronary artery bypass surgery, the finding of APL may be a consequence of tissue damage, however most interestingly those subjects who develop antibodies do appear to have an increased risk of further thrombotic episodes.24+25 Often thrombosis may occur at unusual sites-we have observed an association with ischaemic optic neuropathy and witnessed retinal vein occlusion as a presenting event, for example. Incidental APL APL are not uncommonly found incidentally, usually during preoperative assessment of coagulation. Here, a careful clinical assessment is mandatory and the drug history should be scrutinised in order to determine the significance, if any, of the finding. There is no haemorrhagic risk where prolongation of the KCCT is shown to be due to the presence of LA, although the rare association with specific factor inhibitors, especially to factors II and VIII, and the occasional finding of thrombocytopenia in association with APL, should be noted. The risk of subsequent thromboembolic events is the subject of a current survey. Obstetric Associations Since the first report by Nilsson et a126recurrent fetal loss, stillbirth and pre-eclamptic toxaemia of early onset have been increasingly recognised in association with the detection of APL in the blood of women with SLE,27 as well as in otherwise apparently healthy women.28*2g Our own findings support an independent association of LA and IgG ACL with recurrent fetal loss (3 or more episodes) in the first or second
BLOOD REVIEWS
trimester-up to 30% of such women being positive for one or other of these tests. In contrast, the prevalence of positive tests among women with one or two episodes of fetal loss is 10w.~’ In a study of 501 uncomplicated pregnancies we have noted a low incidence (2.8%) of positive tests for APL. Although positivity identified some of those with previous fetal loss poor outcome in the current pregnancy was not predicted by the finding of positive tests for APL. Our results indicate that therapeutic intervention in pregnancy should be reserved for those women with APL who have suffered more than three consecutive episodes of fetal 10~s.~’
165
agulant system. 35,36 The requirement for phospholipid in protein C activation provides a basis for this interaction, although the association with arterial as well as venous thrombosis is not entirely consistent with this mechanism of action. The influence of antibody interaction with the 50 kDa cofactor (probably l3,-glycoprotein 1) referred to already on these various mechanisms is a potentially fruitful area for further research. It also remains to be determined whether the obstetric complications associated with the presence of these antibodies results from a thrombotic process in the placental vasculature, as placental infarction has not invariably been demonstrated.
The Antiphospholipid Syndrome
The concept of a primary APS encompassing the clinical and laboratory associations of antiphospholipid is useful can be defined as the finding of antiphospholipid (LA or antibodies to cardiolipin), on at least two occasions 8 weeks apart, in a subject with two or more of spontaneous venous thrombosis, arterial thrombosis, recurrent fetal loss or thrombocytopenia, without clinical or serological evidence of systemic lupus erythematosus. However, this definition is somewhat restrictive as subjects with isolated thromboembolic disease or obstetric complications, although not fulfilling the diagnostic criteria for the APS, may have antiphospholipid of prognostic and therapeutic importance. Pathogenetic Mechanisms Several lines of investigation have been pursued to determine the mechanisms of the thrombotic tendency in the presence of APL. No single mode of action fully explains the clinical correlates observed. Although the association with thrombosis appears firm, causality has not been demonstrated. Positive sera and IgG purified therefrom may tend to result in a reduction in vascular endothelial prostacyclin synthesis in in vitro systems although the experimental conditions must be carefully controlled in order to demonstrate this effect.32 However, our own observations also suggest that this inhibition may relate more closely to the titre of antibodies to endothelial antigens than to the ACL titre. Such antibody activity to endothelial cells is common in sera positive for antiphospholipid. Furthermore, it is perhaps simplistic to suggest that a deficiency of prostacyclin could underlie the range of thrombotic manifestations observed clinically. Endothelial cell damage can be suspected in some situations where APL are commonly found33 and an alternative possibility is that such damage, possibly mediated immunologically, could increase the tissue factor procoagulant activity of endothelium or impair fibrinolytic responses.34 IgG preparations from plasma of patients positive for ACL/LA can interfere in the protein C/S antico-
Clinical Management Antithrombotic Therapy
Controlled clinical trial results are not available to provide guidance on thrombosis management in subjects with APL. The incidental finding of APL is not an indication for intervention, although in certain clinical settings; e.g. SLE, a positive test for APL, should alert the clinician to the increased thrombotic tendency and lower the threshold for use of prophylaxis during high risk periods such as the postoperative and postpartum states. Counselling against exposure to other risk factors, smoking and possibly the contraceptive pill, should be given. Acute thrombotic episodes should be managed in the usual way. Recurrent thrombosis on discontinuation of anticoagulants may occur and the duration of anticoagulant treatment may be influenced by positive tests for APL. Such therapeutic decisions must depend upon the site and severity of the event and the perceived risk-benefit ratio in an individual sufferer. The efficacy of aspirin prophylaxis against thrombosis in the presence of APL remains to be determined. Its use could certainly be justified in the presence of transient cerebral ischaemic episodes or myocardial infarction and after thrombotic stroke. Immunosuppressive therapy is not justified for thrombotic disease but may be indicated for other immune-mediated complications, especially against a background of SLE. Management of Recurrent Fetal Loss
Again, published information is largely anecdotal. Apparent good results from intervention should be gauged against the relatively high chance of a successful outcome without intervention, even after several previous episodes of fetal loss. Aspirin, heparin, steroids and high dose intravenous human IgG have all been recommended. Our own approach, in women with 3 or more miscarriages and detectable APL on at least two occasions, is to offer prophylaxis with low-dose subcutaneous heparin and/or low-dose as-
166
LUPUS ANTICOAGULANT
pirin for subsequent pregnancies, combined with close liaison with obstetric colleagues and regular review of progress. In the event of evidence of intrauterine growth retardation or in subsequent pregnancies in the event of fetal loss, full anticoagulation or the use of steroids or intravenous immunoglobulin should be considered. Concluding Remarks Long considered as little more than a laboratory phenomenon of little significance, the finding of LA is now known to be of clinical importance in many instances. The disease associations have been defined but further work is needed to determine the mechanisms of the prethrombotic effect and to establish optimal management strategies in the various clinical circumstances where APL have been detected. Acknowledgements The authors would like to acknowledge Mr R. G. Malia and Mrs L. Wattam for their help in the preparation of this manuscript.
References 1. Conley C L, Hartmann R C 1952 A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. Journal of Clinical Investigation 31: 621-622 2. Bowie W E J, Thompson 3 H, Pascuzzi C A, Owen G A 1963 Thrombosis in systemic lupus erythematosus despite circulating anticoagulant. Journal of Clinical Investigation 62: 416-430 3. Feinstein D I, Rapaport S I 1972 Acquired inhibitors of blood coagulation. Progress in Hemostasis and Thrombosis 1: 75-95 4. Harris E N, Gharavi A E, Boey M L et al 1983 Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet ii:1211-1214 5. Creagh M D, Malia R G, Greaves M 1991 The use of an evacuated collection system in screening for the lupus anticoagulant by the KCCT and DRVVT. British Journal of Haematology 77: 59 6. Machin S J, Giddings J, Greaves M et al 1990 Detection of lupus like anticoagulant: current laboratory practice in the United Kingdom. Journal of Clinical Pathology 43: 73-76 7. Green D, Hougie C, Kazmier F J et al 1983 Report of the Working Party on Acquired Inhibitors of Coagulation: studies of the ‘lupus’ anticoagulant. Thrombosis and Haemostasis 49: 144-146 8. Austin D E G, Rhymes L L 1975 A Laboratory Manual of Blood Coagulation, Blackwell Scientific Publications, London, p. 15 9. Schleider M A, Nachman R L, JaIfe E A, Coleman M 1976 A clinical study of the lupus anticoagulant. Blood 48: 499-509 10. Exner T, Rickard K A, Kronenberg H 1978 A sensitive test demonstrating lupus anticoagulant and its behavioural oatterns. British Journal of Haematology 40: 143-151 11. Thiagarajan P, Pengo V, Shapiro S S, 1986 The use of the dilute Russell viper venom time for the diagnosis, of lupus anticoagulant. Blood 68: 869-874 12. Lo S C L, Oldmeadow M J, Howard M A, Firkin B G 1989 Comparison of laboratory tests used for identification of the lupus anticoagulant. American Journal of Hematology 30: 213-220 13. Forasterio R R, Falcon C R, Carreras L 0 1990 Comparison of various screening tests for the detection of the lupus anticoagulant. Haemostasis u): 208-214
14. Triplett D A, Brandt J T, Kaczor D, Schaeffer J 1983 Laboratory diagnosis of lupus inhibitors: A comparison of the tissue thromboplastin inhibition procedure with a new platelet neutralization procedure. American Journal of Clinical Pathology 79: 678-682 15. Howard M A, Firkin B G 1983 Investigations of the lupuslike inhibitor by-passing activity of platelets. Thrombosis and Haemostasis 50: 775-779 16. Harris E N 1990 Antiphospholipid antibodies. British Journal of Haematology 74: l-9 17. Galli M, Maassen C, Comfurius P et al 1990 Isolation and oharacterisation of a plasmatic factor interacting with anticardiolipin antibodies. Clinical and Experimental Rheumatology 8: 206 18. McNeil H P. Chesterman C N. Krilis S A 1990 Antiphosphdlipid antibodies are directed against a complex antigen which includes a lipid-binding plasma glycoprotein. Clinical and Experimental Rheumatology 8: 209 19. Peter J B 1990 Cardiolipin antibody assays. Lancet i: 13351405 20. Petri M, Hochberg M, Hellman D, Corash L, Goldman D 1990 The Association of the lupus anticoagulant (LA) with thrombotic events (TE) in systemic lupus erythematosus (SLE). Clinical and Experimental Rheumatology 8: 217 21. Pauzer R, Rosner E, Many A 1986 Circulating anticoagulant in systemic lupus erythematosus: Clinical manifestations. Acta Haematologica 76: 90-94 22. Hasselar P, Derksen R H W M, Blokzijl L et al 1989 Risk factors for thrombosis in lupus patients. Annals of Rheumatic Disease 48: 933-940 23. Gastineau D A, Kazmier F L, Nichols W L, Bowie E J W 1985 Lupus anticoagulant: An analysis of the clinical and laboratory features of 219 cases. American Journal of Hematology 19: 265-275 24. Hamsten A, Norberg R, Bjorkholm M, de Faire U, Holm G 1986 Antibodies to cardiolipin in young survivors of myocardial infarction: an association with recurrent cardiovascular events. Lancet i: 113-l 16 25. Morton K E, Gavaghan T P, Krilis S A, Daggard G E, Baron D W, Hickie J B, Chesterman C N 1986 Coronary artery bypass graft failure: an autoimmune phenomenon? Lancet ii: 1353-1357 26. Nilsson I M, Astedt B, Hedner U, Berezin D 1975 Intrauterine death and circulating anticoagulant ‘Antithromboplastin’. Acta Medica Scandinavica 192: 153-159 27. Lubbe W F, Palmer S J, Butler W S, Liggins G C 1983 Fetal survival after prednisone suppression of maternal lupus anticoagulant. Lancet i: 1361-1363 28. Branch D W, Scott J R, Kochenour N K, Hershgold E 1985 Obstetric complications associated with the lupus anticoagulant. New England Journal of Medicine 313: 13221326 29 Lockshin M D, Druzin M L, Goei S et al 1985 Antibody to cardiolipin as a predictor of fetal distress or death in pregnant women with systemic lupus erythematosus. New England Journal of Medicine 313: 152-156 30 Creagh M D, Malia R G, Cooper S M, Smith A R, Duncan S L B, Greaves M 1991 Screening for lupus anticoagulant and anticardiolipin antibodies in women with fetal loss. Journal of Clinical Pathology 44: 45-47 31. Creagh M D, Duncan S L B, McDonnell J M, Greaves M 1991 Failure of the detection of antiphospholipid antibodies alone to predict poor pregnancy outcome. British Journal of Haematology 77: 4 32. Lindsey N, Henderson F, Malia R, Greaves M, Hughes P 1991 Serum masks the inhibition of thrombin-induced . . .. , prostacyclin release produced by anti-cardiolipm antibodies. British Journal of Rheumatology (in press) 33. Holt C M, Lindsey N, Moult J et al 1989 Antibody dependent cellular cytotoxicity of vascular endothelium; characterisation and pathogenic associations in systemic sclerosis. Clinical and Experimental Rheumatology 78: 359-365 34. Angeles-Cano E, Sultan Y, Clauvel J P 1979 Predisposing factors to thrombosis in systemic lupus erythematosus: possible relation to endothelial cell damage. Journal of Laboratory and Clinical Medicine 94: 312-323 35. Cariou R, Tobelin G, Belluci S, Soria J, Soria C, Maclouf J, Caen J 1988 Effect of the lupus anticoagulant on
BLOOD REVIEWS antithrombogenic properties of endothelial cells-inhibition of thrombomodulin dependent protein C activation. Thrombosis and Haemostasis 60: 54-58 36. Malia R G, Kitchen S, Greaves M, Preston F E 1990
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Inhibition of activated protein C and its cofactor protein S by antiphospholipid antibodies. British Journal of Haematology 76: 101-107
M. D. Creagh, M. Greaves S UMMA R Y. Acquired antibodies to phospholipids form a heterogeneous group, which may be detected in vitro by the inhibition of phospholipid dependent tests of coagulation (Lupusanticoagulant) and also by immunological assays, such that a combined approach is required for their reliable detection. While initially described in sufferers from systemic lupus erythematosus, these antibodies are increasingly recognised in a broad spectrum of disease, most importantly in relation to thromboembolism and recurrent fetal loss; occasionally they may also be found in otherwise healthy individuals. The mechanisms underlying the prethrombotic state associated with these antibodies have not been defined, although interference with the natural anticoagulant systems seems possible. Identification of antiphospholipid in subjects with spontaneous thromboembolism may influence therapeutic decisions, while their presence in women with recurrent fetal loss has lead to attempts to alter the outcome of further pregnancies with anticoagulant and immunosuppressive regimens, however the optimum management has not yet been determined. The recognition of these antibodies and their clinical associations is therefore highly relevant to clinical and laboratory haematology.
It is nearly 40 years since Conley and Hartmann’ described inhibitors of coagulation in 2 patients with systemic lupus erythematosus (SLE). Subsequently these ‘lupus-type’ inhibitors were detected in other subjects with SLE and the associated tendency to thrombosis recognised.’ Paradoxically the term ‘lupus anticoagulant’ (LA) was introduced to describe such inhibitors.3 Over the last decade interest in the phenomenon has grown, LA and related antibodies to negatively charged phospholipids, such as cardiolipin, having been found in a variety of disease states as well as in otherwise healthy individuals, such that this area is clinically important to physicians across many specialist fields. The observation that the presence of these antibodies is associated with adverse pregnancy outcome in young women has also generM. D. Creagh, Lecturer, M. Greaves, Reader and Consultant in Haematology, Royal Hallamshire Hospital, Glossop Road, Sheffield SlO 2JF, UK. Correspondence to Dr M. Greaves. Bhd Reviews (1991) 5, 162-167 0 1991 Longman Group UK Ltd
ated considerable interest. The recognition of the diversity of clinical and laboratory findings has led to the use of the term the ‘anti-phospholipid syndrome’ (APS) to describe the association of antibody to phosphohpid with the clinical occurrence of thrombosis, thrombocytopenia or recurrent miscarriage. Lupus anticoagulants are therefore part of a heterogenous group of antibodies with differing specificities for negatively charged phospholipid. The presence of these antibodies may be determined by their ability to inhibit phospholipid dependent coagulation tests (lupus anticoagulant) or to react with negatively charged phospholipid in a solid phase immunoassay (for example antibody to cardiolipin ACL).4 The biological false positive test for syphilis is also a feature. These antibodies are usually polyclonal and they exhibit considerable laboratory heterogeneity, hence a comprehensive methodological approach is required for their determination.
BLOOD REVIEWS
The Laboratory Identification of Antibodies to Phospbolipid Coagulation-based Assays for LA LA act by inhibition of the prothrombinase complex, prolonging clotting times in the intrinsic pathway and less frequently the extrinsic pathway. The determination of the presence of LA thus requires the finding of prolongation of a phospholipid dependent coagulation test, due to an inhibitor directed at phospholipid and not at a specific coagulation protein. Pre-analytical Variables. Several tests have been promoted as being sensitive and specific for LA determination. Common to all tests is the importance of preanalytical variables, as sample collection and handling may influence the results. Disrupted platelets expose phospholipid to which antibodies to phospholipid (APL) may bind, thus quenching the LA effect. Blood should be processed without delay, with separation of the plasma ideally within an hour. Coagulation activation which may occur with the use of evacuated blood collection systems does not seem to influence results and samples collected in this way can be tested.5 Use of a microfilter or double centrifugation ensures maximal removal of residual platelet fragments and such manoeuvres are strongly recommended.6 Choice of Test. Of the screening tests for LA the APTT is used most frequently. Although more sensitive when a reagent with a low phospholipid content is used, nevertheless this test may be inadequate for detection of weak inhibitors. Furthermore, previous recommendations for the diagnosis of the presence of an inhibitor7-that a prolonged APTT should remain so by greater than 4 s over the control in a 1 : 1 mix with normal plasma-are too stringent; often samples will correct with 50% plasma, but not with 20%, yet LA is diagnosed by a more specific test. The modification of the APTT using aluminium hydroxide absorption and heating’ is unreliable. The dilute thromboplastin time’ is sensitive, however its specificity is limited and it may be affected by Factor VIII or Factor IX antibodies and heparin therapy, although the latter should be suspected from prolongation of the thrombin time. Recent recommendations for the detection of LA9 support the use of the APTT with either the kaolin clotting time (KCT)” or the dilute Russell’s viper venom time (DRWT). ” The KCT is a sensitive test, l2 however it utilises PPP with no added phospholipid and being sensitive to residual platelet debris, requires a filtration procedure. It is a manual procedure, in full entailing a series of dilutions of patient to normal plasma, although for screening purposes the ratio of a 1 : 4 mix of patient to normal plasma compared to a control time is suitable. The DRWT is a sensitive test comparable to the KCT,9*‘3 that
163
may be automated. The result is expressed as a ratio of the patient time to that of normal plasma. Confirmation that the inhibitor is directed at phospholipid rather than coagulation factors may be obtained using a platelet neutralisation procedure (PNP) 14,1Jin either the APTT or the DRVVT. Freeze thawed platelets may be used as a source of dilute phospholipid in place of the platelet substitute in the test: a significant shortening of the test plasma relative to normal plasma, also tested using freeze thawed platelets, confirms the presence of an inhibitor. The confirmation of an inhibitor by a neutralisation procedure helps exclude the likelihood of a significant factor deficiency, nevertheless if doubt remains then coagulation factor assays should be performed to exclude a specific inhibitor. Often the apparent concentration of several coagulation factors will be decreased, with non parallel assays. Testing During Anticoagulant Therapy. Assays for LA should be performed after discontinuation of anticoagulant therapy. Where this is not possible, in the subject taking warfarin a 1 : 1 mix with normal plasma may be used to restore the Vitamin K dependent factors to a level such that the DRVVT should be normal, while a strong LA will still be detectable. No acceptable method for testing in the presence of heparin has been devised.
Assays for Cardiolipin Antibody Introduction of the solid phase cardiolipin antibody assay4 has enabled the screening of large numbers of samples. In this test bovine cardiolipin is dissolved in ethanol, applied to microtitre plates and allowed to evaporate overnight. Test serum is added to the plates, incubated and an anti-human globulin is used to determine the titre of the cardiolipin antibody either, as originally described, by radioimmunoassay or more commonly by ELISA.i6 The assay can be adapted for the assessment of cross-reactivity by substitution of other phospholipids for cardiolipin. Positive results are commonly obtained with a range of negatively charged phospholipids only. The clinical relevance of the particular patterns of reactivity is not yet known. It may be explained by the recent important observation, by two independent groups, that antiphospholipid activity is primarily directed against a 50-kDa plasma glycoprotein which itself binds to negatively charged phospholipids,17*** although the importance of this ‘cofactor’ has been disputed. While estimation of cardiolipin antibodies has become widely practised, the interpretation of results has been limited by a lack of standardisation. This is particularly relevant to weakly positive tests. However an international standardisation exercise has been undertaken to improve the correlation between the results of participating laboratories and to enable multinational studies using these standardised tests.
164
LUPUS ANTICOAGULANT
It has also been recognised that the cardiolipin antibody assay is susceptible to non-specific binding. lg The inclusion of appropriate control wells for each sample is essential in order to avoid false positive results. In the presence of high titre rheumatoid factor estimation of IgM cardiolipin antibodies is unreliable. Also, cardiolipin antibody titres on samples from healthy subjects are not normally distributed. In many laboratories the normal range is therefore constructed from the mean value plus three or more standard deviations. In our own clinical practice to determine the presence of antiphospholipid we use an APTT method with a low phospholipid content and the DRWT to screen for LA, confirmed by a platelet neutralisation procedure. Serum is tested for IgG and IgM cardiolipin antibodies using a standardised ELISA. Positive results lead to repeat testing with an interval of at least two months between the tests, to avoid transient positives of doubtful significance. Our indications for screening are listed in Table 1. Clinical Associations of Antibodies to Phospholipid APL have been reported with variable frequency in a wide range of clinical states (Table 2) as well as, very occasionally, as an incidental finding in the apparently healthy. Whilst the detection of LA and/ or ACL may be predictive for arterial or venous thrombotic disease or for poor pregnancy outcome, in many cases the finding appears not to be associated with such risk. Antibody development during acute infections or in response to drug therapy may thus not carry the same significance as that arising as part of a lupus-like illness, although the thrombotic risk
Table 1 Indications for screening for antiphospholipid 1. Spontaneous venous thromboembolism presenting below 45 years of age 2. Arterial thrombosis presenting below 45 years of age in the absence of risk factors 3. Recurrent idiopathic first and second trimester fetal loss, early severe pre-eclampsia 4. For the assessment of thrombotic risk in cases of SLE 5. To investigate disorders with a possible thrombotic component, e.g. ischaemic optic neuropathy, chorea and livid0 reticularis
Table 2 Clinical conditions associated with, or in which APL have been found
1. SLE, rheumatoid arthritis, systemic sclerosis, temporal arteritis, psoriatic arthropathy and Sjogren’s syndrome 2. Acute infection, syphilis, malaria and HIV infection 3. Thrombotic stroke, transient cerebral ischaemia, retinal vein occlusion, ischaemic optic neuropathy and chorea 4. Myocardial infarction and ischaemic heart disease, peripheral arterial occlusion, venous thromboembolism, pulmonary hypertension and valvular disease 5. Recurrent first and second trimester fetal loss, intrauterine growth retardation and early severe eclampsia 6. Medications particularly phenothiazines and also hydralazine, procainamide, phenytoin and quinidine
in each clinical situation has not yet been adequately quantitated. Thrombosis Most frequently phospholipid antibodies have been identified in association with thromboembolic disease. This was first noted by Bowie et al2 in subjects with SLE; here the correlation with LA appears stronger than that with ACL and it is reported that the presence of LA confers between a 2- and 5-fold increase in the incidence of thrombosis compared to SLE sufferers who are negative.20v2’ Paradoxically the presence of thrombocytopenia may increase the risk of thrombosis,22 although APL also commonly occur in patients with immune thrombocytopenia in the absence of overt thrombosis. In those who do not have SLE, but do have APL, a 25% incidence of thrombotic events has been reported 23 although selection bias may contribute to this apparently high incidence. About 70% of thrombosis occurs in the venous system, most commonly in the deep veins of the leg;23 arterial sites include peripheral, coronary and cerebral vessels. In relation to myocardial infarction and after coronary artery bypass surgery, the finding of APL may be a consequence of tissue damage, however most interestingly those subjects who develop antibodies do appear to have an increased risk of further thrombotic episodes.24+25 Often thrombosis may occur at unusual sites-we have observed an association with ischaemic optic neuropathy and witnessed retinal vein occlusion as a presenting event, for example. Incidental APL APL are not uncommonly found incidentally, usually during preoperative assessment of coagulation. Here, a careful clinical assessment is mandatory and the drug history should be scrutinised in order to determine the significance, if any, of the finding. There is no haemorrhagic risk where prolongation of the KCCT is shown to be due to the presence of LA, although the rare association with specific factor inhibitors, especially to factors II and VIII, and the occasional finding of thrombocytopenia in association with APL, should be noted. The risk of subsequent thromboembolic events is the subject of a current survey. Obstetric Associations Since the first report by Nilsson et a126recurrent fetal loss, stillbirth and pre-eclamptic toxaemia of early onset have been increasingly recognised in association with the detection of APL in the blood of women with SLE,27 as well as in otherwise apparently healthy women.28*2g Our own findings support an independent association of LA and IgG ACL with recurrent fetal loss (3 or more episodes) in the first or second
BLOOD REVIEWS
trimester-up to 30% of such women being positive for one or other of these tests. In contrast, the prevalence of positive tests among women with one or two episodes of fetal loss is 10w.~’ In a study of 501 uncomplicated pregnancies we have noted a low incidence (2.8%) of positive tests for APL. Although positivity identified some of those with previous fetal loss poor outcome in the current pregnancy was not predicted by the finding of positive tests for APL. Our results indicate that therapeutic intervention in pregnancy should be reserved for those women with APL who have suffered more than three consecutive episodes of fetal 10~s.~’
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agulant system. 35,36 The requirement for phospholipid in protein C activation provides a basis for this interaction, although the association with arterial as well as venous thrombosis is not entirely consistent with this mechanism of action. The influence of antibody interaction with the 50 kDa cofactor (probably l3,-glycoprotein 1) referred to already on these various mechanisms is a potentially fruitful area for further research. It also remains to be determined whether the obstetric complications associated with the presence of these antibodies results from a thrombotic process in the placental vasculature, as placental infarction has not invariably been demonstrated.
The Antiphospholipid Syndrome
The concept of a primary APS encompassing the clinical and laboratory associations of antiphospholipid is useful can be defined as the finding of antiphospholipid (LA or antibodies to cardiolipin), on at least two occasions 8 weeks apart, in a subject with two or more of spontaneous venous thrombosis, arterial thrombosis, recurrent fetal loss or thrombocytopenia, without clinical or serological evidence of systemic lupus erythematosus. However, this definition is somewhat restrictive as subjects with isolated thromboembolic disease or obstetric complications, although not fulfilling the diagnostic criteria for the APS, may have antiphospholipid of prognostic and therapeutic importance. Pathogenetic Mechanisms Several lines of investigation have been pursued to determine the mechanisms of the thrombotic tendency in the presence of APL. No single mode of action fully explains the clinical correlates observed. Although the association with thrombosis appears firm, causality has not been demonstrated. Positive sera and IgG purified therefrom may tend to result in a reduction in vascular endothelial prostacyclin synthesis in in vitro systems although the experimental conditions must be carefully controlled in order to demonstrate this effect.32 However, our own observations also suggest that this inhibition may relate more closely to the titre of antibodies to endothelial antigens than to the ACL titre. Such antibody activity to endothelial cells is common in sera positive for antiphospholipid. Furthermore, it is perhaps simplistic to suggest that a deficiency of prostacyclin could underlie the range of thrombotic manifestations observed clinically. Endothelial cell damage can be suspected in some situations where APL are commonly found33 and an alternative possibility is that such damage, possibly mediated immunologically, could increase the tissue factor procoagulant activity of endothelium or impair fibrinolytic responses.34 IgG preparations from plasma of patients positive for ACL/LA can interfere in the protein C/S antico-
Clinical Management Antithrombotic Therapy
Controlled clinical trial results are not available to provide guidance on thrombosis management in subjects with APL. The incidental finding of APL is not an indication for intervention, although in certain clinical settings; e.g. SLE, a positive test for APL, should alert the clinician to the increased thrombotic tendency and lower the threshold for use of prophylaxis during high risk periods such as the postoperative and postpartum states. Counselling against exposure to other risk factors, smoking and possibly the contraceptive pill, should be given. Acute thrombotic episodes should be managed in the usual way. Recurrent thrombosis on discontinuation of anticoagulants may occur and the duration of anticoagulant treatment may be influenced by positive tests for APL. Such therapeutic decisions must depend upon the site and severity of the event and the perceived risk-benefit ratio in an individual sufferer. The efficacy of aspirin prophylaxis against thrombosis in the presence of APL remains to be determined. Its use could certainly be justified in the presence of transient cerebral ischaemic episodes or myocardial infarction and after thrombotic stroke. Immunosuppressive therapy is not justified for thrombotic disease but may be indicated for other immune-mediated complications, especially against a background of SLE. Management of Recurrent Fetal Loss
Again, published information is largely anecdotal. Apparent good results from intervention should be gauged against the relatively high chance of a successful outcome without intervention, even after several previous episodes of fetal loss. Aspirin, heparin, steroids and high dose intravenous human IgG have all been recommended. Our own approach, in women with 3 or more miscarriages and detectable APL on at least two occasions, is to offer prophylaxis with low-dose subcutaneous heparin and/or low-dose as-
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LUPUS ANTICOAGULANT
pirin for subsequent pregnancies, combined with close liaison with obstetric colleagues and regular review of progress. In the event of evidence of intrauterine growth retardation or in subsequent pregnancies in the event of fetal loss, full anticoagulation or the use of steroids or intravenous immunoglobulin should be considered. Concluding Remarks Long considered as little more than a laboratory phenomenon of little significance, the finding of LA is now known to be of clinical importance in many instances. The disease associations have been defined but further work is needed to determine the mechanisms of the prethrombotic effect and to establish optimal management strategies in the various clinical circumstances where APL have been detected. Acknowledgements The authors would like to acknowledge Mr R. G. Malia and Mrs L. Wattam for their help in the preparation of this manuscript.
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Inhibition of activated protein C and its cofactor protein S by antiphospholipid antibodies. British Journal of Haematology 76: 101-107
