March 1999
Coagulation Case Study

Jeffrey S. Dlott, MD Douglas A. Triplett, MD

Editor’s note: This is the second in a series of articles focusing on laboratory evaluation of coagulation disorders. Each article contains a clinical history, laboratory results, an algorithm for evaluating either an abnormal coagulation screening test or a clinical abnormality (bleeding or thrombosis), and a final diagnosis. A major goal of this series is to provide an overview of how specific coagulation abnormalities can be evaluated in the clinical laboratory.

Clinical history. The patient is a 39-year-old Caucasian male who at age 30 had a left anterior myocardial infarction. Shortly thereafter the patient experienced his first of several strokes. Residual deficits include memory loss and expressive aphasia. Multiple neurologic and cardiac imaging studies have failed to demonstrate an etiology. Laboratory findings have repeatedly demonstrated a prolonged activated partial throm-boplastin time and borderline thrombocytopenia. The initial laboratory findings are shown in Table 1.

The differential diagnosis was based on lupus anticoagulant plus or minus other an-ti---phospholipid anti-bodies versus specific factor inhibitors or factor deficiency. (See Table 2.)

Due to the limited amount of plasma sample a factor deficiency had to be excluded as part of the hexagonal phospholipid neutralization test (STACLOT LA) instead of the traditional mixing study (see Fig. 1). Evidence against a factor VIII inhibitor included a history of thrombosis (bleeding seen with a factor VIII inhibitor) and abnormal dRVVT studies.

Laboratory test-based algorithm. Evaluating ischemic stroke involves a multidisciplinary approach with an initial emphasis on screening for atherosclerotic disease and a cardiac etiology. In younger patients-less than 45 years-with ischemic stroke, the incidence of atherosclerotic disease is lower than in older patients, and the differential diagnosis is greatly expanded.^1,2 Laboratory evaluation often consists of testing for hereditary thrombophilic conditions, such as deficiencies of proteins C,S, anti-thrombin, and hyperhomocysteinemia as well as antiphospholipid antibodies.^1,2 This case illustrates a systematic approach to diagnosing antiphospholipid syndrome (APS). (See Fig. 2.)

Antibody-mediated thrombosis is perhaps a more accurate and descriptive term to describe the pathophysiology behind the constellation of clinical and laboratory findings known as APS.³ Neither term, however, encompasses the more recent concept that the antibodies target phospholipid-protein complexes and not just phospholipids alone.⁴ This has lead to efforts to investigate the utility of stratifying patients based on antibodies to specific proteins.⁵ Two proteins, beta2glycoprotein I and prothrombin, have emerged as the most common.⁶ While there is strong evidence associating anti-7ß₂GPI antibodies with an increased risk for thrombosis, the data are still forthcoming for anti-prothrombin antibodies.⁷ The diagnostic criteria for APS are outlined in Fig. 3. The approach to testing is divided into evaluation for a lupus anticoagulant (LA) and other APA (Fig. 4). Although this approach was designed with technical considerations in mind, clinical implications exist as well. A recent meta-analysis has shown that patients with an LA have a higher risk for thrombotic events than do those with anticardiolipin antibodies.⁸ The relative risks are 11.1 and 3.2, respectively.⁸

Often the evaluation for an LA is confounded by the acute phase response or anticoagulant therapy. This experience is commonly seen when a patient is admitted through the emergency room and started on anticoagulant therapy before samples for coagulation testing can be drawn. If the patient does indeed have a deep vein thrombosis, many acute phase proteins such as fibrinogen, factor VIII, and C4B binding protein will be elevated and can affect coagulation assays.

For this reason, our approach for patients not anticoagulated is to screen with at least two screening assays: APTT and dRVVT, or APTT and dilute prothrombin time (dPT). For patients on coumadin or with a limited amount of plasma, we begin with the hexagonal phospholipid neutralization test STACLOT LA (Fig. 1).⁹ This inclusive assay incorporates mixing plasma to exclude a factor deficiency, a heparin neutralizing agent, a sensitive partial thromboplastin reagent, and a hexagonal phase phospholipid in one of two tubes to demonstrate partial lipid dependence of the in-hibitor. The limitations are ex-pense and false positive cross-reactivity- with factor VIII inhibitors¹⁰-a potentially catastrophic event-and excesses of heparin.

Mixing studies are rarely performed on minimally prolonged screening tests (i.e. less than 5 seconds) due to difficulty in interpretation.¹¹ It is not uncommon to see a minimally prolonged APTT and a greatly prolonged dRVVT. In this situation, mixing studies should be performed with the dRVVT. If sample volume is limited, performing a 1:1 patient to plasma mix is preferable to a 4:1 mix. The consequence is a weak LA may go undetected. Classically, LAs prolong a mixing study upon immediate mix whereas factor VIII inhibitors require a two-hour incubation before an inhibitor effect is seen, but this is not always true-approximately 30 percent of LAs are time dependent.¹² Confirmatory assays offer similar challenges, and none are 100 percent specific.¹³ Due to the heterogeneity of the LA, more than one confirmatory assay may be required to make the diagnosis. ¹³ Finally, it is necessary to demonstrate persistence of an LA by retesting after eight weeks.

Beginning the evaluation for antiphospholipid antibodies (other than LA) with anticardiolipin antibody (ACA) studies has a historical and practical rationale.14 ELISA assays for ACAs were the first to be developed and the first to have commercial serum calibrators for semiquantification.14 The bulk of the literature evaluating APA and stroke has focused on anticardiolipin antibodies.¹⁵ Several large studies have demonstrated a correlation between high titer IgG ACA-greater than 40 GPL¹⁶-and incidence of stroke.

Until recently, it has been unclear how to interpret the significance of IgG ACAs greater than 40 GPL or isolated non-IgG ACAs. Many of these antibodies have been associated with infection or other inflammatory conditions and are transitory. Positivity to anti-ß₂GPI has been helpful in distinguishing a separate subgroup of this population with an increased risk of thrombosis.¹⁷

Another commonly overlooked criteria for APS is demonstration of persistence, which may also be a pre-dictor for duration of anti-coagulation therapy.18 Finally, a minority of patients with APS will not have ACAs and will require evaluation for other phospholipid antibodies such as phosphatidlyserine or other phospholipids (not commercially available).¹⁹

Diagnosis. The diagnosis was primary antiphospholipid antibody syndrome. The patient has been on coumadin therapy for four months and has not experienced any transient ischemic attacks, although ACAs are still present in high titer.

References

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13. Brandt JT, Barna LK, Triplett DA. Laboratory identification of lupus anticoagulants: results of the Second International Workshop for Identification of Lupus Anticoagulants. On behalf of the Subcommittee on Lupus Anticoagulants/Antiphospholipid Antibodies of the ISTH. Thromb Haemost. 1995;74:1597-1603.
14. Harris EN. Special report. The Second International Anti-cardiolipin Standardization Workshop/the Kingston Anti-Phospholipid Antibody Study (KAPS) group. Am J Clin Pathol. 1990; 94:476-484.
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16. Levine SR, Salowich-Palm L, Sawaya KL, et al. IgG anticardiolipin antibody titer > 40 GPL and the risk of subsequent thrombo-occlusive events and death. A prospective cohort study. Stroke. 1997;28:1660-1665.
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18. Schulman S, Svenungsson E, Granqvist S. Anticardiolipin antibodies predict early recurrence of thromboembolism and death among patients with venous thromboembolism following anticoagulant therapy. Duration of Anticoagulation Study Group. Am J Med. 1998;104:332-338.
19. Wagenknecht DR, McCarty GA, McIntyre JA. Patients with antiphosphatidylethanolamine (aPE) as the sole antiphospholipid antibody (aPA) have clinical characteristics of antiphospholipid antibody syndrome (APS). Lupus. 1998;7:S204.

Dr. Dlott is associate medical director and Dr. Triplett is director of Midwest Hemostasis and Thrombosis Laboratories, Muncie, Ind. Both are members of the CAP Coagulation Resource Committee.