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
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
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
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.
- Grotta J. Cerebrovascular disease in young patients. Thromb
- Tanne D, Triplett DA, Levine SR. Anti-phospholipid-protein antibodies
and ischemic stroke: not just cardiolipin any more. Stroke.
- Roubey RA, Hoffman M. From anti-phospholipid syndrome to antibody-mediate
thrombosis. Lancet. 1997;350: 1491-1493.
- Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies
(ACA) directed not to cardiolipin but to a plasma protein cofactor.
Lancet. 1990; 335:1544-1547.
- Sanmarco M, Soler C, Christides C, et al. Prevalence and clinical
significance of IgG isotype anti-beta 2-glycoprotein I antibodies
in antiphospholipid syndrome: a comparative study with anticardiolipin
antibodies. J Lab Clin Med. 1997;129:499-506.
- Galli M, Finazzi G, Bevers EM, Barbui T. Kaolin clotting time
and dilute Russell’s viper venom time distinguish between prothrombin-dependent
and beta 2-glycoprotein I-dependent anti-phospholipid antibodies.
Blood. 1995; 86:617-623.
- Palosuo T, Virtamo J, Haukka J, et al. High antibody levels
to prothrombin imply a risk of deep venous thrombosis and pulmonary
embolism in middle-aged men-a nested case-control- study. Thromb
Haemost. 1997;78: 1178-1182.
- Wahl DG, Guillemin F, de Maistre E, Perret-Guillaume C, Lecompte
T, Thibaut G. Meta-analysis of the risk of venous thrombosis in
individuals with antiphospholipid antibodies without underlying
autoimmune disease or previous thrombosis. Lupus. 1998;7:
- Triplett DA, Barna LK, Unger GA. A hexagonal (II) phase phospholipid
neutralization assay for lupus anticoagulant identification. Thromb
- Triplett DA. Simultaneous occurrence of lupus anticoagulant
and factor VIII inhibitors. Am J Hematol. 1997;56: 195-196.
- Kaczor DA, Bickford NN, Triplett DA. Evaluation of different
mixing study reagents and dilution effect in lupus anticoagulant
testing. Am J Clin Pathol. 1991;95:408-411.
- Triplett DA. Antiphospholipid-protein antibodies: laboratory
detection and clinical relevance. Thromb Res. 1995; 78(1):1-31.
- 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.
- 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.
- Levine SR, Brey RL, Sawaya KL, et al. Recurrent stroke and thrombo-occlusive
events in the antiphospholipid syndrome. Ann Neurol. 1995;38:
- 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.
- Forastiero RR, Martinuzzo ME, Kordich LC, Carreras LO. Reactivity
to beta 2 glycoprotein I clearly differentiates anticardiolipin
antibodies from antiphospholipid syndrome and syphilis. Thromb
Haemost. 1996;75: 717-720.
- 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.
- 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.
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.