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Thrombosis in Suspected Heparin-induced Thrombocytopenia Occurs More Often with High Antibody Levels

Published:November 09, 2011DOI:https://doi.org/10.1016/j.amjmed.2011.06.025

      Abstract

      Objective

      The study objective was to determine whether higher antiplatelet factor 4 (PF4)/heparin antibody levels using an enzyme-linked immunosorbent assay are associated with more frequent thrombotic events in patients with clinically suspected heparin-induced thrombocytopenia. Heparin-induced thrombocytopenia is an immune-mediated adverse drug reaction. An enzyme-linked immunosorbent assay detects anti-PF4/heparin antibodies to support a suspected clinical diagnosis of heparin-induced thrombocytopenia. The utility of quantitative enzyme-linked immunosorbent assay results is uncertain.

      Methods

      Our single-centered study evaluated quantitative anti-PF4/heparin antibody levels using an enzyme-linked immunosorbent assay in consecutive hospitalized patients with a clinical suspicion of heparin-induced thrombocytopenia and positive anti-PF4/heparin antibody levels between July 2003 and December 2006.

      Results

      Overall, anti-PF4/heparin antibody values were available for 318 patients with clinically suspected heparin-induced thrombocytopenia. The median level was 0.85 optical density units (range 0.31-4.0). The overall rate of arterial or venous thrombosis was 23.3%. A 1-unit increase in anti-PF4/heparin antibody level was associated with an approximate doubling in the odds of thrombosis by 30 days (odds ratio, 1.9; 95% confidence interval, 1.5-2.6; P=.0001). The proportion of patients with pulmonary embolism increased with higher anti-PF4/heparin antibody levels.

      Conclusion

      Higher levels of anti-PF4/heparin antibody are associated with increased thrombosis risk among patients with clinically suspected heparin-induced thrombocytopenia and might have clinical utility for prediction of true heparin-induced thrombocytopenia and the development of thrombosis.

      Keywords

      SEE RELATED EDITORIAL p. 3
      Heparin-induced thrombocytopenia is a prothrombotic immune-mediated adverse drug reaction that occurs after exposure to unfractionated heparin or low-molecular-weight heparin.
      • Arepally G.M.
      • Ortel T.L.
      Clinical practice Heparin-induced thrombocytopenia.
      Binding of heparin-induced thrombocytopenia-inducing antibodies to a complex of platelet factor 4 (PF4) and heparin produces platelet, endothelial cell, and monocyte activation leading to thrombin generation and thrombus formation in either the venous or arterial system.
      • Kelton J.G.
      The pathophysiology of heparin-induced thrombocytopenia: biological basis for treatment.
      • Shantsila E.
      • Lip G.Y.
      • Chong B.H.
      Heparin-induced thrombocytopenia A contemporary clinical approach to diagnosis and management.
      • Warkentin T.E.
      New approaches to the diagnosis of heparin-induced thrombocytopenia.
      The spectrum of adverse events associated with generation of heparin-induced thrombocytopenia-inducing antibodies ranges from asymptomatic thrombocytopenia to life-threatening heparin-induced thrombocytopenia with thrombosis manifested by deep vein thrombosis, pulmonary embolism, or arterial thrombi in the renal, cerebral, splanchnic, peripheral, or coronary circulations.
      • Greinacher A.
      • Eichler P.
      • Lietz T.
      • Warkentin T.E.
      Replacement of unfractionated heparin by low-molecular-weight heparin for postorthopedic surgery antithrombotic prophylaxis lowers the overall risk of symptomatic thrombosis because of a lower frequency of heparin-induced thrombocytopenia.
      • Warkentin T.E.
      • Greinacher A.
      • Koster A.
      • Lincoff A.M.
      Treatment and prevention of heparin-induced thrombocytopenia: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition).
      The presence of anti-PF4/heparin antibodies is an independent predictor of 30-day myocardial infarction after acute coronary syndromes.
      • Williams R.T.
      • Damaraju L.V.
      • Mascelli M.A.
      • et al.
      Anti-platelet factor 4/heparin antibodies: An independent predictor of 30-day myocardial infarction after acute coronary ischemic syndromes.
      • The enzyme-linked immunosorbent assay for the anti-PF4/heparin antibody complex, used in the diagnosis of heparin-induced thrombocytopenia, is best reported as a quantitative value rather than “positive” or “negative.”
      • Increasing anti-PF4/heparin antibodies are associated with increasing odds of thrombosis.
      • The proportion of patients with pulmonary embolism increases with higher anti-PF4/heparin antibodies.
      • Quantitative anti-PF4/heparin antibodies may have utility for detecting patients who are more likely to suffer thrombosis.
      Usually, when heparin-induced thrombocytopenia is clinically suspected, the diagnosis is supported or excluded with an enzyme-linked immunosorbent assay for the anti-PF4/heparin antibody complex.
      • Warkentin T.E.
      New approaches to the diagnosis of heparin-induced thrombocytopenia.
      Studies have shown that the presence of platelet-activating antibodies measured with the serotonin-release assay occurs in only one third to one half of enzyme-linked immunosorbent assay-positive patients who are investigated for heparin-induced thrombocytopenia.
      • Greinacher A.
      • Juhl D.
      • Strobel U.
      • et al.
      Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity of the lgG, IgM, and IgA classes.
      • Bakchoul T.
      • Giptner A.
      • Najaoui A.
      • et al.
      Prospective evaluation of PF4/ heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia.
      Furthermore, the magnitude of a positive enzyme-linked immunosorbent assay strongly predicts the presence of platelet-activating antibodies.
      • Bakchoul T.
      • Giptner A.
      • Najaoui A.
      • et al.
      Prospective evaluation of PF4/ heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia.
      • Warkentin T.E.
      • Sheppard J.I.
      • Moore J.C.
      • et al.
      Quantitative interpretation of optical density measurements using pf4-dependent enzyme-immunoassays.
      We hypothesized that higher anti-PF4/heparin antibody levels are associated with a higher likelihood of platelet-activating antibodies being present, leading to a higher probability of heparin-induced thrombocytopenia and a higher frequency of heparin-induced thrombocytopenia-associated thrombotic events. To explore the possible association between quantitative anti-PF4/heparin antibody levels and development of thrombosis in patients with suspected heparin-induced thrombocytopenia, we analyzed quantitative antibody levels and patient outcomes from a single-center patient registry at Brigham and Women's Hospital.
      • Baroletti S.
      • Piovella C.
      • Fanikos J.
      • et al.
      Heparin-induced thrombocytopenia: clinical and economic outcomes.

      Materials and Methods

      Our cohort included 349 consecutive hospitalized patients with a clinical suspicion of heparin-induced thrombocytopenia between July 2003 and December 2006. Patients were included if there was a platelet decrease to less than 150×109/L or a 50% reduction of platelet count during hospitalization from baseline and a positive anti-PF4/heparin antibody test using enzyme-linked immunosorbent assay after unfractionated heparin or low-molecular-weight heparin therapy. Patients were not screened for asymptomatic thrombosis.
      This antigen assay (Asserachrom HPIA; Diagnostica Stago, Parsippany, NJ) uses a PF4/heparin-coated microwell to incubate the patient's plasma sample for assessment of antibody development. Laboratory test results were reported to ordering clinicians as positive or negative. The threshold for a positive value was determined daily using a positive and negative control, with a median cutoff value of 0.46 optical density (OD) units (range 0.31-0.67 OD units). A result less than the cutoff value but greater than 70% of that value was reported as borderline, and repeat testing was conducted.
      Quantitative anti-PF4/heparin antibody values, which had been calculated but not reported, were obtained for patients in the registry. The primary clinical outcome was imaging study-confirmed arterial or venous thrombosis within 30 days, beginning the day of positive anti-PF4/heparin antibody testing.

      Statistical Analysis

      Data were inspected for distributional assumptions and summarized using medians and quartiles or percents. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for potential predictors of thrombosis, unadjusted and adjusted for anti-PF4/heparin antibody level. To illustrate the relationship between anti-PF4/heparin antibody level and the observed proportion of patients with thrombosis, 9 patient groups were created in approximately equal group sizes, according to increasing anti-PF4/heparin antibody levels. The proportion with thrombotic events and the proportion with pulmonary embolism were plotted as a function of mean anti-PF4/heparin antibody level. Logistic regression was used to generate the predicted proportion with thrombosis by 30 days, plotted as a function of anti-PF4/heparin antibody level, with a 95% confidence band. Statistical analysis was performed using SAS version 9.1 (SAS Institute Inc, Cary, NC).

      Results

      Patient Characteristics

      Overall, anti-PF4/heparin antibody values were available for 318 patients with clinically suspected heparin-induced thrombocytopenia. The level was 0.85 OD units (range 0.31-4.0 OD units). Baseline characteristics are shown in Table 1. The median age was 66 years, and the median length of hospital stay was 20 days. Approximately 50% of registry patients had a diagnosis of coronary artery disease. Patients undergoing cardiac surgery accounted for 40.9% of the registry population.
      Table 1Baseline Characteristics (n=318)
      CharacteristicResult
      Median age (y) [lower-upper quartile]66 [55-75]
      Female, n (%)149 (46.9)
      Median length of stay (d) [lower-upper quartile]20 [12-35]
      Kidney disease, n (%)65 (20.4)
      Cancer, n (%)73 (23.0)
      Coronary artery disease, n (%)146 (45.9)
      Diabetes mellitus, n (%)100 (31.4)
      Pulmonary disease, n (%)100 (31.4)
      Surgery during admission, n (%)193 (60.7)
       Cardiac surgery130 (40.9)
       Orthopedic surgery4 (1.3)
       All other surgeries59 (18.5)
      Median baseline platelets (×109/L) [lower-upper quartile]173 [120-246]
      Median platelet nadir (×109/L) [lower-upper quartile]65 [42-86]

      Clinical Outcomes

      The overall rate of arterial or venous thrombosis was 23.3% (Table 2). The distribution of anti-PF4/heparin antibody levels varied according to thrombosis status. Among patients without thrombosis, there were greater proportions with lower anti-PF4/heparin antibody levels in contrast with patients with thrombosis, in whom there were greater proportions with higher anti-PF4/heparin antibody levels.
      Table 2Thrombosis Rates and Types
      Outcomesn (%)
      Any thrombosis at 30 d74 (23.3)
      Venous thrombosis57 (17.9)
      Deep vein thrombosis alone29 (9.1)
      Pulmonary embolism alone or pulmonary embolism with deep vein thrombosis26 (8.2)
      Other2 (0.6)
      Arterial thrombosis17 (5.4)
      Stroke12 (3.8)
      Aortic3 (0.9)
      Other2 (0.6)
      Logistic regression showed that increases in anti-PF4/heparin antibody levels were associated with significantly greater odds of thrombosis by 30 days (OR, 1.9; 95% CI, 1.5-2.6; P=.0001). Figure 1 illustrates observed proportions of patients with any thrombosis by 30 days and with pulmonary embolism as a function of anti-PF4/heparin antibody level. The proportions of patients with pulmonary embolism also were greater at the higher anti-PF4/heparin antibody levels.
      Figure thumbnail gr1
      Figure 1Association of anti-PF4/heparin antibody level with any arterial or venous thrombosis and pulmonary embolism. The proportion with any thrombosis by 30 days and the proportion with pulmonary embolism are shown as a function of anti-PF4/heparin antibody level. Increases in anti-PF4/heparin antibody level were significantly associated with greater proportions of any thrombosis and pulmonary embolism by 30 days. PE=pulmonary embolism; PF4=platelet factor 4; OD=optical density.
      The effect of covariates on thrombosis rates, unadjusted and adjusted for anti-PF4/heparin antibody level, is shown in Table 3. Notably, the anti-PF4/heparin antibody level was strongly related to thrombosis at 30 days while adjusting for each of the individual comorbidities in Table 3 (P<.0001). In a separate analysis controlling for age, gender, and cancer, higher anti-PF4/heparin antibody levels remained a strong predictor of thrombosis development at 30 days (OR, 2.0; 95% CI, 1.5-2.6; P=.0001).
      Table 3Odds Ratios and 95% Confidence Intervals Predicting Thrombosis by 30 Days, Unadjusted and Adjusted for Anti-PF4/Heparin Antibody Level
      VariableUnadjusted OR (95% CI), P ValueAdjusted OR (95% CI), P Value
      Anti-PF4/heparin antibody level (OD unit)1.94 (1.48-2.56), .0001
      Age (decade)1.01 (0.85-1.21), .871.00 (0.98-1.02), .96
      Female1.10 (0.65-1.85), .721.13 (0.65-1.96), .67
      Cancer1.91 (1.07-3.41), .031.90 (1.03-3.48), .04
      Renal dysfunction0.7 (0.35-1.39), .310.83 (0.41-1.70), .61
      Hepatic dysfunction0.62 (0.27-1.47), .280.70 (0.29-1.69), .43
      Diabetes0.76 (0.43-1.35), .350.75 (0.41-1.36), .34
      Hypertension1.04 (0.62-1.76), .881.10 (0.64-1.91), .72
      Pulmonary disease1.06 (0.61-1.85), .831.20 (0.67-2.14), .55
      Cardiac surgery0.91 (0.54-1.55), .740.95 (0.55-1.66), .86
      Figure 2 shows observed and predicted results for any thrombosis. Our regression model predicts that the proportions with any clinically evident thrombosis at anti-PF4/heparin antibody levels of 1.0, 2.0, and 3.0 OD units are 19%, 32%, and 48%, respectively. The overall mortality rate by 30 days was 16.7%. Anti-PF4/heparin antibody levels were not significantly associated with mortality (OR, 0.89; 95% CI, 0.63-1.26; P=.49).
      Figure thumbnail gr2
      Figure 2Observed and predicted thrombosis proportion according to anti-PF4/heparin antibody level and 95% confidence band. CI=confidence interval; PF4=platelet factor 4.

      Discussion

      We studied a large population of patients with clinically suspected heparin-induced thrombocytopenia over a 3.5-year time period. These patients were severely ill, with a median length of hospital stay of 20 days and 30-day mortality of approximately 17%. Approximately half of the registry population was composed of patients undergoing cardiac surgery. We observed that higher levels of quantitative anti-PF4/heparin antibody levels were associated with increased risk of any arterial or venous thrombosis.
      Quantitative anti-PF4/heparin antibody values have traditionally been used to assist in the clinical diagnosis of suspected heparin-induced thrombocytopenia via a dichotomous “cutoff” or breakpoint value associated with a positive diagnosis.
      • Pouplard C.
      • Amiral J.
      • Borg J.Y.
      • et al.
      Decision analysis for use of platelet aggregation test, carbon 14-serotonin release assay, and heparin-platelet factor 4 enzyme-linked immunosorbent assay for diagnosis of heparin-induced thrombocytopenia.
      Although the sensitivity of this assay is high, the low specificity is a recognized weakness, particularly in those undergoing cardiac surgery.
      • Warkentin T.E.
      • Sheppard J.I.
      • Moore J.C.
      • et al.
      Quantitative interpretation of optical density measurements using pf4-dependent enzyme-immunoassays.
      • Warkentin T.E.
      • Sheppard J.A.
      • Horsewood P.
      • et al.
      Impact of the patient population on the risk for heparin-induced thrombocytopenia.
      Appropriate detection of heparin-induced thrombocytopenia is crucial to minimize the risk of thrombosis and poor clinical outcomes.
      • Kuitunen A.
      • Suojaranta-Ylinen R.
      • Raivio P.
      • et al.
      Heparin-induced thrombocytopenia following cardiac surgery is associated with poor outcome.
      Our findings indicate that quantitative anti-PF4/heparin antibody levels are more important than previously appreciated and have potential clinical utility for detecting groups of patients with heparin-induced thrombocytopenia who are more likely to have heparin-induced thrombocytopenia with thrombosis.
      Detection of platelet-activating antibodies with the serotonin-release assay is a more specific method of diagnosing heparin-induced thrombocytopenia than measurement with the enzyme-linked immunosorbent assay.
      • Greinacher A.
      • Juhl D.
      • Strobel U.
      • et al.
      Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity of the lgG, IgM, and IgA classes.
      The presence of platelet-activating antibodies occurs in one third to one half of enzyme-linked immunosorbent assay-positive patients with suspected heparin-induced thrombocytopenia.
      • Greinacher A.
      • Juhl D.
      • Strobel U.
      • et al.
      Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity of the lgG, IgM, and IgA classes.
      • Lo G.K.
      • Sigouin C.S.
      • Warkentin T.E.
      What is the potential for overdiagnosis of heparin-induced thrombocytopenia?.
      Higher anti-PF4/heparin antibody levels are associated with a higher likelihood of platelet-activating antibodies being present.
      • Greinacher A.
      • Juhl D.
      • Strobel U.
      • et al.
      Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity of the lgG, IgM, and IgA classes.
      • Bakchoul T.
      • Giptner A.
      • Najaoui A.
      • et al.
      Prospective evaluation of PF4/ heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia.
      The presence of these antibodies increases the likelihood of heparin-induced thrombocytopenia and thus increases the likelihood of developing heparin-induced thrombocytopenia with thrombosis.
      • Bakchoul T.
      • Giptner A.
      • Najaoui A.
      • et al.
      Prospective evaluation of PF4/ heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia.
      • Warkentin T.E.
      • Sheppard J.I.
      • Moore J.C.
      • et al.
      Quantitative interpretation of optical density measurements using pf4-dependent enzyme-immunoassays.
      A commercial enzyme-linked immunosorbent assay with a strong positive result (≥2.0 OD units) has been associated with a strong positive serotonin release assay in approximately 90% of patients, and therefore a high likelihood of clinical heparin-induced thrombocytopenia.
      • Warkentin T.E.
      • Sheppard J.I.
      • Moore J.C.
      • et al.
      Quantitative interpretation of optical density measurements using pf4-dependent enzyme-immunoassays.
      • Warkentin T.E.
      • Levine M.N.
      • Hirsh J.
      • et al.
      Heparin-induced thrombocytopenia in patients treated with low-molecular weight heparin or unfractionated heparin.
      The lack of availability of a platelet-activating assay often forces clinicians to consider the diagnosis of heparin-induced thrombocytopenia using clinical data supported by anti-PF4/heparin antibody levels as determined by enzyme-linked immunosorbent assay. To assess clinical likelihood of heparin-induced thrombocytopenia, guideline-based clinical decision rules or a validated scoring tool (eg, the 4T score) are often used.
      • Warkentin T.E.
      • Greinacher A.
      • Koster A.
      • Lincoff A.M.
      Treatment and prevention of heparin-induced thrombocytopenia: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition).
      • Lo G.K.
      • Juhl D.
      • Warkentin T.E.
      • et al.
      Evaluation of pretest clinical score (4 T's) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings.
      High anti-PF4/heparin antibody levels also have been observed to be independently associated with an increased in vivo thrombin generation as measured by thrombin-antithrombin complexes and D-dimers.
      • Chilver-Stainer L.
      • Lammle B.
      • Alberio L.
      Titre of anti-heparin/PF4-antibodies and extent of in vivo activation of the coagulation and fibrinolytic systems.
      The association of thrombosis risk with increasing anti-PF4/heparin antibody levels has been demonstrated by a dichotomous cutoff of1.0 OD units.
      • Altuntas F.
      • Matevosyan K.
      • Burner J.
      • et al.
      Higher optical density of an antigen assay predicts thrombosis in patients with heparin-induced thrombocytopenia.
      • Zwicker J.I.
      • Uhl L.
      • Huang W.-Y.
      • et al.
      Thrombosis and ELISA optical density values in hospitalized patients with heparin-induced thrombocytopenia.
      Our study suggests that the risk and severity of thrombosis continue to increase as anti-PF4/heparin antibody levels increase. Quantitative PF4-heparin antibody levels may help providers to identify patients with true heparin-induced thrombocytopenia who are at high risk of developing thrombosis.
      There was no significant association with mortality and anti-PF4/heparin antibodies in our study. This may be due to similar clinical comorbidities present across the population that predisposed patients to the risk of death. A recent study evaluating patients with thrombocytopenia found that the absence of platelet-activating antibodies was paradoxically associated with a higher mortality risk.
      • Lo G.K.
      • Sigouin C.S.
      • Warkentin T.E.
      What is the potential for overdiagnosis of heparin-induced thrombocytopenia?.
      Such patients with thrombocytopenia without platelet-activating antibodies probably have other high-risk conditions (eg, sepsis, multisystem organ failure, or cancer) that are more life threatening than heparin-induced thrombocytopenia.
      A limitation is that this data set did not include patients who might have developed late-onset heparin-induced thrombocytopenia after discharge or early, rapid-onset, heparin-induced thrombocytopenia due to reexposure to heparin.
      • Rice L.
      • Attisha W.K.
      • Drexler A.
      • Francis J.L.
      Delayed-onset heparin-induced thrombocytopenia.
      • Warkentin T.E.
      • Kelton J.G.
      Temporal aspects of heparin-induced thrombocytopenia.
      • Warkentin T.E.
      • Kelton J.G.
      Delayed-onset heparin-induced thrombocytopenia and thrombosis.
      Instead, we included only those patients with an identifiable heparin exposure, clinical suspicion of heparin-induced thrombocytopenia, and serologic evidence of developing heparin-induced thrombocytopenia during hospitalization. We did not confirm the diagnosis of heparin-induced thrombocytopenia with a specific platelet-activating assay, such as the serotonin release assay. Therefore, we cannot determine whether the strong association we observed between thrombosis and higher anti-PF4/heparin antibody levels is explained by higher anti-PF4/heparin antibody levels predicting the presence of platelet-activating antibodies and confirming true heparin-induced thrombocytopenia. Our clinical decision to suspect heparin-induced thrombocytopenia was based on the American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th edition),
      • Warkentin T.E.
      • Greinacher A.
      • Koster A.
      • Lincoff A.M.
      Treatment and prevention of heparin-induced thrombocytopenia: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition).
      supported by a positive anti-PF4/heparin antibody enzyme-linked immunosorbent assay to identify patients at risk.
      A strength of this report is that our registry represents a “real world” experience with a large number of patients with heparin-induced thrombocytopenia based on clinical and serologic diagnoses. In addition, we were able to obtain 100% follow-up for patients included in this cohort.

      Conclusions

      Improved understanding of serologic testing results may lead to improved patient care. When heparin-induced thrombocytopenia is considered in high-risk patients, rapid and accurate detection of high quantitative levels of anti-PF4/heparin antibody can assist clinicians by identifying a subset of patients at particular risk for developing thrombotic complications. The next management steps for such patients should be to stop the administration of unfractionated heparin or low-molecular-weight heparin and begin treatment with an alternative non-heparin anticoagulant, such as a direct thrombin inhibitor, fondaparinux, or danaparoid.
      • Warkentin T.E.
      • Greinacher A.
      • Koster A.
      • Lincoff A.M.
      Treatment and prevention of heparin-induced thrombocytopenia: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition).
      • Oliveira G.B.
      • Crespo E.M.
      • Becker R.C.
      • et al.
      Incidence and prognostic significance of thrombocytopenia in patients treated with prolonged heparin therapy.
      In contrast, more cost-effective, conservative treatment may be possible in those patients with heparin-induced thrombocytopenia with a low probability of thrombosis development, thereby reducing exposure to unnecessary additional testing or medications. Our findings support the need for a prospective study using quantitative anti-PF4/heparin antibody levels to help guide clinical management in patients with clinically suspected heparin-induced thrombocytopenia.

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