Utility of consecutive repeat HIT ELISA testing for heparin-induced thrombocytopenia

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1 Utility of consecutive repeat HIT ELISA testing for heparin-induced thrombocytopenia Maren Chan, 1 * Elizabeth Malynn, 1 Beth Shaz, 2 and Lynne Uhl 1 Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. Limited data are available regarding repeat HIT antibody testing after an initial negative test. We conducted a retrospective study to determine the utility of repeat testing. Heparin antibodies were detected using the GTI-PF4 enzyme-linked immunoabsorbent assay, ELISA (GTI Diagnostics, Waukesha, WI). Patients (n 5 137) were assigned to one of three groups based upon the initial negative test optical density (OD) range of low , medium , and high A pretest clinical score was retrospectively determined using the 4T s (Thrombocytopenia, Timing of platelet fall, Thrombosis, and the absence of other causes of thrombocytopenia). A subsequent positive ELISA was found in 16% (22/137) of patients who underwent repeat testing. Most of these patients had a low pretest clinical score (62%). Four patients had an interval change in the pretest score between the initial negative and subsequent positive tests. Only these four patients developed HIT with thrombosis (HITT). Eighty percent of patients with a high initial negative test OD value had a positive ELISA on repeat testing; however, the initial negative test OD value could not predict whether a patient developed HITT. In contrast, an increase in the pretest clinical probability between initial and repeat testing better predicted HITT. Consecutive repeat ELISA testing for heparin antibodies may be warranted in patients with an increase in their pretest clinical score after an initial negative test as an adjunct to confirm the diagnosis of HIT. Am. J. Hematol. 83: , VC 2007 Wiley-Liss, Inc. Introduction Heparin-induced thrombocytopenia (HIT) is a clinicopathological syndrome of immune-mediated thrombocytopenia that confers an increased risk of thrombosis (HITT) in patients exposed to heparin [1]. HIT is suspected in patients receiving heparin in the setting of an unexplained fall in platelet count by 50%, skin lesions at the site of heparin injection, or a systemic reaction to heparin infusion [2]. Typically, the thrombocytopenia of HIT occurs within 5 10 days after the onset of heparin therapy, coincident with the development of antibodies against heparin/platelet factor 4 (PF4) complexes. In some patients with a recent heparin exposure the platelet count may drop rapidly within 24 hr of heparin re-exposure [3]. New, recurrent, or progressive thrombosis occurs in at least 50% of patients with HIT, particularly those with severe thrombocytopenia [4]. When isolated HIT is suspected clinically, discontinuation of heparin is recommended and consideration for initiation of anticoagulant therapy with direct thrombin or factor Xa inhibitors is advised to prevent thrombus formation, which can proceed in the absence of further heparin therapy in 20 50% of untreated patients [5,6]. Direct thrombin inhibitor therapy, however, confers a moderate risk for increased morbidity. Bleeding events [7] and fatal anaphylactic reactions [8] have been reported in association with lepirudin, a commonly used direct thrombin inhibitor. These serious adverse events emphasize the importance of making an accurate diagnosis of isolated HIT. The detection of antibodies directed against heparin/platelet factor 4 (PF4) complexes assists in the diagnosis of isolated HIT. Although several assays exist, the commercially available enzyme-linked immunosorbant assay (ELISA), which detects IgG, IgA, and IgM antibodies directed against the heparin/pf4 complex is most commonly used [9]. The advantages of using the ELISA for detection of heparin/ PF4 antibodies include high sensitivity (95 100%), general availability, and ease of use; however, a positive result may not correlate with clinically significant antibodies or an VC 2007 Wiley-Liss, Inc. increased risk of thrombosis. For example, for unknown reasons up to 50% of postoperative cardiac patients will develop heparin/pf4 antibodies, yet their incidence of isolated HIT remains low (2 3%) [10]. Additionally, the degree of positivity of the ELISA measurement is variable; some investigators have suggested that the strength of the optical density (OD) result may correlate with an increased risk of a subsequent thrombosis [9,11]. In cases in question, a 14 C serotonin release assay (SRA), which is performed by reference laboratories and measures washed platelet activation following incubation with the patient s serum in the presence of heparin, may provide greater specificity. A negative result from the highly sensitive PF4-dependent immunoassay has generally been assumed to imply exclusion of isolated HIT in patients with a low pretest probability of HIT [12]; however, it possible the heparin/pf4 antibodies do not become detectable until after initial testing. Furthermore, in rare instances the antibody generated may be directed against a different antigen than the heparin/pf4 complex [13 15]. Though the role of repeat ELISA for detection of heparin/pf4 antibodies after an initial negative test is unknown, we have observed this to be a frequent ordering practice at our institution. Recently, Refaai et al. [16] proposed using the initial negative ELISA OD 1 Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts; 2 Department of Pathology, Grady Health System, Emory University School of Medicine, Atlanta, Georgia *Correspondence to: Maren Chan, Department of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA mmchan@bidmc.harvard.edu Received for publication 1 July 2007; Revised 7 August 2007; Accepted 15 August 2007 Am. J. Hematol. 83: , Published online 1 October 2007 in Wiley InterScience ( wiley.com). DOI: /ajh American Journal of Hematology 212

2 TABLE I. Characteristics of Hospitalized Patients Who Underwent Repeat Heparin-Induced Thrombocytopenia Testing as Determined by ELISA Based Upon Their Subsequent Negative (SN) and Subsequent Positive (SP) Test Results SN Test (n 5 115) SP Test (n 5 22) P-value Age, years (mean) 70 ± ± Gender (% Female) 52 (45%) 11 (50%) 0.68 Plt count at INT (10 9 /L) 82 ± ± D Plt count INT (10 9 /L) a 85 ± ± Surgery (within preceding 60 days) 69 (59%) 15 (68%) 0.47 Cardiac (%) 27 (23%) 5 (23%) Vascular (%) 13 (11%) 4 (18%) Orthopedic (%) 5 (4%) 0 (0%) General/Other (%) 24 (21%) 6 (27%) Heparin b 70 (61%) 20 (91%) ELISA optical density ± ± <0.05 a The change in platelet (Plt) count from 7 days prior to the initial negative test (INT). b Unfractionated heparin (re-) exposure at anytime within 5 days prior to repeat testing (intravenous and/or subcutaneous). Low-molecular weight heparin was given to one patient in the SN Test group. value as an adjunct to decide whether to repeat the test, as more patients with an OD value in the upper third of the negative OD range had subsequent positive tests. Recently, an effort has been made to estimate the probability of HIT based upon clinical parameters and their correlation to both functional and immunologic laboratory data. Warkentin and coworkers in collaboration with the International Society of Thrombosis and Hemostasis Scientific Committee on Platelet Immunology, reported on a pretest clinical scoring system based upon the 4T s including Thrombocytopenia, Timing of platelet fall, Thrombosis (or other sequelae of HIT), and the absence of other causes of thrombocytopenia [17 19]. Recognizing that clinical correlation is essential for the diagnosis of HIT, we sought to further define the relationship of laboratory ELISA OD values with pertinent clinical parameters and the pretest clinical score in the setting of repeat testing, although we acknowledge that the scoring system was not available to clinicians during the entire study period. Results A total of 2,232 ELISA tests were performed from January 2001 to March One hundred thirty-seven patients underwent consecutive repeat laboratory ELISA testing for suspected HIT. Initial and subsequent tests accounted for 12.3% of all tests ordered during the study time period. Of those with a single repeat test, heparin/pf4 antibodies were detected in 16% of the subsequent tests (n 5 22/137). Additionally, there were 15 patients for whom three consecutive tests were ordered within 1 month accounting for 45 tests, none of which yielded a positive result. These latter patients were excluded from the following study analysis. Summary statistics were generated based upon the outcome of the repeat test (i.e. hospitalized patients with a subsequent positive or subsequent negative ELISA). Neither the age, gender, and platelet count at the time of the initial negative test, nor the mean platelet count drop from 7 days prior to the initial negative test differed between patients with a subsequent positive versus a subsequent negative test (Table I). Ninety-seven percent of all patients were thrombocytopenic at the time of the initial negative test (<150,000 ll 21 ). Both the number of patients having a surgical procedure within the preceding 60 days and the type of surgery performed were similar between groups. The two groups did, however, differ with respect to (1) the initial negative test OD value and (2) heparin (re-) exposure just prior to repeat testing. More patients with a subsequent positive test were (re-) exposed to heparin at some point within 5 days of repeat testing. This was in addition to having been exposed to heparin prior to initial testing. Furthermore, the average ELISA OD measurement of the initial negative test was higher in patients with a subsequent positive test (0.163 ± and ± for subsequent negative and subsequent positive groups, respectively, P < 0.05). The average platelet count 7 days prior to the initial negative test, at the time of the initial negative test and at the time of the repeat testing did not differ between patients with a subsequent positive or a subsequent negative HIT test (Fig. 1). Seven days prior to the initial negative test, the average platelet count for both groups was 167,000 ll 21 (167,000 ± 94,000 and 167,000 ± 87,000 ll 21 for subsequent negative and subsequent positive groups, respectively). For both groups the mean platelet count dropped by at least 50%, and the average platelet count at the time of repeat testing was similar between the two groups. A more detailed evaluation of the study parameters in relation to grouping of patients based upon an initial negative test OD reading of Low ( ), Medium ( ), and High ( ) is shown in Table II. On the basis of the initial negative test OD value there was no statistical difference between the numbers of days to repeat the test result. Though patients with a higher initial negative test OD value tended to have a slightly longer time to repeat testing, this trend was not statistically different and was observed in both the subsequent positive and subsequent negative test groups. Overall, the average number of days to a subsequent positive test was 7 days. Platelet counts at the time of the initial negative test were also similar between different initial negative test OD groupings suggesting that neither the platelet count alone nor platelet count in conjunction with initial negative test OD value can predict the outcome of repeat testing. To answer the question of whether the initial negative test OD value itself can predict a subsequent positive ELISA, the number of patients with a subsequent positive test was determined for each initial negative test OD grouping. Figure 2 graphically shows the percent of patients with a subsequent positive test in each initial negative test OD value group. Forty percent of patients with a High initial negative test OD value had a subsequent positive test (n 5 8/20), suggesting that patients with a OD value in the upper third of the negative testing range (High initial negative test OD value) have a greater chance of turning positive. The majority of OD values for all initial negative tests performed American Journal of Hematology DOI /ajh 213

3 TABLE II. Evaluation of Study Parameters Based Upon Grouping of the INT OD Value INT OD INT OD of SP test (n 5 22) INT OD of SN test (n 5 115) Days to SP test Days to SN test Mean platelet count at INT 10 9 /L SP test SN test Low (n 5 55) 4/22 (18%) 51/115 (44%) 8 ± 4 6 ± 5 49 ± ± 42 Medium (n 5 62) 10/22 (46%) 52/115 (45%) 6 ± 6 7 ± 6 58 ± ± 61 High (n 5 20) 8/22 (36%) 12/115 (11%) 9 ± 7 11 ± ± ± 20 INT, initial negative test; OD, optical density; SP, subsequent positive; SN, subsequent negative; Low , Medium , High Figure 1. Average platelet counts 7 days prior to the initial negative test (INT), at the time of the INT and at the time of repeat testing. Platelet counts of patients with subsequent positive (SP) and subsequent negative (SN) tests are similar at each evaluated time point. SN, open diamonds; SP, closed squares. [Color figure can be viewed in the online issue, which is available at Figure 3. HIT ELISA optical density (OD) value of the subsequent positive (SP) tests. The range of SP test OD values were (mean and median ). Most SP results are near the cut-off value of 0.4 (18/22 < 1.0). [Color figure can be viewed in the online issue, which is available at Figure 2. The percent of patients within each assigned initial negative test optical density (INT OD) grouping who had a subsequent positive (SP) test. Forty percent (n 5 8/20) of patients with a High INT OD value had a SP test, compared to only 16% (n 5 10/62) of patients with a Medium INT OD value and 7% (n 5 4/55) of patients with a Low INT OD value. at our institution, however, fell into the Low and Medium range (85%, n 5 117/137) and only 15% (n 5 20/137) of initial negative tests had a High OD value (Table II). In regards to the subsequent positive test itself, the positive ELISAs had a wide range of OD values ( ). However, the mean (0.894) and median (0.569) were in the low range of positivity (Fig. 3). In fact, most of the subsequent positive OD values were near the cut-off value of 0.4 with 18/22 subsequent positive tests having an OD value of less than 1.0. Stratification of the pretest clinical score based upon the 4T s has been described and performed by others [17,18]. We retrospectively calculated the pretest clinical score for the time period prior to the initial negative test, and found that overall 77% of patients had a low pretest clinical score (Group 0); 23% had an intermediate pretest clinical score (Group 1), and no patient had a high pretest clinical score (Group 2). Comparison of the initial negative test OD value with the pretest clinical score showed the following: of patients with a subsequent negative test, 80% (89/111) had a low pretest clinical score (Group 0) while only 20% (22/111) had an intermediate pretest clinical score (Group 1) (Fig. 4). For the subsequent positive test group, 62% (13/21) had a low pretest clinical score and 38% (8/21) had an intermediate pretest clinical score (Group 1) (Fig. 5a). Although the initial negative test OD values tended to be higher in patients with an intermediate pretest clinical score compare to those with a low pretest clinical score in the subsequent positive group (Fig. 5a), there was no apparent correlation between the pretest clinical score and the ELISA OD value in either test group. Interestingly, patients with a low pretest clinical score had initial negative test OD values which spanned the full range of possible negative values below the assay cut-off of 0.4 (Figs. 4, 5a). On the basis of the low HIT ELISA OD values in the subsequent positive patients, the heparin/pf4 antibodies detected may have represented non-pathogenic antibody complexes and not the clinical syndrome of HIT. Thus, we sought to further clarify the clinical scenario at the time repeat testing was requested. We retrospectively calculated the pretest clinical scores for the period immediately preceding repeat testing in the subsequent positive test group to assess whether there was a change in the clinical probability of HIT between the initial negative test and the subsequent positive test. We found an interval change to a higher pretest clinical score in four patients prior to the subsequent positive test (see Fig. 5a,b). Two of these patients had developed a new thrombosis between the time of the 214 American Journal of Hematology DOI /ajh

4 Figure 4. Relationship between pretest clinical score group and the initial negative test (INT) optical density (OD) values for patients with a subsequent negative (SN) test. The 4T s: Group 0, low pretest probability; Group 1, intermediate pretest probability; Group 2, high pretest probability. The area above the upper dashed line 5 High INT OD values, between dashed lines 5 Medium INT OD values, and below the lower dashed line 5 Low INT OD values. initial negative test and the subsequent positive test. One patient had an acute decline in a stable platelet count while undergoing plasmapheresis for thrombotic thrombocytopenic purpura. The fourth patient had further progression of pulmonary emboli while taking warfarin shortly after being treated with heparin. These were the only patients in the subsequent positive HIT testing group to show definitive evidence of new thrombus formation or progression of an existing thrombus at the time of repeat testing (Table III). Interestingly, three of these patients had a low pretest clinical score at the time of the initial negative test and had OD values in the Low to Medium range (Fig. 5a, symbols). Figure 5b shows the comparison between the SP OD values and the pretest clinical score. Of the patients with a change in their pretest clinical score (Fig. 5b, symbols) three of four had subsequent positive OD values less than 1.0. We then reviewed the records of patients with a subsequent negative test and found a single patient with evidence of new thrombus formation between the time of the initial negative test and subsequent negative test. This patient s pretest clinical score also increased from Group 0 to Group 2, while the initial negative test OD value decreased from to The 1 month mortality rate was similar between groups; 7/22 patients (33%) in the subsequent positive group and 30/115 (26%) in the subsequent negative group (P ). SRAs were ordered at the time of repeat testing in only five patients with a subsequent negative test, all of which confirmed the absence of activating heparin antibodies. No SRAs were performed in patients with a subsequent positive test. Discussion Repeat HIT testing accounted for 12% of all ELISAs for detection of heparin/pf4 antibodies performed at our institution with 16% of these having a subsequent positive result. Most patients with a subsequent positive test had a heparin (re-) exposure within 5 days preceding the repeat test. Though more patients with an initial negative test OD value in the High range ( % of the threshold for positivity) had a subsequent positive test, a change in the pretest clinical score was more predictive of HITT. Our findings support the use of pretest clinical scoring alone [20], in particular an increase in the pretest clinical score after an initial negative test to assist in clinical decisions regarding repeat evaluation for heparin dependent antibodies, alternative therapeutic anticoagulation strategies, monitoring of potential thrombotic complications, and continued use of heparin. Regarding the laboratory diagnosis of HIT, there are several reasons why a patient may have an initial negative HIT test. Commercially available serological methods for measuring heparin/pf4 antibodies are highly sensitive, however, a negative ELISA with a concurrent positive SRA can occur when the antibody is not directed against PF4, such as an antibody to interleukin-8 or neutrophil-activating peptide [13 15]. In patients with isolated HIT where an ELISA turns positive on repeat testing, the heparin/pf4 antibodies may be undetectable at the time of the initial test. Additionally, commercially available immunoassays for heparin/pf4 antibodies detect all immunoglobulin classes including IgG, IgM, and IgA [21]. Given the heterogeneity in antibody subtypes observed in patients with HIT [22 24], it is possible that patients with different antibody subtypes may also have variable onset of antibody formation or differing affinity of the assay antibodies for detection especially early in the course of the syndrome [9]. Recently, the role of platelet surface PF4 antigenic complexes has been implicated in the pathogenesis of HIT. Using a transgenic mouse model of human PF4 expression, Rauova et al. have demonstrated a correlation between severity of thrombocytopenia and thrombosis with PF4 expression [25,26]. Furthermore, only human platelets with high levels of surface bound PF4 showed enhanced anti-heparin/pf4 monoclonal antibody binding in the presence of exogenous heparin compared to platelets with low levels of PF4 expression [26]. Not only do these findings have implications for identifying patients at risk for developing HIT, individual variations in platelet expression of PF4 may explain why a subset of patients may have an initial negative ELISA. Patients with higher levels of PF4 expression on their platelets may develop thrombocytopenia in the presence of exceptionally low levels of PF4/heparin antibodies; levels that may initially evade detection by current immunologic assays. The role of repeat testing for HIT has drawn little attention in the medical literature. With regard to the ELISA OD value, our results are similar to those reported by Refaai et al., in which a different commercially available ELISA for detection of heparin/pf4 antibodies was used [16]. Both studies showed that while only 15 20% of all initial negative OD values fall within the upper third of the threshold for positivity, 40% of patients with an initial negative test OD value in this range will have a subsequent positive ELISA. In neither study did the magnitude of the platelet count change or the time to repeat testing correlate with a subsequent positive test. Our study differed from Refaai et al., in that we retrospectively determined the pretest clinical probability of HIT and observed that a change in the pretest clinical score was more predictive of developing HITT than the ELISA OD range. In our study population, neither the initial negative test OD value nor the subsequent positive OD value correlated with the development or progression of HITrelated thromboses. Furthermore, patients with an initial negative test had a wide range of OD values <0.4 irrespective of the pretest clinical score. It is generally accepted that HIT is not purely a laboratory diagnosis, but a clinicopathologic diagnosis made with the assistance laboratory tests [20]. Our study further supports the utility of a clinical assessment for the diagnosis of HIT rather than reflexive serial testing for the presence of heparin/pf4 antibodies. Warkentin and coworkers in collaboration with the International Society for Thrombosis and Haemostasis Scientific Committee for Platelet Immunology, have developed a pre- American Journal of Hematology DOI /ajh 215

5 Figure 5. Relationship between pretest clinical score group and subsequent positive (SP) optical density (OD) values for patients with a SP test. Panel A, initial negative test (INT) OD values. Panel B, SP test OD values. The 4T s: Group 0, low pretest probability; Group 1, intermediate pretest probability; Group 2, high pretest probability. The area above the upper dashed line in Panel A 5 High INT OD values, between dashed lines 5 Medium INT OD values, and below the lower dashed line 5 Low INT OD values. Inset symbols represent patients with an increase in their pretest clinical score group between initial and repeat testing. [Color figure can be viewed in the online issue, which is available at TABLE III. Pretest Clinical Probability Score for Patients with an Increase in the 4T s Between an Initial Negative Test (INT) and Subsequent Positive (SP) Test Patient symbol Clinical Pretest 4T s clinical score INT SP test * 58-year-old M with thrombocytopenia 11 days after coronary artery bypass surgery, splenic vein thrombosis diagnosed after a positive HIT ELISA h 47-year-old M admitted with sepsis, receiving subcutaneous and IV heparin flushes, documented portal vein thrombosis 10 days after heparin exposure * 48-year-old M treated for pulmonary emboli with IV heparin, discharged on coumadin, readmitted with extension of pulmonary emboli ^ 84-year-old F admitted with thrombotic thrombocytopenia purpura, new onset thrombocytopenia after platelet stabilization with plasmapheresis 3 (1,1,0,1) a 5 (2,1,0,2) 4 (2,1,0,1) 6 (2,1,2,1) 2 (1,0,0,1) 6 (1,2,2,1) 1 (0,1,0,0) 5 (2,2,0,1) a (T,T,T,T), 4 T s; Thrombocytopenia, Timing, Thrombosis, other score. test clinical scoring system based upon the 4 Ts including Thrombocytopenia, Timing of platelet fall, Thrombosis, and the absence of other causes of thrombocytopenia (medications, disseminated intravascular coagulopathy, etc.) [17 19]. Using this scoring system, they have concluded that a low pretest clinical score for HIT is reliably suitable for ruling out isolated HIT in most clinical settings. We retrospectively determined the pretest clinical scores in our patients at the time of initial negative test and repeat testing (subsequent positive patients only). Interestingly, three of four patients with a subsequent positive HIT test and strong clinical evidence of HITT that evolved between the time of the initial negative test to the subsequent positive result (i.e. a change in the pretest clinical score), had low pretest clinical scores at the time of initial testing. Our findings emphasize the importance of continuing to assess the clinical probability of HIT, even in the initial setting of a low probability of HIT. A diagnostic algorithm to confirm or rule out HIT in patients who have not undergone bypass surgery has been proposed [27]. In this algorithm if there is a low clinical suspicion of HIT, an alternative diagnosis for thrombocytopenia may be sought and heparin or low molecular weight heparin may be continued. If the patient has an intermediate clinical suspicion of HIT and a negative immunoassay, an alternative diagnosis should be considered and heparin therapy may be restarted. Our findings suggest that patients with a low to intermediate clinical suspicion of HIT should continue to be assessed for changes in their clinical probability of HIT. Furthermore, restarting heparin may be considered with caution as all of our patients who developed HITT (four in the subsequent positive HIT group, and one in the subsequent negative HIT group) were reexposed to heparin prior to repeat testing. In conclusion, we have emphasized the utility of using a clinical model for assessing the pretest probability of isolated HIT and/or HITT as a guide for consecutive repeat laboratory testing for heparin dependent antibodies. In particular, one should consider repeat ELISA testing in patients with an increase in their pretest clinical score after having a recent negative antibody test irrespective of the ELISA OD value at the time of initial testing. Methods This study was approved by the Institutional Review Board at Beth Israel Deaconess Medical Center. A computerized hospital laboratory data base identified patients with a repeat ELISA for heparin/pf4 antibodies performed within 3 weeks after an initial negative test, during which time heparin/pf4 antibodies would become detectable, for a time period from January 2001 to March Heparin-dependent antibody tests were ordered at the discretion of the patient s physician in accordance with an established institutional protocol which includes a platelet count decline of 50% while on heparin therapy. As this was a retrospective study, the ordering physicians were unaware of the present study or the ELISA OD value. 216 American Journal of Hematology DOI /ajh

6 Clinical and laboratory data for patients who underwent repeat testing while hospitalized at Beth Israel Deaconess Medical Center were reviewed. Platelet counts were recorded at 7 days prior to the initial test, and at the time of both the initial negative test and the repeat test. During the interval between initial and repeat testing a patient was considered to have been (re-) exposed to heparin if either intravenous heparin, subcutaneous heparin, or low molecular weight heparin was administered at any time within the 5 days preceeding the repeat test. The presence of vascular thrombosis was determined by review of either electronic physician hospital notes or radiographic imaging prior to and after testing for heparin/pf4 antibodies. Mortality was evaluated within 1 month of the initial test. During the study time period, Warkentin and coworkers in collaboration with the International Society of Thrombosis and Hemostasis Scientific Committee on Platelet Immunology introduced a pretest clinical scoring system based upon the 4T s: Thrombocytopenia, Timing of platelet fall, Thrombosis (or other sequelae of HIT), and the absence of other causes of thrombocytopenia [17 19]. To ascertain if the scoring system would have provided additional support for repeat testing, we retrospectively applied this scoring system to our study population, for whom clinical data was available (21/22 subsequent positive and 111/ 115 subsequent negative patients), and categorized them according to their pretest clinical score into either a low (Group 0), intermediate (Group 1), or high (Group 2) pretest probability group as previously described [17,18]. Laboratory detection of heparin/pf4 antibodies was done using the GTI-PF4 ELISA (GTI Diagnostics, Waukesha, WI). The threshold for a positive result was defined as an OD value cut-off of 0.4 according to the manufacturer s guidelines and run with internal positive and negative controls. Confirmatory testing with heparin neutralization was not standard laboratory protocol during the study time period. A negative ELISA was defined as any value below the cut-off. The negative ELISA OD range was then arbitrarily divided into Low , Medium , and High Patients who underwent repeat testing were assigned to one of these groups based upon their initial negative test OD value. These patient groups were then compared with regard to their clinical data, subsequent test results and their pretest clinical probability of HIT as determined by the 4T s scoring system. Data were analyzed using parametric comparisons including v 2 for categorical variables and Student s t-test for continuous variables. Statistical significance was defined by a two-sided a of References 1. Warkentin TE. Heparin-induced thrombocytopenia, Part 1: The diagnostic clues. J Crit Illn 2002;17: Warkentin TE. New approaches to the diagnosis of heparin-induced thrombocytopenia. Chest 2005;127:35S 45S. 3. Warkentin TE, Kelton JG. Temporal aspects of heparin-induced thrombocytopenia. N Engl J Med 2001;344: Warkentin TE, Levine MN, Hirsh J, et al. Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. N Engl J Med 1995;332: Hirsh J, Heddle N, Kelton JG. Treatment of heparin-induced thrombocytopenia: A critical review. Arch Intern Med 2004;164: Selleng K, Warkentin TE, Greinacher A. Heparin-induced thrombocytopenia in intensive care patients. Crit Care Med 2007;35: Greinacher A, Eichler P, Lubenow N, et al. Heparin-induced thrombocytopenia with thromboembolic complications: Meta-analysis of 2 prospective trials to assess the value of parenteral treatment with lepirudin and its therapeutic aptt range. Blood 2000;96: Greinacher A, Lubenow N, Eichler P. Anaphylactic and anaphylactoid reactions associated with lepirudin in patients with heparin-induced thrombocytopenia. Circulation 2003;108: Warkentin TE, Sheppard JA, Moore JC, et al. Laboratory testing for the antibodies that cause heparin-induced thrombocytopenia: How much class do we need? J Lab Clin Med 2005;146: Warkentin TE, Sheppard JA, Horsewood P, et al. Impact of the patient population on the risk for heparin-induced thrombocytopenia. Blood 2000;96: Zwicker JI, Uhl L, Huang WY, et al. Thrombosis and ELISA optical density values in hospitalized patients with heparin-induced thrombocytopenia. J Thromb Haemost 2004;2: Warkentin TE, Greinacher A. Heparin-Induced Thrombocytopenia, 3rd ed. New York: P. Marcel Dekker; Arepally G, Reynolds C, Tomaski A, et al. Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparininduced thrombocytopenia. Am J Clin Pathol 1995;104: Francis JL. A critical evaluation of assays for detecting antibodies to the heparin-pf4 complex. Semin Thromb Hemost 2004;30: Eichler P, Raschke R, Lubenow N, et al. The new ID-heparin/PF4 antibody test for rapid detection of heparin-induced antibodies in comparison with functional and antigenic assays. Br J Haematol 2002;116: Refaai MA, Laposata M, Van Cott EM. Clinical significance of a borderline titer in a negative ELISA test for heparin-induced thrombocytopenia. Am J Clin Pathol 2003;119: Warkentin TE. Heparin-induced thrombocytopenia: Diagnosis and management. Circulation 2004;110:e454 e Lo GK, Juhl D, Warkentin TE, et al. Evaluation of pretest clinical score (4 T s) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings. J Thromb Haemost 2006;4: Warkentin T. Clinical and laboratory diagnosis of HIT. In: Chong B, editor. Refining the Scoring System. ISTH SSC Subcommittee on Platelet Immunology; Aster RH. Heparin-induced immune thrombocytopenia A clinical or laboratory diagnosis? J Thromb Haemost 2006;4: GTI Diagnostics, PF4 Enhanced, Package Insert, Waukesha, WI, January Suh JS, Malik MI, Aster RH, et al. Characterization of the humoral immune response in heparin-induced thrombocytopenia. Am J Hematol 1997;54: Amiral J, Pouplard C, Vissac AM, et al. Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: Biological characteristics and effects on platelet activation. Br J Haematol 2000;109: Ahmad S, Untch B, Haas S, et al. Differential prevalence of anti-heparin-pf4 immunoglobulin subtypes in patients treated with clivarin and heparin: Implications in the HIT pathogenesis. Mol Cell Biochem 2004;258: Eslin DE, Zhang C, Samuels KJ, et al. Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: Dissociation between anticoagulant and antithrombotic effect of heparin. Blood 2004;104: Rauova L, Zhai L, Kowalska MA, et al. Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: Diagnostic and therapeutic implications. Blood 2006;107: Arepally GM, Ortel TL. Clinical practice. Heparin-induced thrombocytopenia. N Engl J Med 2006;355: American Journal of Hematology DOI /ajh 217