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A Diagnosis of Heparin-Induced Thrombocytopenia with Combined Clinical and Laboratory Methods in Cardiothoracic Surgical Intensive Care Unit Patients

Demma, Linda J. MD, PhD*; Winkler, Anne M. MD; Levy, Jerrold H. MD, FAHA*,‡

doi: 10.1213/ANE.0b013e3182297031
Cardiovascular Anesthesiology: Research Reports
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BACKGROUND: Diagnosing postoperative heparin-induced thrombocytopenia (HIT) in cardiothoracic surgical patients is complicated because of the profound thrombocytopenia that occurs with cardiopulmonary bypass (CPB). CPB predisposes patients to develop a frequent incidence of antibodies directed against platelet factor 4 (PF4)/heparin complexes and HIT. The sensitivity of readily available antibody immunoassays is high, but specificity is quite low. The use of both a clinical probability score and rapid laboratory immunoassay has been shown to increase specificity, which is of particular importance in the CPB setting. Prompt diagnosis is crucial because cessation of heparin and treatment with alternative anticoagulation can reduce the risk of thromboembolic events.

METHODS: We retrospectively reviewed records from cardiothoracic surgical patients whose serum was tested with both the serotonin release assay (SRA) and the PF4/heparin immunoassay from January 2007 through December 2010. We assigned a high, intermediate, or low clinical “4Ts” probability score that quantifies thrombocytopenia, timing of platelet decrease, and thrombotic complications in each patient. We then compared the clinical score and the PF4/heparin immunoassay against the “gold standard” diagnostic test, the SRA.

RESULTS: The sensitivity and specificity for PF4/heparin optical density >0.40 were 100% and 26%, respectively. Sensitivity and specificity for the diagnosis of HIT with a combination of PF4/heparin optical density >0.40 and high/intermediate 4Ts score were 100% and 70%, respectively. The negative predictive value was 100% for low 4Ts score.

CONCLUSIONS: We demonstrated that the use of the 4Ts clinical score combined with the PF4/heparin immunoassay for HIT diagnosis increases the sensitivity and specificity of HIT testing compared with the PF4/heparin immunoassay alone. Furthermore, with an intermediate 4Ts score and positive PF4/heparin antibody test, a confirmatory platelet activation assay such as the SRA is necessary. Physicians treating patients after cardiothoracic surgery should recognize the need for an antibody test and confirmation with a platelet activation assay with even moderate clinical probability of HIT.

Published ahead of print July 25, 2011 Supplemental Digital Content is available in the text.

From the *Department of Anesthesiology, Transfusion Medicine, and Department of Critical Care Medicine, Emory University, Atlanta, Georgia.

Supported in part by Emory University School of Medicine, Emory Healthcare, FAER, and a grant from GlaxoSmithKline.

This study was presented in part at the 2010 American Society of Anesthesiologists FAER presentations, October 19, 2010, San Diego, CA.

Conflict of Interest: See Disclosures at the end of the article.

Reprints will not be available from the authors.

Address correspondence to Jerrold H. Levy, MD, FAHA, Department of Anesthesiology, Emory University Hospital, 1364 Clifton Rd., Atlanta, GA 30322. Address e-mail to jlevy01@emory.edu.

Accepted June 3, 2011

Published ahead of print July 25, 2011

Cardiothoracic surgical patients receive large doses of unfractionated heparin during cardiopulmonary bypass (CPB) and cardiac surgery, and are at risk for postoperative heparin-induced thrombocytopenia (HIT). HIT is an antibody-mediated, prothrombotic adverse event of heparin therapy that occurs in approximately 1% to 3% of cardiothoracic surgical patients treated with heparin.13 Patients who develop HIT antibodies, antibodies that recognize multimeric platelet factor 4 (PF4)/ heparin complexes on platelet surfaces, are considered to be in a procoagulant state and at high risk for adverse thrombotic complications.4 HIT with thrombosis is the clinical syndrome in which patients with HIT antibodies develop myocardial infarction, pulmonary embolism, limb ischemia, or deep vein thromboses secondary to HIT.57 HIT is considered a clinicopathologic diagnosis because both clinical and laboratory criteria should be sought for confirmation.8

Cardiothoracic surgical patients also have an increased likelihood of developing HIT antibodies directed against PF4/heparin complexes9,10 in part because of platelet activation associated with tissue injury, surgery, and CPB.11 The presence of these antibodies before surgery is an independent predictor for death or prolonged hospitalization after adult cardiac surgery.5,7 In a large study of cardiothoracic surgical patients, thrombotic complications from HIT resulted in a mortality rate of 28%.12 There is a high index of suspicion for HIT in the cardiothoracic intensive care unit (CT ICU) because of evidence that this patient population is at increased risk of HIT with thrombosis.

Diagnosis of HIT is often a dilemma in these patients because thrombocytopenia secondary to hemodilution and platelet consumption also occurs after cardiothoracic surgery. A major decrease in platelet count of approximately 40% to 50% occurs during the first 72 hours after cardiothoracic surgery in the majority of patients.13 Clinically evident HIT is characterized by a second episode of thrombocytopenia beginning between postoperative days 5 and 10 (the P1 pattern reported by Pouplard et al.9), or, in some patients, persistent thrombocytopenia beyond the first 5 days (the so-called P2 pattern described by Pouplard et al.9). The PF4/heparin antibody response, measured in optical density (OD) units by immunoassay, measures the presence of HIT antibodies but not the functional ability of these antibodies to activate platelets, resulting in a prothrombotic state.14 The sensitivity of the PF4/heparin OD in the cardiothoracic surgical population is high, but the specificity is quite low. To improve clinical detection of HIT, a clinical scoring system, known as the “4Ts” score was developed.15 The 4 “Ts” in the 4Ts score are: 1thrombocytopenia with a platelet count decrease >50% and platelet nadir ≥20,000/μL; 2timing of platelet decrease with clear onset between days 5 and 10 or platelet decrease ≤1 day (prior heparin exposure within 30 days); 3 new thrombosis (confirmed); and 4 no likely alternative cause for thrombocytopenia.15 The use of both the clinical 4Ts score and PF4/heparin OD has been shown to reduce overdiagnosis of HIT, which is of particular importance in the CPB setting where thrombocytopenia secondary to platelet consumption and hemodilution is common.16 However, prompt recognition of HIT is crucial in the ICU setting because cessation of heparin and treatment with alternative anticoagulation such as direct thrombin inhibitors can substantially reduce the risk of thrombosis.17,18

The purpose of this study was to assess clinical and laboratory diagnosis of HIT in a cohort of patients after cardiothoracic surgery. Because alternative anticoagulation therapy with direct thrombin inhibitors is expensive and has increased potential for bleeding complications, a prompt and accurate diagnosis of HIT is important and often challenging in this patient population. Therefore, we examined the relationships among the magnitude of anti-PF4/heparin antibodies, the 4Ts clinical score, and confirmed HIT cases as defined by a positive serotonin release assay (SRA). We analyzed the sensitivity and specificity of the PF4/heparin OD alone and the combined use of 4Ts score and PF4/heparin OD. To improve early detection of HIT in this clinical setting, we also evaluated additional factors that may be associated with HIT, including baseline preoperative platelet count, magnitude of thrombocytopenia, and on- versus off-pump surgery.

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METHODS

Study Design and Patient Selection

After receiving institutional approval, a retrospective review of patients in the Emory University Hospital Cardiothoracic Surgical ICU was conducted for the period of January 2007 through December 2010. This study included all patients who underwent cardiothoracic surgery and postoperatively had plasma samples sent to the laboratory for PF4/heparin antibody testing. The clinical suspicion of HIT was determined by the attending physician, based on a persistently low or rapid decrease in platelet count, or new evidence of thrombosis after surgery. Only patients who had both screening PF4/heparin antibody and SRA testing, which was performed at the attending physician's discretion, were included.

Detailed data collected included age, gender, medical history, date of admission and discharge, date and cause of death if applicable, surgery date and type, baseline platelet count before surgery, daily platelet counts when available from 2 days before surgery to a maximum of 30 days postsurgery. The postoperative platelet peak was defined as the maximum platelet count reached within the first 10 days after surgery. The postoperative decrease in platelet count, delta platelet (Δp), was defined as the percent decrease in platelets after the postoperative platelet peak. Occurrence of thrombotic complications as documented in the discharge summary was recorded, specifically deep vein thrombosis, pulmonary embolism, myocardial infarction, and limb ischemia. A 4Ts score was calculated from retrospective chart review with the following methodology as described previously15: 1thrombocytopenia with a platelet count decrease >50% and platelet nadir ≥20,000/μL (2 points) or platelet count decrease 30% to 50% and platelet nadir 10 to 19 (1 point); 2timing of platelet decrease with clear onset between days 5 and 10 or platelet decrease ≤1 day (2 points) or onset after day 10 (1 point); 3 new confirmed thrombosis (2 points) or progressive, recurrent, or suspected thrombosis (1 point); and 4 no other likely cause for thrombocytopenia (2 points) or possible other cause (1 point).15 The resulting clinical probability scores are divided into high (6–8 points), intermediate (4–5 points), and low (≤3 points) groups.

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Serologic Analysis

HIT antibody testing was performed by the Special Hemostasis Laboratory at Emory University Hospital. Plasma samples from January 2007 through August 2009 were tested using a commercial enzyme-linked immunosorbent assay (ELISA) for antibodies of immunoglobulin (IgG, IgA, and IgM classes [PF4 Enhanced®; GTI Diagnostics, Waukesha, WI]) as previously described.19 In September 2009, the laboratory changed methodologies in an attempt to increase the detection of IgG HIT antibodies and, as a result, plasma samples collected from September 2009 through December 2010 were tested using a commercial ELISA specific for IgG antibodies (Zymutest HIA IgG, HYPHEN BioMed; Aniara, Mason, OH). For both assays, results were expressed in OD units, and a value of >0.4 was reported as positive for the IgG/A/M ELISA and >0.5 for the IgG ELISA in concordance with the manufacturer's established ranges. The SRA, which is currently considered the gold standard for the diagnosis of HIT, was performed by a designated reference laboratory at the discretion of the ordering physician. A result of ≥20% serotonin release was reported as positive by the reference laboratory.

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Statistical Analysis

The sensitivity and specificity of the PF4/heparin ELISA and the combined use of a positive PF4/heparin OD with a 4Ts score ≥4 were calculated using SRA as the gold standard. Receiver operating characteristic curves, sensitivity versus 1 − specificity, were plotted and compared for the PF4/heparin ELISA assay alone and 4Ts clinical score alone, also using SRA as the gold standard. Mean laboratory values for Δp, PF4/heparin OD, baseline platelet count, and postoperative platelet peaks were compared between positive and negative HIT, as defined by SRA, using the Mann-Whitney U test. Frequencies of patients with positive SRA results were compared for each clinical 4Ts probability group (high, intermediate, and low) by χ2 test. Spearman rank correlation tests were used to test for an association between the clinical 4Ts score and the PF4/heparin OD. All data analyses were performed using SPSS version 19.0 (SPSS, Inc., Chicago, IL). A P value of 0.05 was used as a cutoff for statistical significance for all tests.

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RESULTS

Between January 2007 and December 2010, 50 patients from the CT ICU had both a PF4/heparin immunoassay and SRA results available and were included in the retrospective analysis. HIT testing was conducted during the same hospitalization and within 30 days after cardiothoracic surgery for all patients. None of the patients had a preoperative history of HIT.

Eleven of the patients (22%) had a positive SRA. Clinical and laboratory characteristics of all patients and a comparison between SRA-positive and SRA-negative patients are shown in Table 1. The mean PF4/heparin OD was greater for patients with a positive SRA (1.62 vs 0.65, P < 0.001). In addition, there was a greater frequency of patients with SRA-positive tests with increasing clinical 4Ts probability (χ2, P < 0.001). Baseline (preoperative) platelet counts, postoperative platelet peak, and Δp (percent change in platelets from the postoperative platelet peak to the subsequent platelet nadir) were similar between groups. There were also no differences between groups for number of thrombotic events, mortality, or number undergoing on-pump cardiothoracic surgery.

Table 1

Table 1

The relationship between PF4/heparin OD and 4Ts score is shown in Figure 1, and boundaries are drawn for a 4Ts score of intermediate and higher (≥4) and for PF4/heparin OD ≥0.40. The 4Ts clinical score was significantly associated with PF4/heparin OD (Spearman rank correlation coefficient, P = 0.04). The sensitivity of the PF4/heparin ELISA with a cutoff value of 0.40 was 100% (95% confidence interval [CI], 72%–100%), but specificity was only 26% (95% CI, 13%–42%). The sensitivity for an intermediate to high 4Ts score (cutoff value of 4) was 100% (95% CI, 72%–100%) and specificity 56% (95% CI, 40%–72%). With a high 4Ts score (≥6), sensitivity was 55% (95% CI, 17%–77%) and specificity 100% (95% CI, 91%–100%). The sensitivity of combined 4Ts score ≥4 and PF4/heparin OD >0.40 was 100% (95% CI, 72%–100%) and specificity was 70% (95% CI, 52%–83%), with a false-positive rate of 12 of 50 (24%) and a false-negative rate of 0. None of the patients with a low 4Ts score (≤3) had a positive SRA, resulting in a negative predictive value for low 4Ts score of 100%. Receiver operating characteristic curves (sensitivity versus 1 − specificity) are shown in Figure 2 for the PF4/heparin ELISA and the 4Ts score. The area under the curve, representing the ability of each test to correctly classify those with and without HIT, did not significantly differ between the 2 tests; area under the curve was 0.84 (95% CI, 0.71–0.97) for PF4/heparin ELISA and 0.92 (95% CI, 0.85–1.00) for 4Ts score.

Figure 1

Figure 1

Figure 2

Figure 2

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DISCUSSION

In this study, we found that the combination of positive PF4/heparin ELISA OD and an intermediate or high clinical 4Ts score provides the most accurate diagnosis of HIT, with specificity of 70%, compared with 26% for positive PF4/heparin ELISA OD alone. However, a false-positive rate of 24% for the combined use of PF4/heparin ELISA OD and 4Ts score demonstrates that an SRA should be ordered to confirm HIT antibody activity. These results indicate that the ELISA, despite its widespread use, is only a moderate predictor of HIT in cardiothoracic surgery.

In our study, the PF4/heparin ELISA OD was significantly greater in SRA-positive patients, which is consistent with studies demonstrating that the probability and clinical severity of HIT increases in relation to the magnitude of the PF4/heparin ELISA OD in surgical patients.1,2022 However, a positive antibody ELISA was not always associated with the clinical development of HIT, thus corroborating the increasing number of studies that have reported the presence of PF4/heparin antibodies in patients without clinical HIT, especially in cardiothoracic surgical patients.2326

Thrombocytopenia secondary to hemodilution and platelet consumption occurs after cardiothoracic surgery with persistent thrombocytopenia extending through 72 hours after surgery, findings that can confound HIT recognition. Thrombocytopenia during the first 4 postoperative days is rarely attributable to HIT.10,27 It is the relative decrease in platelets on or after days 5 to 10 after cardiothoracic surgery that suggests HIT,15,16,28 whereby if platelets decrease >50% after the postoperative peak, HIT should be clinically suspected.9,29 Because of the occurrence of platelet activation and thrombocytopenia seen in all cardiothoracic surgical patients and the potential for false positives with the PF4/heparin ELISA alone, a clinical scoring system can be crucial in the diagnosis of HIT. The clinical combinations of specific platelet dynamics and thromboembolic events have been quantified by Lo et al.15 with the 4Ts score, and this scoring system has been validated in several populations.30 In addition, a low 4Ts score has been shown to reliably exclude HIT with a negative predictive value of 100%,31 consistent with the results of this study. Additional clinical probability scores have been used to improve diagnosis of HIT. The Lillo-Le Louet score combines the following variables: 1 a biphasic platelet count from CPB to the first day of suspected HIT, 2 an interval of ≥5 days from CPB to the first day of suspected HIT, and 3 a CPB duration of >118 minutes.32 The score, which can be applied as soon as HIT is suspected after CPB, had good negative predictive value of 97%. A second scoring system, the HIT Expert Probability score, was developed from the opinions of 26 HIT experts based on 8 clinical features of potential importance in the diagnosis of HIT obtained from a comprehensive review of the literature.33 In a retrospective validation of 50 patients, this score was shown to be 100% sensitive and 60% specific for determining the presence of HIT defined by 3 independent HIT adjudicators.33 However, neither the Lillo-Le Louet nor the HIT Expert Probability score has been validated prospectively. Because the 4Ts score has been prospectively validated, is applicable in many clinical settings, and is easily available to physicians, we chose to use this scoring system to measure the clinical probability of HIT. Our study shows that this clinical scoring system can be beneficial for determining the next clinical action in the CT ICU when combined with PF4/heparin ELISA OD. Specifically, a low 4Ts score effectively excludes HIT, but with an intermediate or high 4Ts score and positive PF4/heparin ELISA, heparin should be stopped and anticoagulation with an alternative agent such as a direct thrombin inhibitor initiated, and confirmatory testing with an SRA should be sought. Our study is a comparison between the diagnostic ability of the 4Ts clinical score versus the laboratory PF4/heparin ELISA assay; future studies are needed to directly compare each of the clinical scoring systems.

Several methods are available for the identification of HIT antibodies. Functional laboratory assays use washed platelets and detect their activation as a result of antibody binding (primarily IgG) by measuring aggregation of platelets (heparin-induced platelet aggregation) or release of radiolabeled serotonin (SRA).34 These functional assays are currently considered the gold standard for the laboratory diagnosis of HIT. However, these assays require technical expertise and use of donor platelets, both of which are not available in most hospitals. As a result, the most frequent HIT diagnostic test used is the detection of HIT antibodies with commercially available ELISAs that immobilize PF4 alone or a platelet lysate containing PF4, interleukin-8, and other proteins along with heparin or another polyanion to a microtiter plate. The ELISA that was used for the first half of this study, which detects IgG, IgM, and IgA antibodies, has a lower specificity to detect pathologic (platelet-activating) HIT antibodies than the current IgG-only assay that is used in our institution.35 In addition, there is currently a lack of standardization among commercially available assays. Nonetheless, PF4 is the heparin-dependent protein regardless of the capability of these antibodies to activate platelets, and the negative predictive value for OD <0.4 in both assays is known to be 100%.35

Our study is subject to several limitations. First, it is a retrospective study design that only includes patients in the CT ICU for whom the physician ordered an ELISA for HIT antibodies and SRA, presumably because a subjective threshold of clinical suspicion for HIT had been reached. In addition, some patients may have left the CT ICU before the 5- to 10-day period during which the platelet nadir is observed and the diagnosis of HIT is pursued. This resulted in a small patient population with potential bias. Second, the ELISA that was used for patients in the first half of this study may have a lower specificity to detect pathologic HIT antibodies than the current IgG-only assay that is used in our institution as of September 2009. None of the patients who tested negative for PF4/heparin antibodies had a positive SRA, and analyses that were conducted separately for each assay (IgG-only versus IgG/A/M) demonstrated the same relationship between 4Ts score and PF4/heparin ELISA OD and the same validity with respect to SRA. Third, the SRA is considered the gold standard laboratory test for HIT confirmation in our study, and has been used as such in previous studies but is subject to different cutoff values.32,36 Although the SRA may be the most accurate laboratory diagnostic, it should be emphasized that a confirmatory HIT diagnosis should be made with both laboratory and clinical diagnostic criteria.

In summary, our study provides evidence that use of a clinical scoring system together with quantitation of anti-PF4/heparin antibody development after cardiothoracic surgery should be used to determine the next course of action in the CT ICU, including cessation of heparin and initiation of a direct thrombin inhibitor. The ELISA result is not adequate for diagnosis of HIT, and a clinical assessment and confirmatory SRA should be pursued before HIT can be excluded. Treatment should be initiated until HIT is excluded. Further studies should aim to improve laboratory diagnostics for HIT, compare clinical scoring systems, and to reduce the anti-PF4/heparin immune response and platelet activation after cardiothoracic surgery.

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RECUSE NOTE

Jerrold H. Levy is section Editor of Hemostasis and Transfusion Medicine for the Journal. This manuscript was handled by Steve Shafer, Editor-in-Chief, and Dr. Levy was not involved in any way with the editorial process or decision.

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DISCLOSURES

Name: Linda J. Demma, MD, PhD.

Contribution: Study design, conduct of study, data analysis, and manuscript preparation.

Conflicts of Interest: This author has no conflicts of interest to declare.

Name: Anne M. Winkler, MD.

Contribution: Data acquisition, conduct of study, and manuscript preparation.

Conflicts of Interest: This author has no conflicts of interest to declare.

Name: Jerrold H. Levy, MD, FAHA.

Contribution: Study inception, design, implementation, and manuscript preparation.

Conflicts of Interest: This author is a member of the steering committee at Canyon Pharmaceutical.

This manuscript was handled by: Steven L. Shafer, MD.

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ACKNOWLEDGMENTS

Foundation for Anesthesia Education and Research Anniversary: Funding/Career Development Summary.

Linda J. Demma, MD, PhD.

The Foundation for Anesthesia Education and Research (FAER) has contributed to my career development in several important ways. While trained as a PhD researcher when I entered medical school, I had little to no experience in clinical research. I sought the mentorship of Dr. Jerrold Levy because of his involvement in critical care and anesthesia research as a FAER mentor. The FAER Medical Student Anesthesia Research Fellowship allowed me to spend a summer assisting and eventually leading clinical studies with Dr. Levy. I attended rounds in the intensive care unit regularly during this fellowship, and found that this clinical experience outside of the typical medical school curriculum was vital to my development as an independent clinical researcher.

I traveled to the American Society of Anesthesiologists (ASA) meeting in San Diego, CA, and presented my research findings at a poster session, organized by FAER. The opportunity to receive feedback from established researchers in the field was a unique experience that helped solidify my participation in clinical anesthesia research. Furthermore, the ASA meeting exposed me to the broad array of topics in the field of Anesthesia, from clinical to basic science. Because I was applying to residency programs, this was a key time period for exposure to the field and its leaders. I developed long-term relationships with clinician researchers in the field, which I intend to pursue in the form of continuing research and educational experiences during residency and beyond.

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