Timely diagnosis and management of heparin‐induced thrombocytopenia in a frequent request,low incidence single centre using clinical 4T’s score and particle gel immunoassay

Clinicians must promptly decide which patients suspected of having heparin‐induced thrombocytopenia (HIT) warrant a change in anticoagulation. This single‐centre series of 246 HIT testing referrals assessed the combination of clinical score (thrombocytopenia, timing, thrombosis, other causes of thrombocytopenia not evident; 4T’s), Diamed ID‐Heparin‐PF4 immunoassay (PaGIA) and 14C Serotonin Release Assay (SRA) to develop a practical and safe diagnostic strategy for HIT. A total of 142/256 (58%) referrals were in patients with a low 4T’s score, with 12/246 (5%) in the high scoring group. PaGIA was positive in 24/246 (9·7%) patients, whilst SRA was positive in 9/246 (3·6%). The overall positive predictive value of a positive PaGIA test alone was 37·5%, however this reached 80% for the high scoring group. Both negative PaGIA and low clinical score independently had negative predictive values of 100%. We subsequently developed an algorithm that, when applied to this cohort, would have resulted in 18/246 patients (7%) definitely requiring alternative anticoagulation, whilst a further 7/246 (2·8%) patients would have been considered on an individual basis. Ultimately (based on SRA) this would have resulted in 16/246 (6·5%) patients unnecessarily having a change in their anticoagulation, with 9/246 (3·6%) patients being ‘correctly treated’. The combination of 4T’s scoring and PaGIA permitted a practical and safe approach to rapid HIT diagnosis and management.     

Changes in platelet count after cardiac surgery can effectively predict the development of pathogenic heparin-dependent antibodies

Cardiopulmonary bypass (CPB) induces the release of platelet factor 4 (PF4) and patients are at risk of heparin-induced thrombocytopenia (HIT). This study was aimed to determine whether an abnormal evolution in platelet count (PC) after CPB is predictive of the development of HIT antibodies. Two abnormal PC patterns were defined: pattern P1, characterized by a decrease in PC following previous correction of thrombocytopenia occurring during CPB, and pattern P2, defined as a persistent low PC in the days following CPB. PC was evaluated for 10 d in 305 consecutive patients before and after CPB. Serotonin release assay (SRA) was carried out between days 8 and 10 to detect pathogenic heparin-dependent antibodies. Moreover, antibodies to heparin-PF4 (H-PF4) complexes were assayed by enzyme-linked immunosorbent assay. PC evolution after CPB was normal in 300 patients although antibodies to H-PF4 were frequently present (53.4%). Changes in PC were abnormal in five patients with pattern P1 (n = 4) or P2 (n = 1). As SRA was positive in four of the five cases, the positive predictive value of abnormal PC pattern for pathogenic HIT antibodies was 80%. Careful follow-up of PC after CPB makes it possible to predict with high specificity (99%) for those patients who develop pathogenic HIT antibodies.     

What is the potential for overdiagnosis of heparin‐induced thrombocytopenia?

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4//heparin (PF4/H) complexes. According to the "iceberg model," only a subset of anti-PF4/heparin antibodies of IgG class evincing strong platelet-activating properties cause clinical HIT. Since many centers rely predominantly on an anti-PF4/polyanion enzyme-immunoassay (EIA) to diagnose HIT, we estimated the potential for overdiagnosis when only this single test is available. We examined a database of 100 patients in whom the probability of HIT had been estimated using a clinical scoring system (4Ts), and where patients underwent systematic testing for HIT antibodies using three assays: the platelet serotonin release assay (SRA), an "in-house" EIA that detects IgG anti-PF4/heparin antibodies (EIA-IgG), and a commercial EIA that detects anti-PF4/polyanion antibodies of all three immunoglobulin classes (EIA-GTI). Whereas 16 of 100 patients fulfilled a "classic" definition of HIT (intermediate/high probability plus strong platelet-activating anti-PF4/heparin IgG antibodies), an additional 16 patients fulfilled a "liberal" definition in which any investigated patient (irrespective of the pretest probability) who had a positive EIA-GTI was considered to have HIT. The clinical features of these 16 additional patients--including generally weak antibodies and low risk for thrombosis--suggest underlying non-HIT explanations for thrombocytopenia. Patients with a positive SRA generally corresponded to those with intermediate or high pretest probability of HIT who also had strong EIA-GTI reactivity (>1.20 OD units). We conclude there is the potential to overdiagnose HIT by approximately 100% if any positive EIA is considered to "confirm" the diagnosis of HIT irrespective of the clinical scenario.     

Evaluating heparin-induced thrombocytopenia: the old and the new

Heparin-induced thrombocytopenia (HIT) is a rare but potentially serious complication of heparin use. Prompt diagnosis is crucial and requires the integration of clinical assessment and laboratory testing. Pretest clinical scoring systems (i.e., 4 Ts) have been established. Immunoassays can detect the presence of antibodies directed toward heparin-platelet factor 4 (H-PF4) complexes, but provide no information about their ability to activate platelets. A low clinical score, when combined with a negative immunoassay result obviates the need for further testing. However, immunoassays and 4 Ts scores have only modest specificity. Functional testing (serotonin release assay or heparin-induced platelet activation) remain important in confirming the presence of pathogenic H-PF4 antibodies, but are technically demanding to perform and limited in guiding clinical decisions in the acute setting. This review evaluates current immuno- and functional assays available in the laboratory diagnosis of HIT, and describes recent attempts to improve the specificity of enzyme immunoassays, including adopting an immunoglobulin G-specific assay and raising the optical density value cutoff for a positive result. The importance of donor selection and newer functional assays, including flow cytometry-based assays, are also discussed. A current approach to integrating clinical scoring, immunoassays, and functional testing for HIT is also outlined.     

Suspicion of heparin-induced thrombocytopenia in internal medicine: How appropriate is the ordering of anti-PF4/heparin antibody testing?

Thrombocytopenia is frequent in hospitalized patients, and heparin-induced thrombocytopenia (HIT) is often suspected when a decrease in platelet count is concomitant with heparin treatment. ELISA tests used for anti-PF4/heparin antibodies detection usually have high sensitivity but only fair specificity for HIT. Pre-test probability scores (such as 4Ts or HEP scores) have been validated and a low probability score rules out HIT without anti-PF4/heparin testing. The aims of this study are to evaluate the appropriateness of anti-PF4/heparin testing according to pre-test probabilities of HIT and to compare the abilities of the 4Ts and HEP scores to avoid inappropriate anti-PF4/heparin testing. This retrospective observational study included 74 consecutive patients hospitalized in a general internal medicine division who had anti-PF4/heparin testing for suspicion of HIT. 4Ts and HEP scores were computed retrospectively. About 73% of patients who had ordering of an anti-PF4/heparin were at low risk according to the 4Ts score, and 46% according to the HEP score. Heparin was discontinued in 61% and 62% of low-risk patients according to 4Ts and HEP scores and switched to alternative anticoagulant in 31% and 32% of them, respectively. No case of HIT was diagnosed in patients with a low-risk score. One major bleeding and no thrombosis were observed. For the 4Ts score, the sensitivity was 100%, the negative predictive value (NPV) was 100%, the specificity was 77%, and the positive predictive value (PPV) was 20% (95% CI: 7–44). For the HEP score, the sensitivity was 100%, the NPV was 100%, the specificity was 49%, and the PPV was 10%. In conclusion, pre-test probability scores were vastly underused in this internal medicine population despite their ability to rule out HIT without laboratory testing in a large proportion of patients. Appropriate use of those instruments should be actively promoted.     

Prospective evaluation of a rapid nanoparticle‐based lateral flow immunoassay (STic Expert® HIT) for the diagnosis of heparin‐induced thrombocytopenia

A rapid lateral flow immunoassay (LFIA) (STic Expert^® HIT), recently developed for the diagnosis of heparin‐induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme‐linked immunosorbent assay (Asserachrom^® HPIA IgG) and serotonin release assay. The inter‐reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4‐PaGIA^®) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert^® HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT.     

Prospective multicentre cohort study of heparin-induced thrombocytopenia in acute ischaemic stroke patients

Acute ischaemic stroke patients sometimes receive heparin for treatment and/or prophylaxis of thromboembolic complications. This study was designed to elucidate the incidence and clinical features of heparin-induced thrombocytopenia (HIT) in acute stroke patients treated with heparin. We conducted a prospective multicentre cohort study of 267 patients who were admitted to three stroke centres within 7 d after stroke onset. We examined clinical data until discharge and collected blood samples on days 1 and 14 of hospitalization to test anti-platelet factor 4/heparin antibodies (anti-PF4/H Abs) using an enzyme-linked immunosorbent assay (ELISA); platelet-activating antibodies were identified by serotonin-release assay (SRA). Patients with a 4Ts score ≥4 points, positive-ELISA, and positive-SRA were diagnosed as definite HIT. Heparin was administered to 172 patients (64·4%: heparin group). Anti-PF4/H Abs were detected by ELISA in 22 cases (12·8%) in the heparin group. Seven patients had 4Ts ≥ 4 points. Among them, three patients (1·7% overall) were also positive by both ELISA and SRA. National Institutes of Health Stroke Scale score on admission was high (range, 16-23) and in-hospital mortality was very high (66·7%) in definite HIT patients. In this study, the incidence of definite HIT in acute ischaemic stroke patients treated with heparin was 1·7% (95% confidence interval: 0·4-5·0). The clinical severity and outcome of definite HIT were unfavourable.     

Clinical decision support to reduce unnecessary diagnostic testing for heparin-induced thrombocytopenia

Appropriate evaluation of heparin-induced thrombocytopenia (HIT) is imperative because of the potentially life-threatening complications. However, overtesting and overdiagnosis of HIT are common. Our goal was to evaluate the impact of clinical decision support (CDS) based on the HIT computerized-risk (HIT-CR) score, designed to reduce unnecessary diagnostic testing. This retrospective observational study evaluated CDS that presented a platelet count versus time graph and 4Ts score calculator to clinicians who initiated a HIT immunoassay order in patients with predicted low risk (HIT-CR score 0–2). The primary outcome was the proportion of immunoassay orders initiated but cancelled after firing of the CDS advisory. Chart reviews were conducted to assess anticoagulation usage, 4Ts scores and the proportion of patients who had HIT. In a 20-week period, 319 CDS advisories were presented to users who initiated potentially unnecessary HIT diagnostic testing. The diagnostic test order was discontinued in 80 (25%) patients. Heparin products were continued in 139 (44%) patients, and alternative anticoagulation was not given to 264 (83%). The negative predictive value of the advisory was 98.8% (95% CI: 97.2–99.5). HIT-CR score-based CDS can reduce unnecessary diagnostic testing for HIT in patients with a low pretest probability of HIT.     

Serotonin-release assay-negative heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Pathogenic HIT antibodies can be detected by the serotonin-release assay (SRA), a platelet activation test. We have regarded the SRA performed in our medical community (“McMaster” SRA) as having high sensitivity and specificity. Recently, the concept of “SRA-negative HIT” has been proposed for enzyme-immunoassay (EIA)-positive/SRA-negative patients with a HIT-compatible clinical picture, who test positive in a PF4-enhanced platelet activation assay. After identifying an index case of SRA-negative HIT, we estimated the frequency of this condition by performing the “PF4-SRA” (modified SRA using high concentrations of added PF4 rather than heparin) in EIA-positive patients from a cohort study evaluating clinical and laboratory diagnosis of HIT. We defined SRA-negative HIT as patients meeting three criteria: clinical picture compatible with HIT (4Ts ≥ 4 points); EIA-positive (≥1.00 units); and PF4-SRA-positive. Among 430 patients, 35 were EIA-positive/SRA-positive and 27 were EIA-positive/SRA-negative. Among these 27 SRA-negative patients, three were found to have subthreshold levels of platelet-activating antibodies by PF4-SRA, of whom one met clinical criteria for SRA-negative HIT. Thus, based on identifying one patient with SRA-negative HIT within a cohort study that found 35 SRA-positive HIT patients, we estimate the sensitivity of the McMaster SRA for diagnosis of HIT to be 35/36 (97.2%; 95% CI, 85.8-99.9%). Although the McMaster SRA is highly sensitive for HIT, occasional SRA-negative but EIA-positive patients strongly suspected of having HIT can have this diagnosis supported by a PF4-enhanced activation assay such as the PF4-SRA.     

A differentiated approach to assess the diagnostic usefulness of a rapid particle gel immunoassay for the detection of antibodies against heparin-platelet factor 4 in cardiac surgery patients.

Enzyme-linked immunosorbent assay (ELISA) methods have shown to be of a low specificity for confirming heparin-induced thrombocytopenia (HIT) in cardiac surgery patients. In other patient collectives, a new antigen test [ID-HPF4 Particle Gel Immuno Assay (PaGIA); Diamed, Cressier sur Morat, Switzerland] was recently reported to exhibit a better specificity than the ELISA. Since a more specific test would serve as a useful tool when diagnosing HIT, a prospective study was carried out to elucidate the specificity and diagnostic usefulness of PaGIA in cardiac surgical patients. As assessed in cardiac surgery patients without HIT, the proportion of PaGIA-positive samples was lowest at baseline (16%; 13/74), increasing to 31% (19/61) at postoperative days 5-9 (P = 0.06) and 69% (29/42) at postoperative days 10-18 (P < 0.001 versus postoperative days 5-9). The respective proportions in an ELISA (HPF4 ELISA; Stago, Asnières sur Seine, France) were lower. Because the prevalence of antibody positivity increases during the postoperative course, a differentiated mathematical model was used to assess predictive values and likelihood ratios (LR) of the PaGIA at three different phases of hospitalization. Calculating LR revealed -LR to be 0.07 at baseline, 0.09 during postoperative days 5-9, and 0.19 during postoperative days 10-18, while +LR were 5.9 at baseline, 3.0 during postoperative days 5-9, and 1.4 during postoperative days 10-18. Thus, in cardiac surgery patients, the PaGIA can be regarded as a useful tool in excluding rapid onset and typical onset of HIT, and further provides valuable information in the confirmation of rapid onset of HIT. Using a differentiated approach for calculation of predictive values and LR, the present study demonstrates that PaGIA, despite a lower specificity than the employed ELISA, in some situations is a valuable tool when diagnosing HIT in cardiac surgery patients.     

Antibodies to platelet factor 4-heparin after cardiopulmonary bypass in patients anticoagulated with unfractionated heparin or a low-molecular-weight heparin : clinical implications for heparin-induced thrombocytopenia.

BACKGROUND: Cardiopulmonary bypass (CPB) induces platelet activation with release of platelet factor 4 (PF4), and patients are exposed to high doses of heparin (H). We investigated whether this contributes to the development of antibodies to H-PF4 and heparin-induced thrombocytopenia (HIT). METHODS AND RESULTS: CPB was performed with unfractionated heparin (UFH) in 328 patients. After surgery, patients received UFH (calcium heparin, 200 IU. kg-1. d-1) (group 1, n=157) or low-molecular-weight heparin (LMWH, Dalteparin, 5000 IU once daily) (group 2, n=171). Eight days after surgery, antibodies to H-PF4 were present in 83 patients (25.3%), 46 in group 1 and 37 in group 2 (P=0.12). Most patients (61%) had IgG1 to H-PF4, but only 8 samples with antibodies induced platelet activation with positive results on serotonin release assay. HIT occurred in 6 patients in group 1, but no thrombocytopenia was observed in subjects receiving LMWH, although 2 had high levels of antibodies with positive serotonin release assay results. When antibodies to H-PF4 were present, mean platelet counts were lower only in patients with FcgammaRIIA R/R131 platelets. CONCLUSIONS: These results provide evidence that the development of antibodies to H-PF4 after CPB performed with UFH is not influenced by the postoperative heparin treatment. The antibodies associated with high risk of HIT are mainly IgG1, which is present at high titers in the plasma of patients continuously treated with UFH.     

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

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 = 137) were assigned to one of three groups based upon the initial negative test optical density (OD) range of low = 0-0.132, medium = 0.133-0.267, and high = 0.268-0.399. 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.     

Beneficial effect of exogenous platelet factor 4 for detecting pathogenic heparin‐induced thrombocytopenia antibodies

The laboratory diagnosis of heparin‐induced thrombocytopenia (HIT) is based on an enzyme immunoassay combined with a functional test, and serotonin release assay (SRA) is the gold standard for detecting activating HIT antibodies. However, a recent atypical history of HIT prompted us to evaluate whether addition of platelet factor 4 (PF4) during SRA could improve its ability to detect pathogenic HIT antibodies. Using 5B9, a monoclonal antibody to PF4/H with a human Fc fragment, we first defined the optimal PF4 concentration for detecting low amounts of platelet‐activating IgG with SRA. Plasma samples from 50 patients with suspected HIT were then studied, and SRA was positive in 17 cases (Group SRA^pos), with relatively high levels of PF4‐specific IgG (median optical density = 2·66). SRA was also systematically performed after adding 10 μg/ml of PF4 in the reaction mixture, and significant serotonin release was measured with samples from 9 additional patients (Group PF4‐SRA^pos). Importantly, levels of PF4‐specific IgG were similar in these samples and those from the 24 persistently SRA negative patients. Moreover, the pre‐test probability of HIT was intermediate/high in all ‘SRA^pos’ or ‘SRA‐PF4^pos’ patients. In conclusion, addition of exogenous PF4 might improve the detection of pathogenic HIT antibodies by SRA.     

New platelet functional method for identification of pathogenic antibodies in HIT patients

Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy. The most used functional method for HIT diagnosis is serotonin release assay (SRA). A different functional method based on ATP release with luciferin/luciferase long-life and stable luminescent signal is used here, which is shown to be comparable for accuracy with SRA in both negative (patients 4Ts ≤3, and negative for both anti-PF4/heparin immunoassay and SRA) and positive (4Ts >3, and positive for both PF4/heparin antibodies and SRA) patients.

Our results show that ATP release is higher in washed platelets activated by sera from positive patients than in platelets activated by sera from negative patients.

In conclusion, we demonstrate that ATP release assay is a valid alternative method to SRA for the identification of pathogenic anti-PF4/heparin antibodies.     

Differences in specificity of heparin-dependent antibodies developed in heparin-induced thrombocytopenia and consequences on cross-reactivity with danaparoid sodium

Heparin-induced thrombocytopenia (HIT) is frequently associated with antibodies (Abs) to heparin–PF4 complexes (H-PF4). In order to investigate whether there are variations in specificity of Abs, we studied 63 samples from patients with suspected HIT. Two groups of samples were separated after comparing their reactivity against H-PF4 or recombinant PF4 (r-PF4) using ELISA. In group Ab1 ( n  = 46), Abs only or mainly bound to H-PF4 complexes and thus most of the epitopes recognized probably involved both heparin and PF4. In group Ab2 ( n  = 17), Abs exhibited similar reactivity to r-PF4 and H-PF4, and the antigens recognized were possibly neoepitopes mainly expressed by modified PF4 and by H-PF4 complexes. Platelet activation tests were positive with 56 samples containing high titres of Abs to H-PF4. Most samples ( n  = 59) contained IgG antibodies, often associated with IgA antibodies which were more frequently found in group Ab2, and/or IgM. With unfractionated heparin treatment, HIT was associated with Ab1 or Ab2 antibodies, whereas only Ab1 antibodies were detected after low-molecular-weight heparin (LMWH). Furthermore, cross-reactivity with danaparoid sodium was present only in group Ab1 and mainly involved LMWH-treated patients.     

Computerised risk scores to guide recognition and diagnosis in patients with possible heparin‐induced thrombocytopenia

The heparin‐induced thrombocytopenia computerised risk (HIT‐CR) score is designed to aid in the diagnosis of HIT. We sought to evaluate its potential clinical utility. In this retrospective cohort study, we collected HIT‐CR scores on all inpatients receiving heparin over a 4‐month period and performed chart reviews on the subset who independently underwent clinical diagnostic testing for HIT to identify patients with HIT. In all, 34 342 patients received heparin, 1744 had high‐risk HIT‐CR scores of ≥3 and 220 had the maximal risk score of 4. Only 6% of high‐risk and 10% of maximal‐risk patients underwent testing for HIT. Conversely, among all 317 patients who underwent independent testing for HIT, 67% had low‐risk HIT‐CR scores (<3). Among patients independently tested, the positive predictive value (PPV) was 6·6% [95% confidence interval (CI) 4·9–8·8%] and the negative predictive value (NPV) was 99·5% (95% CI 97·1–99·9%) at a HIT‐CR score cut‐off of 3, and the PPV was 22·7% (95% CI 12·7–37·4%) and NPV was 99·0% (95% CI 97·6–99·6%) at a cut‐off of 4. This study suggests clinicians fail to test most high‐risk patients and unnecessarily test many low‐risk patients for HIT. A reasonable approach to clinical application of HIT‐CR scores would be recommending no testing for patients with a score of <3 and recommend testing for patients with a score of 4.     

Heparin-induced multiple electrode aggregometry is a promising and useful functional tool for heparin-induced thrombocytopenia diagnosis: Confirmation in a prospective study

Heparin-induced thrombocytopenia (HIT) is a potentially lethal adverse effect of heparin therapy. Accurate and rapid HIT laboratory diagnosis when HIT is suspected is crucial. The combination of an immunological assay with a functional test improves the accuracy of HIT, but functional assays are currently limited to a few laboratories. Multiplate® analyzer (Dynabyte, Munich, Germany) is a practical, semi-automated and easy-to-perform platelet aggregation assay. The aim of this study is to explore whether heparin-induced platelet aggregation in whole blood assessed by Multiplate® (Heparin-induced multiple electrode aggregometry, HIMEA) can replace platelet aggregation test (PAT) in platelet-rich plasma. For this purpose, HIMEA performance in HIT diagnosis was prospectively evaluated. HIMEA and PAT were compared to serotonin-release assay (SRA) in 200 well-characterized consecutive patients suspected for HIT. HIMEA was found to be more sensitive (81% vs. 76%) and more specific (99% vs. 96%) than PAT compared to SRA. Both tests showed a high negative predictive value while HIMEA had a better positive predictive value. HIMEA has overall better performance characteristics than PAT for the detection of HIT platelet-activating antibodies. The combination of an immunological assay with HIMEA could be a feasible option in non-specialized laboratories for HIT diagnosis optimization.     

Improving the specificity of the PF4 ELISA in diagnosing heparin-induced thrombocytopenia

Heparin induced thrombocytopenia (HIT) is a serious complication of heparin therapy. The PF4 ELISA is a serologic assay that provides laboratory support for the clinical diagnosis of HIT, but it is often positive in patients who do not have the syndrome. We examined whether the specificity of the PF4 ELISA can be improved by 1) taking antibody potency into consideration, 2) by measuring only IgG antibodies, and 3) by utilizing a high concentration heparin inhibition step. We reviewed clinical information on 116 patients whose samples were referred for HIT antibody testing and assigned each a clinical score related to the likelihood of the patient having HIT. The scores were then correlated with serologic findings. Patients with strongly positive PF4ELISA results (OD ≥ 1.0) using both versions of the assay (IgG/A/M and IgG only) had clinical scores and SRA activity that were significantly higher than those having reactive or negative results. When the IgG-only PF4 ELISA was used, only the strongly positive result group had significantly higher clinical scores and SRA release, and fewer samples were classified as weakly positive or reactive, suggesting that detection of IgG only in the PF4 ELISA improves the assay's specificity. The heparin inhibition step identified "reactive" samples that were associated with clinical scores and SRA release indistinguishable from the "negative" result groups, confirming that this step further improves specificity of the test. This study supports utilizing these 3 modifications of the PF4 ELISA to improve specificity in supporting the clinical diagnosis of HIT.     

Generation of antibodies to heparin-PF4 complexes without thrombocytopenia in patients treated with unfractionated or low-molecular-weight heparin

The incidence of antibodies to heparin-PF4 complexes (H-PF4) has been evaluated in patients who were under heparin therapy for more than 7 days: 109 patients treated with unfractionated heparin (UH) and 100 patients with low-molecular-weight heparin (LMWH). The presence of antibodies was identified in 17% of the former group and 8% of the latter. In both the UH and the LMWH groups, IgM antibodies were found in all but four patients who showed IgA antibodies. IgG isotypes were only detected in five patients and were consistently associated to either IgM or IgA antibodies. The follow-up of H-PF4 antibodies in 76 patients treated with UH from 1 to ≥ 12 days showed a relationship between the incidence of antibodies and the duration of therapy. Despite the presence of anti-H-PF4 antibodies there was no thrombocytopenia (<150 10⁹/L) in the patients. A significant drop of platelets requiring the discontinuation of heparin was observed, however, in three patients, but their platelet count consistently remained >150 10⁹/L. Our study demonstrates that the induction of antibodies to H-PF4 is a frequent phenomenon in patients treated with UH or with LMWH. The absence of thrombocytopenia and of clinical complications in these patients demonstrates that other conditions must be associated with H-PF4 antibodies for inducing type II HIT: optimal concentrations of heparin and PF4 in the blood circulation to allow the formation of macromolecular H-PF4 complexes, presence of activated platelets that present an increased binding of H-PF4 complexes, increased expression of FcγRIIA receptors, or presence of their H 131 phenotype. We conclude that the measurement of antibodies to H-PF4 complexes allows the detection of heparin-treated patients at risk of developing type II HIT. © 1996 Wiley-Liss, Inc.     

Heparin-induced thrombocytopenia: an improved method of detection based on lumi-aggregometry

Summary Heparin-induced thrombocytopenia (HIT) is a recognized complication of heparin administration. Early detection of this syndrome is essential in the prevention of immune-mediated thromboembolic sequelae. The ¹⁴C-sero-tonin release assay (SRA) has been used in reference laboratories to identify sera from patients on heparin therapy capable of inducing platelet dense granule release. In an attempt to improve existing methodologies, we employed luminographic detection of platelet-dense granule ATP release as an endpoint of HIT antibody-mediated platelet activation. Sera tested included 10 SRA confirmed positive and five SRA confirmed negative samples (to establish the assay), five samples from patients with thrombocytopenia not on heparin therapy and 34 patients suspected of HIT syndrome. All SRA confirmed positive sera ( n = 19) were positive by the luminographic procedure. 24/26 SRA confirmed negative sera and five sera from thrombocytopenic patients not on heparin therapy were negative using luminography. Two of four sera yielding equivocal SRA results were found to be positive by the luminographic technique. The data suggest that the use of a lumi-aggregometer in the coagulation laboratory to detect HIT antibody-induced platelet activation is a reliable alternative to the SRA. The luminographic procedure is both rapid and sensitive, and does not require the use of biohazardous radio-isotopes.