Heparin-induced Platelet Activation Assay Research Articles

Heparin-activated procoagulant platelet assay: a flow cytometry-based functional test for heparin-induced thrombocytopenia

BackgroundFunctional platelet activation assays are required for the diagnosis of heparin-induced thrombocytopenia (HIT). Due to their sophisticated methodology, they are only available in reference centers. ObjectivesTo evaluate the diagnostic accuracy of the flow cytometry-based heparin-activated procoagulant platelet (HAPP) assay in the laboratory diagnosis of HIT. MethodsProcoagulant platelets (PCP), defined by the expression of phosphatidylserine and CD62-P, were evaluated by flow cytometry in platelet-rich plasma from healthy donors after incubation with patient sera in the absence and presence of heparin. A sample was considered positive in HAPP assay, if the following 3 criteria were met: 1) the percentage of PCPs was ≥10.3% after incubation with 0.2 IU/mL heparin, 2) the fold increase in presence of 0.2 IU/mL heparin compared with buffer was ≥1.5, and 3) 100 IU/mL of heparin resulted in ≥50% inhibition of PCP. HAPP assay was validated in a prospective cohort (n = 202) of consecutive specimens submitted to our laboratory for serologic diagnosis of HIT. Heparin-induced platelet activation (HIPA) assay was used as the reference standard. ResultsHIT-positive sera induced PCPs in the presence of 0.2 IU/mL heparin, which was inhibited with 100 IU/mL of heparin. In the prospective validation cohort, there were 15 HIPA+ and 187 HIPA- sera. HAPP was positive in 20 samples in this cohort. Using optimized cut-offs, HAPP assay had a sensitivity of 93.3% and specificity of 96.8%. ConclusionHAPP assay is promising as a simple and reliable functional assay for HIT; however, further studies are needed to confirm our results in larger cohorts.

Limited concordance of heparin/platelet factor 4 antibody assays for the diagnosis of heparin-induced thrombocytopenia: an analysis of the TORADI-HIT study

BackgroundAnecdotal reports suggest that the correlation between heparin/platelet factor 4 (PF4) antibody assays for the diagnosis of heparin-induced thrombocytopenia (HIT) is limited. ObjectivesTo investigate the correlation between widely used assays and examine possible factors contributing to variability. MethodsThis is a large, prospective cohort study with 10 participating tertiary care hospitals including 1393 patients with suspected HIT in clinical practice. HIT was defined by a positive heparin-induced platelet activation (HIPA) assay (washed platelet reference standard test). Three different immunoassays were used to measure heparin/PF4 antibodies: chemiluminescent immunoassay, enzyme-linked immunosorbent assay, and particle gel immunoassay. Various factors that could influence the assays were examined: sex (male or female), age (<65 years or ≥65 years), unfractionated heparin exposure, presence of thrombosis, cardiovascular surgery, and intensive care unit. Spearman’s correlation coefficients were calculated. Z-scores and diagnostic odds ratios were determined in the aforementioned subgroups of patients. ResultsAmong 1393 patients, 119 were classified as HIT-positive (prevalence, 8.5%). The median 4Ts score was 5 (IQR, 4-6) in patients with HIT compared with 3 (IQR, 2-4) in patients without HIT. Correlations (rs) between immunoassays were weak (0.53-0.65). Inconsistencies between immunoassays could not be explained by further analyses of z-scored test results and diagnostic odds ratios in subgroups of patients. ConclusionThe correlation between widely used heparin/PF4 antibody assays was weak, and key factors could not explain this variability. Standardization of immunoassays is requested to improve comparability.

A machine-learning model for reducing misdiagnosis in heparin-induced thrombocytopenia: A prospective, multicenter, observational study.

Diagnosing heparin-induced thrombocytopenia (HIT) at the bedside remains challenging, exposing a significant number of patients at risk of delayed diagnosis or overtreatment. We hypothesized that machine-learning algorithms could be utilized to develop a more accurate and user-friendly diagnostic tool that integrates diverse clinical and laboratory information and accounts for complex interactions. We conducted a prospective cohort study including 1393 patients with suspected HIT between 2018 and 2021 from 10 study centers. Detailed clinical information and laboratory data were collected, and various immunoassays were conducted. The washed platelet heparin-induced platelet activation assay (HIPA) served as the reference standard. HIPA diagnosed HIT in 119 patients (prevalence 8.5%). The feature selection process in the training dataset (75% of patients) yielded the following predictor variables: (1) immunoassay test result, (2) platelet nadir, (3) unfractionated heparin use, (4) CRP, (5) timing of thrombocytopenia, and (6) other causes of thrombocytopenia. The best performing models were a support vector machine in case of the chemiluminescent immunoassay (CLIA) and the ELISA, as well as a gradient boosting machine in particle-gel immunoassay (PaGIA). In the validation dataset (25% of patients), the AUROC of all models was 0.99 (95% CI: 0.97, 1.00). Compared to the currently recommended diagnostic algorithm (4Ts score, immunoassay), the numbers of false-negative patients were reduced from 12 to 6 (-50.0%; ELISA), 9 to 3 (-66.7%, PaGIA) and 14 to 5 (-64.3%; CLIA). The numbers of false-positive individuals were reduced from 87 to 61 (-29.8%; ELISA), 200 to 63 (-68.5%; PaGIA) and increased from 50 to 63 (+29.0%) for the CLIA. Our user-friendly machine-learning algorithm for the diagnosis of HIT (https://toradi-hit.org) was substantially more accurate than the currently recommended diagnostic algorithm. It has the potential to reduce delayed diagnosis and overtreatment in clinical practice. Future studies shall validate this model in wider settings. Swiss National Science Foundation (SNSF), and International Society on Thrombosis and Haemostasis (ISTH).

Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies

AbstractHeparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

Organ Donation From a Brain Dead Donor With Vaccine-induced Immune Thrombotic Thrombocytopenia After Ad26.COV2.S: The Risk of Organ Microthrombi.

Organ Donation From a Brain Dead Donor With Vaccine-induced Immune Thrombotic Thrombocytopenia After Ad26.COV2.S: The Risk of Organ Microthrombi.

Multicentre evaluation of 5B9, a monoclonal anti‐PF4/heparin IgG mimicking human HIT antibodies, as an internal quality control in HIT functional assays: Communication from the ISTH SSC Subcommittee on Platelet Immunology

BackgroundFunctional tests for the diagnosis of heparin‐induced thrombocytopenia (HIT) exhibit variable performance. ObjectivesWe evaluated in a multicenter study whether 5B9, a monoclonal anti‐PF4/heparin IgG mimicking human HIT antibodies, could be used as an internal quality control. Methods5B9 was sent to 11 laboratories in seven countries, and six initial concentrations ranging from 10 to 400 μg/mL were tested by heparin‐induced platelet activation assay (HIPA), serotonin release assay (SRA), platelet aggregation test (PAT), flow cytometry (FC), or heparin‐induced multiple‐electrode aggregometry (HIMEA). Each method was evaluated in three different laboratories using experimental procedures identical to those usually applied for the diagnosis of HIT by testing platelets from 10 different healthy donors. ResultsThe procedures used varied among the laboratories, particularly when platelet‐rich plasma and whole blood were used. Nevertheless, positive results were obtained with at least 100 μg/ml of 5B9 for most donors tested by all centers (except one) performing HIPA, SRA, or HIMEA. FC and PAT results were more heterogeneous. FC results from one center that used washed platelets preincubated with PF4 were positive with all donors at 50 µg/ml 5B9, but at least 200 μg/ml of 5B9 were required to activate cells with most donors tested using PAT. ConclusionThis study confirms that HIT functional tests are not well standardized and exhibit variable sensitivity for the detection of platelet‐activating antibodies. However, 5B9 is a potentially useful tool to standardize functional tests, to select responding platelet donors, and consequently to improve the performance of these assays and comparability between laboratories.

Near fatal stent thrombosis in an aneurysmatic RCX as first manifestation of heparin induced thrombocytopenia (HIT) without thrombocytopenia

BackgroundThrombosis resulting from heparin-induced thrombocytopenia (HIT) occurs in about 2% of patients without a significant decrease in platelet counts. We report on such a near fatal thrombotic event caused by coronary intervention.Case presentationA supposedly “completely healthy” 53-year-old patient was admitted to hospital with covered rupture of an aneurysm of the Aorta descendens. He was successfully operated on and underwent coronary angiography due to NSTEMI six days later. Immediately after intervention of a 90% RCX stenosis he developed ventricular flutter, was defibrillated, and re-angiography showed partial occlusion of the RCX stent. Lots of white thrombi could be retrieved by aspiration catheter and gave reason for a HIT without thrombocytopenia. The detection of platelet factor 4/heparin complex antibodies by immunoassay supported and the subsequent Heparin Induced Platelet Activation Assay proved this diagnosis.ConclusionsThe clinical event of an acute stent thrombosis should alarm the interventional team to the diagnosis of HIT even with a normal platelet count.

Heparin-induced Thrombocytopenia Diagnosis: A Retrospective Study Comparing Heparin-induced Platelet Activation Test to 14 C-serotonin Release Assay.

Laboratory confirmation of heparin-induced thrombocytopenia (HIT) is of crucial importance and remains challenging and relies on platelet functional assays highlighting the presence of heparin-dependent platelet-activating antibodies in patient serum or plasma. Platelet functional assays using washed platelets include the 14 C-serotonin release assay (SRA), usually described as the gold standard, and the heparin-induced platelet activation assay (HIPA). Since its first comparison with SRA there has been no additional published study regarding HIPA diagnostic performances compared with SRA. Aim of our retrospective study was to compare the concordance between HIPA and SRA in HIT suspected-patients with positive anti-PF4/heparin antibodies between October 2010 and October 2015. Fifty-five HIT-suspected patients who beneficiated from both HIPA and SRA were included. Positive and negative percent agreements were 83.8% (95% CI 68.0–93.8%) and 66.7% (95% CI 41.0–86.7%), respectively. Overall percent agreement was 78.2% (95% CI 65.0–92.2%). Agreement was higher in patients who underwent cardiopulmonary bypass with extracorporeal circulation circuit for cardiac surgery. We also confirm that the use of a minimum of 2 platelet donors to establish positive HIT diagnosis and 4 platelet donors to exclude HIT diagnosis allows obtaining a good agreement with SRA. Although HIPA and SRA were performed with different platelet donors and in different laboratories, HIPA had a good positive agreement with SRA for HIT diagnosis, showing that HIPA is a useful functional assay that does not require radioactivity and could be developed worldwide to improve HIT diagnosis.

Challenges in Detecting Clinically Relevant Heparin-Induced Thrombocytopenia Antibodies.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated hypercoagulable state featuring high thrombosis risk and distinct pathogenesis involving immunoglobulin G-mediated platelet activation. The target of the immune response is a cationic “self” protein, platelet factor 4 (PF4), rendered antigenic by heparin. A key problem is that only a minority of anti-PF4/polyanion antibodies induced by heparin are pathogenic, i.e., capable of causing platelet activation and thereby clinical HIT. Since thrombocytopenia occurs frequently in hospitalized, heparin-treated patients, testing for “HIT antibodies” is common; thus, the problem of distinguishing between pathogenic and nonpathogenic antibodies is important. The central concept is that those antibodies that have platelet-activating properties demonstrable in vitro correlate well with pathogenicity, as shown by platelet activation tests such as the serotonin-release assay (SRA) and heparin-induced platelet activation assay. However, in most circumstances, immunoassays are used for first-line testing, and so it is important for clinicians to appreciate which immunoassay result profiles—in the appropriate clinical context—predict the presence of platelet-activating antibodies (Bayesian analysis). Clinicians with access to rapid, on-demand HIT immunoassays (e.g., particle gel immunoassay, latex immunoturbidimetric assay, chemiluminescent immunoassay) can look beyond simple dichotomous result interpretation (“negative”/“positive”) and incorporate semiquantitative interpretation, where, for example, a strong-positive immunoassay result (or even combination of two immunoassays) points to a greater probability of detecting platelet-activating antibodies, and hence supporting a diagnosis of HIT. Recent recognition of “SRA-negative HIT” has increased the importance of semiquantitative interpretation of immunoassays, given that strong immunoassay reactivity is a potential clue indicating possible HIT despite a (false) negative platelet activation assay.

Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is typically caused by platelet-activating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin (H). Much less frequent "autoimmune" HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs. To characterize 1E12, 1C12, and 2E1 in comparison to the heparin-dependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3). Interactions of Abs with PF4 and PF4/H were studied by enzyme-linked-immunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method). 1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin. 1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs.

Heparin-induced thrombocytopenia: Diagnostic challenges in intensive care patients especially with extracorporeal circulation

BackgroundHeparin-induced thrombocytopenia (HIT) is a serious drug induced reaction that may be associated with life threatening complications. Platelet-activating antibodies directed against platelet factor 4 (PF4)/heparin complexes cause the disease. The diagnosis of HIT is challenging, as thrombocytopenia is a frequent finding in intensive care (ICU) patient population, especially during extracorporeal membrane oxygenation. ObjectiveTo investigate the performance of a diagnostic algorithm for HIT in ICU patients. MethodsICU patients who developed thrombocytopenia or thrombosis under heparin treatment were included in this study. The pretest probability for HIT was estimated using the 4Ts-score and patient's sera were tested using two rapid immunoassays (RA) LFI-HIT and PaGIA (from Milenia Biotec and DiaMed), and within 72 h using the IgG enzyme immunoassay (EIA) from Hyphen and the heparin induced platelet activation assay (HIPA). Results392 consecutive ICU patients with suspected HIT were enrolled in this study, of whom 83/392 (21.2%) patients had extracorporeal circulation. Sera from 120/392 (30.6%) and 98/392 (25.0%) patients revealed positive results in RA and IgG EIA, respectively. The HIPA test revealed heparin-dependent platelet activation in a total of 15/392 (3.8%) ICU patients (3 medical and 12 surgical patients). In addition, sera from 7 patients revealed indeterminate HIPA results, of whom 2 patients had a clinical course compatible with HIT. ConclusionsData from our study confirm the high frequency of IgG PF4/heparin antibodies in ICU patients under unfractionated heparin and shows that the combination of 4Ts-score and RA does not reduce the laboratory overinvestigation for HIT in these patients.

Progress in laboratory diagnosis of heparin induced thrombocytopenia

Heparin induced thrombocytopenia (HIT) is a severe side effect of heparin with antibody-mediation. Laboratory assays can be divided into two major categories, about functional assays and HIT antibodiesdetection. Thefunctional assays, such as the serotonin release assay (SRA) and heparin-induced platelet activationassay(HIPA), are sensitive and specific for HIT. They arethe reference standard assays generally, but have thedeficiencies of complicated operation and time-consuming, and cannot be used as a routine examination. TheHIT antibodiesdetections, such as ELISA, immune turbidimetry assay, chemiluminescent assay and lateral flow immunoassay, have high diagnostic sensitivityandareavailable at routine laboratories.They can exclude the diagnosis of HIT or beused to diagnose HIT effectively combined with the pre-test probability score(4Ts score) of HIT.(Chin J Lab Med, 2016, 39: 795-800) Key words: Thrombocytopenia; Heparin; Autoantibodies; Serotonin; Platelet activation; Enzyme-linked immunosorbent assay; Nephelometry and turbidimetry; Immunoassay

Clinical and laboratory tests for the diagnosis of heparin-induced thrombocytopenia.

A rapid diagnostic work-up is required in patients with suspected heparin-induced thrombocytopenia (HIT). However, diagnosis of HIT is challenging due to a number of practical issues and methodological limitations. Many laboratory tests and a few clinical scoring systems are available but the individual characteristics and the diagnostic accuracy of these are hard to appraise. The 4Ts score is a well evaluated clinical assessment tool with the potential to rule out HIT in many patients. Still, it requires experience and is subject to a relevant inter-observer variability. Immunoassays such as enzyme-linked immunosorbent assays or recently developed rapid assays are able to exclude HIT in a number of patients. But, accuracy of immunoassays differs depending on type of assay, threshold, antibody specificity and even manufacturer. Due to a comparatively low positive predictive value, HIT cannot be confirmed by immunoassays alone. In addition, only some of them are immediately accessible, particularly in small laboratories. While functional assays such as the serotonin release assay (SRA) and the heparin-induced platelet activation assay (HIPA) are considered as gold standard for diagnosis of HIT, they require a highly specialised laboratory. In addition, some of them are not adequately evaluated. In clinical practice, we recommend an integrated diagnostic approach combining not only clinical assessment (the 4Ts score) but immunoassays and functional assays as well. We propose a clear diagnostic algorithm supporting clinical decision-making. Furthermore, we provide an overview of all current laboratory techniques for HIT and discuss diagnostic pathways and strategies to reduce diagnostic errors, and future perspectives.

Successful intraprocedural anticoagulation with bivalirudin during pulmonary vein isolation in a patient with known heparin-induced thrombocytopenia type II

We report the case of a 56-year-old female who presented with symptomatic paroxysmal atrial fibrillation. Anamnestic heparin-induced thrombocytopenia (HIT) type II was suspected, and a rapid diagnostic test showed antibodies against platelet factor 4. The heparin-induced platelet activation-assay was negative. Radiofrequency pulmonary vein isolation with intraprocedural anticoagulation using bivalirudin was ultimately performed. Dosing was controlled by monitoring the activated clotting time. Post-procedural blood tests were normal. There were no thromboembolic or bleeding events. Bivalirudin is a therapeutic option for anticoagulation during pulmonary vein isolation procedures in patients with a history of HIT type II.

Platelet-activating protamine-heparin-antibodies lead to higher protamine demand in patients undergoing cardiac surgery

ObjectivesPlatelet-activating antibodies against protamine-heparin-complexes were described in patients undergoing cardiac surgery, but their clinical consequences remain unclear. This prospective single-center observational study aimed to describe the prevalence and clinical consequences of protamine-heparin-complex antibodies in patients undergoing cardiac surgery with cardiopulmonary bypass. MethodsA total of 200 patients undergoing cardiac surgery with cardiopulmonary bypass were included. Blood samples were collected preoperatively and 1 hour, 24 hours, and 7 days after weaning from cardiopulmonary bypass. All sera were tested for the presence of protamine-heparin-complex antibodies using a modified heparin-induced platelet-activation assay. Specific Fcγ receptor IIa–dependent platelet activation was confirmed by repeated testing in the presence of the Fcγ receptor IIa–blocking antibody IV.3. ResultsSamples from 185 patients were obtained, of whom 24 patients (13%) were positive for protamine-heparin-complex antibodies preoperatively. In all positive samples, functional reactivity was reversible in the presence of IV.3. Although patients with a preoperative presence of protamine-heparin-complex antibodies were significantly older compared with patients negative for protamine-heparin-complex antibodies (73 ± 9.8 years vs 68 ± 10 years, P = .037), no other potential risk factors were identified at 1 day before operation. Patients with protamine-heparin-complex antibodies required significantly more protamine to neutralize heparin (47.66 mg vs 41.67 mg, P = .027). Protamine-heparin-complex antibodies have no significant influence on perioperative platelet numbers, bleeding complications, transfusion requirement, thromboembolic events, major cardiovascular and cerebrovascular events, inflammation parameters, or kidney function. ConclusionsProtamine-heparin-complex antibodies occur frequently in patients undergoing cardiac surgery on cardiopulmonary bypass, resulting in specific platelet activation in vitro. Protamine-heparin-complex antibodies are associated with increased protamine requirement after cardiopulmonary bypass and possibly slower recovery of platelet numbers.

Frequent off-label use of fondaparinux in patients with suspected acute heparin-induced thrombocytopenia (HIT) – findings from the GerHIT multi-centre registry study

IntroductionIn life-threatening immune heparin-induced thrombocytopenia (HIT), treatment with an approved non-heparin anticoagulant is essential. However, off-label use with fondaparinux has been reported in the literature. The study aim was to collect data on “real-life” management of patients with suspected acute HIT regarding diagnostic and therapeutic strategies. Patients and MethodsIn a national multi-centre registry study, patients with a 4T’s HIT-probability score of ≥4 points and treatment with at least one dose of (A)rgatroban, (L)epirudin, (D)anaparoid, or (F)ondaparinux were retrospectively evaluated. ResultsOf 195 patients, the 4T’s scores were 4/5/6/7/8 points in 46 (23.6%)/50 (25.6%)/74 (38.0%)/13 (6.7%)/7 (3.6%) patients, respectively. During heparin therapy, 47 (24.1%) thromboembolic events, 5 (2.6%) skin lesions, 1 (0.5%) amputation, 24 (12.3%) Hb-relevant bleedings, and 2 (1.0%) fatalities occurred. A functional heparin-induced platelet activation assay was performed in 96.9%, a platelet factor 4/heparin-dependent enzyme immunoassay in 89.2%, a particle gel immunoassay in 12.3%, and a serotonin-release assay in none of the patients. Argatroban was used in 16.4%, lepirudin in 2.1%, danaparoid in 23.6%, fondaparinux in 40.0% of the patients; the sequential therapy strata were: AF (5.6%), DA (5.6%), DF (2.6%), DL (2.1%), ADF (1.5%), and DFL (0.5%). ConclusionsThe current diagnostic laboratory strategy for suspected HIT is mostly (>96%) based on the recommended 2-step strategy (immunoassay plus functional assay). However, there is a wide fondaparinux off-label use (up to 50.3%) for suspected HIT, even in those patients with a high clinical pretest probability. Efficacy and safety of fondaparinux for HIT-treatment require further evaluation.

Clinical and Laboratory Diagnosis of Heparin-Induced Thrombocytopenia: An Integrated Approach

Heparin-induced thrombocytopenia (HIT) is a clinicopathologic disorder that predisposes to thrombosis. Diagnosis rests on a compatible clinical picture and laboratory evidence of antiplatelet factor 4 (PF4)/heparin antibodies that activate platelets in a heparin-dependent manner. Rapid and accurate diagnosis is paramount to avoid the perils of misdiagnosis. Clinical evaluation may be guided by scoring systems such as the 4Ts and HIT Expert Probability (HEP) score. Laboratory tests include immunoassays, such as the PF4/heparin enzyme-linked immunosorbent assay (ELISA) and functional tests such as the 14C-serotonin release assay and heparin-induced platelet activation assay. Clinical scoring systems and commercially available immunoassays have high sensitivity but modest specificity. Functional assays are more specific, but they are technically demanding. Novel laboratory assays with faster turnaround times, greater specificity, and lesser technical complexity are in development. A Bayesian approach that combines the 4T score and the PF4/heparin ELISA result may be used to estimate the probability of HIT and guide clinical decision making.

Heparin-induced thrombocytopenia associated with thrombotic microangiopathy

Some cases of thrombotic microangiopathy (TMA) are refractory to plasma exchange therapy (PE) with persistence or recurrence of thrombocytopenia. We report two patients suffering from TMA of different aetiologies (associated with disseminated malignancy, typical haemolytic uraemic syndrome) with recurrent or persistent thrombocytopenia despite adequate therapy including PE. Since both patients were exposed to unfractionated heparin, heparin-induced thrombocytopenia (HIT) was suspected as a cause. Pretest probabilities for HIT were intermediate. ELISA for PF4/heparin antibodies was strongly positive in both cases, and HIT was confirmed by heparin-induced platelet activation assay. Anticoagulation with lepirudin was initiated, with subsequent rapid increase of the platelet count. TMA might represent a predisposition for HIT. This could be due to TMA-related platelet activation with increased PF4 release. In TMA patients exposed to heparin and with refractory or rapidly recurrent thrombocytopenia HIT should always be considered as a possible cause.

Single Center Experience with the Nanoparticle-Based Flow Immunoassay for Diagnosis of Heparin-Induced Thrombocytopenia (HIT).

Abstract 2189Heparin-induced thrombocytopenia (HIT), caused by antibodies against heparin/platelet factor 4 (HPF4) complex, is a rare but potentially serious side effect of heparin therapy where due to high mortality, rapid diagnosis is crucial. For the detection of HPF4 antibodies we compared the new nanoparticle-based lateral-flow immunoassay (LFI-HIT, Milenia Biotec, Germany) and a particle gel immunoassay (PaGIA, BioRad, Germany) with an IgG-specific-PF4/polyanion enzyme-linked immunosorbent assay (IgG-ELISA, GTI Diagnostics, USA).Sera from 121 patients (54/67 f/m, median 73 years, range 14–94) with suspected HIT were prospectively tested. The LFI-HIT and the PaGIA were evaluated visually, the IgG-ELISA was positive at an optical density (OD) cutoff > 0.4. For most of the positive samples, the functional heparin-induced platelet activation (HIPA) assay was additionally performed to detect false positive serological results and to confirm a clinically relevant HIT by in vitro platelet-activation. Regarding HIT as a clinico-pathological syndrome, characteristics for HIT were evaluated for each patient by the 4Ts scoring system and divided into high, intermediate or low risk.Results of serological analyses and OD values are summarized in the table. Ten of 121 samples were positive in the LFI-HIT, 10/10 positive in the PaGIA and 8/10 positive in the IgG-ELISA. The HIPA was tested in 9/10 samples and was positive in 8/9 samples. Of the 2 samples positive for LFI-HIT and PaGIA but negative in the ELISA, 1 was HIPA positive, 1 HIPA negative, resulting in a specificity of 88.9% for the LFI-HIT assay correlated to the HIPA.From 111/121 LFI-HIT-negative samples, 2 were positive in the PaGIA, the IgG-ELISA (OD 1.318 and 2,019) and in the HIPA. Seven of the 111 LFI-HIT negative samples were positive only in the IgG-ELISA. Due to marginal positive reactions of 5/7 samples in the ELISA with OD values between 0.4 to 0.5, only 2 LIF-HIT negative IgG-ELISA positive samples were tested by HIPA and 1/2 was positive.Based on the ELISA, the sensitivity of the LFI-HIT was 91.9% (102/111 negative samples also negative in the ELISA) in contrast to 93.1% of the PaGIA. The specificity of the LFI-HIT was 80% (LFI-HIT and IgG-ELISA positive), compared to 57.9% of the PaGIA. Notably, the clinical risk estimated by the 4Ts score system (received from 92/121 patients) did not correlate with laboratory diagnosis of HIT, probably due to inadequate evaluation.Concluding our data, a reliable exclusion of HIT by rapid testing with the LFI-HIT only seems possible with additional analysis of HPF4 antibodies by IgG-ELISA and/or HIPA assay.LFI-HITPaGIAIgG-ELISAOD IgG-ELISAHIPA assayMedian (range)Samples n=121Pos 10Pos 10Pos 82.366 (0.902-3.000)7/7 posNeg 20.199 and 0.1701/2 pos, 1/2 negNeg 0----Neg 111Pos 9Pos 21.318 and 2.0192/2 posNeg 70.110 (0.054-0.139)6/6 negNeg 102Pos 70.436 (0.404-1.463)1/2 pos, 1/2 negNeg 950.082 (0.013-0.376) Disclosures:No relevant conflicts of interest to declare.

Guidelines on the diagnosis and management of heparin‐induced thrombocytopenia: second edition

• Patients who are to receive any heparin should have a baseline platelet count (2C). • Post-operative patients including obstetric cases receiving unfractionated heparin (UFH) should have platelet count monitoring performed every 2–3 d from days 4 to 14 or until heparin is stopped (2C). • Post-cardiopulmonary bypass patients receiving low molecular weight heparin (LMWH) should have platelet count monitoring performed every 2–3 d from days 4 to 14 or until heparin is stopped (2C). • Post-operative patients (other than cardiopulmonary bypass patients) receiving LMWH do not need routine platelet monitoring (2C). • Post-operative patients and cardiopulmonary bypass patients who have been exposed to heparin in the previous 100 d and are receiving any type of heparin should have a platelet count determined 24 h after starting heparin (2C). • Medical patients and obstetric patients receiving heparin do not need routine platelet monitoring (2C). • If the platelet count falls by 30% or more and/or the patient develops new thrombosis or skin allergy or any of the other rarer manifestations of heparin-induced thrombocytopenia (HIT) between days 4 and 14 of heparin administration, HIT should be considered and a clinical assessment made (2C). • HIT can be excluded by a low pre-test probability score without the need for laboratory investigation (2B). • If the pre-test probability of HIT is not low, heparin should be stopped and an alternative anticoagulant started in full dosage whilst laboratory tests are performed (1C). • Platelet aggregation assays using platelet-rich plasma (PRP) lack sensitivity and are not recommended (2C). • Platelet activation assays using washed platelets [heparin-induced platelet activation assay (HIPA) and serotonin release assay (SRA)] have a higher sensitivity than platelet aggregation assays using PRP and are regarded as the reference standard, but are technically demanding and their use should be restricted to experienced laboratories (2C). • Non-expert laboratories should use an antigen assay of high sensitivity. Only the IgG class needs to be measured. Useful information is gained by reporting the actual optical density, degree of inhibition by high dose heparin, and the cut-off point for a positive test rather than simply reporting the test as positive or negative (1B). • In making a diagnosis of HIT, the clinician’s estimate of the pre-test probability of HIT, together with the type of assay used and its quantitative result [enzyme-linked immunosorbent assay (ELISA) only] and information on reversal using higher doses of heparin should be used to determine the post-test probability of HIT (2B). • HIT can be excluded in patients with an intermediate pre-test score who have a negative particle gel immunoassay (2B). • HIT can be excluded in all patients by a negative antigen assay of high sensitivity (1A). • Clinical decisions should be made following consideration of the risks and benefits of treatment with an alternative anticoagulant (1C). • For patients with suspected (non-low pre-test probability) or confirmed HIT, heparin should be stopped and full dose anticoagulation with an alternative anticoagulant commenced (1B). • LMWH should not be used in the treatment of HIT (1A). • Warfarin should not be used until the platelet count has recovered to the normal range. When introduced, an alternative anticoagulant must be continued until the International Normalized Ratio (INR) is therapeutic. Argatroban affects the INR and this needs to be considered when using this drug. A minimum overlap of 5 d between non-heparin anticoagulants and vitamin K antagonist (VKA) therapy is recommended (1B). Correspondence: Dr Henry Watson, British Society for Haematology, 100 White Lion Street, London, N1 9PF, UK. E-mail bcsh@b-s-h.org.uk