Laboratory Diagnosis Of Heparin-induced Thrombocytopenia 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.

Optimization of laboratory diagnosis of heparin-induced thrombocytopenia using HemosIL-AcuStar-HIT-IgG assay.

The aim of this study was to determine an optimal cutoff value for the newly available HemosIL-AcuStar-HIT-IgG assay (AcuStar) for the diagnosis of heparin-induced thrombocytopenia (HIT). We evaluated the performance of AcuStar using serotonin release assay (SRA) as the gold standard and incorporated 4T score calculation in a cohort of suspected HIT cases. Statistical analysis was performed to determine optimal cutoff value for the diagnosis of HIT. A diagnosis of HIT can be excluded with a platelet factor 4 (PF4) value of <0.4 U/mL by AcuStar and 4T score in the low-risk category (≤3). All other cases will require confirmation with a functional test. Our study resulted in the implementation of a diagnostic algorithm for laboratory diagnosis of HIT, which incorporates pretest calculation of 4T score and AcuStar as a screening test, with reflex confirmation by SRA. This new algorithm resulted in extended hours of test availability and a more rapid turnaround time in reporting PF4 results.

Laboratory diagnosis of heparin-induced thrombocytopenia: A retrospective experience.

Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy associated with thrombosis that requires a quick diagnosis. Therefore, laboratory assays must provide an accurate and swift answer. This work aims to evaluate the performances of an ELISA assay, especially when combined with 4T risk score, and a functional assay. Data were collected for 894 patients treated by heparin who underwent anticoagulant switch because of HIT suspicion and were examined by a multidisciplinary expert team who confirmed or ruled out HIT diagnosis. All patients were tested for anti-PF4 IgG with Asserachrom HPIA IgG (ELISA), and 307 were tested with a platelet aggregation test done on platelet-rich plasma (PRP-PAT). The 4T risk score was available for 607 of them. HIT was diagnosed in 232 patients. 4T risk score had a 94.2% negative predictive value (NPV) for risk scores ≤3 and 77.3% for risk scores ≤5. The sensitivity of ELISA was 90.9%, its specificity 79.0%, and its NPV 96.1%. When combined with 4T risk score, its NPV reached 100% and 97% for risk scores ≤3 and ≤5, respectively. PRP-PAT sensitivity was 70.4%, and its specificity was 92.3%. Combination of ELISA and PRP-PAT had a 0.7% false-negative rate. This study shows that ELISA can rule out HIT with an excellent NPV, especially when combined with the 4T risk score. Nonetheless, it has low specificity; hence, it needs to be associated with a functional assay.

Application of a clinical decision rule and laboratory assays in pediatrics: Adult heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is rare among pediatric patients. The diagnosis of HIT depends upon clinical decision tools to assess its pretest probability, supported by laboratory evidence of anti-platelet factor 4 (anti-PF4)/heparin antibodies. To compare the use of the 4Ts score clinical decision tool, clinical characteristics, and laboratory findings between pediatric and adult patients with suspected HIT. We compiled all pediatric patients in our center for whom HIT testing was performed during the years 2015-2021. These were compared with a cohort of consecutive adult patients. Laboratory diagnosis of HIT was performed with particle gel immunoassay (PaGIA) as screening test and confirmed by an automated latex-enhanced immunoturbidimetric assay (LIA) and/or by functional flow cytometry assay (FCA). The cohort included 34 children (under 18years) and 105 adults. Adults mostly received heparins for thromboembolism prophylaxis and treatment (72.4%, n=76), and were more frequently treated with low-molecular-weight heparin (LMWH). Children were mostly exposed during cardiopulmonary bypass and extracorporeal membrane oxygenation (ECMO, 61.8%, n=21), and were more frequently treated with unfractionated heparin (UFH). Compared with adults, children had significantly higher 4Ts scores. Nevertheless, adults had a slightly higher rate of a positive diagnosis of HIT. Six out of 16 adults with confirmed HIT presented with thrombosis (37.5%), whereas all three pediatric patients with HIT presented with thrombosis (p= .087). 4Ts scores are higher in children compared with adult patients for whom laboratory tests for HIT were obtained. A potentially higher incidence of thrombosis in children with HIT may be attributable to the severity of underlying illness.

Functional Flow Cytometric Assay for Reliable and Convenient Heparin-Induced Thrombocytopenia Diagnosis in Daily Practice.

Reliable laboratory diagnosis of heparin-induced thrombocytopenia (HIT) remains a major clinical concern. Immunoassays are highly sensitive, while confirmatory functional tests (based on heparin-dependent platelet activation) lack standardization. We evaluated the diagnostic performance of a functional flow cytometric assay (FCA) based on the detection of heparin-dependent platelet activation with an anti-p-selectin. A total of 288 patients were included (131 HIT-positive and 157 HIT-negative) with a HIT diagnosis established by expert opinion adjudication (EOA) considering clinical data and local laboratory results. The FCA was centrally performed in a single laboratory on platelet-rich plasma, using a very simple four-color fluorometer. The results were standardized according to the Heparin Platelet Activation (HEPLA) index. The serotonin release assay (SRA) was performed in the four French reference laboratories. Based on the final HIT diagnosis established by EOA, the sensitivity and specificity of the FCA were 88 and 95%, respectively, values very similar to those of the SRA (88 and 97%, respectively). This study showed that the FCA, based on easily implementable technology, may be routinely used as a reliable confirmatory test for HIT diagnosis.

Optimising the laboratory diagnosis of heparin-induced thrombocytopenia (HIT)

Background: A variety of screening and confirmatory tests are employed for the diagnosis of heparin-induced thrombocytopenia (HIT). To ensure timely diagnosis, accurate and rapid assays are required.

Diagnostic accuracy of IgG‐specific versus polyspecific enzyme‐linked immunoassays in heparin‐induced thrombocytopenia: a systematic review and meta‐analysis

Background There are conflicting data on whether the IgG-specific or polyspecific antiplatelet factor 4/heparin (PF4/H) enzyme-linked immunosorbent assay (ELISA) is preferred for the laboratory diagnosis of heparin-induced thrombocytopenia (HIT). Objectives To directly compare diagnostic accuracy of IgG-specific versus polyspecific ELISA in HIT. Patients/Methods A systematic search yielded nine studies comprising 1948 patients with suspected HIT tested by both IgG-specific and polyspecific ELISAs and a reference standard against which the diagnostic accuracy of the ELISAs could be measured. Study quality was assessed by QUADAS-2 criteria. Results There was identical sensitivity for IgG-specific and polyspecific ELISAs (0.97; 95% confidence interval (CI), 0.95-0.99) and superior specificity of IgG-specific compared with polyspecific ELISA (0.87 [0.85-0.88] vs. 0.82 [0.80-0.84], respectively). Performance was similar in subgroups using the serotonin release assay and a single commercial ELISA manufacturer. The negative predictive values of IgG-specific and polyspecific ELISA were similarly high (0.99, [0.99-1.00], but the positive predictive value was superior with IgG-specific compared with polyspecific ELISA (0.56 [0.52-0.61] vs. 0.32 [0.28-0.35], respectively). The positive likelihood ratio (LR) was higher in IgG-specific than polyspecific ELISA, although negative LRs were similar. There was high risk of quality concerns in domains of index test and reference standard. Conclusions The superior diagnostic accuracy of IgG-specific ELISA reinforces the ISTH-SSC recommendation for standardization of laboratory testing for HIT. Likelihood ratios of individual assays may be used in combination with clinical scoring systems as part of an integrated diagnostic algorithm for HIT.

Current insights into the laboratory diagnosis of HIT

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction and prothrombotic disorder caused by immunization against platelet factor 4 (PF4) after complex formation with heparin or other polyanions. After antibody binding to PF4/heparin complexes, HIT antibodies are capable of intravascular platelet activation by cross-linking Fc gamma receptor IIa (FcγRIIa) on the platelet surface leading to a platelet count decrease and/or thrombosis. In contrast to most other immune-mediated disorders, the currently available laboratory tests for anti-PF4/heparin antibodies show a high sensitivity also for clinically irrelevant antibodies. This makes the diagnosis of HIT challenging and bears the risk to substantially overdiagnose HIT. The strength of the antigen assays for HIT is in ruling out HIT when the test is negative. Functional assays have a higher specificity for clinically relevant antibodies, but they are restricted to specialized laboratories. Currently, a Bayesian approach combining the clinical likelihood estimation for HIT with laboratory tests is the most appropriate approach to diagnose HIT. In this review, we give an overview on currently available diagnostic procedures and discuss their limitations.

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.

Using HitAlert flow cytometry to detect heparin-induced thrombocytopenia antibodies in a tertiary care hospital

We aimed to assess the utility of HitAlert flow cytometry as a diagnostic functional heparin-induced thrombocytopenia (HIT) assay in a tertiary care hospital. The 4Ts score was used to assess pretest probability of HIT in 37 patients. Serum was analysed for HIT antibodies by the flow cytometry HitAlert assay. Results were compared with an antigenic assay, the particle gel immunoassay, PaGIA ID PF4/Hep Ab assay; and two functional assays, the Multiplate whole blood impedance aggregometry assay (WBIA), and the serotonin release assay (SRA). Flow cytometry was positive in 14 out of 37 patients, including zero out of eight, five out of 19 and nine out of 10 in the low, intermediate and high-risk groups by 4Ts score, respectively. Using the SRA as a 'gold standard', flow cytometry has a sensitivity of 81% and a specificity of 100% for the diagnosis of HIT. The other functional assay (WBIA) had similar sensitivity (81%) and specificity (90%) to flow cytometry. In contrast, the PaGIA maintained a high sensitivity of 100% but a specificity of only 20%. The improved specificity of flow cytometry over the antigenic assay was most marked in the 4T intermediate-risk group in which similar results were obtained between all three functional assays. We demonstrate that compared with an immunological assay (PaGIA), flow cytometry can improve the specificity of laboratory diagnosis of HIT without loss of sensitivity using SRA as a standard. Flow cytometry may have a role in the first-line laboratory diagnosis of HIT, especially when combined with an immunological assay such as PaGIA.

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.

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.

Perspective on enzyme-linked immunosorbent assay for detecting antibodies against PF4/heparin complex in laboratory diagnosis of heparin-induced thrombocytopenia

Perspective on enzyme-linked immunosorbent assay for detecting antibodies against PF4/heparin complex in laboratory diagnosis of heparin-induced thrombocytopenia

Heparininduzierte Thrombozytopenie

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that carries an increased risk of thromboembolic complications. HIT is caused by platelet-activating antibodies directed against a complex of platelet factor4 (PF4) and heparin. HIT typically manifests in the second week after initiation of heparin therapy with a platelet count reduction of more than 50% of the highest level after the start of heparin administration as well as thromboembolic events. The clinical probability can be calculated by the 4 T's score. The laboratory diagnosis of HIT is based on confirmation of PF4/heparin antibodies or on functional tests that provide evidence of heparin-dependent platelet-activating antibodies. A low 4 T's score and negative HIT test virtually rule out the presence of HIT. Patients with acute HIT require anticoagulation with a compatible anticoagulant in a therapeutic dose. The drugs currently available for this include the direct thrombin inhibitors argatroban, lepirudin, bivalirudin, and desirudin and the indirect factor Xa inhibitors danaparoid and fondaparinux.

ASH evidence-based guidelines: is the IgG-specific anti-PF4/heparin ELISA superior to the polyspecific ELISA in the laboratory diagnosis of HIT?

You are asked to consult on a 76-year-old man admitted to the hospital with pneumonia and thrombocytopenia. Ten days before the current admission, he had undergone surgery to repair a small bowel obstruction. A preoperative platelet count had been normal. Following surgery, he received subcutaneous unfractionated heparin thromboprophylaxis until his discharge on post-operative day 5. In your differential diagnosis for the patient's thrombocytopenia, you consider heparin-induced thrombocytopenia (HIT) and wish to order laboratory testing. In addition to a polyspecific anti-PF4/heparin ELISA for the diagnosis of HIT, your laboratory has recently begun to offer an IgG-specific ELISA. You wonder which of these assays performs better in the diagnosis of HIT.

Platelet PlA2Polymorphism and the Risk for Thrombosis in Heparin-Induced Thrombocytopenia

Platelet glycoprotein (GP) IIb/IIIa has an important role in platelet aggregation. A polymorphism of platelet GPIIIa (PlA2, also called HPA1b) has been associated with a higher risk of thrombosis, but its implication in heparin-induced thrombocytopenia (HIT) is unclear. To investigate the hypothesis that the PlA2 polymorphism influences the prothrombotic effects of HIT, we conducted a prospective study of 66 consecutive patients with a laboratory diagnosis of HIT. The end point of the study was the diagnosis of a thrombus within 30 days of the positive HIT test result. The Diagnostica Stago (Asnières, France) enzyme-linked immunosorbent assay was used to detect HIT antibodies, and a polymerase chain reaction assay was used to detect the PlA2 polymorphism. Of the 66 patients, thrombotic complications developed in 27 (41%). Patients with the PlA2 allele demonstrated a significantly higher thrombosis risk than did patients without (69% vs 32%; P = .0088; odds ratio, 4.68; 95% confidence interval, 1.39-15.72). The risk was stronger for arterial thrombosis and for patients 60 years or older. There was a significant association between the PlA2 polymorphism of GPIIIa and the risk of thrombosis in patients with HIT antibodies.

Serologic Results in &gt;1000 Patients With Suspected Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) can lead to life-threatening and limb-threatening thrombosis. HIT is thought to be initiated by the interaction of pathogenic antibodies toward a complex platelet factor 4 (PF4) and heparin (PF4:H), which can activate platelets and predispose to thrombosis. As such, the laboratory diagnosis of HIT includes antigenic and functional assays to detect antibodies directed at PF4:H complexes. We performed a retrospective analysis of 1017 consecutive samples tested by serotonin-release assay and by enzyme-linked immunosorbent assay (ELISA). Most samples showed no serologic evidence of HIT, whereas 4% to 5% of samples demonstrated both antigenic and functional serological evidence for HIT. Approximately 12% to 18% of samples showed immunologic evidence of anti-PF4:H antibodies but without functional evidence of serotonin release in vitro. Interestingly, a small minority of samples (0.7%) caused serotonin release but were negative in the ELISA. The results are presented using cutoff values established at our hospital and for the ELISA manufacturer. This study provides a pretest probability of the serologic results from an antigenic assay (ELISA) and a functional assay (serotonin-release assay) in patients clinically suspected of having HIT.

Multiplex Assay for Profiling Plasma Protein Biomarkers for Improved Diagnosis and Prognosis of Heparin Induced Thrombocytopenia (HIT).

Background: Laboratory diagnosis of heparin-induced thrombocytopenia (HIT) is currently done by using immunologic, platelet aggregation, and serotonin-release assays; however, the diagnostic efficacy of these tests is variable. Although, various clinical scoring systems are currently used to attempt to differentiate patients with HIT from those with other causes of thrombocytopenia, a definite predictive test for HIT is still not available. The pathogenesis of HIT could involve dysregulation of inflammatory mediators (and other immunomodulators) and growth factors from platelets and endothelial cells. Accordingly, with the goal of identifying potential biomarkers of disease, the present study examined the profiles of cytokines, chemokines, and selected growth factors in HIT patients. We are testing a novel multiplex microbead immunoassay approach, developed by Luminex (Austin, TX), for simultaneous detection of multiple plasma protein analytes.Methods: Plasma samples were obtained from 20 patients diagnosed with HIT and from 15 healthy controls. Patient plasma samples in this study were positive for heparin-platelet factor 4 (PF4) antibodies by ELISA (GTI, Mulwaukee WI). Using the multiplex microbead immunoassay, measurements were made on plasma levels of thirty four cytokines/chemokines, which included tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, IL-1 beta, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p40), IL12(p70), IL-16, Eotaxin, RANTES, MCP-1, MIP-1alpha, MIP-1beta (CCL4), IP-10, MCP-3 (CCL7), GM-CSF, TNF-beta, IL-1RA, soluble form of interleukin (IL) 2 receptor (sIL-2R)alpha, IFNalpha2a, sFAS ligand, GRO, MDC(CCL22), G-CGF in HIT patients and healthy controls; commercial multiplex detection panels were used (Upstate, Lake Placid, NY). In addition, the levels of six growth factors were measured, including FGF-2, VEGF, EGF, Flt-3 Ligand, PDGF-AA and PDGF-AB/BB by the multiplex method (by detection panels also from Upstate).Results: Multiplex data were analyzed by methods of computational biology to establish hierarchical clustering. In these surveys, the level of sIL-2R, an in vivo marker of T-cell activation was higher in patient plasma than in healthy control subjects (p≤0.002). Plasma levels of the chemokines, MCP-1, MIP-1alpha and IP-10, were significantly reduced in patients compared to controls. Among the growth factors tested, EGF appeared to be significantly decreased in HIT patients compared to the control group. In addition, patients showed lower PDGF-AA and PDGF-AB/BB levels.Conclusions: Various immunomodulators and growth factors were either increased or decreased in HIT patient plasma. Accordingly, our studies are providing leads for further characterization of plasma biomarkers of HIT in a larger number of patients, including longitudinal samples. In combination with the detection of anti-PF4-heparin antibodies, and other criteria, multiple microbead immunoassays of plasma protein profiles may enhance the accuracy of HIT diagnosis and prognosis. Additionally, the multiplex method, enabling simultaneous detection of multiple protein factors, is also providing novel leads for further studies on the molecular and cellular mechanisms of HIT.

Prospective Evaluation of Laboratory Tests for the Diagnosis of Heparin-Induced Thrombocytopenia.

The laboratory diagnosis of heparin-induced thrombocytopenia (HIT) relies on the demonstration of antibodies to the heparin-platelet factor 4 (H-PF4) complex. Assays are based on the functional ability of H-PF4 antibodies to activate platelets, or detect the antibody directly by immunological methods. Multiple assays in each category are currently in clinical use and newer, rapid immunological assays are becoming available. The aim of this study was to compare available methods for detecting H-PF4 antibodies in a prospective study of patients with clinically suspected HIT. Functional assessment included serotonin release assay (SRA) and lumi-aggregometry (LA). Immunological assessment included ELISA (GTI), and particle gel immunoassay (PGIA; Diamed and Akers). Circulating platelet microparticles (PMP) were assessed by flow cytometry. Patients were also assessed for the pre-test probability of HIT using the Warkentin 4-T scoring system. 151 patients were enrolled. 54/151 patients (35.8%) had a positive GTI ELISA, while 53/151 (35.1%) and 39/151 (25.8%), respectively, had positive Akers and Diamed PGAI tests. Only 15/149 (10.1%) patients had a positive SRA, while only 5/150 (3.3%) gave a positive result by lumi-aggregometry. There was a strong correlation between the ELISA OD values obtained in serum and plasma using both fresh (r=0.98) and frozen (r=0.99) samples, although slightly more positive results were obtained using serum. Differences were only seen with OD values around the cut-off of 0.4. The majority (77.8%) of H-PF4 antibodies detected by ELISA were neutralized by heparin in the ‘confirmatory' procedure. Weak antibodies (OD 0.4–0.5) were more likely to be non-neutralizable (5/12; 42%) than strong antibodies (OD>1.0; 4/23; 17%). 47 patients positive by ELISA were retested to determine the predominant immunoglobulin subclass. 15/47 (32%) were positive (OD>0.4) for IgG; 27/47 (57%) for IgM, and 12/47 (25%) for IgA. The Diamed assay more closely correlated with the GTI ELISA than the Akers test (82.1% vs. 56.7%, respectively). The PGIAs were only moderately correlated with each other (64%) with the Akers assay giving more “false positive” results relative to the ELISA. PMP were higher in patients with a positive ELISA (6.2 vs 4.7 × 106/ml) or positive SRA (5.5 vs. 5.1 ×106/ml) but this was not statistically significant due to the wide range of results. Of 119 patients assessed, 87 had a low pre-test probability of HIT (4-T score 0–3), 27 had an intermediate probability (4–5), and 5 had a high probability (6–8). The GTI ELISA was positive in 24, 56 and 80% of low, intermediate and high probability cases. The Akers PGIA was positive in 39, 41 and 40% respectively; the Diamed assay in 21, 33 and 40%, and the SRA in 7, 11 and 40%, respectively. This study was conducted in a patient population biased towards cardiovascular surgery, and confirms previously reported observations that immunoassays are more frequently positive than functional assays. The ELISA correlated better than the PGIA tests with the pre-test probability of HIT, although the Diamed test showed acceptable correlation with the ELISA. In contrast, the Akers assay correlated poorly with the ELISA, often producing positive results when the latter test was negative. We conclude that while the PGIA tests are rapid and convenient, further studies are needed to determine the basis for disparate results relative to the widely used ELISA.

Does the Platelet Factor 4 (PF4) ELISA Correlate with the Clinical Diagnosis of Type II Heparin-Induced Thrombocytopenia?.

Type II heparin-induced thrombocytopenia (HIT) is an antibody-mediated complication of heparin therapy that is associated with a consumptive thrombocytopenia and a high risk of thromboembolism. It is now known that antibodies associated with type II HIT recognize sites on PF4 when complexed with heparin. Due to technical challenges associated with the serotonin release assay, most clinicians rely on the PF4 ELISA to establish a laboratory diagnosis of HIT. However, it is unclear whether the ELISA has predictive value in identifying those patients who have clinically apparent disease and whether ELISA optical density (OD) measurements correlate with the development of thrombosis. We retrospectively reviewed the medical records of 103 sequential patients who tested positive for HIT by a commercial PF4 ELISA from 2000–2002. While blinded to ELISA OD values, we recorded patient age and gender, admission diagnosis, hospital course, type of heparin administered, route of administration, baseline and nadir platelet counts, timing of thrombocytopenia, HIT therapy, clinical thrombotic events within 30 days, and other potential causes of thrombocytopenia to determine the clinical likelihood of HIT. The association between OD values and both the clinical likelihood of HIT and objectively confirmed thrombotic events was analyzed using the Pearson Chi-square test. Median patient age was 62 yrs and 52% of patients were female. Those with cardiac disease, cancer, and venous thromboembolism constituted 44%, 19% and 9% of the study population, respectively. Median platelet count nadir was 40K/mL (range, 2K–90K), and average onset of thrombocytopenia was 6.5 days. Median ELISA OD value was 0.77 (0.40–3.81). The clinical likelihood of HIT was noted in 24/103(23%) patients, and 22(21%) suffered a thrombotic event. An ELISA OD &gt;1.0 was significantly associated with the clinical likelihood of HIT (p=0.03), though OD values ranged from 0.41 to 3.81 among these patients. Neither an OD threshold of 1.0 or 1.5 was associated with thromboembolism (p=0.2 and p=0.1, respectively). In contrast, the clinical likelihood of HIT was significantly associated with subsequent thrombosis (p=0.002). ELISA results were not significantly associated with the extent or onset of thrombocytopenia, type of heparin or route administered, patient age or gender, admission diagnosis, or hospital course. In conclusion, PF4 ELISA OD values are significantly associated with the clinical likelihood of HIT but are not predictive of HIT on an individual patient basis. Clinical judgment but not ELISA OD values is associated with subsequent thromboembolism. In summary, the clinical likelihood of HIT, and not ELISA results, remains the best parameter by which to modify anticoagulation therapy in patients being assessed for HIT. Figure Figure