scholarly journals Heparin-induced thrombocytopenia diagnosis: a retrospective study comparing heparin-induced platelet activation test to 14C-serotonin release assay

TH Open ◽  
2021 ◽  
Author(s):  
Marie-Caroline Gonthier ◽  
Nicolas Gendron ◽  
philippine Eloy ◽  
Marie-Charlotte Bourrienne ◽  
martine alhenc-gelas ◽  
...  
Keyword(s):  
Retrospective Study ◽  
Platelet Activation ◽  
Serotonin Release ◽  
Functional Assay ◽  
Heparin Induced Thrombocytopenia ◽  
Functional Assays ◽  
Laboratory Confirmation ◽  
Activation Assay ◽  
Circulation Circuit ◽  
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 14C-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 to 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.

scholarly journals Is There an Alternative to Serotonin Release Assay for the Diagnosis of Heparin Induced Thrombocytopenia (HIT)? Evaluation of 5 Other Functional Methods Using 5B9 a Monoclonal IgG That Mimics HIT Human Antibodies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3742-3742
Author(s):  
Eve-Anne Guéry ◽  
Caroline Vayne ◽  
Cloé Derray ◽  
Joévin Besombes ◽  
Wayne Corentin Lambert ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Atp Release ◽  
Serotonin Release ◽  
The Other ◽  
Functional Assay ◽  
Heparin Induced Thrombocytopenia ◽  
Functional Methods ◽  
Release Assay

Abstract Introduction: Serotonin Release Assay (SRA) is today considered as the "gold standard" to detect pathogenic Heparin-Induced Thrombocytopenia (HIT) antibodies. However, this method is time-consuming, expensive and necessitates the use of 14C-radio-labelled serotonin, this implicating a specific agreement and secured premises, with a non-negligible environmental impact. These limitations explain that the use of SRA is restricted to a few laboratories worldwide. Finding a more accessible method with similar performances is therefore a challenge, and other different functional assays, such as Heparin-Induced Multiple Electrode Aggregometry (HIMEA), Light Transmission Aggregometry (LTA) using platelet rich plasma (PRP) or washed platelet (WP), ATP release, and Flow Cytometry (FC), are available. However, the sensitivity of these assays has never been comparatively evaluated with a standardized reagent. Objectives: The objective of our study was therefore to evaluate the sensitivity of these 5 functional methods for the detection of HIT antibodies in comparison with SRA, using 5B9, a monoclonal chimeric anti-PF4/H IgG recently developed in our laboratory, which fully mimics the effects of human HIT antibodies (Kizlik-Masson et al, J Thromb Haemost, 2017). Material and Methods: Platelet activation induced by 5B9 with heparin was assessed by the 6 following methods with blood samples from 10 consecutive unselected healthy donors:HIMEA performed with whole blood (Multiplate Analyzer® Roche),LTA performed with PRP (Chronolog®, Chrono-Log corporation),FC based on the assessment of P-selectin expression and performed with PRP (HIT Confirm®, Emosis on AccuriC6 plus®, Becton Dickinson),ATP release performed with WP (Chronolog®, Chrono-Log corporation),LTA performed with WP (Chronolog®, Chrono-Log corporation),SRA performed with WP (LSC scintillation counter, Perkin Elmer). For each method, different concentrations of 5B9 (10-20-50 µg/mL) were tested without heparin, and with "therapeutic" or high concentrations of unfractionated heparin (ranging from 0.1 to 1 and from 10 to 200 IU/mL respectively, according to the functional assay performed). The 3 concentrations of 5B9 were previously defined as "low" (10 µg/mL inducing in most cases a serotonin release <50% and no platelet aggregation in PRP), "high" (50 µg/mL always inducing a serotonin release >50% and platelet aggregation in PRP) or "intermediate" (20 µg/mL yielding variable results). Results: With the highest concentration of 5B9 (50 µg/mL), a strong platelet activation was detected with all methods and donors tested. HIMEA exhibited similar sensitivity (Ss 100%) than SRA to detect the activation induced by 20 μg/mL 5B9. FC was also able to detect the effect induced by 20 μg/mL 5B9 with 9/10 donors tested (90%). Alternatively, the measurement of ATP release, and LTA performed with WP or PRP failed to detect the effect of 20 μg/mL 5B9 in 30, 30 and 40 % of donors tested, respectively. SRA was the only method able to detect platelet activation induced by 10 μg/mL 5B9 with all donors tested, and the other methods were less sensitive (table). LTA performed with PRP was always negative (Ss= 0%). Platelet washings increased LTA sensitivity for detecting 10 or 20 μg/mL 5B9 (40% and 70% with WP vs. 0 and 60% with PRP, respectively), and the measurement of ATP release exhibited similar sensitivity. When platelet activation was evaluated in whole blood by HIMEA or in PRP using FC, the sensitivity to detect HIT antibodies was also improved (60% and 50%, respectively). Conclusion: These results confirm that SRA is likely the more sensitive functional assay to detect low concentrations of HIT antibodies. Indeed, apart from SRA, none of the other methods was able to detect the lowest concentration of 5B9 with 100% of donors. Interestingly, FC or HIMEA, which are rapid assays, also exhibit a high sensitivity, close to 100%, for detecting "intermediate" concentrations of HIT antibodies (i.e. corresponding to 20 μg/mL 5B9). We will further study the performances of these functional tests, including their specificity, by assessing patient's samples with confirmed HIT or having developed non-pathogenic antibodies (study in progress). Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Correlation Of Heparin Confirmatory Test (HCT) With Optical Density (OD) and Serotonin Release Assay (SRA) In The Diagnosis Of Heparin Induced Thrombocytopenia (HIT)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4754-4754 ◽  
Author(s):  
Ravneet Thind ◽  
Danielle Heidemann ◽  
Sundara Raman ◽  
Philip Kuriakose
Keyword(s):  
Optical Density ◽  
Predictive Value ◽  
Serotonin Release ◽  
Thrombin Inhibitors ◽  
Confirmatory Test ◽  
Functional Assay ◽  
Heparin Induced Thrombocytopenia ◽  
Laboratory Confirmation ◽  
Confirmation Test ◽  
Release Assay

Introduction Heparin-induced thrombocytopenia (HIT) is a potentially fatal, thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin. Accurate and rapid diagnosis with prompt commencement of therapy are imperative as delays in treatment are associated with an increasing risk of thrombosis, amputation, or death. On the flip side, initiation of therapy with direct thrombin inhibitors without laboratory confirmation carries a significant risk of bleeding. Two types of laboratory tests are available for detection of these antibodies: a widely available immunoassay (ELISA), which is very sensitive to the presence of anti-heparin/PF4 antibodies, but is less specific to the clinical syndrome of HIT because of detection of non-pathological antibodies. The Serotonin Release Assay (SRA) is a functional assay that is now considered the gold standard for confirmatory diagnosis of HIT due to its high specificity. However, the downside of SRA is the cost involved, limited availability and a turnaround time of 5-7 days. As such, a heparin confirmatory test (HCT) with excess heparin has been in use since mid 2011 on positive ELISA samples in our laboratory to improve test specificity. This test is more cost and time efficient, with a turnover time of no more than 48 hours. As noted in prior studies, inhibition of a positive ELISA result by 50% or more in the presence of excess heparin is considered confirmatory of heparin-dependent antibodies. Likewise a negative confirmatory test is defined as a decrease of 50% or less in antibody binding in the presence of heparin. Aim a) Correlation of Heparin Confirmatory test (HCT) with strength of HIT ELISA, vis-à-vis optical density (OD) of 0.4 - 0.99 and OD of >/= 1.0. b) Correlation of HCT results with SRA, to see if the latter can be replaced by the heparin confirmatory test. Patients and Methods A retrospective chart review of adult patients hospitalized at our institution with suspected HIT from July 2011 until January 2013 was done. There were 101 such patients. All patients who had a positive HIT ELISA, then had HCT as per our standard lab practice, with an SRA test done for diagnosis/confirmation of HIT, as per standard clinical practice. Historically, the major strength of SRA assay is its specificity. The optical density on HIT ELISA and SRA results were then compared with the Heparin Confirmatory test to establish clinical significance. Results Of the 101 patients tested for HIT ELISA, 49 were positive. HCT and SRA were performed on all 49 samples, 1 out of which was reported as indeterminate. Hence 48 samples were used for primary analysis, comparing HCT to the OD as well as the SRA results. Out of 48 patients, 6 had positive SRA with Heparin inhibition of >50% (sensitivity 6/6 = 100%). Remaining 42 patients had negative SRA, 7 out of which had Heparin inhibition of <50% (specificity 7/42 = 16.6%). All 7 patients with a negative HCT had a negative SRA, making the negative predictive value of the HCT 100%; however positive predictive value was only 14.6% (6/41). There was no correlation between the OD and Heparin Confirmation test. Conclusions Although there is data suggesting that there might be some value to the Heparin Confirmation test, we were unable to show a significant correlation between HCT and OD or between HCT and SRA. The prospect of having a cost effective and rapid assay for laboratory confirmation of HIT will always be a relevant need. We feel that a larger, prospective study should be conducted to definitively assess the relationship between HCT and SRA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Challenges in Detecting Clinically Relevant Heparin-Induced Thrombocytopenia Antibodies

2020 ◽  
Vol 40 (04) ◽  
pp. 472-484
Author(s):  
Theodore E. Warkentin
Keyword(s):  
Platelet Activation ◽  
False Negative ◽  
Serotonin Release ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Thrombosis Risk ◽  
Activation Assay ◽  
Release Assay ◽  
Chemiluminescent Immunoassay

AbstractHeparin-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.

Pathophysiology of Heparin-Induced Thrombocytopenia

2000 ◽  
Vol 124 (11) ◽  
pp. 1657-1666 ◽  
Author(s):  
Fabrizio Fabris ◽  
Sarfraz Ahmad ◽  
Giuseppe Cella ◽  
Walter P. Jeske ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Platelet Factor ◽  
Cellular Activation ◽  
Heparin Induced Thrombocytopenia ◽  
New Information ◽  
Study Selection ◽  
Release Assay

Abstract Objective.—This review of heparin-induced thrombocytopenia (HIT), the most frequent and dangerous side effect of heparin exposure, covers the epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment of this disease syndrome. Data Sources and Study Selection.—Current consensus of opinion is given based on literature reports, as well as new information where available. A comprehensive analysis of the reasons for discrepancies in incidence numbers is given. The currently known mechanism is that HIT is mediated by an antibody to the complex of heparin–platelet factor 4, which binds to the Fc receptor on platelets. New evidence suggests a functional heterogeneity in the anti-heparin-platelet factor 4 antibodies generated to heparin, and a “superactive” heparin-platelet factor 4 antibody that does not require the presence of heparin to promote platelet activation or aggregation has been identified. Up-regulation of cell adhesion molecules and inflammatory markers, as well as preactivation of platelets/endothelial cells/leukocytes, are also considered to be related to the pathophysiology of HIT. Issues related to the specificity of currently available and new laboratory assays that support a clinical diagnosis are addressed in relation to the serotonin-release assay. Past experience with various anticoagulant treatments is reviewed with a focus on the recent successes of thrombin inhibitors and platelet GPIIb/IIIa inhibitors to combat the platelet activation and severe thrombotic episodes associated with HIT. Conclusions.—The pathophysiology of HIT is multifactorial. However, the primary factor in the mediation of the cellular activation is due to the generation of an antibody to the heparin-platelet factor 4 complex. This review is written as a reference for HIT research.

Serologic Results in >1000 Patients With Suspected Heparin-Induced Thrombocytopenia

2007 ◽  
Vol 14 (4) ◽  
pp. 410-414 ◽  
Author(s):  
Suresh G. Shelat ◽  
Anne Tomaski ◽  
Eleanor S. Pollak
Keyword(s):  
Laboratory Diagnosis ◽  
Serotonin Release ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Life Threatening ◽  
Release Assay ◽  
Pathogenic Antibodies

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.

scholarly journals Easily Performed Functional Flow Cytometry Assay for Heparin-Induced Thrombocytopenia and Thrombosis (HIT): Correlation with Clinical Presentation, Immunoassay and Serotonin-Release Assay

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2601-2601
Author(s):  
Michal Cipok ◽  
Varda R Deutsch ◽  
Ismail Elalamy ◽  
Ilya Kirgner ◽  
Sigi Kay ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Clinical Presentation ◽  
Medical Center ◽  
High Sensitivity ◽  
Relative Sensitivity ◽  
Serotonin Release ◽  
Double Positive ◽  
Functional Assays ◽  
Release Assay ◽  
Positive Results

Abstract Background: Reliable diagnosis of HIT is mandatory for patient management. Commonly used antibody-detecting immunoassays carry certain limitations compared to functional assays which determine antibody-mediated platelet activation. However, current functional assays are limited by feasibility. The functional test for detecting platelet-activating antibodies is the [14C] serotonin-release (14C-SRA), considered the gold standard assay, (Sheridan D et al, Blood1986;67:27), is not commonly performed as it includes the use of a radiolabeled C14 and requires expertise to obtain reliable. Aims: We aimed to overcome these limitations by developing a simple functional flow cytometric assay (FCA) and assess its sensitivity and specificity as compared to a widely used immunoassay and the radioactive serotonin-release assay (SRA) in correlation with clinical presentation of HIT. Methods: Samples from patients clinically suspected for HIT were routinely tested by the PF4/H-PaGIA immunoassay (DiaMed, Switzerland), followed by the functional FCA which determines the capacity of the patient's serum to induce platelet activation in the presence of heparin, and the results were compared to those of the SRA. The assays results were correlated with the HIT presentation based on the 4Ts score. Results: Of 649 samples sent to the routine coagulation laboratory , 99 (15.3%) were positive by the H/PF4-PaGIA and 31 (4.8%) by the FCA. Out of 23 double positive samples that were further tested, 19 were positive by the radioactive SRA (82.6%, relative sensitivity). Of 32 PaGIA-positive but FCA-negative samples, 30 (93.8%) were also negative by the SRA, and 10 samples negative by both tests were all negative by the SRA. Overall, of 42 negative samples by FCA, 40 were negative by the SRA showing relative specificity of 95.2%. Following sample dilution of 1:32 (Nellen V. et al, Haematologica 2012;97:89), the H/PF4-PaGIA-positive results decreased to 29 (4.5%), constituting a 70.6% reduction, whereas the number of FCA-positive results (4.8%) remained consistent, indicating the specificity and the high sensitivity of the assay following high dilution. Forty normal samples were all negative by both assays.The overall agreement between the FCA and the PF4/H-PaGIA immunoassay following dilution was 93.1% for positive (sensitivity) and 94.3% for negative results (specificity). Overall, the FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score) compared to the PF4/H-PaGIA (ROC-plot analysis, AUC 0.93 vs. 0.63, p<0.001). At a cut-off level of 92% sensitivity, the respective specificity of the FCA was 96%. Conclusions: Our findings demonstrate that the easily performed functional FCA is practical for routine daily use, providing reliable results for initial diagnosis, as well as confirmation of HIT. Disclosures Kirgner: Tel Aviv Sourasky Medical Center: Consultancy. Tomer:Aaron Tomer: Patents & Royalties: Pending patent.

scholarly journals Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia

2020 ◽  
Vol 9 (4) ◽  
pp. 1226 ◽  
Author(s):  
Brigitte Tardy ◽  
Thomas Lecompte ◽  
François Mullier ◽  
Caroline Vayne ◽  
Claire Pouplard
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Membrane Expression ◽  
Platelet Factor ◽  
Strict Adherence ◽  
Heparin Induced Thrombocytopenia ◽  
Functional Assays ◽  
Healthy Donors ◽  
Sensitivity Specificity

Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and serotonin release assay (SRA) is considered to be the current gold standard, but functional assays suffer from certain limitations regarding their sensitivity, specificity, complexity, and/or accessibility. However, the strict adherence to adequate preanalytical conditions, the use of selected platelet donors and the inclusion of positive and negative controls in each run are key points that ensure their performances.

The Impact of Non-H:PF4 Antibodies on Results of the Serotonin Release Assay for Heparin-Induced Thrombocytopenia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3952-3952
Author(s):  
M. Margaret Prechel ◽  
Maura A. Woznica ◽  
Meredith K. McDonald ◽  
Walter P. Jeske ◽  
Jeanine M. Walenga
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Hla Class I ◽  
Serotonin Release ◽  
Class I ◽  
Heparin Induced Thrombocytopenia ◽  
Platelet Antibodies ◽  
Group I ◽  
Release Assay ◽  
Group Ii

Abstract Background: The Serotonin Release Assay (SRA) is frequently used to test for the presence of heparin-platelet factor 4 (H:PF4) antibodies as part of a diagnosis of Heparin-Induced Thrombocytopenia (HIT). In this test, immune complexes formed when antibodies bind to soluble or membrane-bound H:PF4 complexes, activate platelets via FcγIIa receptors. A HIT-positive SRA demonstrates donor platelet activation when test sera are incubated with low concentrations (0.1–1.0 U/ml) of heparin, but not when the incubation includes excess, neutralizing levels (10–100 U/ml) of heparin. Platelet activation at both low and high heparin is designated an indeterminate result. This heparin-independent platelet activation can be caused by immune complexes unrelated to HIT, by other anti-platelet antibodies or by platelet agonists. The present studies were conducted to study the occurrence of anti-platelet antibodies in specimens with marginal or inconclusive SRA activity. Methods: The study included two populations of specimens that had been previously tested by both SRA and H:PF4 antibody ELISA (GTI, Brookfield, WI). Group I included 44 specimens that tested SRA positive in spite of the absence of measurable H:PF4 antibodies. Most were relatively weak in the SRA: 51% ± 3.4 % (S.E.) serotonin release with 0.1 U/ml heparin. Group II consisted of 18 specimens that gave an indeterminate SRA response: heparin-independent platelet activation. Of these, 5 were positive for H:PF4 antibodies and 13 were negative. All specimens were analysed by PakPlus ELISA screening (GTI, Brookfield, WI) to determine if antibodies to HLA Class I and/or to common platelet specific glycoproteins (GPs) were present. Results: In Group I, 19 of the 44 (43%) specimens tested positive for one or more anti-platelet antibody. 18 of the 19 (95%) had either anti-GP IIb/IIIa (n=10)(53%) and/or anti- HLA Class I (n=11)(58%) antibodies. One specimen had antibodies to GP Ib/IX and to GP IV. The remaining 25 (57%) specimens tested negative. In Group II, 13 of the 18 (72%) SRA-indeterminate specimens had detectable anti-platelet antibodies. All but one of the 13 (92%) included antibody to HLA Class I. The anti-glycoprotein antibodies were less frequent in this group: anti-GP IIb/IIIa (n=2)(15%), GP Ia/IIa (n=3)(23%), GP Ib/IX (n=2)(15%) or GP IV (n=3)(23%). Conclusion: Non-H:PF4 anti-platelet antibodies, especially antibodies to GP IIb/IIIa or to HLA Class I, are not uncommon in sera referred for SRA testing. Specimens containing anti-platelet antibodies can give a positive or an indeterminate response in the two-point SRA. Specimens without H:PF4 antibodies that test positive in the SRA should be scrutinized for anti-platelet antibodies as an alternative to the diagnosis of HIT. Also, an indeterminate SRA should not be considered a negative test result. Anti-platelet antibodies alone can cause a non-heparin dependent platelet activation: however, their presence may also mask a positive, heparin-dependent, SRA response to H:PF4 antibodies. Finally, it is not uncommon for specimens to test positive by the 2-point SRA in the absence of antibodies to H:PF4 or to other platelet antigens. The mechanism for this response, and its significance to diagnosis of HIT, requires further investigation.

A New Enzyme Immuno-Assay That Only Detects IgG Antibodies to Heparin-PF4 Complexes (Zymutest HIA IgG®) Improves the Specificity of the Diagnosis of HIT without Loss of Sensitivity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2096-2096
Author(s):  
Claire Pouplard ◽  
Sandra Regina ◽  
Jean Baptiste Valentin ◽  
Yves Gruel
Keyword(s):  
Platelet Activation ◽  
High Specificity ◽  
Serotonin Release ◽  
Igg Antibodies ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Release Assay ◽  
Immuno Assay ◽  
Enzyme Immuno Assay ◽  
Test Probability

Abstract Heparin-induced thrombocytopenia (HIT) is associated in most patients with the development of antibodies to heparin-modified platelet factor 4 (PF4). Commercial immuno-assays frequently detect these antibodies after cardiac surgery but only few patients develop clinical HIT. Therefore, platelet activation tests such as serotonin release assay (SRA) are necessary to ensure the diagnosis of HIT with high specificity. Another approach to increase diagnosis specificity could be to detect IgG antibodies which are of major clinical relevance since they are the only class able to directly activate platelets in the presence of heparin. Therefore, we evaluated the performances of a new commercial immuno-assay specific to IgG for the diagnosis of HIT Abs (Zymutest HIA IgG®, Hyphen Biomed, Neuville sur Oise, France). Samples from 101 patients with suspected HIT were analysed. 40 cases had developed significant levels of Abs to PF4 measured with PVS/PF4 ELISA (HAT45®,GTI, Brookfiled, WI, USA) and the diagnosis of HIT had been confirmed since SRA was positive. Every sample was then tested with a global assay named Zymutest HIA G/A/M® as followed: each diluted plasma (200 μl) was incubated for 1 hour with 50 μl of platelet lysate providing PF4 into wells previously coated with unfractionated heparin. After washings and incubation (1 hour) with anti IgG/A/M-HRP immunoconjugate, the enzyme activity was developed and absorbance was read at 450nm. In case of positive result (A450 ≥ 0.5), the isotype distribution was analysed with a specific and standardized assay using monospecific anti-IgG-, anti-IgA- and anti IgM-HRP conjugates (Zymutest HIA-IgG® or -IgA® or -IgM®). A450 values ≥ 0.5 were also considered as positive. GTI assay that detected IgG/A/M Abs to PVS/PF4 complexes in the 40 patients with HIT (Ss 100%) was also positive in 30 of the 61 cases with no HIT (Sp 50.8%). Comparatively, Zymutest HIA® global assay was positive in 39 of the 40 patients with HIT (Ss 97.5%) and in 14 of 61 cases without HIT (Sp 77%). On the other hand, significant levels of heparin-dependent IgG antibodies were also measured in these 39 HIT patients using Zymutest HIA IgG® assay (mean A450: 1.81; range A450: 0.5 – 2.76), and only in 6 patients without HIT (Sp: 90%). However, IgG levels measured in patients without HIT were significantly lower (mean A450: 0.60; range A450: 0.08 – 2.20) than in those with HIT (p &lt; 0.0001). In addition, IgA or IgM heparin-dependent antibodies were only present, i.e. without IgG, in samples from patients for whom the diagnosis of HIT had been ruled out (n = 3). In conclusion, this study supports that the detection of significant levels of IgG heparin-dependent antibodies improves the diagnosis specificity in patients with a suspicion of HIT without loss of sensitivity. Nonetheless, whether IgG-specific immunoassays could avoid to perform platelet activation tests in patients with a strong pre-test probability of HIT warrant further study.

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