Heparin-induced Thrombocytopenia ELISA Research Articles

Cross Reactivity of Heparin-Induced PF4 Antibodies with Heparin- Bound IL-8.

Background: Heparin-Induced Thrombocytopenia (HIT) is caused by polyclonal antibodies, formed to conformational neoepitopes exposed in platelet factor 4 (PF4) bound to heparin (H). HIT antibodies are commonly measured by one of several commercially available ELISAs. The literature reports cases of clinical HIT and/or positive HIT antibody-mediated platelet activation tests in patients without antibodies detectable in PF4 based ELISAs, and suggests that antibodies to other chemokines, such as interleukin-8 (IL-8), may be involved. The current study was undertaken to characterize the frequency of such antibodies in a population of consecutive serum specimens submitted for HIT diagnosis by 14C-serotonin release assay (SRA), and also in a group of retrospectively selected SRA-positive specimens.Methods: Specimens (n=90) analyzed by SRA between March and July 2008 were subsequently tested by PF4 Enhanced ELISA (GTI, Waukesha, WI), and the Zymutest HIA ELISA (Hyphen BioMed, Neuville sur Oise, France). An additional ELISA was carried out on the specimens using the Zymutest heparin-bonded plate coated with purified, recombinant human interleukin-8 (IL-8) (72 aa form, Prospec, Rehovot, Israel). The second group consisted of archived specimens (n=32) that tested positive by SRA but negative by GTI HAT ELISA; these specimens were subsequently tested by the Zymutest HIA and the H:IL-8 ELISA.Results: Recombinant IL-8 (1μg/well) added to heparin-bound ELISA plates was recognized by anti-IL-8 (BD Biosciences), but not anti-PF4 (Leinco Technologies, Inc.) antibodies and was not displaced from the plate by addition of PF4 from normal serum at the dilution (1:100) used in the ELISA. Of the 90 consecutive specimens, 21 (23.3%) tested positive in both the GTI and the Zymutest HIA tests; 15 of the 90 (16.6%) tested positive by one but not both of the PF4-based ELISAs (Table 1). In this group of 90 specimens, 17 (18.9%) had OD > 0.500 on the heparin-bound IL-8 plate. Only 3 of the specimens bound to the H:IL-8 exclusively, while 14 were also positive in the GTI and/or Zymutest HIA ELISA. Thus, of the 36 specimens that tested positive in one or both of the PF-4-based ELISAs, 14 (38.9%) had antibodies that also targeted heparin-bound IL-8. Ten of the 90 specimens tested positive in the SRA. Six of SRA-positives had PF4-specific antibodies that did not cross react with H:IL-8, 3 had H:IL-8 reactive antibodies in addition to PF4 reactive antibodies, and 1 SRA positive specimen tested negative in all ELISAs.Table 1. Comparison of GTI HAT vs Zymutest HIA results: n = 90 consecutive patientsNumbers in parentheses indicate how many specimens in each subgroup contained antibodies that bound to H:IL-8.Zymutest HIA+−Total+21 (11)9 (1)30GTI HAT−6 (2)54 (3)60Total276390Among the set of 32 specimens that tested positive by SRA and negative by GTI ELISA, 8 tested positive in the Zymutest ELISA, including 4 that had OD > 0.500 on the H:IL-8 plate. The 24 specimens that tested negative by both HIT ELISAs ranged in percent serotonin release (tested with 0.1 U/ml H) between 21–84% with a median of 45.6%, and did not contain antibodies that bound the H:IL-8 plate.Conclusions: The data agree with other studies that show differences between results of commercially available HIT ELISA kits. The study also shows that over one third of specimens that test positive in the HIT ELISAs contain antibodies that are not specific for PF4, but also recognize heparin-bound IL-8. These results do not prove that the antibodies that bind PF4 targets are the same ones that bind IL-8. However, the significant degree of homology (40%) between the peptides suggests that the same antibodies may cross react with either target. The relevance of these antibodies is as yet unclear. Only 3% of specimens bound H:IL-8 plates but not PF4 plates, and these were not associated with SRA activity. Also HIT ELISA negative specimens with SRA activity, which showed only moderate percent serotonin release, did not contain H:IL-8-binding antibodies. The study did not find evidence of a role for H:IL-8 antibodies in HIT-related platelet activation tests.

Argatroban Associated Bleeding Complications.

Background: Heparin induced thrombocytopenia (HIT) is a frequently observed side effect observed with heparin administration. Treatment for this condition includes direct thrombin inhibitor (DTI) therapy. Major hemorrhagic complications are considered rare events in this setting.Methods: This is a case series of patients diagnosed with HIT in a tertiary medical referral center. The diagnosis of HIT was established on the base of clinical criteria. All included patients had positive HIT ELISA based antibody testing during the time interval from July 2006 to July 2007 and all patients were treated with argatroban. A systematic review for hemorrhagic and thrombotic complications of argatroban therapy was performed. Major hemorrhage was defined as clinically identified bleeding with the demand for transfusion therapy. Thrombotic complications were defined as thrombosis evidenced by imaging studies developing during argatorban therapy. We performed bivariate and multivariate logistic regression analysis to identify demographic and clinical factors that influence the development of complications of argatroban therapy as treatment for HIT. Examined covariates were gender, age, type of service the patient was admitted under (surgical versus medical), amount of comorbidities, preexisting history of gastrointestinal bleeding, documented thrombotic or thrombembolic event at the time point of HIT diagnosis and coexisting coexisting coagulopathy other than HIT.Results: We identified 102 patients with the diagnosis of HIT. The median optical density (OD) of the ELISA HIT immunoassay was 1.23 (95% CI 0.93–1.43). We identified 11 (10.7%) major hemorrhagic events in patients on argatroban. Four patients (3.9%) died as a consequence of major hemorrhagic complications. No thrombotic events were identified in our study cohort. As statistically significant; predictors for clinically relevant hemorrhagic complications in our model remained male gender, preexisting GI bleeding history and being patient on a surgical service.Conclusion: The incidence of major hemorrhagic episodes with agatroban therapy is significantly higher then generally reported and the mortality rate is very disturbing. These data suggest that alternatives to standard direct thrombin inhibitor therapy should be aggressively pursued.

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

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. Limited data are available regarding repeat HIT antibody testing after an initial negative test. We conducted a retrospective study to determine the utility of repeat testing. Heparin antibodies were detected using the GTI-PF4 enzyme-linked immunoabsorbent assay, ELISA (GTI Diagnostics, Waukesha, WI). Patients (n = 137) were assigned to one of three groups based upon the initial negative test optical density (OD) range of low = 0-0.132, medium = 0.133-0.267, and high = 0.268-0.399. A pretest clinical score was retrospectively determined using the "4T's" (Thrombocytopenia, Timing of platelet fall, Thrombosis, and the absence of oTher causes of thrombocytopenia). A subsequent positive ELISA was found in 16% (22/137) of patients who underwent repeat testing. Most of these patients had a low pretest clinical score (62%). Four patients had an interval change in the pretest score between the initial negative and subsequent positive tests. Only these four patients developed HIT with thrombosis (HITT). Eighty percent of patients with a high initial negative test OD value had a positive ELISA on repeat testing; however, the initial negative test OD value could not predict whether a patient developed HITT. In contrast, an increase in the pretest clinical probability between initial and repeat testing better predicted HITT. Consecutive repeat ELISA testing for heparin antibodies may be warranted in patients with an increase in their pretest clinical score after an initial negative test as an adjunct to confirm the diagnosis of HIT.