Serotonin‐release assay‐positive but platelet factor 4‐dependent enzyme‐immunoassay negative: HIT or not HIT ?

2020 ◽  
Author(s):  
Theodore E. Warkentin ◽  
Maureen A. Smythe ◽  
Mona A. Ali ◽  
Naveed Aslam ◽  
Jo‐Ann I. Sheppard ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Release 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.

Frequency and Clinical Impact of Platelet Factor 4-Specific Antibodies in Patients Undergoing Extracorporeal Membrane Oxygenation

2019 ◽  
Vol 119 (07) ◽  
pp. 1138-1146 ◽  
Author(s):  
Caroline Vayne ◽  
Marc-Antoine May ◽  
Thierry Bourguignon ◽  
Eric Lemoine ◽  
Eve-Anne Guery ◽  
...  
Keyword(s):  
Platelet Count ◽  
Extracorporeal Membrane Oxygenation ◽  
Serotonin Release ◽  
Clinical Impact ◽  
Platelet Factor 4 ◽  
Ecmo Circuit ◽  
Platelet Factor ◽  
Specific Antibodies ◽  
Release Assay ◽  
Specific Igg

Introduction/Objectives Extracorporeal membrane oxygenation (ECMO) provides circulatory support in patients with severe heart failure, but the frequent use of unfractionated heparin exposes patients to high risk of heparin-induced thrombocytopenia (HIT). We prospectively evaluated the development and clinical impact of platelet factor 4 (PF4)-specific antibodies (Abs) during ECMO and whether specific biological characteristics could predict HIT. Materials and Methods From 2014 to 2018, we studied 57 adults who underwent an ECMO for at least 5 days. The plasma samples collected daily were tested for PF4-specific Abs using immunoassays to detect immunoglobulin (Ig) G, A, and M isotypes or only IgG. Serotonin release assay was performed without and with PF4 to detect pathogenic Abs. Results Twenty-nine patients (50%) were positive for PF4-specific Abs (IgG, A, M), with IgG in 17/57 (30%) and 16 of them (94%) were immunized within 10 days. PF4-specific IgG Abs did not affect the clinical or biological course of most patients. HIT was suspected in only two patients with ECMO circuit dysfunction and unexpected platelet count decrease after day 5. High levels of PF4-specific IgG were detected in both patients, and HIT was confirmed by a serotonin release assay, which was also more sensitive when exogenous PF4 was present. Conclusion PF4-specific Abs are common during ECMO but are mostly non-pathogenic and not associated with a less favorable prognosis. However, an abnormal platelet count evolution, in particular if associated with ECMO circuit dysfunction, should prompt the search for pathogenic PF4-specific IgG.

Antibodies to the Platelet Factor 4-Heparin Complex and Mortality in Chronic Hemodialysis Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 274-274
Author(s):  
Marc Carrier ◽  
Greg A. Knoll ◽  
Dean Fergusson ◽  
Steven Doucette ◽  
Michael J. Kovacs ◽  
...  
Keyword(s):  
Cardiovascular Events ◽  
Hemodialysis Patients ◽  
Serotonin Release ◽  
Chronic Hemodialysis ◽  
Platelet Factor 4 ◽  
Elisa Assay ◽  
Platelet Factor ◽  
Risk Of Death ◽  
Asymptomatic Patients ◽  
Release Assay

Abstract Background: Heparin-induced thrombocytopenia is a serious complication of heparin therapy that can lead to thromboembolism, cardiovascular events or death. Patients with this disorder develop antibodies to the platelet factor 4-heparin (PF4-H) complex. Hemodialysis patients are repeatedly exposed to heparin and are at risk for developing PF4-H antibodies. The clinical impact of asymptomatic PF4-H antibodies in patients on chronic hemodialysis is not known. Objective: To determine the association between asymptomatic PF4-H antibodies and mortality in a cohort of chronic hemodialysis patients repeatedly exposed to heparin. Methods: Pre-dialysis blood samples were drawn from 419 asymptomatic patients. All patients received unfractionated heparin (Baxter) while on dialysis. All samples were screened for PF4-H antibodies using an ELISA assay (GTI PF4 Enhanced, GTI Diagnostics). All positive and indeterminate samples were then tested using an IgG-specific PF4-H ELISA assay and a platelet serotonin-release assay. Participants were then followed up prospectively for thromboembolic events, cardiovascular events, or death. Results: During a median follow-up of 2.5 years there were 129 deaths. After controlling for important potential confounding variables, the relative risk of death was 2.92 (95% CI: 1.18-7.25; P= 0.02) in patients with IgG-specific PF4-H antibodies and 4.08 (95% CI: 1.26–13.2; P= 0.02) in patients with IgG-specific antibodies and an indeterminate serotonin-release assay. Conclusions: PF4-H antibody formation is associated with increased all-cause mortality in patients on chronic hemodialysis. Further investigation is needed to determine if anticoagulation with alternative agents would improve survival in this population.

Prevalence of Heparin-Dependent Platelet Antibodies in Children after Cardiopulmonary Bypass Surgery.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3019-3019
Author(s):  
Rowena C. Punzalan ◽  
Sheila J. Hanson ◽  
Nancy Ghanayem ◽  
Brian R. Curtis ◽  
Kathleen Murkowski ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Unfractionated Heparin ◽  
Pediatric Intensive Care ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Platelet Antibodies ◽  
Release Assay

Abstract Heparin is used during cardiopulmonary bypass (CPB) surgery in children. Upon exposure to heparin, heparin-dependent platelet antibodies (HDPA) may form against complexes of platelet factor 4 and heparin on platelet surfaces. Heparin-induced thrombocytopenia (HIT) or heparin-induced thrombocytopenia and thrombosis (HITT) may then ensue. Among adult CPB patients, up to 50% demonstrate HDPA, although only 2% develop HIT and 1% develop HITT. In previous studies done among non-CPB newborns and pediatric intensive care patients, the incidence of HDPA has been zero to minimal. Objective: To determine prevalence of HDPA among children undergoing CPB who are exposed to unfractionated heparin for >120 hours after CPB. Methods: We designed a prospective pilot study. All patients ≤ 12 years old who were to receive heparin during CPB were eligible. The presence of HDPA was assessed by heparin-platelet factor 4 enzyme-linked immunosorbent assay (ELISA); positive and equivocal results were confirmed by serotonin release assay. Blood samples were obtained at the time of cessation of heparin or after 10 days on heparin, whichever came first. Results: Thirty patients were enrolled: 15 were aged 2–19 days (median 6 days); 15 were aged 1.2 to 50 months (median 4.5 months). One patient had borderline positive and 1 had positive results by heparin-platelet factor 4 ELISA; both had negative results by serotonin release assay; neither developed clots. Eighteen patients developed thrombocytopenia, which is common after CPB, including the 2 with equivocal results by ELISA. Six patients, all with negative ELISAs, developed symptomatic thromboses. Conclusion: There were no HDPA identified among children who received unfractionated heparin for CPB. The specificity of heparin-platelet factor 4 ELISA among children should be assessed.

Platelet factor-4 concentration in adult veno-arterial ECMO patients

Perfusion ◽  
2020 ◽  
pp. 026765912096510
Author(s):  
Michael Mazzeffi ◽  
Madeline Clark ◽  
Alison Grazioli ◽  
Colleen Dugan ◽  
Raymond Rector ◽  
...  
Keyword(s):  
Antibody Formation ◽  
Critical Factor ◽  
Epidemiologic Studies ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Release Assay ◽  
Va Ecmo ◽  
Control Plasma

Background: Heparin induced thrombocytopenia (HIT) is reported at a variable rate in extracorporeal membrane oxygenation (ECMO) patients. A critical factor impacting platelet factor-4 (PF4)-heparin antibody formation is plasma PF4 concentration. We hypothesized that PF4 concentration would be increased during veno-arterial (VA) ECMO. Methods: Plasma PF4 concentration was measured during the first 5 ECMO days in 20 VA ECMO patients and 10 control plasma samples. PF4-heparin ratios were estimated using an assumed heparin concentration of 0.4 IU/mL. This correlates with an activated partial thromboplastin time of 60 to 80 seconds, which is the anticoagulation target in our center. Results: Twenty VA ECMO patients were enrolled, 10 of which had pulmonary embolism. Median PF4 concentration was 0.03 µg/mL [0.01, 0.13] in control plasma. Median PF4 concentration was 0.21 µg/mL [0.12, 0.34] on ECMO day 1 or 2, 0.16 µg/mL [0.09, 0.25] on ECMO day 3, and 0.12 µg/mL [0.09, 0.22] on ECMO day 5. Estimated median PF4-heparin ratios were 0.04, 0.03, and 0.02 respectively. Two patients (10%) developed HIT that was confirmed by serotonin release assay. PF4 concentration did not differ significantly in these patients compared to non-HIT patients (p = 0.37). No patient had an estimated PF4-heparin ratio between 0.7 and 1.4, which is the reported optimal range for PF4-heparin antibody formation. Conclusion: Our data suggest that PF4 concentration is mildly elevated during VA ECMO compared to control plasma. Estimated PF4-heparin ratios were not optimal for HIT antibody formation. These data support epidemiologic studies where HIT incidence is low during VA ECMO.

scholarly journals False-Negative Platelet Factor 4 Antibodies and Serotonin Release Assay and the Utility of Repeat Testing in the Diagnosis of Heparin-Induced Thrombocytopenia and Thrombosis

2019 ◽  
Vol 2019 ◽  
pp. 1-4 ◽  
Author(s):  
Tarig Omer ◽  
Naresh Mullaguri ◽  
Pravin George ◽  
Christopher R. Newey
Keyword(s):  
False Negative ◽  
Serotonin Release ◽  
Clinical Suspicion ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Repeat Testing ◽  
Tandem Occlusion ◽  
Release Assay ◽  
The Right

Objective. To report a case of false-negative serological tests in the diagnosis of heparin-induced thrombocytopenia (HIT) followed by a brief review of the literature on this topic. Case Presentation. A 75-year-old Caucasian female patient was admitted with a traumatic right ankle fracture that required open reduction and internal fixation. Despite postoperative subcutaneous heparin chemoprophylaxis, she developed deep vein thrombosis (DVT) and pulmonary embolism (PE) on day 4 and subsequently started on continuous heparin infusion. On day 5, she suffered a stroke from a complete occlusion of the right common carotid artery with tandem occlusion of the right middle cerebral artery. She underwent successful thrombectomy of both arteries. The proposed stroke mechanism was paradoxical embolism through a patent foramen ovale. Over the next few days, thrombocytopenia was noted, the heparin drip was stopped, and HIT antibodies (antibodies targeting the complex of platelet factor 4 and heparin; PF4-H AB) and serotonin release assay (SRA) tests were sent. Because of the suspicion for HIT, she was started on bivalirudin with subsequent improvement in platelet count. Initial PF4-H AB and SRA tests were negative, bivalirudin was stopped, and heparin was restarted. Subsequently, her platelets trended down, again raising clinical suspicion of HIT. Repeat PF4-H AB and SRA testing resulted positive. Conclusions. A positive SRA in the appropriate context is considered for the diagnosis of heparin-induced thrombocytopenia. This case report highlights that false-negative serological evaluation is possible early in the course of the disease. Repeat testing is recommended in patients with high clinical suspicion.

Investigations of the Immunoglobulin Subtype Transformation of Anti–Heparin-Platelet Factor 4 Antibodies During Treatment With a Low-Molecular-Weight Heparin (Clivarin) in Orthopedic Patients

2003 ◽  
Vol 127 (5) ◽  
pp. 584-588
Author(s):  
Sarfraz Ahmad ◽  
H. Peter Bacher ◽  
Michael R. Lassen ◽  
Debra A. Hoppensteadt ◽  
Helen Leitz ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
Platelet Factor ◽  
Significant Difference ◽  
Antibody Titers ◽  
Orthopedic Patients

Abstract Context.—It is now widely accepted that the pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated by the generation of a wide array of functional and molecularly heterogeneous anti–heparin-platelet factor 4 (AHPF4) antibodies that may mediate platelet and/or endothelial cell activation/destruction. Objective.—We investigated the differential prevalence and functionality of AHPF4 immunoglobulin subtypes (IgA, IgG, and IgM) in plasmas obtained from orthopedic patients immobilized with Plaster-Cast and treated with clivarin (a low-molecular-weight heparin) in comparison to a placebo for the prophylaxis of deep-vein thrombosis. Design and Methods.—Clivarin was administered subcutaneously at a fixed daily dosage of 1750 U without any adjustment or loading dosage. Citrated plasmas were obtained at baseline, at 10 to 14 days, and at postbrace procedure (5–12 weeks). An enzyme-linked immunosorbent assay (ELISA) was used to quantitate the AHPF4 antibody titers. The functionality of the ELISA-positive samples was determined by a 14C-serotonin release assay (SRA). Results.—In the ELISA test, 16 of 1073 samples (1.5%; 6 in clivarin and 10 in placebo groups) were positive for AHPF4 antibodies (mean optical density [OD] = 0.46 ± 0.02). None of the ELISA-positive samples for AHPF4 antibodies could mediate platelet activation responses as determined by the SRA (0%–3% serotonin release, P > .10, n = 16). Through differential immunoglobulin subtype analysis of the samples positive for (cumulative) AHPF4 antibodies, we determined that their relative prevalence in plasma were as follows: IgM (mean OD = 0.71 ± 0.13) > IgG (0.31 ± 0.08) > IgA (0.14 ± 0.02). Although there was no significant difference in the total antibody titers between clivarin and placebo groups, the antibody subtyping data showed conversion trends (ie, IgA [clivarin to placebo], IgG [placebo to clivarin], and IgM [clivarin to placebo]). Conclusion.—These observations indicate that even at reduced dosages, clivarin can shift the immunogenic up-regulation toward the IgG subpopulation; however, the IgG subtype is of a nonfunctional type of AHPF4 antibody and thus may not cause any HIT-related pathogenic responses.

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.

HIT: Anti-Platelet Factor 4-Heparin (PF4-H) Assay Validation and Comparison Study

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S124-S124 ◽  
Author(s):  
Mark De Luna
Keyword(s):  
Statistical Data ◽  
Instrument Validation ◽  
Poor Performance ◽  
Serotonin Release ◽  
Comparison Study ◽  
Elisa Method ◽  
Platelet Factor ◽  
Good Comparison ◽  
Release Assay ◽  
Positive Agreement

Abstract Objectives The aims of the study were to perform an instrument validation of the HemosIL HIT-Ab (PF4-H) Assay and to compare the sensitivities and specificities of the three HIT tests (HemosIL HIT-Ab [PF4-H] Assay, PIFA Heparin/PF4 Assay, and Immucor PF4 ELISA) versus the gold standard Serotonin Release Assay performed in ARUP Laboratories and versus each other. Methods The HemosIL HIT-Ab (PF4-H) Assay was validated on the ACL TOP 750 using calibrators, controls, and samples from UF Pathology Laboratories and ARUP Laboratories that were previously tested for the HIT-Ab IgG via an ELISA method. EP Evaluator was used to calculate statistical data. Results A total of 116 samples were assayed using the HemosIL HIT-Ab (PF4-H) Assay and the PIFA Heparin/PF4 Rapid Assay. Overall, there were 125 positive tests and 255 negative tests for anti-PF4-H (ACL TOP 39 positive, 77 negative; PIFA 11 positive, 105 negative; ELISA 60 positive, 56 negative). There were 15 positive and 17 negative for the SRA. The comparison between the ACL TOP and SRA had a sensitivity of 80.0% and specificity of 35.3%. ELISA vs SRA had a sensitivity of 100.0% but had a 0.0% specificity. The PIFA vs SRA correlation had a sensitivity of 0.0% and specificity of 88.2%. The ACL TOP versus UF Health’s current HIT ELISA method had a good comparison as well, with a sensitivity of 61.7% and a specificity of 96.4%. Conclusion The three HIT methods (ACL TOP, ELISA, and PIFA) compared to the SRA showed that the ACL TOP had a good positive agreement, but poor negative agreement; however, the ACL TOP had a very good negative agreement with the ELISA method. The PIFA cartridge test had an overall poor performance with no positive agreement with the SRA and poor sensitivity versus the ACL TOP and ELISA methods.

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