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.

Defibrotide Does Not Cross-React with HIT Antibodies. Implications in the Management of HIT.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1054-1054
Author(s):  
Jawed Fareed ◽  
Debra A. Hoppensteadt ◽  
Massimo Iacobelli ◽  
Jeanine M. Walenga
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Antithrombotic Agent ◽  
Platelet Factor ◽  
Prolonged Incubation ◽  
Heparin Induced Thrombocytopenia ◽  
Orally Bioavailable ◽  
Microparticle Formation ◽  
Heparin Exposure

Abstract Defibrotide is a mammalian DNA derived antithrombotic and anti-ischemic agent that does not produce systemic anticoagulation. Heparin-induced thrombocytopenia (HIT) is an immune disease related to heparin exposure, in which patients are at risk of developing life- and limb-threatening thrombosis. Studies were performed to determine whether defibrotide cross-reacts with HIT antibodies. Sera from 141 clinically confirmed HIT patients were tested for platelet activation in the presence of defibrotide (1 – 100 μg/ml) and unfractionated heparin (1 – 100 μg/ml). 103 sera (73%) produced platelet aggregation and serotonin release activities with heparin. Only 4 samples (2%) showed a weak reactivity with defibrotide which was eliminated by heparinase treatment indicating heparin contamination in the patient sample. Further studies revealed that defibrotide does not complex with platelet factor 4 (PF4), and that prolonged incubation of defibrotide with platelet rich plasma does not mobilize PF4. Studies of patients treated with extended dosing of intravenous or oral defibrotide (n=270) demonstrated that HIT antibodies are not generated with defibrotide treatment. Studies carried out on the effect of purified IgG from HIT patients revealed that defibrotide blunts platelet activation and microparticle formation as measured by flow cytometry. These studies suggest that defibrotide may be a useful antithrombotic agent for the management of patients with HIT. Moreover, unlike heparin, defibrotide does not promote platelet activation, rather it is capable of suppressing the hypercoagulable state associated with HIT. Defibrotide is orally bioavailable and can be used for extended anticoagulant management of HIT patients.

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.

scholarly journals Comparison of an IgG-Specific Enzyme-Linked Immunosorbent Assay Cutoff of 0.4 Versus 0.8 and 1.0 Optical Density Units for Heparin-Induced Thrombocytopenia

2016 ◽  
Vol 23 (3) ◽  
pp. 282-286 ◽  
Author(s):  
Brianne M. Ritchie ◽  
Jean M. Connors ◽  
Katelyn W. Sylvester
Keyword(s):  
Optical Density ◽  
Immunosorbent Assay ◽  
Predictive Value ◽  
Retrospective Chart Review ◽  
Serotonin Release ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Institutional Review ◽  
Sensitivity Specificity

Background: Previous studies have demonstrated optimized diagnostic accuracy in utilizing higher antiheparin–platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) optical density (OD) thresholds for diagnosing heparin-induced thrombocytopenia (HIT). We describe the incidence of positive serotonin release assay (SRA) results, as well as performance characteristics, for antiheparin–PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units in the diagnosis of HIT at our institution. Methods: Following institutional review board approval, we conducted a single-center retrospective chart review on adult inpatients with a differential diagnosis of HIT evaluated by both antiheparin–PF4 ELISA and SRA from 2012 to 2014. The major endpoints were to assess incidence of positive SRA results, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy at antiheparin–PF4 ELISA values ≥0.4 OD units when compared to values ≥0.8 and ≥1.0 OD units. Clinical characteristics, including demographics, laboratory values, clinical and safety outcomes, length of stay, and mortality, were collected. Results: A total of 140 patients with 140 antiheparin–PF4 ELISA and SRA values were evaluated, of which 23 patients were SRA positive (16.4%) and 117 patients were SRA negative (83.6%). We identified a sensitivity of 91.3% versus 82.6% and 73.9%, specificity of 61.5% versus 87.2% and 91.5%, PPV of 31.8% versus 55.9% and 63.0%, NPV of 97.3% versus 96.2% and 94.7%, and accuracy of 66.4% versus 86.4% and 88.6% at antiheparin–PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units, respectively. Conclusion: Our study suggests an increased antiheparin–PF4 ELISA threshold of 0.8 or 1.0 OD units enhances specificity, PPV, and accuracy while maintaining NPV with decreased sensitivity.

scholarly journals Clinical Features of Heparin-Induced Thrombocytopenia Diagnosed By Lumi-Aggregometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4185-4185
Author(s):  
Elona Turley ◽  
Artur J. Szkotak ◽  
Irwindeep Sandhu ◽  
Cynthia M Wu
Keyword(s):  
Platelet Activation ◽  
Light Flash ◽  
Patient Characteristics ◽  
Serotonin Release ◽  
Radioactive Isotopes ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Platelet Counts ◽  
Dense Granules ◽  
Heparin Exposure

Abstract Background: Heparin-induced thrombocytopenia (HIT) is associated with anti-platelet factor-4 (PF4)/heparin antibodies that activate platelets, resulting in thrombocytopenia and a pro-thrombotic state. At our institution antibody-mediated platelet activation is demonstrated by lumi-aggregometry, which is a method previously validated against the gold standard serotonin-release assay (SRA). Lumi-aggregometry does not involve radioactive isotopes, which is its major advantage over the SRA. The clinical course of HIT diagnosed via SRA and ELISA has been previously described, and clinical prediction tools such as the 4-T score were validated using these diagnostic tests. However, the clinical picture of HIT diagnosed by lumi-aggregometry has not been previously described. Aims: The objective of this study is to describe the clinical and laboratory presentation of patients diagnosed with HIT by lumi-aggregometry. Methods: Patients with clinically suspected HIT and quantitative anti-PF4 IgG-specific ELISA OD ≥0.400 (Gen-Probe, San Diego) received confirmatory HIT testing by lumi-aggregometry. Briefly, HIT antibody-induced activation of washed healthy donor platelets was tested at therapeutic (0.1U/mL and 0.5U/mL) and high (100U/mL) porcine heparin concentration. The degree of platelet activation was quantitated luminographically based on the light flash reaction of ATP (released from platelet dense-granules) with luciferin luciferase reagent. A ratio of therapeutic to high heparin luminescence amplitude of >5.0 and platelet aggregation at therapeutic, but not high, concentrations was considered a positive result. The results of assays performed by our regional HIT testing referral laboratory from June 2009 to July 2012 were reviewed to identify patients with positive HIT testing by lumi-aggregometry. Patient records were retrospectively reviewed to obtain predefined data on baseline patient characteristics, heparin exposure, platelet counts, and thrombotic events occurring in the 5 days preceding or the 30 days following the date of positive HIT testing. Results: We identified 43 patients diagnosed with HIT by lumi-aggregometry (median age 68.0, 49% male) while under the care of local academic (46%) or urban community hospitals (37.2% medical; 53.5% surgical; 9.3% intensive care). Median baseline platelet count was 187 (14-349). Median date of platelet drop post-heparin exposure was 6 days (range 3-14) in patients without prior heparin exposure or platelet transfusions (Figure 1). Platelet drop >50% and platelet nadir ≥20x109/L were present in the majority of patients (Table 1). Thrombocytopenia occurred prior to (70.5%) or the same day (23.5%) as thrombosis in 16/17 patients with serial platelet counts who developed HIT-associated thromboembolism. Conclusion: Patients diagnosed with HIT by lumi-aggregometry present with similar findings to those described in SRA-confirmed HIT. These findings lend support to the use of lumi-aggregometry as an accurate diagnostic assay for the clinico-pathologic syndrome of HIT. Figure 1 Figure 1. Table 1. Percentage platelet drop from baseline and platelet nadir Percent platelet drop Platelet nadir >50% 30-50% <30% ≥20 x 109/L 28 3 2 10-19 x 109/L 5 1 0 <10 x 109/L 1 1 0 Disclosures Szkotak: Alexion Pharmaceuticals: Research Funding. Sandhu:Celgene: Honoraria; Jansen: Honoraria; Novartis: Honoraria.

scholarly journals PF4-dependent immunoassays in patients with vaccine-induced immune thrombotic thrombocytopenia (VITT): results of an inter-laboratory comparison

2021 ◽  
Author(s):  
Ulrich J. Sachs ◽  
Nina Cooper ◽  
Andreas Czwalinna ◽  
Jens Müller ◽  
Bernd Pötzsch ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Functional Testing ◽  
Initial Screening ◽  
Antibody Testing ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Functional Assays ◽  
Diagnostic Work ◽  
Work Up ◽  
Diagnostic Work Up

Background. COVID-19 vaccine ChAdOx1 nCov-19 may rarely lead to vaccine-induced thrombotic thrombocytopenia (VITT). Antibody-mediated, platelet factor 4 (PF4) dependent platelet activation appears to resemble a key mechanism in VITT, partially comparable to heparin-induced thrombocytopenia (HIT). The use of PF4/heparin immunoassays has been proposed as part of a diagnostic approach, but their sensitivity has not been established. Methods. Sera from 12 well-defined VITT patients were first studied by two different laboratories in functional assays. Sera where then used for an inter-laboratory comparison, in which 5 different PF4/heparin immunoassays were used by 4 laboratories. Results. Results for functional testing were highly concordant. VITT antibodies were also reliably detected by PF4/heparin ELISAs (92-100%). In contrast, only 25% of VITT antibodies were reactive in a particle gel immunoassay (PaGIA), and 8% in a lateral flow assay (LFA). An automated chemiluminescence assay (CLIA) was negative for all sera tested (0%). Conclusion. It seems feasible to establish functional antibody testing for the confirmation of VITT. For the initial screening of suspected VITT cases, PaGIA, LFA, and CLIA are useless when applied as single tests. Only ELISA-based PF4/heparin immunoassays are sensitive enough to be incorporated in the diagnostic work-up. However, a combination of a positive ELISA and a negative CLIA may be useful to identify VITT antibodies in the absence of confirmatory functional assays.

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.

Low Level Platelet-Activating Antibodies in Patients Suspected of Having Heparin-Induced Thrombocytopenia but Testing Negative in the ”Gold Standard” Functional Test

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2757-2757
Author(s):  
Ishac Nazi ◽  
Donald M Arnold ◽  
James W Smith ◽  
Theodore E. Warkentin ◽  
Jane C Moore ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Research Funding ◽  
Heparin Therapy ◽  
Serotonin Release ◽  
Patient Specific ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Increased Risk ◽  
Increased Sensitivity ◽  
Clinical Conditions

Abstract Background: Heparin-induced thrombocytopenia (HIT) is a common drug reaction that causes arterial or venous thrombosis as a result of heparin therapy. Platelet-activating antibodies, against complexes of platelet factor 4 (PF4) and heparin, cause intense platelet activation, ultimately leading to an increased risk of thrombosis, limb-loss and even death. Most patients exposed to heparin will produce non-pathogenic anti-PF4/heparin antibodies while only a small number will produce platelet-activating and HIT-causing antibodies (pathogenic HIT antibodies). Among HIT tests, the functional assays, such as the serotonin release assay (SRA), correlate best with the disease because they can specifically identify the pathogenic HIT antibodies whereas the enzyme immunoassays (EIAs) cannot. We have previously shown that anti-PF4/heparin antibody production precedes thrombocytopenia in HIT patients (Warkentin et al., Blood 2009 113: 4963-4969) possibly indicating the need for a threshold plasma level of pathogenic HIT antibody, among other factors, to cause the disease. The objective of this study was to investigate the presence of low levels of pathogenic HIT antibodies in samples from patients suspected of HIT who had detectable anti-PF4/heparin antibodies in the EIA (EIA-positive), but who did not have platelet-activating antibodies in the standard SRA (SRA-negative). Methods: We used an in-house IgG-specific EIA to detect the presence of anti-PF4/heparin antibodies (EIA-positive: OD405nm> 0.45) and the standard SRA to detect the presence of heparin-dependent platelet-activating antibodies (SRA-positive: release >20% with 0.1-0.3 IU/mL of unfractionated heparin). We developed an enhanced SRA (eSRA) by adding increasing concentrations of exogenous PF4 (0-100 μg/mL) to detect sub-threshold levels of platelet activating antibodies undetectable in the standard SRA (eSRA-positive: release >20%). Samples tested were referred for HIT testing by the McMaster Platelet Immunology Laboratory (Hamilton, Canada). Results: Sera from healthy individuals (n=10) and from suspected HIT patients with a negative anti-PF4/heparin EIA (n=15) did not demonstrate platelet activation in the eSRA at any dose of exogenous PF4 added. SRA-positive sera (n = 7), diluted sufficiently that they were non-reactive in the standard SRA, demonstrated PF4 dose-dependent platelet activation in the eSRA. This confirmed the increased sensitivity of the eSRA in detecting low-titre platelet-activating antibodies. Reactivity in the eSRA was inhibited by high heparin (100 U/mL) and by blocking the platelet FcgRIIa receptor with the monoclonal antibody IV.3. We then tested samples (n=24) referred for HIT testing that were positive in the anti-PF4/heparin EIA (optical densities OD405nm 0.7 to 2.4) but negative in the standard SRA. Heparin-dependent platelet activation (20-99% release) was demonstrated in 11 of 24 (46%) in the eSRA. This reactivity directly correlated with the amount of PF4 added to the platelets (optimal concentration of PF4 12.5 - 100 μg/mL) but not with the strength (OD405nm) of the anti-PF4/heparin EIA. In further investigations, we concentrated (4-fold) 7 of the 11 eSRA-positive samples in an attempt to increase the concentration of the antibodies. Of those 7 samples, 5 (71%) became positive in the standard SRA upon testing of the concentrated sample. Conclusions: These data indicate that low-titre platelet-activating antibodies may be found in some patients suspected of having HIT that test negative in the standard SRA irrespective of the strength (OD405nm) of the anti-PF4/heparin EIA. The immune response during heparin therapy can produce both families of pathogenic and non-pathogenic anti-PF4/heparin antibodies but it is the titre of the pathogenic antibody that may be necessary for platelet activation. Perhaps under permissive clinical conditions and with patient-specific factors, the titre of the pathogenic HIT antibodies may increase and lead to HIT. Disclosures Warkentin: Pfizer Canada: Honoraria; Instrumentation Laboratory: Honoraria; GlaxoSmithKline: Consultancy, Research Funding; W.L. Gore: Consultancy, Research Funding.

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

2020 ◽  
Author(s):  
Caroline Vayne ◽  
Thi-Huong Nguyen ◽  
Jérôme Rollin ◽  
Noémie Charuel ◽  
Anne Poupon ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Single Molecule ◽  
Binding Sites ◽  
Serotonin Release ◽  
Titration Calorimetry ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Activation Assay ◽  
Group 3

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

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.

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