Heparin‐induced thrombocytopenia: an improved method of detection based on lumi‐aggregometry

Stewart, Michael W.; Etches, Wai S.; Boshkov, Lynn K.; Gordon, Philip A.

doi:10.1111/j.1365-2141.1995.tb05265.x

Cited by 54 publications

(27 citation statements)

References 18 publications

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“…Addition of mAb aE11 reduced platelet ATP release to background levels in the presence of pure APA and normal serum, but had no effect on platelet ATP release induced by heparin-induced thrombocytopenia (HIT)-positive serum (Fig 1). Therefore, inhibition of platelet activation by mAb aE11 was not due to interference of an Fc receptor-mediated response, since the HIT serum studied has been shown to activate platelets in an Fc-dependent manner (Stewart et al, 1995). Sepharose-purified APA IgG (1 mg/ml), 1 mM CaCl 2 and normal serum (NS) as a source of complement factors in the presence and absence of anti-C5b-9 antisera (mAb aE11, 10 mg/ml, final concentration).…”

Section: Effect Of Mab To C5b-9 On Apa-induced Platelet Activationmentioning

confidence: 96%

“…A luciferin/ luciferase reagent, Chronolume (Chronolog Corporation, Havertown, Pa.) was added to the suspension after initiating stirring at 1000 rpm at 37ЊC in a Chronolog 560 VS lumi-aggregometer linked to a model 810 Aggrolink data reduction system (Chronolog). The resulting light flash was proportional to the amount of ATP released and indicative of the degree of platelet activation (Stewart et al, 1995).…”

Section: Measurement Of Platelet Activation In Response To Apa Seramentioning

confidence: 99%

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Antiphospholipid antibody‐dependent C5b‐9 formation

1997

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The relationship between the presence of antiphospholipid antibodies (APA) and the production of the terminal membrane attack complex (MAC) of complement (C5b‐9) was studied. Serum samples from known high positive APA patients induced platelet activation and destruction which was inhibited by heat‐inactivation of the sera. The response was restored if the heat‐inactivated APA‐positive sera were supplemented with normal sera. Adsorption of the APA‐positive sera with phospholipid (PL)‐coated polystyrene beads inhibited platelet destruction. Addition of monoclonal antibody (mAb) to C5b‐9 (aE11) also inhibited platelet destruction, suggesting that the APA‐dependent platelet destruction might be complement‐mediated. Purified APA, in the presence of normal serum, induced C5b‐9 formation and binding to PL‐coated beads in a dose‐dependent manner as detected by flow cytometry. Prospective analysis of 200 serum samples for C5b‐9 production showed that all sera testing negative for the presence of APA also tested negative for C5b‐9 production. All sera with high levels of IgG binding to PL (GPL) showed evidence of C5b‐9 production. Sera with low or moderate GPL values showed varying levels of C5b‐9 production. These data suggest that complement may play a key role in APA‐dependent platelet activation, in vivo.

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Section: Effect Of Mab To C5b-9 On Apa-induced Platelet Activationmentioning

confidence: 96%

Section: Measurement Of Platelet Activation In Response To Apa Seramentioning

confidence: 99%

Antiphospholipid antibody‐dependent C5b‐9 formation

1997

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“…This method, also known as platelet aggregation test (PAT), is classically performed with PRP but can also be carried out with WP. Whole blood platelet aggregation can also be assessed using heparin‐induced multiple electrode aggregometry (HIMEA) for HIT diagnosis, as well as measurement of adenosine triphosphate (ATP) released from dense granules using chemiluminescence . Moreover, several assays based on flow cytometry (FC) have been proposed recently, most of them assessing P‐selectin (CD62P) expression on platelet surface .…”

Section: Introductionmentioning

confidence: 99%

Evaluation of functional assays for the diagnosis of heparin induced thrombocytopenia using 5B9, a monoclonal IgG that mimics human antibodies

Vayne

Guéry

Charuel

et al. 2020

Journal of Thrombosis and Haemostasis

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Background Serotonin release assay (SRA) is considered as the “gold standard” for detecting pathogenic heparin‐induced thrombocytopenia (HIT) antibodies. However, this method is time consuming, expensive, and uses radioelements. Heparin‐induced multiple electrode aggregometry (HIMEA), light transmission aggregometry (LTA) with platelet rich plasma (PRP) or washed platelets (WP), adenosine triphosphate (ATP) release, and flow cytometry (FC) are available alternatives. Objectives To evaluate the performance of these assays, comparatively with SRA, for detecting HIT antibodies, using 5B9, a monoclonal IgG fully mimicking human HIT antibodies. Patients/Methods Heparin‐dependent platelet activation induced by 5B9 (50/20/10 µg/mL) was evaluated by all assays performed on the same day using platelets from 20 healthy donors. The three methods exhibiting the highest sensitivity to 5B9 were then assessed by testing samples from patients with either likely (n = 10), or indeterminate/unlikely HIT (n = 10). Results All methods exhibited good sensitivity for detecting 5B9 50 µg/mL, but only SRA and HIMEA were positive with 100% of donors using 5B9 20 µg/mL, followed by FC (83%). SRA detected 5B9 10 μg/mL with 90% of donors, while HIMEA and FC were positive in 45% and 44% of cases, respectively. Whereas SRA was positive with 9/10 samples from likely HIT, HIMEA and FC were positive with 6 and 7 of them, respectively. Neither SRA nor HIMEA was positive with indeterminate/unlikely HIT samples, while FC was positive or doubtful in three cases. Conclusions Serotonin release assay likely remains the most sensitive and specific assay for detecting platelet activating HIT antibodies, but HIMEA or FC are potential alternatives, despite being less performant.

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“…As for every laboratory assay, the clinician should be aware of the specificity and sensitivity of the assay used. A large number of studies comparing the sensitivities and specificities of immunological and functional assays in detecting heparin-induced drug-dependent antibodies have been published (6,9,12,14,15,22,26,27,37,39,43,47,49,51,54,58,59,62,78). In general it should be pointed out that defining specificity and sensitivity in diagnostic assays depends on the definition of reference groups expressing or not expressing the value to be calculated.…”

Section: Laboratory Assays For Hit II Diagnosismentioning

confidence: 99%

“…Widely used, at least in Europe, is the heparin-induced platelet activation assay (HIPA) (19). Platelet aggregometry with determination of ADP release is utilized in lumiaggregometry (51). Flow cytometry is applied in the detection of platelet microparticles (33), the expression of the platelet activation marker P-selectin (CD62p) (28), or annexin V binding to phosphatidylserine (43,54).…”

Section: Laboratory Assays For Hit II Diagnosismentioning

confidence: 99%

Laboratory Diagnosis of Heparin-Induced Thrombocytopenia and Monitoring of Alternative Anticoagulants

Leo

Winteroll

2003

Clin Vaccine Immunol

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The major complication in the therapeutic or prophylactic use of heparin in medical treatment is type II heparin-induced thrombocytopenia (HIT II), a unique form of drug-induced immune-mediated thrombocytopenia. Due to the extensive use of heparin, this side effect is widespread; up to 3% of patients treated with unfractionated heparin (UFH) develop HIT II (69). Clinically, HIT II is characterized by thrombocytopenia which paradoxically is associated with thrombosis (HIT IIinduced thrombosis [HITTS]) (i.e., deep vein thrombosis, pulmonary embolism, and venous gangrene) in about 50% of the cases.In the pathogenesis of HIT II, the formation of platelet factor 4 (PF4)/heparin complexes seems to be the major determinant (2, 3, 20). The binding of PF4 to heparin induces antibody production directed against a neoepitope created by the three-dimensional assembly of PF4 and heparin. Subsequently, PF4/heparin-antibody complexes induce platelet activation, followed by the shedding of platelet microparticles, thrombin generation, and the involvement of endothelial cells (Fig. 1).Remarkably, the incidence of HIT II, HIT II-associated thrombotic complications, and PF4/heparin-antibodies (HIT II antibodies) in patients receiving therapy with UFH is clearly dependent on the clinical setting of the patient. Cardiosurgical patients are at a high risk to develop HIT II antibodies, while orthopedic patients undergoing hip replacement have a high risk for developing thromboembolic complications (Iceberg model [66]). In medical patients the risk for developing HIT II is substantially lower. Comparing low-molecular-weight heparins (LMWH) with UFH, the use of LMWH coincides with a substantially lower risk of HIT II, but this effect holds true only within the same group of patients (77,78).It is generally accepted that the clinical diagnosis of HIT II should be followed by immediate cessation of heparin therapy and initiation of therapy with alternative anticoagulants. Negative results in the concomitant laboratory testing should not serve as the sole criterion for restarting heparin therapy.In almost every case of HIT II, administration of alternative anticoagulants is essential either to continue prophylactic anticoagulation or to treat and prevent thrombotic complications due to HIT II. Three substances (danaparoid, hirudin, and argatroban) are available. Since these substances have not been compared directly with each other in the treatment of HIT II, criteria such as half-life and/or route of excretion should be considered for selection. This review focuses on (i) the present laboratory methods for diagnosing HIT II, including their principles, time consumption, and significance, and (ii) the alternative anticoagulants, including their characteristics and indications in certain clinical settings. LABORATORY ASSAYS FOR HIT II DIAGNOSISLaboratory testing in the diagnosis of HIT II is based on either the immunological detection of antibodies directed against the PF4/heparin complex or the functional plateletactivating potential of t...

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Heparin‐induced thrombocytopenia: an improved method of detection based on lumi‐aggregometry

Cited by 54 publications

References 18 publications

Antiphospholipid antibody‐dependent C5b‐9 formation

Antiphospholipid antibody‐dependent C5b‐9 formation

Evaluation of functional assays for the diagnosis of heparin induced thrombocytopenia using 5B9, a monoclonal IgG that mimics human antibodies

Laboratory Diagnosis of Heparin-Induced Thrombocytopenia and Monitoring of Alternative Anticoagulants

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