A sensitive and specific functional flow cytometric assay for the diagnosis of heparin-induced thrombocytopenia

A functional flow cytometric assay (FCA) for the immediate diagnosis of heparin-induced thrombocytopenia (HIT), with simultaneous compatibility testing for alternative anticoagulant therapies, has been developed to provide rapid and reliable results which effectively support patient management. The assay provides results within 1–2 h, uses readily available non-radioactive reagents, and employs standard equipment. Using the highly sensitive annexin V protein probe, the method detects activated platelets induced by heparin immune-complexes, with 300-fold increased binding to activated platelets. Twenty-five samples from patients clinically-suspected of having HIT (131 tests) and 10 normal control (NC) samples (36 tests) were simultaneously tested with unfractionated heparin (UH) and low-molecular-weight heparin (LMWH), and by the radioactive serotonin-release assay (SRA) (62 and 16 tests respectively). The FCA highly correlated with the SRA, showing 100% specificity and 95% sensitivity. Moreover, the FCA exhibited higher resolution between positive and negative samples (an average value of 8.6-fold the NC versus 4.0-fold the NC by SRA). The LMWH showed concordant results with UH ( r =0.95). We conclude that the functional FCA for HIT is practical, specific and sensitive, thereby permitting the rapid diagnosis of HIT and the suitability of alternative therapies.     

Laboratory diagnosis of heparin-induced thrombocytopenia: advantages of a functional flow cytometric test in comparison to the heparin-induced platelet-activation test

Nearly one third of patients with heparin-induced thrombocytopenia (HIT) will progress to overt thrombosis. Owing to the severity of HIT, a reliable prompt diagnosis is mandatory. In this study 248 consecutive samples from patients referred to our laboratory for HIT diagnosis and 97 specimens from normal controls were prospectively evaluated in parallel using the heparin-induced platelet aggregation (HIPA) test and a flow cytometric (FC) test. The HIPA test resulted in 214 negative, 17 indeterminate and 17 positive samples of patients. The FC method detects activated platelets induced by heparin-immune complexes using the highly sensitive recombinant probe annexin V and pooled platelets from multiple donors. The criteria for positive FC test results included an increase in platelet activiation of at least 11% at 0.3 IU/mL heparin concentration in the tube, and a ratio of more than 1.5 between platelet activation at 0.3 and 200 IU/mL heparin. According to the cut-off level 17 patients who showed indeteminate HIPA test results had 14 negative and 3 indeterminate corresponding FC test results. Only one of these patients (HIPA test indeterminate, FC test indeterminate) had no other obvious medical cause for thrombocytopenia than HIT. Infections or inflammations did not show any association with the FC test results, whereas thromboembolic events displayed a significant patelet activation at pharmacological heparin concentration. Therefore the FC test is associated to the complications of HIT. In conclusion, the FC test, which is fast and practical, showed a good agreement with the HIPA test and may be an accurate and useful test for HIT.     

Investigation of platelet function and platelet disorders using flow cytometry

Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard–Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard–Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.     

Simultaneous binding of heparin and platelet factor-4 to platelets: Further insights into the mechanism of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is mediated by antibody against complexes of platelet factor-4 (PF4) and heparin. Although it has been assumed that these complexes bind to platelets and provide a target for the antibody, this has never been demonstrated. Furthermore, there is evidence suggesting that heparin-PF4 complexes do not bind to platelets. We have analyzed the effect of each ligand on the platelet binding of the other. We particularly focused on the result when heparin and PF4 are in equimolar concentration because we had previously shown that this was the condition under which HIT-IgG increased on the platelet surface. We found that when the molar concentration of PF4 approximates or exceeds that of heparin, the ligands bind simultaneously to the cells and HIT-IgG binds also. However, when heparin is in molar excess, both PF4 binding and HIT-IgG binding are diminished. Our data are consistent with the hypothesis that heparin-PF4 complexes bind via their heparin component to heparin binding sites on the platelet membrane rather than by their PF4 component to PF4 sites. The conditions promoting the binding of the complexes also lead to binding of HIT-IgG. Am. J. Hematol. 58:24–30, 1998. © 1998 Wiley-Liss, Inc.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation

Antibodies to heparin platelet factor 4 (H-PF4) complexes were purified from the plasma of three patients with heparin-induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 M NaCl at pH 7.5 (peak 1) and the remainder was obtained using 0.1 M glycine/0. 5 M NaCl at pH 2.5 (peak 2). In an enzyme-linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H-PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H-PF4 complexes. Finally, IgG in patient 3 only bound to H-PF4 and was unreactive with PF4 alone. Using [14C]-serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H-PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.     

Evaluation of platelet turnover by flow cytometry

The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K₂EDTA, was incubated with 0.6?µg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n?=?23) was 6.13?±?3.09%. RPs were 10.41?±?9.02% in patients (n?=?10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45?±?6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81?±?18.79 (P?<?0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n?=?21; 21.04?±?16.21%, P?<?0.001 vs. controls) or systemic lupus erythematosus (n?=?6, 29.08?±?15.57%; P?<?0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.     

The complicated relationships of heparin-induced thrombocytopenia and platelet factor 4 antibodies with COVID-19

COVID-19 (coronavirus disease 2019) represents a prothrombotic disorder, and there have been several reports of platelet factor 4/heparin antibodies being present in COVID-19-infected patients. This has thus been identified in some publications as representing a high incidence of heparin-induced thrombocytopenia (HIT), whereas in others, findings have been tempered by general lack of functional reactivity using confirmation assays of serotonin release assay (SRA) or heparin-induced platelet aggregation (HIPA). Moreover, in at least two publications, data are provided suggesting that antibodies can arise in heparin naïve patients or that platelet activation may not be heparin-dependent. From this literature, we would conclude that platelet factor 4/heparin antibodies can be observed in COVID-19-infected patients, and they may occur at higher incidence than in historical non-COVID-19-infected cohorts. However, the situation is complex, since not all platelet factor 4/heparin antibodies may lead to platelet activation, and not all identified antibodies are heparin-dependent, such that they do not necessarily reflect “true” HIT. Most recently, a “HIT-like” syndrome has reported in patients who have been vaccinated against COVID-19. Accordingly, much more is yet to be learnt about the insidious disease that COVID-19 represents, including autoimmune outcomes in affected patients.     

Biological and clinical features of low-molecular-weight heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of unfractionated heparin (UFH) therapy. In contrast, only a few patients have been reported with HIT following low-molecular-weight heparin (LMWH) therapy (LMW-HIT). To define the clinical and biological characteristics of LMW-HIT, 180 patients treated for suspected HIT at 15 French centres were investigated. Clinical history was recorded and HIT was confirmed in 59 patients with positive serotonin release assay results: 57 of them had high levels of antibodies (Abs) to heparin-platelet factor 4 complexes (H/PF4) and two had Abs to interleukin 8. Eleven patients were treated exclusively with LMWH (LMW-HIT) and 48 with UFH either alone (UF-HIT, n = 34) or combined with LMWH (UF/LMW-HIT, n = 14). The LMW-HIT and UF-HIT groups were similar with respect to sex, age, platelet count before heparin therapy, frequency of bleeding and occurrence of disseminated intravascular coagulation. The interval to onset of HIT was longer in LMW-HIT patients compared with UF-HIT patients (P = 0.03). Severe thrombocytopenia (platelets < 15 x 10(9)/l) was more frequent in the LMW-HIT group (P = 0.04). Thrombosis occurred in three of 11 LMW-HIT patients, i.e. as frequently as in UF-HIT patients. LMW-HIT is potentially severe and may be observed after longer heparin treatment compared with UF-HIT. It is highly recommended, therefore, that platelet counts be monitored carefully whenever LMWH is administered.     

Danaparoid for heparin-induced thrombocytopenia: an analysis of treatment failures

BACKGROUND: Patients with heparin-induced thrombocytopenia (HIT) (with or without thrombosis) require alternative anticoagulation because of their extreme risk of new thromboembolic complications. The first effective agent for this purpose may be danaparoid, a less-sulfated low molecular weight heparinoid. Recently, direct thrombin inhibitors have been used. OBJECTIVE: Five HIT patients, who developed new thromboembolic complications while receiving danaparoid, were analyzed to consider possible reasons for treatment failure and to promulgate strategies that improve efficacy. RESULTS: Three patients had acute HIT, one had recent HIT, and one with remote HIT was re-exposed to heparin during heart surgery. Danaparoid was started as intravenous bolus and infusion in one patient, and as 1250 units subcutaneously twice daily in four patients. The new complications that emerged on danaparoid were new venous thrombi in three patients (one with pulmonary emboli), lower extremity arterial thrombosis in one, myocardial ischemia in one, thromboembolic cardiovascular accidents in one, and fatal bowel necrosis in one (two patients suffered more than one complication). Platelet counts did not improve or worsened in four, improved partially in the other, and parameters of disseminated intravascular coagulation failed to improve in one patient. Four patients responded relatively dramatically when direct thrombin inhibitors were substituted. Possible reasons for danaparoid failure include that: 1) no treatment is expected to completely prevent complications, 2) antithrombin III consumption can blunt efficacy in some patients, 3) low or intermediate doses may be insufficient, and 4) there was clinically significant cross-reactivity of the pathogenic HIT antibodies. CONCLUSIONS: It is emphasized that the possibility of clinically significant antibody cross-reactivity and that low or intermediate dosage may be inadequate when using danaparoid in therapy of HIT. The latter problem probably extrapolates to other anticoagulants used for HIT.     

Detection of microparticles from human red blood cells by multiparametric flow cytometry

BackgroundDuring storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as “storage lesions”. These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC.ResultsWe demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected.DiscussionMultiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies.     

流式细胞术研究特发性血小板减少性紫癜患者血小板相关抗体

目的：探讨特发性血小板减少性紫癜（ITP）患者血小板表面相关抗体（PAIg）在诊断及预后的价值。方法：应用流式细胞术（FCM）检测84例ITP患者及20例正常人PAIgG、PAIgM、PAIgA。结果：初发ITP和复发ITP患者组与正常对照组比较，PAIgG、PAIgA差异有统计学意义（P〈0．01），PAIgM差异无统计学意义（P〉0．05）。PAIgM与PAIgA之间有显著相关性，r=0．451（P〈0．01）。结论：①PAIg增高可作为诊断初发ITP的重要指标之一；②FCM检测ITP患者血小板表面PAIg敏感性好、特异性高，适用于临床，对ITP的诊断及预后评价有较好的实用价值；③初发型ITP患者以PAIgG和PAIgA增高为主，预后较好；复发型以PAIgM增高为主，预后较差，易复发。     

Pathogenesis of chronic immune thrombocytopenia: increased platelet destruction and/or decreased platelet production

Chronic immune thrombocytopenia (ITP) is a haematological disorder in which patients predominantly develop skin and mucosal bleeding. Early studies suggested ITP was primarily due to immune-mediated peripheral platelet destruction. However, increasing evidence indicates that an additional component of this disorder is immune-mediated decreased platelet production that cannot keep pace with platelet destruction. Evidence for increased platelet destruction is thrombocytopenia following ITP plasma infusions in normal subjects, in vitro platelet phagocytosis, and decreased platelet survivals in ITP patients that respond to therapies that prevent in vivo platelet phagocytosis; e.g., intravenous immunoglobulin G, anti-D, corticosteroids, and splenectomy. The cause of platelet destruction in most ITP patients appears to be autoantibody-mediated. However, cytotoxic T lymphocyte-mediated platelet (and possibly megakaryocyte) lysis, may also be important. Studies supporting suppressed platelet production include: reduced platelet turnover in over 80% of ITP patients, morphological evidence of megakaryocyte damage, autoantibody-induced suppression of in vitro megakaryocytopoiesis, and increased platelet counts in most ITP patients following treatment with thrombopoietin receptor agonists. This review summarizes data that indicates that the pathogenesis of chronic ITP may be due to both immune-mediated platelet destruction and/or suppressed platelet production. The relative importance of these two mechanisms undoubtedly varies among patients.     

A diagnostic test for heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia can be a serious and difficult-to- diagnose complication of heparin therapy. Serum from patients with heparin-induced thrombocytopenia can cause heparin-dependent platelet aggregation, but the low sensitivity and specificity of this test limit its clinical usefulness. In this report we describe an assay for heparin-induced thrombocytopenia that is both sensitive and specific. The improvement in the assay was accomplished by measuring platelet release instead of aggregation and by measuring platelet release at two heparin concentrations. The rationale for the use of two heparin concentrations was that sera from patients with heparin-induced thrombocytopenia caused release at therapeutic but not at high concentrations of heparin. Twenty-eight sera samples from patients suspected of having heparin-induced thrombocytopenia and 573 controls were coded and tested in the assay. The patients with possible heparin- induced thrombocytopenia were ranked according to the likelihood of having this disorder by using prospectively defined criteria. The test had a high specificity (99%); only one of 573 controls showed a positive result. The test was also very sensitive, and the likelihood of a positive test result was directly correlated with the clinical likelihood of the patient having heparin-induced thrombocytopenia. Six of six patients with definitive heparin-induced thrombocytopenia had positive test results, whereas zero of four patients in whom the diagnosis was unlikely had positive test results. The two-point test for heparin-induced thrombocytopenia represents a sensitive and specific test for this disorder. This test may be useful not only in confirming the diagnosis of this disorder but also may provide information about its pathogenesis.