Incidence and clinical relevance of heparin-induced antibodies in patients with deep vein thrombosis treated with unfractionated or low-molecular-weight heparin

The frequency of heparin-induced thrombocytopenia (HIT) varies between different clinical treatment settings and remains unknown for patients treated with unfractionated (UFH) or low-molecular-weight heparin (LMWH) because of deep vein thrombosis. In this multicentre, open-label study, 1137 patients with deep vein thrombosis were randomly assigned to UFH for 5-7 d, reviparin, a LMWH, for 5-7 d (short-treated group) or reviparin for 28 d (long-treated group). Heparin-platelet factor 4 antibodies (HPF4-A) were determined on d 5-7 and d 21. Heparin-induced thrombocytopenia was defined by clinical evaluation. Two patients in the UFH group (incidence: 0.53%, 95% confidence interval (CI): 0.06-1.91) and two patients in the long-treated LMWH group (incidence: 0.53%, 95% CI: 0.06-1.92) had HIT, while no HIT was observed in the short-treated LMWH group. Pulmonary embolism developed in one of the HIT-patients, who had HPF4-A and was treated with UFH. On d 5-7 the incidence of HPF4-A was 9.1% in the UFH group, 2.8% in the short-treated LMWH group and 3.7% in the long-treated LMWH group, with a significant increase to 20.7% in the UFH group and to 7.5% in the long-treated LMWH group on d 21. Therefore the incidence of HPF4-A and heparin-induced thrombocytopenia was lower in patients treated with LMWH compared with UFH for the same duration of treatment.     

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.     

Determination of heparin–platelet factor 4–IgG antibodies improves diagnosis of heparin-induced thrombocytopenia

Only a few patients with heparin-induced antibodies develop heparin-induced thrombocytopenia (HIT). In this study, we investigated whether different immunglobulin classes can be used to differentiate between antibody-positive patients with and without HIT. Four different patient populations were investigated: 32 patients with the immune type of HIT with thromboembolic complications, 13 patients with HIT without thromboembolism, 24 patients with heparin-platelet factor 4 (PF4) antibodies without clinical symptoms of HIT, and 20 heparin-treated patients with thrombocytopenia caused by other reasons. In all patients the immunglobulin mixture of IgG, IgM and IgA, and the single immunglobulin classes of heparin-PF4 antibodies, were investigated. No significant differences between HIT patients with thromboembolic complications and patients with isolated HIT were found concerning the different immunglobulin classes. Antibody-positive patients with HIT had significantly higher levels of IgG antibodies than those without HIT (P < 0.05), while they did not differ concerning IgM and IgA antibodies. By determining IgG antibodies, the specificity of the enzyme-linked immunosorbent assay (ELISA) system was increased without loss of sensitivity. Heparin-PF4-IgG antibodies can identify patients at risk of developing life-threatening HIT. Future ELISAs should only include this immunglobulin class, as the determination of the antibody mixture may lead to overestimation of HIT.     

Pathogenicity of IgA and/or IgM antibodies to heparin–PF4 complexes in patients with heparin-induced thrombocytopenia

Antibodies to heparin–PF4 (H-PF4) complexes have been tested and isotyped in 38 patients who developed severe heparin-induced thrombocytopenia (type II HIT). All the patients had a platelet count < 120 × 10⁹/l or a reduction of >30% of the initial value, occurring at least 5 d after the onset of heparin. Thrombocytopenia, which rapidly reversed following the withdrawal of heparin, was associated with thrombosis in nine patients. Although IgG isotypes were found in most cases ( n  = 26), the presence of only IgM and/or IgA was observed in 12 patients, including three cases showing a thrombotic complication. Our results indicate that type II HIT may be induced by IgA and/or IgM anti-H-PF4 antibodies even in the absence of IgG isotypes. This finding demonstrates that platelet Fc receptors (FcγRII) are not necessarily involved in the pathogenicity of heparin-dependent antibodies and emphasizes the major role of platelet PF4 receptors. The increased expression of the latter following a slight activation by thrombin, and the subsequent binding of IgM and IgA antibodies to H-PF4 on the platelet surface, may directly trigger platelet activation, aggregation and thrombosis. Alternatively, thrombocytopenia could be indirectly induced through the mediation of neutrophils, monocytes and lymphocytes which expose receptors for IgA (FcαR) or IgM (FcμR). IgM–platelet complexes may also bind and activate complement, leading to platelet activation or destruction. Moreover, the reactivity of the antibodies with glycosaminoglycans–PF4 complexes present on the endothelial surface could also induce endothelial lesions and promote procoagulant activity and predisposition to thrombosis.     

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.     

Pathogenicity of IgA and/or IgM antibodies to heparin–PF4 complexes in patients with heparin-induced thrombocytopenia

Antibodies to heparin–PF4 (H-PF4) complexes have been tested and isotyped in 38 patients who developed severe heparin-induced thrombocytopenia (type II HIT). All the patients had a platelet count < 120 × 10⁹/l or a reduction of >30% of the initial value, occurring at least 5 d after the onset of heparin. Thrombocytopenia, which rapidly reversed following the withdrawal of heparin, was associated with thrombosis in nine patients. Although IgG isotypes were found in most cases (n = 26), the presence of only IgM and/or IgA was observed in 12 patients, including three cases showing a thrombotic complication. Our results indicate that type II HIT may be induced by IgA and/or IgM anti-H-PF4 antibodies even in the absence of IgG isotypes. This finding demonstrates that platelet Fc receptors (FcγRII) are not necessarily involved in the pathogenicity of heparin-dependent antibodies and emphasizes the major role of platelet PF4 receptors. The increased expression of the latter following a slight activation by thrombin, and the subsequent binding of IgM and IgA antibodies to H-PF4 on the platelet surface, may directly trigger platelet activation, aggregation and thrombosis. Alternatively, thrombocytopenia could be indirectly induced through the mediation of neutrophils, monocytes and lymphocytes which expose receptors for IgA (FcαR) or IgM (FcμR). IgM–platelet complexes may also bind and activate complement, leading to platelet activation or destruction. Moreover, the reactivity of the antibodies with glycosaminoglycans–PF4 complexes present on the endothelial surface could also induce endothelial lesions and promote procoagulant activity and predisposition to thrombosis.     

Heparin-induced thrombocytopenia with thrombosis: Incidence,analysis of risk factors,and clinical outcomes in 108 consecutive patients treated at a single institution

Heparin-induced thrombocytopenia with thrombosis (HITT) can lead to serious morbidity and may be potentially fatal. We reviewed our experience with this entity over a 4-year period, to determine the following: 1) incidence and type of thrombosis in patients with heparin-induced thrombocytopenia (HIT), 2) clinical consequences of thrombosis, i.e., amputation, cerebrovascular accidents and death, 3) risk factors associated with development of thrombosis, and 4) impact of therapy on clinical outcomes in patients with HITT. Between 1991–1994, 108 patients were diagnosed to have HIT by heparin-induced platelet aggregation test. Thirty-two (29%) of these developed thrombotic complications, of which 20 were venous, 8 arterial, and 4 both. Five of the 32 died, 3 underwent amputations, and 3 had cerebrovascular accidents. The patients who developed thrombotic complications, when compared to those with HIT alone, were older (68.7 ± 11.5 vs. 63.3 ± 16 years, P = .05), had more severe thrombocytopenia (platelet count 46,300 ± 30,400/mm³ vs. 62,500 ± 34,400/mm³, P = .02), and developed it earlier (6.0 ± 2.9 vs. 7.4 ± 3.1 days, P = .03). Multivariate analysis showed that severity of thrombocytopenia and early fall in platelet count were independent risk factors for development of thrombotic complications. We did not find an association between development of thrombosis and clinical events (myocardial infarction, cardiac procedures or surgery, noncardiac surgery, and sepsis) that occurred immediately prior to onset of thrombocytopenia. Heparin was stopped in all 32 patients with HITT. Six received no additional therapy, and one received a single dose of aspirin. Three of these 7 died. The other 25 received anticoagulant or multiagent therapy, with 2 deaths. The death rate was lower in those who were treated with anticoagulant or multiagent therapy (P = .05). We conclude that: 1) Thrombotic complications occur in about 29% of hospitalized patients who develop HIT. 2) Early, severe fall in platelet count in elderly patients receiving heparin appears to be associated with development of thrombotic complications. 3) Our data do not show an association between development of thrombotic complications and clinical events immediately preceding the diagnosis of HIT. 4) In addition to discontinuation of heparin, anticoagulant or thrombolytic therapy should be considered in patients with HITT. Am. J. Hematol. 56:12–16, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Spontaneous heparin-induced thrombocytopenia syndrome without any proximate heparin exposure,infection, or inflammatory condition: Atypical clinical features with heparin-dependent platelet activating antibodies

Recent studies suggest that a thromboembolic disorder resembling heparin-induced thrombocytopenia (HIT), so-called spontaneous HIT syndrome, can occur in patients without any history of heparin exposure. It is likely due to anti-platelet factor 4 (PF4)/polyanion antibodies induced by other polyanions, such as bacterial surfaces and nucleic acids. We describe an atypical case of spontaneous HIT syndrome. A 70-year-old man suddenly presented with acute cerebral sinus thrombosis (CST). Soon after the initiation of unfractionated heparin (UFH) for the treatment of CST, his platelet count fell precipitously and he developed deep vein thrombosis, a clinical picture consistent with rapid-onset HIT but without any proximate episodes of heparin exposure, infection, trauma, surgery, or other acute illness. Antigen assays and a washed platelet activation assay indicated that the patient already possessed anti-PF4/heparin IgG antibodies with heparin-dependent platelet activation properties on admission. Cessation of UFH and initiation of argatroban resulted in prompt recovery of his platelet count without further thromboembolic events. We identified two similar cases in the literature. However, these patients do not meet the recently proposed criteria for spontaneous HIT syndrome. Even in atypical cases, however, inappropriate or delayed diagnosis of HIT appears to be associated with worse outcomes. We propose that these atypical cases should be included in the category of spontaneous HIT syndrome.     

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.     

The new ID-heparin/PF4 antibody test for rapid detection of heparin-induced antibodies in comparison with functional and antigenic assays

Heparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin treatment. Several in vitro assays are available to detect the causative HIT antibodies: functional assays, usually requiring freshly prepared platelets and immunological tests based on the enzyme-linked immunosorbent assay (ELISA) principle. We compared a new, simple and rapid test based on the ID-microtyping particle agglutination system with 14C-serotonin release assay, heparin-induced platelet activation (HIPA) test and two ELISAs. Sera from 100 confirmed HIT patients, 20 serologically negative suspected HIT patients and 20 healthy blood donors were used. The specificity and sensitivity of the new test was similar to the functional assays. Compared with the ELISAs, specificity was better at the cost of reduced sensitivity. As in all other immunological tests, HIT antibodies against less typical antigens, such as interleukin (IL)-8 or neutrophil-activating peptide (NAP) 2 could not be detected. Thus, although the ID-Heparin/PF4 antibody test seems to be a quick, reliable and robust test to determine the presence of HIT antibodies, it should still be combined with a functional assay if possible. Evaluation of the test in a prospective setting as well as interlaboratory variation should be assessed as a next step.     

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.     

Beneficial effect of exogenous platelet factor 4 for detecting pathogenic heparin‐induced thrombocytopenia antibodies

The laboratory diagnosis of heparin‐induced thrombocytopenia (HIT) is based on an enzyme immunoassay combined with a functional test, and serotonin release assay (SRA) is the gold standard for detecting activating HIT antibodies. However, a recent atypical history of HIT prompted us to evaluate whether addition of platelet factor 4 (PF4) during SRA could improve its ability to detect pathogenic HIT antibodies. Using 5B9, a monoclonal antibody to PF4/H with a human Fc fragment, we first defined the optimal PF4 concentration for detecting low amounts of platelet‐activating IgG with SRA. Plasma samples from 50 patients with suspected HIT were then studied, and SRA was positive in 17 cases (Group SRA^pos), with relatively high levels of PF4‐specific IgG (median optical density = 2·66). SRA was also systematically performed after adding 10 μg/ml of PF4 in the reaction mixture, and significant serotonin release was measured with samples from 9 additional patients (Group PF4‐SRA^pos). Importantly, levels of PF4‐specific IgG were similar in these samples and those from the 24 persistently SRA negative patients. Moreover, the pre‐test probability of HIT was intermediate/high in all ‘SRA^pos’ or ‘SRA‐PF4^pos’ patients. In conclusion, addition of exogenous PF4 might improve the detection of pathogenic HIT antibodies by SRA.     

Established venous thromboembolism therapies: heparin, low molecular weight heparins, and vitamin K antagonists, with a discussion of heparin-induced thrombocytopenia

For a majority of patients with venous thromboembolism (VTE), initial treatment is straightforward and necessitates the immediate initiation of a parenteral anticoagulant (eg, heparin or low molecular weight heparin), simultaneous initiation of long-term therapy (eg, vitamin K antagonist), and discontinuation of the parenteral anticoagulant after 5 days assuming that the vitamin K antagonist is therapeutic. This standardized approach is based on numerous pivotal clinical trials completed over the past 3 decades. Yet, advances in standardized VTE treatment continue to evolve and include issues related to the selection and dosing of parenteral anticoagulants (eg, relative efficacy and dosing in the obese patient, patients with renal impairment, and pregnant patients), optimal location of initial care delivery, use of dosing initiation nomograms for vitamin K antagonists with the potential of gene-based dosing, and demonstration that longterm low molecular weight heparin therapy may be optimal for some patient populations (eg, those with active cancer). Further, in parallel with the evolution of VTE treatment, there have been remarkable advances in our understanding of heparin-induced thrombocytopenia, a prothrombotic complication of parenteral anticoagulant use.     

Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies

Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin.     

Recurrent pneumococcal infections in a patient with lack of specific IgG and IgM pneumococcal antibodies and deficiency of serum IgA, IgG2 and IgG4

Disseminated pneumococcal infections in a young woman are described. Serum from the patient showed reduced opsonic capacity for pneumococci and absence of pneumococcal anticapsular IgG and IgM antibodies even after pneumococcal immunization. Total serum IgG level was normal, but IgG2, IgG4, and IgA were deficient. The possible location of pneumococcal IgG antibodies in the IgG2 subclass is discussed as well as the existence of serious antibody deficiencies in patients with normal total IgG levels.     

Analysis of immunoglobulin class, IgG subclass and titre of HPA-1a antibodies in alloimmunized mothers giving birth to babies with or without neonatal alloimmune thrombocytopenia

We analysed the titre and isotype composition of antibodies produced by mothers giving birth to babies with or without neonatal alloimmune thrombocytopenic purpura (NAITP) and patients with post-transfusion purpura (PTP). All these individuals produced an antibody specific for the HPA-1a allotype present on the platelet glycoprotein IIb-IIIa (GPIIb-IIIa). Sera from mothers who gave birth to thrombocytopenic babies (group 1, n = 36), non-thrombo-cytopenic babies (group 2, n = 4) or from PTP patients (group 3, n = 3) were tested by an indirect-ELISA. Results indicated no evident differences in the isotype composition or titre of the antibodies from the three groups of sera. The antibody titre ranged from 1: 120 to 1: 3500. Antibodies with the IgGl subclass were present in all sera. Most sera contained IgGl alone (24/43 sera tested) or in combination with IgG3 (10/43). IgG2 was never present and only three sera showed intermediate reactivity with anti-IgG4 MAb. Few sera (nine sera from groups 1 and 2) were weakly positive when tested with the anti-IgM antibodies. These results suggest that neither the titre nor the isotype composition can be used to predict the severity or the occurrence of thrombocytopenia in newborns.