Immunologic Effects of Heparin Associated With Hemodialysis: Focus on Heparin-Induced Thrombocytopenia.

Intermittent hemodialysis (HD) is almost invariably performed with heparin, and thus HD patients are at risk of developing the immune-mediated adverse effect heparin-induced thrombocytopenia (HIT), caused by anti-platelet factor 4/heparin IgG, which strongly activates platelets. HIT patients develop hypercoagulability with greatly increased risk of thrombosis, both venous and arterial. Certain HIT-associated complications are more likely to develop among HD patients, including hemofilter thrombosis despite heparin, intravascular catheter and/or arteriovenous fistula-associated thrombosis, post-heparin bolus anaphylactoid/anaphylactic reactions, and thrombotic stroke and acute limb artery thrombosis (reflecting the high frequency of underlying arteriopathy in many patients with renal failure). Management of HIT in HD usually requires use of an alternative (non-heparin) anticoagulant; for example, danaparoid sodium (outside the USA) or argatroban (USA and elsewhere). Whether heparin-grafted hemodialyzers (without systemic heparin) can be used safely in acute HIT is unknown. The HIT immune response is remarkably transient and usually not retriggered by subsequent heparin administration. Accordingly, since renal failure patients often require long-term HD, there may be the opportunity-following seroreversion (loss of platelet-activating HIT antibodies)-to restart heparin for HD, a practice that appears to have a low likelihood of retriggering HIT.

Survey of Practice Pattern in Patients With Heparin-Induced Thrombocytopenia Requiring Cardiopulmonary Bypass.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse reaction to heparin. Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are routinely anticoagulated with heparin before the initiation of bypass. Heparin is contraindicated, however, in patients with acute HIT, and alternatives to routine practice are often used. While guidelines have recently been published addressing this topic 10, there remains variance between institutions in how these cases are treated. Our goal was to better delineate practice trends in the diagnosis and management of HIT patients requiring CPB. METHODS: We surveyed members of the Society of Cardiovascular Anesthesiologists (SCA) and the American Society for Extracorporeal Technology (AmSECT) using an online survey tool. RESULTS: We received 304 completed surveys (5.8% response rate), 75% completed by an anesthesiologist, and 24% by a perfusionist. The majority of respondents used clinical history and/or antibody testing (71% and 63%, respectively) to diagnose HIT. Seventy-five percent of respondents reported using an institutional protocol for HIT-CPB cases. Most respondents (89%) reported having at least 1 case in the last 3 years, with a total case experience of at least 785 cases (785 = the minimum number of cases in each case volume category × the number of respondents choosing that category). The strategy recommended in published guidelines, bivalirudin, was the most commonly reported alternative anticoagulation strategy (75%) used by respondents in HIT cases, with most (83%) using the activated clotting time (ACT) to monitor anticoagulation. CONCLUSIONS: Most responding SCA and AmSECT members reported that their institution used a protocol or guideline for HIT/CPB cases, and most guidelines directed the use of bivalirudin as an alternative anticoagulant. Various other methods such as plasmapheresis are also being used with success in this patient population. Further research, including comparison studies of alternative anticoagulant strategies, is required to elucidate the best approach to these difficult cases.

Vaccine-induced immune thrombotic thrombocytopenia: what we know and do not know.

The development of vaccines to fight COVID-19 has been a remarkable medical achievement. However, this global immunization effort has been complicated by a rare vaccine-related outcome characterized by thrombocytopenia and thrombosis in association with platelet-activating anti-platelet factor 4 antibodies. In this Spotlight, we will discuss the recently described complication of vaccine-induced immune thrombotic thrombocytopenia (VITT) occurring in response to certain COVID-19 vaccines. Although information about this clinical condition is rapidly evolving, we will summarize our current understanding of VITT.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex-mediated thrombotic disorders.

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.

A prospective, blinded study of a PF4-dependent assay for HIT diagnosis.

Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14-labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.

Near fatal stent thrombosis in an aneurysmatic RCX as first manifestation of heparin induced thrombocytopenia (HIT) without thrombocytopenia.

BACKGROUND: Thrombosis resulting from heparin-induced thrombocytopenia (HIT) occurs in about 2% of patients without a significant decrease in platelet counts. We report on such a near fatal thrombotic event caused by coronary intervention. CASE PRESENTATION: A supposedly "completely healthy" 53-year-old patient was admitted to hospital with covered rupture of an aneurysm of the Aorta descendens. He was successfully operated on and underwent coronary angiography due to NSTEMI six days later. Immediately after intervention of a 90% RCX stenosis he developed ventricular flutter, was defibrillated, and re-angiography showed partial occlusion of the RCX stent. Lots of white thrombi could be retrieved by aspiration catheter and gave reason for a HIT without thrombocytopenia. The detection of platelet factor 4/heparin complex antibodies by immunoassay supported and the subsequent Heparin Induced Platelet Activation Assay proved this diagnosis. CONCLUSIONS: The clinical event of an acute stent thrombosis should alarm the interventional team to the diagnosis of HIT even with a normal platelet count.

Heparin-Induced Thrombocytopenia: A Focus on Thrombosis.

Heparin-induced thrombocytopenia is an immune-mediated disorder caused by antibodies that recognize complexes of platelet factor 4 and heparin. Thrombosis is a central and unpredictable feature of this syndrome. Despite optimal management, disease morbidity and mortality from thrombosis remain high. The hypercoagulable state in heparin-induced thrombocytopenia is biologically distinct from other thrombophilic disorders in that clinical complications are directly attributable to circulating ultra-large immune complexes. In some individuals, ultra-large immune complexes elicit unchecked cellular procoagulant responses that culminate in thrombosis. To date, the clinical and biologic risk factors associated with thrombotic risk in heparin-induced thrombocytopenia remain elusive. This review will summarize our current understanding of thrombosis in heparin-induced thrombocytopenia with attention to its clinical features, cellular mechanisms, and its management.

Prospective evaluation of two specific IgG immunoassays (HemosIL® AcuStar HIT-IgG and HAT45G® ) for the diagnosis of heparin-induced thrombocytopenia: A Bayesian approach.

INTRODUCTION: The accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential to ensure adequate treatment and prevent complications. First step diagnosis test are immunoassays including enzyme-linked immunosorbent assays (ELISAs) and rapid immunoassays. METHODS: Using a Bayesian approach, we prospectively evaluated the performance of the IgG PF4/polyvinylsulfonate ELISA and a chemiluminescent immunoassay (CLIA), which are specific for IgG and use the same antigenic target to detect HIT antibodies. RESULTS: One hundred and eighty-four 184 consecutive patients with an intermediate (n = 159) or high (n = 25) clinical pretest probability of HIT based on the 4Ts score or platelet pattern were included. Both immunoassays (IAs) were performed on all 184 samples, and definite HIT was confirmed with a positive serotonin release assay in 29 patients (12.7%). The sensitivity (Ss) and negative predictive value (NPV) of ELISA were excellent (100%) allowing HIT to be excluded with good confidence when the test was negative. In addition, the Ss and NPV of the CLIA equalled 93.1% and 98.6%, respectively, as it was negative in two definite HIT. When the CLIA was negative, the post-test probability of HIT was 0.7% in case of intermediate risk. Although there was excellent agreement between CLIA and ELISA results, the quantitative values provided by the two IAs were not correlated. CONCLUSION: AcuStar HIT® detects more than 90% of HIT, as do all rapid IAs, and appears to be a good tool for excluding HIT when the pretest probability is intermediate. A chemiluminescent signal higher than 10 IU/mL is highly predictive of definite HIT with a PPV of 100%.

Heparin-induced thrombocytopenia: ELISA optical density value and 4T score in correlation with panel donor platelets activation in functional flow cytometric assay.

BACKGROUND: Serological assays for the diagnosis of heparin-induced thrombocytopenia (HIT) detect both platelet-activating and platelet non-activating anti-heparin/platelet factor 4 (PF4) antibodies and have therefore a limited positive predictive value. Functional assays confirm the presence of platelet-activating antibodies but require platelets from healthy donors, whose response to patient serum can differ. Our aim was to investigate the correlation between the level of anti-heparin/PF4 antibodies, 4T score, and the extent of panel donor platelet activation in the functional assay. MATERIALS AND METHODS: In total, 38 sera from enzyme immunoassays (ELISA) positive patients were tested against panel platelets obtained from 10 healthy, randomly selected donors, using our routine flow cytometry functional test for CD62P expression. Levels of anti-heparin/PF4 antibodies from medical and surgical patients and 4T pretest probability scores (where available) were correlated with the number of activated panel platelets. RESULTS: Sera with low ELISA optical density (OD) values (0.4-1) activated on average 5.6, sera with intermediate ELISA OD values (>1-2.5) activated on average 7.3, and sera with high ELISA OD values (>2.5) activated on average 8.6 out of 10 panel platelets. One serum with low 4T score did not activate donor platelets, 12 sera with intermediate 4T score activated on average 6.3 donors, 8 sera with high 4T score activated on average 8.5 panel platelets. DISCUSSION: Sera with higher ELISA OD values activated platelets from a higher number of platelet donors, independently of patient type (medical or surgical). The average number of activated panel platelets increased with rising 4T score. Results indicate that both donor platelet reactivity and quantity of anti-heparin/PF4 antibodies affect the result of the functional assay, meaning special attention is needed in platelet donor selection when testing sera with low levels of antibodies.

Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

Rapid and accurate Bayesian diagnosis of heparin-induced thrombocytopenia.

Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.

Production of a specific monoclonal antibody and a sensitive immunoassay for the detection of diphacinone in biological samples.

Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC50) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 µg/L, 0.32 µg/L, and 0.55 µg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis. Graphical abstract.

Heparin-induced thrombocytopenia: An international assessment of the quality of laboratory testing.

BACKGROUND: Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential to ensure timely treatment and prevent complications. Current diagnostic assays include enzyme-linked immunosorbent assays (ELISAs) and rapid immunoassays (RIs). RIs offer fast turnaround times but were not significantly represented in previous external proficiency testing challenges. OBJECTIVES: To use external proficiency testing to assess qualitative concordance for heparin/PF4 antibody detection. METHODS: From 2013 to 2017, the External Quality Control for Assays and Tests (ECAT) Foundation distributed 10 samples internationally. RESULTS: In total, 437 laboratories submitted 3149 results. ELISAs accounted for 1484 (47%) responses with RIs accounting for 1665 (53%) responses. RI use increased over the 5-year period. ELISAs classified 96% of both consensus positive and consensus negative samples concordantly. The coefficient of variation (CV) for positive sample optical densities (ODs) ranged from 35% to 50% when combining ELISA assay methods together. Quantitative RIs classified 97% of consensus-positive and 98% of consensus-negative samples concordantly. Qualitative RIs had a higher proportion of discordant responses and classified 88% of consensus-positive samples and 73% of consensus-negative samples concordantly. Of RIs only latex immunoassays and IgG specific chemiluminescent assays identified > 95% of samples concordantly with consensus. CONCLUSION: Quantitative RIs and ELISAs classify > 95% of samples concordantly. The ODs from different ELISA methods vary considerably and are not interchangeable. Qualitative RI use is increasing despite a greater proportion of discordant classifications. This includes a higher than expected number of negative classifications for consensus-positive samples among many RIs, challenging their use as "rule out" tests.

Reduced ß2-GPI is associated with increased platelet aggregation and activation in patients with prolonged isolated thrombocytopenia after allo-HSCT.

We aimed to measure platelet function and its relationship with ß2-GPI in prolonged isolated thrombocytopenia (PT) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fifty-six patients with PT and 60 allo-HSCT recipients without PT (non-PT controls) were enrolled. Platelet aggregation and activation, ß2-GPI and anti-ß2-GPI antibody levels, vWF antigen, and vWF activity were analyzed. The effect of ß2-GPI on platelet aggregation was also measured ex vivo. Results showed that ADP-induced platelet aggregation significantly increased (39%±7.5% vs. 23%±8.5%, P=0.032), and the platelet expression of both CD62p (33.6%±11.6% vs. 8.5%±3.5%, P<0.001) and PAC-1 (42.4%±7.6% vs. 6.8%±2.2%, P<0.001) was significantly higher in patients with PT than in those without PT. Significantly lower ß2-GPI levels (164.2±12 µg mL-1 vs. 234.2±16 µg mL-1, P<0.001), higher anti-ß2-GPI IgG levels (1.78±0.46 U mL-1 vs. 0.94±0.39 U mL-1, P<0.001), and increased vWF activity (133.06%±30.50% vs. 102.17%±25.90%, P<0.001) were observed in patients with PT than in those without PT. Both ADP-induced platelet aggregation (n=116, r2=-0.5042, P<0.001) and vWF activity (n=116, r2=-0.2872, P<0.001) were negatively correlated with ß2-GPI levels. In summary, our data suggested that platelet aggregation and activation were significantly higher in patients with PT than in those without PT, which might be associated with reduced ß2-GPI levels. The reduced ß2-GPI levels might be due to the existence of anti-ß2-GPI IgG.

Refractory Heparin-Induced Thrombocytopenia With Cerebral Venous Sinus Thrombosis Treated With IVIg, Steroids, and a Combination of Anticoagulants: A Case Report.

Heparin-induced thrombocytopenia (HIT) type II is caused by antibody production that bind complexes between heparin and platelet factor 4 leading to platelet consumption and thrombosis. In a small subset of cases referred to as autoimmune HIT, the antibodies activate platelets even in the absence of heparin. Refractory HIT is a type of autoimmune HIT in which thrombocytopenia persists for weeks after heparin discontinuation and carries increased risk for thrombosis and more severe thrombocytopenia. We present a case of refractory HIT with cerebral venous sinus thrombosis (CVST) that was successfully treated with a change in anticoagulant alongside steroids and a second trial of intravenous immunoglobulin (IVIg).

A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro.

Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.