Clinical Validation of a Radiolabeled PF-4 Staph-a Binding Assay for Confirming Heparin Induced Thrombocytopenia

Abstract Introduction:Confirmatory laboratory assays for heparin induced thrombocytopenia (HIT) can broadly be classified as functional which have high specificity and rely on activation of platelets by the platelet factor 4 (PF4)-heparin-IgG immune complex or as immune based assays that are relatively more sensitive. At the time when the clinician is evaluating heparin as a cause of thrombocytopenia the 4 "T" criteria are helpful. However, an increase in the patient's platelet count after the heparin has been stopped is critical for confirmation of the diagnosis. Methods: We developed a variation of a previously described technique (Newman Thromb Haemost 1998;80:292) and used clinical criteria as the standard for comparison to evaluate the assay. Radiolabeled 125-I-PF4 is incorporated into the immune complex of PF-4-heparin-immunoglobulin and the amount of radiolabeled immune complex is measured after binding to staphylococcal A protein sepharose (Staph-A). The hospitalized subjects medical record was reviewed to: measure a 4 "T" score, to determine if the patient's platelet count increased after heparin was stopped and to exclude other plausible causes of thrombocytopenia. Aim: The assay relies on the binding of the heparin immune complexes to Staph A. Staph A preferentially binds to larger as compared to smaller immune complexes and the larger complexes produce a greater degree of F(c) mediated platelet activation when compared with the smaller complexes. This suggests the hypothesis that a Staph A based assay will have have better specificity and sensitivity than the currently used methodologies. Results: 28 patient samples were evaluated. True positives were observed in 4 hospital patients and 4 known positives. 19 were true negatives and included 7 from hospital patients and 12 from thrombocytopenic patients who were not treated with heparin. 1 sample was negative in our assay, and was judged as false negative. Concordance between the radiolabeled PF-4 assay and the commercial PF-4 assay was observed in all the 28 patients. Conclusions: To date when judged using clinical criteria, the radiolabeled PF-4 assay correctly distinguished true positives and true negatives in 27 of the 28 samples. Disclosures No relevant conflicts of interest to declare.

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A Radiolabeled PF-4 Staph-a Binding Assay Proposed As a Solitary Method for Confirming Heparin Induced Thrombocytopenia

Blood ◽

10.1182/blood.v124.21.5011.5011 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5011-5011

Author(s):

Kenneth A. Schwartz ◽

John Davis ◽

Hemasri Tokala

Keyword(s):

Platelet Count ◽

Immune Complex ◽

Conflicts Of Interest ◽

High Specificity ◽

Care Hospital ◽

Platelet Factor ◽

Clinical Criteria ◽

T Score ◽

Specificity And Sensitivity ◽

Hospital Patients

Abstract Introduction:Heparin induced thrombocytopenia (HIT) is present in over 30% of normal subjects receiving heparin and the incidence of HIT may even be higher in patients with occlusive atherosclerotic disease. Confirmatory laboratory assays can broadly be classified as functional which have high specificity and rely on activation of platelets by the platelet factor 4 (FP4)-heparin-IgG immune complex or as immune based assays that are relatively more sensitive. At the time when the clinician is evaluating heparin as a cause of thrombocytopenia the 4 ”T” criteria are helpful. However, after the episode of decreased platelets is resolved, an increase in the patient’s platelet count is critical for confirmation of the diagnosis. Methods: We developed a variation of a previously described technique (Newman Thromb Haemost 1998;80:292) and used clinical criteria as the standard for comparison to evaluate the assay. Radiolabeled 125-I-PF4 is incorporated into the immune complex of PF-4-heparin-immunoglobulin and the amount of radiolabeled immune complex is measured after binding to Staphylococcal A Protein Sepharose. Our working hypothesis is that since this assay has both immune and binding components it would have better specificity and sensitivity than the currently used assays and as a single assay be more cost effective. The hospitalized subjects medical record was reviewed to; measure a 4 “T” score, to determine if the patient’s platelet count increased after heparin was stopped, to record the results of the hospitals’ assays for HIT and to ascertain what other causes of thrombocytopenia might be possible. Results: To date we have evaluated 20 samples. True positives were observed in 3 hospital patients and 4 known positives that were a gift from the Blood Center of S.E. Wisconsin. 10 true negatives included 5 from hospital patients and 5 from thrombocytopenic patients who were not treated with heparin. 3 samples were negative in our assay, had a 4 ”T” score of 6 and 5 and NA and had increased platelet counts after heparin was stopped and were judged as false negatives. However, these samples had high relative binding to the Staph-A-Sepharose in the sample without the addition of heparin suggesting that either the sample already had enough heparin from the patient’s treatment to form immune complexes or as has been recently reported another negatively charged molecule formed a complex with PF-4.(Warkentin Blood;2014;123:3651) Concordance between the radiolabeled PF-4 assay and the commercial PF-4 hospital assay was observed in 10 of 11 patients. Conclusions:To date when judged using clinical criteria, the radiolabeled PF-4 assay correctly distinguished true positives and true negatives in 17 of the 20 samples. This is an ongoing study with the goal of studying another 30 patients before the ASH December meeting. The addition of clinical criteria including the 4 “T” score and the increase in platelet count after stopping heparin to a review of the patient’s acute care hospital record is helpful in evaluating the reliability of the radiolabeled PF-4 assay as a predictor of HIT. Disclosures No relevant conflicts of interest to declare.

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Atomic Level Description of the Immune Complex That Causes Heparin-Induced Thrombocytopenia (HIT)

Blood ◽

10.1182/blood.v124.21.465.465 ◽

2014 ◽

Vol 124 (21) ◽

pp. 465-465

Author(s):

Zheng Cai ◽

Serge V. Yarovoi ◽

Zhiquiang Zhu ◽

Lubica Rauova ◽

Tatiana Lebedeva ◽

...

Keyword(s):

Immune Complex ◽

Immune Complexes ◽

Conflicts Of Interest ◽

Structural Basis ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

New Model ◽

Tetramer Formation ◽

Asymptomatic Patients

Abstract Heparin-induced thrombocytopenia (HIT) is thrombotic disorder caused by immune complexes containing antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). The structure of these immune complexes and how their composition might contribute to the difference between pathogenic and non-pathogenic anti-PF4 antibodies are unknown. To address these questions, we solved the crystal structures of human recombinant PF4 in complex with Fabs derived from KKO (a murine monoclonal HIT-like antibody that competes with pathogenic human HIT antibodies) and RTO (an isotype-matched non-HIT anti-PF4 antibody) combined with the crystal structure of PF4 complexed with the heparin-mimic pentasaccharide fondaparinux as a model sugar. The PF4 tetramer is asymmetric and is capable of accommodating only two fondaparinux molecules. Fondaparinux binds between monomers A, B and C or between monomers A, C, and D, which stabilizes the AB/CD and AC/BD associations and the resultant tetramer. KKO-Fab binds to the PF4 tetramer by making contacts with now identified residues within each of three PF4 monomers, indicating that tetramerization of PF4 is a critical initiating step in antigen formation. Mutations in the putative KKO epitopes in PF4 abolished antibody binding.Unexpectedly, RTO-Fab binds to the PF4 monomer between the AB dimer interface. Importantly, the amino acid sequence recognized by RTO and KKO show considerable overlap. However, the epitope for RTO is obscured upon tetramer formation, in direct contrast to binding of KKO, which requires tetramer formation to bind. Binding of RTO to the PF4 monomer prevents formation of AB dimers and subsequent tetramerization. In support of these findings, preincubation of PF4 with RTO inhibits KKO induced platelet activation and platelet aggregation in vitro. Based on the analyses of crystal lattices, we propose a new model of the heparin/PF4 complex, in which PF4 tetramers cluster around a semi-rigid linear heparin subunit. Clustering of PF4 on heparin might be required for apposition of sufficient HIT antibodies to induce persistent activation of cellular FcgIIA receptors. Heparin and pathogenic HIT antibodies collaborate to stabilize the ternary immune complex, which leads to the disappearance of binding sites for at least some non-pathogenic HIT antibodies. The balance between anti-monomer and anti-tetramer PF4 antibodies may help determine the probability of clinical disease. This model also helps to explain why RTO-like anti-PF4 antibodies are found so commonly in asymptomatic patients exposed to heparin and why fondaparinux may be antigenic but rarely causes HIT, whereas longer heparin fragments and GAGs extend and render the holo-complex more stable and thereby foster the formation of pathogenic immune complexes. In summary, these crystallographic studies lead to a new model to explain the formation of pathogenic immune complexes that lead to HIT. The inhibitory effect of the anti-PF4 antibody RTO provides a structural basis for the development of new diagnostics and non-anticoagulant therapeutics. Disclosures No relevant conflicts of interest to declare.

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Spontaneous Heparin-Induced Thrombocytopenia and COVID-19 Infection

Blood ◽

10.1182/blood-2021-151590 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4216-4216

Author(s):

Suneetha Amara

Keyword(s):

Platelet Count ◽

Conflicts Of Interest ◽

Hospitalized Patients ◽

Target Range ◽

Low Grade ◽

Primary Therapy ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

The Right ◽

D Dimer

Abstract Background and Objective: Heparin-induced thrombocytopenia (HIT) can develop if immune responses to infections become pathologic in the presence of heparins. Low molecular weight heparin or unfractionated heparin are recommended for prophylaxis and treatment of venous thromboembolic disease in hospitalized patients with Covid-19 infection but may trigger HIT. Our aim is to alert clinicians that HIT occurs in association with Covid-19 infections even in the absence of prior exposure and may not be easily recognized without a high index of suspicion. Case Summary: A 33-year-old previously healthy male was initially evaluated for low grade fever, dyspnea without hypoxia and cough. A Covid-19 PCR swab was negative despite a recent exposure. He was treated with azithromycin. However, his symptoms did not improve, he then developed right leg swelling and hypoxia, so he was re-evaluated. CTA of the chest showed bilateral pulmonary emboli and ground-glass opacities at the lung bases. Venous Duplex Ultrasound showed non-occlusive thrombus in the deep veins of right lower extremity. He was hospitalized and placed on oxygen and heparin. Covid-19 swab was negative again. Laboratory tests before heparin showed a decreased platelet count of 64,000 k/ul, elevated prothrombin time of 16.4 seconds, normal aPTT at 30.8 seconds, decreased serum fibrinogen at 120 mg/dl and markedly elevated D-dimer at 59,966 ng/ml. Lupus anticoagulant and anti-phospholipid antibody tests were negative. On heparin at the desired therapeutic aPTT target range, the right leg became significantly swollen and painful by day five. Platelet count had decreased further to 39,000 k/ul. Repeat doppler examination of the right leg now showed more severe and extensive deep venous thrombosis. D-dimer had increased to 125,133 ng/ml. The HIT 4T score was 4, suggesting intermediate probability. Rapid HIT immunoassays on 2 separate samples were positive. Heparin was discontinued and he was placed on argatroban. Serotonin release assays on 2 separate samples came back positive. Suspicion for Covid-19 infection remained high and so a Covid-19 serology sample was obtained which was positive for IgG. A repeat nasopharyngeal swab at this time turned positive. He did not receive any COVID specific treatments. As viability of his leg appeared threatened, he underwent right iliofemoral vein thrombectomy with arteriovenous fistula creation. He improved on argatroban and was transitioned to apixaban with gradual normalization of hemostasis laboratory parameters, improvement in hypoxemia and fading clinical symptoms, he was discharged home on day 15. Conclusion: Current consensus guidelines for thromboprophylaxis and treatment of thromboembolism in hospitalized patients with Covid-19 infection recommend heparins as primary therapy to reduce morbidity and mortality. However, our report in addition to the two previous reports of HIT in Covid-19 patients illustrate that HIT can be a complication in the setting of Covid-19 infection. Further, our report also highlights that HIT with thrombosis can occur in a spontaneous manner in the absence of prior heparin exposure, which has been so far studied only in bacterial infection with the hypothesis that Platelet factor 4 (PF4) can bind to negatively charged polysaccharides on the surface of bacteria, triggering an immune response. Disclosures No relevant conflicts of interest to declare.

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A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽

10.1182/blood.v126.23.10.10 ◽

2015 ◽

Vol 126 (23) ◽

pp. 10-10

Author(s):

Jose Perdomo ◽

Jaa Yien New ◽

Zohra Ahmadi ◽

Xing-Mai Jiang ◽

Beng H Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Immune Complexes ◽

Whole Blood ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Single Chain ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Micro Fluidics

Abstract Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions. Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec. Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1). Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition. Disclosures No relevant conflicts of interest to declare.

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Outcomes in Heparin-Induced Thrombocytopenia Managed with Direct Oral Anticoagulants

Blood ◽

10.1182/blood-2019-129485 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4975-4975 ◽

Cited By ~ 1

Author(s):

Nwabundo Anusim ◽

Filip Ionescu ◽

Anish S Konde ◽

Vishal Jindal ◽

Ruby Gupta ◽

...

Keyword(s):

Platelet Count ◽

Conflicts Of Interest ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Beaumont Hospital ◽

Coagulation Cascade ◽

Bleeding Events ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia

Background: Heparin-induced thrombocytopenia (HIT) occurs as a result of autoantibodies to the platelet factor 4 (PF4)-heparin complex, which activate the coagulation cascade with subsequent thrombosis. HIT can be fatal if not diagnosed and treated promptly with replacement of all heparin with non-heparin anticoagulants. Classic options are parenteral direct anti-thrombin agents which require intravenous administration and can prolong hospital stay. Direct oral anticoagulants (DOACs) address these inconveniences and are an interesting alternative. However, data regarding their efficacy and safety in HIT is limited. Methods: We retrospectively identified patients with HIT using ICD code 9: 289.84 and ICD code 10: D75.82 at Beaumont Hospital (Royal Oak) between December 2013 and December 2018. Only patients with HIT confirmed by positive serotonin release assay and managed with DOACs were included. Data regarding diagnostic tests, bleeding and thrombosis during the 30-day follow-up were recorded. Results: A total of 229 patients were identified using the ICD codes; only 8 patients had confirmed diagnosis of HIT and were treated with DOACs. The average age was 70 years (51-92 years); most were male (5, 62.5%) and Caucasian (6, 75%). The median optical density of PF4-heparin antibody was 1.97 (0.85-3.108). Six patients (75%) had confirmed HIT-associated thrombosis; one had negative Doppler ultrasonography of the lower extremities (upper extremities were not assessed) and one patient was not assessed for thrombosis. Seven patients (87.5%) received apixaban and one patient received rivaroxaban. The lowest platelet count prior to initiating DOAC was 40,000/microL, while three patients started DOACs when their platelet count was above 150,000/microL. Within the follow-up period, none of the patients on apixaban had bleeding episodes or clot progression. The only patient treated with rivaroxaban was re-admitted within one week of discharge for right upper extremity deep vein thrombosis. Unfortunately, this was the one patient who was not evaluated for thrombosis at the time of HIT diagnosis. Conclusion: In the 30 days following HIT diagnosis, treatment with apixaban resulted in adequate anticoagulation and was not associated with increased bleeding events despite relative thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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Neutrophil Activation and Netosis Are the Key Drivers of Thrombosis in Heparin-Induced Thrombocytopenia

Blood ◽

10.1182/blood-2018-99-116421 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 378-378

Author(s):

Jose Perdomo ◽

Halina HL Leung ◽

Zohra Ahmadi ◽

Yan Feng ◽

Freda H. Passam ◽

...

Keyword(s):

Immune Complexes ◽

Conflicts Of Interest ◽

Negative Impact ◽

Thrombus Formation ◽

Significant Proportion ◽

Extracellular Dna ◽

High Morbidity ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Histone 3

Abstract Adverse drug effects are common in clinical practice and often have a negative impact on patient safety. Heparin and heparin-derived drugs may induce an immune reaction, termed heparin-induced thrombocytopenia (HIT). HIT is mediated by IgG antibodies with specificity for heparin/platelet factor 4 (PF4) antigenic complexes. HIT is a hypercoagulable state which often causes severe and extensive thrombosis that results in high morbidity and mortality. The prevailing view is that these immune complexes activate platelets via FcγRIIa receptors leading to thrombocytopenia and thrombosis. Neutrophil extracellular traps (NETs) are DNA-containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non-immune disorders. Here we show that HIT immune complexes directly activate neutrophils via FcγRIIa and induce NETs formation. In addition, NETosis is also induced by activated platelets/neutrophil interactions mediated by P-selecting and PSGL-1. Ex vivo reconstitution of the HIT condition in a microfluidics system demonstrated the formation of thrombi rich in platelets, neutrophils, extracellular DNA and citrullinated histone 3 (Figure 1A, left panels). Neutrophil depletion abolished thrombus formation. Conversely neutrophil reconstitution restored thrombus deposition (Figure 1A, middle panels and right panels). Moreover, neutrophils alone treated with HIT IgG plus heparin formed thrombi containing extracellular DNA networks and citrullinated histone 3 on P-selectin coated channels (Figure 1B). Establishment of HIT in hFcγRIIa+/hPF4+ transgenic mice (HIT mice) using HIT patient's IgG or the HIT-like monoclonal antibody KKO recapitulated the hallmarks of NETosis: citrullinated histone 3, cell free DNA and MPO were detected in plasma and the presence of neutrophils, extracellular DNA and citrullinated histone 3 was found in lung thrombi. Low density neutrophils were also present in HIT mice treated with HIT IgG plus heparin but not in animals treated with control IgG. Treatment of HIT mice with DNase I or NETs formation inhibition with the PAD4 inhibitor GSK484 led to a dramatic decrease in thrombosis. This was corroborated by deletion of PAD4 in HIT mice. No thrombi were detected in hFcγRIIa+/hPF4+/PAD4-/- mice treated with HIT IgG and heparin (Figure 1C), indicating that NETs formation is required for thrombosis. Unlike thrombosis, thrombocytopenia was not affected by the absence of NETs formation, suggesting that these are separable processes. However, they are FcγRIIa-mediated mechanisms as anti-FcγRIIa antibodies abolished both processes. Analyses of sera from HIT patients revealed the presence of NETs markers and a significant proportion of neutrophils from patients with active HIT were undergoing NETosis. Our observations demonstrate that NETs formation is present in HIT and that it is essential for the development of thrombosis. Thrombocytopenia is not affected by the absence of NETosis. These findings suggest a new concept of the pathogenesis of thrombosis in HIT and as such are of clinical significance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Platelet Trends Are Not Predictive of Heparin Induced Thrombocytopenia in Patients on Extra Corporeal Membrane Oxygenation

Blood ◽

10.1182/blood-2018-99-115396 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3767-3767

Author(s):

Daniel Stapor ◽

Anil Rengan ◽

Mark Weiner ◽

Huaqing Zhao ◽

Nadia Ali

Keyword(s):

Platelet Count ◽

Conflicts Of Interest ◽

Small Sample ◽

Clinical Suspicion ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Membrane Oxygenation ◽

Increased Risk ◽

Test Result ◽

Over Time

Abstract Introduction: Extracorporeal membrane oxygenation (ECMO) is a form of a cardiopulmonary bypass circuit that is utilized for patients in dire need of cardiac or pulmonary support. The circuit exposes blood to artificial surfaces which leads to platelet activation, clotting and thrombocytopenia. The addition of heparin is required in order to prevent clots from forming along the circuit. Heparin-induced thrombocytopenia (HIT) and heparin induced thrombocytopenia and thrombosis (HITT) are serious adverse effects of heparin treatment characterized by formation of antibodies to heparin platelet factor 4 (PF4) complexed with heparin which leads to thrombocytopenia and a greatly increased risk of arterial and venous thrombosis. The well-established incidence of HIT ranges from 0.5-5% in patients exposed to heparin for more than four days. The reported incidence of HIT in ECMO patients ranges from less than 1% to 8.3%. However this data comes from only a few studies with small sample sizes. Our institution has a large lung center and as a result ECMO is utilized frequently. When patients are suspected to have HIT, they are taken off heparin and switched to a different anticoagulant. For patients without HIT, the switch to a different anticoagulant is unnecessary and exposes the patient to additional risks such as a higher risk of bleeding. Our aim was to calculate the incidence of HIT and HITT at our institution. Furthermore we wanted to examine the platelet trends of all patients on ECMO specifically comparing and contrasting the patients who are deemed true HIT positive vs. all other patients on ECMO. Methods: We conducted a retrospective analysis of all patients on ECMO at Temple University Hospital from July 1, 2012 to December 31, 2017. The data for this study was drawn from the T'PAU (Temple Population Access Utility) research data warehouse that has successfully merged data from the Epic EMR and the McKesson administrative system that houses important summary data on inpatient admissions going back through July 2012. Our inclusion criteria consisted of all patients on ECMO who had a clinical suspicion of HIT and thus had a screening test sent for HIT, the ELISA polyclonal IgG testing against PF4. The diagnosis of HIT or HITT was confirmed by PF4 antibody positivity with an optical density (OD) >1.0, serotonin release assay (SRA) positivity or a positive PF4 antibody test >0.4 and the presence of thrombosis. Linear mixed-effects model was used to estimate and compare platelet count change over time between true HIT patients and patients who tested negative for the HIT ELISA. Results: There were 164 patients on ECMO between July 1st, 2012 and December 31, 2017. A total of 77 patients had the HIT ELISA sent while on ECMO. Of those, 15 patients had a positive test result (OD > 0.4) and 62 had a negative test result. Of the patients with positive results, 5 patients had an OD > 1.0. Three of the 10 patients with an OD < 1.0 were found to have thrombosis. Therefore 8 patients had true HIT out of a total of 164 patients on ECMO, for an incidence of 4.88%. There was no difference of the change of platelet count over time between the HIT (n=8) and non-HIT (n=62) groups (P=0.98). Conclusion: The incidence of HIT in our ECMO patient population is similar to the incidence of HIT in all patients who have been exposed to heparin for four or more days. Patients requiring ECMO are very ill and with the accompanying thrombocytopenia that develops with the initiation of ECMO, the clinical suspicion for HIT is high. Unlike the majority of hospitalized patients, patients on ECMO are a unique population and platelet count trends are not necessarily a clinically reliable indicator for HIT. Our research supports that the full clinical picture should be taken in account when suspecting HIT in patients on ECMO, especially when considering a change in anticoagulation. Disclosures No relevant conflicts of interest to declare.

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Heparin-induced thrombocytopenia: when a low platelet count is a mandate for anticoagulation

Hematology ◽

10.1182/asheducation-2009.1.225 ◽

2009 ◽

Vol 2009 (1) ◽

pp. 225-232 ◽

Cited By ~ 14

Author(s):

Thomas L. Ortel

Keyword(s):

Platelet Count ◽

Heparin Therapy ◽

Severe Thrombocytopenia ◽

Drug Induced ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Immune Mediated ◽

Number Of Patients ◽

Increased Risk ◽

Antibody Levels

Abstract Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder caused by the development of antibodies to platelet factor 4 (PF4) and heparin. The thrombocytopenia is typically moderate, with a median platelet count nadir of ~50 to 60 × 109 platelets/L. Severe thrombocytopenia has been described in patients with HIT, and in these patients antibody levels are high and severe clinical outcomes have been reported (eg, disseminated intravascular coagulation with microvascular thrombosis). The timing of the thrombocytopenia in relation to the initiation of heparin therapy is critically important, with the platelet count beginning to drop within 5 to 10 days of starting heparin. A more rapid drop in the platelet count can occur in patients who have been recently exposed to heparin (within the preceding 3 months), due to preformed anti-heparin/PF4 antibodies. A delayed form of HIT has also been described that develops within days or weeks after the heparin has been discontinued. In contrast to other drug-induced thrombocytopenias, HIT is characterized by an increased risk for thromboembolic complications, primarily venous thromboembolism. Heparin and all heparin-containing products should be discontinued and an alternative, non-heparin anticoagulant initiated. Alternative agents that have been used effectively in patients with HIT include lepirudin, argatroban, bivalirudin, and danaparoid, although the last agent is not available in North America. Fondaparinux has been used in a small number of patients with HIT and generally appears to be safe. Warfarin therapy should not be initiated until the platelet count has recovered and the patient is systemically anticoagulated, and vitamin K should be administered to patients receiving warfarin at the time of diagnosis of HIT.

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Heparin-induced thrombocytopenia occurring after surgical treatment of atrial myxoma: A case report

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh0910540d ◽

2009 ◽

Vol 137 (9-10) ◽

pp. 540-544 ◽

Cited By ~ 1

Author(s):

Irena Djunic ◽

Dragica Tomin ◽

Nebojsa Antonijevic ◽

Sinisa Gradinac ◽

Mirjana Kovac ◽

...

Keyword(s):

Differential Diagnosis ◽

Platelet Count ◽

Correct Diagnosis ◽

Corticosteroid Treatment ◽

Clinical Score ◽

Atrial Myxoma ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Unfractioned Heparin ◽

Left Atrial Myxoma

Introduction Heparin-induced thrombocytopenia (HIT) is an acquired, prothrombotic disorder, caused by antibodies to a complex of heparin and platelet factor 4 (PF4) that activates platelets, resulting in the release of procoagulant microparticles, thrombocytopenia occurrence, generation of thrombin, and frequent thromboses. Case Outline We present a case of severe HIT in a 68-year-old female, which occurred after cardiosurgery of the left atrial myxoma with the aim to point out the importance of differential diagnosis of thrombocytopenia in patients recently exposed to heparin. Platelet count dropped on the eleventh postoperative day, six days after unfractioned heparin and enoxaparine treatment, to 4?109/l. The correct diagnosis failed to be made at first. Since thrombocytopenia remained refractory to a corticosteroid treatment and platelet transfusion, the patient was hospitalized on the 13th postoperative day at the Institute of Hematology. The diagnosis of HIT was confirmed with the high-probability clinical score (4T's) and strongly positive antiheparin-PF4 (PaGIA) test as well as positive platelet aggregation test. The treatment started with a smaller therapeutic doses of danaparoid than recommended of 750 U intravenous bolus and was followed by continuous infusions of 100 U per 1 h and intravenous gammaglobulins in full dosage for four days. The platelet count started to rise on the third day and it was completely normalized on the 5th day of the therapy. Conclusion Treatment of severe HIT with small doses of danaparoid supplemented by intravenous gamma globulin was successful. Additional awareness of heparin-induced thrombocytopenia is needed, especially of HIT in differential diagnosis of thrombocytopenia in patients recently exposed to heparin.

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Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms21072556 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2556

Author(s):

Elmira R. Mordakhanova ◽

Tatiana A. Nevzorova ◽

Gulnaz E. Synbulatova ◽

Lubica Rauova ◽

John W. Weisel ◽

...

Keyword(s):

Platelet Activation ◽

Immune Complexes ◽

Gel Filtration ◽

Human Platelets ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Mitochondrial Membrane Depolarization ◽

Low Platelet Count ◽

Apoptotic Pathways

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-XL, and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.

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