Heparin Antibodies Research Articles

7648 Bilateral Adrenal Hemorrhage After Laminectomy: A Rare Complication

Abstract Disclosure: M. Rizk: None. S. Sharma: None. H. Qadeer: None. R. Paudel: None. C. Sherpa: None. Introduction: Bilateral Adrenal Hemorrhage (BAH) is an exceedingly uncommon complication of laminectomy, with few cases reported in the literature. In patients with history of any surgical procedure who present with unexplained symptoms of abdominal pain, back pain, hypotension, fever, confusion, or electrolyte abnormalities especially hyponatremia, acute adrenal insufficiency (AI) should be highly suspected. We herein describe a case of BAH following laminectomy. Clinical Case: 63-year-old male with past medical history of hypertriglyceridemia was admitted for post traumatic L1 burst fracture and was treated with T11-L3 fusion and T12-L3 laminectomy. The patient had normal bilateral adrenal glands after the fall on CT thoracic/lumbar spine imaging. The course was complicated by thrombocytopenia initially thought to be induced by therapeutic heparin since heparin antibody was positive. Serotonin-release assay returned negative weeks later ruling out heparin induced thrombocytopenia. Two weeks later, he developed unexplained tachycardia and CT angiogram was done which showed segmental pulmonary embolism and bilateral adrenal nodules measuring at least 3.9 cm on right side and 3.3 cm on left side. He was treated with Eliquis and was discharged home. Two weeks later, he was readmitted with weight loss of 30 lbs, weakness and altered mental status. His vital signs were significant for hypotension and tachycardia. Initial work up showed profound hyponatremia (sodium 121 mEq/L), hyperkalemia (potassium 6.1 mEq/L), hypoglycemia (Glucose 58 mg/dL), hypercalcemia (calcium 11.7 mg/dL) and acute kidney injury (creatinine 2.58 mg/dL). Repeat CT scan of chest/abdomen/pelvis without contrast showed bilateral hyperdense 4 cm adrenal mass/hematoma. Since initial CT of thorax/spine done after the fall showed normal adrenal glands, the new bilateral hyperdense adrenal masses noted post procedure is suggestive of adrenal hemorrhage/hematoma. Undetectable cortisol and high ACTH confirmed AI. Patient was treated with stress dose hydrocortisone followed by slow taper. MRI of pituitary gland showed partial empty Sella however since ACTH was high, empty sella was only an incidental finding and not the cause of AI. He is maintained on hydrocortisone 15 mg in the morning and 5 mg in the evening plus fludrocortisone 0.1 mg daily. His fatigue has improved, he is gaining weight and electrolytes are normal. Conclusions: This case report emphasizes the significance of timely diagnosis and management of BAH, which leads to primary AI. BAH is a rare complication following laminectomy. Clinicians should be aware of this possibility and consider it in the differential diagnosis of postoperative abdominal pain and electrolyte abnormalities, especially hyponatremia. Timely recognition and high index of suspicion should prompt empiric glucocorticoid replacement to decrease mortality. Presentation: 6/1/2024

Subsequent Vaccination against SARS-CoV-2 after Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but severe complication following vaccination with adenovirus vector-based COVID-19 vaccines. Antibodies directed against platelet factor 4 (PF4) are thought to be responsible for platelet activation and subsequent thromboembolic events in these patients. Since a single vaccination does not lead to sufficient immunization, subsequent vaccinations against COVID-19 have been recommended. However, concerns exist regarding the possible development of a new thromboembolic episode after subsequent vaccinations in VITT patients. Methods: We prospectively analyzed follow-up data from four VITT patients (three women and one man; median age, 44 years [range, 22 to 62 years]) who subsequently received additional COVID-19 vaccines. Platelet counts, anti-PF4/heparin antibody level measurements, and a functional platelet activation assay were performed at each follow-up visit. Additionally, we conducted a literature review and summarized similar reports on the outcome of subsequent vaccinations in patients with VITT. Results: The patients had developed thrombocytopenia and thrombosis 4 to 17 days after the first vaccination with ChAdOx1 nCoV-19. The optical densities (ODs) of anti-PF4/heparin antibodies decreased with time, and three out of four patients tested negative within 4 months. One patient remained positive even after 10 months post first vaccination. All four patients received an mRNA-based vaccine as a second vaccination against SARS-CoV-2. No significant drop in platelet count or new thromboembolic complications were observed during follow-up. We identified seven publications reporting subsequent COVID-19 vaccination in VITT patients. None of the patients developed thrombocytopenia or thrombosis after the subsequent vaccination. Conclusion: Subsequent vaccination with an mRNA vaccine appears to be safe in VITT patients.

Association of Metal Cations with the Anti-PF4/Heparin Antibody Response in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD405] of 0.93 (SD = 1.21) units, and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD405 ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody-negative patients. We observed statistical differences between antibody-positive and -negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody-positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts.

Combination of 2 Quantitative Immunoassays and Clinical Score Algorithm to Reduce False-Negative Results in Heparin-Induced Thrombocytopenia: Prevalence Study of Mauriziano Hospital in Turin, Italy.

Heparin-induced thrombocytopenia (HIT) is a serious adverse effect of heparin treatment caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Accurate diagnosis of HIT is essential but remains challenging. The aim of our study was to explore the performance of our optimized diagnostic laboratory algorithm, based on Chemiluminescence (CliA) and ELISA immunoassays, on suspected HIT patients. The study compared the prevalence of HIT diagnosis in A.O. Mauriziano with the literature. 163 consecutive patients were investigated for suspected HIT with CliA HemosIL Acustar HIT-IgG, Werfen. HIT was ruled out in all patients with CliA <0.13 U/mL. All patients with CliA >0.13 U/mL were further investigated with Zymutest-HIA anti-PF4 IgG ELISA immunoassay. In these patients, HIT was ruled out on the combination of CliA between 0.13 and 1.0 U/mL followed by ELISA assay <0.300 OD. HIT was ruled in patients whose plasma tested positive or doubtful with CliA and positive with ELISA immunoassay and confirmed positive with a platelet aggregation test (PAT). Suspicion of HIT was revealed with clinical 4Ts score or recent suggestive anamnestic history. Our diagnostic algorithm ruled out HIT diagnosis in 144/163 patients (88%) and predicted a positive PAT in 5/19 (26%) of CliA positive (4/5) or ELISA positive and CliA doubtful (1/5) patients. Our prevalence was 3.1%, comparable with the literature. The approach combining 2 quantitative immunoassays' (CliA and ELISA) results and 4Ts score probability was able to rule out the diagnosis within 1 h in 66% of patients with suspected HIT and within 24 h in 88% of patients. In the remaining 12% of cases, management decisions have to be based on individualized judgment while awaiting functional confirming results (48-72 h).

Low Incidence of Anti-PF4/Heparin Antibodies in Patients with Acute Myelogenous Leukemia.

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Association of Metal Cations with the Anti-PF4/Heparin Antibody Response in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD405] of 0.93 (SD = 1.21) units and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD405 ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody negative patients. We observed statistical differences between antibody positive and negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts.

The Binding of the SARS-CoV-2 Spike Protein to Platelet Factor 4: A Proposed Mechanism for the Generation of Pathogenic Antibodies.

Pathogenic platelet factor 4 (PF4) antibodies contributed to the abnormal coagulation profiles in COVID-19 and vaccinated patients. However, the mechanism of what triggers the body to produce these antibodies has not yet been clarified. Similar patterns and many comparable features between the COVID-19 virus and heparin-induced thrombocytopenia (HIT) have been reported. Previously, we identified a new mechanism of autoimmunity in HIT in which PF4-antibodies self-clustered PF4 and exposed binding epitopes for other pathogenic PF4/eparin antibodies. Here, we first proved that the SARS-CoV-2 spike protein (SP) also binds to PF4. The binding was evidenced by the increase in mass and optical intensity as observed through quartz crystal microbalance and immunosorbent assay, while the switching of the surface zeta potential caused by protein interactions and binding affinity of PF4-SP were evaluated by dynamic light scattering and isothermal spectral shift analysis. Based on our results, we proposed a mechanism for the generation of PF4 antibodies in COVID-19 patients. We further validated the changes in zeta potential and interaction affinity between PF4 and SP and found that their binding mechanism differs from ACE2-SP binding. Importantly, the PF4/SP complexes facilitate the binding of anti-PF4/Heparin antibodies. Our findings offer a fresh perspective on PF4 engagement with the SARS-CoV-2 SP, illuminating the role of PF4/SP complexes in severe thrombotic events.

Flow cytometry vs conventional methods for the evaluation of anti-PF4/heparin antibodies:a single center study.

Aim: Heparin-induced thrombocytopenia (HIT) is a rare, life-threatening, immune-mediated adverse effect of heparin administration. This study compares frequently used laboratory assays in terms of their effectiveness in HIT diagnosis.Materials & methods: Fifty patients with suspected HIT were tested by gel immunoassay and solid phase PF4/heparin antibody ELISA. On positive results, platelet activation markers P-selectin and Annexin V were assayed using flow cytometry.Results: Thirty/50 patients were negative for both immunoassays. Flow cytometry was performed in the 20 immunoassay positive patients. Platelet activation was observed in 7/20 in the presence of low heparin concentration (0.2IU/ml).Conclusion: The results are in accordance with the currently available literature and flow cytometry seems a promising alternative in HIT laboratory investigation.

Anti-PF4/Heparin Monoclonal Antibodies Induce Thrombus Formation in an Ex Vivo Endothelialized Microfluidic System

Introduction: Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin. Antibodies against platelet factor 4 (PF4) or a heparin/PF4 complex have been reported to activate platelets, neutrophils and possibly endothelial cells, resulting in thrombocytopenia, hypercoagulability and thromboembolic complications. While the impact of anti-PF4/heparin antibodies on platelet, neutrophil and monocyte activation has been extensively studied, the role of endothelial cells in HIT-associated thrombosis remains underexplored. In particular, the interactions between HIT-induced procoagulancy and endothelial cells under dynamic conditions is not completely analyzed. In this report, we investigated how HIT antibodies induce thrombosis in an endothelialized microfluidic system. Methods: Fibrinogen microfluidic channels were coated with confluent monolayer of human umbilical vein endothelial cells (HUVECs). Cells were primed with low-dose TNF-α to induce a sub-thrombotic endothelial activation, prior to perfusion of whole blood. Recalcified unstimulated or Thrombin Receptor Activator Peptide 6 (TRAP-6) activated whole blood was perfused at a venous flow rate. HIT-thrombosis model was established and tested with monoclonal anti-PF4/heparin antibodies in whole blood. In brief, whole blood was pre-incubated with unfractionated heparin (UFH, 0.2 IU/mL and 100 IU/mL). Next, anti-PF4/heparin antibodies were introduced to the whole blood mixture and incubated for 30 minutes. Whole blood was recalcified and perfused over resting or primed endothelial cells at a venous shear stress. Thrombus formation was recorded over time. Results: The endothelialized microfluidic model successfully captures sub-thrombotic conditions under venous shear. Activated platelets induced a procoagulant shift and three-dimensional thrombi. We applied our thrombosis model to investigate thrombus formation induced by a HIT mimicking monoclonal antibody. We show that the heparin-dependent platelet activation predicts the thrombotic response in the microfluidic system: HIT antibodies in absence of heparin did not exert a pro-thrombotic effect on activated endothelial cells. Co-incubation with 0.2 IU/mL UFH induced thrombosis, embolization of thrombus material and channel occlusion only when endothelial cells were primed with TNF-α. The pro-thrombotic effect of HIT antibodies was fully reversed at the super-therapeutic dose of 100 IU/mL UFH. Conclusions: HIT antibodies induce thrombosis and embolization in a system emulating physiological environment. For the first time, we present a comprehensive thrombosis model that incorporates the established thrombogenicity of HIT-mimicking antibodies. We show that our thrombosis model incorporates both endothelial- and blood-based control of thrombosis. Primed endothelial cells and antibody-induced procoagulancy trigger thrombosis in a concerted fashion, allowing investigation of the underlying mechanisms and possible preclinical evaluation of potential inhibitors of HIT-thrombosis. Figure 1, upper panel: Maximal thrombus surface area coverage of TNF-α primed endothelial cells, perfused with unstimulated, TRAP-6 activated or HIT-like IgG/heparin challenged whole blood. HIT-like IgG did not increase thrombus formation in absence of heparin. Addition of low-dose heparin exerted a strong pro-thrombotic effect, that was fully reversible with a super-therapeutic concentration of heparin. Lower panel: representative images of thrombi formed by HIT-like IgG in absence and under low or high-dose heparin. Platelets: green, Calcein-AM. Scale bar: 200 µm.

Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure

AbstractPlatelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

Laboratory and demographic predictors of functional assay positive status in suspected heparin-induced thrombocytopenia: A multicenter retrospective cohort study

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against platelet factor 4 (PF4) bound to heparin anticoagulants. A priori identification of patients at-risk for HIT remains elusive and a number of risk factors have been identified, but these associations and their effect sizes have limited validation in large cohorts of suspected HIT patients. The aim of this study was to investigate existing anti-PF4/heparin antibody thresholds and model the relationship of demographic variables and anti-PF4/heparin antibody levels with functional assay positivity across multiple institutions in the absence of detailed clinical data. In a large collection of suspected HIT patients (n = 8904), we tested for associations between laboratory and demographic variables and functional assay positive status as well as anti-PF4/heparin antibody levels. We also tested for correlation between IgG-specific and polyspecific (IgG/IgA/IgM) anti-PF4/heparin antibody values and their ability to predict functional assay positive status using area under the receiver operating characteristic (AUROC). Logistic regression identified increasing anti-PF4/heparin antibody OD levels (OR = 51.84 [37.27–74.34], p < 2.0 × 10−16) and female sex (OR = 1.47 [1.19–1.82], p = 3.5 × 10−4) as risk factors for positive functional assay in the largest cohort with consistent effect sizes in two other cohorts. In a subset of 1175 patients, polyspecific and IgG-specific anti-PF4/heparin antibody values were heterogeneous (mean coefficient of variation = 31.9 %), but strongly correlated (rho = 0.878; p < 2 × 10−16) with similar prediction of functional assay positivity (polyspecific AUROC = 0.976 and IgG-specific AUROC = 0.980). Thus, we recapitulate previously identified risk factors of functional assay positivity, providing precise effect sizes in a large observational population of suspected HIT patients. Our data reinforce the necessity of functional assay confirmation and suggest that, despite heterogeneity, polyspecific and IgG-specific anti-PF4/heparin antibody assays predict functional assay positive status similarly, even in the absence of 4Ts scores and detailed clinical data.

Reducing Inappropriate Simultaneous Ordering of Heparin Antibody and Serotonin Release Assays.

A 4T score with intermediate or high probability of heparin-induced thrombocytopenia prompts ordering of anti-platelet 4 heparin complex. If positive, a serotonin release assay (SRA) is recommended to confirm diagnosis. Despite these recommendations, overtesting of both anti-platelet 4 and SRA is highly prevalent. This was a quality improvement initiative using two forms of clinical decision support across 11 acute care hospitals. First, a 4T calculator was incorporated into anti-platelet 4 orders. Second, a Best Practice Advisory fired when anti-platelet 4 and SRA were ordered simultaneously, prompting the provider to remove the SRA order. Data were analyzed by a quasi-experimental interrupted time series linear regression comparing weekly average laboratory tests per 1,000 patient-days pre- and postintervention. Average ordering frequency of anti-platelet 4 changed from 0.508 to 0.510 per 1,000 patient-days (0.5%, p = 0.42) without significant slope or level differences. Average ordering frequency of SRA decreased from 0.430 to 0.289 per 1,000 patient-days (32.8%, p < 0.001) with a significant level difference of -0.128 orders per 1,000 patient-days (-31.2%, p < 0.05). A simultaneous Best Practice Advisory was effective in reducing SRA orders, but not anti-platelet 4 orders.

Abstract Number ‐ 233: Vaccine‐Induced Thrombotic Thrombocytopenia presenting as Massive Stroke: Case Report and Literature Review

Introduction Vaccines have been pivotal for the COVID‐19 pandemic. Rare adverse effects of the vaccines such as thrombosis have been observed. Here we report a case of an acute malignant ischemic stroke in a young healthy patient caused by thrombosis due to Vaccine‐Induced Thrombotic Thrombocytopenia (VITT). Methods Electronic chart review for a case report. Results A 43‐year‐old Caucasian female with a medical history of hypertension was found unresponsive on the morning of presentation. Her Last known normal was the night before. On arrival in the emergency department, she was globally aphasic, with left eye deviation, right‐sided neglect, right facial droop, and right‐side hemiplegia. Pupils were equal and reactive to light. NIHSS was 23. Head CT showed large left middle cerebral artery and anterior cerebral artery strokes with significant cerebral edema and midline shift. Head and neck CTA showed left ICA and left MCA occlusions. She was taken for decompressive craniectomy immediately. Ten days prior to the stroke, she received her first COVID vaccination. She smoked but did not take oral contraceptives. She did not have a family history of hypercoagulability. Stroke workup showed LDL 141, A1C 4.8%, and a negative COVID test. She had normal white and red blood cell counts but low platelet counts at 73,000, which was 233,000 one month prior. 2D echocardiogram showed an ejection fraction of 54% and no patent foramen ovale or thrombus. Lower extremity doppler showed deep vein thrombosis. Because of arterial and venous thrombosis with new thrombocytopenia, hematology was consulted. She was found to have positive anti–PF4–heparin antibody, leading to a VITT diagnosis. She received IVIG, rituximab, and steroid treatments, and her platelets gradually returned to the baseline. She was later transferred to a rehabilitation facility. Conclusions VITT is characterized by thrombosis and thrombocytopenia with positive PF4 antibodies after a median of 14 days post‐vaccination. It was reported in COVID vaccines from all manufacturers. The mechanism remains unclear. The current hypothesis describes a two‐hit process through which the vaccine triggers neoantigen formation (the first hit) followed by a systemic inflammatory response (the second hit). Incidence of VITT from COVID vaccinations is unknown but the reported cases were rare. VITT‐caused acute stroke, especially malignant strokes, is even rarer.Although the COVID vaccines can cause rare life‐threatening adverse events, they are essential for controlling the pandemic. When encountering an ischemic stroke patient with thrombocytopenia, we should consider VITT. Further study with post‐vaccination registration and monitoring is warranted.

Heparin-Induced Thrombocytopenia in Patients Undergoing Venoarterial Extracorporeal Membrane Oxygenation.

Background: Heparin-induced thrombocytopenia (HIT) is a serious, immune-mediated adverse drug reaction to unfractionated heparin (UFH) affecting also patients undergoing venoarterial extracorporeal membrane oxygenation (VA-ECMO). Although the association between VA-ECMO support and the development of thrombocytopenia has long been known and discussed, HIT as one underlying cause is still insufficiently understood. Therefore, the purpose of this study was to further investigate the epidemiology, mortality, diagnosis, and clinical management of HIT occurring in VA-ECMO patients treated with UFH. Methods: We conducted a retrospective single-center study including adult patients (≥18 years) with VA-ECMO support in the cardiac intensive care unit (ICU) of the University Hospital of Munich (LMU) between January 2013 and May 2022, excluding patients with a known history of HIT upon admission. Differences in baseline characteristics and clinical outcome between excluded HIT (positive anti-platelet factor 4 (PF4)/heparin antibody test but negative functional assay) and confirmed HIT (positive anti-PF4/heparin antibody test and positive functional assay) VA-ECMO patients as well as diagnosis and clinical management of HIT were analysed. Results: Among the 373 patients included, anti-PF4/heparin antibodies were detected in 53/373 (14.2%) patients. Functional HIT testing confirmed HIT in 13 cases (3.5%) and excluded HIT in 40 cases (10.7%), corresponding to a prevalence of confirmed HIT of 13/373 (3.5%) [1.6, 5.3] and a positive predictive value (PPV) of 24.5% for the antibody screening test. The platelet course including platelet recovery following argatroban initiation was similar between all groups. One-month mortality in patients with excluded HIT was 14/40 (35%) and 3-month mortality 17/40 (43%), compared to 5/13 (38%) (p > 0.999), and 6/13 (46%) (p > 0.999) in patients with confirmed HIT. Neurological outcome in both groups measured by the cerebral performance category of survivors on hospital discharge was similar, as well as adverse events during VA-ECMO therapy. Conclusions: With a prevalence of 3.5%, HIT is a non-frequent complication in patients on VA-ECMO and was not associated with a higher mortality rate. HIT was ultimately excluded by functional essay in 75% of VA-ECMO patients with clinical suspicion of HIT and positive anti-PF4/heparin antibody test. Argatroban seems to be an appropriate and safe therapeutic option for confirmed HIT-positive patients on VA-ECMO support.

Heparin-Induced Thrombocytopenia in Chronic Hemodialysis Patients.

Heparin-induced thrombocytopenia (HIT) is a disorder originating from exposure to heparin and has a spectrum of presentation ranging from asymptomatic positive antibodies to thrombotic complications. When symptomatic, it is associated with morbidity and mortality. The incidence of HIT in the ESRD population is yet to be defined. End-stage renal disease (ESRD) patients are at particular risk due to constant exposure to heparin. The main treatment of HIT is to avoid heparin and pursue alternative anticoagulants. Since 1 of the main advantages of heparin in ESRD patients is the ease of its use due to non-renal clearance, the use of alternative anticoagulants poses yet another challenge for this population due to cost, availability, and adverse effects on ESRD patients. Argatroban seems like the best alternative to heparin in hemodialysis (HD) patients due to its liver clearance. Despite having limited studies in HIT, direct oral anticoagulants (DOACs) were added as a potential treatment for HIT, with apixaban favored in kidney dysfunction as it is the least dependent on kidney clearance. Other treatment modalities exist but are still being studied in ESRD patients. The presence of HIT antibodies is not always associated with clinical syndrome, and some studies suggested that heparin antibodies are transient, and the reintroduction of heparin is still being evaluated as a treatment option. Hence, HIT is a challenging diagnosis in ESRD patients, a population that has frequent exposure to anticoagulants, and a risk/benefit ratio should be weighed between the risk of progression to symptomatic HIT and the benefit of switching to a non-heparin anticoagulant bearing in mind the difficulties associated with the latter.

Longitudinal Characteristics of Anti-Platelet Factor 4 Immunoglobulin G in Vaccine-Induced Immune Thrombotic Thrombocytopenia

Introduction: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenoviral vector-based vaccines against the SARS-CoV-2 virus. VITT typically presents as moderate to severe thrombocytopenia often accompanied by arterial and/or venous thrombi occurring in unusual locations. The thrombocytopenia and thrombosis seen in VITT are triggered by antibodies against platelet factor 4 (PF4, CXCL4), leading to the activation of platelets and inducing a hypercoagulable state, similar to what is observed in heparin-induced thrombocytopenia (HIT). In a previous study, we identified a unique VITT antibody binding site. involving amino acids located at the heparin binding region of PF4. In this study, we describe the change in VITT anti-PF4 IgG binding regions and binding response, as well as the ability of longitudinal patient samples to activate platelets at initial presentation and up to 109 days after vaccination with Vaxzevria AZD1222 (AstraZeneca ChAdOx1 nCoV-19). Methods: We longitudinally studied sera from VITT patients (n = 7) sent for confirmatory testing to the McMaster Platelet Immunology Laboratory. These VITT patient samples were received at time of diagnosis and follow up samples up to 109 days after vaccination (range 8 - 109 days). Confirmed VITT patients presented with clinical features including thrombocytopenia (platelet count < 150 x 109/L) with thrombotic manifestations and tested positive in both the anti-PF4 IgG/A/M immunoassay (positive optical density (OD) > 0.4) and in the PF4-serotonin release assay (PF4-SRA, positive release > 20%). Alanine scanning mutagenesis was used to identify the changes in antibody binding sites on PF4 from VITT patients at diagnosis and one follow-up time point. We also determined the change in binding responses and dissociation rates at time of diagnosis and follow-up anti-PF4 antibodies using biolayer interferometry (BLI). Results: Anti-PF4 immunoglobulin G antibody levels are positive and high at diagnosis (OD median 2.35, range 1.895 - 3.347) and decreased but remain relatively high and positive (OD >1.0, median 2.09, range 1.178 - 2.631) in all 7 VITT patients, longitudinally. In addition, most follow-up samples were weaker at activating platelets with or without exogenous PF4 compared to their initial sample. However, follow-up sera beyond 45 days still strongly activated platelets with PF4 in 2/7 (28.5%) VITT patients in the PF4-SRA. In 4/7 (57.1%) VITT patients, the number of amino acids on PF4 that continued to be important for VITT antibody binding decreased from 7 on average at time of diagnosis to 2 amino acids in the defined VITT binding site at follow-up. Binding responses to PF4 and PF4/heparin measured by BLI also decreased from initial diagnosis (PF4, median 1.872 nm, range 0.587 - 2.443 nm; PF4/heparin, median 1.362 nm, range 0.450 - 2.072 nm) when compared to follow-up (PF4, median 0.356 nm, range 0.052 - 1.770 nm; PF4/heparin, median 0.159 nm, range 0.006 - 1.206 nm) in all 7 VITT patient samples. Binding responses are a measure of abundance of antigen-specific antibodies present in a given sample. Lastly, we also measured dissociation rates of the antibodies with BLI, which reflects the avidity of an antibody for a given antigen. In similar trend as the binding responses, the dissociation rates of the anti-PF4 and anti-PF4/heparin antibodies decreased in 4/7 (57.1%) VITT patients at follow-up when compared to initial diagnosis. Conclusion: Despite persistent anti-PF4 antibody levels in VITT patients over time, there is reduced platelet activation possibly due to the loss of antibodies to the VITT binding region necessary for platelet activation, or due to a decrease in binding response and avidity. This study suggests that VITT is similar to HIT because over time, there is a decline in platelet activating anti-PF4 antibodies. Clinical implications of persistent anti-PF4 antibodies in VITT require further study.

Reduced risk of death in people with SARS-CoV-2 infection treated with remdesivir: a nested case–control study

Introduction Since the start of the SARS-CoV-2 pandemic, several treatment options have been proposed (e.g. steroids, heparin, antivirals and monoclonal antibodies). Remdesivir was the first antiviral approved for the treatment of COVID-19, even though controversial evidence exists concerning the efficacy. Therefore, we aimed to conduct a study to evaluate whether the use of remdesivir was associated with lower mortality in patients with COVID-19. Methods We conducted a nested case–control study of a retrospective cohort collecting medical records of people with SARS-CoV-2 infection admitted in the infectious Disease Unit of Sassari University Hospital (S.C. Clinica di Malattie Infettive, AOU di Sassari, Italy), or in the Infectious Disease Unit of Foggia (AOU “Ospedali Riuniti” Foggia), between 1 July 2020 and 10 November 2021. The outcome considered was death; thus, we matched death (cases) to survivors (controls) by sex and age (1:1). Results We included in the study 342 patients, with 171 deaths (cases) and 171 survivors (controls). Remdesivir was administered to 60 people in the control group and to 18 people in the case group (35.1% vs. 10.5%, p < .0001). In the multivariate analysis, treatment with remdesivir and heparin was associated with lower mortality (OR: 0.19 [95% CI :0.10–0.38], p <.0001; OR: 0.39 [95% CI: 0.21–0.74] p = .004, respectively). On the contrary, diabetes, oxygen therapy and CPAP/NIV were associated with higher mortality. Conclusion Our study showed lower mortality in people with SARS-CoV-2 infection treated with remdesivir.

Anti‐platelet factor 4/heparin antibodies in patients with Hantaan virus infection

BackgroundHemorrhagic fever with renal syndrome (HFRS) induced by Hantaan virus infection and heparin‐induced thrombocytopenia (HIT) are associated with symptoms such as thrombocytopenia and thrombosis. However, related molecules, such as anti–platelet factor 4 (PF4)/heparin antibodies, in patients with HFRS have not been evaluated. ObjectivesTo test plasma levels of anti‐PF4/heparin antibodies and study the possible role of these antibodies in HFRS pathogenesis. MethodsIndirect ELISA was used to determine plasma levels of anti‐PF4/heparin antibodies in 75 patients with HFRS and 20 normal controls. The 4Ts (thrombocytopenia, timing of platelet count fall, thrombosis or other sequelae, and other causes of thrombocytopenia) scoring system was used to determine the probability of HIT occurrence. A PF4‐enhanced platelet activation assay was used to detect the pathological effects of anti‐PF4/heparin antibodies. The laboratory/clinical features and viral load of all the patients were also assessed. ResultsOf the 75 patients with HFRS enrolled instudy, 69 had thrombocytopenia. Platelet count was negatively correlated with Hantaan viral load. Moreover, the optical density (OD) values of plasma antibodies against PF4/heparin in normal controls were less than 0.65, 4 patients tested strongly positive for anti‐PF4/heparin antibodies (OD values, 1.51–3.87), 21 patients were weakly positive (OD values, 0.66–0.74), and 50 patients were negative (OD values, 0.16–0.65). Moreover, all 4 patients who tested strongly positive for anti‐PF4/heparin antibodies showed a low probability of HIT (4Ts score of 3 or less) and had negative results in the PF4‐enhanced platelet activation assay. ConclusionsHantaan virus infection produces nonpathogenic antibodies against PF4/heparin; however, the generation mechanism of these antibodies requires further study.

Combinations of rapid immunoassays for a speedy diagnosis of heparin‐induced thrombocytopenia

BackgroundEarly recognition and treatment of heparin‐induced thrombocytopenia (HIT) are key to prevent severe complications. ObjectiveTo assess the diagnostic performance of rapid immunoassays (IA) in detecting anti‐PF4/heparin‐antibodies. MethodsDiagnostic performances of lateral‐flow IA (LFIA; STic Expert HIT) and latex IA (LIA; HemosIL HIT‐Ab) were analyzed in pilot (n = 74) and derivation cohorts (n = 267). Two novel algorithms based on the combination of HIT clinical probability with sequentially performed LIA and chemiluminescent IA (CLIA; HemosIL AcuStar‐HIT‐IgG) were compared with published rapid diagnostic algorithms: the “Lausanne algorithm” sequentially combining CLIA and particle‐gel IA (PaGIA) and the “Hamilton algorithm” based on simultaneously performed LIA and CLIA. ResultsLFIA missed 6/30 HIT. The sensitivity and specificity of LIA were 90.9% and 93.5%. The Lausanne algorithm correctly predicted HIT in 19/267 (7.1%), excluded it in 240/267 (89.9%), leaving 8/267 (3%) cases unsolved. The algorithm sequentially combining CLIA and LIA predicted HIT in 19/267 (7.1%) with 1/19 wrong prediction, excluded it in 236/267 (88.4%), leaving 11/267 (4.1%) cases unsolved. The algorithm employing LIA as a first assay predicted HIT in 22/267 (8.2%), excluded it in 235/267 (88%), leaving 9/267 (3.4%) cases unsolved. Finally, the Hamilton algorithm correctly predicted HIT in 10/267 (3.7%), excluded it in 229/267 (85.7%), leaving 28/267 (10.5%) cases unsolved. ConclusionLFIA cannot be used to exclude or predict HIT when using frozen plasma. A Bayesian approach sequentially employing two rapid immunoassays for anti‐PF4/heparin antibodies is most effective for the accurate diagnosis of HIT. Based on retrospective data, the combination LIA/CLIA is a candidate for a prospective validation.

The potential impact of Covid-19 on the capacity of routine laboratory tests to detect heparin-induced thrombocytopenia.

In Covid-19, anticoagulation with heparin is often administered to prevent or treat thromboembolic events. Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin treatment, caused by heparin-dependent, platelet activating anti-platelet factor 4 (PF4)/heparin antibodies. Diagnosis of HIT is based on the combination of clinical parameters, allowing to determine the pretest probability, and laboratory testing for anti-PF4/heparin antibodies and confirmatory functional assays, such as the heparin-induced platelet activation (HIPA) test.We report the case of a patient with severe Covid-19 pneumonia requiring ECMO treatment, who developed recurrent clotting of the ECMO filter and a drop in platelet count under heparin treatment. He was therefore suspected to have HIT and the anticoagulation was switched to argatroban. Despite high clinical probability and high titres of anti-PF4/heparin antibodies, the functional HIPA test was negative. Nevertheless, argatroban was continued rather than to reinstate anticoagulation with heparin. Reevaluation 7 days later then demonstrated a strongly positive functional HIPA test and confirmed the diagnosis of HIT. Under anticoagulation with argatroban the patient gradually improved and was finally weaned off the ECMO.In conclusion, this case highlights the critical importance of clinical judgement, exploiting the 4 T score, given that Covid-19 patients may present a different pattern of routine laboratory test results in HIT diagnostics. The possibility of a false negative HIPA test has to be considered, particularly in early phases of presentation. In cases of a discrepancy with high clinical probability of HIT and/or high titre anti-PF4/heparin antibodies despite a negative HIPA test, a reevaluation within 3 to 5 days after the initial test should be considered in order to avoid precipitant reestablishment of unfractionated heparin, with potentially fatal consequences.