High Heparin Research Articles

Heparinized self‐healing polymer coating with inflammation modulation for blood‐contacting biomedical devices

The current techniques for antithrombotic coating on blood‐contacting biomedical materials and devices are usually complex and lack practical feasibility with weak coating stability and low heparin immobilization. Here, a heparinized self‐healing polymer coating with inflammation modulation is introduced through thermal‐initiated radical copolymerization of methacrylate esterified heparin (MA‐heparin) with methyl methacrylate (MMA) and n‐butyl acrylate (nBA), followed by the anchoring of reactive oxygen species (ROS)‐responsive polyoxalate containing vanillyl alcohol (PVAX) onto the coating through esterification. The aspirin, which is readily dissolved in the solution of MMA and nBA, is encapsulated within the coating after copolymerization. The copolymerization of MA‐heparin with MMA and nBA significantly increases the heparin content of the coating, effectively inhibiting thrombosis and rendering the coating self‐healing to help maintain long‐term stability. ROS‐responsive PVAX and aspirin released in a temperature‐dependent manner resist acute and chronic inflammation, respectively. The heparinized self‐healing and inflammation‐modulated polymer coating exhibits the ability to confer long‐term stability and hemocompatibility to blood‐contacting biomedical materials and devices. Statement of significanceSurface engineering for blood‐contacting biomedical devices paves a successful way to reduce thrombotic and inflammatory complications. However, lack of effectiveness, long‐term stability and practical feasibility hinders the development and clinical application of existing strategies. Here we design a heparinized self‐healing and inflammation‐modulated polymer coating, which possesses high heparin level and self‐healing capability to maintain long‐term stability. The polymer coating is practically feasible to varied substrates and demonstrated to manipulate inflammation and prevent thrombosis both in vitro and in vivo. Our work provides a new method to develop coatings for blood‐contacting biomedical materials and devices with long‐term stability and hemocompatibility.

When HIT Testing Hurts- the Financial and Clinical Repercussions of Heparin Induced Thrombocytopenia Testing: A Single Center Analysis

Introduction: Thrombocytopenia is a common inpatient hematologic concern. Most often, thrombocytopenia is a multifactorial phenomenon, without one clear cause. Heparin Induced Thrombocytopenia (HIT) is a rare but serious hematologic consequence of exposure to heparin. In many hospitalized patients, heparin is routinely used as venous thromboembolism (VTE) prophylaxis or for therapeutic anticoagulation. The incidence of HIT is approximately 0.5-1.0% in patients exposed to unfractionated heparin (UFH). Though rare, there seems to be over-testing of HIT antibody in the inpatient setting. As such, there is potential for high cost with low yield and false positives that alter patient management, resulting in prolonged hospital stays and further complications in patient care. Methods: This was a retrospective, observational study reviewing the indications, results, and patient characteristics of cases in which HIT testing was ordered. Indications for testing and influence of results on management were determined based on clinical documentation. The tests of interest included HIT antibody testing and Seratonin Release Assay (SRA). We analyzed how many times a HIT antibody was positive with a negative result in the SRA. We also determined whether there was an increased length of stay in patients who had HIT testing ordered. The study population included patients who were admitted to University of Florida Shands Medical Center, an academic institution in Jacksonville, FL in 2022. All patients were >18 years of age. Results: 190 patients were identified, for whom 190 HIT antibody tests were ordered and 75 SRAs. All patients who had a SRA performed had exposure to low dose heparin or therapeutic high dose heparin. Out of 190 patients, HIT testing was positive in 30 patients (15.8%). SRA testing was performed on 75 patients despite only 30 positive HIT tests. The true positives were 10 patients who had positive SRA confirmatory testing out of 190 patients with suspected HIT. Based on the results of confirmatory testing, the false positive fraction of HIT testing was 66%. When analyzing the 190 patients who underwent HIT testing, the average length of stay was 14 days. On average, a hospital stay in the US is 5.5 days and evaluation of HIT results in 8.5 additional days of hospital admission at our institution. The prolonged length of stay for HIT testing is likely impacted by awaiting SRA results along with other factors. Conclusion: This analysis examines the relative over-testing for HIT at a single institution. Compared with the known incidence of positive HIT tests, there is a significant number of positive HIT antibody tests at our hospital. The data further reveals a high probability of a false positive HIT antibody test results in the hospitalized patient, possibly due to immune reactivity. Of 190 patients suspected with HIT, only 10 patients had a positive confirmatory SRA, indicating that only 5% of suspected patients truly had HIT. This demonstrates that 95% of the time HIT assays ordered did not provide clinically useful information. From the time work up was initiated to the time of discharge, the average stay length was 14 days in a patient suspected with HIT. Cost burden of HIT testing was evaluated. Each HIT panel is $153.83 and each confirmatory SRA test is approximately $250, at a commercial laboratory. Taken together, the total average cost per patient with suspected HIT is $413.83 in diagnostic testing. Prolonged hospital stay contributes to approximately $25,000 increase in hospital stay per patient being evaluated for HIT. This does not include the cost of further work up or interventions. Changing to an alternate anticoagulant such as Argatroban increased costs by nearly $1,500 per case and this number increased further when adding the daily coagulation panel required while on this drug. In other studies, this cost has been estimated to add $90,000 to the hospital stay. Prolonged hospital stays are also associated with unintended incidents such as GI bleeding leading to blood transfusions, invasive testing (such as endoscopy/colonoscopy) and increased morbidity to the patient. We will use this information to implement a tool in our Electronic Medical Record (EMR) that will require answering the 4T score in order to establish high pre-test probability, prior to ordering HIT antibody testing at the University of Florida in Jacksonville.

Decrease of prothrombin level during thrombolysis in acute myocardium infarction.

Previously, the direct interactions of Bβ26-42 fibrin residues with prothrombin were demonstrated. It was also shown that forming prothrombin complexes with E- or DDE-fragments causes non-enzymatic prothrombin activation. The direct measuring of the prothrombin level in the blood plasma of patients with acute myocardial infarction (AMI) allowed us to find a situation where such an activation can occur in vivo. Blood coagulation parameters in the blood plasma of patients with AMI were measured at 2 hours, three days, and seven days after the thrombolysis by streptokinase accompanied with intravenous administration of anticoagulants: unfractionated high molecular weight heparin (HMWH) and low-molecular-weight heparin (LMWH). The prothrombin level in the blood plasma of patients with AMI was normal before thrombolytic therapy and substantially decreased after streptokinase administration. This effect was prominent in the case of concomitant anticoagulant therapy with LMWH and was not observed when HMWH was applied. It can be explained by the fact that LMWH preferentially inhibits factor Xa, while the HMWH is an effective inhibitor of both factor Xa and thrombin. This observation suggested that the prothrombin level decrease was caused by the thrombin-like activity and possible autolysis of prothrombin by thrombin. Also, thrombolytic therapy with streptokinase caused the accumulation of fibrin degradation products (FDPs), some of which were able to bind prothrombin. The dramatic decrease of prothrombin level in the blood plasma of patients with AMI during thrombolysis allowed us to conclude the non-enzymatic prothrombin activation with the following autolysis of prothrombin that contributes to the pathology.

ELISA-Negative Platelet-Activating Anti-PF4 Antibodies Can Cause HIT and Monoclonal Gammopathy of Thrombotic Significance (MGTS)

We identified a patient with a recently described syndrome, monoclonal gammopathy of thrombotic significance (MGTS; Blood. 2023 Apr 6;141(14):1772-1776), and three additional patients with high 4Ts scores who were consistently negative in the PF4-polyanion HIT ELISA (Lifecodes PF4 IgG, Immucor). The MGTS patient, with a monoclonal IgG kappa, had recurrent thrombosis, and transient mild thrombocytopenia (nadir in the low 100s) over a period of years, and was noted to have a HIT ELISA optical density (OD) of 0.117. The three HIT patients experienced thrombocytopenia and thrombosis with ELISA ODs of 0.37, 0.18 and 0.20. All patients were positive in platelet-activation (functional) testing demonstrating PF4 dependence and inhibited with both high concentrations of heparin (100U/mL) and the FcγRIIa blocking monoclonal antibody (mAb) IV.3, consistent with the presence of anti-PF4 antibodies in the patient samples ( data not shown). Because the serologic profile of these patients suggested a novel HIT subtype characterized by negative HIT ELISA results, we undertook a functional platelet assay screen of 500 consecutive HIT ELISA-negative patients tested in our diagnostic laboratory. Research studies were approved by the Institutional Review Board of Mayo Clinic. Of the 500 patients tested, 20 (4.0%) stimulated platelet activation in the PF4-dependent P-selectin Expression Assay (PEA ≥19%; data not shown). To exclude non-specific activation that can be induced by HLA antibodies, additional testing was performed to evaluate whether platelet activation was PF4-dependent using buffer or PF4-treated platelets. Three patients demonstrated PF4-dependent activation that was inhibited by high heparin concentrations and mAb IV.3, as would be expected for HIT antibodies (ELISA-negative HIT, EN-HIT1-3; Figure 1). On retrospective review, all three patients thus identified had clinical histories and courses consistent with high clinical probability for HIT. To develop tools to study this new class of antibodies, monoclonal HIT antibodies were generated in mice after PF4/heparin immunizations and screened using a novel approach that included simultaneous testing of hybridoma supernatants in the PEA and PF4-polyanion ELISA. This strategy was designed to enabled us to identify mAbs that activated platelets (in the PEA), but that did not bind antigenic targets in the HIT ELISA. A novel mAb 431.4 that was developed using this approach, and the well-characterized HIT-like mAb KKO were evaluated for their abilities to both bind PF4-polyanion targets in ELISA, and to activate PF4-treated platelets in the PEA (Figure 2). In significant contrast to KKO, mAb 431.4 did not bind PF4/polyanion complexes, however, like KKO, strongly activated platelets in a PF4-dependent manner. Activation induced by both KKO and mAb 431.4 was inhibited by high heparin concentrations, as would be expected for HIT antibodies. In vivo murine studies using this novel mAb are pending at this time. In summary, the non-canonical diagnostic profile of HIT ELISA-negative but platelet activation-positive antibodies were found at a relatively high incidence in HIT-suspected patients (0.6%; 3 of 500 ELISA-negative patients). These findings have important diagnostic/management implications and suggest a re-evaluation of ASH's HIT management guidelines as noted in recommendations 2.7 and 2.8 of that document (“In patients with an intermediate (Recommendation 2.7) or high (Recommendation 2.8)-probability 4Ts score and a negative immunoassay, the ASH guideline panel recommends discontinuation of the non-heparin anticoagulant and resumption of heparin, if indicated ”; Blood Adv (2018) 2 (22): 3360-3392). Figure 1. A subset of ELISA-negative HIT-suspected patients harbor anti-PF4 platelet-activating antibodies. Three ELISA-negative (EN) HIT patient samples from a screen of 500 samples demonstrate PF4-dependent platelet activation and inhibition with high heparin concentration and mAb IV.3 incubation. PBS, phosphate buffered saline; IC, isotype control; HDH- high dose heparin (100U/mL). Figure 2. Murine mAb 431.4 serologically mimics pathogenic ELISA-negative HIT/MGTS antibodies. ELISA and PEA testing was performed using the “HIT-like” mAb KKO, novel mAb 431.4, or an isotype control (IC) antibody. PVS-polyvinyl sulfonate (polyanion used in the Immucor IgG ELISA assay).

Competence of Health Care Providers on Clinical Management of Myocardial Infarction in Selected Hospitals with Intensive Care Unit Western Kenya

Background: The capacity of healthcare providers to execute an accurate initial assessment is crucial for recognizing the urgency and treatment needs of patients and for creating baseline data from which any changes in patient status may be monitored. In Western Kenya, there is little evidence of studies carried out on the competence of healthcare providers in the clinical management of myocardial infractions thus this study's main aim was to investigate the competence of healthcare providers in the clinical management of myocardial infractions.
 Material and Methods: A cross-sectional analytical study design was used. The study population comprised a total sample of 114 healthcare providers. A structured questionnaire and observation checklist were used. Participants will be picked using a systematic sampling method for those who meet the inclusion criteria. The sample size was determined using Fisher's Method. The data collected was compiled using SPSS software version 22. The data was analyzed through the application of descriptive statistical analysis.
 Findings: Variables that positively influenced the competency are specialization (AOR: 15.1; 95% CI: 2.6 – 87.3; p = 0.002), knowledge (AOR: 5.4; 95% CI: 1.1 – 26.5; p = 0.04) and having a protocol in patient's file (AOR: 10.2; 95% CI: 0.9 – 112.6.5; p = 0.06), although the latter was marginally statistically significant. Healthcare provider's qualification (KRCHN) (AOR: 0.04; 95% CI: 0.008 – 0.219; p = 0.002), filling of the protocol (AOR: 0.03; 95% CI: 0.001 – 0.585; p = 0.02) and use of high dose heparin as opposed to the use of other thrombolytic like Metalase (AOR: 0.1; 95% CI: 0.01 – 0.63; p = 0.02) were all negatively associated with healthcare providers competency.
 Recommendation/Conclusion: The study recommends the need of specialized training and knowledge advancement for health care providers on critical care nursing. This study found marginal statistical significance between the factors that influence competence among healthcare providers on clinical management of myocardial infraction.

The HEPDOSE pilot study: the use of the PRODOSE algorithm to reverse the effects of the administration of initial high dose unfractionated heparin.

The HEPDOSE pilot study: the use of the PRODOSE algorithm to reverse the effects of the administration of initial high dose unfractionated heparin.

End-on PEGylation of heparin: Effect on anticoagulant activity and complexation with protamine

Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.

Sample stability and heparin interference in ionized calcium and ionized magnesium measurements in horses using the Stat Profile Prime Plus co-oximetry electrolyte analyzer.

The determination of iCa and iMg is important in veterinary medicine, but their immediate determination in whole blood is not always possible. Their stability in other sample types and the existence of interferences must be evaluated before its use. We aimed to analyze the effects of storage time on the stability of iCa, iMg, and other analytes in whole blood, plasma, and serum samples in horses and assess the interference of heparin in these measurements. Whole blood, heparin-plasma, and serum samples from 10 horses were stored at 4°C and analyzed 1, 2, 3, 4, 5, 6, 7, 8, 24, 48, and 168 hours after sample collection using the Stat Profile Prime Plus Vet equipment (Nova Biomedical, Waltham, MA, USA). Results were analyzed by ANOVA or mixed-effect models. The concentration of iCa, iMg, total calcium (tCa), total magnesium (tMg), and the ratios iCa/tCa and iMg/tMg did not differ up to 168 hours when compared to the initial time. Total Ca, iMg, and tMg were not significantly different among sample types, but iCa concentrations were slightly but significantly lower in plasma. Freezing at -20°C did not affect iCa, iMg, tCa, and tMg. The pH increased in serum and plasma after 8 hours, and a mild negative correlation existed between plasma iCa concentration and pH. A negative correlation was observed also between the ratios iCa/tCa or iMg/tMg and pH in plasma and serum. A significant decrease in iCa and iMg was detected when comparing homemade syringes at high heparin concentration (~200-300 U heparin/mL) and commercial lithium-heparin tubes (20-30 U/mL). Samples stored at 4°C can be used to determine iCa and iMg concentrations up to 7 days after collection. Other metabolites are stable for up to 8 hours; heparin interference should be taken into account if using homemade heparin syringes.

Standardization of HIT Diagnostic Assays, with K070, a chimeric human-mouse antibody, mimicking heparin dependent pathogenic antibodies

This study reports the K070 monoclonal antibody, a human-mouse recombinant chimeric monoclonal antibody, IgG1 isotype, targeted to Heparin and PF4 complexes, which mimics Heparin Induced Thrombocytopenia. This antibody has a strong reactivity to Heparin-PF4 complexes, and a much weaker one to PF4 alone. When plasmas spiked with this antibody at concentrations ≥ 0.5 µg/ml are tested at the standard immunoassay dilution, a dose dependent reactivity is obtained. This opens the perspective to use this material to calibrate immunoassays for a quantitative measurement of heparin dependent antibodies, when the disease is suspected. Furthermore, when plasmas are spiked at a higher concentration, from 20 to 200 µg/ml, they test positive in functional assays, like Platelet Aggregation/Activation Tests or Flow Cytometry methods, in presence of a low (0.1-1.0 IU/ml) but not a high (10-100 IU/ml) heparin concentration. In functional assays K070 reactivity level depends on the platelet rich plasmas used. In Platelet Aggregometry Tests some platelet rich plasmas are already positive when tested with plasmas supplemented with 20 µg/ml, whilst others require 100 to 200 µg/ml. At this latter concentration, all tested plasmas generate a strong positive responses with functional assays. K070 is then a useful antibody for calibration of immunoassays and standardization of functional methods used to diagnose Heparin Induced Thrombocytopenia.

Manifestation of High Endogenous Heparinization in Postpartum Hemorrhage Patient using Thromboelastography: New Avenue of Coagulopathy Monitoring

Manifestation of High Endogenous Heparinization in Postpartum Hemorrhage Patient using Thromboelastography: New Avenue of Coagulopathy Monitoring

560: A CASE REPORT ON SUPERIOR MESENTERIC ARTERY THROMBOSIS IN A COVID-19 PATIENT

Introduction: This case report discussed and reviewed an atypical presentation of COVID 19 involving superior mesenteric artery thrombosis with associated ischemic colitis. Thrombosis had been reported in up to 50 percent of patients with severe COVID -19. The pathophysiology of thrombosis in COVID 19 infection may include increasing blood viscosity and endothelial damage. Description: Case: A 59-year-old male with GERD, hiatal hernia, and diverticulitis was presented with ten days history of abdominal pain and vomiting, which later became coffee ground in nature. He was diagnosed with COVID 19 seven days before his presentation. Lab work showed hypokalemia with lactic acidosis, polycythemia, leukocytosis, thrombocytosis, and elevated D dimer. Esophagogastroduodenoscopy demonstrated actively bleeding Mallory Weiss tear successfully treated with bipolar circumactive probe cautery (BICAP). The patient’s abdominal pain worsened, and a repeat CTA abdomen revealed a superior mesenteric artery thrombosis with thickening of the distal small bowel and ascending colon. Vascular surgery was consulted, and performed catheter-assisted tPA thrombolysis to the SMA. Atrial fibrillation, diverticulitis, and other possible causes were eliminated as etiologies. Discussion: Acute mesenteric ischemia is a rare abdominal emergency. Due to rapid deterioration, early diagnosis and treatment are momentous for management. Severe abdominal pain and hematemesis are the keys to starting the evaluation. Initial investigation should include basic labs with a coagulation profile; the most common abnormalities are polycythemia, metabolic acidosis, lactic acidosis, and leukocytosis. CTA provides exquisite detail of the vascular anatomy and beneficial information regarding other bowel pathologies. After diagnosis, definitive management with fluid resuscitation, antibiotics, and IV high dose unfractionated heparin, if not contraindicated, should be initiated immediately. Recently, catheter-directed procedures for intravascular thrombectomy have been used with tPA. CONCLUSION: In our case, we attempted to emphasize the importance of a high index of suspicion with proper history, physical examination, and appropriate imaging for proper diagnosis and management of this life-threatening incident.

Plasma‐Assisted Surface Modification and Heparin Immobilization: Dual‐Functionalized Blood‐Contacting Biomaterials with Improved Hemocompatibility and Antibacterial Features

AbstractThe inferior hemocompatibility or antibacterial properties of blood‐contacting materials and devices are restraining factors that hinder their successful clinical utilization. To highlight these, a plasma‐enhanced modification strategy is favored for surface tailoring of an extensively used biomaterial, polypropylene (PP). The surface activation of the PPs is achieved by oxygen plasma etching and subsequent surface functionalization through amine‐rich precursor mediated coating by plasma glow discharge. After optimum plasma processing parameters are decided, heparin (anticoagulant and antithrombic drug) is either attached or covalently conjugated on the PPs’ surfaces. The aminated films produced at 75 W plasma power with 15 min exposure time are highly hydrophilic (34.72 ± 5.92°) and surface active (65.91 mJ m−2), facilitating high capacity heparin immobilization (≈440 µg cm−2) by covalent linkage. The kinetic‐blood coagulation rate and protein adhesion amount on the plasma‐mediated heparinized PPs are decreased about tenfold and 15‐fold, and platelet adhesion is markedly lowered. In addition, heparinized‐PP surfaces comprise superior antibacterial activity against gram‐positive/‐negative bacteria conveyed particularly by contact‐killing (99%). The heparin‐coating did not cause cytotoxicity on fibroblast cells, instead enhanced their proliferation, as shown by the (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide) assay. Overall, this simple methodology is highly proficient in becoming a universal strategy for developing dual‐functionalized blood‐contacting materials.

Rethinking the Gold Standard: Real-World Performance of the Serotonin Release Assay (SRA) and PF4-Dependent P-Selectin Expression Assay (PEA) in the Diagnosis of Heparin-Induced Thrombocytopenia (HIT)

Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin exposure with potential for significant morbidity and mortality. The serotonin release assay (SRA) has been considered the gold standard for HIT diagnosis. However, the SRA is technically challenging and results can be influenced by specimen handling and preparation. Therefore, the performance of the real-world assays may differ from published standards but reports of real-world performance are not widely available. A new assay, the PF4-dependent P-selectin expression assay (PEA), has demonstrated comparable accuracy in HIT diagnosis employing a simpler laboratory technique. Herein, we report the real-world performance of a reference laboratory SRA and PEA and describe how incorporation of the PEA into the testing algorithm positively influenced the HIT diagnostic process at a large academic medical center. Methods At the University of Alabama at Birmingham, we incorporated the PEA into HIT testing at provider discretion in patients with inconclusive SRA results starting January 1, 2020. Since July 15, 2020, we solidified the use of PEA in our algorithm (Figure 1) in which all patients with an inconclusive SRA had a PEA on the same specimen. A PEA could also be ordered ad hoc if the SRA result did not correlate with clinical suspicion for HIT. We retrospectively compared patients tested for HIT for 2 years before PEA use (January 1, 2018 to December 31, 2019; pre-PEA period) to the >2 years since using the PEA (January 1, 2020 to March 1, 2022; PEA period). All patients tested with PEA at our institution are included in Table 1. Laboratory testing: Enzyme immunoassay (EIA) was performed at our institution using Asserachrom® HPIA, Diagnostica Stago (France). The SRA and PEA assays were performed at Versiti (Milwaukee, WI). SRA was positive if there was >20% release of serotonin with low dose heparin (0.1 U/mL) and <20% release in with high dose heparin (100 U/mL). An inconclusive result was defined as a percentage of serotonin release not meeting criteria for a positive result (e.g., >20% release of serotonin with both low and high dose heparin, or some release but <20% with low dose along with inhibition with high dose). Statistical analysis Descriptive statistics were employed to examine the sample of patients with an inconclusive SRA result, comparing those with and without PEA testing. Bivariate analyses were conducted to compare testing patterns in the pre-PEA and PEA periods, and T-tests of significance were conducted to analyze assay results. All models were estimated using Stata 17 (StataCorp. 2019. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC). ResultsSRA results: In the pre-PEA and PEA periods, there were 1,252 and 1,403 EIAs performed, respectively. The rate of EIA positivity was similar in both groups (14% vs 15%, p=0.813). A total of 379 SRAs were sent in both periods, with 24 inconclusive results (6%, 15 pre-PEA and 9 PEA). PEA performance: In the 24 patients with inconclusive SRA results, HIT was diagnosed in 7 of 15 patients (47%) pre-PEA, compared to 3 of 9 patients (33%) in the PEA period. Table 1 presents the details of each case where PEA was used due to diagnostic uncertainty. Of the 9 patients with an inconclusive SRA result in the PEA period, PEA provided a definitive result in 8 cases (89%). In 2 cases, the PEA confirmed the SRA result, 1 positive and 1 negative. In 1 case with a positive SRA and low clinical suspicion, the PEA was negative. Conclusion Evaluation of the real-world performance of the SRA reveals that although inconclusive results are rare (6%), they can present a significant diagnostic challenge in centers with high testing volume. The PEA demonstrated reliable performance in patients with an inconclusive SRA, producing a definitive result in 89% of cases. The percentage of patients diagnosed with HIT after an inconclusive SRA result decreased with incorporation of the PEA (47% to 33%), suggesting that PEA use helped refute a HIT diagnosis when there was clinical uncertainty. Further studies are needed to determine the optimal testing algorithm to produce rapid, reliable HIT diagnosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rapidly evolving necrotic skin lesions of the face

Rapidly evolving necrotic skin lesions of the face

Heparin-induced thrombocytopenia: effect of heat inactivation of patient's serum on functional flow cytometric assay results.

Heat inactivation of a patient's sample is not systematically performed in the diagnostics of heparin-induced thrombocytopenia (HIT). Some authors recommend that the patient's sample is heat-inactivated to avoid the effect of thrombin on platelet activation in a functional assay. Others do not find this additional step essential or even advise against it. An enzyme-linked immunosorbent assay (ELISA) and flow cytometry-based functional assay with CD62P as a marker of platelet activation were performed. Forty-seven patients with suspected HIT and three healthy controls were included in the study. Each serum sample was divided into two aliquots: one was heat-inactivated and the other was not. Both aliquots were tested in parallel using the same donor platelets from four randomly selected individuals. We designed an index of platelet activation for both protocols to assess platelet activation in the assay and to compare the results. We observed a higher percentage of platelet activation in heat-inactivated compared to non-heat-inactivated sera. This phenomenon was seen in low and high heparin steps, although it did not occur for all samples. There were discrepant results in seven samples, which tested negative in the non-heat-inactivated protocol and positive in the heat-inactivated protocol. There was no case in which the result of a non-heat-inactivated aliquot was positive and the corresponding heat-inactivated aliquot was negative. Due to the higher percentages of donor platelet activation, all seven non-compliant cases met our current criteria for a positive result. However, those results were probably false-positive based on ELISA optical density value and 4T score. Therefore, in the current settings, heat inactivation of serum is not suitable for our flow cytometric functional assay since it can cause an elevated risk of creating false-positive results.

Modulation of assembly of TDP-43 low-complexity domain by heparin: From droplets to amyloid fibrils

TAR DNA-binding protein 43 (TDP-43) is an RNA-regulating protein that carries out many cellular functions through liquid-liquid phase separation (LLPS). The LLPS of TDP-43 is mediated by its C-terminal low-complexity domain (TDP43-LCD) corresponding to the region 267–414. In neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia, pathological inclusions of the TDP-43 are found that are rich in the C-terminal fragments of ∼25 and ∼35 kDa, of which TDP43-LCD is a part. Thus, understanding the assembly process of TDP43-LCD is essential, given its involvement in the formation of both functional liquid-like assemblies and solid- or gel-like pathological aggregates. Here, we show that the solution pH and salt modulate TDP43-LCD LLPS. A gradual reduction in the pH below its isoelectric point of 9.8 results in a monotonic decrease of TDP43-LCD LLPS due to charge-charge repulsion between monomers, while at pH 6 and below no LLPS was observed. The addition of heparin to TDP43-LCD solution at pH 6, at a 1:2 heparin-to-TDP43-LCD molar ratio, promotes TDP43-LCD LLPS, while at higher concentration, it disrupts LLPS through a reentrant phase transition. Upon incubation at pH 6, TDP43-LCD undergoes gelation without phase separation. However, in the reentrant regime in the presence of a high heparin concentration, it forms thick amyloid aggregates that are significantly more SDS resistant than the gel. The results indicate that the material nature of the TDP43-LCD assembly products can be modulated by heparin which is significant in the context of liquid-to-solid phase transition observed in TDP-43 proteinopathies. Our findings are also crucial in relation to similar transitions that could occur due to alteration in the molecular level interactions among various multivalent biomolecules involving other LCDs and RNAs.

Benefits and harms of direct oral anticoagulation and low molecular weight heparin for thromboprophylaxis in patients undergoing non-cardiac surgery: systematic review and network meta-analysis of randomised trials

ObjectiveTo systematically compare the effect of direct oral anticoagulants and low molecular weight heparin for thromboprophylaxis on the benefits and harms to patients undergoing non-cardiac surgery.DesignSystematic review and network meta-analysis...

Diagnostic Utility of High Dose Heparin Confirmation Step in Heparin Induced Thrombocytopenia ELISA Assay

Introduction:Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening condition that could occur following exposure to heparin. Accurate and timely diagnosis is critical for appropriate clinical management. Laboratory testing for suspected cases is based on screening for the presence of serum anti-PF4/heparin antibodies using solid-phase enzyme-linked immunosorbent assay (ELSIA) which is known to be sensitive but less specific. A positive ELISA test is followed by functional testing to demonstrate the platelet activating properties and heparin dependence of the pathogenic antibodies. Serotonin release assay (SRA) is considered the gold standard functional test for the diagnosis of HIT. In most anti-PF4/heparin ELISA assays, a high-dose heparin buffer (100U/mL) confirmation step is recommended to demonstrate heparin dependence of detected antibodies and increase the specificity of the assay. The necessity of this confirmation step is controversial with some reports suggesting it could lead to misinterpreting positive ELISA results as negative or indeterminate, especially in cases of very strong and high titer HIT activating antibodies. We hence aimed to investigate the utility of applying this confirmation step as part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA).Materials:Three hundred archived serum samples were tested using anti-PF4/heparin IgG antibody ELISA (Immucor Diagnostics, GA, USA). High-dose heparin (100U/mL) confirmation step was performed on all samples with OD units ≥0.4 as recommended by the manufacturer. Samples with OD ≥ 0.4 and ≥50% OD inhibition in the high dose heparin confirmation step are interpreted positive. Mayo-SRA results were compared to a reference 14C SRA method. The 4T clinical score was retrospectively calculated for all patient (range 0-8 points).Results:Of the 300 tested samples, 57 samples were interpreted positive by the anti-PF4/heparin screening ELISA. 33 of the 57 samples were positive using the reference 14C SRA method, whereas 43 samples were positive by Mayo-SRA assay (≥20% serotonin release). Three additional samples were positive by Mayo-SRA, but negative by both screening ELISA and the reference 14C SRA method. All samples with OD units ≥0.4 displayed >50% inhibition in the high-dose heparin regardless of the intensity of the initial OD value or the HIT 4T score, with the exception of one that was negative by both SRA methods and of 1.35 OD value and 6 4T HIT score (Fig-1A). Importantly, thirteen samples were anti-PF4/heparin positive, but SRA negative (by Mayo-SRA and reference method). These samples also displayed positive %heparin inhibition (≥50% OD inhibition) (Fig-1B). Lastly, there were no differences in the degree of %inhibition in samples positive by both reference and Mayo-SRA or Mayo-SRA only (Fig-1B).Conclusion:In our patient cohort, addition of the high dose heparin inhibition confirmation step to the screening anti-PF4/heparin ELISA assay was of no additional diagnostic utility. We hence propose eliminating the heparin inhibition step which would improve laboratory turnaround time, reduce costs, and importantly speed up urgent clinical management decisions.Figure Legend:Fig-1A. No correlation between initial OD values and %OD inhibition using high dose heparin. Scatter plot of all ELISA positive samples (OD ≥0.4) grouped according to OD values. Samples include all SRA-positive and thirteen SRA-negative ELISA-positive samples. Fig-1B. Scatter plot of %OD inhibition comparing samples positive by Mayo-SRA and reference SRA method, positive by Mayo-SRA only, and ELISA-positive SRA-negative by both methods. [Display omitted] DisclosuresPruthi: Bayer Healthcare AG: Honoraria; CSL Behring: Honoraria; Merck: Honoraria; Genentech: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Flow Cytometric Detection of Procoagulant Properties of Plasma from Patients with Clinically Confirmed Vaccine-Induced Immune Thrombotic Thrombocytopenia

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines including ChAdOx1 nCoV-19 (AstraZeneca) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor to drive thrombosis and the associated thrombocytopenia. Functional assays are important in the VITT diagnostic pathway as not all detectable PF4 antibodies are pathogenic. Detection of procoagulant platelets (platelets supporting thrombin generation) in presence of PF4 has been proposed as a diagnostic assay for VITT (Althaus et al). Procoagulant platelets are not typically generated in response to low level agonist stimulation; however, combination of ligand binding of G-protein coupled receptors (GPCR) (eg. PAR1) and ITAM linked receptors (eg. GPVI, CLEC2 and FcγRIIa) synergistically induce procoagulant platelet formation. Here, we describe an alternative flow cytometric assay to diagnose VITT. We hypothesized that priming of platelets with a PAR1 agonist at a level sufficient to release PF4, but insufficient to generate a significant procoagulant response in donor platelets, would provide a platform in which procoagulant response would be dependent on presence of FcγRIIa dependent procoagulant antibodies in patient plasma, without requirement for additional PF4.Methods: Our previously established flow cytometry-based procoagulant platelet assay (using cell death marker GSAO and P-selectin) was modified to incorporate exogenous patient plasma and performed on whole blood from healthy donors screened for FcγRIIa responsiveness (aggregation response to anti-CD9 antibody, ALB6), primed with 5 μM SFLLRN. The assay was performed on Australian patients referred for confirmatory VITT testing with probable VITT (confirmed thrombosis within 4-42 days of ChAdOx1 nCov-19 vaccination, D-Dimer > 5x ULN, platelets < 150 x 10 9/L or falling platelet count) after screening on PF4/heparin ELISA (Asserachrom HPIA IgG Assay, Stago Diagnostics). Procoagulant response was also measured in presence of 0.5 U/mL and 100 U/mL heparin, monoclonal FcγRIIa blocking antibody, IV.3, and intravenous immunoglobulin, IVIg. Some plasmas were incubated with ChAdOx1 nCoV-19 or SARS-CoV-2 spike protein. Flow cytometry positive patients were also tested by serotonin release assay (SRA) and multiplate aggregometry. Clinical correlation was obtained.Results: Citrated plasma from 49 unique patients with suspected VITT are reported. Plasma from ELISA+ve patients with clinical picture consistent with VITT (n=31), significantly increased the procoagulant platelet proportions in healthy donors in presence of 5 μM SFLLRN (p<0.0001, Figure 1A). This increase was not seen with plasma from healthy donors (n=14); or individuals exposed to ChAdOx1 nCov-19 vaccine without VITT: thrombocytopenic thrombosis patients who were ELISA-ve and SRA-ve (n=14); or low-level ELISA+ve patients without thrombocytopenia who were negative by either multiplate or SRA (n=4). The procoagulant platelet response induced by VITT positive plasma was reduced with low dose heparin (0.5 U/mL, p<0.01) except for 3 patients who showed a heparin-enhancing effect (Figure 1B). High dose heparin (100 U/mL, p<0.0001), IV.3 (10 µg/mL, p<0.0001) or IVIg (10 mg/mL, p<0.0001) abolished the procoagulant response (Figures 1C-D). The in vitro effect of IVIg was predictive of the in vivo response to IVIg therapy (Figure 1E). Addition of SARS-CoV-2 spike protein had no effect on the procoagulant platelet response. ChAdOx1 nCov-19 had an inconsistent effect on procoagulant platelet formation in presence of VITT plasma. Use of donors without a robust aggregation response to ALB6 resulted in false negative results.Conclusion: Induction of FcγRIIa dependent procoagulant response by patient plasma, suppressible by high dose heparin and IVIg, is highly indicative of VITT in the correct clinical circumstance. This assay modification of priming donor platelets from known FcγRIIa responsive donors with a GPCR agonist to potentiate the ITAM signaling from platelet activating immune complexes, results in a sensitive and specific assay. This may represent a functional platform that can be adopted into diagnostic laboratories to identify patients with platelet-activating antibodies and potentially predict treatment responses. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

No Diagnostic Utility of Zero Heparin Control Buffer in Serotonin Release Assay: Findings from a Validation Study

Background: Heparin induced thrombocytopenia (HIT) is a rare immune mediated complication that is triggered in a subset of patients temporal to therapeutic heparin exposure. Laboratory testing is based on screening for the presence of serum anti-PF4 antibodies using sensitive solid-phase immunoassays. If antibodies are detected, functional testing to demonstrate the platelet activating properties and heparin dependence of these immune complexes is then performed. Previous studies have reported possible clinical utility in identifying non heparin dependent platelet activating antibodies using a buffer control with zero heparin in the serotonin release functional assay (SRA). These reports suggested a correlation between reactivity in the zero heparin buffer control and pathogenicity of HIT antibodies which may define a subtype of HIT, referred to as autoimmune HIT. We aimed to investigate the utility of zero heparin buffer control as a part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA).Methods: Three hundred archived serum samples were tested using anti-PF4 IgG antibody enzyme-linked immunosorbent assay (ELISA; Immucor Diagnostics, GA, USA). SRA was preformed on all samples using Mayo-SRA and reference 14C SRA methods. Zero heparin control buffer was included in the Mayo-SRA assay. Serotonin release >20% in the low dose heparin (0.1U/mL, LDH) and ELISA optical density (OD) >0.4 were considered positive. Drug interference studies were performed by spiking known SRA-positive samples with increasing concentrations of unfractionated heparin (UFH), low molecular weight heparin (LMWH) or fondaparinux in the LDH and zero heparin SRA. The clinical 4T score was calculated retrospectively calculated for all patients.Results: Of the 300 tested samples, 57 were anti-PF4 ELISA positive. 33 of the 57 samples were positive using the reference 14C SRA method. Whereas 43 samples were positive by Mayo-SRA assay. Three additional samples were positive by Mayo-SRA, but negative by both screening anti-PF4 ELISA and the reference SRA method (Fig- 1A). Lastly, 13 samples were anti-PF4 ELISA positive, but SRA negative by both methods in comparison. Interestingly, 44 of 46 (95%) samples interpreted positive by LDH were also interpreted positive (serotonin release >20%) under zero heparin conditions. These included the 3 samples that were positive by Mayo-SRA but negative by both screening anti-PF4 ELISA and the reference SRA. The overall % serotonin release using zero heparin control was significantly lower (P= 0.003, paired Student T-test) compared to LDH (Fig-1B). In addition, zero heparin followed a similar pattern as LDH, with highest levels at ELISA OD units >2 (Fig-1C).Strikingly, drug interference studies showed artifactual serotonin release in the zero heparin reaction, which was not detected in the absence of spiked drugs. For UFH, serotonin release in zero heparin control occurred at very low spiked concentrations, ≥0.063 U/mL. LMWH and fondaparinux spiking experiments also displayed similar zero heparin serotonin release patterns (Fig-1D). Of note, none of these interferences were detected in UFH spiked SRA-negative samples (data not shown).Conclusion: Contrary to prior results suggesting that less than 50% of LDH SRA positive samples are also positive in the zero heparin SRA, our results show high zero heparin SRA positivity rate of >95%. Zero heparin SRA showed a pattern with highest levels at ELISA OD units >2 suggesting that reactivity in this condition is a function of antibody strength rather than a qualitative difference (i.e. “autoimmune” HIT antibodies vs “non-autoimmune” HIT antibodies). In addition, contamination of patient sera with small amounts of remnant heparin can significantly impact platelet activation in the zero heparin SRA test. Thus, zero heparin SRA positive results may be artifactual and represent residual heparin contained in the patient sample.Figure legend:Fig-1A. Scatter plots of SRA positive samples grouped by 4T scores. Red dots are Mayo-SRA only positive samples. Fig-1B and Fig-1C. Scatter plots of % serotonin release of Mayo-SRA positive samples at LDH (circles) vs. zero heparin buffers (squares), and grouped by ELISA OD values, respectively. Fig-1D. % Serotonin release of known SRA-positive samples spiked with increasing concentrations of UFH, LMWH, or fondaparinux at LDH (white) or zero heparin buffers (red). [Display omitted] DisclosuresPruthi: CSL Behring: Honoraria; Genentech: Honoraria; Bayer Healthcare AG: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.