Donor and Storage Duration Impact Cold-Stored Platelet Function More Than Handling Protocols

Cold storage of platelets has the potential to mitigate the logistical and financial burdens associated with platelet inventory management. In addition, cold-stored platelets (CS-PLT, stored at 2-6°C) have better preserved hemostatic function than room temperature (RT)-stored platelets (RT-PLT), suggesting that CS-PLT may provide improved hemostatic resuscitation in actively bleeding patients as compared to RT-PLT when transfused. With CS-PLT clinical use on the rise, questions regarding CS-PLT inventory management have been raised - specifically how should CS-PLT be stored and handled over the course of storage (after collection and prior to transfusion). RT-PLT are currently stored flat on specialized porous racks and agitated at 18-22°C, which allows for oxygenation. These products are stored out to 5 days, with 7 days allowed for large volume delayed sampling collections. In contrast, refrigerated blood products (i.e., packed red blood cells and whole blood) are stored vertically in pull out drawers, with the goal of reducing harmful oxidizing damage to red blood cells. Notably, refrigeration is known to induce aggregation of platelets, a feature historically associated with bacterial contamination in RT-PLT. In order to diminish concerns regarding aggregate formation, it may be beneficial to massage CS-PLT briefly each day to reduce aggregate formation, yet how this may impact hemostatic function is unknown. To this end, optimal storage and handling conditions for CS-PLT remain to be determined. The objective of our study was to measure CS-PLT hemostatic function in response to two major storage and handling variables: agitation and massage to reduce aggregation in the bag. Single donor apheresis platelet units (single Trima collection in plasma, split equally into two bags to control for donor variability; n=20 donors in 40 bags) were purchased from our regional blood center and delivered to our lab on day of collection. Upon arrival, units were spiked and sampled under sterile conditions for baseline (day 0) profiling, assigned to one of the study groups, and stored accordingly. Our study groups were as follows: "Agitated" (n=10 units), "Not Agitated" (n=10 units, paired with Agitated donors), "Massaged Daily", (massaged 60s daily, n=10 units), and "Massaged at Sampling" (massaged only at sampling, n=10 units, paired with Massaged Daily donors). "Agitated" platelet units were stored in a custom refrigerated shaker (courtesy Helmer Scientific). For the other groups, platelet units were stored in a walk-in cold room. All units were stored on the same style perforated agitator rack, with agitation either powered on or off as assigned. Units from all groups were sampled under sterile conditions at days 2, 5, 7, 14, and 21 of storage, using 8 mL draws at each time point to ensure equal volume removal over the course of storage. Hemostatic function was assayed using light transmission aggregometry (LTA; ADP, collagen, epinephrine agonists), rotational thromboelastometry (ROTEM; ExTEM, InTEM agonists), and thrombin generation in response to 5 pM tissue factor. Platelet counts (x10 3/µL) were obtained using a hematology analyzer. During the first week of storage, there were no significant differences in the hemostatic profiles between study groups. While platelet counts, endogenous thrombin potential, and ROTEM clot formation time and maximum clot firmness were fairly stable over the first 7 days of storage across all groups, there was a > 50% reduction from baseline in aggregation responses to both ADP and collagen across all 4 groups, suggesting that platelet aggregation, as detected by LTA, may not be the best representative of CS-PLT function. By day 21 of storage, there was a robust increase in endogenous thrombin potential in all study groups when compared to baseline (Agitated, 22%; Not Agitated, 70%; Massaged Daily, 51%; Massaged at Sampling, 47%). Despite this increase in thrombin generation, after extrinsic activation day 21 CS-PLT in all groups had two-fold increased clot formation times compared to baseline (Agitated, 185%; Not Agitated, 223%; Massaged Daily, 236%; Massaged at Sampling, 190%), and reduced maximum clot firmness compared to baseline (Agitated, -44%; Not Agitated, -36%; Massaged Daily, -54%; Massaged at Sampling, -43%). These data suggest that storage duration, and not agitation nor massaging to reduce aggregates, has the most impact on CS-PLT hemostatic function. Disclosures Spinella: Cerus Corporation: Consultancy, Research Funding; Secure Transfusion Services: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Increased Histone-DNA Complexes and Endothelial-Dependent Thrombin Generation in Severe COVID-19

Objective: Coagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. Approach: We studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Blood samples were assayed for levels of histone-DNA complexes. Confocal microscopy was used to evaluate fibrin structure in clots formed from recalcified plasma samples using fluorescently-labeled fibrinogen. Endogenous thrombin potential was measured by calibrated automated thrombin assays in the presence of tissue factor and phospholipid (PCPS) or cultured human endothelial cells. Results: Circulating nucleosomes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). COVID-19 patient plasma thrombin generation was also altered. Despite having an increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. Control plasma samples did not generate measurable thrombin (lag time >60 min); in contrast, plasma samples from COVID-19+ patients generated thrombin (mean lag time ~20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. Conclusions: Elevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation in COVID-19 may contribute to coagulopathy.

Thrombin Generation and Platelet Function in ICU Patients Undergoing CVVHD Using Regional Citrate Anticoagulation

Background: To investigate pro- and anticoagulant alterations in uremic critically ill patients prior to and during continuous renal replacement therapy. In addition to the conventional thrombin generation assay (TGA), we performed a thrombomodulin-modified variant to better elucidate procoagulant imbalances. Platelet function was determined via multiple electrode aggregometry (MEA) to round off hemostatic analysis.Methods: We prospectively enrolled patients at surgical intensive care units (ICU) with acute kidney injury undergoing continuous veno-venous hemodialysis using regional citrate anticoagulation. TGA and platelet function testing were performed at baseline (≤ 12 h prior to continuous renal replacement therapy) and on 3 consecutive days (day A–C) of extracorporeal therapy.Results: We did not observe significant changes in thrombin generation after start or during renal replacement therapy. Ratios of endogenous thrombin potential in patients were significantly increased (p < 0.001) compared to standardized plasma of healthy donors confirming the assumed procoagulant alterations in ICU patients. Test results of the conventional TGA differed significantly (p < 0.05) from those of the thrombomodulin-modified assay. The area under the curve remained below MEA reference values during the entire observation period, indicating a persistent reduction in platelet function.Conclusion: In summary, in-depth analysis using standard and modified TGA, as well as calculation of endogenous thrombin potential (ETP) ratios, revealed no further aggravation of the procoagulatory shift in the critically ill patient during CVVHD using regional citrate anticoagulation. MEA ruled out the potential impact of platelets.Clinical Trial Registration: German Clinical Trials Register (DRKS00004336), 29 August 2012; www.drks.de.

INDEPENDENT PREDICTORS AND PROGNOSIS OF LONG-TERM MYOCARDIAL INFARCTION IN PATIENTS WITH UNSTABLE ANGINA AFTER CORONARY ARTERY STENTING

Objective. to develop independent predictors for predicting long-term myocardial infarction (MI) in patients (pts) with unstable angina (UA) after coronary artery stenting (PCI) based on the results of a seven-year follow-up. Materials and Methods. The study involved 165 pts with UA and coronary artery stenting (PCI). PCI was performed in 3.2±1.6 days after admission to the in-patient department. Drug-coated stents (Xience V and Biomatrix) were used, the average number of stents was 2.1±0.8 per person, the average length of the stented area was 43.12±25.6 mm, and the average diameter of the implanted stents was 3.12±0.5 mm. All patients were assessed for troponin I, myeloperoxidase, and C-reactive protein levels; coagulation hemostasis was assessed; and a thrombin generation test was performed. The aggregatogram was performed on the analyzer Multiplate (ASPI-test, ADP-test). The patients underwent echocardiography, coronary angiography. Double antithrombotic therapy with clopidogrel 75 mg and acetylsalicylic acid 75 mg was prescribed for 12 months. The follow-up period was 7±1.6 years. Results. Repeated UA developed in 91 (55.2%) pts during a 7-year follow-up period, myocardial infarction was registered in 21 (12.7%) pts. Cardiovascular mortality was 7.3%. Independent predictors of MI risk included: baseline D-dimer level ≥796 ng/ml AUC 0.766 (RR 5.272; 95% CI 2,125-13,082), endogenous thrombin potential ≥2294.5 nM*min AUC 0.912 (RR 4,769; 95% CI 2,457-10,546), N-terminal fragment of brain natriuretic peptide (NTproBNP) ≥816 pg/ml AUC 0.794 (RR 1,935; 95% CI 1,218-3.075), homocysteine level ≥16 µmol/l AUC 0.707 (RR 1.971; 95% CI 1.140-3.406), highly sensitive C-reactive protein ≥6.4 g/l AUC 0.790 (RR 1.333; 95% CI 1.081-1.644), number of affected arteries≥ 3 AUC 0.714 (RR 2.129; 95% CI 1.237-2.664). The developed model for predicting myocardial infarction included the initial level of endogenous thrombin potential≥2294.5 nM * min, D-dimers ≥796 ng / ml, and the number of affected coronary arteries≥ 3. For the developed model, the AUC was 0.964, which corresponds to the excellent quality of the model. Conclusion. The prognosis of myocardial infarction in patients with unstable angina and stenting of the coronary arteries receiving the standard antiplatelet therapy involves laboratory criteria that reflect the activation of the hemostatic system and the residual thrombogenic risk.

Genetics and Hemostatic Potential in Persons with Mild to Moderate Hemophilia A with a Discrepancy between One-Stage and Chromogenic FVIII Assays

Background Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them. Methods The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential. Results Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays. Conclusion There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8, and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA.

Delayed Thrombin Generation Is Associated with Minor Bleedings in Venous Thromboembolism Patients on Rivaroxaban: Usefulness of Calibrated Automated Thrombography

Bleeding is the most feared and difficult to predict adverse event of anticoagulation. We sought to investigate whether calibrated automated thrombography (CAT) parameters are associated with minor bleeding (MB) in anticoagulated patients following venous thromboembolism (VTE). Enrolled were 132 patients on rivaroxaban, 145 on vitamin K antagonists (VKA) and 31 controls who stopped anticoagulation. Prior to the next dose of the anticoagulant, we measured CAT parameters, along with rivaroxaban concentration and INR. During a median follow-up of 10 months, we recorded minor and major bleedings. On rivaroxaban, 27 (20.5%) patients with MB had longer time to start thrombin generation, lower peak thrombin generation and lower endogenous thrombin potential compared with subjects without MB (all p < 0.001). All CAT parameters, except for peak thrombin generation (p = 0.049), were similar in VKA patients with (n = 25, 17.2%) vs. without MBs. By logistic regression, time to start thrombin generation (p = 0.007) and unprovoked VTE (p = 0.041) independently predicted MBs on rivaroxaban. Major bleedings were more frequent in patients with MBs (17.3% vs. 1.8%, p < 0.001). Abnormal CAT parameters characterize VTE patients prone to MBs on rivaroxaban, but not on VKA. Time to start thrombin generation measured about 24 h since the last rivaroxaban dose might help predict MBs.

A Pilot Randomized Study of Atorvastatin As Adjunct Therapy in Patients with Acute Venous Thromboembolism

Background: Venous thromboembolism (VTE) is a considerable public health threat. Improving the management of VTE may achieve better long-term outcomes. In this single-center pilot study, we sought to investigate the effects of statins in addition to anticoagulation in patients with acute VTE.Methods: We enrolled patients over the age of 18 presenting with an acute proximal lower extremity deep vein thrombosis with or without pulmonary embolism. Patients were randomized to anticoagulation alone (with either warfarin or rivaroxaban) or anticoagulation plus atorvastatin 40 mg daily and followed for 9 months. The primary objective was to determine if adjunct atorvastatin reduced thrombin generation, estimated by endogenous thrombin potential and/or peak thrombin concentration. Secondary endpoints included recurrent VTE, arterial thrombosis, bleeding events, and more.Results: A total of 21 patients (11 randomized to anticoagulation only and 10 to anticoagulation plus atorvastatin) were enrolled over 3.5 years. The addition of atorvastatin significantly reduced the mean low-density lipoproteins at 3 months, but did not reduce either endogenous thrombin potential, peak thrombin, D-dimer, or high sensitivity-C reactive protein. Given the low recruitment rate, continuation of the study was deemed futile and the study was terminated early. Barriers to enrollment and completion of study included the many ineligible patients due to the exclusion criteria (e.g. pre-existing statin use, active malignancy, etc) and high rate of lost follow-up.Conclusions: The pilot study did not enroll sufficient number of patients, but was able to inform obstacles for future similar studies investigating the effects of statins in the management of patients with VTE.Trial registration: The study was registered with ClinicalTrials.gov (NCT02331095), January 6, 2015. https://clinicaltrials.gov/ct2/show/NCT02331095