Effects of frozen storage conditions and freezing rate on the stability of coagulation proteins in human plasma.

OBJECTIVES: Degradation of coagulation proteins in frozen plasma may influence assay results. The aims of this study were to explore the changes in coagulation parameters in patient plasma and internal quality control (IQC) after different freezing and storage conditions during the short-term and long-term periods. METHODS: Platelet poor plasma was prepared from citrated peripheral blood collected from a group of healthy donors. The plasma was pooled, frozen and stored in a variety of freezing and storage conditions. The changes were monitored using routine coagulation assays, as well as factor VIII (FVIII) and protein S (PS) assays. RESULTS: Plasma stored in liquid nitrogen (LN 2 ) or in -80°C showed long-term stable values for routine tests for a period of over 12 months, and 6 months for FVIII. Interestingly, the activated partial thromboplastin time (aPTT) showed a temporary significant prolongation over the first two weeks. Plasma frozen and stored in -40°C is not viable for aPTT and FVIII testing, otherwise it can be used for other parameters for up to 4 months. PS showed a significant increase in all frozen samples. Freezing rate has a significant impact on plasma quality and the final storage temperature influences the long-term stability. CONCLUSION: The optimal storage conditions are ultra-low temperatures (LN 2 or -80°C) and the highest freezing rate possible. However, frozen plasma is not viable for IQC of aPTT during a period of two weeks after freezing. This study is unique in its conception as a practical guide for the handling of frozen plasma samples in modern laboratory settings.

The impact of PROS1 mutation position on thrombotic risk in protein S-deficient patients.

Background: Inherited protein S deficiency is a thrombophilic risk factor associated with venous thromboembolism. However, there is not much data on the impact of mutation position on thrombotic risk. Objectives: The aim of this study was to evaluate the risk of thrombosis due to mutations located in the sex hormone-binding globulin (SHBG)-like region as opposed to the rest of the protein. Methods: Genetic analysis of PROS1 was performed in 76 patients with suspected inherited protein S deficiency, and the effect of missense mutations present in the SHBG region on thrombosis risk was analyzed by statistical methods. Results: We found 30 unique mutations (13 of them novel), of which 17 were missense mutations, in 70 patients. Patients with missense mutations were then divided into 2 groups: the "SHBG-region" mutation group (27 patients) and the "non-SHBG" group (24 patients). The multivariable binary logistic regression analysis showed that mutation position in the SHBG region of protein S is an independent risk factor for thrombosis in deficient patients (OR, 5.17; 95% CI, 1.29-20.65; P = .02). The patients with a mutation in the SHBG-like region also developed a thrombotic event at a younger age compared to the "non-SHBG" group in the Kaplan-Meier analysis (median thrombosis-free survival of 33 vs 47 years, respectively; P = .018). Conclusion: Our findings show that a missense mutation located in the SHBG-like region may contribute to higher thrombotic risk rather than a missense mutation located elsewhere in the protein. However, as our cohort was relatively small, these findings should be taken with this limitation.

Extracorporeal membrane oxygenation seems to induce impairment of primary hemostasis pathology as measured by a Multiplate analyzer: An observational retrospective study.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) support is often associated with bleeding complications caused by secondary or primary hemostasis pathology. However, there are limited data investigating primary hemostasis using Multiplate aggregometry with specific diagnostics tests for vWF (von Willebrand factor) deficiency. AIMS: The aim of this study was to find out whether short-term ECMO produces the pathology of primary hemostasis that is detected by Multiplate aggregometry and to investigate the pathology of vWF. METHODS: In this study, blood samples of 20 patients undergoing lung transplantations with short-term perioperative ECMO support were analyzed. The multimeric structure, the levels of von Willebrand factor antigen (vWF), ristocetin cofactor (RCo), collagen-binding protein (CB), and the results of multiple electrode aggregometry RISTO (ristocetin), ADP (adenosine diphosphate), ASPI (Aspirin®; arachidonic acid), and TRAP (thrombin receptor activating peptide) tests were compared to the samples obtained before and after ECMO support. RESULTS: The Multiplate ADP and RISTO tests showed the presence of significant pathology in primary hemostasis after surgery (p < 0.05), suggesting the presence of acquired platelet dysfunction. Although the RISTO tests suggest the presence of acquired vWF deficiency, laboratory tests for vWF antigen and RCo and CB tests showed an increase in this case. The multimeric structure of vWF did not show clinically significant deterioration. CONCLUSIONS: Multiple aggregometry ADP, ASPI, and TRAP tests seem to be able to detect primary hemostasis pathology (platelets aggregation and adhesion pathology) that is present during short-term perioperative ECMO support in lung transplantation procedures. Interestingly, RISTO tests seem to be more suitable for the diagnosis of platelet dysfunction than the diagnosis of acquired vWF deficiency in this situation.

Evaluation of von Willebrand factor with a fully magnetically levitated centrifugal continuous-flow left ventricular assist device in advanced heart failure.

BACKGROUND: Contemporary continuous-flow left ventricular assist devices (CF-LVADs) are associated with degradation of von Willebrand factor (vWF) high-molecular-weight multimers (HMWMs), a critical factor supporting platelet function. We hypothesized that the HeartMate 3 fully magnetically levitated LVAD, designed to reduce circulatory shear stress, favorably influences these hemostatic parameters. METHODS: Fifteen consecutive HeartMate 3 LVAD patients were compared with 11 consecutive HeartMate II controls. Serial plasma samples were collected pre-implant and on Days 2, 7, 30 and 45 post-operatively. Changes in vWF HMWMs were evaluated by 2 independent, study-blind hematologists and confirmed using densitometry-based computerized software. Ristocetin cofactor (RiCO) and vWF antigen (vWF Ag) were measured using standard protocols with enzyme-linked immunosorbent assay. RESULTS: HeartMate 3 patients and HeartMate II controls had a mean age of 67.3 ± 1.4 and 52.8 ± 2.5 years, respectively (INTERMACS Profiles 2 to 4 in 93.3% and 91%, respectively). HeartMate 3 group demonstrated a significantly greater preservation of HMWMs compared with the HeartMate II group, with the most prominent decrease occurring by Day 2 post-operatively and sustained through 45 days (71.94% vs 31.16%, p = 0.001). Laboratory values (normalized to baseline) for RiCO activity, vWF Ag and RiCO:vWF Ag ratio remained in the functional range with no statistically significant differences observed between groups. CONCLUSION: The HeartMate 3 LVAD is associated with enhanced hemocompatibility compared with the HeartMate II LVAD, as demonstrated by the improved preservation of vWF HMWMs, In contrast, effects on HMWM degradation appeared to be dissociated from functional attributes. Further confirmation of these findings in randomized clinical trials is warranted.