Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity.

OBJECTIVE: In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. MATERIALS AND METHODS: Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. RESULTS: The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014). CONCLUSION: Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary Study in Turkey.

OBJECTIVE: Transfusion of platelet suspensions is an essential part of patient care for certain clinical indications. In this pioneering study in Turkey, we aimed to assess the in vitro hemostatic functions of platelets after cryopreservation. MATERIALS AND METHODS: Seven units of platelet concentrates were obtained by apheresis. Each apheresis platelet concentrate (APC) was divided into 2 equal volumes and frozen with 6% dimethyl sulfoxide (DMSO). The 14 frozen units of APCs were kept at -80 °C for 1 day. APCs were thawed at 37 °C and diluted either with autologous plasma or 0.9% NaCl. The volume and residual numbers of leukocytes and platelets were tested in both before-freezing and post-thawing periods. Aggregation and thrombin generation tests were used to analyze the in vitro hemostatic functions of platelets. Flow-cytometric analysis was used to assess the presence of frozen treated platelets and their viability. RESULTS: The residual number of leukocytes in both dilution groups was <1x106. The mean platelet recovery rate in the plasma-diluted group (88.1±9.5%) was higher than that in the 0.9% NaCl-diluted group (63±10%). These results were compatible with the European Directorate for the Quality of Medicines quality criteria. Expectedly, there was no aggregation response to platelet aggregation test. The mean thrombin generation potential of post-thaw APCs was higher in the plasma-diluted group (2411 nmol/L per minute) when compared to both the 0.9% NaCl-diluted group (1913 nmol/L per minute) and the before-freezing period (1681 nmol/L per minute). The flow-cytometric analysis results for the viability of APCs after cryopreservation were 94.9% and 96.6% in the plasma and 0.9% NaCl groups, respectively. CONCLUSION: Cryopreservation of platelets with 6% DMSO and storage at -80 °C increases their shelf life from 7 days to 2 years. Besides the increase in hemostatic functions of platelets, the cryopreservation process also does not affect their viability rates.

In vitro efficacy of frozen erythrocytes: implementation of new strategic blood stores to alleviate resource shortage (issue revisited).

BACKGROUND/AIM: Currently, the provision of blood products largely depends on walking blood banks and limited amounts of stored blood with short shelf lives. We aimed to compare the efficacy of erythrocyte concentrate (ECs) by pre- and postfreezing in vitro tests. MATERIALS AND METHODS: In our study, 10 ECs were glycerolized, frozen, thawed, and then deglycerolized using the Naval Blood Research Laboratory method. In addition to using the standard tests, ATP and 2,3-DPG levels and the viability of erythrocytes were also determined. RESULTS: The prefreezing mean viability rates of erythrocytes changed from 89.7 ± 13.7% to 98.6 ± 1.8% after thawing and deglycerolization. Prefreezing and day 0 ATP levels (1.64 ± 0.15 µmol/g Hb and 1.81 ± 0.14 µmol/g Hb, respectively) were similar. The 2,3-DPG levels decreased from 18.09 ± 4.78 µmol/g Hb measured before the procedure to 10.41 ± 4.58 µmol/g Hb on day 0. The mean hemolysis rates and supernatant Hb levels changed from 0.21 ± 0.11% to 0.36 ± 0.12% and 1 ± 0.5 g/L to 1.5 ± 0.5 g/L, respectively. CONCLUSION: The test results showed the efficacy of the frozen-thawed ECs to be used in humans for a broad spectrum of clinical indications. As a part of a contingency plan, national frozen blood reserves need to be established.