STUDY OF SURVIVAL AND FUNCTIONALITY IN CRYOPRESERVED PLATELETS

SURVIVAL AND FUNCTIONALITY EVALUATION IN VITRO IN CRYOPRESERVED PLATELETS

Platelets correspond to one of the most widely used blood components in transfusion medicine, given their importance in the prevention of bleeding disorders in patients with multifactorial thrombocytopenia and non-immunological etiology, as well as in the management of traumatic or blood-related bleeding. surgical treatments. Given its wide margin of use and the constant demand for it for use within the different management schemes and procedures associated with the medical act, it is the objective of both hemotherapy services and blood banks and blood products to have availability component constant to meet such requirements. The main problem of this resource is its short useful life (5 to 7 days) which limits its availability, especially in the Latin American context where the production of blood components as well as the resources allocated for this purpose by state entities to blood banks are generally limited. Cryopreservation at -80 ° C using Dimethylsulfoxide as a preservative solution is a technique used since the 1950s to prolong platelet survival, the improvement of which has been remarkable in recent years, thus constituting an alternative for the resolution of this problem.

HYPOTHESIS: Through cryopreservation it is possible to prolong the survival time of platelets up to 4 times, compared to those preserved under the conventional method, maintaining a viability equal to or greater than 50%.

There is a direct relationship between survival time, viability and functionality of cryopreserved platelets depending on the model of cryoprotective solution used.

PRIMARY OUTCOME: survival of cryopreserved platelets greater than 28 days SECUNDARY OUTCOME: viability of cryopreserved platelets greater than 50%

INCLUSION CRITERIA: Concentrates from donors over 18 years of age who meet the requirements established by law 22990 (Of Argentine Republic) for donating blood and blood components will be included in this study, from whom a number of platelets greater than or equal to 3 x10 E11 can be collected.

EXCLUSION CRITERIA: Platelet concentrates that present any of the following characteristics will be excluded from the investigation:

Concentrates that have not met all of the standards required for use in humans or that at the time of the study more than 24 hours have passed since they were obtained.

A blind experimental study will be carried out (those who evaluate viability and functionality of platelets will not know the cryopreservation times or the concentration of the cryopreservative)

Aliquots of platelets with group and factor A +, A-, B +, B-, AB +, AB-, O + and O- obtained from those people who come to the hemotherapy center as voluntary donors and who in turn express their decision to participate in this protocol by signing an informed consent designed for this purpose. Obtaining platelets will be performed by apheresis using the Trima ACCEL cell separator, version 7.0, TERUMO BCT brand.

A minimum total of 3.3 x1011 platelets will be obtained in a volume between 200 - 300 ml, for each donor.

Group A: It will consist of 70 aliquots of 3 ml each with 1.9 x109 platelets to which a solution composed of: 1 ml obtained from the following mixture will be added (0.8 ml of 100% Dimethylsulfoxide (DMSO) and 4 ml of 0 sodium chloride , 9%). They will then be frozen at -80 ° C.

B Group: It will be made up of 70 aliquots of 3 ml each with 1.9 x109 platelets to which will be added a solution composed of: 0.4 ml of 20% UNC brand albumin and 0.6 ml obtained from the following mixture (1.4 ml of Dimethylsulfoxide (DMSO) 100% and 4 ml of Dextrose 5%). They will then be frozen at -80 ° C.

Group C: It will be made up of 70 aliquots of 3 ml each with 1.9 x109 platelets to which will be added a solution composed of: 0.4 ml of 20% UNC brand albumin and 0.6 ml obtained from the following mixture (1.4 ml of Dimethylsulfoxide (DMSO) 100% and 4 ml of 0.9% sodium chloride). They will then be frozen at -80 ° C.

A group of 71 aliquots of 3 ml each with 1.9 x109 irradiated platelets kept at 22 ° C under constant stirring for 5 days (twenty-four aliquots for each period of time evaluated) will be used as a reference for the comparison of results (group N).

Viability:

It will be evaluated through the platelet count carried out by the Beckman-Coulter system, the measurement of the pH of the cryopreserved platelets after being thawed (expected values between 6.4 and 7.4) and the immunophenotyping of the platelets by flow cytometry (CMF) using fluorescent anti CD61-PE antibodies.

Functionality:

It will be evaluated from the immunophenotyping of platelets by flow cytometry (CMF) using fluorescent anti-CD41 and anti-CD62-FITC antibodies.

Before carrying out the CMF analysis, 4 microliters of a 50 mmol ADP solution will be added to each sample in order to induce platelet activation.

Both the viability analysis and the functionality of the cryopreserved product will be carried out by 3 biochemists, collaborating members of the research in the biochemical analysis laboratory of the hospital.

Inclusion Criteria:

• Concentrates from donors over 18 years of age who meet the requirements established by law 22990 for donating blood and blood components will be included in this study, from whom a number of platelets greater than or equal to 3 x10 E11 can be collected

Exclusion Criteria:

• Platelet concentrates that present any of the following characteristics will be excluded from the investigation:

Concentrates that have not met all of the standards required for use in humans or that at the time of the study more than 24 hours have passed since they were obtained.