Baboons that were subjected to systemic hypothermia at 32 C had an arm skin temperature of 27.3 C and bleeding time of 5.8 minutes. With local warming of the arm skin to 34 C, the bleeding time was 2.4 minutes. In normothermic baboons with arm skin temperature of 34.6 C, the bleeding time was 3.1 minutes. Local cooling of the arm skin to 27.6 C produced a bleeding time of 6.9 minutes. Increasing the skin temperature of the arm in hypothermic baboons to 38.9 C and in normothermic baboons to 40.1 C reduced bleeding times to 2.1 and 2.3 minutes, respectively. In both hypothermic and normothermic baboons there was a negative and significant correlation between the bleeding time and the arm skin temperature and the thromboxane B2 level in the shed blood obtained at the template bleeding time site. There was a significant positive correlation between the thromboxane B2 level in the shed blood and the arm skin temperature. Both in-vivo and in-vitro studies have shown that the production of thromboxane B2 by platelets is temperature-dependent, and that a cooling of skin temperature produces a reversible platelet dysfunction. Data also suggest that when a hypothermic patient bleeds without surgical cause, skin and wound temperature should be restored to normal before the administration of blood products that are not only expensive but may also transmit disease.

Platelet response to hypotonic stres can be used to estimate the SlCr recovery in u k o of liquid-and frceze-preserved platelets. This simple in rtitro test may prove helpful in determining and controlling the quality of preserved platelets. I n our study, the response to hypotonic stress was not related to the "ICr T-W value of the preserved platelets, but was related to the number of irrevcmibly damaged platelets removed within two hours after transfu- sion.A SIMPLE in uilro test for predicting the survival in vivo and hemostatic effectiveness of preserved platelets would be extremely useful in assessing methods of preservation. Suggested in uitro tests for estimating platelet viability include: serotonin uptake,l4. 1 8 . 2 3 response to hypotonic stress,50 99 11, 16-l7-19 ability to promote clot retraction,6,13 platelet adhesion and w egation,3.4. lo. 20.24 morphology,2; platelet factor 320 and measurement of platelet enzymes such as aminopeptidase, nucleoside diphosphokinase, 3-phosphoglycerate kinase, and enolase, in the supernatant of the platelet concentrate.7.*. 21.23 We report here on the "Cr survival and the response to hypotonic stress of liquid-antl freezepreserved human platelets.

Human platelets were preserved by freezing them with 6% DMSO in a mechanical refrigerator maintained at -80° C. The selection of 6% DMSO was arbitrary. After postthaw washing, the 51Cr recovery in vivo was about 45%, the in vitro recovery was about 75%, and the lifespan was about 8.5 days. The mean residual DMSO of 165 mg produced no adverse side effects. The number of platelets in the circulation 2 hr after the infusion of washed freeze-preserved platelets was about half that found with fresh platelets. The prolonged bleeding time induced by aspirin administration to healthy volunteers was corrected within 24 hr of transfusion of a single unit of previously frozen washed platelets; in nine of 12 studies a 50% reduction in the bleeding time occurred within 2 hr of infusion. We do not know though whether washed freeze-preserved platelets will be as effective in correcting the bleeding diathesis in thrombocytopenic patients.

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Platelet studies were done in healthy male volunteers and in thrombocytopenic patients. Some of the platelets used in the study were isolated by mechanical apheresis using either the Haemonetics blood processor 30, the IBM blood processor 2997 or the Fenwal CS-3000 blood processor before freezing. Other platelets were isolated from individual units of whole blood and pooled before freezing. The platelets were frozen with a 6% cryoprotectant (DMSO) in a polyvinylchloride (PVC) plastic bag or a polyolefin plastic bag at -80 degrees C in a mechanical freezer and stored for as long as 3 years. Some of the frozen platelets were transported in dry ice in polystyrene foam containers to determine whether they would be adversely affected by such treatment. Platelet recovery after freezing, thawing and washing was about 75%. In the healthy male volunteers, in vivo recovery of autologous platelets 1-2 h after transfusion was about 33%, and the life span was about 8 days. In the thrombocytopenic patients, in vivo recovery values were 50% of those from fresh platelets. The transfusion of previously frozen washed platelets reduced clinical bleeding in the thrombocytopenic patients with bleeding. There was no evidence of quality deterioration in platelets after storage at -80 degrees C for at least 2 years, as determined from in vivo recovery and in vivo survival values, nor was there any adverse effect as a result of shipment of the frozen platelets in dry ice in polystyrene foam containers from one facility to another.

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