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Red blood cell volume was measured directly in baboons by infusion of 51Cr-labeled autologous RBC, and was indirectly estimated from the plasma volume measured with 125I-labeled albumin and the total body hematocrit. The total body hematocrit was calculated from the peripheral venous hematocrit multiplied by a correction factor; for nonanemic baboons the correction factor was 0.87, and for anemic baboons, 0.75. Within 2 weeks after the phlebotomy (150 ml of blood), the baboon's RBC volume was restored to normal. Posttransfusion survival of baboon RBC can be measured accurately in nonanemic baboons; the preserved RBC can be labeled with 51Cr and the RBC volume of the baboon can be measured indirectly from the plasma volume measured with 125I-labeled albumin and the total body hematocrit.

Frozen red cells have been field-tested at the Mobile Fleet Hospital deployed at 29 Palms, Calif., and at Bridgeport, Calif., and at the PACOM Blood Program Office, Okinawa, Japan, to evaluate the feasibility of using these products to support the US Navy and Marines in combat areas. We studied both nonrejuvenated and rejuvenated cryopreserved red cell concentrates. Red cells stored at 4 degrees C in CPD or CPDA-1 for 3-6 days (nonrejuvenated), and red cells stored at 4 degrees C in CPD for 22-26 days or in CPDA-1 for 36-38 days before biochemical modification (rejuvenated), were frozen with 40% w/v glycerol at -80 degrees C. A multiple-bag collection system with a 600 or 800 ml primary bag was used for blood collection, and the primary bag was used for processing from preparation of the red cell concentrate through prewash dilution. The thawed red cells were washed with 1.5 liters of a sodium chloride-glucose-phosphate solution. As part of this feasibility study, frozen red cells were shipped by air in dry ice (-79 degrees C), and previously frozen washed red cells were shipped in wet ice (4 degrees C). In vitro red cell recovery values were at least 90%. After postwash storage at 4 degrees C for as long as 3 days, the red cells had 24-hour posttransfusion survivals of at least 70%, normal or slightly impaired oxygen transport function, and minimal hemolysis.

When equine RBC were frozen with 20% (w/v) glycerol and stored at -150 C for as long as 5 years, there were no adverse effects on freeze-thaw or freeze-thaw-wash recovery or oxygen transport function. The manner in which the glycerol was added to, and removed from, the equine RBC was shown to be an important consideration in ensuring optimal freeze-thaw-wash recovery values.

Red blood cells of rhesus macaques cryopreserved with 40% (w/v) glycerol and stored at -80 C had freeze-thaw-wash recovery values of 87%, 24-hour posttransfusion survival values of 85%, and life-span values of 13 days. Liquid and freezing methods of preserving RBC were studied in the macaques.

Autologous baboon RBC stored at 4 C in acid-citrate-dextrose (ACD) or in citrate-phosphate-dextrose (CPD) for 3 weeks after collection had 24-hour 51Cr posttransfusion survival values of about 77%. When 20-day-old ACD and CPD baboon RBC were washed and then stored at 4 C for 24 hours before autotransfusion, the 24-hour 51Cr posttransfusion survival values were about 81%. These values were similar to those seen in studies of human RBC preserved in an identical manner. Our results indicated that the baboon can be used to evaluate RBC preservation techniques before human volunteers are studied.

Nonrejuvenated and rejuvenated baboon RBC were freeze-preserved with 40% (w/v) glycerol at -80 C. To prepare rejuvenated RBC, a 50-ml solution containing pyruvate, inosine, glucose, phosphate, and adenine was used and RBC were incubated with this solution before glycerolization and freezing. Appropriate steps were taken to minimize osmotic damage to the RBC during glycerolization and deglycerolization. Nonrejuvenated and rejuvenated cryopreserved RBC had freeze-thaw recovery values of 98%, freeze-thaw-wash recovery values of 92%, and 24-hour post-transfusion survival values of 85%. Some units of cryopreserved RBC were autotransfused after thawing, washing, and storage at 4 C for 24 hours. Other units were perfused in vitro before autotransfusion. After 24 hours of postwash storage, the RBC were concentrated by centrifugation and suspended in a plasma protein fraction and/or an electrolyte solution, and then were exposed to extracorporeal perfusion. Serious adverse effects were not observed on posttransfusion survival, function, or hemolysis in nonrejuvenated or rejuvenated baboon RBC as a result of perfusion in vitro.

We report here on a new approach to washing red blood cells frozen with a high concentration of glycerol in a special freezing container. The wash solution consists of a 150-ml volume of 12% sodium chloride and 2 liters of 0.9% sodium chloride-0.2% glucose-25 mEq/l disodium phosphate. Both the Haemonetics Blood Processor 115 and the IBM Blood Processor 2991 have been used with this protocol, with similar results. The in vitro recovery of red blood cells frozen with 8.6M glycerol was 89%, and that of red blood cells frozen with 6.2M glycerol was 93%. The 24-hour posttransfusion survival values averaged 88% for eight units of outdated-rejuvenated previously frozen red blood cells washed by this protocol and stored at 4 degrees C for 3 days before autotransfusion.