C. P. Emerson

The safety and therapeutic effectiveness of human red cells stored at — 80°C for as long as 21 years

Human red cells frozen by various methods have been stored in the frozen state at —80°C for as long as 21 years. This report discusses: red cells frozen with 42 percent weight per volume (wt/vol) glycerol in an ionic medium in a polyvinylchloride (PVC) plastic bag using the Cohn method; red cells frozen with 45 percent wt/vol glycerol in a low ionic medium in a PVC plastic bag using the Huggins method; red cells frozen with 40 percent wt/vol glycerol in an ionic medium in a polyolefin plastic bag using the Meryman‐Hornblower method; and red cells frozen with 40 percent wt/vol glycerol in an ionic medium in a standard 600‐ml or an elongated 800‐ml PVC plastic primary collection bag with an adapter port using the Naval Blood Research Laboratory (NBRL) method. After frozen storage for as long as 21 years by the four methods described above, the thawed red cells were deglycerolized with 50 to 150 ml of 12 percent sodium chloride and 1.5 to 2.0 l of sodium chloride‐glucose or sodium chloride‐glucose‐phosphate solution. After washing and storage at 4°C for 24 hours, the red cells had a mean freeze‐thaw‐wash recovery value of 90 percent, a mean 24‐hour posttransfusion survival value of 85 percent, a mean index of therapeutic effectiveness of 75 percent, normal or slightly impaired oxygen transport function, and minimal hemolysis. When red cells frozen by the NBRL method in the standard 600‐ml or the elongated 800‐ml primary collection bag for as long as 5.7 years were stored after washing at 4°C for up to 3 days, these units had a mean freeze‐thaw‐wash recovery value of 90 percent, a mean 24‐hour posttransfusion survival value of 85 percent, a mean index of therapeutic effectiveness of 75 percent, normal or slightly impaired oxygen transport function, and minimal hemolysis. Cultures done after storage at 4°C for 1 week showed that the red cells remained sterile. The incidence of container breakage for red cells frozen in the standard 600‐ml or elongated 800‐ml primary collection bag was about 3 percent for units subjected to shipment and less than 1 percent for units that were not transported.

Freezing in the Primary Polyvinylchloride Plastic Collection Bag: A New System for Preparing and Freezing Nonrejuvenated and Rejuvenated Red Blood Cells

Red blood cells were stored at 4 C in the primary bag with an integrally attached empty transfer pack so that the red blood cells could be rejuvenated or not, as desired before glycerolization and freezing. The rejuvenation and glycerol solutions were added through ports in the system. After glycerolization, the red blood cells were concentrated by centrifugation to remove the supernatant glycerol before freezing with 40% w/v glycerol in the primary polyvinylchloride (PVC) plastic container at −80 C.

Therapeutic Effectiveness and Safety of Outdated Human Red Blood Cells Rejuvenated to Restore Oxygen Transport Function to Normal, Frozen for 3 to 4 Years at −80 C, Washed, and Stored at 4 C for 24 Hours Prior to Rapid Infusion

Human red blood cell concentrates with hematocrit values of 75 V% were prepared from citrate‐phosphate‐dextrose (CPD) blood, stored at 4 C for 20 to 28 days, and biochemically modified with a solution containing pyruvate, inosine, glucose, phosphate, and adenine (PIGPA Solution A). The rejuvenated red blood cells were frozen with 40% W/V glycerol in a polyolefin plastic bag and were stored at −80 C. After three to four years of frozen storage, the units were thawed, washed, and stored at 4 C in a sodium chloride‐glucose‐phosphate solution for 24 hours prior to transfusion. Red blood cell recovery was 97 per cent after thawing and 90 per cent after washing. An automated differential agglutination procedure (ADA) showed 24‐hour survival values of about 80 per cent, and long‐term survival values of about 85 days depending on the disease state of the recipient. The red blood cells had normal affinity for oxygen on the day of transfusion. Plasma hemoglobin levels measured immediately after transfusion indicated extravascular removal of nonviable donor red blood cells. There was no increase in the uric acid level during the 24‐hour posttransfusion period. A pool of three to ten units of rejuvenated washed previously frozen red blood cells was transfused rapidly to each of 19 anemic elderly patients. The red blood cells which had normal oxygen delivery capacity immediately upon transfusion increased the recipients red blood cell mass and produced no untoward effects.

Cryopreservation of Human Platelets Isolated by Discontinuous‐Flow Centrifugation Using the Haemonetics Model 30 Blood Processor

Platelets were isolated from normal volunteers by discontinuous‐flow centrifugation using the Haemonetics Model 30 Blood Processor. The numerical equivalent of about five single units of platelets collected at each pheresis were frozen together in a −80 C mechanical freezer with a 6% final concentration of dimethylsul‐foxide (DMSO) as the cryoprotectant. Platelet freeze‐thaw‐wash recovery in vitro was about 80 per cent and the platelet recovery value depended upon the method used to enumerate the platelets. The 51Cr survival values in vivo were about 50 per cent less than those in fresh platelets. These values were not significantly different from those seen when platelets were isolated from single units of blood by differential serial centrifugation. Transfusion of two and one‐half units of freeze‐preserved platelets provided an increase in the recipients circulating platelet count comparable with that from one unit of fresh platelets. The hemostatic effectiveness of freeze‐preserved platelets isolated by discontinuous‐flow centrifugation has not yet been studied.

Leukocyte-poor red blood cells prepared by the addition and removal of glycerol from red blood cell concentrates stores at 4 C

Glycerol was added to and removed from red blood cells to prepare red blood cells free of white blood cells, platelets, and plasma protein. The red blood cells were stored in the primary polyvinylchloride (PVC) plastic collection bag for up to eight days. Red blood cell concentrates not treated with glycerol were washed either within four to six hours of collection or after seven days of storage at 4 C. Red blood cells mixed with glycerol were either washed immediately after addition or were equilibrated for 15 minutes at room temperature before washing. Leukocyte removal was influenced by the length of storage of the red blood cell concentrate at 4 C after collection and by the equilibration of the red blood cell‐glycerol mixture prior to washing. The greatest number of leukocyte was removed when red blood cell concentrates were stored at 4 C for at least five days, mixed with glycerol solution, and the red blood cell‐glycerol mixture equilibrated for 15 minutes before washing the Haemonetics Blood Processor 115. Leukocyte‐poor red blood cells prepared by this procedure have been shown to be safe and effective in eliminating febrile transfusion reactions.

Circulation and function of human platelets isolated from units of CPDA‐ 1, CPDA‐2, and CPDA‐3 anticoagulated blood and frozen with DMSO

Platelet concentrates (PC) were isolated by serial differential centrifugation from units of blood anticoagulated with one of the citrate‐phosphate‐dextrose‐adenine solutions (CPDA‐1, CPDA‐2, CPDA‐2). The platelet concentrates were frozen with six percent dimethylsulfoxide at 2–3 degrees C per minute and stored in a −80 degrees C mechanical freezer in polyvinyl chloride or polyolefin plastic containers. After frozen storage at −80 degrees C for up to three months, the concentrates were thawed at 42 degrees C within 2.5 to 4.0 minutes, washed with autologous plasma, two percent dimethylsulfoxide and 10 percent acid‐citrate‐dextrose solution, and then resuspended in plasma. The washed platelets were labeled with 51Cr and transfused back to the donor from whom they had been obtained. In vitro recovery from whole blood to platelet concentrate was 70.5 ± 17 percent (mean ± one SD). In vitro freeze‐thaw‐wash recovery determined by phase microscopy was 78.5 ± 12.8 percent, in vivo 51Cr platelet recovery two hours after transfusion was 41.3 ± 13.5 percent, and the platelets had a linear lifespan of about eight days. A single unit of previously frozen platelets shortened an aspirin‐ prolonged bleeding time two and 24 hours after infusion. Results were similar with platelets isolated from all three anticoagulants and stored in both plastics. The results also were comparable to previous findings in this laboratory with platelets isolated from ACD and CPD anticoagulated blood.

Enumeration of previously frozen platelets using the Coulter Counter, phase microscopy, and the technicon optical system

The Coulter Counter®, phase microscopy, and the Technicon® optical system were used to enumerate platelets in samples of whole blood, platelet‐ rich plasma, platelet concentrates before and after addition of DMSO, and on platelet concentrates that had been frozen, thawed, and washed. We observed agreement among the three counting methods when platelet counts were determined in whole blood, platelet‐rich plasma, and platelet concentrate before and after DMSO addition. Enumeration after the cryopreservation process, however, showed highly significant differences among the counting systems. Platelet counts on platelet concentrates after freezing the thawing performed with the Coulter Counter were 25 per cent greater than with phase microscopy and 55 per cent greater than with Technicon. Counts with phase microscopy were 30 per cent greater than Technicon values. These data indicate that the method used to enumerate previously frozen platelets affects the apparent platelet count.

Viability and Function of Outdated Human Red Blood Cells after Biochemical Modification to Improve Oxygen Transport Function, Freezing, Thawing, Washing, Postthaw Storage at 4 C, Perfusion in Vitro Through a Bubble Oxygenator, and Autotransfusion

The quality of transfused blood is especially important during cardiac surgery, and red blood cell viability and function may be adversely affected during perfusion through the artificial blood oxygenator used during extracorporeal bypass. In this study, we administered 10 ml aliquot autotransfusions of rejuvenated red blood cells to 13 healthy volunteers after perfusion through an infant bubble oxygenator for one to three hours. Twenty‐three other volunteers received rejuvenated red blood cells that had not been perfused. The red blood cells were biochemically modified after they had reached their outdating period, a process used to increase 2,3 DPG and ATP levels and improve oxygen transport function. The rejuvenated red blood cells were frozen with 40% W/V glycerol, stored frozen at ‐80 C for about 3 months, thawed, washed, and stored in a sodium chloride‐glucose‐phosphate solution at 4 C for as long as three days. Freeze‐thaw recovery was about 97 per cent, and freeze‐thaw‐wash recovery about 90 per cent. Twenty‐three units were transfused after 1 to 3 days of post‐wash storage, and 13 units were perfused through an infant bubble oxygenator for as long as three hours before transfusion. The 24‐hour posttransfusion survival values were about 80 per cent and oxygen transport function was either normal or improved whether or not the units were perfused before transfusion.

Cryopreserved red blood cells for pediatric transfusion. Frozen storage of small aliquots in polyvinyl chloride (PVC) plastic bags.

Human nonrejuvenated and rejuvenated red bood cells were prepared for cryopreservation and subsequent pediatric transfusion. Glycerol was added to the red blood cells in the primary polyvinyl chloride plastic collection bag to achieve a concentration of 40 per cent W/V. The red blood cells were concentrated by centrifugation, and the supernatant glycerol was discarded. Each glycerolized unit was divided into four equal aliquots in the individual 600‐ml bags of a dry quadruple polyvinyl chloride plastic system, and each aliquot was frozen and stored at −80 C. After thawing, sodium chloride solutions were used to wash the aliquots in the IBM Blood Processor 2991‐1 or 2991‐2 or the Haemonetics Blood Processor 115, and the washed aliquots were stored in a sodium chloride‐glucose‐phosphate solution at 4 C for 24 hours. Freeze‐thaw recovery of the red blood cells was about 97 per cent, and freeze‐thaw‐wash recovery was about 84 per cent. Twenty‐four‐hour posttransfusion survival values were about 92 per cent for both nonrejuvenated and indated‐rejuvenated red blood cells. Nonrejuvenated red blood cells, those frozen within three to five days of collection without biochemical modification, had normal oxygen transport function at the time of transfusion; rejuvenated red blood cells, those biochemically treated with PIGPA Solution A after three to five days of storage at 4 C, had improved oxygen transport function at the time of transfusion.