A.J. Melaragno

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.

Cryopreservation of Human Platelets Using 6% Dimethyl Sulfoxide and Storage at ‐80°

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°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°C for at least 2 years, as determined from in vitro 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.

Cryopreservation of platelets isolated with the IBM 2997 blood cell separator: a rapid and simplified approach.

The equivalent of 5–7 units of platelets, isolated from a single donor with the IBM Blood Cell Processor 2997 using a dual stage separation chamber, was frozen with the cryoprotectant dimethylsulfoxide (DMSO). The DMSO‐saline solution was added directly to the platelets, and the platelets were frozen in a polyvinyl chloride plastic bag by storage in a ‐80°C mechanical freezer. Washing the thawed platelets with a phosphate‐buffered sodium chloride‐dextrose solution, pH of 5.0, removed about 95% of the DMSO. In vitro freeze‐thaw‐wash recovery was 80%, and in vivo 51Cr platelet recovery was 31%. Platelet dense body granules were well maintained after freezing, thawing, and washing.

Liquid and freeze preservation of baboon platelets

Abstract Baboon platelet concentrates preserved in the liquid state at 4 or 22 °C or in the frozen state with 4 or 5% DMSO at −80 °C exhibited post-transfusion suvival values similar to those of human platelets preserved in an identical manner. The baboon can be used to study the viability of liquid-preserved and freeze-preserved platelets before studies in humans are done.

Progressive Loss of Fibronectin-Mediated Opsonic Activity in Plasma Cryoprecipitate with Storage

Abstract. Septic injured patients often manifest a deficiency of plasma fibronectin. Several studies have shown improvements in organ function in such patients following infusion of fibronectin‐rich plasma cryoprecipitate, while other studies found no improvement. One explanation for these differences may be the use of plasma crypoprecipitate which has been stored for various time intervals prior to its use as a source of fibronectin. This investigation tested the hypothesis that the opsonic activity of fibronectin in cryoprecipitate may decline with increased storage duration. Using a bioassay of opsonic activity, we evaluated human plasma cryoprecipitate that was stored at either ‐20 or ‐80°C for various intervals (2 weeks to 12 months) after its preparation from fresh donor plasma. Our findings demonstrated that the opsonic activity of fibronectin in cryoprecipitate declined with increasing time of storage. Significant loss (p < 0.05) of opsonic activity was first evident after 2 months of storage. Storage at ‐80°C did not prevent this decline in opsonic activity as compared to storage at ‐20 °C. Immunoblot analysis revealed extensive fragmentation of the dimeric fibronectin (440 kdaltons) and the presence of lower molecular weight fragments in 4‐ to 12‐month‐old plasma cryoprecipitate. Therefore, plasma cryoprecipitate of varying ages (storage time) when used as a source of fibronectin for replacement therapy to support phagocytic function in septic injured patients may result in different fibronectin‐mediated responses. The decline in activity may be due, in part, to fragmentation of the fibronectin molecule.

Morphology of glutaraldehyde-fixed preserved red blood cells and 24-hr post-transfusion survival

Liquid-stored red blood cells and washed, previously frozen red blood cells were studied to determine whether a correlation existed between morphology and post-transfusion survival. Red cell concentrates were stored at 4 °C in citrate-phosphate-dextrose (CPD) for 21 days or in CPD-adenine (CPDA-1, CPDA-2, or CPDA-3) for as long as 35 days as liquid-preserved red cells. Both nonrejuvenated and rejuvenated red blood cells were frozen with 40%wv glycerol at −80 °C and were washed prior to testing. Samples of fresh, liquid-stored, and washed, previously frozen red blood cells were fixed with a 2% veronal glutaraldehyde solution. Phase, light, and electron microscopy were used to measure the numbers of discocytes, discoechinocytes, echinocytes, echinospherocytes, and spherocytes in each sample. A morphology score was assigned, with 100 representing all discocytes and 500 all spherocytes. In all samples phase and light microscopy gave nearly identical scores (r = 0.94), and phase and electron microscopy gave highly similar scores (r = 0.83). The morphology score proved to be a good indicator of 24-hr post-transfusion survival in liquid-stored red blood cells but not in washed, previously frozen red blood cells. Red blood cells stored in the liquid state at 4 °C in CPD, CPDA-1, CPDA-2, or CPDA-3 showed a significant inverse correlation between morphology and 24-hr post-transfusion survival (r = −0.611) and a significant correlation between red cell ATP and 24-hr post-transfusion survival (r = 0.742). We saw no significant correlation between morphology scores and 24-hr post-transfusion values or between ATP levels and post-transfusion survival values in nonrejuvenated or rejuvenated washed, previously frozen red blood cells.

Cryopreservation of dog platelets with dimethyl sulfoxide: Therapeutic effectiveness of cryopreserved platelets in the treatment of thrombocytopenic dogs, and the effect of platelet storage at −80 °C☆

Dog platelets were frozen with 6% dimethyl sulfoxide at 2-3 degrees C per minute in a -80 degrees C mechanical freezer. The frozen platelets were stored at -80 degrees C for as long as 39 months. After storage at -80 degrees C for less than 1 year, platelet in vitro freeze-thaw-wash recovery values were 70%, and in vivo survival values 1 to 2 hr after transfusion were 40% those of fresh platelets. After 2 years or longer storage, in vitro freeze-thaw-wash recovery values were 60%, and in vivo survival values 1 to 2 hr after transfusion were 20% those of fresh platelets. These results indicate that significant deterioration of the dog platelets occurred between the first and second year of storage at -80 degrees C. Platelets that were stored frozen at -80 degrees C for less than 1 year and washed before transfusion into lethally irradiated thrombocytopenic dogs were hemostatically effective.

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.

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.