A. Gray

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.

A comparison of methods of determining the 100 percent survival of preserved red cells

Studies were done to compare three methods to determine the 100 percent survival value from which to estimate the 24‐hour posttransfusion survival of preserved red cells. The following methods using small aliquots of 51Cr‐labeled autologous preserved red cells were evaluated: First, the 125I‐albumin method, which is an indirect measurement of the recipients red cell volume derived from the plasma volume measured using 125I‐labeled albumin and the total body hematocrit. Second, the body surface area method (BSA) in which the recipients red cell volume is derived from a body surface area nomogram. Third, an extrapolation method, which extrapolates to zero time the radioactivity associated with the red cells in the recipients circulation from 10 to 20 or 15 to 30 minutes after transfusion. The three methods gave similar results in all studies in which less than 20 percent of the transfused red cells were nonviable (24‐hour posttransfusion survival values of between 80–100%), but not when more than 20 percent of the red cells were nonviable. When 21 to 35 percent of the transfused red cells were nonviable (24‐hour posttransfusion survivals of 65 to 79%), values with the 125I‐albumin method and the body surface area method were about 5 percent lower (p less than 0.001) than values with the extrapolation method. When greater than 35 percent of the red cells were nonviable (24‐hour posttransfusion survival values of less than 65%), values with the 125I‐albumin method and the body surface area method were about 10 percent lower (p less than 0.001) than those obtained by the extrapolation method.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

A new approach to washing red blood cells frozen with a high concentration of glycerol in a special freezing container.

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°C for 3 days before autotransfusion.