Therapeutic effectiveness and safety of outdated human red blood cells rejuvenated to restore oxygen transport function to normal, frozen for 3 to 4 years at −80 C, washed, and stored at 4 C for 24 hours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 recipient's red blood cell mass and produced no untoward effects.     

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

Human red cells frozen by various methods have been stored in the frozen state at -80 degrees 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 degrees C for 24 hour, 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

The 24-hour posttransfusion survival, oxygen transport function, and residual hemolysis of human outdated-rejuvenated red cell concentrates after washing and storage at 4 degrees C for 24 to 72 hours

Red cell concentrates with hematocrit values of 80 +/- 5 percent were stored at 4 degrees C either in citrate-phosphate-dextrose for 22 to 28 days or in citrate-phosphate-dextrose-adenine-one for 35 to 39 days. After storage, the red cells had reduced 2,3-diphosphoglycerate and adenosine triphosphate levels, and biochemical treatment with a solution called PIPA was used to restore these levels. The red cells were not preserved further, but instead were washed after rejuvenation with an unbuffered sodium chloride-glucose solution, pH 5.0, for in vivo studies, and with a buffered sodium chloride-glucose-phosphate solution, pH 6.8, for comparative in vitro studies. The red cells were stored in the wash solution at 4 degrees C for 72 hours after washing. Red cell recovery after washing was about 95 percent. Twenty-four-hour posttransfusion survival value was about 80 percent, and the index of therapeutic effectiveness was greater than 75 percent. These biochemically modified washed red cells exhibited higher than normal P50 values, even after 3 days of postwash storage at 4 degrees C. The units that were washed with the sodium chloride-glucose solution with a pH of 5.0 exhibited a greater degree of hemolysis after 3 days of postwash storage at 4 degrees C than did the units that were washed with the sodium chloride-glucose-phosphate solution with a pH of 6.8.(ABSTRACT TRUNCATED AT 250 WORDS)     

The survival, function, and hemolysis of human RBCs stored at 4 degrees C in additive solution (AS-1, AS-3, or AS-5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4 degrees C in sodium chloride and glucose solution for 24 hours

BACKGROUND: A study was done to assess the quality of RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. STUDY DESIGN AND METHODS: RBCs were stored at 4 degrees C for 42 days in AS-1, AS-3, or AS-5 and then biochemically modified with pyruvate, inosine, phosphate, and adenine solution (Rejuvesol), frozen with 40-percent (wt/vol) glycerol, and stored at -80 degrees C for at least 2 months. The RBCs were deglycerolized by the use of a cell washer (Haemonetics 115), and stored for 24 hours at 4 degrees C in a 0.9-percent sodium chloride and 0.2-percent glucose solution before the autologous transfusion. RESULTS: The mean freeze-thaw-wash recovery process produced RBC recovery values of 85 percent, with the mean 24-hour posttransfusion survival at 75 percent, and the mean index of therapeutic effectiveness at 64 percent for the RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. All the units exhibited normal or slightly higher than normal 2,3 DPG levels after deglycerolization and postwash storage at 4 degrees C for 24 hours. CONCLUSION: RBCs stored in AS-1, AS-3, or AS-5 at 4 degrees C for 42 days and then biochemically modified with pyruvate, inosine, phosphate, and adenine and glycerolized, frozen, washed, and stored at 4 degrees C for 24 hours before autologous transfusion had acceptable in vitro and in vivo measurements.     

The viability of autologous human red cells stored in additive solution 5 and exposed to 25°C for 24 hours

BACKGROUND: No data exist on the viability of red cells (RBCs) stored in modern additive solution systems and allowed to warm above 10 degrees C. STUDY DESIGN AND METHODS: In a randomized crossover study, 3 units of blood were collected at least 8 weeks apart from 11 volunteer donors and stored in additive solution 5 (AS-5). Of 3 units from each volunteer, 1 was stored for 6 weeks at 4 degrees C, 1 for 5 weeks at 4 degrees C except for 24 hours at 25 degrees C on Day 14, and 1 for 5 weeks at 4 degrees C except for 24 hours at 25 degrees C on Day 28. Units were sampled periodically during storage; at the end of storage, viability was measured by the 99mTc/51 Cr double-label method. RESULTS: RBC viability was not significantly different in the storage protocols. Less than 1 percent of stored cells hemolyzed. RBC ATP concentrations at the end of storage correlated with viability and were approximately equal in the warmed units after 30 days' storage and the conventionally stored units after 42 days. CONCLUSIONS: The data suggest that RBCs stored in AS-5 and allowed to warm to 25 degrees C for 24 hours lose about 12 days of their shelf life.     

Transfusion experience with platelet concentrates stored for 24 to 72 hours at 22 degrees C. Importance of storage time

We studied the transfusion response from random donor platelet concentrates in 15 stable multitransfused, thrombocytopenic patients by comparing the platelet counts measured before and 20 hours after transfusion. The observed platelet increments were corrected (corrected increment, C.I.) for the number of units of platelet concentrate transfused and the patient's body surface area in square meters (platelets/microliter per unit/m2). Using platelet concentrates stored for less than 24 hours, the patients achieved a median C.I. of 9500 (range: 5000–18,000). When platelet concentrates stored for 24 to 48 hours or 48 to 72 hours were given, the median C.I. markedly decreased to 1000 (range: 0–4800) and 0 (range: 0–5100), respectively (p less than 0.001). These differences could not be explained by further recipient alloimmunization. Transfusion with platelet concentrates less than 24 hours old on a second occasion, bracketing the transfusions of older platelet concentrates, resulted in a median C.I. of 7200 (range: 5400–14,500). Similar results were obtained in three patients when HLA- identical sibling platelet concentrates were employed. In vitro tests, including pH, morphology, and aggregation, demonstrated no statistically significant differences among the platelet concentrates stored for less than 24 hours, 24 to 48 hours, and 48 to 72 hours. These studies suggest that, although platelet concentrates can be stored for 72 hours without loss of in vitro function, the in vivo recovery is significantly diminished after 24 hours of storage, and preferably patients should not be transfused prophylactically with platelet concentrates greater than 24 hours old.     

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.     

Survival and biochemical characteristics of stored red cells preserved with citrate-phosphate-dextrose-adenine-one and two and prepared from whole blood maintained at 20 to 24 degrees C for eight hours following phlebotomy

Studies were conducted to evaluate the characteristics of red cells stored for 35 days following preparation from units of whole blood anticoagulated with citrate-phosphate-dextrose-adenine-one and two (CPDA-1 and CPDA-2) and maintained at 20 to 24 degrees C for 8 hours after phlebotomy. The mean (+/− 1 SD) 24-hour survival for transfused CPDA-1 autologous red cells with hematocrit levels of 78.1 +/− 2.3 percent was 78.0 +/− 8.1 percent (n = 9). The 24-hour survival of red cells from units preserved with CPDA-2 with hematocrit levels of 79.3 +/− 4.5 percent was 74.8 +/− 8.6 percent (n = 15). This difference in survival was not significant. Red cells from control units stored for 1 day showed a 24-hour survival of 91.9 +/− 4.2 percent (n = 7). During the 8-hour holding, red cell adenosine triphosphate levels increased by 15 to 25 percent in units drawn into both CPDA-1 and CPDA-2. After the initial 8-hour period, the red cell 2,3 diphosphoglycerate levels were 54 +/− 12 percent (mean +/− 1 SD) of initial levels in units drawn into CPDA-1 and 58 +/− 8 percent of initial levels in units drawn into CPDA- 2. Following 35 days of storage, units of red cells prepared from whole blood drawn into CPDA-1 and CPDA-2 had comparable plasma cation and ammonia levels and similar amounts of cell-free hemoglobin. These data indicate that red cells can be stored satisfactorily for 35 days when prepared from whole blood held at 20 to 24 degrees C for 8 hours.     

Viability and function of platelets frozen at 2 to 3 C per minute with 4 or 5 per cent DMSO and stored at −80 C for 8 months

Each of 15 healthy male volunteers was treated with 650 mg of aspirin 24 hours before the autologous transfusion of one unit of freeze- preserved platelets. Freeze-thaw-wash recovery values in vitro, viability and function in vivo, and the bleeding time and platelet aggregation response were measured. The platelets were frozen with 4 or 5 per cent dimethylsulfoxide (DMSO) at an overall rate of 2 to 3 C per minute and were stored at −80 C in a mechanical freezer for up to eight months. They were washed by dilution/centrifugation. The mean recovery in vitro of platelets frozen with 4 per cent DMSO was 76+/−16%; the value was 64+/−16% for platelets frozen with 5% DMSO. The mean in vivo 51Cr recovery of autologous platelets frozen with 4% DMSO was 34+/−6%, and for platelets frozen with 5% DMSO it was 33+/−7%. In both groups the platelet lifespan was normal. There was a significant reduction in bleeding time after the transfusion of a single unit of autologous platelets preserved with either 4 or 5% DMSO, but no improvement in the aspirin-induced platelet aggregation pattern.     

The in vitro quality of red blood cells frozen with 40 percent (wt/vol) glycerol at -80 degrees C for 14 years, deglycerolized with the Haemonetics ACP 215, and stored at 4 degrees C in additive solution-1 or additive solution-3 for up to 3 weeks

BACKGROUND: Red blood cells (RBCs) frozen with 40 percent (wt/vol) glycerol, stored at -80 degrees C (mean temperature; range, -65 to -90 degrees C) for 14 years, deglycerolized in the Haemonetics automated cell processor (ACP) 215 with the 325-mL disposable bowl, and stored at 4 degrees C in additive solution (AS)-1 or AS-3 for 21 days were evaluated. STUDY DESIGN AND METHODS: A total of 106 units of citrate phosphate dextrose adenine-1 RBCs were frozen with 40 percent (wt/vol) glycerol in the original 800-mL polyvinylchloride plastic bag and stored in corrugated cardboard boxes at -80 degrees C for 14 years. The thawed units were deglycerolized with the ACP 215 with a 325-mL disposable bowl and stored in AS-1 or AS-3 at 4 degrees C for 21 days. RESULTS: The freeze-thaw recovery value was 94 +/- 4 percent (mean +/- SD), the freeze-thaw-wash recovery value was 80 +/- 7 percent, and there was no breakage. Thirty-eight units were processed as 19 pairs. Two units of ABO-matched units were thawed, pooled, divided equally into two units, and deglycerolized. One unit was stored in AS-1 and the other in AS-3 at 4 degrees C for 21 days. Units stored in AS-1 exhibited significantly greater hemolysis than those stored in AS-3. CONCLUSIONS: Acceptable results were achieved when RBCs frozen at -80 degrees C for 14 years were deglycerolized in the ACP 215. Deglycerolized RBCs in AS-1 exhibited significantly higher hemolysis than those in AS-3 after storage at 4 degrees C for 7 to 21 days.     

The effect of storing whole blood at 22 degrees C for up to 24 hours with and without rapid cooling on the quality of red cell concentrates and fresh-frozen plasma

BACKGROUND: Storage of whole blood (WB) for less than 24 hours at ambient temperature is permitted in Europe, but data directly comparing storage with and without active cooling are lacking, which was investigated and compared to current standard methods. STUDY DESIGN AND METHODS: WB was stored in one of four different ways for 24 hours after donation before processing on Day 1 to red cell concentrates (RCCs) in saline‐adenine‐glucose‐mannitol and fresh‐frozen plasma (FFP; n = 20 each): 1) at 22°C in plastic trays, 2) in cooling devices (Compocool II, NPBI), 3) at 4°C, or 4) processed from WB without storage less than 8 hours from donation (Day 0). RESULTS: 2,3‐Diphosphoglycerate (2,3‐DPG) in RCCs were lower after ambient storage compared with those processed on Day 0 or after 4°C storage. Rapid cooling slowed the loss of 2,3‐DPG but levels were undetectable by Day 21 with any method. On Day 42 of RCC storage, there was no significant difference between storage methods in levels of adenosine triphosphate or hemolysis. Potassium levels were lower in RCCs from WB stored at ambient compared with those produced on Day 0, regardless of the use of cooling plates. FFP produced from WB on Day 0 or after storage at ambient with or without active cooling met UK specifications (>75% of units >0.70 IU/mL Factor VIII). CONCLUSION: These data suggest that RCCs and FFP produced from WB that has been stored at ambient temperature with or without active cooling are of acceptable quality compared with those produced using current standard methods in the United Kingdom.     

Recovery, lifespan, and function of CPD-Adenine (CPDA-1) platelet concentrates stored for up to 72 hours at 4 C

We studied the in vivo recovery, lifespan, and hemostatic effectiveness of CPDA-1 platelet concentrates stored for up to 72 hours at 4 C. A total of 120 CPDA-1 concentrates containing an average (+/− 1 S.D.) of 6.6 +/− 2.0 × 10(10) platelets were prepared. The pH of the units following storage at 4 C was 6.8 +/− 0.2; no unit had a pH below 6.3. Autologous transfusion of six normal volunteers showed that platelets stored at 4 C for 72 hours had an in vivo recovery of 40 +/− 18 per cent and a lifespan of 5.1 +/− 1.5 days. The hemostatic effectiveness of CPDA-1 platelets was determined by platelet counts and template bleeding time measurements in 10 thrombocytopenic patients. Patients receiving 48-hour-stored platelets had a four- to six-hour posttranfusion corrected platelet increment averaging 15,300 +/− 3,200/microliter which was 67 +/− 34 per cent of expected recovery. Four of the five patients transfused with this preparation showed an improved bleeding time. In contrast, three patients receiving 72-hour- stored platelets had a four- to six-hour posttransfusion increment of 5,800 +/− 2,400/microliter that was only 26 +/− 13 per cent of the expected recovery; furthermore, only one of these patients showed any correction of the bleeding time. These data indicate that CPDA-1 platelets are hemostatically effective when stored at 4 C for up to 48 hours.     

In vivo survival of apheresis RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215, and stored at 4°C in AS-3 for up to 21 days

BACKGROUND: The FDA has approved the storage of frozen RBCs at -80 degrees C for 10 years and the postwash storage at 4 degrees C for no more than 24 hours. The 4 degrees C postwash storage period is limited to 24 hours, because the current deglycerolization systems are functionally open systems. STUDY DESIGN AND METHODS: Two units of RBCs were collected from each of 13 healthy male volunteers. The RBCs were collected in CP2D by the FDA-approved protocol for an automated apheresis device (MCS, LN8150, Haemonetics) and were stored at 4 degrees C in AS-3 for 6 days. Using a single disposable glycerolization set in an automated, functionally closed system (ACP 215, Haemonetics) each unit was transferred to a 1000-mL PVC plastic bag and glycerolized to a concentration of 40-percent (wt/vol) glycerol and frozen at -80 degrees C. A single disposable deglycerolization set in the ACP 215 was used to deglycerolize the 2 units from the same donor. The deglycerolized RBCs were stored at 4 degrees C in AS-3 for as long as 21 days. RESULTS: The mean +/- SD freeze-thaw-wash recovery value was 89.4 +/- 3 percent. The residual hemolysis in the RBCs stored at 4 degrees C in AS-3 for 21 days after deglycerolization was 0.9 +/- 0.2 percent, and the units were negative for both aerobic and anaerobic bacteria. The mean Nageotte WBC count was 9 x 10(6) per unit. When the deglycerolized RBCs were given as an autologous transfusion after storage at 4 degrees C in AS-3 for the 7- to 18-day period, the mean +/- SD 24-hour posttransfusion survival was 77 +/- 7 percent, and the index of therapeutic effectiveness was 69 +/- 8 percent. CONCLUSION: Two units of human RBCs collected from a single donor by apheresis in the MCS using an LN8150 set can be glycerolized sequentially with a single disposable set and deglycerolized sequentially with another single disposable set in the ACP 215. The previously frozen RBCs stored in AS-3 for 7 to 18 days at 4 degrees C had acceptable hemolysis and an acceptable mean 24-hour posttransfusion survival value and index of therapeutic effectiveness.     

Posttransfusion survival (24-hour) and hemolysis of previously frozen, deglycerolized RBCs after storage at 4 degrees C for up to 14 days in sodium chloride alone or sodium chloride supplemented with additive solutions

BACKGROUND: Previously frozen human RBCs currently are glycerolized and deglycerolized by the use of open systems that limit storage of the deglycerolized RBCs at 4 degrees C to only 24 hours. STUDY DESIGN AND METHODS: Healthy male volunteers who met AABB requirements for blood donors (n = 38) were studied. A volume of 450 mL of blood was collected into CPDA-1. The RBC concentrates were stored at 4 degrees C for 3 to 6 days before being frozen with 40-percent (wt/vol) glycerol and stored at -80 degrees C. The RBCs were deglycerolized, resuspended in 0.9-percent sodium chloride and 0.2-percent glucose (SG) solution or SG solution supplemented with AS-1, AS-3, or AS-5, and stored in the resuspension medium at 4 degrees C for 14 days. RESULTS: The mean +/- SD freeze-thaw-wash process recovery was 90.0 +/- 4.0 percent for all 38 units. The mean 24-hour posttransfusion survival value was 79 percent for deglycerolized RBC stored at 4 degrees C for 7 days in SG alone, SG plus AS-3, or SG plus AS-5. Deglycerolized RBC that were stored at 4 C for 14 days in SG supplemented with AS-1, AS-3, or AS-5 had a mean 24-hour posttransfusion survival of 74 percent. After 7 days of storage of deglycerolized RBCs in SG alone, the mean hemolysis was 3. 7 percent. After 14 days of storage of deglycerolized RBCs in SG supplemented with AS-1, AS-3, or AS-5, the mean hemolysis was 2.5 percent. CONCLUSIONS: The levels of hemolysis did not correlate with the 24-hour posttransfusion survival values.