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.     

A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4°C in AS-3: assessment of RBC processing in the ACP 215

BACKGROUND: The FDA has approved the storage of frozen RBCs at -80 degrees C for 10 years. After deglycerolization, the RBCs can be stored at 4 degrees C for no more than 24 hours, because open systems are currently being used. Five laboratories have been evaluating an automated, functionally closed system (ACP 215, Haemonetics) for both the glycerolization and deglycerolization processes. STUDY DESIGN AND METHODS: Studies were performed at three military sites and two civilian sites. Each site performed in vitro testing of 20 units of RBCs. In addition, one military site and two civilian sites conducted autologous transfusion studies on ten units of previously frozen, deglycerolized RBCs that had been stored at 4 degrees C in AS-3 for 15 days. At one of the civilian sites, 10 volunteers received autologous transfusions on two occasions in a randomized manner, once with previously frozen RBCs that had been stored at 4 degrees C in AS-3 for 15 days after deglycerolization and once with liquid-preserved RBCs that had been stored at 4 degrees C in AS-1 for 42 days. RESULTS: The mean +/- SD in vitro freeze-thaw-wash recovery value was 87 +/- 5 percent; the mean +/- SD supernatant osmolality on the day of deglycerolization was 297 +/- 5 mOsm per kg of H(2)O, and the mean +/- SD percentage of hemolysis after storage at 4 degrees C in AS-3 for 15 days was 0.60 +/- 0.2 percent. The paired data from the study of 10 persons at the civilian site showed a mean +/- SD 24-hour posttransfusion survival of 76 +/- 6 percent for RBCs that had been stored at 4 degrees C for 15 days after deglycerolization and 72 +/- 5 percent for RBCs stored at 4 degrees C in AS-1 for 42 days. At the three sites at which 24-hour posttransfusion survival values were measured by three double-label procedures, a mean +/- SD 24-hour posttransfusion survival of 77 +/- 9 percent was observed for 36 autologous transfusions to 12 females and 24 males of previously frozen RBCs that had been stored at 4 degrees C in AS-3 for 15 days after deglycerolization. CONCLUSION: The multicenter study showed the acceptable quality of RBCs that were glycerolized and deglycerolized in the automated ACP 215 instrument and stored in AS-3 at 4 degrees C for 15 days.     

In vitro and in vivo measurements of human RBCs frozen with glycerol and subjected to various storage temperatures before deglycerolization and storage at 4 degrees C for 3 days

BACKGROUND: This study was designed to assess the effects of changes in storage temperature of frozen RBCs such as might occur during a malfunction of the -80 degrees C mechanical freezer or during shipment. STUDY DESIGN AND METHODS: Fifteen participants donated blood for autologous transfusion of RBCs; all RBCs were frozen with 40-percent (wt/vol) glycerol. Five subjects received RBCs that were stored at -80 degrees C alone before transfusion. Five subjects received RBCs that were stored initially at -80 degrees C, then at -40 degrees C for 4 weeks, and finally at -80 degrees C before transfusion. Five subjects received RBCs that were stored at -80 degrees C, then at -20 degrees C for 2 weeks, and finally at -80 degrees C before transfusion. After deglycerolization, the RBCs were stored at 4 degrees C in a sodium chloride-glucose solution for 3 days before transfusion. RESULTS: No significant differences were observed in freeze-thaw recovery, freeze-thaw-wash recovery, 24-hour posttransfusion survival, index of therapeutic effectiveness, or RBC ATP levels. Greater hemolysis and reduced RBC K+ levels were observed in the units stored at -80 degrees C/-40 degrees C/-80 degrees C and in those stored at -80 degrees C/ -20 degrees C/-80 degrees C compared with the units stored at -80 degrees C alone, but these differences did not affect the 24-hour posttransfusion survival. CONCLUSIONS: The results of this study indicated that RBCs frozen with 40-percent (wt/vol) glycerol can be stored at -40 degrees C for 4 weeks or at -20 degrees C for 2 weeks between periods of frozen storage at -80 degrees C with satisfactory results.     

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.     

Stability of blood coagulation factors in deep frozen fresh plasma by storage at -20 degrees C and -40 degrees C

Fresh frozen plasma (FFP), was shock-frozen to -25 degrees C within six hours after blood donation. The platelet count was reduced to 20 000/mm3. Aliquots were stored at -20 degrees C and -40 degrees C up to 24 months. Quick, PTT, factor V, VIII, IX, thrombin, antithrombin III, plasminogen, plasma-prekallikrein and kallikrein were determined monthly. With respect to the parameters investigated there was no significant difference between storage at -20 degrees C and -40 degrees C. Factor VIII loss was 10% after 12 months of storage. The activity of factor IX and V remained unchanged during 12 months, then factor V increased during storage. The other parameters did not change. Our study indicates quality of FFP seems not primarily depend on storage temperature, but an optimal preparation technique is much more important.     

Uncontrolled-rate freezing and storage at -80 degrees C, with only 3.5-percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations

BACKGROUND: Although controlled-rate freezing and storage in liquid nitrogen are the standard procedure for peripheral blood progenitor cell (PBPC) cryopreservation, uncontrolled-rate freezing and storage at -80 degrees C have been reported. STUDY DESIGN AND METHODS: The prospective evaluation of 109 autologous PBPC transplantations after uncontrolled-rate freezing and storage at -80 degrees C of apheresis products is reported. The cryoprotectant solution contained final concentrations of 1-percent human serum albumin, 2.5-percent hydroxyethyl starch, and 3.5-percent DMSO. RESULTS: With in vitro assays, the median recoveries of nucleated cells (NCs), CD34+ cells, CFU-GM, and BFU-E were 60.8 percent (range, 11.2-107.1%), 79.6 percent (6.3-158.1%), 35.6 percent (0.3-149.5%), and 32.6 percent (1.7-151.1%), respectively. The median length of storage was 7 weeks (range, 1-98). The median cell dose, per kg of body weight, given to patients after the preparative regimen was 6.34 x 10(8) NCs (range, 0.02-38.3), 3.77 x 10(6) CD34+ cells (0.23-58.5), and 66.04 x 10(4) CFU-GM (1.38-405.7). The median time to reach 0.5 x 10(9) granulocytes per L, 20 x 10(9) platelets per L, and 50 x 10(9) reticulocytes per L was 11 (range, 0-37), 11 (0-129), and 17 (0-200) days, respectively. Hematopoietic reconstitution did not differ in patients undergoing myeloablative or nonmyeloablative conditioning regimens before transplantation. CONCLUSION: This simple and less expensive cryopreservation procedure can produce successful engraftment, comparable to that obtained with the standard storage procedure.     

Long-term storage at −80 degrees C of hematopoietic progenitor cells with 5-percent dimethyl sulfoxide as the sole cryoprotectant

BACKGROUND: Hematopoietic progenitor cells (HPCs) can be cryopreserved and stored below -120 degrees C in liquid nitrogen or at -80 degrees C in mechanical freezers. STUDY DESIGN AND METHODS: The feasibility of long-term storage of HPCs at -80 degrees C was investigated. The studies included a comparison of 5- and 10-percent dimethyl sulfoxide (DMSO) as cryoprotectant at various lengths of storage time. Mononuclear cell (MNC) recovery and viability and colony-forming unit- granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU- E) recovery assays were performed. The peripheral blood HPCs of 24 consecutive patients included in the program of autologous transplantation were studied. RESULTS: The MNC viability decreased progressively with the length of time from cryopreservation, reaching 32 percent after 31 months of storage. The recovery rates of CFU-GM and BFU-E also decreased progressively with the duration of frozen storage, to 50 and 43.5 percent, respectively, after 12 months and to 0 percent (both) after 24 months. At 6 months of storage, MNC viability was 80 percent, and CFU-GM and BFU-E recovery was 63.5 and 80.5 percent, respectively. There were no differences between MNCs cryopreserved with 5- or 10-percent DMSO in terms of cell viability. There were no differences between CFU-GM recovery or BFU-E recovery from HPCs cryopreserved in 5- or 10-percent DMSO. Patients given HPCs stored in these conditions for periods ranging between 123 and 202 days showed a complete and rapid hematologic recovery. CONCLUSION: HPCs can be cryopreserved at -80 degrees C with 5-percent DMSO and stored at -80 degrees C no longer than 6 months. A 5-percent DMSO concentration is comparable to a with 10-percent concentration in terms of recovery and MNC viability.     

Long-term stability of endogenous B-type natriuretic peptide after storage at -20 degrees C or -80 degrees C

BACKGROUND: B-type natriuretic peptide (BNP) is a biomarker used in the investigation of heart failure and other cardiovascular diseases. Our aim was to analyze the degradation of BNP in plasma samples kept at -20 degrees C or at -80 degrees C after 3 months or 1 year of storage. METHODS: Using a Biosite assay-Triage BNP test, we evaluated 20 subjects with plasma BNP concentrations ranging from normal to high. Plasma aliquots were separated in five eppendorfs, one for immediate measurement after centrifugation, two for storage at -20 degrees C and two at -80 degrees C. We measured BNP after 3 months and 1 year. The significance level was set at p<0.05 (one-tailed test). RESULTS: There was a significant decrease of 1% per month in BNP concentration. However, there was a significant interaction between time and group (high vs. low BNP). Low BNP values did not change over time, while high BNP decreased by 2% per month. The decrease over time did not depend on the storage temperature. CONCLUSIONS: The storage of the samples at -20 degrees C or at -80 degrees C did not prevent BNP degradation, especially for high BNP values. For low BNP values, we did not observe a significant decay at either 3 months or 1 year. These results are valid for the Biosite assay-Triage BNP test.     

Cryopreservation of hematopoietic progenitor cells with 5-percent dimethyl sulfoxide at −80 degrees C without rate-controlled freezing

BACKGROUND: Cryopreservation of hematopoietic cells with the rate- controlled method is used in the majority of centers. In recent years, there has been a trend toward the simplification of the process. STUDY DESIGN AND METHODS: A simplified method for cryopreservation was developed with 5-percent dimethyl sulfoxide (DMSO) as the sole cryoprotectant without rate-controlled freezing. Experiments were done with progressive concentrations of DMSO, ranging from 0 to 10 percent. With DMSO concentrations from 5- to 10-percent, the best recovery and viability for hematopoietic progenitor cells were observed. Hematopoietic progenitor cells with plasma and 5-percent DMSO were frozen and stored in a -80 degrees C mechanical freezer. Ten patients with solid and hematologic malignancies underwent transplantation with autologous hematopoietic progenitor cells. RESULTS: The median number of transfused mononuclear cells and CD34+ cells was 3.70 (3.1-8.2) × 10(8) per kg and 1.70 (0.8-6.5) × 10(6) per kg, respectively. The median number of transfused colony-forming units-granulocyte-macrophage was 12.45 (3.4-55.3) × 10(4) per kg. All patients showed rapid and sustained engraftment. The mean times to reach a neutrophil count of 0.5 × 10(9) per L and a platelet count of 50 × 10(9) per L were 11.50 +/− 1.70 and 13.90 +/− 3.98 days, respectively. All patients are alive and without transfusion requirements in complete remission 2 to 8 months after transplantation. CONCLUSION: This simplified cryopreservation technique will be useful for institutions without rate- controlled freezing facilities. Moreover, this method diminishes the amount of DMSO infused to patients, as well as its toxicity.     

Comparison of different X-ray films for 32P-autoradiography using various intensifying screens at -20 degrees C and -70 degrees C

The amplifier effect of different intensifying screens on commercially available X-ray films was tested in autoradiography with 32P at -20 degrees C and -70 degrees C. For the films X-Omat AR, RX and HR-G in combination with the screens G 12 and Lgy-S 600, there was no significant enhancement of blackening at -70 degrees C compared with -20 degrees C. Therefore exposure at -20 degrees C is generally feasible, if an appropriate film-screen combination is used.     

Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at -80 degrees C without postthaw processing

BACKGROUND: Platelets (PLTs) can be frozen with 6 percent dimethyl sulfoxide (DMSO) at -80 degrees C for up to 2 years. This method has been modified by concentrating the PLTs and removing the supernatant before freezing. STUDY DESIGN AND METHODS: High-yield leukoreduced PLTs stored at 22 degrees C for up to 5 days were divided into three equal volumes: one was frozen with 6 percent DMSO at -80 degrees C, thawed, washed, and resuspended in plasma (old method with DMSO); the second was treated with 6 percent DMSO, concentrated to remove the supernatant DMSO, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method with DMSO); and the third was treated with 0.9 percent NaCl without DMSO, concentrated to remove the supernatant solution, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method without DMSO). RESULTS: Freeze-thaw-wash recovery of PLTs frozen by the old method with DMSO was 74 +/- 2 percent with 5 percent PLT microparticles. Freeze-thaw recovery was 94 +/- 2 percent with 7 percent PLT microparticles (new method with DMSO) and 69 +/- 9 percent with 15 percent PLT microparticles (new method without DMSO). Total DMSO in washed PLTs was 400 and 600 mg in PLTs concentrated before freezing. In vivo recovery of PLTs frozen by the new method with DMSO and transfused into normal volunteers was 30 percent and the life span was 7 days. CONCLUSION: Concentrating PLTs before freezing simplified the procedure by eliminating postthaw washing. PLTs frozen by this method had more PLTs with reduced GPIb and increased annexin V binding than those frozen by the old method.     

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)     

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.     

The immunocompetence of residual lymphocytes at various stages of red cell cryopreservation with 40% w/v glycerol in an ionic medium at −80 C

Potentially immunocompetent cells have been found in washed liquid-stored red blood cells, in washed liquid-stored red blood cells to which a 40% W/V glycerol concentration was added, and in washed red blood cells freeze-preserved with 40% W/V glycerol at -80 C. A glycerol concentration of 40% W/V in an ionic medium, in addition to its cryoprotective effect on red blood cells, has a damaging effect on leukocytes. The freeze-thaw-wash process appears to produce the most damage to leukocytes and the remaining lymphocytes can be categorized into two groups: one that is capable of responding to phytohemagglutinin (PHA), and one that is not. Our study confirmed the presence of PHA-responsive lymphocytes in red blood cells freeze-preserved with 40% W/V glycerol in an ionic medium at -80 C. Although the relative proportions of these cells were variable and the results of the study somewhat erratic, we continue to recommend that liquid-preserved and freeze-preserved red blood cells be irradiated before transfusion to patients in whom graft-versus-host disease is a possible complication.