Effect of WBC reduction and storage temperature on PLTs frozen with 6 percent DMSO for as long as 3 years

BACKGROUND: PLTs frozen with 6 percent DMSO can be stored at -80 degrees C for 2 years, while those frozen with 5 percent DMSO at -150 degrees C can be stored for at least 3 years. The more rapid deterioration seen in PLTs frozen at -80 degrees C may be due to the presence of granulocytes. The effects of storage temperature and WBC reduction on PLTs frozen with DMSO and the breakage of the PVC plastic bags stored at -80 and -135 degrees C were assessed. STUDY DESIGN AND METHODS: Apheresed PLT-rich plasma (PRP) was either divided into two equal volumes where one volume was WBC-reduced and the other volume was not or filtered or not and then divided into two equal volumes. PLTs frozen with 6 percent DMSO were stored in PVC plastic bags at either -80 or -135 degrees C for as long as 3 years. RESULTS: After 2 years of storage at -80 degrees C, the PLTs exhibited satisfactory freeze-thaw-wash values regardless of whether or not they were WBC-reduced, but after 2 to 3 years of storage at -80 degrees C, the PLTs had significantly reduced freeze-thaw-wash values. Freeze-thaw-wash values were not reduced in PLTs stored at -135 degrees C for up to 3 years. CONCLUSIONS: WBC reduction did not improve freeze-thaw-wash recovery values in PLTs stored at -80 or -135 degrees C for up to 3 years, but reducing the storage temperature from -80 to -135 degrees C did. Breakage of PVC plastic bags stored at -135 degrees C was excessive.     

Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide

BACKGROUND: There has recently been renewed interest in freezing platelets (PLTs) in dimethyl sulfoxide (DMSO) for the treatment of major traumatic injuries, especially in military situations. This study examined PLTs that were frozen in small volumes of 6 percent DMSO at ?80°C. STUDY DESIGN AND METHODS: Buffy coat–derived pooled leukoreduced PLT concentrates were frozen in 6 percent DMSO and stored at ?80°C. Assays included hypotonic shock response (HSR); aggregation; glycoprotein (GP)Ibα and P‐selectin binding sites; annexin V binding to phosphatidylserine, glycocalicin, and lactate dehydrogenase (LDH). Cone and plate technology (DiaMed Impact‐R, DiaMed) was used to test PLT function under near physiologic conditions. RESULTS: The freeze‐thaw loss of PLTs was 23 percent. HSR was 17 ± 7 percent. Cytometry demonstrated two populations of PLTs: one with normal levels of GPIbα binding sites (27 × 10³ ± 3 × 10³/PLT) and one with reduced levels (5.5 × 10³ ± 1.2 × 10³/PLT). There were 1.4 × 10³ ± 0.2 × 10³ P‐selectin binding sites per PLT. Annexin V binding to phosphatidylserine was 50 ± 9 percent and LDH was 496 ± 207 IU per 10¹² PLTs. Surface coverage and aggregate size, as measured by the DiaMed Impact‐R, were similar to those observed with PLTs stored for 2 days at 22°C. CONCLUSION: Some degree of activation was demonstrated by the proportion of PLTs with reduced levels of GPIbα binding sites, increased P‐selectin expression, and increased Annexin V binding. LDH concentrations indicated a degree of lysis. The DiaMed Impact‐R results showed that the PLTs were still capable of adhering to surfaces and forming aggregates under shear force.     

A randomized controlled trial evaluating recovery and survival of 6% dimethyl sulfoxide–frozen autologous platelets in healthy volunteers

BACKGROUND: Availability of platelets (PLTs) is severely limited by shelf life in some settings. Our objective was to determine and compare to Food and Drug Administration (FDA) criteria the PLT recovery and survival of autologous PLTs cryopreserved at ?65°C or less in 6% dimethyl sulfoxide (DMSO) reconstituted with a no‐wash method (cryopreserved PLTs [CPPs]) compared to autologous fresh PLTs. STUDY DESIGN AND METHODS: This was a randomized, Phase I study analyzing PLT viability and in vitro function in consenting healthy subjects. Apheresis PLTs (APs) were collected in plasma. APs were suspended in 6% DMSO, concentrated, and placed at not more than ?65°C for 7 to 13 days. Frozen CPPs were thawed at 37°C and resuspended into 25 mL of 0.9% NaCl. Control PLTs (fresh autologous) and CPPs were labeled with ¹¹¹In or ⁵¹Cr, and recovery and survival after reinfusion were determined using standard methods. A panel of in vitro assays was completed on APs and CPPs. RESULTS: After frozen storage, CPPs retained 82% of AP yield and showed increased PLT associated P‐selectin and reduced responses to agonists. CPP 24‐hour recovery (41.6 ± 9.7%) was lower than for fresh PLTs (68.4 ± 8.2%; p < 0.0001) and did not meet the current FDA criterion. CPPs had diminished survival compared to fresh PLTs (7.0 ± 2.1 days vs. 8.4 ± 1.2 days, respectively; p = 0.018), but did meet and exceed the FDA criterion for survival. CONCLUSION: While 24‐hour recovery does not meet FDA criteria for liquid‐stored PLTs, the CPP survival of circulating PLTs was surprisingly high and exceeded the FDA criteria. These data support proceeding with additional studies to evaluate the clinical effectiveness of CPPs.     

Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: effect of agonists, pH, and plasma and saline resuspension

BACKGROUND: Some of the tests used to assess the quality of fresh and preserved platelets (PLTs) include PLT number, PLT morphology, pH of the PLT medium, PLT response to hypotonic stress, and PLT aggregation to agonists. This study was performed to assess the function of fresh and preserved PLTs by their response to aggregation and their production of thromboxane A2 after in vitro stimulation with agonists. STUDY DESIGN AND METHODS: PLTs isolated by apheresis procedures were stored at 22 degrees C for as long as 5 days and then frozen with 6 percent dimethyl sulfoxide, stored at -80 degrees C, thawed, washed, and resuspended in medium. The effects of agonists and the pH and composition of the medium on PLT aggregation and PLT production of thromboxane A2 after stimulation were measured. RESULTS: The agonists and the pH and composition of the medium affected both the aggregation response and the production of thromboxane A2 by the fresh and preserved PLTs. PLT aggregation response to arachidonic acid (AA) and adenosine diphosphate (ADP) was significantly lower in the cryopreserved PLTs than in the fresh and preserved PLTs. After stimulation with AA and ADP, the cryopreserved PLTs produced more thromboxane than did the fresh and liquid-preserved PLTs. CONCLUSIONS: The agonists and the pH and composition of the medium affected the response to aggregate and produce thromboxane in vitro in both the fresh and the liquid-preserved PLTs. PLT thromboxane A2 production may be a better in vitro test than PLT aggregation to assess PLT function in vivo.     

The extension of 4 degrees C storage time of frozen-thawed red cells

Blood was drawn from volunteer donors and frozen using the high glycerin, mechanical freezing procedure accepted by the United States Navy. Subsequently, the units of blood were thawed and washed. Various anticoagulants were added, and the red cells were stored in a refrigerator at 4 degrees C for periods of up to 28 days. Chemical analyses were performed periodically. These showed that the addition of the anticoagulants ACD, CPD and CPDA-1 caused the red cells to be preserved better than the currently accepted 0.9-percent NaCl, 0.2-percent glucose solution. In vivo 51Cr viability studies performed on blood stored with CPDA-1 for 14 days showed a 24-hour viability of 78.8 +/- 8.4 percent. In a subsequent study, the blood was stored for 21 days prior to freezing and then was rejuvenated and frozen. The cells were thawed, washed, and stored at 4 degrees C with CPDA-1 for an additional 14 days. The 24-hour viability of these cells was determined to be 74.0 +/- 5.1 percent. These findings show that the postthaw storage time of red cells can be increased greatly over the now-accepted 24 hours, if bacterial sterility can be assured.     

Controlled-rate versus uncontrolled-rate freezing as predictors for platelet cryopreservation efficacy

BACKGROUND: Cryobiologic variables responsible for cell injuries and freezing techniques applicable in medical cryopractice should be revised and/or reengineered for minimizing cryoinjuries and maximizing cell recovery. In this study, the efficacy of different cryopreservation protocols based on platelet (PLT) recovery was evaluated. STUDY DESIGN AND METHODS: PLTs (n = 33) were prepared from whole-blood units. Cell count and viability, PLT morphologic score (PMS), and hypotonic shock response were determined. PLT surface antigens were measured by flow cytometry. Controlled-rate (with compensated fusion heat) and uncontrolled-rate freezing methods combined with 6 percent dimethyl sulfoxide were used. RESULTS: PLT recovery was superior in the controlled-rate setting (91.0 +/- 5.5 vs. 86.0 +/- 6.5; p < 0.05). PMS was significantly better in controlled-rate freezing (p < 0.01). GPIb/CD42b expression was reduced in both freezing groups versus control. GP140/CD62p expression was significantly (p < 0.05) lower in the controlled-rate group and in both frozen groups was significantly higher than in the control groups. CONCLUSION: The use of strictly equalized (1 degrees C/min) controlled-rate freezing, combined with an intensified cooling rate (2 degrees C/min) during the liquid-to-solid-phase transition period, allows advanced quantitative and qualitative PLT recovery, even though the minor intergroup differences for some variables were observed.     

A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment

BACKGROUND: Cryopreservation and storage permitting multiple treatments with single donations is of practical importance to cellular therapies. HES and DMSO, used successfully in simple clinical procedures for freezing marrow and peripheral blood progenitor cells at -80 degrees C, was tested on antigen-presenting dendritic cells (DCs) and cells used in their derivation. STUDY DESIGN AND METHODS: DCs cultured in serum-free media from adherent or CD14+ apheresis MNCs (n = 36) in the presence of GM-CSF + IL4 +/- TNFalpha were frozen and stored at -80 degrees C in 6-percent HES, 5-percent DMSO, and 4-percent HSA. Apheresis MNCs, CD14+ monocytes, and lymphocytes were similarly frozen and later thawed for culture. Cells were assayed for viability, DC phenotype, mixed lymphocyte reaction, and antigen presentation before and 3, 6, 9, 12 or more months after freezing. RESULTS: DCs retained viability (82 +/- 2.3%) for at least 24 months. Mature and immature phenotype and function were preserved. Thawed MNCs and CD14+ cells differentiated to DCs and lymphocytes maintained high functional viability (92 +/- 3%) comparable to prefreeze levels. CONCLUSION: A simple -80 degrees C freezing and storage method that combines extracellular (HES) and intracellular (DMSO) agents is practical and preserves functional viability of DCs, MNCs, CD14+ monocytes, and lymphocytes.     

Incidence of breakage of human RBCs frozen with 40-percent wt/vol glycerol using two different methods for storage at -80 degrees C

BACKGROUND: We reported previously that the incidence of breakage was 34.2 percent when human RBCs were frozen with 40-percent wt/vol glycerol in polyolefin plastic bags stored in aluminum containers at -80 degrees C and subjected to transportation. When human RBCs were frozen with 40-percent wt/vol glycerol at -80 degrees C in PVC plastic bags placed in polyester plastic bags and stored in rigid corrugated cardboard boxes, transportation resulted in a 2.4-percent incidence of breakage. The present study was done to confirm this incidence of breakage. STUDY DESIGN AND METHODS: The Meryman- Hornblower freezing method was compared to the Naval Blood Research Laboratory (NBRL) method of freezing for incidence of bag breakage. Human RBCs frozen by the Meryman-Hornblower method with 40-percent wt/vol glycerol with supernatant glycerol and stored in polyolefin plastic bags in aluminum containers at -80 degrees C were stored at the NBRL from 1974 to 2002. With the NBRL method, human RBCs frozen at -80 degrees C without supernatant glycerol in the 800-mL PVC plastic primary bag inside a polyester plastic bag in a rigid corrugated cardboard box were stored at the NBRL from 1984 to 2002. RESULTS: The incidence of breakage for 532 units of RBCs that had been frozen by the Meryman- Hornblower method and stored in aluminum containers was 47.3 percent for nontransported units. RBCs that had been frozen by the NBRL method and stored in rigid corrugated cardboard boxes exhibited breakage of 2.4 percent for 2424 nontransported units and 6.7 percent for 633 transported units. DISCUSSION: The incidence of breakage was significantly lower for RBCs frozen by the NBRL method than for the RBCs frozen by the Meryman-Hornblower method.     

Cryopreserved buffy-coat-derived platelets reconstituted in platelet additive solution: A safe and available product with sufficient haemostatic effectiveness

BackgroundPlatelets (PLTs) stored at 20–24 °C have a short shelf life of only 5 days, which can result in their restricted availability. PLT cryopreservation extends the shelf life to 2 years.MethodsWe implemented a method of PLT freezing at ?80 °C in 5–6% dimethyl sulfoxide. Buffy-coat-derived leucodepleted fresh PLTs blood group O (FP) were used for cryopreservation. Cryopreserved pooled leucodepleted PLTs (CPP) were thawed at 37 °C, reconstituted in PLT additive solution SSP + and compared to FP regarding PLT content, PLT concentration, pH, volume, PLT loss, anti-A/B antibody titre, total protein, plasma content, and PLT swirling. Clot properties were evaluated via rotational thromboelastometry. PLT microparticle number and surface receptor phenotype were assessed via flow cytometry.ResultsCPP met the required quality parameters. The mean freeze-thaw PLT loss was 22.24 %. Anti-A/B antibody titre and plasma content were significantly lower in CPP. CPP were characterised by faster clot initiation and form stable PLT clots. The number of PLT microparticles increased 25 times in CPP and there were more particles positive for the activation marker CD62 P compared to FP.ConclusionThawing and reconstitution are easy and fast processes if platelet additive solution is used. Low anti-A/B antibody titre and plasma content make possible the use of CPP of blood group O reconstituted in SSP + as universal ABO products, including clinical situations where washed PLTs are required. Clot properties evaluated via rotational thromboelastometry demonstrated that CPP retain a significant part of their activity compare to FP and are haemostatically effective.     

Effects of the temperature, the duration of frozen storage, and the freezing container on in vitro measurements in human peripheral blood mononuclear cells

Background: Bone marrow, peripheral blood, and umbilical cord blood have been used to prepare autologous and allogeneic pluripotential mononuclear cells for use in the repopulation of bone marrow. Study Design and Methods: The purpose of this study was to evaluate how the temperature and duration of frozen storage of human peripheral blood mononuclear cells (PBMCs), as well as the freezing container, affected the in vitro recovery and viability of the mononuclear cells and their growth in colony-forming unit-granulocytic-erythroid-monocytic- megakaryocytic (CFU-GEMM) tissue culture assay. PBMCs were isolated from ficoll-hypaque-treated cellular residue obtained during the plateletpheresis of blood from 15 healthy donors. The PBMCs were treated with dimethyl sulfoxide (DMSO) to achieve a final DMSO concentration of 10 percent. Each unit was then separated into six aliquots: one stored in a polyvinylchloride (PVC) plastic bag, one in a polyolefin plastic bag, and four in polyethylene cryostorage vials. Each aliquot was frozen in a -80 degrees C mechanical freezer at a freezing rate of 2 to 4 degrees C per minute. The frozen PBMCs in PVC bags were stored in a -80 degrees C mechanical freezer and those in polyolefin bags in a -135 degrees C mechanical freezer. Each of the four frozen samples in a vial was stored at a different temperature: one in the -80 degrees C freezer, one in the -135 degrees C freezer, one in the vapor phase of liquid nitrogen at -150 degrees C, and one in liquid nitrogen at -197 degrees C. Some of the frozen PBMCs were stored for periods of 1 to 1.5 years and others for 2 to 2.4 years, after which they were thawed, washed, and tested. Results: The samples stored in PVC bags and those stored in polyolefin bags exhibited in vitro recoveries that were 90 percent of the recovery of fresh PBMCs and viabilities of 90 percent after 2.4 years of frozen storage. The PBMCs stored in PVC bags exhibited no loss of CFU-GEMM activity after 1 to 1.5 years, but a 40-percent loss of activity was observed after 2 to 2.4 years. PBMCs stored in polyolefin bags, however, exhibited no loss of CFU-GEMM activity, even after 2 to 2.4 years of storage. In vitro recovery was significantly lower in PBMCs stored in vials at -80 degrees C or -135 degrees C than in cells stored in PVC or polyolefin bags at these temperatures, both in the 1- to 1.5-year and the 2- to 2.4-year time frames. In vitro recovery and viability were similar in PBMCs stored in vials at -80 degrees C, -135 degrees C, -150 degrees C, and -197 degrees C. The growth patterns in the CFU-GEMM assay in PBMCs stored in vials were significantly lower after storage at -80 degrees C than after storage at -135 degrees C, -150 degrees C, or -197 degrees C. Conclusion: PBMCs isolated by leukapheresis and ficoll-hypaque treatment can be frozen with 10-percent DMSO in a -80 degrees C mechanical freezer. When a PVC bag is used for freezing and storage of PBMCs at -80 degrees C, the duration of frozen storage should not exceed 1.5 years, whereas PBMCs frozen in a polyolefin bag can be stored in a -135 degrees C freezer for as long as 2.4 years. When these guidelines were followed, in vitro recovery was 90 percent that of fresh PBMCs, viability was 90 percent, and growth in the CFU-GEMM tissue culture assay was similar to that of fresh PBMCs. The PBMCs frozen and stored in PVC or polyolefin bags exhibited satisfactory results, whereas those stored in cryostorage vials did not.     

Bulk cryopreservation of lymphocytes in glycerol

The authors describe a method for freezing large amounts of peripheral blood lymphocytes (PBL) in a 20 percent glycerol solution. Between 0.6 and 4.3 X 10(9) cells in autologous plasma were frozen in polyethylene freezing bags in a final volume of 50 ml. The recovery after thawing averaged 89 +/- 14 percent with a mean viability by trypan blue dye exclusion of 80 +/- 7 percent (n = 11). In aliquots of fresh and frozen-thawed PBL from the same subjects radiolabeled with 111In, the radiolabeling efficiency for both fresh and thawed cells was 49 +/- 15 percent (p = 0.98, n = 5). The mitogen mean stimulation indices for glycerol-frozen cells (471 with phytohemagglutinin-M, 176 with pokeweed mitogen, and 380 with concanavalin A) were superior to those of cells frozen by a standard technique with dimethylsulfoxide (DMSO) (141, 47, and 123; p less than 0.05) and comparable to those of fresh PBL (403, 75, and 147). In a mixed lymphocyte culture, glycerol-frozen PBL showed significantly greater responsiveness to a pool of stimulator cells than did PBL frozen in DMSO (p = 0.03). Thawed cells are viable and functional as demonstrated by their response to mitogens and their ability to stimulate and respond in mixed lymphocyte culture.     

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.     

Use of supernatant osmolality and supernatant refraction to assess the glycerol concentration in glycerolized and deglycerolized previously frozen RBC.

BACKGROUND: Human RBC are frozen at a mean temperature of -80 degrees C (with a range of -65 degrees C to -90 degrees C) with a mean concentration of 40% w/v glycerol (with a range from 36% w/v to 45% w/v) for at least 10 years. After thawing and deglycerolization the RBC should have a residual glycerol concentration of about 1%. We conducted three studies to measure the supernatant osmolality and supernatant refraction in RBC frozen with 40% w/v glycerol and stored at -80 degrees C for as long as 16 years. The measurements were made before and after deglycerolization. STUDY DESIGN AND METHODS: In the first study, one hundred and three (103) units of RBC were glycerolized to achieve a concentration of 40% w/v glycerol in an open system and frozen at -80 degrees C for as long as 16 years. In the second study, 106 units of RBC were glycerolized to achieve a concentration of 40% w/v glycerol and in an open system and frozen at -80 degrees C for a mean of 14 years. In the second study, the RBC were deglycerolized using the Haemonetics ACP215 instrument before being stored at 4 degrees C in the AS-1 or AS-3 additive solution. In the third study, fifty-five (55) units of RBC were glycerolized to achieve a 40% w/v glycerol concentration in the functionally closed system of the Haemonetics ACP215 instrument containing the high separation bowl and frozen at -80 degrees C for at least 2 months. These RBC also were deglycerolized using the Haemonetics ACP215 and were stored at 4 degrees C in the AS-3 additive solution. Before and after deglycerolization, measurements also were made of the freeze-thaw recovery and the freeze-thaw-wash recovery values, the percent hemolysis, supernatant hemoglobin level, supernatant osmolality and supernatant refraction. RESULTS: The supernatant osmolality provided an accurate estimate of the glycerol concentration in the thawed RBC before deglycerolization but the supernatant refraction did not. However, after deglycerolization, both the supernatant osmolality and the supernatant refraction gave accurate estimates of the glycerol concentration in the RBC. CONCLUSION: The osmolality measured in the osmometer of the thawed supernatant of the glycerolized RBC provided an accurate estimate of the glycerol concentration but the percent refraction measured in the Palm Abbe refractometer did not. Both the osmolality and percent refraction in the deglycerolized washed RBC provided accurate estimates of the residual glycerol.     

Interruption of agitation of platelet concentrates: a multicenter in vitro study by the BEST Collaborative on the effects of shipping platelets

BACKGROUND: Transported platelets (PLTs) are not under continuous agitation. The aim of this study was to determine whether PLTs shipped between 24 and 48 hours would be able to maintain a pH(22 degrees C) value of 6.5 at the end of 7 days of storage. STUDY DESIGN AND METHODS: Six laboratories prepared leukoreduced PLTs. PLT pools were divided into low and high PLT concentration with paired shipped (20-43 hr) and unshipped controls. Units were under continuous agitation at 22 +/- 2 degrees C when not being transported. In vitro measures including pH, pO(2), and pCO(2) were determined over 7 days. RESULTS: Ninety-two PLT components from 24 pools were eligible for analysis. One unshipped control and three shipped products failed to maintain a pH(22 degrees C) value of 6.5 through 7 days. In vitro characteristics were maintained slightly better over 7 days of storage in the unshipped control arms. PLT concentration, shipping time, and their interaction were significant determinants of the final pH at the end of storage (p < 0.05). Lactate generation rate increased by 35 +/- 2 (mean +/- SE) micromol per 10(12) PLTs per hour over baseline during shipping (p < 0.0001). After restoration of standard blood banking conditions with agitation, this rate dropped 24 +/- 2 micromol per 10(12) PLTs per hour (p < 0.0001). CONCLUSION: PLTs in plasma shipped for at least 20 to 24 hours maintain a pH(22 degrees C) value of 6.5 for 7 days. A longer shipping time may result in a pH(22 degrees C) value of 6.5. During shipping, glycolysis was up regulated in these PLTs resulting in increased lactic acid production. After restoration of agitation, shipped products down regulated glycolysis. These effects should be accounted for in the development of PLT storage and transportation systems.     

Platelet membrane integrity during storage and activation

BACKGROUND: The platelet cell membrane appears to undergo a lipid-phase transition on cooling from 23 degrees C to 4 degrees C. Consequences of this phase transition are leakage of cellular material and irreversible cellular damage. Whether agents, of known benefit in protecting membranes and proteins from cooling and drying injury, could also protect platelets was investigated. Leakage of cytosolic components was assessed by measuring the release of fluorescein into the surrounding medium. STUDY DESIGN AND METHODS: Fresh platelets were suspended in 5 percent dimethyl sulfoxide (DMSO) or in 5 mM of one the following agents: glucose, trehalose, sucrose, glycerol, ethylene glycol, 1,2-propanediol, or L-proline. Platelets were loaded with 10 nMfluorescein diacetate (FD), chilled at 4 degrees C for 24 hours or frozen at -1 degree C per minute to -70 degrees C, warmed rapidly at 37 degrees C, and centrifuged, and the supernatant was measured for the presence of fluorescein. The effect of FD on platelets was assessed by agglutination with ristocetin, aggregation with thrombin and ADP, platelet-induced clot retraction, and expression of p-selectin. Platelet function and activation before and after freezing or cooling were measured by the same methods. RESULTS: By flow cytometry, 98 percent of the platelets incorporated FD. The trapped fluorescein resulted in neither platelet activation (p = 0.9) nor reduction of platelet function (p = 0.12-0.94) from that in control platelets. Freezing of platelets in DMSO caused far less release of fluorescein than did freezing with other agents (p<0.001) or chilling of platelets at 4 degrees C for 24 hours (p<0.0001). Supernatant levels of fluorescein correlated inversely with platelet function. Fluorescein was also shown to be released during aggregation with thrombin or ADP but not during agglutination with ristocetin. CONCLUSIONS: Release of fluorescein into the surrounding medium indicated a loss of platelet membrane integrity and function. Cellular loading with FD is a simple method of studying membrane integrity of platelets and other cells.     

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.     

Storage of buffy coat-derived platelets in additive solutions: in vitro effects of storage at 4 degrees C

BACKGROUND: The aims of this in vitro study were to compare the storage of platelets (PLTs) at 4 degrees C with those stored at 22 degrees C and to determine the in vitro effects of preincubation at 37 degrees C for 1 hour before the analysis on the basis of the maintenance of PLT metabolic and cellular integrity. STUDY DESIGN AND METHODS: PLT concentrates (PCs) were prepared from pooled buffy coats (BCs) for paired studies (total eight pools from 160 BCs). Each pool was divided into four PCs and stored under different conditions: at 20 to 24 degrees C on a flatbed agitator, at 20 to 24 degrees C on a flatbed agitator and with incubation of the samples at 37 degrees C for 1 hour before the analysis, at 4 degrees C, and at 4 degrees C and with incubation of the samples at 37 degrees C for 1 hour before the analysis. RESULTS: Storage of PLTs at 4 degrees C resulted in reductions in the rate of glycolysis and better retention of pH after Day 10 than in PCs stored at 22 degrees C (Day 14, 7.003 +/- 0.047 vs. 7.201 +/- 0.146). Hypotonic shock response and extent of shape change were higher at 22 degrees C than at 4 degrees C and in preincubated PCs stored at 22 degrees C than in reference PCs stored at the same temperature (Day 5, 45.6 +/- 2.7 vs. 36.5 +/- 3.9 and 24.1 +/- 2.0 vs. 15.5 +/- 1.8). The concentration of RANTES was higher in PCs stored at 22 degrees C than at 4 degrees C (Day 7, 179 +/- 25 vs. 79 +/- 32). CONCLUSION: PLTs stored at 4 degrees C without agitation maintain metabolic and cellular characteristics to a great extent during 21 days of storage. These studies confirm the view that PLTs lose their discoid shape and that this loss with storage at 4 degrees C is associated with reductions in metabolic rate and in their release of alpha-granule content.     

Viability and function of 8-day-stored apheresis platelets

BACKGROUND: Methods of bacterial detection and pathogen inactivation of platelets (PLTs) may allow extended storage of PLTs as long as PLT quality is maintained. STUDY DESIGN AND METHODS: Twenty normal volunteers had their PLTs collected with an apheresis machine (Haemonetics Corp.). A variety of in vitro PLT function and metabolic assays were performed both on Day 0 and after 8 days of storage. On Day 8, a small blood sample was drawn from each donor to obtain fresh PLTs. The fresh and stored autologous PLTs were labeled with either (51)Cr or (111)In, and the radiolabeled PLTs were transfused. Posttransfusion serial blood samples were drawn to determine the relative posttransfusion recoveries and survivals of the fresh versus the stored PLTs. RESULTS: Although the in vitro assays showed some differences between the two trial sites, the results were generally within the ranges expected for fresh and stored PLTs. Overall, PLT recoveries averaged 66 +/- 16 percent versus 53 +/- 20 percent and survivals averaged 8.5 +/- 1.6 days versus 5.6 +/- 1.6 days, respectively, for fresh compared to 8-day-stored PLTs. There were no significant differences in the in vivo PLT data between the trial sites or based on the radiolabel used for the measurements. CONCLUSION: After 8 days of storage, the in vivo posttransfusion recovery and survival of autologous Haemonetics apheresis PLTs meet the proposed standards for poststorage PLT quality.     

Leukoreduced buffy coat-derived platelet concentrates photochemically treated with amotosalen HCl and ultraviolet A light stored up to 7 days: assessment of hemostatic function under flow conditions

BACKGROUND: Amotosalen plus ultraviolet A light photochemical treatment (PCT) inactivates high titers of bacteria, and other pathogens, in platelet concentrates (PCs) potentially allowing the storage of platelets (PLTs) for up to 7 days. Adhesion and aggregation of PLTs to injured vascular surfaces are critical aspects of PLT hemostatic function. STUDY DESIGN AND METHODS: Two ABO-identical leukoreduced buffy coat-derived PCs in additive solution were mixed and divided: one-half underwent PCT (PCT-PCs) and the other was kept as a control (C-PCs); both were stored under standard conditions. The total number of paired PCs studied was nine. Samples were taken on Day 1 (before PCT) and after 5 and 7 days of storage. The adhesion and aggregation capacities were evaluated under flow conditions in a ex vivo perfusion model. RESULTS: Compared to control, PCT resulted in a decrease in PLT count of 6.5 percent (p = 0.004) and 10.2 percent (p = 0.008) after 5 and 7 days' storage, respectively (n = 9). PLT interaction with subendothelium was mainly in form of adhesion. The surface covered by PCT PLTs on Day 1 was 26.0 +/- 4.2 percent (mean +/- SEM). On Day 5, PCT-PCs showed a covered surface of 20.9 +/- 2.2 percent, and the C-PCs, 20.6 +/- 1.6 percent. After 7 days, PCT-PCs produced a nonsignificant higher PLT deposition compared to control (27.1 +/- 2.9% vs. 21.2 +/- 2.8%, p = 0.06). CONCLUSION: PCT of PCs and storage up to 7 days was associated with a 10.2 percent decrease in PLT count due to processing losses compared to C-PC. PLT adhesive and aggregating capacities under flow conditions of PCT-PCs were similar to C-PCs and remained well preserved for up to 7 days of storage.     

Paired in vitro and in vivo comparison of apheresis platelet concentrates stored in platelet additive solution for 1 versus 7 days

BACKGROUND: To improve clinical access to platelet concentrates (PCs), prolonging the storage period is one alternative, provided that they are free from bacteria. The quality of platelets (PLTs) stored for 1 versus 7 days was compared by in vitro analyses and in vivo recovery and survival in blood donors. STUDY DESIGN AND METHODS: Apheresis PCs from 10 donors were divided and stored in PLT additive solution in 2 equal units for a paired comparison. PLTs in one unit were (111)In-labeled at 1 day of storage, and PLTs in the other unit were labeled after 7 days of storage. PLTs were injected into the donor after labeling and samples were drawn after 30, 60, and 150 minutes and thereafter once a day for 14 days for recovery and survival measurements. RESULTS: PLT recovery on Day 7 was 80 percent of the recovery on Day 1 (p<0.05), and the survival on Day 7 was 65 percent of survival on Day 1 (p<0.005). No significant differences were seen regarding mean PLT volume (MPV), pH, pCO2, pO2, bicarbonate, or hypotonic shock response. Lactate increased and lactic dehydrogenase increased slightly, whereas glucose and ATP decreased, but not to a critical level. A significant increase in RANTES (110.7+/-76.6 vs. 277.6+/-50.8 pg/10(6) PLTs [p<0.005]) and PLT factor 4 (19.9+/-9.6 vs. 59.8+/-7.5 IU/10(6) PLTs [p<0.0001]) was noticed during storage. CONCLUSION: Recovery and survival of PCs stored for 7 days decreased, but met suggested criteria. Analyzed in vitro parameters showed acceptable results. Randomized patient transfusion studies will provide additional verification of the suitability of 7-day storage of PLTs.