Circulation and function of human platelets isolated from units of CPDA- 1, CPDA-2, and CPDA-3 anticoagulated blood and frozen with DMSO

Platelet concentrates (PC) were isolated by serial differential centrifugation from units of blood anticoagulated with one of the citrate-phosphate-dextrose-adenine solutions (CPDA-1, CPDA-2, CPDA-2). The platelet concentrates were frozen with six percent dimethylsulfoxide at 2–3 degrees C per minute and stored in a -80 degrees C mechanical freezer in polyvinyl chloride or polyolefin plastic containers. After frozen storage at -80 degrees C for up to three months, the concentrates were thawed at 42 degrees C within 2.5 to 4.0 minutes, washed with autologous plasma, two percent dimethylsulfoxide and 10 percent acid-citrate-dextrose solution, and then resuspended in plasma. The washed platelets were labeled with 51Cr and transfused back to the donor from whom they had been obtained. In vitro recovery from whole blood to platelet concentrate was 70.5 ± 17 percent (mean ± one SD). In vitro freeze-thaw-wash recovery determined by phase microscopy was 78.5 ± 12.8 percent, in vivo 51Cr platelet recovery two hours after transfusion was 41.3 ± 13.5 percent, and the platelets had a linear lifespan of about eight days. A single unit of previously frozen platelets shortened an aspirin- prolonged bleeding time two and 24 hours after infusion. Results were similar with platelets isolated from all three anticoagulants and stored in both plastics. The results also were comparable to previous findings in this laboratory with platelets isolated from ACD and CPD anticoagulated blood.     

The in vivo survival of red blood cells stored in modified CPD with adenine: report of a multi-institutional cooperative effort

In order to provide data in support of licensure applications for citrate-phosphate-dextrose (CPD) supplemented with adenine, a multi- institutional cooperative effort was organized to determine survivability of red blood cells subjected to prolonged liquid storage. Two manufacturers supplied plastic multiple bag blood storage containers prefilled with modified CPD (glucose 25/ greater than the normal concentration) supplemented with adenine (17.0 to 17.3 mg per 63 ml of anticoagulant; 0.25 millimolar approximate final concentration when diluted with 450 ml of whole blood for 35 days showed a mean survival of 80.53 +/- 6.44 per cent (1 SD). Both red blood cell and supernatant plasma biochemical characteristics were comparable to those reported for whole blood stored for 21 days in either acid-citrate- dextrose (ACD) or CPD. Red blood cells from 19 units stored as concentrates for 35 days (Hct 75.03 +/− 3.74%) had a mean survival of 71.38 +/− 10.3 per cent with considerable interdonor variation in survival and interlaboratory variation in some biochemical characteristics. Red blood cells from eight units stored as concentrates (Hct 75.38 +/− 4.30%) for 28 days showed a mean survival of 83.97 +/− 6.10 per cent and biochemical characteristics comparable to those reported for red blood cell concentrates stored in CPD or ACD for 21 days. Modified CPD with adenine as formulated offers an improved anticoagulant for blood banking by extending the permissible red blood cell storage period.     

Di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylexyl phthalate (MEHP) accumulation in whole blood and red cell concentrates

Plasma DEHP concentrations were measured weekly in whole blood and red cell concentrates (RCC) during 21 days of storage in standard CPD within PL-130 blood bags. In addition, DEHP and MEHP accumulation patterns were investigated in blood stored for 42 days in modified CPD with adenine within PL-146 and BB-69 storage containers. Total per-unit plasma DEHP of RCC units was 49 to 71 per cent of the total in plasma of whole blood units (PL-130). From 28 to 42 days, mean DEHP levels were 12 to 19 per cent higher in whole blood stored in PL-146 than in BB-69. Although MEHP was not found in any blood bag plastic, MEHP accumulated in plasma during whole blood storage. MEHP concentrations were 2.8 to 3.8 times higher in plasma stored in BB-69 than in PL-146. It is postulated that MEHP arises from hydrolysis of DEHP by plasma lipase, even in frozen plasma sample, and that the rate of this reaction is influenced by blood bag plastic surface characteristics.     

An in vivo comparison of CPD and CPDA-2 preserved platelet concentrates after an 8-hour preprocess hold of whole blood

To see if citrate-phosphate-dextrose-adenine-two (CPDA-2) anticoagulant- preservative had an effect on the viability of platelets, we studied autologous in vivo recovery and survival in humans for platelet concentrates prepared from six units of blood drawn into CPDA-2 and compared them to six units drawn into citrate-phosphate-dextrose (CPD). These units were prepared from whole blood held at room temperature for 8 hours after collection and were then stored for 3 days at 22 ± 2 degrees C. The recovery for platelets preserved in CPD was 39.0 +/0 4.8 percent and for platelets preserved in CPDA-2, 32.5 ± 4.4 percent. The difference was not significant (p greater than 0.10). In order to estimate population differences, in vitro effects on in vivo viability were also evaluated. Six in vitro variables were studied but only pH at 72 hours (r = 0.77), platelet count (r = 0.64), and morphology score (r = 0.66) correlated to recovery. Only pH at 72 hours significantly influenced recovery (p = 0.007). By adjusting for individual pH differences, mean recovery for platelets stored in CPD was 37.5 percent, and for platelets stored in CPDA-2, 34.0 percent. The mean lifespan was 6.7 ± 0.7 days for platelets preserved in CPD and 6.1 ± 1.0 days for those preserved in CPDA-2. Although hemostatic function was not studied, these data support in vitro observations that platelets preserved with CPDA-2 are not different from platelets preserved with CPD, even after 8-hours of storage of whole blood at room temperature prior to platelet concentrate preparation.     

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.     

Di‐2‐ethylhexyl Phthalate, a Plasticizer Contaminant of Platelet Concentrates

Di‐2‐ethylhexyl phthalate (DEHP), a plasticizer used in polyvinyl chloride plastic blood bags, is extracted by human blood during storage at 4 C. The rate of extraction (0.25 mg/100 ml/day) suggested that after only two days of storage, a concentration of 0.5 mg/100 ml might be found in human blood. In contrast to this, five platelet concentrates that were stored at 22 C for two days had a calculated DEHP concentration of approximately 19 mg/100 ml. The concentration of DEHP in platelet‐poor plasma was 16.7 mg/100 ml while the platelets were found to contain 37.7 mg/100 ml packed platelet volume. This result might suggest that platelets can preferentially accumulate DEHP. It is not known if such concentrations of DEHP are injurious to humans who receive platelet transfusions.     

用AGN保存浓缩血小板悬液的效果观察

本文观察了用一种新的血小板保存添加剂——酸化葡萄糖营养液(AGN)于22℃水平振摇条件下,在国产PVC塑料袋内保存从ACD全血制备的浓缩血小板血浆悬液(PC)的保存效果。结果表明,保存5天的PC其血小板计数、pH值、血小板聚集率及血小板低渗休克反应等指标皆优于对照组(未加AGN的ACD-PC),用新鲜血浆孵育2小时后,其聚集率和低渗休克反应有较明显的恢复。透射电镜显示,在保存过程中,血小板的形态完整、无伪足出现。用上述方法保存120小时的PC,其保存效果与国外一些实验室用第二代血小板保存袋保存的CPD-PC的效果相似。     

Factors Influencing the 24-Hour Posttransfusion Survival and the Oxygen Transport Function of Previously Frozen Red Cells Preserved with 40 Per Cent W/V Glycerol and Frozen at —80 C

A 6.2 M glycerol solution was added directly to concentrated red blood cells before storage at — 80 C for at least two and one-half years. The glycerol was removed from the thawed red blood cells by one of four different washing procedures, and the washed cells were stored at 4 C for an additional 24 hours before transfusion. Recovery in vitro was about 90 per cent, and the posttransfusion survival was about 85 per cent. The CPD anticoagulant maintained the oxygen transport much better than ACD during storage of the red blood cells at 4 C for one week prior to freezing. Results were similar whether glycerolized red blood cells were washed in reusable stainless steel bowls, disposable polycarbonate bowls, or collapsible disposable polyvinylchloride plastic bags. The composition of the wash solution had no significant effect on the posttransfusion survival or oxygen transport function. When the washed red blood cells were stored in a sodium chloride-glucose-phosphate solution at 4 C for 24 hours before transfusion, the 24-hour posttransfusion survival and oxygen transport function was satisfactory. Freeze-preservation of red blood cells with hematocrits of about 40 V per cent and postthaw storage at 4 C for 24 hours resulted in an accumulation of supernatant hemoglobin and extracellular potassium. At the time of transfusion, the red blood cells were concentrated by centrifugation, the supernatant medium was removed, and the hematocrit adjusted to 70 V per cent.     

Platelet storage. Effects of leachable materials on morphology and function

A polyolefin plastic (PL 732) bag formulated without liquid plasticizer allows storage of platelets for 5 and, now, up to 7 days. In order to assess the leaching of compounds from this new plastic, extracts of the supernatant from platelet concentrates stored in these bags were analyzed by high-performance liquid chromatography, mass spectrometry, and gas-liquid chromatography. A leachable material was detected and identified as di(2-ethylhexyl) phthalate (DEHP). During the sterilization process, migration of the DEHP occurs from the polyvinylchloride (PVC) bags into the PL 732 plastic bag. The level of DEHP was 12-fold less in the extracts of PC supernatant stored in the PL 732 bag than those in the polyvinyl chloride (PL 146) plastic bags which were used previously for platelet storage. Platelets stored in low DEHP concentrations in the PL 732 bags were composed of 10 to 35 percent of unclassifiable shapes. These shape changes were not observed in higher concentrations of plasticizer, although the morphology scores decreased during storage in PL 146 as well. This effect on morphology was not related directly to the dose of DEHP. When platelet membranes were isolated from platelets stored in the presence of radiolabeled DEHP, the amount of bound 14C-DEHP was found to be directly proportional to the concentration of DEHP in the plasma supernatant. However, while there was a linear relationship between the protein concentration in the membrane fraction and the amount of bound DEHP, no specific DEHP binding site could be identified by electrophoresis of the solubilized platelet membranes.     

Physical Characteristics of Microaggregates in Stored Blood

The volume of microaggregates 13 to 80 μ in size which developed in blood components stored under varying conditions was measured with a particle size analyzer. The microaggregates were found to develop progressively during storage of ACD whole blood at 4 to 6 C. Coincident with this there was a drop in the platelet count during the first week of storage and a progressive reduction in the absolute granulocyte count. Microaggregate development after storage of various components of ACD blood was proportional to the concentration of platelets and leukocytes prior to storage. The microaggregates settled into the buffy coat after centrifugation and became larger. In vitro studies indicated that they were resistant to dissociation in vitro in comparison to platelet aggregates induced in fresh blood by adenosine diphosphate. Microaggregate formation was greater in CPD than in ACD anticoagulated blood stored at 4 to 6 C for 24 hours, but was not different after seven days of storage. A greater volume of microaggregates was formed in aliquots of ACD blood stored at 4 to 6 C than at room temperature, while no differences were noted after storage of blood in plastic bags or glass vacuum bottles.     

Behavior of Previously Frozen Erythrocytes Used during Open‐heart Surgery

Glycerolized human red blood cells that had been stored in the frozen state at ‐80 C up to six months, thawed, and washed by the dilution‐agglomeration procedure of Huggins, were administered during extracorporeal circulation along with nonfrozen acid‐citrate‐dextrose‐(ACD) stored blood. Eleven patients who received both nonfrozen ACD‐stored blood and the previously frozen, washed, concentrated cells were compared with 14 patients who were given whole blood stored only in ACD. No significant differences were observed between the two groups in respect to platelet count, partial thromboplastin time, prothrombin time, or fibrinogen level. Higher plasma and urine hemoglobin levels were observed in the patients who received both blood stored in ACD and washed, previously frozen, concentrated red blood cells. Hemoglobinuria was observed in both groups but was not accompanied by significant differences in BUN or creatinine levels. There was no significant difference in morbidity or mortality between the two groups of patients.     

The In Vivo Survival, Mode of Removal of the Non-Viable Cells, and the Total Amount of Supernatant Hemoglobin in Deglycerolized, Resuspended Erythrocytes

The data presented indicate that with the use of the Cohn fractionator to add and remove the intracellular additive, glycerol, and using the slow freeze technic with storage in the frozen state at —80 C or —120 C, the viability of erythrocytes can be maintained for at least two months. The thawed, deglycerolized erythrocytes can then be resuspended in either autologous plasma or in an artificial 5% Hyland albumin resuspension medium, stored up to 8 days at 4C, and transfused to the original donor with acceptable red blood cell survival. ACD blood can be stored for up to seven days at 4 C prior to glycerolization with acceptable 24-hour posttransfusion survival. The mode of removal of the non-viable erythrocytes is primarily through an extravascular mechanism without detectable hemoglobinemia. A limiting factor in the clinical acceptability of deglycerolized, resuspended erythrocytes is the amount of free supernatant hemoglobin present in the unit on the day of transfusion. Washing units of deglycerolized erythrocytes resuspended in autologous plasma and stored for up to 12 days at 4 C is effective in reducing the total amount of supernatant hemoglobin, and the saline washed erythrocytes show acceptable posttransfusion survival. However, deglycerolized red blood cells resuspended initially in the 5% Hyland albumin medium and stored at 4 C for five days or longer do not tolerate saline washing; 24-hour survival of less than 70 per cent was observed and intravascular liberation of hemoglobin was noted in this group only.
This paper reports the total in vitro loss of hemoglobin associated with preservation and with the saline wash used to remove the excessive supernatant hemoglobin which accumulates during the post-thaw period at 4C.     

Storage of platelet concentrates harvested from blood collected into dextrose-free preservative without agitation

Summary. This study addresses the possibility of platelet quality being maintained during storage by an endogenous metabolic fuel, while avoiding dextrose-induced lactate accumulation. This was achieved by harvesting platelet concentrates from blood donations collected into a dextrose-free anticoagulant. Adequate maintenance of all metabolic and functional parameters was observed in platelets from blood collected into 4% citrate. The requirement for platelets stored in CPD plasma to be agitated during storage was confirmed, but agitation could be omitted for dextrose-free platelets without increased lactate generation and a drop in pH. These results indicate that platelet concentrates from dextrose-free blood may be stored without some of the constraints accompanying platelet storage in conventional media, and may thus result in improved delivery of this product.     

Enumeration of previously frozen platelets using the Coulter Counter, phase microscopy, and the technicon optical system

The Coulter Counter, phase microscopy, and the Technicon optical system were used to enumerate platelets in samples of whole blood, platelet- rich plasma, platelet concentrates before and after addition of DMSO, and on platelet concentrates that had been frozen, thawed, and washed. We observed agreement among the three counting methods when platelet counts were determined in whole blood, platelet-rich plasma, and platelet concentrate before and after DMSO addition. Enumeration after the cryopreservation process, however, showed highly significant differences among the counting systems. Platelet counts on platelet concentrates after freezing the thawing performed with the Coulter Counter were 25 per cent greater than with phase microscopy and 55 per cent greater than with Technicon. Counts with phase microscopy were 30 per cent greater than Technicon values. These data indicate that the method used to enumerate previously frozen platelets affects the apparent platelet count.     

The Purification of Red Cells for Transfusion by Freeze Preservation and Washing

In the study of 125 units of washed, previously frozen red blood tells approximately 96.6 per cent of the leukocytes and 98.9 per cent of the platelets were removed. Less than 0.5 mg of plasma protein remained per unit. The length of storage as whole blood at 4 C prior to glycerolization of the red blood cells influenced the number of leukocytes in the washed, previously frozen red blood cells. Red blood cells frozen on the day of collection had 7.2 per cent residual white cells, whereas those stored at 4 C for 10 days or longer prior to glycerolization, freezing, and washing had 1.1 per cent residual leukocytes. When red blood cells were stored at 4 C for longer than one week, glycerolized, and washed without freezing, they had about 2 per cent residual leukocytes and platelets. The washing procedure removed at least 95 per cent of the platelets from nonfrozen and previously frozen red blood cells. Because of the removal of large numbers of leukocytes and platelets during the freeze-preserva-tion procedure and the excellent recovery of the red blood cells, washed, previously frozen glycerolized red cells are recommended for patients requiring red blood cells depleted of leukocytes, platelets, and plasma protein.     

Effect of delayed blood processing on the yield of factor VIII in cryoprecipitate and factor VIII concentrate

Current standards for the preparation of factor VIII (FVIII) concentrates from human plasma recommend separation of plasma from red cells (RBCs) within 6 hours of blood donation, thereby reducing the volume of plasma from donated whole blood available for processing to FVIII concentrate. The decay of FVIII clotting activity (FVIII:C) in whole blood and plasma stored at 22 and 4 degrees C and the recovery of FVIII:C in cryoprecipitate and FVIII concentrate prepared from plasma separated from whole blood stored overnight at 4 degrees C were investigated. In whole blood stored at 22 degrees C and plasma stored at either 4 or 22 degrees C, 90 percent of the original FVIII:C was present at 6 hours, 80 percent at 12 hours, and 65 to 70 percent at 18 hours. At these times lower levels of FVIII:C were recovered from whole blood stored at 4 degrees C, that is, 84, 68, and 56 percent, respectively. In cryoprecipitates prepared from plasma separated from RBCs after 18 hours' storage at 4 degrees C (18-hour plasma), 43 percent of FVIII:C activity was recovered, as compared with 61 percent recovered from standard plasma separated within 6 hours of donation (6-hour plasma), p less than 0.05. With large-scale preparation of FVIII concentrates, however, the yield of FVIII:C was similar whether 18- or 6-hour plasma was used. Thus FVIII concentrates--but not cryoprecipitates--can be prepared from plasma separated from whole blood stored at 4 degrees C for up to 18 hours without undue loss of potency.     

Manufacture of red cells in additive solution from whole blood refrigerated for 5 days or remanufactured from red cells stored in plasma

Background and Objectives: To investigate methods for the production of red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAG‐M), from whole blood or red cells stored in plasma for 5 or 6 days and to provide evidence that exchange transfusion RCC in citrate phosphate dextrose (CPD) plasma or citrate, phosphate, dextrose, adenine (CPDA‐1) plasma are of comparable quality. Methods and Materials: Ten RCC in SAG‐M were produced following the remanufacture of red cells in CPD plasma on day 5/6 or after 5 days hold as leucodepleted CPD whole blood. In addition, 10 RCC in CPD plasma and 9 in CPDA‐1 plasma were stored without further processing. Units were assessed for red cell parameters including haemolysis, adenosine triphosphate (ATP), 2,3‐diphosphoglycerate (2,3‐DPG) and extracellular potassium. Results: Units in SAG‐M produced by remanufacture of RCC in plasma or by delayed manufacture of whole blood had comparable levels of haemolysis, ATP and 2,3‐DPG. Furthermore, these units underwent biochemical changes similar to reference SAG‐M units, with the exception of haemolysis which was greater at the end of shelf life and supernatant potassium which was lower following remanufacture. As expected, the decline in ATP was greater in red cells stored in CPD plasma compared with CPDA‐1 plasma. In general, units in CPD plasma were of similar quality at day 28 compared to those in CPDA‐1 plasma at day 35. Conclusions: RCC produced following the remanufacture of RCC in plasma or the delayed manufacture of whole blood are of acceptable in vitro quality and should be assigned the same shelf life as standard RCC in SAG‐M.     

The effect of platelets and red cells on granulocytes stored at 22 degrees C

Granulocyte concentrates contain varying numbers of platelets and red cells depending upon the method of collection. Either platelet or red cell concentrations may be as high as 2.0 × 10(12) per I. Studies were done on unwashed and washed granulocyte concentrates and on pure granulocyte suspensions with known numbers of platelets or red cells added. These suspensions or concentrates were stored for 72 hours at 22 degrees C. In both experiments, the following were measured: leukocyte and absolute granulocyte counts, dye exclusion, chemotaxis, plasma glucose, plasma pH, and osmolality. Red cell contamination did not adversely effect granulocyte storage. Platelets, however, did contribute to the functional deterioration of stored granulocytes. In the presence of high concentrations of platelets, both granulocyte dye exclusion and chemotaxis were adversely affected at 48 hours of storage. In another experiment, fresh autologous granulocytes incubated for 18 hours in hydroxyethyl starch-citrated-plasma obtained from stored granulocyte concentrates showed a progressive decrease in chemotaxis related to the age of the stored plasma. Glucose supplementation of the spent plasma maintained chemotactic activity. Contamination of granulocyte concentrates with other cells and the availability of glucose to granulocytes are variables affecting the short-term liquid storage of granulocytes at 22 degrees C.     

Effects of Adenine on Clotting Factors in Fresh Blood, Stored Blood, and Stored Fresh Frozen Plasma

The addition of small quantities of adenine to whole blood may prolong the useful shelf life of bank blood. The present study was undertaken to evaluate the effects of adenine on the clotting factors in blood containing ACD and CPD. Units of whole blood were collected in ACD, ACD-adenine, CPD and CPD-adenine, and each was stored 42 days under standard blood bank conditions. Samples of fresh frozen plasma containing these anticoagulants were stored three to four months at -30 C. Assays of Factors V, VIII (AHF), IX (PTC), X (Stuart Factor), fibrinogen, and prothrombin were performed on fresh blood, stored blood and stored fresh frozen plasma. The presence of small quantities of adenine did not appear to produce any appreciable alteration in the activity of the clotting factors in fresh blood. Further, adenine did not appear either to improve or worsen the survival of the procoagulants in whole blood stored 42 days or in fresh frozen plasma stored three to four months. There was significant deterioration of Factors V and VIII in whole blood stored 42 days in ACD, ACD-adenine, CPD, and CPD-adenine, but the degree of storage loss was independent of the anticoagulant employed. Factor X, fibrinogen, and prothrombin remained stable in blood stored 42 days regardless of the anticoagulant used, but Factor IX activity increased during storage possibly as the result of contact activation. Fresh frozen plasma stored three to four months showed a uniform slight loss of Factor VIII in all four anticoagulants, but Factors V, IX, X, fibrinogen, and prothrombin remained stable in stored fresh frozen plasma regardless of the anticoagulant employed.     

Preparation of white-cell-poor blood components using a quadruple bag system

A method for the preparation of white-cell-poor red cells from 400 ml of blood collected in a quadruple bag with one 80-ml and two 300-ml satellite bags is described. In this procedure, a platelet concentrate was prepared from the buffy coat fraction obtained by the first centrifugation of whole blood. After centrifugation of whole blood for 5 minutes at 3500 X g, the plasma was transferred into the 300-ml bag until the interface of red cells and plasma reached a level 32 mm from the top of the bag; then approximately 70 g of plasma and buffy coat were collected into the 80-ml bag. The buffy coat fraction was centrifuged further for 5 minutes at 170 X g, and the supernatant (concentrated platelets in plasma) was transferred into the second 300-ml bag. In this blood processing, the recovery of red cells into the packed red cells and of platelets into the platelet concentrate was 93 +/- 4 percent and 52 +/- 13 percent, respectively, of the original value. White cells in the packed red cells were 70 +/- 28 X 10(7) with recovery of 32 +/- 9 percent of the original value, and the lymphocytes in the white cells were only 7 +/- 5 X 10(7) (7 +/- 4% of the original value). White cell contamination of platelet concentrate was below the threshold of white cell detection by the microcell counter (less than 300 cells per microliter of concentrate).