Viability and function of platelet concentrates stored in CPD-adenine (CPDA-1)

The effective use of CPDA-1 as an anticoagulant in routine blood banking practice requires demonstration that platelet concentrates prepared in this solution meet both in vitro quality control standards and maintain posttransfusion viability and function after storage. In this study of 138 units of CPDA-1 platelet concentrates, the average platelet count was 8.0 +/− 0.2 × 10(10) with 81 per cent of the units having greater than 5.5 × 10(10) platelets. The mean poststorage pH was 6.68 +/− 0.03 and only four of the units had a pH of less than 6.0 (3%). Residual plasma volume averaged 75 +/− 1 ml. Platelet viability was determined in 16 normal volunteers by measuring survival of 51Cr- labeled autologous platelets after storage for 72 hours at 22 +/− 2 C. Platelet recovery averaged 50 +/− 4 per cent, while survival was 7.3 +/− 0.4 days for the 15 units with a pH above 6.0. Measurements of posttransfusion platelet viability and function were made in 12 paients with thrombocytopenia secondary to marrow failure. Their mean pretransfusion platelet count was 17,000 +/− 2,000/microliter, and their standardized template bleeding times were all greater than 30 minutes. Platelet recovery averaged 44 +/− 5 per cent and survival 3.3 +/− 0.5 days. In seven of the patients with the best posttransfusion increments, bleeding time was improved. Five patients with poor posttranfusion platelet increments showed no improvement in bleeding time with CPDA-1; two of these patients were also transfused with CPD platelets and had no response. Our studies indicate that platelet concentrates prepared in CPDA-1 meet in vitro quality control standards and after transfusion, maintain viability and function comparable to that of CPD collected platelets.     

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.     

Recovery of in vitro functional activity of platelet concentrates stored at 4 degrees C and treated with second-messenger effectors

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. Refrigerated storage at 4 degrees C would abrogate this problem but would also result in a rapid loss of in vitro viability and functional activity and in vivo viability. The inhibition of platelets during storage by a combination of specific, reversible, second-messenger effectors has been investigated to allow prolonged storage at 4 degrees C with significant retention of in vitro viability and functional activity. STUDY DESIGN AND METHODS: The combination of effectors was added directly to platelet concentrates, and this step was followed by storage at 4 degrees C. Control units were incubated at 4 degrees C without the effectors and at 22 degrees C according to standard blood-banking techniques. At 1, 5, and 9 days, the units were tested for recovery of cell number, recovery of in vitro functional activity and viability, and expression of platelet surface markers. RESULTS: Treated platelets stored at 4 degrees C for 9 days, while spherical in shape, displayed no loss of cell number and had a recovery of viability and functional activity, as compared with control platelets stored at 22 degrees C for 5 days, as follows: ADP and collagen aggregation responses of 250 and 100 percent, respectively; a 70-percent recovery of hypotonic shock response; and a 60-percent recovery of extent of shape change. The treated platelets also expressed an equivalent amount of the surface marker glycoprotein lb and a lower amount of the activation marker alpha-granule membrane protein-140 on the membrane surface. CONCLUSION: Second-messenger effectors added to platelets significantly maintained in vitro functional activity with storage at 4 degrees C. In vitro analysis demonstrates the potential for extended 4 degrees C storage of platelets with numerical and functional recovery comparable to that achieved with current methods. Refrigerated storage of platelet concentrates has the potential to reduce the risk of bacterial contamination.     

P-selectin mRNA is maintained in platelet concentrates stored at 4°C

BACKGROUND: Platelets (PLTs) contain mRNA and synthesize proteins in response to activation. Most guidelines for PLT concentrates (PCs) recommend ambient temperature for storage but the impact of the storage temperature on PLT mRNA content has not yet been investigated.
STUDY DESIGN AND METHODS: Ten leukoreduced apheresis PCs were split and stored at 22 and 4°C. P-selectin mRNA, its expression on PLTs, and its soluble form were quantified. In parallel, cellular (cell count, mean PLT volume), metabolic (pH, pO₂, pCO₂, HCO₃, glucose), and functional markers (swirling, hypotonic shock response, aggregation to collagen) were analyzed. Rotation thrombelastography was used to monitor the hemostatic potential of PLTs. All measurements were performed on Days 1 and 5 of storage.
RESULTS: After 5 days of storage at 4°C, only 31 ± 27 percent of P-selectin mRNA and 29 ± 41 percent of glyceraldehyde-3-phosphate dehydrogenase mRNA were lost, while minute amounts of the mRNAs were detectable at 22°C. In PCs stored at 4°C the percentage of P-selectin–positive PLTs was significantly higher when compared to PCs stored at 22°C. Soluble P-selectin concentrations did not significantly differ between both storage temperatures. Thrombelastography revealed significantly shorter reaction times in PLTs kept at 4°C.
CONCLUSION: Our data indicate that storage at 4°C is accompanied by maintained mRNA levels. PLTs with intact mRNA levels and short reaction times in thrombelastography might be functionally superior to PLTs that are devoid of mRNA and show less augmented P-selectin surface expression. In therapeutic applications, that is, if PLTs are transfused to control acute bleeding, PLTs kept at 4°C may be advantageous.     

Growth of bacteria in platelet concentrates obtained from whole blood stored for 16 hours at 22 degrees C before component preparation

BACKGROUND: Previous studies have shown that cooling whole blood to 22 degrees C immediately after collection allows it to be held for up to 16 hours before component preparation (overnight-hold method) without a significant decrease in the quality of components obtained. A study was designated to evaluate the effect of the overnight-hold method on the growth of bacteria in experimentally contaminated blood units. STUDY DESIGN AND METHODS: Twenty whole-blood units were inoculated with Staphylococcus epidermidis (300 colony-forming units [CFU]/mL; n = 10) or Escherichia coli (50 CFU/mL; n = 10) immediately after collection. Half the units of each group were fractionated 6 hours after collection and the other half after storage for 16 hours at 22 degrees C. Twenty additional whole-blood units were divided in two equal parts, one of which was white cell reduced before inoculation. These 40 half-units were inoculated with S. epidermidis or E. coli and processed by the overnight-hold method. The growth of bacteria was assessed in platelet concentrates on the second and fifth days of storage, in packed red cells on Day 35, and in fresh-frozen plasma after 60 days. RESULTS: No bacteria growth was detected in plasma or red cell units. On the second day of storage, both bacteria strains grew more slowly in platelet concentrates obtained from blood processed by the overnight-hold method. This difference disappeared for S. epidermidis on the fifth day. When white cell-reduced and non-white cell-reduced whole-blood units were compared, platelet concentrates from the latter showed a delayed growth of both bacterial strains on the second and fifty days of storage. CONCLUSION: Prolonged storage of whole-blood units at 22 degrees C before component preparation delays bacteria growth. This effect seems to be mediated by white cells.     

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)     

Effects of the addition of second-messenger effectors to platelet concentrates separated from whole-blood donations and stored at 4 degrees C or -80 degrees C

BACKGROUND: Platelet concentrates (PCs) are currently stored at 22 degrees C under continuous agitation. Because of the potential risk of the overgrowth of bacteria in case of contamination, PC shelf life is limited to 5 days. A mixture of second-messenger effectors is being evaluated to determine if it has benefits for cold liquid storage and cryopreservation of platelets. STUDY DESIGN AND METHODS: PCs separated from whole-blood donations by the buffy coat method were randomly assigned (n = 6 each) to be stored for 5 days at 22 degrees C under continuous agitation or at 4 degrees C after treatment with a platelet storage medium (ThromboSol, LifeCell Corp. ). PCs were also cryopreserved with 6-percent DMSO (final concentration) or with ThromboSol plus 2-percent DMSO (final concentration) (TC). After storage, platelets were analyzed by flow cytometry, transmission electron microscopy, and aggregation and perfusion techniques. RESULTS: Cold liquid storage of ThromboSol-treated platelets resulted in a lower binding of coagulation factor Va on the platelet surface than on platelets stored at 22 degrees C. In transmission electron microscopy, a conversion to spherical morphology was seen in the case of cold liquid storage. No difference between ThromboSol-treated platelets stored at 4 degrees C and platelets stored at 22 degrees C was seen in perfusion studies. Cryopreservation in the presence of TC prevented the reduction in glycoprotein Ib and IV expression on platelet surface that is seen in 6-percent DMSO-cryopreserved platelets. Platelets cryopreserved in TC covered, by thrombus, a significantly greater percentage of the perfused surface after the freezing and thawing process. CONCLUSION: ThromboSol-treated PCs separated from whole-blood donations by the buffy coat method, stored at 4 degrees C for 5 days, or cryopreserved in the presence of TC, maintained in vitro functional activity comparable to that achieved by current methods of storage, although discoid morphology was not preserved during cold liquid storage with ThromboSol.     

Platelet apoptosis and activation in platelet concentrates stored for up to 12 days in plasma or additive solution

Background: Several studies suggest that apoptosis of platelets occurs during storage of platelet concentrates (PC). We sought to determine whether storage of PC in additive solution alters levels of apoptosis during storage beyond the current shelf life (5–7 days). Study design and methods: Pooled buffy coat PC (n = 7) were prepared in either 100% plasma or 70% Composol and stored at 22 °C for 12 days. A third arm of the study stored PC in 100% plasma at 37 °C, which is thought to induce apoptosis. PC were tested for mitochrondrial membrane potential, annexin V binding, microparticles, caspase‐3/7 activity and decoy cell death receptor 2, as well as standard platelet quality tests. Results: Composol units remained ≥pH 6·88, with 36% lower lactate and higher pH vs plasma by day 12 (P < 0·001). Platelet function was better maintained, and activation and apoptotic markers tended to be lower in Composol units towards the end of storage. However, levels of all apoptosis markers assessed were not significantly different in units stored in Composol. Storage at 37 °C saw stronger correlation of apoptotic markers with standard quality tests compared to 22 °C, but loss of correlation of caspase‐3/7 activity with other apoptosis markers. Conclusion: We conclude that storage of platelets in 70% Composol vs 100% plasma does not increase the rate of platelet apoptosis. Our data agree with other studies suggesting that platelet apoptosis is sequential to high levels of activation, but share a significant degree of overlap.     

The fate of bacteria introduced into whole blood from which platelet concentrates were prepared and stored at 22 or 4C

Bacteria were intentionally introduced into units of whole blood. Platelet concentrates (PC) which were made from these units were stored at either room temperature (22 C) or at 4 C. In order to isolate small numbers of bacteria from a PC (i.e., 1 to 10 organisms per ml), substantial contamination (42 to 125 organisms per ml) of the whole blood was required. If the PC were stored at room temperature, all organisms except Pseudomonas aeruginosa, which was apparently killed, grew out of control within 48 hours. Storage of PC at 4 C resulted in the general maintenance of bacterial numbers. Since gross contamination of PC has only occasionally been reported, we conclude that past reports of modest contamination of platelet concentrates are primarily sampling artifacts.     

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.     

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.     

Therapeutic effectiveness and safety of outdated human red blood cells rejuvenated to improve oxygen transport function, frozen for about 1.5 years at 80 C, washed, and stored at 4 C for 24 hours prior to rapid infusion

After storage at 4 C for 20 to 28 days, red blood cells were biochemically modified to improve their oxygen transport function which had deteriorated during liquid storage. The solution used for rejuvenation contained pyruvate, inosine, glucose, phosphate, and adenine (PIGPA Solution B). The rejuvenated red blood cells were frozen with 40% W/V glycerol in a polyolefin plastic bag and were stored in the frozen state for about 1.5 years at −80 C. After thawing and washing the red blood cells were stored at 4 C in a sodium chloride- glucose-phosphate solution for 24 hours before transfusion. A pool of four to ten units was rapidly transfused to each of 14 elderly anemic recipients, 11 of whom had cardiopulmonary insufficiency. Recovery of the red blood cells after the freeze-thaw process was about 97 per cent, and after the freeze-thaw-wash process about 90 per cent. The 24− hour posttransfusion survival values were about 75 per cent, and the long-term survival values were about 85 days depending on the disease state of the recipient. The red blood cells had 1.5 times normal 2.3- DPG levels and a decreased affinity for oxygen at the time of transfusion and were able to delivery oxygen at high oxygen tension immediately after the rapid infusion of pools of from four to ten units through a 40-or 170-micron filter. Plasma hemoglobin levels were consistent with extravascular sequestration of nonviable red blood cells, and uric acid levels were not increased during the immediate 24− hour posttransfusion period.     

Transfusions of CPDA-1 red blood cells stored for up to 28 days decrease donor exposures in very low-birth-weight premature infants

The goal of this research was to study the safety and the efficacy of transfusing citrate-phosphate-adenine anticoagulant-preservative (CPDA-1) RBC stored for up to 28 days to reduce donor exposures in premature infants. A prospective randomized two-group study was conducted with very low-birth-weight premature infants that received at least one RBC transfusion during hospital stay. Neonates randomly assigned to Group 1 (26 infants) were transfused with CPDA-1 RBC stored for up to 28 days; those assigned to Group 2 (26 infants) received CPDA-1 RBC stored for up to 3 days. Demographic and transfusion-related data were collected. Neonates from both groups showed similar demographics and clinical characteristics. The number of transfusions per infant transfused was 4.4 +/- 4.0 in Group 1 and 4.2 +/- 3.1 in Group 2, and the number of donors per infant transfused was 1.5 +/- 0.8 (Group 1) and 4.3 +/- 3.4 (Group 2), P < 0.001. RBC transfusions containing 29.7 +/- 18.3 mmol L(-1) of potassium (RBC stored for up to 28 days) did not cause clinical or biochemical changes and reduced donor exposures by 70.2%, compared to transfusions containing 19.8 +/- 12.3 mmol L(-1) of potassium (RBC stored for up to 3 days), P < 0.001. In conclusion, RBC stored for up to 28 days safely reduced donor exposures in premature infants.     

The effect of prestorage white cell reduction on the function and viability of stored platelet concentrates

The role of residual donor white cells (WBCs) in producing the storage lesion of platelets used for transfusion was studied. The effect of prestorage WBC reduction on in vitro and in vivo measurements of the quality of stored platelet concentrates (PCs) was examined by using a newly developed WBC-reduction filter capable of preparing PCs with a mean residual WBC concentration of less than 1 per microL. For in vitro studies, a triplet study design was used, in which WBC-reduced PCs were matched to standard PCs and to WBC-enriched PCs obtained from the same donor at the same phlebotomy. Twelve donors were studied. Prestorage WBC reduction resulted in a higher pH and pO2 and a lower pCO2 than in standard PCs. In accord with previous in vitro studies, a significant rise in plasma glycocalicin and lactate dehydrogenase was measured during storage, but the levels were not significantly different in WBC-reduced PCs and standard PCs. Platelet aggregation and ATP release in response to graded doses of thrombin was similar in WBC-reduced and standard PCs. In vivo recovery and survival studies were comparable in WBC-reduced and standard PCs. Although the residual donor WBC content of PCs has a significant impact on storage pH, pO2, and pCO2, prestorage WBC reduction does not affect platelet structure, function, or viability as assessed by in vitro or in vivo measurements.     

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.