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.     

Apoptotic markers are increased in platelets stored at 37 degrees C

BACKGROUND: PLTs for transfusion lose viability during storage in blood banking. This loss of viability is accelerated at 37 degrees C, as is the risk of bacterial contamination, and has led to the selection of 22 degrees C as the routine storage temperature. Because PLTs contain an intact apoptotic mechanism, we sought to determine whether PLTs undergo apoptosis during storage and whether storage at 37 degrees C accelerated this process. STUDY DESIGN AND METHODS: PLT-rich plasma from PLT concentrates was stored at 37 or 22 degrees C in small aliquots or whole bags, with and without cell-permeable caspase inhibitors. Number of PLTs, pH, LDH level, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium activity were analyzed over time. PLT lysates were prepared and tested for the presence and activation of apoptotic proteins by enzyme assay and Western blotting. RESULTS: PLT viability was greatly reduced after 1 to 2 days of storage at 37 degrees C; however, signs of apoptosis were evident by 3 hours after temperature shift. In temperature-stressed PLTs only, a gradual rise in caspase-3 activity was detected that correlated with the appearance of the 17- to 20-kDa cleavage products of caspase-3. Gelsolin, a caspase-3 substrate, underwent cleavage within the same time frame. Bcl-xL and caspase-2 also declined significantly; caspase-9 activity rose. Specific caspase inhibitors could prevent caspase activation but did not improve PLT cellular viability at 37 degrees C. CONCLUSIONS: PLTs contain apoptotic proteins that are activated during PLT storage at 37 degrees C and may account for the rapid decline in PLT cellular viability. Although ineffective here, inhibition of PLT apoptosis may improve PLT cellular viability.     

Aggregation response of human platelets stored at 22 C as platelet-rich plasma

The aggregating response of human platelets stored at 22 C for 72 hours has been studied. Platelets were stored as platelet-rich plasma in order to maintain the plasma pH essentially constant. The response to ADP and collagen decreased with time, but the decrease was less with relatively high concentrations of the aggregating agents. The response to the ionophore A23187 was essentially unaltered during the storage period. Synergistic aggregation with combinations of ADP, collagen and ionophore A23187 was observed with platelets stored for 72 hours under conditions where singly each of the stimuli caused little or no effect. Stored platelets underwent reversible aggregation over a wide range of ADP concentrations but irreversible aggregation was observed in the presence of nonaggregating concentrations of collagen or ionophore A23187. The inhibition of aggregation by PGE1 or cyclic AMP was facilitated as a result of storage. It is suggested that the decreased response toward ADP and collagen reflects a reduced ability on the part of the platelets to mobilize calcium.     

Studies on platelets exposed to or stored at temperatures below 20 degrees C or above 24 degrees C

BACKGROUND : Platelet concentrates (PCs) may be subjected to temperatures outside 20 to 22 degrees C during shipping or storage, which may have an adverse effect on platelet quality. STUDY DESIGN AND METHODS : These studies systematically evaluated the effect of short- term exposure (≤ 24 hours) of platelets to temperatures above 22° or below 20° C as part of standard 5-day PC storage at 22° C, as well as the effect of long-term storage (5 days) at 24 and 26° C. For the short-term exposure studies, up to 6 units of Day 1 standard PCs were mixed, split, and returned to the containers. Test units were then stored without agitation in an incubator at a specific temperature (4, 12, 16, or 18° C) for various times up to 24 hours, after which they were stored with agitation at 22° C. One unit acted as control and was stored at 20 to 22° C throughout the 5-day storage period. Loss of platelet discoid shape was determined photometrically by the extent of shape change assay, by an increase in apparent platelet size by morphologic evaluation, and by swirling. RESULTS : A gradual loss of platelet discoid shape occurred at temperatures below 20° C. For similar periods, a greater difference between test and control PCs was observed in units held at 4° C than in those held at 16° C. The data were fitted to an equation to relate platelet discoid shape (% of control) to exposure temperature and time. Assuming that a 20-percent decrease or more in the extent of shape change assay represents a significant loss in platelet viability, the equation predicts that such a loss occurs when the platelets are exposed to 16° C for ≥16 hours, to 12° C for ≥10 hours, or to 4°C for ≥6 hours, whereas exposure to 18° C for ≤24 hours has no significant effect. Storage for 5 days at temperatures ≤26° C was not associated with any significant reduction in platelet discoid shape or other measures of platelet quality. CONCLUSION : There was a gradual loss of platelet discoid shape at exposure temperatures < 20°C, which worsened as temperatures decreased and exposure times increased to 24 hours. This relationship can be described in an equation that could be used as a guideline for allowable exposure conditions.     

Release of platelet-derived growth factors and proliferation of fibroblasts in the releasates from platelets stored in the liquid state at 22 degrees C after stimulation with agonists

BACKGROUND: Fresh platelet (PLT)-rich plasma (PRP) treated with thrombin plus calcium chloride (CaCl(2)) is used to prepare a PLT gel to promote hemostasis and wound healing in a variety of surgical procedures. The effects of various agonists on stimulating the release of growth factors from liquid-preserved PLTs and the effects of the PLT releasate on the growth of fibroblasts in tissue culture were investigated. STUDY DESIGN AND METHODS: Plateletpheresis PLTs stored at 22 degrees C as high-yield PLTs for 3 to 6 days or outdated PLTs for 9 days were treated with agonists to assess release of platelet-derived growth factor (PDGF) AA, PDGF AB, PDGF BB, transforming growth factor-beta1 (TGF-beta1), and osteocalcin and the proliferation of fibroblasts treated with the PLT releasates in tissue culture. RESULTS: All treatments except for CaCl(2) alone and zeolite-CaCl(2) produced significant increases in PDGF AA compared to PRP. Thrombin-CaCl(2) produced significant increases in PDGF BB. Treatment by all the agonists produced similar increases in PDGF AB. TGF-beta1 and osteocalcin levels after treatment were similar to those in PRP. PRP releasate before and after stimulation with different agonists increased proliferation of fibroblasts in tissue culture. CONCLUSION: High-yield and outdated liquid-preserved PLTs released PDGF AA, AB, and BB but not TGF-beta1 or osteocalcin. The releasate from untreated PRP stimulated the proliferation of fibroblasts in tissue culture similar to the releasates from PRP treated with the different agonists. Further studies are needed to assess whether or not high-yield and outdated PLTs may be useful in wound healing.     

Stability of imipenem in Mueller-Hinton agar stored at 4 degrees C.

The purpose of the present study was to measure the stability of imipenem in Mueller-Hinton agar stored at 4 degrees C over time. MICs for Staphylococcus aureus ATCC 25923, Streptococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were determined in triplicate daily for up to 15 days. The calculated mean time to observe a shift of one dilution in MIC endpoints was 4.33 +/- 1.25 days. For routine work, imipenem agar dilution plates should be prepared within 48 to 72 h of the test.     

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.     

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.     

Pretransfusion incubation of apheresis platelets at 37 degrees C improves posttransfusion recovery

The effect of brief 37 degrees C pretransfusion incubation of apheresis platelets (n = 25) was studied in 14 allogenic bone marrow transplant recipients (5 children, 9 adults). Apheresis platelets were collected on a cell separator, divided in two aliquots, and stored 1 to 5 days at 22 degrees C during agitation. One aliquot was incubated at 37 degrees C for 1 hour before transfusion, and the other was transfused as a paired control. When the patient's peripheral platelet count fell below 30 x 10(9) per L, one aliquot was transfused. The other aliquot was transfused the following day. Each patient received the two aliquots in random order. Corrected count increment (CCI) at 1 hour after transfusion of 37 degrees C incubated platelets was 12.2 +/- 9.5 (mean +/- SD) compared to 7.5 +/- 5.4 for paired control platelets (p < 0.05). CCIs the next day were 4.4 +/- 6.1 and 2.4 +/- 2.7, respectively (not significant). It can be concluded that 37 degrees C incubation of apheresis platelets improves posttransfusion CCI.     

Logistics of platelet production: platelet concentrate from one-day-old blood stored at 10 degrees C

Platelet concentrates were prepared from blood stored at 18 to 20 hours at either 10 ± 2 degrees C or 16 ± 2 degrees C. Platelets prepared from blood stored at 10 degrees C had good in vitro properties. The results were not distinguishable from those obtained with freshly- isolated platelets. The in vivo effectiveness of platelets prepared in this manner must be established.     

The quality of fresh-frozen plasma produced from whole blood stored at 4 degrees C overnight

BACKGROUND: The aim of this study was to assess whether the quality of FFP produced from whole blood stored at 4 degrees C overnight is adequate for its intended purpose. STUDY DESIGN AND METHODS: Fresh-frozen plasma (FFP) separated from whole blood (n = 60) leukodepleted (LD) after storage at 4 degrees C overnight (18-24 hr from donation, Day 1 FFP) was compared with that LD within 8 hours of donation (Day 0 FFP, the current standard method). RESULTS: In more than 95 percent of Day 1 FFP units, levels of factor (F) II, FV, FVII, FVIII, F IX, FX, FXI, and FXII were greater than 0.50 U per mL except for von Willebrand factor (VWF) antigen and FVIII, where 92 and 87 percent of units, respectively, contained greater than 0.50 IU per mL. Compared with historical data on FFP stored for 8 hours, fibrinogen, FV, FVIII, and FXI were reduced by 12, 15, 23, and 7 percent, respectively, but other factors were not significantly reduced. Levels of VWF-cleaving protease activity were not different between FFP prepared from paired units of blood (n = 3) held for 8 or 24 hours, but were below the reference range in an additional 2 of 6 units held for 24 hours. The activities of protein S, protein C, antithrombin III, and alpha(2)-antiplasmin were reduced by less than 10 percent in Day 1 FFP (n = 20), but with final levels above the lower limit of the normal range in greater than 95 percent of units. Activated FXII antigen was not significantly raised in plasma stored for 18 to 24 hours, but levels of prothrombin fragment 1 + 2 were slightly increased (0.88 ng/mL, 18-24 hr; 0.65 ng/mL, < 8 hr). CONCLUSION: These data suggest that there is good retention of relevant coagulation factor activity in plasma produced from whole blood stored at 4 degrees C for 18 to 24 hours and that this would be an acceptable product for most patients requiring FFP.     

Stability of plasma fibrinogen during storage of platelet concentrates at 22 degrees C

Platelet concentrates (PC) are stored for up to 5 days at 22 degrees C prior to infusion. Since considerable suspending plasma is infused with the platelets, we examined the integrity of plasma fibrinogen from stored PC. The concentration of fibrinogen after storage was normal. After purification, fibrinogen from stored PC had normal thrombin time and rate of polymerization of fibrin monomer, and after reduction, its A alpha, B beta, and gamma chains had normal mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Assays of plasma from stored PC for fibrinogen-fibrin degradation products were negative. When 125I-fibrinogen was added to PC prior to storage and supernatant plasma was filtered in a sepharose 4B column after storage, radioactivity eluted in a single, symmetrical peak with no evidence for formation of low or high molecular weight material. These results make it unlikely that thrombin, plasmin, or other proteolytic activity is generated during storage. The levels of factors V and VIII fell to 40 to 65 percent of control values while the activities of factors IX, X, and XI did not change significantly during storage. We conclude that suspending plasma fibrinogen and other coagulation factors are remarkably stable during PC storage. They should be of value during the support of patients with massive hemorrhage.     

Evaluation of red blood cells stored at -80 degrees C in excess of 10 years

BACKGROUND: RBCs frozen in 40 percent (wt/vol) glycerol are currently approved by the FDA and the AABB for storage at -80 degrees C for up to 10 years. STUDY DESIGN AND METHODS: This study examined 20 RBC units that had been cryopreserved in 40 percent (wt/vol) glycerol and stored at -80 degrees C for up to 22 years. Measures of the freeze-thaw-wash (FTW) recovery, ATP, 2,3-DPG, methemoglobin, RBC indices, morphology, and osmotic fragility were made immediately after deglycerolization and after 24 hours of storage at 4 degrees C. RESULTS: RBCs frozen for longer than 10 years had acceptable mean FTW recovery, normal oxygen transport function, RBC morphology, RBC indices, methemoglobin, and osmotic fragility. Statistical analysis indicated that the in-vitro viability and function of cryopreserved RBCs was not dependent on the length of frozen storage or postthaw storage at 4 degrees C but did correlate with the storage length at 4 degrees C before cryopreservation. CONCLUSION: The data reported in this study demonstrate that RBCs can be stored at -80 degrees C beyond 10 years with acceptable in-vitro quality and suggest that more defined criteria for the cryopreservation process be adopted.     

Morphological classification by scanning electron microscopy of erythrocytes stored at 4 degrees C in citrate-phosphate-dextrose anticoagulant

Scanning electron microscopy of human blood stored at 4 degrees C in CPD buffer shows a variation of red cell morphology over a period of 21 days. Our results show that at least 10 significantly different red cell shapes are present in stored whole blood.     

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.     

Generation of annexin V-positive platelets and shedding of microparticles with stimulus-dependent procoagulant activity during storage of platelets at 4 degrees C

BACKGROUND: The ability of propyl gallate to activate platelet factor 3 has been determined through the activated partial thromboplastin time, but its effect on phosphatidylserine has not been established. STUDY DESIGN AND METHODS: A novel platelet activator, propyl gallate, was introduced to a study of platelets stored at 4 degrees C. The effects of storage on platelet coagulation activity, on phosphatidylserine, and on the shedding of activated and activable membrane particles (microparticles) were examined by activated plasma clotting time, and the effect on annexin V binding was examined by gated flow cytometry. The ratios of annexin V binding and microparticle shedding in stored platelet samples were compared with those in fresh platelets stimulated with propyl gallate. RESULTS: Microparticle shedding by stored platelets compensated for the diminished procoagulant potential of intact platelets (shown as the total propyl gallate-dependent platelet factor 3 activity), which did not change during prolonged (20-day) storage, but levels of phosphatidylserine confined to microparticles increased dramatically as platelet counts fell. Both annexin V binding and microparticle shedding increased spontaneously with storage and artificially with propyl gallate stimulation. However, at the same level of annexin V binding, stored platelets shed more microparticles than did fresh platelets stimulated with propyl gallate. CONCLUSION: Propyl gallate induces platelet procoagulant activity and annexin V binding. Stored platelets differ from fresh platelets in a lower reactivity to propyl gallate activation and a higher rate of microparticle shedding.     

Granulocyte concentrates. Glucose concentrations and glucose utilization during storage at 22 degrees C

Neutrophil metabolism is dependent on the available stores of either exogenous or endogenous glucose. We studied 38 granulocyte concentrates collected by four different centrifugal cytapheresis systems: Haemonetics model 30, IBM 2997, Fenwal CS-3000, and Fenwal Celltrifuge II. Extracellular glucose concentrations were measured immediately following collection and after 24 hours of storage. The Fenwal CS-3000 produced the highest concentrations of platelets and leukocytes, and the units had the lowest initial glucose levels and pH. After 24 hours of storage, there was a mean glucose of 1.7 mg per dl in the Fenwal CS-3000 granulocyte concentrates and 9 of 12 units had no detectable glucose. In contrast, concentrates collected from other systems had sufficient glucose available to support neutrophil metabolism for a longer period.     

Postfiltration factor VIII and fibrinogen levels in cryoprecipitate stored at room temperature and at 1 to 6 degrees C

The 13th edition of the standards of the American Association of Blood Banks specified storage at 1 to 6 degrees C for cryoprecipitated anti-hemophilic factor (Cryo) administered up to 6 hours after thawing if the Cryo is used for factor VIII (FVIII) content (Standard J4.210). Previous editions specified room-temperature (RT) storage for up to 6 hours. Currently, the temperature specification has been deleted. There are few data addressing the optimal storage temperature and maximum storage time for FVIII and fibrinogen in thawed Cryo. Thirty bags of Cryo were assayed for FVIII and fibrinogen. Each bag was divided into two aliquots; one was stored at RT and the other at 1 to 6 degrees C. Assays were performed immediately after thawing (Base) and 6 and 24 hours after thawing, respectively. All samples were filtered through 200-mu blood component infusion sets before assay. Three hundred analyses were performed, 150 each for FVIII and fibrinogen by conventional clotting technique. Data were analyzed by using a paired t test. Cryo stored at 1 to 6 degrees C for 6 and 24 hours showed an FVIII loss of 35 percent (p less than 0.0001) and 63 percent (p less than 0.0001), respectively. Cryo stored at RT for 6 and 24 hours had an FVIII loss of 8 percent (p greater than 0.05) and 20 percent (p less than 0.0001). Cryo stored at 1 to 6 degrees C for 6 and 24 hours had a fibrinogen loss of 20 percent (p less than 0.0001) and 43 percent (p less than 0.0001). Cryo stored at RT for 6 hours had no fibrinogen loss and a 2 percent loss at 24 hours (p greater than 0.05). These preliminary data show a significant loss of FVIII and fibrinogen activity in Cryo stored at 1 to 6 degrees C and filtered before assay. The FVIII and fibrinogen activity at RT is clearly maintained up to 6 hours after thawing.