Storage of pools of six and eight platelet concentrates

Platelet concentrates (PCs) prepared from units of whole blood are routinely stored singly at 20 to 24 degrees C and pooled prior to transfusion. Studies have been conducted to evaluate the in vitro properties of pools of six (n = 19) and eight (n = 17) ABO-identical PCs after storage, with comparative studies involving single units (n = 33). The pools were prepared using the sterile connecting device. One- day-old and 3-day-old PCs were pooled and stored for a total of 5 days in a container system consisting of two 1000-mL polyolefin containers. The pooled platelet suspension was divided approximately equally between the two containers. The platelet count was reduced by less than 5 percent during storage of the pools, which is similar to the reduction found with storage of control units of single PCs. The volume loss due to pooling was 9.6 +/− 1.9 percent (mean +/− 1 SD). The pH of the PC pools was approximately 7.0 after 5 days of storage, with no pool having a pH below 6.2. In vitro platelet properties, such as morphology score, extent of shape change induced by ADP, total ATP, aggregation response to ADP and collagen, response to hypotonic stress, lactate dehydrogenase discharge, and beta-thromboglobulin release, were similar for pools and control single PCs. In addition, comparable low levels of thymidine uptake were detected in the mononuclear leukocyte fraction of pooled and unpooled PCs that were stored for 5 days at 20 to 24 degrees C, which indicates that the mixing of lymphocytes in the pool did not stimulate in vitro immunologic reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Storage of pooled platelet concentrates. In vitro and in vivo analysis

The use of sterile connecting devices will permit up to 5-day storage of pooled platelet concentrates (PCs). However, there are no data evaluating long-term storage of PCs pooled from multiple donors. Four units of ABO-compatible or -incompatible PCs were pooled and stored in single 300-ml PL-732 storage bags for up to 5 days. Results of in vitro assays showed acceptable storage values regardless of the ABO types in the pool. Pool pH on Day 5 was 6.83 +/- 0.3 (mean +/- 1 SD). The in vitro storage characteristics were comparable to those of unpooled age-matched platelets reported previously from our laboratory. For in vivo studies, 4-unit pools of ABO-compatible random-donor PCs stored for up to 96 hours in 1000-ml PL-732 bags were transfused into patients who were thrombocytopenic due to bone marrow failure, and the correct count increments (CCI) were determined. In vivo results showed a mean 1-hour CCI of 11,368 +/- 5824 for the pooled stored platelets and 7819 +/- 5189 for unpooled controls (p greater than 0.05). To evaluate the possibility that passenger lymphocytes in the concentrates would generate mixed lymphocyte reactions (MLR) in the pooling bag during storage, lymphocytes were studied over 5 days of storage by the use of monoclonal antibodies against activated T-cell markers and by 3H thymidine uptake. Results failed to show evidence of either the generation of activated T-cell markers or the uptake of 3H thymidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteria levels in components prepared from deliberately inoculated whole blood held for 8 or 24 hours at 20 to 24 degrees C

BACKGROUND : An increase from 8 to 24 hours in the time that units of whole blood can be held at room temperature after phlebotomy would give blood centers more flexibility in component manufacturing and might allow receipt of many infectious disease test results prior to component preparation. However, the potential for bacterial growth during prolonged holding periods requires further study. STUDY DESIGN AND METHODS : In the Phase I study, 2-unit pools of ABO-identical whole blood were deliberately inoculated on Day 0 with Staphylococcus aureus or Pseudomonas fluorescens. They were then divided in half and stored at 20 to 24 degrees C. Red cells (RBCs) with additive solution, platelet concentrates (PCs), and frozen plasma were prepared after 8 and 24 hours. Bacteria levels in PCs and RBCs were monitored on Day 1; bacteria levels were measured in plasma after thawing. In the Phase II study, the same basic design as in Phase I was used, except that 10 bacterial species were studied, lower inocula were used, and RBCs prepared after a 24-hour room-temperature whole-blood hold were white cell-reduced by filtration. Bacterial growth was monitored during 42- day storage of RBCs (1 – 6 degrees C) and 5-day storage of PCs (20 – 24 degrees C) and after thawing of frozen plasma. RESULTS : For Phase I, significantly higher bacteria levels were observed in RBCs prepared after a prolonged hold (p < 0.05); higher levels were not observed in PCs and thawed plasma units. In Phase II, prior to white cell reduction by filtration, 8 of 10 organisms had significantly higher levels in RBCs prepared after a 24-hour hold than in RBCs prepared after an 8- hour hold, when both were examined on Day 1 (p < 0.05). For seven of eight organisms examined on Days 1, 21, and 42, filtration (white cell reduction) reduced the bacteria in RBCs prepared from 24-hour whole blood units to those levels found in unfiltered RBCs prepared from whole blood units held at 8 hours. A prolongation of the holding time from 8 to 24 hours resulted in significantly lower bacteria levels (p < 0.05) in PCs early in storage (Days 1, 1 – 2, or 1 – 3) for seven organisms, with no significant difference for two organisms, and a small but significant increase for one organism (Day 3, p < 0.05). There was no difference in bacteria or endotoxin levels in thawed units of plasma prepared from whole blood after 8- or 24-hour holding times. CONCLUSION : The levels of bacteria present in components after deliberately inoculated whole blood units are held for 8 and 24 hours depended on the organisms tested, the whole-blood holding period, and the blood component assayed; for RBCs, they also depended on whether WBC reduction by filtration was performed.     

Removal of soluble biologic response modifiers (complement and chemokines) by a bedside white cell-reduction filter

BACKGROUND: Biologic response modifiers infused with stored platelet concentrates (PCs) are believed to contribute to symptoms seen during transfusion reactions. Although prestorage white cell reduction is known to decrease the production of some biologic response modifiers during storage, the possibility that poststorage (bedside) white cell reduction could reduce the amount of biologic response modifiers already present in stored PCs during bedside filtration has not been well studied. STUDY DESIGN AND METHODS: Individual PCs were pooled on storage Days 2 and 5 and passed through a third-generation white cell- reduction filter. The results from a series of in vitro PC assays were studied, before and immediately after filtration, as were levels of C3a and interleukin 8 (n = 5). Levels of other biologic response modifiers- C5a, interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and RANTES-were also studied. Removal of interleukin 8 and RANTES was studied further by using serial filtration of units of PC. RESULTS: For the in vitro platelet assays studied, pH was unchanged after filtration from prefiltration values in units of PCs pooled on storage Day 2 or 5. A 4 log10 reduction in white cells was reliably seen after filtration in Day 2 and 5 pooled PCs. Postfiltration platelet loss was 14.8 percent for Day 2 pooled PCs and 9.6 percent for Day 5 pooled PCs. For pools of both Day 2 and Day 5 platelets, postfiltration levels of CD62 (P-selectin, CD62P) were unchanged from prefiltration levels, as were results for morphology scores. Levels of C3a decreased after filtration in both the Day 2 pooled PCs (448 ng/mL before filtration vs. 20 ng/mL after filtration) and the Day 5 pooled PCs (1976 ng/mL before filtration vs. 124 ng/mL after filtration). Levels of interleukin 8 were similarly reduced after filtration in the Day 2 pooled platelets (188 pg/mL before filtration vs. 27 pg/mL after filtration) and the Day 5 pooled platelets (2234 pg/mL before filtration vs. 799 pg/mL after filtration). Levels of interleukin 8 in other components evaluated after filtration declined similarly. However, levels of the proinflammatory cytokines interleukin 1 beta and interleukin 6 did not decline after filtration. Serial filtration studies showed that, although levels of interleukin 8 and RANTES were initially lowered by filtration, they returned to prefiltration values with increases in the volume of filtration. CONCLUSION: The third-generation bedside filter used in this study reliably reduced the level of white cell contamination to 4 log10 white cells per PC. It also lowered the levels of interleukin 8, RANTES, and C3a. The filter did not, however, remove (scavenge) the proinflammatory cytokines interleukin 1 beta and 6. The mechanism of chemokine and C3a removal by the filter is unknown, but it may be related to ionic interactions between these biologic response modifiers and the filter medium.     

Maintenance of in vitro properties of leukoreduced whole blood–derived pooled platelets after a 24-hour interruption of agitation

BACKGROUND: Continuous agitation during platelet concentrate (PC) storage is frequently interrupted during shipping. Studies have evaluated the effects of interrupted agitation in apheresis and single whole blood–derived PCs, but not PC pools. This study evaluated in vitro properties of pooled whole blood–derived platelets (PLTs) after a 24-hour interruption of agitation.
STUDY DESIGN AND METHODS: Eleven ABO-identical leukoreduced whole blood–derived PCs (Leukotrap RC-PL, Pall), pooled in a transfer container, were equally divided into each of two CLX-HP containers (Acrodose PL, Pall). One pool (test) was held in a shipping container unagitated for 24 hours between Day 2 and Day 3, while the other (control) was continuously agitated.
RESULTS: Ten pairs underwent in vitro assays after 5 and 7 days' storage. Pools contained a mean (±SD) of 5.0 × 10¹¹ ± 0.4 × 10¹¹ PLTs. Interrupting agitation for 24 hours reduced test pool pH versus control after 5 days' storage (6.77 ± 0.15 vs. 6.98 ± 0.06, p = 0.0005). Test and control pH differences were greater after 7 days' storage (6.17 ± 0.29 vs. 6.65 ± 0.14, p < 0.0001); 5 of 10 test pool pHs were less than 6.2 (vs. 0 of 10 controls). Other test pool key in vitro variables were reduced compared with controls after 5 days' storage, with greater differences after 7 days.
CONCLUSION: After 5 days' storage, pooled leukoreduced whole blood–derived-PCs in CLX-HP containers adequately maintained pH and other key in vitro variables after a 24-hour interruption of agitation. After 7 days' storage, 5 of 10 pools did not maintain a pH value of 6.2 or greater while matched continuously agitated units did.     

Prestorage pooled whole-blood-derived leukoreduced platelets stored for seven days, preserve acceptable quality and do not show evidence of a mixed lymphocyte reaction

BACKGROUND: Prestorage pooling of whole-blood-derived PCs (WBD-PCs) would be advantageous to transfusion services in that it would make the product available in a more timely manner, reduce wastage of untransfused pools, and simplify bacterial screening by allowing testing of the pool rather than each single PLT concentrate (PC). STUDY DESIGN AND METHODS: Four to six individual leukoreduced PCs were pooled into a 1.5-L CLX-HP PLT storage bag using a sterile connecting device. Controls were individual prestorage leukoreduced PCs that were stored as single products. Products were sampled on Days 5 and 7 for measures of PLT quality; coagulation, fibrinolytic and complement activation; and for evidence of a mixed lymphocyte reaction. RESULTS: The pH level was well maintained to Day 7 with no prestorage pool having a pH below 6.7. Day 7 studies showed no evidence of coagulation or difference in complement activation. F1.2 levels did not differ between Days 5 and 7, but a 10- to 15-percent increase in C3a des-Arg was observed between these days in all product types. Day 7 activated lymphocyte surface markers (CD69, CD71, HLA-DR) were all at lower limits of detection in the prestorage pooled products, and levels of supernatant cytokines were either not different between product types on either study day or, if different, were lower in the prestorage pooled products. CONCLUSION: There is no evidence of a deterioration in quality, activation of coagulation or complement, or a mixed lymphocyte reaction attributable to the prestorage pooling process with up to 7 days of storage.     

Storage of platelet-rich plasma-derived platelet concentrate pools in plasma and additive solution

BACKGROUND: Prestorage pooling of platelet (PLT)-rich plasma (PRP)-derived PLT concentrates (PCs) and storage in either plasma (PS) or an additive solution (AS) is logistically feasible and would result in a product similar to buffy-coat or apheresis PLTs. STUDY DESIGN AND METHODS: On Day 0, PS PRP PCs were pooled with a sterile connecting device into a new 1.3-L storage container (ELX, PALL Medical). AS-PCs were prepared by addition of a new low-pH glucose-containing AS to the PLT sediment. AS-PCs were pooled into a 1.3-L ELX bag containing four tablets of NaHCO3. PC pools were sampled on Days 1, 5, and 7. RESULTS: PS pools containing 5 units had a mean PLT yield similar to the AS pools (39 x 10(10) +/- 3 x 10(10) vs. 37 x 10(10) +/- 6 x 10(10); p = 0.25). All pools had WBC counts of less than 1 x 10(6). pH and HCO3 decreased in PS pools with storage, but either increased or remained constant in the AS pools. On Day 7, no differences were seen in morphology score or extent of shape change. Hypotonic shock response was better preserved in the plasma pools (71 +/- 12% vs. 56 +/- 13%, p < 0.01); however, surface P-selectin was expressed less in the AS pools (6 +/- 4% vs. 18 +/- 10%, p < 0.01). CONCLUSION: Manufacture and storage of PRP-PCs in pools either in plasma or in a glucose-containing AS in this new container are feasible, and there is good preservation of PLT quality to Day 7.     

WBC content of platelet concentrates prepared by the buffy coat method using different processing procedures and storage solutions

BACKGROUND: The number of WBCs in platelet concentrates (PCs) prepared by the buffy coat (BC) method with different storage solutions can result in low (5 x 10(6)/unit) WBC levels by the use of careful centrifugation techniques without filtration. At present, most blood banks use filtration steps to meet these requirements. The difference in processing methods and suspension solutions prompted the investigation of the influence of the various procedures on the WBC and platelet content of PCs. STUDY DESIGN AND METHODS: PCs from 5 BCs were harvested without or with inline filtration (AutoStop BC, Pall Corp.) in either plasma (PCs-plasma) or platelet additive solution (PCs-PAS-2). After preparation, samples were taken for counting WBCs and platelets and for analyzing WBC subsets by flow cytometry using specific MoAbs. The WBCs were concentrated before analysis of the WBC subsets. Results less than 2.5 cells per microL were considered below the limit of accuracy of the subset analysis. RESULTS: All filtered PCs met the AABB standard of 5 x 10(6) per unit and the European guidelines of 1 x 10(6) per unit. None of the nonfiltered PCs met the European guidelines, but all met the AABB guidelines. All filtered units gave residual WBC counts below the detection limit for subset analysis. Filtered PCs-plasma gave significantly higher platelet counts than filtered PCs-PAS-2 or nonfiltered PCs (p<0.01, ANOVA). CONCLUSION: Careful centrifugation of pooled BCs, with plasma or PAS-2, can result in PCs with low WBC contamination levels. However, filtered PCs are superior, because of better WBC removal and higher platelet counts.     

Storage of platelets in additive solution for up to 12 days with maintenance of good in-vitro quality

BACKGROUND: Storage of PLT concentrates (PCs) may be extended beyond 5 days, provided in-vitro and in-vivo variables allow longer storage and bacterial screening is performed. The aim of this study was to examine in-vitro storage characteristics of PCs in various storage solutions: plasma only, or mixtures of plasma with PAS-II, PAS-III, PAS-IIIM, and Composol. STUDY DESIGN AND METHODS: PCs from five pooled buffy-coats and WBCs reduced by filtration were stored in 1.3-L butyryl-tri-hexyl-citrate-plasticised PVC containers. First, a paired comparison was made between PAS-II and Composol, with 35-percent final plasma concentration (n = 10). Then, plasma, PAS-III with 30-percent plasma, PAS-IIIM with 20- and 30-percent plasma, and Composol with 20 and 30-percent final plasma concentration were compared (n = 5 pairs). Finally, 50 PCs in Composol with 35-percent final plasma concentration were studied. RESULTS: PCs in PAS-II or Composol had a mean +/- SD pH of 6.95 +/- 0.09 and 6.96 +/- 0.08 at Day 12, respectively. For PCs in PAS-IIIM and Composol with 30-percent final plasma concentration, pH on Day 7 was 7.00 +/- 0.02 and 6.83 +/- 0.05. With 20-percent final plasma concentration, pH was 6.98 +/- 0.02 and 6.81 +/- 0.03 for PAS-IIIM and Composol, respectively. PCs in PAS-III with 30-percent plasma had a pH on Day 7 of 6.87 +/- 0.03, whereas PCs in 100-percent plasma had a pH of 7.05 +/- 0.03. PCs in Composol with 35-percent plasma maintained pH greater than 6.8 in 48 of 50 of the units (96%), averaging 7.00 +/- 0.10 on Day 8. CONCLUSION: In-vitro quality of PCs in AS with at least 35-percent plasma can be maintained for 7 to 12 days after collection.     

Evaluation of bacterial inactivation in prestorage pooled, leukoreduced, whole blood-derived platelet concentrates suspended in plasma prepared with photochemical treatment

BACKGROUND: Photochemical treatment (PCT) with amotosalen and ultraviolet light was developed to inactivate pathogens in platelet (PLT) components suspended in 35 percent plasma and 65 percent additive solution (AS). Because PLT additive solutions (ASs) are not used in the United States, this study evaluated the ability of the PCT process to inactivate low levels of bacteria in pooled whole blood-derived PLTs (RDP) suspended in 100 percent plasma. STUDY DESIGN AND METHODS: Four replicate experiments were performed with two Gram-positive organisms, Staphylococcus epidermidis and Staphylococcus aureus, and two Gram-negative organisms, Klebsiella pneumoniae and Escherichia coli. For each experiment, 6 ABO-identical RDP units were pooled, leukoreduced before or after pooling, inoculated to approximately 1 to 10 colony-forming units per mL with plasma-resistant bacteria, and treated with the PCT process. Residual viable bacterial levels were measured before and after each step and 4 and 6 days after inoculation. For each bacterium studied, a fifth RDP pool was prepared and contaminated, but not treated. These units served as controls for bacterial growth. RESULTS: Growth of S. epidermidis, S. aureus, and K. pneumoniae was eliminated in all four treated pools while growth continued in the control pools. There was no growth of E. coli in the treated pools and the control pool. CONCLUSION: These pilot experiments demonstrate inactivation of bacteria in PLTs suspended in plasma, suggesting that the PCT process may address contamination in conventional RDPs. Additional experiments with a wider range of bacteria and evaluation of PLT function in 100 percent plasma will be needed before implementation.     

Effects of filtration and gamma radiation on the accumulation of RANTES and transforming growth factor-β1 in apheresis platelet concentrates during storage

BACKGROUND: Platelet-derived biologic response modifiers (BRMs) including RANTES and transforming growth factor (TGF)-beta1 accumulate in platelet components during storage because of platelet activation, and they may play a causative role in nonhemolytic febrile transfusion reactions. The majority of PCs with high unit values are provided by single donor apheresis in Japan. STUDY DESIGN AND METHODS: RANTES and TGF-beta1 levels in platelet units prepared from single-donor apheresis platelet concentrates (apheresis PCs) and units from whole blood (buffy coat PCs) were investigated. The effects of prestorage and poststorage filtration and gamma radiation on the levels of RANTES and TGF-beta1 in the supernatant of apheresis PCs during storage were also examined. RESULTS: The levels of RANTES and TGF-beta1 increased during storage from Day 0 to Day 5. The levels of RANTES and of TGF-beta1 correlated with the platelet concentration (p<0.01), but not with the residual white cell concentration in apheresis PCs that were not white cell reduced by filtration (p>0.05). In addition, there was a correlation between RANTES and TGF-beta1 levels (p<0.01). In white cell-reduced apheresis PCs using negatively charged filters as well as in gamma-radiated apheresis PCs, the levels of these two BRMs-did not differ at any storage time from those of untreated apheresis PCs. Filtration of apheresis PCs with negatively charged filters after 3 days of storage significantly (p<0.05) reduced the levels of RANTES, but not of TGF-beta1. There was no reduction in the levels of RANTES and TGF-beta1 levels by positively charged filters. The RANTES levels in buffy coat PCs were slightly higher than but not significantly different from those of apheresis PCs during storage, except for the level on Day 1. There were no differences in the TGF-beta1 levels in apheresis and buffy coat PCs during storage. CONCLUSION: Prestorage filtration and gamma radiation had neither preventive effects on the accumulation of RANTES and TGF-beta1 nor adverse effects on platelet activation. Negatively charged filters might be useful for the reducing the levels of RANTES in stored apheresis PCs.     

Evaluation of BacT/ALERT plastic culture bottles for use in testing pooled whole blood-derived leukoreduced platelet-rich plasma platelets with a single contaminated unit

BACKGROUND: In certain countries, whole blood-derived platelet (PLT)-rich plasma PLTs can only be pooled within 4 hours of transfusion. One prerequisite for prestorage pooling is the ability to detect low levels of bacteria from a single unit (approx. 10 colony-forming units [CFUs]/mL) once pooled (10/6 approx. 2 CFUs/mL). This study evaluated the BacT/ALERT (bioMérieux) for detection of bacteria in 1 unit of a 6-unit pool. STUDY DESIGN AND METHODS: Bacillus cereus, Clostridium perfringens, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Streptococcus viridans, and Propionibacterium acnes were inoculated into single PLT units (target, 10 and 100 CFUs/mL; mean recovered, 5 and 92 CFUs/mL) and then pooled with 5 sterile units. Four milliliters was inoculated into both plastic aerobic and anaerobic bottles, and 0.5 mL was plated (10 sets). RESULTS: All cases were detected when the single unit had at least 6 CFUs per mL. With B. cereus (< or =2 CFUs/mL), all bottles were reactive. With K. pneumoniae and S. viridans (< or =3 CFUs/mL), all samples were detected with a two-bottle set, but not all bottles were reactive. With S. marcescens (< 2 CFUs/mL), only 7 of the 10 sets were reactive. With C. perfringens (0.2 CFUs/mL), only 3 of 10 samples were detected in the anaerobic bottles. CONCLUSIONS: This study evaluates the use of the BacT/ALERT system for detection of bacteria in PLT pools. Overall, the BacT/ALERT detected all contaminated pooled PLTs when the single inoculated unit had a calculated or recovered concentration at least 3 CFUs per mL with 10 different species of bacteria. Low recovered concentrations (< or =2 CFUs/mL) were, in some cases, only detected with a two-bottle set.     

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.     

Sterilization of platelet concentrates at production scale by irradiation with short-wave ultraviolet light

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) is recognized as a serious threat to transfusion safety. We developed a simple method for sterilization of PCs with short-wave ultraviolet light (UVC). The effects of treatment on the sterility of contaminated PCs and in vitro platelet (PLT) variables were evaluated.
STUDY DESIGN AND METHODS: Plasma-reduced PCs were prepared from pools of five buffy coats. Irradiation with UVC (wavelength, 254 nm) under vigorous agitation was from both sides of the irradiation bags. Kinetics of the inactivation of Bacillus cereus , Propionibacterium acnes , and Staphylococcus epidermidis were determined. PCs spiked with approximately 10 to 100 colony-forming units (CFUs)/mL of 10 bacteria species (n = 12/species) were irradiated with UVC doses between 0.25 and 0.4 J/cm² and tested for sterility by a commercially available bacterial detection system (BacT/ALERT, bioMérieux) after storage at 22°C for 3 or 6 days. The influence of a dose of 0.3 J/cm² on PLT variables was investigated on Days 1, 4, and 6 after irradiation.
RESULTS: At 0.3 J/cm² all bacteria species tested were inactivated by more than 4 log. At this dose the influence of UVC on in vitro PLT variables was marginal; the storage stability for up to 6 days after treatment was maintained. PCs spiked with approximately 10 to 100 CFUs/mL were reproducibly sterilized in the dose range tested. In individual experiments with the spore former B. cereus , PCs were, however, unsterile after treatment.
CONCLUSION: Irradiation at UVC doses not detrimental to in vitro PLT variables sterilizes PCs contaminated with a wide range of different bacteria species.     

First results using automated epifluorescence microscopyto detect Escherichia coli and Staphylococcus epidermidisin WBC-reduced platelet concentrates

BACKGROUND: Many methods have been tested for the detection of bacterial contamination in platelets. However, only those using molecular biology or cell culturing consistently detect contamination at levels below 10(5) bacteria per mL. This report describes the initial investigation into an alternative method that offers the possibilities of high sensitivity and rapid response while using available laboratory equipment and supplies. This method relies on a fluorescent nucleic acid stain, which preferentially stains bacteria but not platelets, and automated epifluorescence microscopy for rapid analysis. Measurements in WBC-reduced platelet concentrates (PCs) contaminated with bacteria are reported at concentrations between 10(3) and 10(6) bacteria per mL. STUDY DESIGN AND METHODS: Staphylococcus epidermidis or Escherichia coli was inoculated into aliquots of WBC-reduced PCs on Days 2 through 5 of storage. Bacterially inoculated and control PCs were stained, platelets and residual WBCs were lysed, and 200 microL of sample was filtered onto black polycarbonate filters. All preparations were done in triplicate. An automated epifluorescence microscope examined approximately 2 percent of the area of each filter and used image analysis to select the fluorescent particles that should be counted as bacteria. RESULTS: Samples containing 3 to 5 x 10(3) bacteria per mL produced about three times as many fluorescent particles classified as bacteria as the controls. Lower concentrations of S. epidermidis were detected because of higher fluorescence intensity. Simultaneous preparation of six samples requires about 35 minutes. Analysis of each prepared sample takes 10 minutes, for a total preparation and analysis time of about 95 minutes for 6 samples. CONCLUSION: Low concentrations (<5 x 10(3) bacteria/mL) of deliberately inoculated S. epidermidis or E. coli can be measured quickly in WBC-depleted PCs by using a fluorescent nucleic acid stain, differential lysis, and automated microscopy. Continued refinement of the method, studies employing other bacterial strains, and further validations of assay performance are warranted.     

The effect of interruption of agitation on in vitro measures of platelet concentrates in additive solution

BACKGROUND: Interruption of agitation results in lower in vitro quality of platelet concentrates (PCs). The rates at which the deleterious effects occur, however, are unknown. Therefore, in vitro parameters of PCs in additive solution (AS) during various periods without agitation have been investigated. STUDY DESIGN AND METHODS: PCs from five buffy coats in AS (Composol, Fresenius HemoCare) were white cell (WBC)-reduced by filtration. Four PCs were pooled and divided to obtain paired samples. Beginning immediately after processing, three PCs were stored without agitation and placed on an agitator after 16, 20, and 24 hours. The fourth PC was agitated throughout storage and served as reference (n = 10 paired experiments). RESULTS: pH(37 degrees C) on Day 7 was greater than 6.8 in reference PCs, and in PCs that were not agitated for 16 hours, longer interruption resulted in lower pH values. During interruption of agitation, metabolic rates were significantly higher in the study groups: glucose consumption was 12.5 +/- 1.6 micromol per 10(11) platelets (PLTs) per hour in PCs during the first 24 hours without interruption versus 2.0 +/- 0.4 micromol per 10(11) PLTs per hour in the reference group (p < 0.01). Lactate formation was 24.7 +/- 4.2 versus 3.9 +/- 0.4 micromol per 10(11) PLTs per hour in the above-mentioned groups, respectively (p < 0.01). Once replaced on the agitator, the metabolic rates lowered, but remained significantly elevated during consecutive storage days compared to the reference. CONCLUSION: WBC-reduced PCs in Composol AS may experience 16 hours without agitation with no permanent effects on in vitro measures compared to reference units. During interruption of agitation, glucose and lactate metabolism is elevated, resulting in lower pH values in the subsequent storage period.     

Pathogen inactivation of platelets using ultraviolet C light: effect on in vitro function and recovery and survival of platelets

BACKGROUND: We evaluated the effect of treating platelets (PLTs) using ultraviolet (UV)C light without the addition of any photosensitizing chemicals on PLT function in vitro and PLT recovery and survival in an autologous radiolabeled volunteer study. STUDY DESIGN AND METHODS: For in vitro studies, pooled or single buffy coat–derived PLT concentrates (PCs) were pooled and split to obtain identical PCs that were either treated with UVC or untreated (n = 6 each) and stored for 7 days. PLT recovery and survival were determined in a two‐arm parallel autologous study in healthy volunteers performed according to BEST guidelines. UVC‐treated or untreated PCs (n = 6 each) were stored for 5 days and were compared to fresh PLTs from the same donor. RESULTS: There were no significant differences on Day 7 of storage between paired UVC‐treated and control PC units for pH, adenosine triphosphate, lactate dehydrogenase, CD62P, CD63, PLT microparticles, and JC‐1 binding, but annexin V binding, lactate accumulation, and expression of CD41/61 were significantly higher in treated units (p < 0.05). Compared with control units, the recovery and survival of UVC‐treated PC were reduced after 5 days of storage (p < 0.05) and when expressed as a percentage of fresh values, survival was reduced by 20% (p = 0.005) and recovery by 17% (p = 0.088). CONCLUSION: UVC‐treated PLTs stored for 5 days showed marginal changes in PLT metabolism and activation in vitro and were associated with a degree of reduction in recovery and survival similar to other pathogen inactivation systems that are licensed and in use.     

Removal of Yersinia enterocolitica from AS-1 red cells

The growth of Yersinia enterocolitica in AS-1 red cells was investigated so as to study the organism's proliferation kinetics and to evaluate the effect of prestorage white cell (WBC) reduction on bacterial multiplication. Twenty-four 2-unit pools of ABO-compatible whole blood were prepared and inoculated with Y. enterocolitica to final concentrations ranging from 0.3 to 132 organisms per mL. After inoculation, pools were split equally, AS-1 red cells were prepared, and 1 unit of each pair (test unit) was WBC-reduced with a WBC-reduction filter. Quantitative bacterial cultures of both WBC-reduced and control units were performed at several points throughout preparation and storage. Less than 10 percent of the inoculated organisms was recovered from blood samples taken after a 7-hour room-temperature holding period. By the end of 42 days of storage, Y. enterocolitica was recovered from unfiltered red cells in 2 of the 6 units inoculated at the lowest levels (0.3 and 0.7 organism/mL), from 8 of the 12 units inoculated at the intermediate levels (2.8, 5.2, 30.7, and 43 organisms/mL) and from 6 of the 6 units inoculated at the highest levels (98.8 and 132 organisms/mL). Positive cultures were seen as early as Day 7. In contrast, filtered units inoculated at all levels less than or equal to 98.8 organisms per mL (21/21 units) were sterile at the end of the 42-day storage period, while 2 units (2/3) inoculated at 132 organisms per mL showed growth despite filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of white cell reduction on the resistance of blood components to bacterial multiplication

BACKGROUND: While prestorage white cell (WBC) reduction by filtration may improve platelet and red cell quality, it also may remove an important anti-bacterial defense mechanism, especially if blood is WBC- reduced shortly after collection. STUDY DESIGN AND METHODS: The question of whether WBC reduction of platelet concentrates and red cells altered bacterial proliferation kinetics in components prepared from deliberately contaminated, freshly collected blood was investigated. Two-unit pools of whole blood were inoculated, at a concentration of approximately one colony-forming unit per mL, with one of 17 bacterial species reported to have caused septicemia in transfusion recipients. Each pool was divided after inoculation, and components were prepared from the 2 units after a 7-hour room- temperature holding period. One unit of each AS-1 red cell or platelet pair was WBC-reduced, and the pairs were then stored for 42 days at 4 degrees C (red cells) or for 10 days at 22 degrees C (platelets). Quantitative bacterial cultures were performed at periodic intervals. RESULTS: In red cells, clinically significant bacterial proliferation occurred in only one instance (Serratia marcescens), and growth was less rapid in the WBC-reduced unit than in the control. Three patterns of growth were seen in platelet concentrates. In four cases, there was rapid proliferation in both test and control units, while on 13 occasions there was minimal replication in either pair. On six occasions, substantial growth was noted in control units, while few or no bacteria could be found in the WBC-reduced units. There was no evidence in either red cells or platelets that bacteria proliferated more rapidly in units that had been WBC-reduced before storage than they did in units in which WBCs were retained. CONCLUSION: Rather than increasing the risk of bacterial proliferation through removal of active phagocytic cells, WBC reduction by filtration before blood storage may act to reduce the likelihood of significant bacterial proliferation, possibly by removal of microorganisms along with WBCs.