Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries.

BACKGROUND: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). METHODS: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. RESULTS: The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. DISCUSSION: These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.

Interlaboratory comparison of red-cell ATP, 2,3-diphosphoglycerate and haemolysis measurements.

BACKGROUND AND OBJECTIVES: Red blood cell (RBC) storage systems are licensed based on their ability to prevent haemolysis and maintain RBC 24-h in vivo recovery. Preclinical testing includes measurement of RBC ATP as a surrogate for recovery, 2,3-diphosphoglycerate (DPG) as a surrogate for oxygen affinity, and free haemoglobin, which is indicative of red cell lysis. The reproducibility of RBC ATP, DPG and haemolysis measurements between centres was investigated. MATERIALS AND METHODS: Five, 4-day-old leucoreduced AS-1 RBC units were pooled, aliquotted and shipped on ice to 14 laboratories in the USA and European Union (EU). Each laboratory was to sample the bag twice on day 7 and measure RBC ATP, DPG, haemoglobin and haemolysis levels in triplicate on each sample. The variability of results was assessed by using coefficients of variation (CV) and analysis of variance. RESULTS: Measurements were highly reproducible at the individual sites. Between sites, the CV was 16% for ATP, 35% for DPG, 2% for total haemoglobin and 54% for haemolysis. For ATP and total haemoglobin, 94 and 80% of the variance in measurements was contributed by differences between sites, and more than 80% of the variance for DPG and haemolysis measurements came from markedly discordant results from three sites and one site, respectively. In descending order, mathematical errors, unvalidated analytical methods, a lack of shared standards and fluid handling errors contributed to the variability in measurements from different sites. CONCLUSIONS: While the methods used by laboratories engaged in RBC storage system clinical trials demonstrated good precision, differences in results between laboratories may hinder comparative analysis. Efforts to improve performance should focus on developing robust methods, especially for measuring RBC ATP.

Comparison of total nucleated cell measurements of UC blood samples using two hematology analyzers.

BACKGROUND: The total nucleated cell (TNC) content of umbilical cord blood (UCB) units currently serves as the most important measure for determining suitability for transplantation. Hence it is important that TNC measurements are performed in an accurate manner. TNC content is evaluated routinely by hematology analyzers (HA) as WBC counts. The objective of the study was to compare TNC content utilizing two different HA, one utilizing an impedance channel and optical channel, and the other using only an optical channel. METHODS: The HA utilized in this study used two different modes of operation for lysis, regular mode (RM) and extended lysis mode (ELM). Cell-Dyn 3200 (CD3.2) utilizes optical technology for WBC measurements, involving WBC optical count (WOC) and nuclear optical count (NOC), whereas the Cell-Dyn 3700 (CD3.7) utilizes both the impedance (WIC) and optical technology (WOC) for WBC measurements. TNC content was determined with 17 identical samples using CD3.2 in one laboratory and CD3.7 in the other laboratory. Cord blood samples processed to concentrate nucleated cells by either of the laboratories were sent by overnight courier and assays were performed on the same day by both laboratories. RESULTS: For CD3.7, the WOC values were consistently lower than the WIC using the regular mode, but showed no significant differences (P>0.05). The WIC and WOC values were comparable on using the ELM and RM. For CD3.2, WOC values using RM and NOC values using ELM showed no significant differences (P>0.05), even though the WOC measurement was lower than the NOC values for most samples. The best comparison of TNC measurement between the two HA could be achieved by comparing CD3.7-WIC with CD3.2-NOC values. The results were equivalent (P>0.05) and 12 of 17 samples had equal to or less than 10% difference (mean 9.5%). DISCUSSION: TNC measurements of UCB samples were essentially identical using the WIC channel of the Cell-Dyn 3700 and the NOC channel of the Cell-Dyn 3200.

A multicenter study evaluating three methods for counting residual WBCs in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorometry.

BACKGROUND: A multicenter study was conducted to evaluate the performance characteristics of flow cytometry and microfluorimetry for counting low concentrations of WBCs and to compare the results with Nageotte hemocytometry. STUDY DESIGN AND METHODS: A two-phase study involving 10 centers located in the United States and in Europe was performed. Coded samples of RBCs and platelets were distributed by 24-hour (Phase 1) or 2-day (Phase 2) courier service to each test site for analysis. Samples were prepared to include concentrations of WBCs slightly above and below the concentration corresponding to the threshold standards for WBC-reduced RBCs and platelets. All centers tested samples by Nageotte hemocytometry plus one or both of two automated methods. RESULTS: Both flow cytometry and microfluorometry gave better results than Nageotte hemocytometry in testing freshly prepared samples. At WBC concentrations >5 per microL (RBCs) or >3 per microL (platelets), the intersite CV was <20 percent for the automated methods but >30 percent for the Nageotte hemocytometer method (p<0.001). Accuracy was greater for the automated methods than for the Nageotte hemocytometer method (p<0. 001). Nageotte hemocytometry showed a bias to underestimation relative to the results obtained with the automated methods. All methods had poorer performance in testing samples that required > or =2 days' shipment than in testing of those requiring overnight shipment. CONCLUSION: Automated methods for counting residual donor WBCs in WBC-reduced cellular components offer advantages of improved precision and greater accuracy than are seen with the Nageotte hemocytometer method. Automated methods are less labor-intensive but more costly than microscopic methods. Preparation and shipping methods will need further refinement for samples to be counted more than 24 hours after sample collection.

Retention of coagulation factors in plasma frozen after extended holding at 1-6 degrees C.

BACKGROUND AND OBJECTIVES: The ability to use plasma, isolated from units of whole blood and frozen within 24 h of phlebotomy, as a substitute for plasma frozen within 8 h of phlebotomy would have several advantages for blood centers. It should provide increased flexibility pertaining to the freezing of plasma for clinical use. We have conducted studies to assess the influence of an extended holding time for separated plasma, prior to freezing, on the retention of coagulation factor activity. STUDY DESIGN AND METHODS: Freshly harvested plasma from each of 10 units of CPD-whole blood was divided into four equal aliquots. These aliquots were held in plastic packs at 1-6 degrees C for a total of 0, 8, 15 and 24 h. Subsequently, the plasma aliquots were frozen rapidly and stored at -20 degrees C for 4 months. The thawed plasma was tested for coagulant factors V and IX, factor VIII coagulant activity (factor VIII:C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor of von Willebrand factor. RESULTS: The levels of factor V, factor vWF:Ag, factor IX and ristocetin cofactor were not influenced by holding the plasma for up to 24 h prior to freezing. Factor VIII:C activity was reduced with extended holding at 1-6 degrees C; the percentage at time zero activity was 75.9+/-2.4% for samples frozen immediately after a 24-hour period. CONCLUSIONS: The data indicate that coagulation factor properties of harvested plasma are retained except for factor VIII for at least 24 h prior to freezing.

Irradiation of platelet components: inhibition of lymphocyte proliferation assessed by limiting-dilution analysis.

BACKGROUND: Using a limiting-dilution analysis (LDA) assay that measures clonigenic T cells, it has been demonstrated that, with 2500 cGy, there is no T-cell growth in red cell components irradiated in blood bags. In the current study, the LDA assay was used to investigate the effect of gamma radiation on the proliferative capacity of T cells in plateletpheresis components. STUDY DESIGN AND METHODS: Platelets were collected by using an apheresis instrument and settings that provided sufficient mononuclear cells for the LDA assay. Platelet components (n = 8) were irradiated in 1-L plastic bags 24 hours after collection with 500, 1500, and 2500 cGy of gamma radiation in a stepwise manner. Mononuclear cells were isolated after each irradiation dose by the use of ficoll-hypaque. A density separation medium was used to reduce the platelet numbers. T cells were enumerated by fluorescence-activated cell sorter and functionally assessed by LDA assay, which quantified T cells proliferating in the presence of polyclonal stimuli and cytokines. The frequency of T-cell growth (f) was visually scored after 4 weeks of incubation at 37 degrees C. Data were calculated as f(experimental)/f(control) and expressed as log(10) reduction. RESULTS: The T-cell content of the mononuclear cell population was 17 +/- 10.5 percent, which was unaltered by irradiation. After 500-cGy irradiation, functional T cells were reduced by 2.09 log(10). Irradiation with 1500 cGy resulted in a 3. 96 log(10) reduction, but viable clonable T cells were detected in all experiments. With 2500-cGy irradiation, no T-cell growth was detected; this represented a greater than 4.86 log(10) reduction. CONCLUSION: As the dose of gamma radiation delivered to plateletpheresis components increased, the number of residual functional T cells decreased exponentially. Irradiation with 2500 cGy inactivates T cells in apheresis platelets, as measured by an LDA assay.

Viability and in vitro properties of AS-1 red cells after gamma irradiation.

BACKGROUND: Irradiation has been shown to adversely affect both in vivo 24-hour recovery (recovery [%]) and in vitro properties of stored red cells (RBCs). There is uncertainty as to how these changes are related to the day of irradiation and the length of storage after irradiation. STUDY DESIGN AND METHODS: Four protocols used day of irradiation and storage time after irradiation as the independent variables. At the conclusion of the storage period, viability was measured with radiolabeled RBCs as the recovery and the long-term survival time for RBCs that were circulating beyond 24 hours. In addition, in vitro values including RBC ATP, hemolysis level, and supernatant potassium were measured. Each subject donated 2 units of whole blood (CPD) and received autologous irradiated and untreated control RBCs (AS-1) on two separate occasions. RESULTS: Reduced recovery in irradiated units was noted when compared to that in control units, and the reduction was most apparent with long periods of storage after irradiation, irrespective of the day of irradiation. With irradiation on Day 1 of storage and a total storage period of 28 days, mean +/- SD recovery (single label) was 84.2 +/- 5.1 percent for control RBCs and 78.6 +/- 5.9 percent for irradiated RBCs (n = 16; p<0.01). With irradiation on Day 14 and storage through Day 42, the recoveries were 76.3 +/- 7.0 percent for control RBCs and 69.5 +/- 8.6 percent for irradiated RBCs (n = 16; p<0.01). Less reduction in recovery was observed with shortening of the postirradiation storage time. When the total storage period was reduced to 28 days after Day 14 irradiation, the recoveries were not significantly different. With an additional 2-day storage period after irradiation on Day 26, the recoveries were also comparable. Long-term survival times for control and irradiated RBCs were not significantly different in any of the four protocols. RBC ATP levels and hemolysis were minimally, but significantly influenced by irradiation. Supernatant potassium levels, however, were substantially increased after irradiation in each of the four protocols. CONCLUSION: Irradiation has only a small effect on the properties of RBCs treated and stored according to the utilized protocols. Longer storage times after irradiation resulted in progressively reduced recovery while long-term survival remained unaffected.

Evaluation of the Amicus Separator in the collection of apheresis platelets.

BACKGROUND: A new apheresis instrument, the Amicus Separator with software versions 2.13 and 2.34, was evaluated for component yields, collection efficiency, and incidence of donor and transfusion recipient reactions. The Amicus was also compared to the Spectra Leukocyte Reduction System (LRS) with version 5 software. STUDY DESIGN AND METHODS: Single and double apheresis platelets (APs) were collected at two locations. The targeted platelet yields were 4.0 x 10(11) for single APs and 6.8 x 10(11) for double APs. One location used a double-needle procedure, and the other used a single-needle procedure. Along with 28 of the Amicus procedures (14 at each of two locations), the same donors underwent single or double AP collections on the Spectra LRS. APs were tested for platelet yields and residual white cells. APs were transfused in three hospitals. Donor and transfusion recipient reactions and technical problems were documented. RESULTS: The Amicus Separator efficiently collected single APs (n = 59) and double APs (n = 62) with mean platelet yields of 4.2 x 10(11) and 6.5 x 10(11), respectively. When inlet line alarms occurred in single-needle procedures, platelet yields were lower and collection times were longer. All APs were white cell-reduced below 5.0 x 10(6), and all but one AP were white cell-reduced below 1.0 x 10(6) without filtration. Component yields from the paired Amicus and Spectra LRS procedures were comparable. Collection times (excluding reinfusion/rinseback) were 20 to 23 minutes faster on the Amicus Separator. No serious donor or transfusion recipient reactions occurred. CONCLUSION: The Amicus Separator provided satisfactory platelet yields and collection efficiency, with shorter collection times than did the Spectra LRS, and it white cell-reduced components without filtration.

A multi-laboratory evaluation of in vitro platelet assays: the tests for extent of shape change and response to hypotonic shock. Biomedical Excellence for Safer Transfusion Working Party of the International Society of Blood Transfusion.

BACKGROUND: There is no consensus regarding the use of specific in vitro tests for the assessment of the quality of platelet components. A literature review found that the platelet discoid shape as measured photometrically by the extent of shape change (ESC) and hypotonic shock response (HSR) correlated well with in vivo viability. The purpose of this study was to determine whether multiple research laboratories can perform the ESC and HSR assays in an accurate, reproducible manner, with acceptable sensitivity and comparable results. STUDY DESIGN AND METHODS: Eleven laboratories conducted five identical experiments, each with a different unit of platelet-rich plasma (PRP). For each experiment, 2 half-units of PRP were prepared and stored overnight: 1 half-unit at 20 to 24 degrees C in CPD (CPD-PRP) and the other at 1 to 6 degrees C with 2 mg per mL of EDTA (cold EDTA-PRP) added to produce spherical platelets with reduced HSR. Platelet suspensions having different proportions of the two PRPs were prepared and evaluated in duplicate by ESC and HSR assays, and morphologically scored by microscopy. One-way ANOVA and Duncan multiple-range tests were performed to determine significant differences in assay results for suspensions having different proportions of CPD-PRP. RESULTS: Comparable ESC (mean range: 20-28% for CPD-PRP and 1-6% for cold EDTA-PRP) and HSR (mean range: 58-81% for CPD-PRP and 12-31% for cold EDTA-PRP) measurements were obtained by nine laboratories. Duplicate testing showed high reproducibility of ESR and HSR results /in all laboratories. A 25-percent difference in the proportion of CPD-PRP (indicative of a difference of approximately 25% in the proportions of discoid and spherical platelets) was detected with a sample size of five (p<0.05) for both the ESC and HSR assays. A high correlation was found for the ESC assay and morphology score (r = 0.93, n = 345). CONCLUSION: Multiple laboratories were able to obtain comparable results with the ESC and HSR tests. They were able to show that the tests can be performed in an accurate, reproducible manner and with acceptable sensitivity.

The irradiation of blood and blood components to prevent graft-versus-host disease: technical issues and guidelines.

In recent years, there have been several advances in blood irradiation practice. These include a better definition of the most appropriate dose level that should be used when irradiating blood components. Commercial innovation has provided the tools for a quality assurance program to assess the dose that is delivered throughout the canister in a free-standing irradiator, and, through the use of radiation-sensitive indicator labels, to confirm that the irradiation process has taken place. With the apparent increased use of linear-accelerators to irradiate blood components, appropriate quality assurance measures need to be developed. The maximum storage period for irradiated red cells should be shorter than for nonirradiated red cells if the treatment is performed early during the storage period because irradiation reduces the in vivo 24-hour red cell recovery parameter. The storage period for irradiated platelets does not need to be modified. Some questions are being raised regarding whether fresh-frozen plasma should be irradiated to inactivate a small number of immunocompetent progenitor cells that may be present. Table 4 summarizes the practices that should be followed in connection with the technical issues that have been addressed in this article. These guidelines follow the recommendations issued in July 1993 by the FDA in the United States. This article and Tables 1 and 2 contain additional guidelines.

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.

Comparison of bacteria growth in single and pooled platelet concentrates after deliberate inoculation and storage.

BACKGROUND: The ability to store pools of platelet concentrates (PCs) for extended periods would provide logistical flexibility. However, reports of severe adverse reactions due to the transfusion of contaminated PCs led to an examination of whether the total bacteria levels after storage of pools containing a deliberately inoculated platelet unit would be significantly different than the levels in paired unpooled concentrates. STUDY DESIGN AND METHODS: A single PC was deliberately inoculated on Day 0 with one of three bacterial species (0.1-8.0 colony-forming units/mL). On Day 1, the deliberately inoculated PC was divided into three equal parts and either 1) pooled with 5 half-volume, ABO- and Rh-identical PCs; 2) similarly pooled and white cell reduced; or 3) kept as a control. Sterile connections were used during pooling; modified storage containers were used to ensure the correct surface-to-volume ratio of the single unit. RESULTS: Between Day 2 and Day 5 of storage, in 26 of 36 paired samples, nonfiltered pools containing Escherichia coli had greater total numbers of bacteria than did the paired single PCs. Day 2 pools had total bacteria levels approximately five times higher (colony-forming units/mL x container volume) than those in single units (p < 0.05). There was rapid growth of Staphylococcus aureus by Day 2 in pooled and unpooled PCs; by Day 3, total bacteria levels were approximately five times higher in pools than in single units (p < 0.05). Between Days 3 and 5 of storage, in 23 of 27 paired samples, nonfiltered pools containing S. aureus had greater total bacteria levels than the single PCs. By Day 5, 15 of 16 non-white-cell reduced pools had total levels of Staphylococcus epidermidis bacteria approximately five times those in the paired single PCs. Greater total bacteria levels in pooled units than in single units generally occurred when bacteria in pools reached the stationary phase of growth (when bacteria concentration became constant), and they were well correlated with the sixfold volume of pooled units. White cell reduction did not substantially affect the time required to attain stationary phase. CONCLUSION: The potential during storage for greater total bacteria levels in pools than in single PCs is a consequence of the greater volume of the pool.

Storage of apheresis platelets after gamma radiation.

BACKGROUND: There are conflicting data on the effect of irradiation and subsequent storage on the quality of platelet components. STUDY DESIGN AND METHODS: The retention of platelet properties during storage of gamma-irradiated apheresis suspensions was studied in 22 apheresis components obtained on a cell separator with a specialized centrifugation chamber. Immediately after collection, each suspension was divided equally into two 1-L polyolefin containers. On Day 1 (n = 12) and Day 3 (n = 10) one of each pair of suspension containers was gamma radiated with 2500 cGy. All platelet suspensions were stored for 5 days at 20 to 24 degrees C. Samples were drawn on Day 5 from each of the 22 pairs of containers for evaluation of an array of in vitro properties. Samples were taken from 10 pairs of containers for platelet labeling with either 51Cr or 111In for subsequent transfusion and concurrent in vivo measurement of recovery and survival. Posttransfusion samples were drawn after 24 hours for ex vivo whole blood aggregation. RESULTS: Comparable in vitro and in vivo properties were measured in irradiated and control platelets, whether irradiation was performed on Day 1 or Day 3. The mean +/- 1 SD in vivo recovery and survival time for controls and platelets irradiated on Day 1 was 52 +/- 14 percent and 146 +/- 34 hours and 51 +/- 7 percent and 147 +/- 36 hours, respectively. For Day 3 irradiation, the values were 46 +/- 12 percent and 150 +/- 60 hours and 47 +/- 9 percent and 151 +/- 53 hours, respectively. A small, but measurable adverse effect of irradiation on ex vivo platelet aggregation was present. CONCLUSION: These data indicate that storage of apheresis platelets after gamma radiation is without clinically significant, demonstrably adverse effects on platelet quality.

Effect on platelet properties of exposure to temperatures below 20 degrees C for short periods during storage at 20 to 24 degrees C.

BACKGROUND: When platelet concentrates (PCs) are shipped over long distances, it is not always possible to ensure that their temperature is maintained at 20 to 24 degrees C. In addition, PCs are not agitated as during routine storage. STUDY DESIGN AND METHODS: Studies have been conducted to evaluate how exposure to temperatures below 20 degrees C in the absence of agitation influences properties of platelets. In initial studies, exposure to 4 degrees C for 3 or 5 hours or to 12 degrees C for 5 or 17 hours on Day 2 of a 5- to 6-day storage period was associated with a loss of discoid shape. This was reflected by slightly lower but statistically different morphology scores after storage compared to those observed with control platelets that were stored only at 20 to 24 degrees C. In addition, a qualitative difference in morphology was noted in controls and PCs held at 16 degrees C for 17 hours. In more detailed studies, both the in vivo viability and in vitro properties of platelets exposed between Day 1 and Day 2 to either 12 degrees C or 16 degrees C for 17 hours were evaluated. The protocol involved a paired study design (n = 4 for each exposure temperature) with the simultaneous storage of two identical PCs, one exposed to 12 or 16 degrees C and the other one maintained at 20 to 24 degrees C throughout the 5-day storage. RESULTS: Exposure to 12 degrees C significantly reduced (p < 0.05 by paired t test) the in vivo recovery to 37.6 +/- 13.8 percent (mean +/- 1 SD) from 47.8 +/- 11.5 percent and the survival time to 2.0 +/- 0.3 days from 6.5 +/- 1.4 days. On exposure to 16 degrees C, the differences in viability from those of control units were much less but still significant. The in vivo recovery was 42.7 +/- 3.8 percent compared to 49.2 +/- 3.0 percent and the survival time was 3.5 +/- 1.2 days compared to 6.6 +/- 0.3 days. The loss of in vivo viability of the test platelets was associated with a loss of discoid shape, as reflected by morphology scores, extent of shape change, and mean platelet volume. In addition, platelet metabolism also appeared to be affected, as suggested by increased lactate production. All of the in vitro properties except for total ATP and residual glucose that were statistically different from those of controls on exposure to 12 degrees C were also significantly different on exposure to 16 degrees C. CONCLUSION: These findings demonstrate that platelets undergo substantial changes in in vivo viability and in vitro properties when they are exposed to temperatures below 20 degrees C for short periods.

Effect of gamma irradiation of red blood cell units on T-cell inactivation as assessed by limiting dilution analysis: implications for preventing transfusion-associated graft-versus-host disease.

Transfusion-associated graft-versus-host disease can be prevented by treating cellular blood products with gamma irradiation. A wide range of gamma irradiation dose levels has been used in routine practice. We used limiting dilution analysis, which measures clonable T cells, to assess the influence of 500 to 3,000 cGy of gamma irradiation delivered from a 137Cs source on T cells when delivered in situ to ADSOL-preserved red blood cell (RBC) units in blood bags. In a series of experiments using RBC units irradiated within 24 hours after collection, 1,500 cGy inactivated > 4 log10 of T cells; however, viable T cells were detected in all experiments. With 2,000 cGy, a > or = 4.7 log10 decrement in T-cell growth occurred in 7 of 8 experiments. With 2,500 or 3,000 cGy, no T-cell growth (> 5 log10 depletion) was detected. Comparable effects were observed with ADSOL-preserved RBC units in the standard PL 146 plastic container and in the recently developed PL 2209 plastic container. T-cell inactivation, as a function of gamma irradiation dose, was similar when either a 137Cs or a linear accelerator source was used. T cells isolated from ADSOL-preserved RBC units after storage for 7 and 21 days, although reduced in number as compared with a fresh unit stored for 24 hours, were viable, capable of proliferation, and susceptible to inactivation by gamma irradiation. Using a sensitive in vitro assay for T-cell proliferation, we found that a gamma irradiation dose of 2,500 cGy may be required to completely inactivate T cells in RBC units.

Validation of use of the Nageotte hemocytometer to count low levels of white cells in white cell-reduced platelet components.

BACKGROUND: Determination of the white cell (WBC) count in WBC-reduced platelet components requires methods that have a detection limit in the range of approximately 5.0 x 10(2) to 5.0 x 10(4) per mL. STUDY DESIGN AND METHODS: With a 50-microL Nageotte hemocytometer and bright-field microscopy (200x magnification), studies were conducted to develop and validate a method that could be used routinely with filtered and apheresis-harvested platelets. A 1-in-5 dilution of sample with a commercially available blood-diluting fluid was used because, with a lower (1-in-2) dilution, the observed number of WBCs was substantially less than the number expected at relatively high platelet counts (> 1.9 x 10(9)/mL). RESULTS: The observed and expected WBC counts in WBC-reduced platelet samples correlated well at levels between approximately 5 and 1100 WBCs per counting area (5.0 x 10(2)-1.1 x 10(5)/mL). At levels of more than 300 to 400 WBCs per counting area, accurate counts were obtained when 10 of the 40 rectangles were counted. CONCLUSION: These studies provide data to confirm that the 50-microL Nageotte hemocytometer can be used to accurately count low levels of WBCs in platelet components.

Evaluation of platelet concentrates stored for 5 days with reduced plasma volume.

BACKGROUND: Currently, platelet concentrates (PCs) are stored in a suspending plasma volume of 45 to 65 mL. Previous studies using second-generation containers indicated that PCs stored for 5 days at volumes less than 30 mL have reduced in vivo percentage recoveries as compared to PCs stored at volumes of 50 mL or more. STUDY DESIGN AND METHODS: This study has evaluated the effect of PC plasma volume on the maintenance of in vivo and in vitro platelet properties following 5 days of storage, with the purpose of establishing the minimum plasma volume in the range of 30 to 50 mL. Twenty paired studies were performed in which identical populations of platelets from the same donor (obtained by double manual apheresis) were stored in a normal volume (55-60 mL, control) and reduced volume (30-50 mL, test) of plasma. Comparison of in vivo viability between test and control PCs was performed after random radiolabeling of 1 unit with 51Cr and of the other with 111In, with simultaneous transfusion and with calculation of percentage recovery and the area below the survival curve (integral) as measures of viability. RESULTS: When test unit volumes were > or = 35 mL, essentially identical platelet survival curves and in vitro results were obtained for test and control. The integral and the percentage recovery for the test units were (mean, 95% confidence interval) 98.7 (96.3-101.0) and 99.0 percent (94.7-103.3) of those values in the control units, respectively. Test units with volume < or = 34 mL demonstrated reduced in vivo viability with integral and percentage recovery of 81.1 (68.9-93.3) and 80.4 (69.3-91.5), respectively, as compared to the control units. This loss was associated with increased metabolic activity (lactate production), which may suggest platelet activation due to the increased surface-to-PC volume ratio. CONCLUSION: These results show that the storage volume of PCs may be reduced from 50 to 60 mL to 35 to 40 mL without any significant decrease in in vivo or in vitro platelet quality.