Multinational comparison study of aircraft pilot healthcare avoidance behaviour.

BACKGROUND: US and Canadian pilots are required to meet medical standards to secure their active flying status, but a subgroup exhibit healthcare avoidance behaviour due to fear of loss of that status. This phenomenon has the potential to impact pilot health, aeromedical screening and aviation safety. No international comparison study of pilot healthcare avoidance currently exists between US and Canadian pilots. AIMS: To compare the rate and subtypes of healthcare avoidance behaviour secondary to fear for loss of flying status between US and Canadian pilots. METHODS: A comparison analysis of data collected during two independent, non-probabilistic, cross-sectional internet surveys including any individual certified to perform flying duties in the USA (US survey) or Canada (Canadian survey). RESULTS: There were 4320 US pilots and 1415 Canadian pilots who completed informed consent and 3765 US pilots and 1405 Canadian pilots were included in the results. There were 56% of US pilots who reported a history of healthcare avoidance behaviour compared to 55% of Canadian pilots (P = 0.578). A multivariable logistic regression that included age, pilot type and gender showed that US pilots were slightly more likely than Canadian pilots to report this behaviour (odds ratio 1.22, 95% confidence interval 1.06-1.4). CONCLUSIONS: Healthcare avoidance behaviour due to fear of loss of flying status has a relatively high prevalence in both US and Canadian pilot populations.

Effect of cryopreservation on a rare McLeod donor red blood cell concentrate.

Units of red blood cell (RBC) concentrates with rare phenotypes are typically not included in method validation studies for cryopreservation processes; rather, they are reserved for patients with rare blood needs. Some rare RBC phenotypes may demonstrate membrane abnormalities, like acanthocytosis as observed for RBCs with the McLeod phenotype, and are specifically banked for these rare attributes; however, the impact that rare RBC phenotypes have on post-thaw quality has not been well studied. To evaluate how a rare RBC phenotype is affected by the cryopreservation process, 4 RBC units, cryopreserved in 1993 using manual methods, were selected for evaluation. These RBCs included one with the McLeod phenotype and three with phenotypes not known to cause significant membrane changes. Post-thaw, an altered deglycerolization protocol, implemented to reduce supernatant glycerol after cryopreservation, was used before processing RBCs on an automated closed system (ACP 215; Haemonetics, Boston, MA) to accommodate the use of a closed system cell processor not available when the RBC units were previously cryopreserved. RBC quality was tested at 24 hours, 7 days, and 14 days post-deglycerolization. Before deglycerolization, an extracted sample from the thawed glycerolized RBC unit was used to obtain genetic material for phenotype confirmation. Genotyping confirmed the McLeod phenotype. When comparing McLeod with non-McLeod units, RBCs from the McLeod donor exhibited acanthocytosis, higher rigidity, and lower morphology scores than RBCs from the non-McLeod units post-deglycerolization. Hemolysis, however, was comparable across all 4 units, meeting regulatory standards. Therefore, McLeod RBCs can withstand cryopreservation, suggesting that units from these donors, glycerolized using older methods, can be deglycerolized using the ACP 215 and stored hypothermically for 14 days. It was also determined that genotyping can be performed on non-leukocyte-reduced cryopreserved RBCs, allowing for confirmation of genetic profiles of donor units banked before the implementation of molecular methods.Units of red blood cell (RBC) concentrates with rare phenotypes are typically not included in method validation studies for cryopreservation processes; rather, they are reserved for patients with rare blood needs. Some rare RBC phenotypes may demonstrate membrane abnormalities, like acanthocytosis as observed for RBCs with the McLeod phenotype, and are specifically banked for these rare attributes; however, the impact that rare RBC phenotypes have on post-thaw quality has not been well studied. To evaluate how a rare RBC phenotype is affected by the cryopreservation process, 4 RBC units, cryopreserved in 1993 using manual methods, were selected for evaluation. These RBCs included one with the McLeod phenotype and three with phenotypes not known to cause significant membrane changes. Post-thaw, an altered deglycerolization protocol, implemented to reduce supernatant glycerol after cryopreservation, was used before processing RBCs on an automated closed system (ACP 215; Haemonetics, Boston, MA) to accommodate the use of a closed system cell processor not available when the RBC units were previously cryopreserved. RBC quality was tested at 24 hours, 7 days, and 14 days post-deglycerolization. Before deglycerolization, an extracted sample from the thawed glycerolized RBC unit was used to obtain genetic material for phenotype confirmation. Genotyping confirmed the McLeod phenotype. When comparing McLeod with non-McLeod units, RBCs from the McLeod donor exhibited acanthocytosis, higher rigidity, and lower morphology scores than RBCs from the non-McLeod units post-deglycerolization. Hemolysis, however, was comparable across all 4 units, meeting regulatory standards. Therefore, McLeod RBCs can withstand cryopreservation, suggesting that units from these donors, glycerolized using older methods, can be deglycerolized using the ACP 215 and stored hypothermically for 14 days. It was also determined that genotyping can be performed on non-leukocyte­reduced cryopreserved RBCs, allowing for confirmation of genetic profiles of donor units banked before the implementation of molecular methods.

Quality control of apheresis platelets: a multicentre study to evaluate factors that can influence pH measurement.

BACKGROUND AND OBJECTIVES: Blood operators routinely monitor the pH of apheresis platelets as a marker of the so-called storage lesion, which can result from manufacturing problems. It is also suspected that some donor characteristics can increase the risk of poor platelet storage. To explore this hypothesis, we analysed a large, multinational data set of quality control (QC) pH test results on apheresis platelets. MATERIALS AND METHODS: For the period between September 2011 and August 2014, seven blood operators in Canada, the USA, the Netherlands, the United Kingdom, France and Australia provided pH QC test results and donor characteristics on a total of 21,671 apheresis platelets. RESULTS: Some variations in pH distribution between blood operators were in part explained by differences in collection, processing and testing methods. Younger age and female gender were significantly associated with a pH value below the 10th percentile. Among donors who had two or more pH measurements (n = 3672), there was a strong correlation between pH results (r = 0·726; P < 0·0001). CONCLUSION: The strong intradonor correlation of pH measurements and the association between donor characteristics and pH results suggest that donor factors play a role in the quality of platelets.

Assessing the influence of component processing and donor characteristics on quality of red cell concentrates using quality control data.

BACKGROUND AND OBJECTIVES: Quality control (QC) data collected by blood services are used to monitor production and to ensure compliance with regulatory standards. We demonstrate how analysis of quality control data can be used to highlight the sources of variability within red cell concentrates (RCCs). MATERIALS AND METHODS: We merged Canadian Blood Services QC data with manufacturing and donor records for 28 227 RCC between June 2011 and October 2014. Units were categorized based on processing method, bag manufacturer, donor age and donor sex, then assessed based on product characteristics: haemolysis and haemoglobin levels, unit volume, leucocyte count and haematocrit. RESULTS: Buffy-coat method (top/bottom)-processed units exhibited lower haemolysis than units processed using the whole-blood filtration method (top/top). Units from female donors exhibited lower haemolysis than male donations. Processing method influenced unit volume and the ratio of additive solution to residual plasma. CONCLUSIONS: Stored red blood cell characteristics are influenced by prestorage processing and donor factors. Understanding the relationship between processing, donors and RCC quality will help blood services to ensure the safety of transfused products.

Manufacturing method affects mitochondrial DNA release and extracellular vesicle composition in stored red blood cells.

BACKGROUND AND OBJECTIVES: Damage-associated molecular patterns (DAMPs) are found in transfusion products, but their potential impacts are not fully understood. We examined the influence of manufacturing method on levels of mitochondrial (mt) DNA and extracellular vesicle (EV) DAMPs in red cell concentrates (RCCs). MATERIALS AND METHODS: Eighty-seven RCCs were prepared using nine different methods (6-15 units/method), including three apheresis, five whole blood (WB)-derived leucoreduced (LR) and one WB-derived non-LR method. On storage days 5 and 42, levels of mtDNA (by PCR) and number and cell of origin of EVs (by flow cytometry) were assessed in RCC supernatants. RESULTS: There was a 100-fold difference in mtDNA levels among methods, with highest levels in non-LR, followed by MCS+ and Trima apheresis RCCs. There was a 10-fold difference in EV levels among methods. RBC-derived CD235a+ EVs were found in fresh RCCs and increased in most during storage. Platelet-derived CD41a+ EVs were highest in non-LR and Trima RCCs and did not change during storage. WBC-derived EVs were low in most RCCs; CD14+ EVs increased in several RCCs during storage. CONCLUSION: DAMPs in RCCs vary by manufacturing method. MtDNA and EV could be informative quality markers that may be relevant to RCC immunomodulatory potential.

An investigation of red blood cell concentrate quality during storage in paediatric-sized polyvinylchloride bags plasticized with alternatives to di-2-ethylhexyl phthalate (DEHP).

BACKGROUND AND OBJECTIVES: Di-2-ethylhexyl phthalate (DEHP) is a blood bag plasticizer. It is also a toxin, raising concerns for vulnerable populations, for example, neonates and infants. Here, the in vitro quality of red cell concentrates (RCC) stored in paediatric bags formulated with alternative plasticizers to DEHP was compared. MATERIALS AND METHODS: RCC were pooled and split into polyvinylchloride (PVC)/DEHP, PVC/1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH) or PVC/butyryl trihexyl citrate (BTHC) bags. Quality was assessed on storage days 5, 21, 35 and 43. RESULTS: Metabolism differed among the bags: pCO2 levels were lowest and pO2 were highest in BTHC bags. Glucose consumption and lactate production suggested higher metabolic rates in BTHC bags. ATP levels were best maintained in DINCH bags (day 43 mean level: 2·86 ± 0·29 µmol/g Hb). RCC in BTHC bags had the greatest potassium release (54·6 ± 3·0 mm on day 43). From day 21, haemolysis was higher in BTHC bags (P < 0·01) and by day 43 had exceeded 0·8% (0·85 ± 0·10%). RCC in BTHC bags showed more microparticle formation than RCC in DEHP or DINCH bags. CONCLUSION: The results suggest that the BTHC formulation used was detrimental to RBC quality. DINCH bags could be a viable alternative to DEHP: they outperformed DEHP bags energetically, with better maintenance of ATP levels.

Buffy coat (top/bottom)- and whole-blood filtration (top/top)-produced red cell concentrates differ in size of extracellular vesicles.

BACKGROUND AND OBJECTIVES: The influence that blood component separation methods have on changes to the red blood cell membrane during storage is not well understood. In Canada, red cell concentrates (RCCs) are produced using the buffy coat (BC, top/bottom) and the whole-blood filtration (WBF, top/top) methods, and this study aimed at comparing their influence on the characteristics of the extracellular vesicles (EV) which accumulated in the respective products during storage. MATERIALS AND METHODS: Using flow cytometry, dynamic light scattering and mass spectrometry, we assessed RCC EVs for concentration, size, lipid composition and correlation with supernatant haemoglobin (Hb). RESULTS: Accumulation of RBC EVs (CD235a(+) ) with storage time was similar in WBF and BC RCCs. The size of the EVs changed from <100 nm at d5 to near 200 nm by d42, with the EVs from WBF being smaller (P < 0·001) than BC RCCs at all storage times. The amount of EV-bound Hb in the WBF and BC units was similar (about 10% of total supernatant Hb). WBF EVs and BC EVs displayed similar lipid composition. CONCLUSION: Haemolysis and EVs increase in BC and WBF RCCs during storage. Differences in the size characteristics of the EVs in WBF and BC RCCs suggest that non-RBC EVs are more prevalent in WBF products. Understanding the impact that manufacturing has on the characteristics of the different populations of EVs in RCCs will aid quality improvement efforts.

The effect of processing method on the in vitro characteristics of red blood cell products.

BACKGROUND AND OBJECTIVES: While the clinical impact of differences in red blood cell (RBC) component processing methods is unknown, there are concerns they may be confounding variables in studies such as the ongoing 'age of blood' investigations. Here, we compare the in vitro characteristics of red cell concentrates (RCCs) produced by several different processing methods. MATERIALS AND METHODS: Nine processing methods were examined: three apheresis methods (Alyx, MCS+ and Trima), as well as leucoreduced whole blood-derived RCCs produced by buffy coat and whole blood filtration and non-leucoreduced RCCs. RCCs were stored in saline-adenine-glucose-mannitol or additive solutions (AS) 1 or 3 for 42 days, with quality tested on day 5 and day 42. RESULTS: Many significant product differences were observed both early in and at the end of storage. Mean haemoglobin (Hb) ranged from 52 to 71 g/unit and mean Hct from 59·5 to 64·8%. Most RCC passed regulated quality control criteria according to Canadian Standards Association guidelines, although there were some failures relating to Hb content and residual WBC counts. CONCLUSION: Processing method impacts RCC characteristics throughout storage; better understanding of these differences and reporting of processing method details is critical.

The effect of timing of gamma-irradiation on hemolysis and potassium release in leukoreduced red cell concentrates stored in SAGM.

While irradiation of red cell concentrates (RCC) prevents graft-versus-host disease in susceptible transfusion recipients, it also damages red blood cells (RBC). To understand the ability of irradiation regulations to prevent transfusion of inferior units, we irradiated 980 RCC in saline-adenine-glucose-mannitol (SAGM) using various combinations of pre-irradiation age and post-irradiation storage times, and measured hemolysis and extracellular potassium levels. We observed unacceptably high hemolysis (>0·8%) in some RCC and elevated extracellular potassium levels in all gamma-irradiated RCC. This suggests that more restrictive storage times should be considered for RCC in SAGM.

Microfluidic approach to genotyping human platelet antigens.

Centralised laboratories routinely determine blood types by serological and molecular methods. Current practices have limitations in terms of cost, time and accessibility. Miniaturised microfluidic platforms offer an alternative to conventional genotyping methods, since they consume fewer reagents, provide faster analysis and allow for complete integration and automation. As these 'lab-on-a-chip' devices have been used for bacterial and viral detection, the authors investigated blood group genotyping as a novel application of microfluidic technology. To demonstrate the feasibility of microfluidic chip-based genotyping, the authors compared human platelet antigen 1 (HPA-1) genotype results from conventional and chip-based analysis for 19 blood donor specimens. DNA purification was performed with ChargeSwitch™ magnetic beads, DNA amplification (PCR), restriction length polymorphism (RFLP) and capillary electrophoresis (CE) for identification of the DNA on microfluidic chips. It was found that nine donors were HPA-1a/1a and ten were HPA-1a/1b. Concordance between the conventional and on-chip methods was achieved for all but one sample. All the steps were demonstrated for complete blood group genotyping analysis of patient whole blood specimens on separate microfluidic chips. Future work will focus on integration of all the genotyping protocols on a single microfluidic chip.

Eadie-Hofstee analysis of red blood cell deformability.

In recent years, linear data transformation has become an accepted method to simplify the analysis of red blood cell (RBC) deformability curves obtained by ektacytometry. In this study, we introduce the Eadie-Hofstee transformation as an alternative linearization method for the analysis of RBC deformability. RBCs were treated with hydrogen peroxide (H(2)O(2)), tert-butyl-hydroperoxide (t-BuOOH), or methyl ß-cyclodextrin (MßCD) and analyzed via ektacytometry (LORCA). RBC hemopathological clinical isolates (hereditary spherocytosis and α-thalassemia) were also analyzed by LORCA. Following ektacytometry, Eadie-Hofstee linearization was performed to obtain the maximum deformability (EI(max)) and shear stress at half maximal deformation (K(EI)) parameters. Significant changes in deformability parameters were observed with all agents tested. For H(2)O(2) and t-BuOOH, the K(EI) values increased significantly accompanied by marginal changes in EI(max), while treatment with MßCD resulted in a dose dependant decrease in EI(max). Contrasting deformability profiles were also observed in the two hematological disorders tested. In this study we have demonstrated the ability of Eadie-Hofstee linearization to detect and resolve changes in RBC deformability induced in vitro as well as deformability changes associated with in vivo hematological disorders. This technique shows promise in basic research, blood bank and clinical hematology settings.

Spectrophotometric measurement of intraliposomal trehalose.

Trehalose, a non-reducing glucose disaccharide found at high concentrations in many species of anhydrobiotic organisms, shows significant promise in protecting cellular viability and structural integrity during freezing and desiccation. As mammalian cell membranes are impermeable to trehalose, extensive efforts have been taken to introduce trehalose into mammalian cells. In this study, we report on the characterization of trehalose-containing liposomes, with focus on the entrapment of trehalose inside liposomes, as the first step in establishing liposomes as a delivery system in the biopreservation field. Liposomes were synthesized by hydrating a phospholipid/cholesterol lipid bilayer with 200-400 mM trehalose buffer and repeatedly extruding the lipid suspension to form unilamellar vesicles. The trehalose content of the liposomal lysate was determined spectrophotometrically using a commercial kit Megazyme and confirmed with HPLC measurements. The number of liposomes was calculated from the phosphate content of the liposomal preparation and an estimated number of lipid molecules in a 401+/-8 nm liposome. Based on an intraliposomal trehalose content, the calculated liposomal encapsulation efficiency of 200 mM trehalose liposomes was of 92+/-0.7%. This value was in agreement with the 300 and 400 mM trehalose liposomes (91.1+/-8.2% and 102.1+/-9.4%, respectively). The Megazyme method for trehalose measurement is an inexpensive and sensitive technique that does not require specialized instrumentation or extensive technical expertise. Therefore, it can be used to enhance current efforts in the development of alternative strategies for the cryo- and lyoprotection of mammalian cells.

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood.

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.

The effect of cell size distribution on predicted osmotic responses of cells.

An understanding of the kinetics of the osmotic response of cells is important in understanding permeability properties of cell membranes and predicting cell responses during exposure to anisotonic conditions. Traditionally, a mathematical model of cell osmotic response is obtained by applying mass transport and Boyle-vant Hoff equations using numerical methods. In the usual application of these equations, it is assumed that all cells are the same size equal to the mean or mode of the population. However, biological cells (even if they had identical membranes and hence identical permeability characteristics--which they do not) have a distribution in cell size and will therefore shrink or swell at different rates when exposed to anisotonic conditions. A population of cells may therefore exhibit a different average osmotic response than that of a single cell. In this study, a mathematical model using mass transport and Boyle-van't Hoff equations was applied to measured size distributions of cells. Chinese hamster fibroblast cells (V-79W) and Madin-Darby canine kidney cells (MDCK), were placed in hypertonic solutions and the kinetics of cell shrinkage were monitored. Consistent with the theoretical predictions, the size distributions of these cells were found to change over time, therefore the selection of the measure of central tendency for the population may affect the calculated osmotic parameters. After examining three different average volumes (mean, median, and mode) using four different theoretical cell size distributions, it was determined that, for the assumptions used in this study, the mean or median were the best measures of central tendency to describe osmotic volume changes in cell suspensions.

Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment.

In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34(+) cells, and granulocyte-macrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34(+) cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P=0.136 and 0.417, respectively) or platelets (P=0.88 and 0.126, respectively). However, the reinfused viable CD34(+) cells/kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P=0.0001 and 0.001, respectively) and platelets (P=0.023 and 0.010, respectively), whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P=0.011 and 0.007, respectively) but not to platelets (P=0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34(+) cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34(+) cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.

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.

Pre- and post-thaw assessment of intracellular ice formation.

Intracellular ice formation (IIF) refers to the formation of ice crystals within cells during rapid freezing. To develop an understanding of the means by which intracellular ice forms and the mechanisms by which it damages cells and tissues requires techniques that combine real-time assessment of ice nucleation and ice crystal growth with detailed assessments of cell structure and function. Intracellular ice formation has been detected in live samples using light scattering, freeze substitution and fluorescent detection. In this study we develop a method to correlate IIF with post-thaw structural analyses by combining low temperature microscopy and freeze substitution. V79-4 hamster fibroblasts were frozen on a low temperature microscope at various temperatures, IIF was visualized using the nucleic acid-specific fluorophore SYTO 13, then the samples were fixed (10% formaldehyde, 85% ethanol, 5% acetic acid) while still frozen. The monolayers were then thawed and stained with routine histological stains haematoxylin and eosin and assessed. Fixation allowed for the post-thaw assessment of IIF and for subsequent histological processing to examine in detail the structural consequences of IIF. The post-thaw identification of cells that form intracellular ice during freezing is a significant improvement to current methods used in low temperature biology.

Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation.

Three widely used viability assessments were compared: (1) membrane integrity of nucleated cells using trypan blue (TB) exclusion and a fluorometric membrane integrity assay (SYTO 13 and propidium iodide), (2) enumeration of viable CD34+ cells, and (3) clonogenic assay (granulocyte-macrophage colony-forming units, CFU-GM). Post thaw peripheral hematopoietic progenitor cells (HPC) were incubated at 0, 22, and 37 degrees C for 20-min intervals before assessment. The recovery of viable nucleated cells assessed by TB and SYTO/PI decreased significantly with time at incubation temperatures of 22 and 37 degrees C (P<0.05), and correlated with the concentration of mononuclear cells (MNC) (r=0.936, P<0.05). The decrease in recovery of viable nucleated cells was slower when thawed cells were incubated at 0 degrees C compared with 22 degrees C or 37 degrees C. The recovery, measured by absolute viable CD34+ or CFU-GM, was not affected by 2 h post thaw incubation (P>0.05) at 0, 22, and 37 degrees C (P>0.05). There were no significant differences in the measured recovery of viable CD34+ cells and CFU-GM at all incubation times (P>0.05) and temperatures (P>0.05). Both CFU-GM and absolute CD34+ cells can be used as post thaw viability assays for HPC cryopreserved for transplantation.