Characterization of transfusion-relevant bacteria reference strains in a lyophilized format.

BACKGROUND AND OBJECTIVES: Blood safety measures used by blood establishments to increase blood component safety can be validated using Transfusion-Relevant Bacterial Reference Strains (TRBRS). Ultra-cold storage conditions and manual preparation of the current TRBRS may restrict their practical use. To address this issue, the ISBT Transfusion-Transmitted Infectious Diseases Working Party's Bacterial Subgroup organized an international study to validate TRBRS in a user-friendly, lyophilised format. MATERIALS AND METHODS: Two bacterial strains Klebsiella pneumoniae PEI-B-P-08 and Staphylococcus aureus PEI-B-P-63 were manufactured as lyophilised material. The lyophilised bacteria were distributed to 11 different labs worldwide to assess the robustness for enumeration, identification and determination of growth kinetics in platelet concentrates (PCs). RESULTS: Production of lyophilised TRBRS had no impact on the growth properties compared with the traditional format. The new format allows a direct low-quantity spiking of approximately 30 bacteria in PCs for transfusion-relevant experiments. In addition, the lyophilised bacteria exhibit long-term stability across a broad temperature range and can even be directly rehydrated in PCs without losing viability. Interlaboratory comparative study demonstrated the robustness of the new format as 100% of spiked PC exhibited growth. CONCLUSION: Lyophilised TRBRS provide a user-friendly material for transfusion-related studies. TRBRS in the new format have improved features that may lead to a more frequent use in the quality control of transfusion-related safety measures in the future.

Proliferation of psychrotrophic bacteria in cold-stored platelet concentrates.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PC) are stored at 20-24°C to maintain platelet functionality, which may promote growth of contaminant bacteria. Alternatively, cold storage of PC limits bacterial growth; however, data related to proliferation of psychotrophic species in cold-stored PC (CSP) are scarce, which is addressed in this study. MATERIALS AND METHODS: Eight laboratories participated in this study with a pool/split approach. Two split PC units were spiked with ~25 colony forming units (CFU)/PC of Staphylococcus aureus, Klebsiella pneumoniae, Serratia liquefaciens, Pseudomonas fluorescens and Listeria monocytogenes. One unit was stored under agitation at 20-24°C/7 days while the second was stored at 1-6°C/no agitation for 21 days. PC were sampled periodically to determine bacterial loads. Five laboratories repeated the study with PC inoculated with lyophilized inocula (~30 CFU/mL) of S. aureus and K. pneumoniae. RESULTS: All species proliferated in PC stored at 20-24°C, reaching concentrations of ≤109 CFU/mL by day 7. Psychrotrophic P. fluorescens and S. liquefaciens proliferated in CSP to ~106 CFU/mL and ~105 CFU/mL on days 10 and 17 of storage, respectively, followed by L. monocytogenes, which reached ~102 CFU/mL on day 21. S. aureus and K. pneumoniae did not grow in CSP. CONCLUSION: Psychrotrophic bacteria, which are relatively rare contaminants in PC, proliferated in CSP, with P. fluorescens reaching clinically significant levels (≥105 CFU/mL) before day 14 of storage. Cold storage reduces bacterial risk of PC to levels comparable with RBC units. Safety of CSP could be further improved by implementing bacterial detection systems or pathogen reduction technologies if storage is beyond 10 days.

Quality assessment of red blood cell concentrates from blood donors at the extremes of the age spectrum: The BEST collaborative study.

BACKGROUND: Blood donors at the extremes of the age spectrum (16-19 years vs. ≥75 years) are characterized by increased risks of iron deficiency and anemia, and are often underrepresented in studies evaluating the effects of donor characteristics on red blood cells (RBC) transfusion effectiveness. The aim of this study was to conduct quality assessments of RBC concentrates from these unique age groups. STUDY DESIGN: We characterized 150 leukocyte-reduced (LR)-RBCs units from 75 teenage donors, who were matched by sex, and ethnicity with 75 older donors. LR-RBC units were manufactured at three large blood collection centers in the USA and Canada. Quality assessments included storage hemolysis, osmotic hemolysis, oxidative hemolysis, osmotic gradient ektacytometry, hematological indices, and RBC bioactivity. RESULTS: RBC concentrates from teenage donors had smaller (9%) mean corpuscular volume and higher (5%) RBC concentration compared with older donors counterparts. Stored RBCs from teenage donors exhibited increased susceptibility to oxidative hemolysis (>2-fold) compared with RBCs from older donors. This was observed at all testing centers independent of sex, storage duration, or the type of additive solution. RBCs from teenage male donors had increased cytoplasmatic viscosity and lower hydration compared with older donor RBCs. Evaluations of RBC supernatant bioactivity suggested that donor age was not associated with altered expression of inflammatory markers (CD31, CD54, and IL-6) on endothelial cells. CONCLUSIONS: The reported findings are likely intrinsic to RBCs and reflect age-specific changes in RBC antioxidant capacity and physical characteristics that may impact RBC survival during cold storage and after transfusion.

The Fc-effector function of COVID-19 convalescent plasma contributes to SARS-CoV-2 treatment efficacy in mice.

COVID-19 convalescent plasmas (CCPs) are chosen for plasma therapy based on neutralizing titers and anti-Spike immunoglobulin levels. However, CCP characteristics that promote SARS-CoV-2 control are complex and incompletely defined. Using an in vivo imaging approach, we demonstrate that CCPs with low neutralizing (ID50 ≤ 1:250), but moderate to high Fc-effector activity, in contrast to those with poor Fc function, delay mortality and/or improve survival of SARS-CoV-2-challenged K18-hACE2 mice. The impact of innate immune cells on CCP efficacy depended on their residual neutralizing activity. Fractionation of a selected CCP revealed that IgG and Ig(M + A) were required during therapy, but the IgG fraction alone sufficed during prophylaxis. Finally, despite reduced neutralization, ancestral SARS-CoV-2-elicited CCPs significantly delayed Delta and Beta-induced mortality suggesting that Fc-effector functions contribute to immunity against VOCs. Thus, Fc activity of CCPs provide a second line of defense when neutralization is compromised and can serve as an important criterion for CCP selection.

Assessment of the soluble proteins HMGB1, CD40L and CD62P during various platelet preparation processes and the storage of platelet concentrates: The BEST collaborative study.

BACKGROUND: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. This international study investigates the effects of platelet (PLT) processing and storage conditions on HMGB1, sCD40L, and sCD62P protein levels in platelet concentrate supernatants (PCs). STUDY DESIGN/METHODS: PC supernatants (n = 3748) were collected by each international centre using identical centrifugation methods (n = 9) and tested centrally using the ELISA/Luminex platform. Apheresis versus the buffy coat (BC-PC) method, plasma storage versus PAS and RT storage versus cold (4°C) were investigated. We focused on PC preparation collecting samples during early (RT: day 1-3; cold: day 1-5) and late (RT: day 4-7; cold: day 7-10) storage time points. RESULTS: HMGB1, sCD40L, and sCD62P concentrations were similar during early storage periods, regardless of storage solution (BC-PC plasma and BC-PC PAS-E) or temperature. During storage and without PAS, sCD40L and CD62P in BC-PC supernatants increased significantly (+33% and +41%, respectively) depending on storage temperature (22 vs. 4°C). However, without PAS-E, levels decreased significantly (-31% and -20%, respectively), depending on storage temperature (22 vs. 4°C). Contrastingly, the processing method appeared to have greater impact on HMGB1 release versus storage duration. These data highlight increases in these parameters during storage and differences between preparation methods and storage temperatures. CONCLUSIONS: The HMGB1 release mechanism/intracellular pathways appear to differ from sCD62P and sCD40L. The extent to which these differences affect patient outcomes, particularly post-transfusion platelet increment and adverse events, warrants further investigation in clinical trials with various therapeutic indications.

Humoral immune responses against SARS-CoV-2 Spike variants after mRNA vaccination in solid organ transplant recipients.

Although SARS-CoV-2 mRNA vaccination has been shown to be safe and effective in the general population, immunocompromised solid organ transplant recipients (SOTRs) were reported to have impaired immune responses after one or two doses of vaccine. In this study, we examined humoral responses induced after the second and the third dose of mRNA vaccine in different SOTR (kidney, liver, lung, and heart). Compared to a cohort of SARS-CoV-2 naïve immunocompetent health care workers (HCWs), the second dose induced weak humoral responses in SOTRs, except for the liver recipients. The third dose boosted these responses but they did not reach the same level as in HCW. Interestingly, although the neutralizing activity against Delta and Omicron variants remained very low after the third dose, Fc-mediated effector functions in SOTR reached similar levels as in the HCW cohort. Whether these responses will suffice to protect SOTR from severe outcome remains to be determined.

Long-term Stability of Epitheliotropic Factors in Frozen Serum Eye Drops.

The objective of this experimental study was to investigate the stability of functional proteins in human serum eye drops, which are used for the treatment of ocular surface disorders, after prolonged storage of 6 months at -20°C. After obtaining whole blood from 3 volunteers and preparing 100% (S100), 50% (S50), and 20% (S20) serum eye drops, fibronectin was quantified before and after storage for 6 months at -20°C using appropriate enzyme-linked immunoassay kits. The pH and microbial contamination of preparations were also evaluated longitudinally. The fibronectin concentration showed no significant reduction in undiluted (S100) or diluted (S50 and S20) serum after 6 months of frozen storage at -20°C. None of the preparations showed any microbial contamination and no significant changes in pH were noted during storage. Frozen serum eye drops appear stable after prolonged storage at -20°C. Fibronectin in serum is temperature and time resistant after prolonged storage at -20°C. While the impact of individual serum proteins on ocular surface health remains unclear, our results suggest that freezing up to 6 months provides adequate preservation of epitheliotropic factors and a minimal risk of microbial contamination.

Residual risks of bacterial contamination for pathogen-reduced platelet components.

Platelet components are commonly transfused to patients for a variety of indications, including patients with low platelet counts or patients with platelet dysfunction who are bleeding or at high risk of bleeding. Although the risk of pathogen contamination of platelet components has declined significantly over the last 40 years, it remains a concern for the patients, for blood banks and for physicians. Pathogen inactivation (PI) technologies have been developed to mitigate this risk. This review focuses on the residual risks of transfusion-transmitted bacterial infections by platelet transfusion after PI. We describe and assess the relationship between the bacterial load and the timing and capacity of reduction of the different PI technologies, as well as the risks that could represent spore-forming microorganisms and the possible introduction of microorganisms after PI.

Comparison of the Effect of Holder Pasteurization and High-Pressure Processing on Human Milk Bacterial Load and Bioactive Factors Preservation.

OBJECTIVES: This project aims at comparing the impact of Holder pasteurization (HoP) and high-pressure processing (HPP) on bacterial load and retention of immunological components in human milk. METHODS: Human milk samples discarded by the Public Mothers' milk bank (Montreal, Canada) for bacterial purpose were pooled (n = 6) and pasteurized either by heating in a water bath (62.5°C, 30 minutes) or by HPP treatment (425 MPa, four cycles of 6 minutes, initial milk temperature of 4°C or 37°C). Bacterial load, lysozyme activity, and levels of immunoglobulins, lactoferrin, lipase, and 26 cytokines were analyzed. Untreated milk samples from same pools served as control. RESULTS: HPP treatment of milk allows a similar elimination of bacteria than HoP; bacterial counts were under the detection limit [<3 colony-forming units (CFU)/mL] in 50% of milk pools after HPP treatment, compared to 17% for HoP. With initial heating of samples to 37°C before HPP treatment, inactivation to an extent under the detection limit was reached in 67% of pools. There is no significant difference in IgA, lysozyme, and cytokines concentrations between untreated milk and all treatment methods. While no significant difference was observed in the amount of lipase (P > 0.07) and IgG (P > 0.11) between untreated milk and HPP-treated milk samples, HoP seems to be damaging for these factors (P < 0.04). IgM is well preserved in HPP-4°C samples compared to untreated milk (P = 0.07) whereas a decrease is observed for this immunoglobulin levels in HPP-37°C and HoP samples (P < 0.01). Lactoferrin activity, is well maintained in HPP-37°C milk samples in comparison to untreated milk samples (P = 0.52). A decrease in activity of this molecule is noted for samples treated with HPP at 4°C (P = 0.02) and this decrease is even more pronounced for HoP samples (P = 0.004). CONCLUSIONS: HPP is a promising alternative to HoP for treatment of human milk intended to preterm babies. Our results demonstrate that HPP treatment of human milk provides safe milk with less detrimental effects on the biochemically and immunologically active milk components than HoP.

Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2.

Characterization of the humoral response to SARS-CoV-2, the etiological agent of COVID-19, is essential to help control the infection. The neutralization activity of plasma from patients with COVID-19 decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglobulin isotype in the overall neutralizing capacity is still not well understood. In this study, we select plasma from a cohort of convalescent patients with COVID-19 and selectively deplete immunoglobulin A, M, or G before testing the remaining neutralizing capacity of the depleted plasma. We find that depletion of immunoglobulin M is associated with the most substantial loss of virus neutralization, followed by immunoglobulin G. This observation may help design efficient antibody-based COVID-19 therapies and may also explain the increased susceptibility to SARS-CoV-2 of autoimmune patients receiving therapies that impair the production of immunoglobulin M (IgM).

Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Evaluation of the Tempo® System: Improving the Microbiological Quality Monitoring of Human Milk.

Background: Bacteriological testing of donor human milk is mostly done both before and after pasteurization to control contamination in the end-product and meet the microbiological standards. Although the plate count method represents a reliable and sensitive technique and is considered the gold standard for bacteriological testing, it is recognized for being time-consuming and requiring qualified personnel. Recently, faster testing technologies, mostly geared toward the food industry, have been developed. Among these, the bioMérieux TEMPO® system uses the most probable number method to assess microbiological content in a semi-automated fashion. Objective: The performances of the TEMPO® system in enumerating bacterial quality indicators in human milk were assessed and compared to the reference plate count method. Methods: Naturally and artificially contaminated human milk samples were used to compare the analytical performances of the TEMPO® system to the plate count technique. More specifically, bacteria belonging to the genera Bacillus, Enterobacteriaceae, Staphylococcus aureus, and total aerobic flora were screened using both methods. Bacteria isolated on agar plates containing selective media were identified by supplemental testing. Bacterial testing results and method parameters were compared using linear regression analyses and Bland-Altman approaches. Results: Naturally contaminated milk samples (n = 55) tested for total aerobic flora showed < 1 log (CFU/ml) discrepancy between the two methods in the output results for 98% of the samples. Comparative linear regression analyses demonstrate good correlations between the two methods (R 2 > 0.9). At lower levels of bacterial contamination, the TEMPO® method precision (C.V. < 8%) and accuracy (> 83%) were comparable to plate counts. Conclusions: The analytical performances of the TEMPO® system for human milk bacteriological testing are equivalent to the reference plate count method. Results from the TEMPO® system are available within a 24-h turnaround time from sample inoculation without the need for further supplemental testing, suggesting that this semi-automated method could be implemented within milk bank operations as an in-process monitoring technology to optimize end-product quality and safety.

Lack of the multidrug transporter MRP4/ABCC4 defines the PEL-negative blood group and impairs platelet aggregation.

The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3',5'-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.

Reevaluating immunization delays after red blood cell transfusion.

BACKGROUND: Current American and Canadian guidelines recommend delaying live vaccine immunization by 5 to 6 months after transfusion of red blood cells (RBCs) due to potential interference by serum antibodies. Due to lack of data, the recommendations may be unfounded and prevent valuable vaccination opportunities for children with frequent blood transfusions. The primary aim of this study was to determine measles, mumps, rubella (MMR) vaccine immunogenicity in patients chronically transfused with RBCs. A secondary aim was to quantify vaccine antibodies in RBC donations. STUDY DESIGN AND METHODS: MMR-specific antibodies were quantified in 25 pediatric patients who received both doses of MMR vaccine while on a chronic RBC transfusion program (median, 6.6 years after vaccination). MMR antibodies were also quantified in 30 samples of supernatants from RBC donations. RESULTS: Immunity to each of the MMR components was 68% to 76%. In blood donors born before 1976 (pre-MMR vaccine), 67% of MMR serologies in the supernatants of RBC donations were at the immune level versus 7% in blood donors born after 1976. CONCLUSION: This is the first study of the impact of RBC transfusions on MMR vaccine immunogenicity. Although lower than in the literature (immunity rates, ≥90%), the results show a high rate of immunogenicity of the MMR vaccine in chronically transfused patients. Additionally, results suggest that MMR antibody content in transfusions will continue to decrease with time. Weighing the risks and benefits of disease prevention in a vulnerable population, a reevaluation of immunization delays after RBC transfusions is called for.

The impact of red blood cell manufacturing variables on bacterial growth dynamics: a pilot study.

BACKGROUND AND OBJECTIVES: Bacterial contamination of red blood cells (RBC) remains a rare but serious clinical concern. Despite the low temperature storage of RBC, some bacteria can proliferate. The impact of RBC additive solutions (AS), manufacturing method or donor sex on bacterial growth/survival in RBC was addressed in this pilot study. MATERIALS AND METHODS: Using a partial pool-and-split design, bacterial growth/survival was assessed in intentionally inoculated RBC, manufactured separately from male and female donors using three different manufacturing methods (two whole blood [WB] filtration methods; one RBC filtration method), and resuspended in one of four AS: SAGM, PAGGSM, AS-1 or AS-3. At the beginning of storage, RBC were inoculated with 10 CFU/ml of either Klebsiella pneumoniae, Staphylococcus epidermidis, Yersinia enterocolitica or Propionibacterium acnes. Manufacturing, inoculation, storage (until day 42) and monitoring of bacterial growth were conducted at two sites: Canadian Blood Services and Héma-Québec. RESULTS: Yersinia enterocolitica was the only bacterium that proliferated during storage at both sites. RBC tested at Canadian Blood Services had higher bacterial concentrations than those at Héma-Québec (P = 0·0044). At Héma-Québec, where two different manufacturing methods were used, Y. enterocolitica reached significantly higher bacterial concentrations in AS-3 RBC (WB filtration method) compared to units prepared in the other three AS (RBC filtration method; P < 0·05). Bacterial survival/growth dependent on donor sex was not uniformly noted. CONCLUSION: Only one of four bacteria grew under RBC storage conditions. The results indicate that RBC manufacturing variables, rather than AS or donor sex, affect bacterial growth in RBC.

Bordetella holmesii Contamination of Platelet Concentrates: Revisiting the Definition of a Positive Culture.

Bacterial contamination remains the most important infectious risk of platelet transfusion. After an initially positive result, a second test is performed on the blood products and the initial culture bottle to confirm the contamination. Based on the blood center's decision algorithm used, results can be either confirmed negative, positive, or indeterminate, or be unconfirmed or discordant. Here, we report the first cases of platelet concentrates contaminated with Bordetella holmesii The in vitro growth characteristics of this unusual contaminant in platelet concentrate were investigated. Two B. holmesii strains isolated from platelet concentrates, as well as a control strain (Serratia marcescens), were spiked into platelet concentrates (PCs) at 1 and 10 CFU/ml. PCs were stored at 20 to 24°C under agitation. Samples were collected on days 2, 3, 4, and 7 for colony count and for bacterial screening using the BacT/Alert 3D system. Two PCs were detected as being positive for B. holmesii However, recultures were negative. In vitro, B. holmesii did not grow but remained detectable in PCs. Its viability diminished rapidly in contact with human plasma. Upon screening using the BacT/Alert 3D system, the majority of products spiked with B. holmesii were negative. This is the first description of PCs contaminated with B. holmesii This bacterium survives in blood products and remains dormant at low concentrations in blood products stored at room temperature, thus making difficult its detection with the BacT/Alert 3D system. The present definition of a true-positive culture of PCs may be overly restrictive for certain bacterial strains.

An alternative to dextran for the thawing of cord blood units.

BACKGROUND: Preparation of umbilical cord blood units (CBUs) for infusion requires a step of dilution or washing to reduce the toxicity of the dimethyl sulfoxide present in the freezing solution. However, the worldwide shortage of clinical-grade dextran 40, the most widely used cord blood dilution or washing solution, prompted us to search for an alternative solution. We elected to evaluate the performance of alternative solutions that could be used as potential replacements for the dextran 40-based solution. STUDY DESIGN AND METHODS: Frozen CBUs were rapidly thawed and immediately diluted 1:4 in each of 10 dilution solution variants of dextran 40, Plasma-lyte, or Hespan. We compared these alternatives by assessing viability, CD34, CD45, and colony-forming units recovery postthaw. RESULTS: A significantly lower CD34 and CD45 recovery was observed in all solutions without human serum albumin (HSA). All solutions with 5% HSA gave recovery, viability, and potency figures similar to the dextran 40/HSA solution. CONCLUSION: Based on our results and considering that Plasma-lyte A (PA)/HSA is already used for the thawing of CD34 from mobilized blood, we conclude that PA/HSA could be a safe and efficient solution for the replacement of dextran 40-based dilution solutions.

In Vitro Culture of Human Hematopoietic Stem Cells in Serum Free Medium and Their Monitoring by Flow Cytometry.

Hematopoietic stem cells can be isolated from human blood cells trapped in leukoreduction systems. The leukoreduction systems filters or chambers are usually discarded from routine blood or platelet donations in blood banks around the world. These CD34+ cells are a good source of normal stem cells and can be used as models to characterize the blood stem cells before and after culture in vitro. This chapter contains detailed methodologies for the isolation of stem cells from peripheral blood, the culture of these cells in a medium exempt of animal proteins and for the flow cytometry analysis of the resulting cell population for the characterization of their differentiation.

Heterogeneity of in vitro-cultured CD34+ cells isolated from peripheral blood.

BACKGROUND AIMS: For transplantation, hematopoietic stem cells (HSC) are obtained from bone marrow, cord blood and mobilized adult peripheral blood. HSCs are present in the blood of healthy adults and can be recovered in leuko-reduction system chambers, with a potential yield of 1 to 4 × 10(6) CD34+ cells per unit. Some groups have investigated this valuable source of stem cells; however, investigations are still needed to support their use. METHODS: CD34+ cells were purified from leuko-reduction system chambers and cultured with a defined custom medium without animal protein and supplemented with interleukin-3, interleukin-6, Fms-like tyrosine kinase 3, stem cell factor and thrombopoietin. Cells were cultured under 8% and 21% oxygen levels. With the use of multiparametric flow cytometry analysis, the phenotypes of emerging populations were compared between oxygen levels and resting CD34+ cells. Both conventional gating and clustering analysis were used to visualize the cellular outcome. RESULTS: A maximum expansion of 20-fold was obtained without major differences in viability, number of cells or cellular heterogeneity between atmospheric and physiologic oxygen conditions. Worthy of note, phenotype analysis revealed that megakaryocyte and erythrocyte progenitors were favored, albeit more moderately when submitted to 8% O2. CONCLUSIONS: This study suggests that the bias of cultured blood CD34+ cells toward megakaryocyte and erythrocyte progenitor cells can be reduced by use of 8% pO2. It also shows how clustering software, such as SPADE, can help visualize the complexity of stem cell differentiation.