Liquid Storage of Hematopoietic Stem Cells Versus Proliferative Potential Colony-Forming Unit Granulocyte-Monocytes: Validation of Cell Processing.

BACKGROUND: The material for transplantation must be of the highest quality. As far as we know, short-term storage is one of the crucial points of stem cell banking. According to the quality assurance system in a stem cell bank, each step of cell processing must be validated. The aim of this study was to assess the influence of short-term storage conditions into a clonogenic assay. METHODS: Material was collected from mobilized peripheral blood by means of leukapheresis from 15 patients. Samples were stored at 4°C and 20°C; samples were evaluated on the day of leukapheresis and after 24 hours and after 48 hours of storage. The number of colony-forming unit granulocyte-monocyte (CFU-GM) precursors was analyzed with the use of in vitro culture. The material was evaluated before freezing and after thawing. RESULTS: The average number of CFU-GM precursors in the material stored at 4°C before freezing on the day of collection was 84/10(5) nuclear cells (nc) and after 24 hours and 48 hours of storage was, respectively, 62/10(5) nc (P = .011719) and 36/10(5) nc (P = .02088). The average of the CFU-GM precursors in material stored at 20°C after 24 hours and 48 hours of storage amounted to 33/10(5) nc (P = .004439) and 2/10(5) nc (P = .00346), respectively. CONCLUSIONS: In our study, the number of colonies of CFU-GM after 24 hours and 48 hours of storage, both at 4°C and 20°C, was significantly reduced compared with the number of colonies on the day of collection. Significantly greater numbers of CFU-GM precursors were observed in the material stored before freezing at 4°C in comparison with the material stored at 20°C.

Banking of Hematopoietic Stem Cells: Influence of Storage Time on Their Quality Parameters.

BACKGROUND: Banking of hematopoietic stem cells (HSCs) is a rapidly growing part of the transplant field. The essence of the banking process is to maintain the optimal quality parameters throughout the storage period, allowing successful transplantation. METHODS: Our laboratory research was carried out on 126 HSC samples that were collected by means of leukapheresis from patients with lymphoproliferative diseases. The samples were frozen in a controlled rate and stored up to 76 months in containers in vapor phase of liquid nitrogen. The evaluation was performed after thawing the probes. Viability of nuclear cells was assessed after incubation in Trypan blue, CD34+ phenotype cells were determined by means of cytometry with the use of 7-aminoactinomycin D (7-AAD), and an analysis of the proliferative potential of granulocyte-monocyte precursors was performed. For comparative statistical analysis, the material was divided into 3 groups according to storage time: A: <1 month (n = 45); B: 1-12 months (n = 50); C: >12 months (n = 31). RESULTS: In the examined groups, similar median values were observed of nuclear cell viability (A, 86%; B, 87%; and C, 83%) and CD34+ cells (95%, 94.5%, and 95.8%, respectively). A gradual, nonsignificant, reduction in the median of granulocyte-monocyte precursors was found: 68 × 10(4)/kg of body weight (kg bw), 48.5 × 10(4)/kg bw, and 47 × 10(4)/kg bw, respectively. Statistical analysis with the use of the Kruskal-Wallis test showed a P value of >.05 for all variables. CONCLUSIONS: There were no significant differences in the viability of nuclear cells, CD34+ cells, and proliferative potential granulocyte-monocyte precursors between groups. Storage for up to 76 months does not change the essential quality parameters, and HSCs could be qualified for distribution.

Analysis of the effect of cryoprotectant medium composition to viability of autologous hematopoietic cells collected by leukapheresis.

OBJECTIVES: Cryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system. MATERIAL AND METHODS: Two hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (-160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 10(8)/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland). RESULTS AND CONCLUSIONS: The results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P < .05). To assess the viability of CD34+, cells frozen using RPMI 1640 results showed a large span of at 16.4% to 99.1% living cells.

Microbial contamination of peripheral blood and bone marrow hematopoietic cell products and environmental contamination in a stem cell bank: a single-center report.

Hematopoietic stem cells (HSC) derived from peripheral blood (PB) and bone marrow (BM) are frequently used for autologous and allogenic transplantations. Establishing quality control at appropriate steps of the stem cell preparation process is crucial for a successful transplantation. Microbial contamination of haematopoietic stem cells is rare but could cause a potentially mortal complication of a stem cells transplantation. We investigated the microbiological contamination of PB (291 donations) and BM (39 donations) products. Microbial cultures of 330 donations between January 2012 and June 2013 were retrospectively analyzed after the collection and preparation steps. The microbiological analysis was performed with an automated system. Hematopoietic stem cells were processed in a closed system. Additionally, in this report the environment of the working areas of stem cell preparation was monitored. We analyzed microbial contamination of the air in a class I laminar air flow clean bench at the time of preparation and in the laboratory once per month. We reported 9 (2.73%) contaminated HSC products. The most frequent bacteria isolated from PB and BM products were Bacillus species. Coagulase-negative staphylococci and Micrococcus species were the most frequent micro-organisms detected in the air microbial control. Microbial control results are necessary for the safety of hematopoietic stem cell products transplantation. Microbial control of hematopoietic stem cell products enables an early contamination detection and allows for knowledgeable decision making concerning either discarding the contaminated product or introducing an efficient antibiotic therapy. Each step of cell processing may cause a bacterial contamination. A minimum of manipulation steps is crucial for increasing the microbial purity of the transplant material. Also, the air contamination control is essential to ensure the highest quality standards of HSC products preparation.