PubMed HealthSearch

PubMed · 9480133

[Flow cytometry quality control in stem cell separation].

Abstract

Peripheral blood-derived haematopoietic stem cells (PBSC) are used as an alternative to bone marrow stem cells for autologous transplantation. One of the most important prerequisites for successful PBSC separation is the precise determination of the optimal separation days. As we previously demonstrated that neither PLT counts nor WBC counts in the peripheral blood (PB) are of predictive value for the amount of colony-forming cells (CFC) in the patients' PB, we started a daily monitoring of CD-34-positive cells to determine the beginning of the separation series. In addition to routine cell counts and CFU testing we assessed the number of CD 34+ in the PBSC concentrates to ascertain the number of separations needed for each patient. Due to the strong correlation of CD 34+ cells to CFC it is possible to predict the number of CFC collected within 2 h after finishing the PBSC separation and to calculate the efficiency of the separation for quality control.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

J Zingsem, S Serke, V Weisbach, R Zimmermann, T Zeiler, D Kampe, O Kunhardt, R Eckstein. 1994. [Flow cytometry quality control in stem cell separation].. https://pubmed.ncbi.nlm.nih.gov/9480133/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

Primitive hematopoietic progenitors within mobilized blood are spared by uncontrolled rate freezing.

Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P< or =0.02; bags: 14x10(9) vs. 11x10(9), P< or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P< or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.

Colony-Forming Units Assay

Quality assurance of CFU-GM assays: inter-laboratory variation despite standard reagents.

To investigate the hypothesis that commercial kits for CFU-GM (colony forming unit granulocyte-macrophage) assay will reduce the interlaboratory variation noted by many workers, we carried out a quality assurance exercise in 2 parts. There were 8 participants in the first study and each performed CFU-GM assays using their in-house method and a commercial kit (Stem Cell CFU Kit, Gibco) in parallel. In the second exercise there were 10 participants and each performed CFU-GM with in-house methods and with a different commercial medium (Methocult GF H4534, Stem Cell Technologies). Twelve samples of cryopreserved peripheral blood progenitor cells (PBPC) were analysed by each participant in each part of the study. A very wide range of results was found for the different in-house methods, but standardizing the clonogenic assay with the commercial kits did not reduce the variation seen. To improve the reproducibility of CFU-GM assays between laboratories, scrupulous attention should be paid to all the steps involved in the assays, as little progress will be made by using commercial medium in isolation from efforts to reduce other sources of variation.

Colony-Forming Units Assay