title: Validation of a single-platform method for hematopoietic CD34+ stem cells enumeration according to accreditation procedure.
abstract: BACKGROUND
  Stem cells for autologous and allogenic transplantation are obtained from several sources including bone marrow, peripheral blood or cord blood. Accurate enumeration of viable CD34+ hematopoietic stem cells (HSC) is routinely used in clinical settings, especially to monitor progenitor cell mobilization and apheresis. The number of viable CD34+ HSC has also been shown to be the most critical factor in haematopoietic engraftment. The International Society for Cellular Therapy actually recommends the use of single-platform flow cytometry system using 7-AAD as a viability dye.
  
  
  OBJECTIVE
  In a way to move routine analysis from a BD FACSCaliburTM instrument to a BD FACSCantoTM II, according to ISO 15189 standard guidelines, we define laboratory performance data of the BDTM Stem Cell Enumeration (SCE) kit on a CE-IVD system including a BD FACSCanto II flow cytometer and the BD FACSCantoTM Clinical Software. InterQCTM software, a real time internet laboratory QC management system developed by VitroTM and distributed by Becton DickinsonTM, was also tested to monitor daily QC data, to define the internal laboratory statistics and to compare them to external laboratories.
  
  
  METHODS
  Precision was evaluated with BDTM Stem Cell Control (high and low) results and the InterQC software, an internet laboratory QC management system by Vitro. This last one drew Levey-Jennings curves and generated numeral statistical parameters allowing detection of potential changes in the system performances as well as interlaboratory comparisons. Repeatability, linearity and lower limits of detection were obtained with routine samples from different origins. Agreement evaluation between BD FACSCanto II system versus BD FACSCalibur system was tested on fresh peripheral blood, freeze-thawed apheresis, fresh bone marrow and fresh cord blood samples.
  
  
  RESULTS
  Instrument's measure and staining repeatability clearly evidenced acceptable variability on the different samples tested. Intra- and inter-laboratory CV in CD34+ cell absolute count are consistent and reproducible. Linearity analysis, established between 2 and 329 cells/μl showed a linear relation between expected counts and measured counts (R2=0.97). Linear regression and Bland-Altman representations showed an excellent correlation on samples from different sources between the two systems and allowed the transfer of routine analysis from BD FACSCalibur to BD FACSCanto II.
  
  
  CONCLUSIONS
  The BD SCE kit provides an accurate measure of the CD34 HSC, and can be used in daily routine to optimize the enumeration of hematopoietic CD34+ stem cells by flow cytometry. Moreover, the InterQC system seems to be a very useful tool for laboratory daily quality monitoring and thus for accreditation.
authors.affiliation: Plate-forme de Cytométrie en Flux, CHU de Nancy, Vandœuvre-lès-Nancy, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Massin
authors.affiliation: Service d'Hématologie Biologique, CHU de Nancy, Vandœuvre-lès-Nancy, France.
authors.forename: Cai
authors.initials: C
authors.lastname: Huili
authors.affiliation: Unité de Thérapie Cellulaire et Tissulaire, CHU de Nancy, Vandœuvre-lès-Nancy, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Decot
authors.affiliation: Unité de Thérapie Cellulaire et Tissulaire, CHU de Nancy, Vandœuvre-lès-Nancy, France CNRS IMoPA Unit 7365, Faculté de Médecine, Vandœuvre-lès-Nancy, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Stoltz
authors.affiliation: Unité de Thérapie Cellulaire et Tissulaire, CHU de Nancy, Vandœuvre-lès-Nancy, France CNRS IMoPA Unit 7365, Faculté de Médecine, Vandœuvre-lès-Nancy, France.
authors.forename: Danièle
authors.initials: D
authors.lastname: Bensoussan
authors.affiliation: Plate-forme de Cytométrie en Flux, CHU de Nancy, Vandœuvre-lès-Nancy, France Service d'Hématologie Biologique, CHU de Nancy, Vandœuvre-lès-Nancy, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Latger-Cannard
mesh_terms: D018952:Antigens, CD34 / Q000276:immunology; D002452:Cell Count / Q000379:methods / Q000592:standards*; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000592:standards*; D005602:France; D017408:Guidelines as Topic; D018380:Hematopoietic Stem Cell Transplantation / Q000592:standards*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D038622:Internationality; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D033581:Stem Cell Transplantation / Q000592:standards
chemical_list: D018952:Antigens, CD34
keywords: CD34+ hematopoietic stem cells; flow cytometry; hematopoietic transplantation