title: Thawed autologous peripheral blood stem cells require modified quantification methods for hematopoietic progenitor cell evaluation.
abstract: OBJECTIVE
  The aim of this study was first, to analyze the post-thaw progenitor assays usually performed on peripheral blood stem cell autografts and second, to achieve standardization with improved flow cytometric and CFU-GM assays.
  
  
  METHODS
  In the first part of the study (n=79), recovery and Intraclass Correlation Coefficient (ICC) of total nucleated cells, CD34 and CFU-GM were analyzed before and after cryopreservation. In the second part (n=20), evaluation methods were modified : the washing step was suppressed in the flow cytometric method and 500 CD34 were plated compared to 4×10(4) total nucleated cells in the CFU-GM assay. The recovery rates were analyzed and the CFU-GM results were regarded as reliable when 30-100 colonies were observed, according to the manufacturer recommendation.
  
  
  RESULTS
  The analysis of the first part showed an ICC that was perfect for total nucleated cells (0.93), substantial for CD34 (0.67) and fair for CFU-GM (0.25). Median CD34 recovery was 112.6% (29.9-222%). The CFU-GM median recovery was 31.7% (0.19-142%) leading to reliable results for 27 grafts. In the second part, the median CD34 recovery was 85.75% (54-99%). No recovery over 100% was observed. The CFU-GM assay led to 18 out of 20 evaluable autografts when 500 CD34 were seeded, compared to 10 out of 20 when total nucleated cell were seeded.
  
  
  CONCLUSIONS
  Avoiding cell washing in the flow cytometric method limited the overestimate of the CD34 percentage. Plating 500 thawed CD34 improved reliability of the results and allowed a better standardization of the assay.
authors: Cell Therapy and Tissues Unit, CHU Nancy, Vandoeuvre-les-Nancy, France. v.decot@chu-nancy.fr
authors: Véronique
authors: V
authors: Decot
authors: François
authors: F
authors: Alla
authors: Véronique
authors: V
authors: Latger-Cannard
authors: Sorin
authors: S
authors: Visanica
authors: Brigitte
authors: B
authors: Witz
authors: Jean-François
authors: JF
authors: Stoltz
authors: Danièle
authors: D
authors: Bensoussan
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D018952:Antigens, CD34 / Q000032:analysis*; D002648:Child; D003114:Colony-Forming Units Assay / Q000379:methods*; D015925:Cryopreservation; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D055815:Young Adult
chemical_list: D018952:Antigens, CD34