title: Comparison of ISHAGE protocol CD34 cell enumeration with a lineage negative backgating technique.
abstract: BACKGROUND
  CD34(+) cell enumeration in PBSC apheresis products has become the standard for assessing graft hematopoietic potential.
  
  
  METHODS
  An in-house, three color, lineage negative-gating technique [University of Nebraska Medical Center (UNMC) protocol] for CD34 cell enumeration was compared with the ISHAGE protocol over 100 apheresis products. Cell doses determined by both methods were compared with each other and to colony-forming units-granulocyte/macrophage (CFU-GM) assay results.
  
  
  RESULTS
  Overall, the assays compared well with each other for samples with CD34 cell doses > 0.2 2 10(6)/kg (r values > 0.8). The ISHAGE method showed a constant negative bias, with a mean of 38% in comparison to the UNMC protocol, which was more linear at lower cell doses. Both assays showed similar correlation with CFU-GM doses after log conversion (UNMC, r = 0.915; ISHAGE, r = 0.917). When comparing integer values, however, the ISHAGE method correlated with CFU-GM only in the high dose range (CFU-GM > 2 2 10(4)/kg), while the UNMC method correlated across the entire measured range of CFU-GM doses. Finally, an inter-technologist gating reproducibility study (n = 6) yielded a 23% coefficient of variation (CV) for the ISHAGE method and a 7% CV for the UNMC method, when the same two sets of CD34 histograms were analyzed to calculate cell dose.
  
  
  CONCLUSIONS
  In this study the lineage negative protocol (UNMC) had a larger dynamic range, correlated better with CFU-GM results and showed better inter-technologist reproducibility than the ISHAGE method.
authors.affiliation: University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Pirruccello
authors.forename: C J
authors.initials: CJ
authors.lastname: Page
authors.forename: M R
authors.initials: MR
authors.lastname: Bishop
authors.forename: B A
authors.initials: BA
authors.lastname: Letheby
authors.forename: P I
authors.initials: PI
authors.lastname: Warkentin
authors.forename: J D
authors.initials: JD
authors.lastname: Jackson
authors.forename: A
authors.initials: A
authors.lastname: Kessinger
mesh_terms: D018952:Antigens, CD34 / Q000096:biosynthesis*; D001781:Blood Component Removal; D018929:Cell Culture Techniques / Q000379:methods*; D019070:Cell Lineage; D017690:Cell Transplantation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008954:Models, Biological; D012044:Regression Analysis; D015203:Reproducibility of Results; D013234:Stem Cells / Q000166:cytology
chemical_list: D018952:Antigens, CD34; D016178:Granulocyte-Macrophage Colony-Stimulating Factor