Comparison of ISHAGE protocol CD34 cell enumeration with a lineage negative backgating technique

Samuel Jay Pirruccello, C. J. Page, M. R. Bishop, B. A. Letheby, Phyllis Irene Warkentin, J. D. Jackson, A. Kessinger

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 × I06/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 × 104/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. Discussion: 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.

Original languageEnglish (US)
Pages (from-to)279-286
Number of pages8
JournalCytotherapy
Volume1
Issue number4
DOIs
StatePublished - Jan 1 1999

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Granulocyte-Macrophage Progenitor Cells
Blood Component Removal
Color
Transplants

Keywords

  • CD34
  • Flow cytometry
  • ISHAGE
  • Lineage gating

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Oncology
  • Genetics(clinical)
  • Cell Biology
  • Transplantation
  • Cancer Research

Cite this

Comparison of ISHAGE protocol CD34 cell enumeration with a lineage negative backgating technique. / Pirruccello, Samuel Jay; Page, C. J.; Bishop, M. R.; Letheby, B. A.; Warkentin, Phyllis Irene; Jackson, J. D.; Kessinger, A.

In: Cytotherapy, Vol. 1, No. 4, 01.01.1999, p. 279-286.

Research output: Contribution to journalArticle

Pirruccello, Samuel Jay ; Page, C. J. ; Bishop, M. R. ; Letheby, B. A. ; Warkentin, Phyllis Irene ; Jackson, J. D. ; Kessinger, A. / Comparison of ISHAGE protocol CD34 cell enumeration with a lineage negative backgating technique. In: Cytotherapy. 1999 ; Vol. 1, No. 4. pp. 279-286.
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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 × I06/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 × 104/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. Discussion: 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.",
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