A NEW DQB1 ALLELE DQB1*0308. K. Balakrishnan1, G.Mersch2, J.R. Rugg 1, K. Lebeer2, M. Blanton1, G. Van Reybroeck2, L.R. Whitacre1 and R. Rudi2, Hoxworth Blood Center, University of Cincinnati1 and Innogenetics2, Belgium.

METHODS FOR COMPARISON OF CD34 ENUMERATION.
K. Balakrishnan, M. Blanton. Histocompatibility Laboratory, Hoxworth Blood Center, University of Cincinnati.

The aim of the study was to enumerate CD34 cells as a percentage of CD45 cells using ProCOUNT Progenitor Cell Enumeration software. The results obtained were compared with those obtained using ISHAGE techniques. Cord blood samples and stem cell from apheresis collections and bone marrow were sources of stem cells. The methodology utilizes the granularity (SSC), nucleic acid dye expression (FL1), CD45 expression (FL3) and antigen expression of the CD34 (FL2) cells to aid in distinguishing them from other cells. CD34 cells can be expressed as a percentage of nucleated cells as well as a percentage of CD45 cells. The necessity to do manual absolute counts is obviated in the ProCOUNT technique. The ISHAGE methodology expresses CD34 as a percentage of CD45 cells and requires a manual count. We evaluated the results obtained utilizing the new software for 20 cord blood samples, four peripheral blood stem cells and five bone marrow samples. No formalized proficiency testing was available at the time of the study. We also found that each laboratory that does CD34 assays appears to have its own individual methodology although the majority are modifications of the ISHAGE technique. Results obtained on analyzing cord blood samples were comparable and there was no statistical difference between the two methods. The stem cells obtained by apheresis (stimulated and with more passes) were easily manageable. However, bone marrow samples presented difficulties, the auto-gating was not always accurate. The new software though permitting manual gating cannot adequately address the problem on debris; large numbers of nucleated cells present a problem since a nuclear stain is used. 1) It was concluded that there was no statistically significant difference between the two methods. 2) The ProCOUNT software does auto-gating, although the new software does permit manual-gating. 3) The analysis software was not accurate when marrow specimens contained large numbers of nucleated cells.