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I thought there are thousands of experts would know and could share their
experience with you. I have not read any responses to you since your
posting. Perhaps, I can be of a little help to you and share some of my
findings from the development of absolute CD34 count.
I have developed a rapid and accurate method to enumerate the number of CD34
positive cells in peripheral blood, bone marrow, and leukopheresis samples.
We talked about it in the ISHAGE workshop last November, and the manuscript
(for Journal of Hematotherapy) is in printing. The method consists of a two
tube (one IgG1 control tube, one CD34 tube) assay and a dedicated software
program for automatic instrument setup, data acquisition, and data analysis.
It is a 3-color assay: a DNA dye to identify nucleated cells; CD45 to
discriminate progenitor cells from mature lymphoid, myeloid, and erythoid
cells; and CD34-PE to determine the CD34+ cells. You can get the percentage
of CD34+ cells in nucleated cells, the absolute CD34+ cell counts (per 5L),
and the absolute nucleated cell counts within one hour, from sample staining
(20' staining, 30' FACSLyse lysing and fixing, and analysis on a FACScan) to
results printout. This method is sensitive enough to detect CD34+ cells in
peripheral blood of normal donors and can reliably determine an increase in
CD34+ cells in the peripheral blood of patients treated with chemotherapy
and/or growth facters. Over 50 normal periperal blood we have tested, we
detected 0 - 0.07% (We have not seen any exceeds 0.1%), or 0 - 5 CD34+
cells/5L. We have tested hundreds of patient samples (> 700 peripheral
blood, > 100 leukopheresis samples, 10-20 bone marrow and fetal bone marrow
samples), it ranges from 0 to 1% CD34+ cells for peripheral blood samples; 0
to 3-4% for leukopheresis samples; and 0 to 1-2% for bone marrow samples.
I hope these have answered your question. If you would like to try our this
automated system, you can call me at 408-954-2160 (I'll be on vacation from
3/28 - 4/8/94) or Leon Terstappen at 408-954-2494. Good Luck!
Chia-Huei Chen
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