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It also relies upon an isotype-matched control to set the positive cell
analysis region for CD34+ cells. An increasing number of investigators have
concurred that this is a dangerous practice for rare event analysis such as
CD34+ cell detection in PB etc., where CD34+ cell numbers can be in the
0.02%-o.04% range. Some isotype matched controls stain more events
non-specifically than are specifically stained by the CD34 antibody used.
Other isotypes may stain virtually nothing in a particular sample, leading
to the detection of many more CD34+ events than in fact are contained in a
particular sample.
We have developed a two-colour (CD34/CD45), four parameter CD34+ cell
detection protocol for ISHAGE (Journal of Hematotherapy 5:213-226, 1996)
that addresses a number of these issues and is more broadly useful than the
single fluorescence parameter analysis at the heart of 'Milan'-type
protocols. While the recommended gating strategy may require a little more
attention, accurate rare event analysis warrants this extra effort and in
our experience, the ISHAGE protocol is far more accurate, sensitive and
reliable compared to relatively unsophisticated FL1- (or FL2-) versus side
scatter analysis. In the ISHAGE protocol, the isotype control is simply
used to enumerate non-specifically stained events that otherwise exhibit
the staining and light scatter characteristics of true CD34+ cells.
We have also recently demonstrated that dead cells are 'gated out' by the
sequential, cumulative gating strategy at the heart of this protocol.
While some (vested interest) groups have suggested that this gating
strategy using CD45 as a counter stain results in the ISHAGE protocol
'missing' CD34+/CD45- cells, we have yet to detect such events in the
hundreds of samples of normal marrow, PB, cord blood or cytokine-mobilised
blood. Like others, we too have noted occasional pre-B type leukemias that
express very low levels of CD45. However, non-malignant CD34+ cells all
appear to express CD45, albeit at levels lower than expressed by
lymphocytes.
The ISHAGE protocol can be adapted to include a third antibody conjugate so
that the qualitative composition of bona fide CD34+ cells can be assessed
(for example see Experimental Hematology 23:1619-1627, 1995).
Alternatively, in an allotransplant setting, we can incorporate CD3 in a
three-colour analysis so that we can enumerate CD34+ cells and
contaminating lymphocytes and T lymphocytes in for example, Cellpro
selected CD34+ cell suspensions.
We can also add fluorescent microspheres to the basic two/three colour
protocol to generate an absolute CD34+ cell count from a single instrument
platform, without having to use an automated hematology analyser. Thus
this protocol is very flexible and can be used in a number of different
clinical and research laboratory settings on both normal and abnormal
hematologic samples.
D. Robert Sutherland
ISHAGE Stem Cell Enumeration Committee,
Oncology Research, Toronto Hospital,
67 College Street,
Ontario M5G 2M1, Canada.
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web