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We (my lab) have gained very good experience over the years with the blast
gating you described using CD45 versus side scatter
dot plots. We have used CD45 in all fluorochrome combinations including
CD45-PE-Cy5 tandem and CD45-PerCP. Both work well
in our hands depending on the quality of the batch esp. for PE-Cy5. There are
very few good ones out therefore we did not
recommend the use of three colour so far in our German guidelines for
immunophenotysping of hematological malignancies (Leukemia,
in press).
We (in my lab) use however a whole blood procedure in order to describe the
myeloid maturation as well (see work of Mike Loken,
Leon Teerstappen and Bernd Woermann). Differently to their approach we use whole
blood or marrow, stain and lyse (BD procedure
including prewashing steps eg. for kappa and lambda). We also use the CD45/SSC
gating for cytoplasmic antigens (An der Grub
method).
In all cases the position of the clusters including the blasts remain constant,
excluding combinations with CD15.
The major hints from my side are:
1. Carefully saturate unspecific binding with serum of the species where the
detection reagent is derived from when using ficollated
or washed blood cells. Whole blood makes life much easier.
2. Carefully titrate your reagents (save money and reduce unspecific binding).
3. The CD45/SSC blast gate works well in most cases of acute leukemia but less
with NHL cases. It is contaminated with basophils,
large platelets and proerythroblasts (when CD45 density on blasts is very low
also with normablasts).
4. Unspecific binding of PE-Cy5 and PerCP is higher to myeloid cells and higher
than FITC or PE, higher in marrow than in blood.
5. For marker settings comparison with normal marrow is better than with isotype
controls.
I hope these comments are helpful. In case you find them unsatisfying, please
contact me again and send me your dot plots
and protocol via FAX.
With kind regards
Thomas Nebe
Central Laboratory
Klinikum Mannheim
Faculty for Clinical Medicine University of Heidelberg
D-68135 Mannheim / Germany
FAX +49 621 383-3819, Phone 383-3485
e-mail 0621339221-0001@t-online.de
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