Dear Jill,
I have done extensive work on this, to the point of making 2 patent
applications, one current.
Most of the measurement issues are technical:
Flow rates used
RBC thresholding
Nucleated and WBC discrimination/gating
Coincidence effects on scatter signals
Coincidence effects on fluorescence of positives and negatives...
Basically, you need to run at high flow rates, of >50,000 cells/s and then
you need to threshold out most RBCs (say 90%, when 0.1% are WBCs), but the
remainder comprise WBCs at or below 1/1000 cells. Every white cell has 1, 2
, 3, 4,.. RBCs coincident except for a MoFlo MLS system with 5 microsecond
dead-times, where you get mainly none, one or two coincident RBCs.
My patent protocols generally use a pulse width and a DNA dye to add an
exclusion gate for non-nucleated cells, but then you must resolve the issue
of RBC coincidences and how they affect gates for the WBC subsets and the
dispersion in fluorescences of positives and negatives!
If you don't have a MoFlo, then buy one; else forget it!
You dig?
Try me out for more, though I am not a masochist.
Bob
-----Original Message-----
From: Martin, Jill V. [SMTP:martin.jill@mayo.edu]
Sent: Tuesday, October 14, 1997 4:12 AM
To: Cytometry Mailing List
Subject: Staining of Whole Blood.
Hi to everyone in Flower Land. I have an investigator who wants
to look at leucocyte markers using unlysed whole blood. He says that was
the protocol used at the institution where he used to work. I have
never run samples prepared this way, nor have I heard of anyone doing it.
The first trial was not a success. If anyone who reads this knows if
and how the samples can be stained and analysed I would appreciate hearing
from them. I would also like to hear if you think that it is impossible
so I can pass that information along.
Jill Martin, , Manager
Molecular Biology & Flow Cytometry Core
Johnson Research Building
Mayo Clinic Scottsdale
602-301-6071 (Voice)
602 301-7017 (FAX)
email: jmartin@mayo.edu or martin.jill@mayo.edu
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