PMN Markers ( and that pesky CCC article)

Gerhard Nebe-von-Caron Gerhard.Nebe-von-Caron at unilever.com
Tue Nov 5 12:22:54 EST 1996



          The increased autofluorescence of the eosinophils can indeed 
          be used to separate them from the neutrophils. When CD45 
          stain is used, that autofluorescence should be swamped by 
          the CD45 FITC which is 10 times higher. Thus the eosinophils 
          must also express a higher cd45 density.
          When useing a fixative lyse such as the Becton Dickinson 
          lysing solution one can (even on a Coulter cytometer) 
          observe the eosinophils as a separate cluster in light 
          scatter. It shows very nice in the "log scatterspace".

          By the way, having read that pesky CCC article I really 
          wonder what data people can produce. I am amazed to see that 
          the bandwith between 564 and 606nm is stated to be 
          equivalent to a 570nm long pass in terms of what is seen of 
          the PE spectrum. To mention the effect of the clever angular 
          arangement of the dichroics in the Facsscan to change their 
          filter characteristics migth be too much to grasp then 
          anyhow. I also wonder if anyone read the article apart from 
          (or even not) the reviewers that it has passed without any 
          more comments. I haven't even heard anything with respect to 
          the calibration procedures.

          By the way, I am also still looking for the missing 135mW of 
          my aircooled laser in the Elite. Anybody got them?

          Gerhard.Nebe-von-caron at unilever.com

           


______________________________ Reply Separator _________________________________
Subject: RE: PMN Markers
Author:  Kbahjat at nwu.edu at INTERNET
Date:    01/11/96 23:43


Forgive me if I am oversimplifying, and please correct me if I'm wrong, but:

I have always had the impression that when staining a fresh peripheral
blood with CD45 FITC and CD14 PE, that a five part differential could be
performed from this 2 color plot?

With FITC (CD45) on the X axis and PE (CD14) on the Y, the Lymphocytes,of
course, are the bright CD45+, CD14 negative population. Monocytes are the
CD45 bright, CD14 bright population, with your PMNs being CD45 moderate,
CD14+(somewhat dim). The eosinophils seperate to the right of the PMNs, as
their azurphillic granules autofluoresce enough that they are brighter in
the FITC channel than the neutrophils. Basophils do not bind CD14 at all
and will sit directly below the PMNs (same CD45 intensity as PMNs, with no
CD14 binding).

As far as precursor cells (blasts, metamyelocytes, myelocytes...), I'm not
sure of their location in 45-14 plots,  but have used CD45 vs SSC according
to articles published by Dr. Greg Stelzer to identify these populations. I
of course confirmed these gates by comparing with sorted cell morphology.

>From all the replies/discussion, I'm thinking I've oversimplified things?

Please correct or confirm these hypotheses...

kb


Keith Bahjat
Kbahjat at nwu.edu



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