[Cytometry] staining method for diagnostic immunophenotyping

Sandy Smith ssmith at stvincents.com.au
Sun Feb 22 21:13:25 EST 2009


In our diagnostic lab we use a stain/lyse/no-wash method for our lymphocyte
subsets (basic enumeration), and a stain/lyse/wash/fix for the oncology
phenotyping; we use FACSLyse for both.

The no-wash method is much quicker and easier; we have to use CD45 to threshold
out the significant amount of un-lysed RC/debris, but we always use CD45 anyway.
The only 'drawback' was that our sample probe had a red tinge from these samples
on our FACSCalibur, even with the bleach tube in every carousel; but now we use
the 'SIT flush' between tubes on the new FACSCanto II and don't have that issue
any more. A few times a year (if that) we have to perform the lyse/wash method on
a sample that doesn't show clear separation of populations, doesn't happen often
enough for us to see a pattern in these samples to explain why. We used to use
lyse/wash for subsets, but we compared methods and the results were very similar
so we switched. Not washing also reduces risk of staff exposure to sample.

We use the lyse/wash method for all oncology samples; takes longer but cleans up
the CD45 neg area so we don't have to use a CD45 threshold (which would exclude
some neoplastic cells, esp B-ALLs). With the CD45 neg mess we see from the no-wash
method, we haven't investigated switching methods.

Hope this is useful,

Sandy Smith

Flow Cytometry, SydPath
St Vincents Hospital, Sydney
(02) 8382 9176

>>> "Lydene McArthur" <Lydene.McArthur at cdhb.govt.nz> 23/02/2009 9:22:18 am >>>
Hi everyone


I'd like to get an idea of staining approaches used by other
laboratories for diagnostic immunophenotyping for leukaemia/lymphoma.
We use a lyse/wash/stain/wash technique for the majority of samples
(peripheral blood, bone marrow, lymph node and cellular FNA samples).
I've been asked to investigate whether it would be suitable to use a
stain/lyse/no-wash technique instead, but would like to get feedback
from other diagnostic labs and find out which stain method you are
using, if you are using (or have tried) a no-wash technique, and whether
you have a preference for any method over another.  (See attached file
for brief summary of techniques mentioned).


Many thanks

Lydene McArthur


Haematology Surface Markers

Canterbury Health Laboratories


New Zealand




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