CD34 test

ctnebe ctnebe at t-online.de
Thu Nov 28 17:09:00 EST 1996


Dear collegues,

in extension of the discussion I want to emphasize that the determination of 
absolute counts add to the list of errors.

Cord blood contains more nucleated red cells and it depends on your hematology 
system whether you count them or not.  Leukapheresis concentrates contain in 
addition cell debris where you can argue about counting thresholds.  At least 
the thresholds should be the same in your hematology and fluorescence flow 
cytometer.

Background also depends on the nature of your sample.  We found it very useful 
to counterstain CD34 (PE) with CD3,16,19 (FITC).  The CD34 progenitor cells form 
a clear cluster in the FITC vs PE dot plot where as the unspecific debris forms 
a diagonal.  The backgating shows the clear and typical position in the FCS/SSC 
dot plot. The percentage of debris is often in the same range as the true CD34 
progenitor cells.

Theoretically the bead count method should be ideal because it does not combine 
the errors of two instruments.  However the practical experience with the 
commercial product was less satisfying (no good cluster, sticky tubes).

The lysing method influences the result too.  FACS lyse reduces the FSC distance 
between debris and leukocytes compared to ammonium chloride.  The latter however 
shows more often incomplete lyse and nucleated reds.  

CD45 as the third colour reagent reduces this problem. It excludes erythroblasts 
and giant platelets from the analysis.  LDS751 or other DNA stains do not 
penetrate monocytes well, stain nucleated reds and in part giant platelets and 
are therefore unreliable.

I have seen consensus protocols on CD34 but they are either rather general or 
cannot substantiate some of their recommendations.  However, in good blood 
samples a good agreement was seen by Stefan Serke in the INSTAND EQA in Germany 
over 35 labs.

Good EQA schemes with problem samples will hopefully help on this issue.

Thomas Nebe, ctnebe at t-online.de
Klinikum Mannheim, D-68135 Mannheim



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