unusual T cell phenotype/absolute counts

ctnebe ctnebe at t-online.de
Tue Nov 19 18:34:00 EST 1996

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The question was:
>We have recently examined a lymph node excised from a piece of jejunum
>and the larger cells in the suspension(25% of all cells) had the
>atypical T cell immunophenotype - CD2+, CD3+, CD7+, CD4-, CD8-, CD5-,
>CD56-, CD1a-, CD10-, I3- and CD71+.  The cells also displayed strong
>intensity CD45.

This phenotype is consistent with TCR1 T cells (gamma delta TCR).  They occur at 
low frequency in the peripheral blood as well.

Absolute counts
The discussion needs some fuel.  Volumetric metering also suffers from problems 
like demixing of cells in the tubings to the flow cell and requires calibration 
(Ortho uses beads for calibration!).  The whole blood approach without lyse and 
wash using dilution only is preferred by us for these cases of small sample 
volumes (10 ul blood + 2 ml PBS).
LDS 751 does not enter monocytes well and also takes nucleated red cells.  
Monocytes are also relatively resistant to PI and 7AAD.
We prefer CD45 (in combination with CD14 and CD16 or SSC only) using clean 
reagents and solutions meaning either sterile filtration or high speed 
centrifugation (conjugated mabs). Some beads tend to stick to tubes or cells or 
they undergo a sedimentation different to that of leukocytes.  Both needs to be 
controlled.  We use the BD calibrites (pooled empty bottles and coulter counter) 
or absolute count beads and compare the CD45 counts with the WBC from a 
calibrated hematology analyzer (factor of 1.05 in between for 350 cases).  In 
cases of disagreement we use the manual hemocytometer count.  This approach 
combined with CD41 or similar helps also for low platelet counts (where 
hemocytometer counts are a nightmare).  
We also used the quoted approach from Leon Teerstappen, in addition the red 
cells for count beads and the RBC from the coulter counter.  However, this 
method is fairly slow.

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

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