lymph.function assay by CD69 and CD71.

brettT@qimr.edu.au
Fri, 30 Jun 95 14:49:19 EST

We have just started using a CD69 and CD71 based lymphocyte function
assay (based on some work I've previously done and the BDIS paper in Cytometry
20:127-133 (1995)).
Generally I am seeking a group opinion on whether this method is valid
to replace the tritiated Thymidine method, and, what ways can it be improved
to make it more viable an alternative.

The method uses ficolled lymphocytes (1million) stimulated for 4 hours
with PWM, Con A, CD2, PHA, SEB, Candida A., PMA as a maximal control and an
unstimulated control. These are then FACSed 4 hours using CD3/CD69 for
activation and again at 16hours with CD3/CD71 for proliferation.

My Questions:

1. Is there a need for an additional proliferation marker (eg. CD25) or is
CD71 alone acceptable.

2. Would more co-stimulatory mitogens (eg. CD2 and 28) be more relevant
clinically to test activation/function than mitogens like PWM and ConA.

3. Would DNA profiles be of any real advantage in this case.

4 Are there any conditions/disorders where this assay may not perform.

and
5. Am I just chasing my tail, or is this truly an alternative to the tritiated
thymidine.

Brett Teale
Queensland Institute of Medical Research/Dept.of Immunology,
Royal Brisbane Hospital
Queensland
Australia

brettT@qimr.edu.au

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