ZAP-70 and CD38 revisited

Dear All,

I have been performing ZAP-70 and CD38 on some cases of CLL on hand. So far
I have done already 10 cases and all turned out to be ZAP-70 negative. I
wonder whether I am doing it correctly or really all my cases at hand are
cases with mutated IgVH.

Complicating the issue further. We recently performed a quality assurance
sample from the Australian quality assurance program. The consensus result
for the CD38 in this case was positive. (though the results were quite
scattered and one third of the participants, including us, reported this
case as CD38 negative). We have at the same time performed a ZAP-70 on this 
case and it was a definite negative. This make me thinks that our ZAP-70 
might have some problem.

Here is what I am doing.

- FASCalibur, 3 color with a single 488nm laser
- using peripheral blood from CLL patients
- using Caltag Fix and Perm for membrane permeation
- gated the lymphocyte population using the SSC vs CD45 plot
- plotting the tube with ZAP-70 FITC(ebioscience), CD19 PE and CD45 PE-Cy5
as in http://www.dslserver.net/ZAP-70.gif
- using the T cell population (CD19 negative population) as marker to
determine the percentage of ZAP-70 positive cells

The problem which I encountered were

1. Should we use the T cell population as the marker as describe in NEJM
article? The Lancet article use isotype control for marker instead of the T
cell population.

2. As I assume that even if the CLL population is ZAP-70 is positive, it
must be very weak. How close your marker should be to the T cell population
or isotype control? In the example at http://www.dslserver.net/ZAP-70.gif ,
the B cell population has 19.1% in the positive region. So just by moving
the line slightly will turn this case from negative to positive.

3. How can I validate my results if I do not have the resources to
perform the IgVH mutation status for my cases? Is there somewhere I can get
some typical ZAP-70 positive or negative case to perform on my system to see
the result? Any ZAP-70 quality assurance peer group?


Thanks.

 Michael