Comments I have recieved about ZAP-70

Markestad, Sara SMarkestad at
Wed Jun 16 08:25:07 EST 2004

Here are a few of the emails I have gotten from others about the ZAP-70. It seems that I am not the only one having problems with this antibody. If anyone finds out anything new with this procedure do you mind sharing with all of us.

Sara Marekstad, MT (ASCP)
St. Joseph's of Atlanta 

Sara -

We have been trying to develope this assay for several months and have not found
it to be reliable.  We don't have any problem combining surface
antibodies/Zap-70, however determining whether or not Zap-70 is truly postivie
has been questionable.  If you have any luck, we would be interested in hearing
your method.  Good luck!!

Abby Kelliher
Clinical Flow Cytometry Lab Supervisor
Massachusetts General Hospital
Boston, MA  02114


We're using ZAP-70 Alexa 488 from Caltag, along with 5 PE 19 ECD and 3+/56 +- PC5. The PC5 antibodies are actually a combination of 2 antibodies. We're having problems though, with every sample staining for ZAP-70. I'm looking to adjust the fix and perm method. We use Caltags' Fix and Perm. If you receive any messages in response to your querry, I'd appreicate it if you could share them with me. 
Michael Schwartzm MT(ASCP) 
mschwartz3 at <mailto:mschwartz3 at>


We have run many samples of CLL patients using both the reagents and
protocols of UPSTATE and of BIOSCIENCE, and have several questions and
1-When setting the quadrants, what percent of negative T and NK cells (left
upper quadrant) is permissible? We find that even the slightest shift of
quadrant can change CLL cells from ZAP positive to negative and vice versa.
I have not seen any publication defining the upper limits of ZAP negativity
2-Is anyone performing the ZAP assay by using quadrant based on isotype
control?	When we set the quadrant  using isotype control instead of
T/NK cells we consistently find that all CLL cells are ZAP positive. I
cannot understand why we cannot use isotype control as we do with other
We decided to perform the ZAP assay on normal controls and found the same
phenomenon: all B cells were ZAP positive when using the isotype control for
quadranting, and when we used T/NK cells for quadranting we could get any
result we wanted depending on where we moved the cursor.
I would greatly appreciate comments and suggestions, and as of now we have
ceased to perform ZAP assays as we do not feel, that in our hands,  we can
rely on results.
What is your experience?


A difficult issue but if we treat this new assay like any new assay then do the following:
1. Test for reproducibility: test your procedure on CD38[-] cases and CD38[+] cases, 10 replicates for each case, 3 cases each. If your results have a CV of more than 20% within a case, then you need a new method.
2. Test for accuracy: get the above cases tested for IgVh mutational status [expensive].
or Test for accuracy: assay at least 100 CLL cases for ZAP70 [at least 20% should be CD38[+]. Generate a frequency analysis of all the results [how many patients have the same results]. A good test will have two populations of ZAP70, one you will assign to be negative and one positive. Deciding where the cutoff should be is the tricky part but if you have enough positives you can use a visual cutoff.
3. Use an assay that has already been validated somewhere else and perform parallel testing on 10 negatives an 10 positives.
Hope this helps.

Mark L. Shenkin, Ph.D. 
Director, Flow Cytometry 
Ameripath, Inc. 
mshenkin at 

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