APC detection anomaly

Pál Jáksó pal.jakso at aok.pte.hu
Thu Nov 24 08:14:56 EST 2005


Dear Flowers,

To set up a new compensation matrix I used BD Compbeads stained with  
different florochrome conjugated antibodies. Unfortunately I  
experienced very strange result in the APC channel.

First I tried to measure positive beads stained with CD7 APC antibody  
and I surprised because only a fraction of the events showed  
positivity with APC. Previous to this I adjusted the gain using  
negative unstained beads so that the events took place at the 1st  
quadrant of the four decade axis. I used the antibody in the  
concentration what the manufacturer suggested.
I could reproduce this anomaly with T-lymphocytes too. I stained a  
blood sample with CD2 PE, CD3 PE-Alexa700 and CD7 APC. The ratios of  
the CD2 and CD3 positive cells were the same but the CD7 positive  
ratio was lower significantly (19 percent of the CD2/CD3 positives).  
Then I stained this sample with CD2 PE and CD7 FITC and the ratio was  
the same.

Second, as I lowered the gain for the FL4 channel the ratio of the  
positive beads decreased! I've never seen such a thing before.

I checked the beads with FITC staining and 99,5% of them were  
positive in addition adjusting the gain didn't modify the ratio of  
the positive events in the FL1 channel.

Had anybody such an experience before ?  Something may be wrong with  
the photomultiplier ?

Thanks,


Pal Jakso

University of Pecs
Faculty of Medicine
Dept. of Pathology
12. Szigeti str.
H-7643 Pecs, HUNGARY





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