Abstract
Background:
In this study we developed a method to measure the amount of ZAP‐70 [zeta accessory protein] in B‐CLL cells without relying on the ZAP‐70 expression of patient B or T cells to normalize fluorescence intensity.
Methods:
B‐CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP‐70 antigen was blocked in one tube with unlabeled antibody to ZAP‐70 [clone 1E7.2]. Zap‐70‐PE was then added to this tube. ZAP‐70‐PE was added to a second tube without unlabeled antibody to ZAP‐70. The mean fluorescence intensity of the ZAP‐70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP‐70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non‐specific ZAP‐70 fluorescence in the B‐CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP‐70 fluorescence intensity in the tube with unlabeled antibody to ZAP‐70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP‐70 cells in the tube without unlabeled antibody to ZAP‐70. The brighter the ZAP‐70 fluorescence above the non‐specific background, the higher the %POS.
Results:
Due to the varying amount of non‐specific staining between patient B‐CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP‐70 in B‐CLL cells than other methods, which use the ratio of B‐CLL fluorescence to normal B or T‐cell fluorescence. Using this improved method, ZAP‐70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP‐70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut‐off value lower than previous values published, due to exclusion of non‐specific staining. Both cut‐offs were based upon patient specimen distribution profiling.
Conclusions:
Use of a blocking antibody resulted in a robust, reproducible clinical B‐CLL assay that is not influenced by the need to measure the amount of ZAP‐70 in other cells. ZAP‐70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. © 2006 International Society for Analytical Cytology