Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome.
BACKGROUND
ZAP-70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP-70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP-70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories.


METHODS
Intracellular ZAP-70 levels in CLL and normal B cells were assessed by molecules of equivalent soluble fluorochrome (MESF), employing Quantum FITC MESF calibration beads to establish a standard curve relating channel value to fluorescence intensity in MESF units and the QuickCal v. 2.2 program (www.bangslabs.com) and clinical relevance of the data was determined.


RESULTS
The average ZAP-70 expression value in the CD19(+)/CD5(+) cells from 35 CLL patients was 103,701 MESF when compared with 12,621 MESF in B cells from 20 normal blood samples. "Low" and "high" ZAP-70 CLL subgroups were defined. Patients with "high ZAP-70 MESF" CLL had a shorter time to disease progression (P = 0.0005) and a more advanced clinical stage (P = 0.0018) when compared with patients in the "low ZAP-70 MESF" CLL subgroup.


CONCLUSIONS
This quantitative analysis method can be employed to obtain a more specific and highly accurate assessment of ZAP-70 levels in CLL cells. The method can easily be standardized, in any routine flow laboratory, thereby improving reproducibility and reliability of ZAP-70 analysis.
Kay
Sigi
S
The Hematology Institute, Tel Aviv Sourasky Medical Center,Tel Aviv, Israel.
Herishanu
Yair
Y
Pick
Marjorie
M
Rogowski
Ori
O
Baron
Shoshana
S
Naparstek
Elizabeth
E
Polliack
Aaron
A
Deutsch
Varda R
VR
D000368:Aged; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D001402:B-Lymphocytes / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis; D018450:Disease Progression; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D005500:Follow-Up Studies; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D053208:Kaplan-Meier Estimate; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000188:drug therapy / Q000378:metabolism; D008297:Male; D009367:Neoplasm Staging; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012995:Solubility; D015996:Survival Rate; D016896:Treatment Outcome; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000096:biosynthesis
D014408:Biomarkers, Tumor; D005456:Fluorescent Dyes; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human