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Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome.
Cytometry B Clin Cytom. 2006 Jul 15; 70(4):218-26.CB

Abstract

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.

Authors+Show Affiliations

The Hematology Institute, Tel Aviv Sourasky Medical Center,Tel Aviv, Israel.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

16456869

Citation

Kay, Sigi, et al. "Quantitative Flow Cytometry of ZAP-70 Levels in Chronic Lymphocytic Leukemia Using Molecules of Equivalent Soluble Fluorochrome." Cytometry. Part B, Clinical Cytometry, vol. 70, no. 4, 2006, pp. 218-26.
Kay S, Herishanu Y, Pick M, et al. Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome. Cytometry B Clin Cytom. 2006;70(4):218-26.
Kay, S., Herishanu, Y., Pick, M., Rogowski, O., Baron, S., Naparstek, E., Polliack, A., & Deutsch, V. R. (2006). Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome. Cytometry. Part B, Clinical Cytometry, 70(4), 218-26.
Kay S, et al. Quantitative Flow Cytometry of ZAP-70 Levels in Chronic Lymphocytic Leukemia Using Molecules of Equivalent Soluble Fluorochrome. Cytometry B Clin Cytom. 2006 Jul 15;70(4):218-26. PubMed PMID: 16456869.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome. AU - Kay,Sigi, AU - Herishanu,Yair, AU - Pick,Marjorie, AU - Rogowski,Ori, AU - Baron,Shoshana, AU - Naparstek,Elizabeth, AU - Polliack,Aaron, AU - Deutsch,Varda R, PY - 2006/2/4/pubmed PY - 2007/12/7/medline PY - 2006/2/4/entrez SP - 218 EP - 26 JF - Cytometry. Part B, Clinical cytometry JO - Cytometry B Clin Cytom VL - 70 IS - 4 N2 - 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. SN - 1552-4949 UR - https://www.unboundmedicine.com/medline/citation/16456869/Quantitative_flow_cytometry_of_ZAP_70_levels_in_chronic_lymphocytic_leukemia_using_molecules_of_equivalent_soluble_fluorochrome_ L2 - https://doi.org/10.1002/cyto.b.20078 DB - PRIME DP - Unbound Medicine ER -