PMID- 16906581
OWN - NLM
STAT- MEDLINE
DCOM- 20071206
LR  - 20151119
IS  - 1552-4949 (Print)
IS  - 1552-4949 (Linking)
VI  - 70
IP  - 4
DP  - 2006 Jul 15
TI  - An optimized whole blood method for flow cytometric measurement of ZAP-70 protein 
      expression in chronic lymphocytic leukemia.
PG  - 259-69
AB  - BACKGROUND: ZAP-70 protein expression has been proposed as a marker for 
      immunoglobulin heavy chain mutational status, which some studies have correlated 
      with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies 
      published to date measuring levels of expression of ZAP-70 intracellular protein 
      using flow cytometry have demonstrated poor performance, as defined by the 
      difference in signal in known positive and negative lymphocyte populations. 
      METHODS: A recently published method (Chow S, Hedley DW, Grom P, Magari R, 
      Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular 
      phospho-epitopes was optimized using a design of experiments (DOE) approach to 
      provide the best separation of ZAP-70 expression in positive T- or NK-cells as 
      compared to negative B-cells in peripheral blood samples. A number of 
      commercially available anti-ZAP-70 antibody-conjugates were screened using this 
      methodology, and the antibody-conjugate showing the best performance was chosen 
      to develop a four-color, five antibody assays to measure ZAP-70 levels in whole 
      blood specimens. RESULTS: Using the optimized fixation and permeabilization 
      method, improvement in assay performance (signal-to-noise, S/N) was seen in most 
      of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used 
      in conjunction with this optimized fixation /permeabilization method. In 
      conjunction with carefully standardized instrument set-up protocols, we obtained 
      both intra- and interlaboratory reproducibility in the analysis of ZAP-70 
      expression in whole blood samples from normal and CLL patients. CONCLUSIONS: The 
      development of a sensitive, specific and highly reproducible ZAP-70 assay 
      represents only the first essential step for any clinical assay. The universal 
      implementation of a validated data analysis method and the establishment of 
      methodology-based cutoff points for clinical outcomes must next be established 
      before ZAP-70 protein analysis can be routinely implemented in the clinical 
      laboratory.
CI  - (c) 2006 International Society for Analytical Cytology.
FAU - Shankey, T Vincent
AU  - Shankey TV
AD  - Beckman Coulter Incorporated, Miami, FL 33196, USA. vincent.shankey@coulter.com
FAU - Forman, Meryl
AU  - Forman M
FAU - Scibelli, Paul
AU  - Scibelli P
FAU - Cobb, Jeffrey
AU  - Cobb J
FAU - Smith, Cecilia M
AU  - Smith CM
FAU - Mills, Rhonda
AU  - Mills R
FAU - Holdaway, Karen
AU  - Holdaway K
FAU - Bernal-Hoyos, Elizabeth
AU  - Bernal-Hoyos E
FAU - Van Der Heiden, Mafalda
AU  - Van Der Heiden M
FAU - Popma, Jan
AU  - Popma J
FAU - Keeney, Mike
AU  - Keeney M
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Cytometry B Clin Cytom
JT  - Cytometry. Part B, Clinical cytometry
JID - 101235690
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Biomarkers, Tumor)
RN  - EC 2.7.10.2 (ZAP-70 Protein-Tyrosine Kinase)
RN  - EC 2.7.10.2 (ZAP70 protein, human)
SB  - IM
MH  - Antibodies, Monoclonal/immunology
MH  - Antigen-Antibody Reactions
MH  - B-Lymphocytes/*chemistry/immunology
MH  - Biomarkers, Tumor/analysis/biosynthesis/immunology
MH  - Cell Membrane Permeability
MH  - Flow Cytometry/*methods
MH  - Humans
MH  - Killer Cells, Natural/*chemistry/immunology
MH  - Leukemia, Lymphocytic, Chronic, B-Cell/*diagnosis/immunology
MH  - Reproducibility of Results
MH  - Staining and Labeling
MH  - T-Lymphocytes/*chemistry/immunology
MH  - Tissue Fixation/*methods
MH  - ZAP-70 Protein-Tyrosine Kinase/*analysis/biosynthesis/immunology
EDAT- 2006/08/15 09:00
MHDA- 2007/12/07 09:00
CRDT- 2006/08/15 09:00
PHST- 2006/08/15 09:00 [pubmed]
PHST- 2007/12/07 09:00 [medline]
PHST- 2006/08/15 09:00 [entrez]
AID - 10.1002/cyto.b.20135 [doi]
PST - ppublish
SO  - Cytometry B Clin Cytom. 2006 Jul 15;70(4):259-69. doi: 10.1002/cyto.b.20135.