Clinical Laboratory Validation of an Improved Flow Cytometry Assay for Detection of ZAP-70
Edward H Hart, Michael Liew, Sally Hill, Ramsey A Mohl, Todd W Kelley, David W Bahler. University of Utah and ARUP Laboratories, Salt Lake City, UT
Background: Expression of ZAP-70 by chronic lymphocytic leukemia (CLL) is associated with a more aggressive disease course and with cases that express unmutated immunoglobulin heavy chain variable genes segments (VH). In theory, flow cytometric detection of ZAP-70 offers many advantages over other methodologies, but has not been standardized and typically shows poor reproducibility among different laboratories. We recently described a novel flow cytometric method for detecting ZAP-70 that employs an experimentally optimized isotypic control antibody (Clinical Cytometry 2012, 82B:78-84) that shows a very high degree of correlation with IgVH mutational status and generates a bimodal distribution of ZAP-70 values among CLL cases. This study examines how this assay performs in a clinical laboratory setting.
Design: All cases sent to ARUP laboratories for both ZAP-70 analysis and VH mutational status testing since November 2008 were identified. Cases submitted for ZAP-70 analysis with cell viabilities less than 95% were excluded. ZAP-70 and VH mutational status results were reviewed for the remaining cases.
Results: ZAP-70 expression among the 110 identified cases showed a discontinuous almost bimodal distribution, with the majority of cases being either in the ZAP-70 low to negative group (38) or ZAP-70 positive group (65). Expression of ZAP-70 was highly correlated with mutational status since 35/38 ZAP-70 low to negative cases had mutated VH genes and 57/65 ZAP-70 positive cases had unmutated VH genes.
Conclusions: Use of our optimized isotypic control method for setting a negative threshold to determine ZAP-70 positivity works well in a clinical flow cytometry laboratory setting and still generates a higher degree of correlation with IgVH mutational status compared to other methods. The discontinuous distribution of ZAP-70 values generated could increase the reliability of flow based ZAP-70 detection among labs and improve prognostic value.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 283, Wednesday Morning