Related Experiment Video
Updated: May 28, 2026
Flow Cytometric Characterization of Murine B Cell Development
Published on: January 22, 2021
Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic
Sergey N Preobrazhensky1, Philippe Szankasi, David W Bahler
1ARUP (Associated Regional and University Pathologists) Institute of Clinical and Experimental Pathology, University of Utah, Salt Lake City, Utah, USA.
Insights
Optimizing ZAP-70 detection in chronic lymphocytic leukemia (CLL) using isotypic controls improves reliability. This method accurately differentiates mutated and unmutated immunoglobulin heavy chain variable genes (VH), crucial for predicting CLL prognosis.
Area of Science:
- Hematology
- Immunology
- Oncology
Background:
- ZAP-70 expression in chronic lymphocytic leukemia (CLL) correlates with aggressive disease and immunoglobulin heavy chain variable gene (VH) mutational status.
- Current flow cytometric detection of ZAP-70 in CLL exhibits significant variability, limiting its clinical significance due to inconsistent correlation with VH mutational data and outcomes.
Purpose of the Study:
- To evaluate ZAP-70 expression in chronic lymphocytic leukemia (CLL) using an optimized flow cytometry staining procedure.
- To compare two distinct methods for eliminating nonspecific background staining in ZAP-70 detection.
- To assess the correlation between ZAP-70 levels and immunoglobulin heavy chain variable gene (VH) mutational status in CLL.
Main Methods:
- Seventy CLL cases were analyzed for ZAP-70 expression via flow cytometry.
- Two background elimination techniques were employed: optimized isotypic control antibodies and ZAP-70 stained normal B-cells.
- Isotypic control antibody concentrations were adjusted to ensure negative ZAP-70 staining on normal B-cells.
Main Results:
- An optimized isotypic control threshold resulted in a bimodal ZAP-70 distribution in CLL cases (30 low, 40 high).
- This optimized threshold strongly correlated with VH mutational status: low ZAP-70 cases predominantly had mutated VH genes, while high ZAP-70 cases had unmutated VH genes.
- A continuous ZAP-70 distribution was observed when using the ZAP-70 stained normal B-cell threshold, lacking clear correlation with VH status.
Conclusions:
- Employing an optimized isotypic control threshold enhances the reliability of flow cytometry-based ZAP-70 detection in CLL.
- This refined method demonstrates a strong correlation with immunoglobulin heavy chain variable gene (VH) mutational status, aiding in disease stratification.
Introduction:
Expression of ZAP-70 by chronic lymphocytic leukemia (CLL) is associated with more aggressive disease and can help differentiate CLL using mutated immunoglobulin heavy chain variable genes (VH) from cases expressing unmutated VH genes. However, flow cytometric detection of ZAP-70 in CLL shows considerable variability and may be of questionable significance because most laboratories cannot correlate their results to clinical outcome or VH mutational data.
Methods:
Seventy cases of CLL were evaluated for ZAP-70 using a previously optimized staining procedure and two different methods to eliminate nonspecific background staining. One method, not previously reported, used isotypic control antibodies, where the concentrations were adjusted/optimized so that normal B-cells stained negatively for ZAP-70. The other used ZAP-70 stained peripheral blood B-cells from normal donors. The percentages of ZAP-70 stained CLL cells above the two thresholds were compared.
Results:
Concentrations of isotypic control antibodies had to be increased from manufacture's recommendations to insure normal B-cells were ZAP-70 negative. ZAP-70 levels among the CLL cases formed a bimodal distribution using the optimized isotypic control threshold, with 30 having low values (0-32% positive) and 40 high values (60-99% positive). In contrast, a continuous distribution was obtained with the ZAP-70 stained B-cell threshold. VH mutational status strongly correlated with the optimized control values as 29/30 low ZAP-70 cases had mutated VH genes and 37/40 high ZAP-70 cases used unmutated VH genes.
Conclusions:
Use of an optimized isotypic control threshold could increase the reliability of flow based ZAP-70 detection and correlates well with VH mutational status.
