An optimized whole blood method for flow cytometric measurement of ZAP‐70 protein expression in chronic lymphocytic leukemia

Shankey, T. Vincent; Forman, Meryl A.; Scibelli, Paul; Cobb, J. R.; Smith, Cecilia M.; Mills, Rhonda; Holdaway, Karen M.; Bernal-Hoyos, Elizabeth; Heiden, Mafalda Van Der; Popma, Jan; Keeney, Mike

doi:10.1002/cyto.b.20135

Cited by 46 publications

(63 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The large subpopulation with high levels of ZAP-70 (median fluorescence ratio ¼ 45) most likely represents T lymphocytes, and the small subpopulation with the highest levels of the molecule (median fluorescence ratio ¼ 188) is consistent with the expression levels of NK cells. Although we did not specifically identify these subpopulations with multi-color staining, this interpretation conforms with the published results from other laboratories (16,21).…”

Section: Zap-70 Expression Among Peripheral Blood Mononuclear Cellssupporting

confidence: 90%

“…demonstrated that phospho-ZAP-70, phospho-Syk, and p21 cip1 are stable in cells maintained at room temperature ex vivo for 24 hours (Table 3). It should be noted that others have shown that ZAP-70 levels decline over this period of time (15)(16)(17)(18)(19), but we have found they are relatively stable for 5 hours at room temperature, which is the time limit for processing in our procedure. Thus, the data we have obtained is reproducible, quantitative, stable upon freezing/thawing, and stable under the parameters of sample collection used in our procedure.…”

Section: Flow Cytometric Analysismentioning

confidence: 56%

“…For the most part, the median fluorescence ratios determined by EAS correlated well with the percent positive cells obtained by standard methodology; however, two samples were significantly discrepant. Although we cannot be certain of the explanation for these two discrepancies, the detection of ZAP-70 is known to be significantly affected by cellular fixative, reporting methods, and deterioration of the signal (15)(16)(17)(18)(19)32). Additionally, the sample size for the analytical method comparison was relatively small.…”

Section: Discussionmentioning

confidence: 90%

See 2 more Smart Citations

Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

Kaplan

Meyerson

et al. 2009

Cytometry Part B Clinical

View full text Add to dashboard Cite

Section: Zap-70 Expression Among Peripheral Blood Mononuclear Cellssupporting

confidence: 90%

Section: Flow Cytometric Analysismentioning

confidence: 56%

Section: Discussionmentioning

confidence: 90%

See 1 more Smart Citation

Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

Kaplan

Meyerson

et al. 2009

Cytometry Part B Clinical

View full text Add to dashboard Cite

“…Among proposed methods, calculating the ZAP70 T/B ratio is one of the earliest and most common approaches (12,(15)(16)(17)(18). Other methods based on ratios calculated on normal B-cell MFIs have drawbacks such as the very low percentage of residual B-cells or the difficulty of separating them from CLL cells by flow cytometry in the case of weak CD5 expression (11,13,32).…”

Section: T/b Ratio Does Not Reflect Zap70 Expression Levelsmentioning

confidence: 99%

“…Regarding the antibody and fluorochrome, various authors agree that the phycoerythrin-conjugated (PE-conjugated) SBZAP monoclonal antibody (mAb) is one of the best reagents for this method (11)(12)(13). Currently most groups express FCM results of ZAP70 expression in CLL B-cells as a ratio between mean fluorescence intensities (MFI) of ZAP70 in CLL B-cells and in normal residual or externally added B-cells (11,13,14) or, more frequently, as a ratio between ZAP70 MFIs in residual T-cells and CLL B-cells (ZAP70 T/ B ratio) (12,(15)(16)(17)(18)(19). The main disadvantage of the former method is that residual normal B-cells may be virtually absent in peripheral blood of patients with CLL and external addition of normal B-cells to the blood sample prior to staining supposes that ZAP70 levels in normal subjects are biologically stable.…”

Section: Introductionmentioning

confidence: 99%

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Rizzo

Bouvier

Youlyouz-Marfak

et al. 2012

Cytometry Part B Clinical

View full text Add to dashboard Cite

Flow cytometry is the reference technique for assessing ZAP70 expression, a marker of poor prognosis in CLL. One of the most common methods is to assess ZAP70 levels in CLL cells by calculating the ratio between ZAP70 mean fluorescence intensities (MFIs) in residual T-cells and CLL B-cells (ZAP70 T/B ratio). In this study, we developed a new method for ZAP70 labeling. Cells were labeled with a combination of anti ZAP70 phycoerythrin-conjugated SBZAP monoclonal antibody (mAb) and mAbs against CD45, CD19, and CD5. The latter three were used to specifically gate on different lymphocyte subsets. Staining was performed in absence (test) or in presence of excess unconjugated SBZAP mAb (isoclonic control). A so-called ZAP70 isoclonic ratio between SBZAP MFIs in the test and isoclonic control was calculated. A series of 32 patients with CLL and 10 normal controls were studied. Prediction of IGHV mutation status by ZAP70 isoclonic and T/B ratios was similar. By using the ZAP70 isoclonic ratio, we showed that ZAP70 expression was increased in T-cells from CLL patients. Nearly all cases with increased ZAP70 expression in CLL cells were associated with high ZAP70 expression in cognate T-cells. Therefore, the ZAP70 isoclonic ratio was more likely to closely reflect the biology of ZAP70 dysregulation rather than the T/B ratio. These results also explained why ZAP70 T/B ratios were artefactually close to normal in cells from CLL patients with high levels of ZAP70. V C 2012 International Clinical Cytometry Society

show abstract

Optimized Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression

2007

Self Cite

View full text Add to dashboard Cite

Optimized whole-blood assay for measurement of ZAP-70 protein expression chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. While CLL can be relatively indolent, in some individuals it can progress rapidly into a life-threatening disease. A number of different markers have been proposed as useful in predicting the disease course (Binet et al., 2006). Although ZAP-70 protein expression has been correlated with the disease course Orchard et al., 2004;Rassenti et al., 2004;Del Giudice et al., 2005;Schroers et al., 2005;Krober et al., 2006), there is currently no agreement on a uniform methodology, reagents, or data-analysis techniques to perform a clinically useful measurement of ZAP-70 protein expression using flow cytometry.Using modifications to a recently published technique that was developed to fix and permeabilize whole-blood samples for measurement of intracellular signal-transduction proteins (Chow et al., 2005), the authors have developed a technique to measure ZAP-70 protein levels in whole-blood samples. The protocols used here include: (1) an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP-70 protein with significantly greater signal-to-noise ratio (SNR) compared to other reported fix/perm methods, (2) an optimized combination of antibodiesfluorophores to maximize ZAP-70 expression levels, (3) standardized methodology to set up flow cytometers, including compensation, to improve inter-and intra-laboratory reproducibility, and (4) a method to index ZAP-70 protein expression levels to internal positive and negative cell populations (see Shankey et al., 2006). This protocol describes a technique to measure ZAP-70 protein expression in wholeblood specimens from CLL samples. It includes the use of cell surface-specific antibodies essential for the identification of key cell populations in the sample, including the residual normal T and B cells, which are used as internal positive and negative controls. These are used to index ZAP-70 protein expression levels in the CLL population. The Basic Protocol is designed for a flow cytometer equipped with a single (488-nm) laser.An important strength of this optimized assay is the use of standardized instrumentation and compensation set up. This enhances the ability to perform ZAP-70 measurements in a reproducible manner, both within a laboratory and among different laboratories. The use of calibrated bead standards to normalize fluorescence intensities for each fluorescence channel allows standardization and reproducible results when using the same or multiple instruments. This is particularly important when trying to standardize results from different laboratories. It should be noted that the protocol described here is specific for use with a particular flow cytometer (FC-500, Beckman Coulter, Inc.). However, a similar approach should be used regard...

show abstract

An optimized whole blood method for flow cytometric measurement of ZAP‐70 protein expression in chronic lymphocytic leukemia

Cited by 46 publications

References 29 publications

Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Optimized Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression

Contact Info

Product

Resources

About