ZAP-70 by Flow Cytometry: A Comparison of Different Antibodies, Anticoagulants, and Methods of Analysis

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1 Cytometry Part B (Clinical Cytometry) 70B: (2006) ZAP-70 by Flow Cytometry: A Comparison of Different Antibodies, Anticoagulants, and Methods of Analysis O. G. Best, R. E. Ibbotson, A. E. Parker, Z. A. Davis, J. A. Orchard,* and D. G. Oscier Background: The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement. Methods: We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated. Results: Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin. Conclusion: Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression. q 2006 International Society for Analytical Cytology Chronic lymphocytic leukaemia (CLL) is a disease of great clinical heterogeneity: it may stay stable for decades or progress rapidly, and treatment options span the possibilities from a wait and watch policy to allogeneic transplantation. As the diagnosis is increasingly made in early Stage A disease, and as the possibility of inducing complete remission at a molecular level increases, the need for prognostic markers becomes correspondingly stronger (1). IgVH gene mutational status is a reliable and reproducible prognostic marker (2,3) but remains expensive and technically unsuited to a routine diagnostic laboratory; aberrant expression of ZAP-70 in CLL emerged as a marker of poor prognosis from cdna microarray studies that compared the IgVH mutated and unmutated groups (4). ZAP-70 protein expression by flow cytometry has been shown to correlate well with IgVH gene mutational status (5 7), although discordant cases are well described, and to be a marker of both treatment free (7,8) and overall (6,9,10) survival. Since flow cytometry is routinely used to diagnose CLL, the measurement of ZAP-70 by flow should be a widely used prognostic marker. Its relatively slow uptake as a routine prognostic marker reflects the difficulties involved in its measurement. Two main antibody clones to ZAP-70 have been used for flow cytometry: the 2F3.2 clone, raised against the two SH2 domains of human ZAP-70 (amino acids 1 254), and the 1E7.2 clone, raised against an epitope *Correspondence to: Jennifer Orchard, rbhpathology@lds.co.uk Received 4 May 2006; Accepted 8 May 2006 Published online in Wiley InterScience ( com). DOI: /cyto.b q 2006 International Society for Analytical Cytology

2 236 BEST ET AL. within the tyrosine kinase domain (amino acids ). The protein is intracellular and several methods for accessing it have been described, the principal ones being a commercial kit (5), and 2 in-house methods, paraformaldehyde and ethanol (6), and paraformaldehyde and saponin (7). T and NK cells express ZAP-70 protein strongly but are easily defined by surface antibodies; the principal problem with ZAP-70 measurement by flow cytometry is that the protein is weakly expressed in CLL (11). In addition, in positive cases the whole CLL population moves homogeneously to the right; this creates problems with the conventional definition of a positive population that measures the percentage of test cells staining more strongly than a negative population, since a very small shift in the negative marker may create significant differences in percentage values. The present study aims to evaluate a commercial method for ZAP-70 measurement by flow cytometry against our previously published method. The 1E7.2 antibody conjugated to Alexa-488 with intracellular access via the Fix and Perm kit (Caltag Laboratories, Burlington, CA) was assessed against the unconjugated 2F2.3 antibody (Upstate Biotechnology, Charlottesvile, VA), fixed, and permeabilized with paraformaldehyde and ethanol. The commercial kit from Caltag, if satisfactory, could offer a relatively simple method of measuring ZAP-70 by flow cytometry that might be more widely taken up at the time of flow diagnosis of CLL. We explore a method of analysis comparing the geometric mean of the test CLL population with that of an isotype control, which we show to be much more reproducible than the conventional percentage method. We examine in detail those cases in which there is a discrepancy between antibody results or analysis methods, and aim to resolve these using data on IgVH gene mutational status and clinical information. METHODS Patients and Samples Patients (72) with CLL diagnosed by standard NCI criteria were studied. IgVH gene mutational status was as follows: 27 unmutated (98 100% germline homology), 36 highly mutated (<96% homology), and 9 with low levels of mutation ( % homology). Mean age was 63 years; 45 patients were untreated and no patients were studied within 3 months of treatment. Twentyseven patients had progressive disease. Peripheral blood samples were taken into preservative free heparin and assayed or fixed within 24 h. We compared our standard assay using the unconjugated 2F3.2 clone and fixation/permeabilization by paraformaldehyde and ethanol (hereafter called the Upstate method), with the conjugated 1E7.2 clone, using a commercial fixation/permeabilization kit (hereafter called the Caltag method.). For each antibody, results were analyzed by four different methods, and all analyses were carried out independently by three different observers. Flow Cytometry For labeling with the Caltag antibody, *10 6 cells from whole blood were incubated with 3 ml anti-cd2-pe (DakoCytomation, Denmark) prior to use of the Fix and Perm kit (Caltag) according to the manufacturer s instructions. Five microliter of anti-zap-70 Alexa Fluor 488 antibody (test) or mouse IgG1-Alexa Fluor 488 antibody (control) was added with reagent B. Labeling with the Upstate antibody was as previously described: ficolled lymphocytes were placed in 0.5% paraformaldehyde for 30 min, washed with phosphate-buffered saline containing 0.05% Tween and 2% Human serum albumin (PBSAT), and stored in 80% ethanol at 208C for periods between 48 h and 4 weeks. Cells were washed with PBSAT prior to staining, and then incubated with the primary antibody (ZAP-70, clone 2F32, Upstate) or isotype control (Mouse IgG2a, Dako) for 20 min. Subsequent staining was carried out with the secondary antibody (Sheep anti-mouse FITC, BD Biosciences, San Diego, CA) for 15 min and then 1 ml anti-cd2 PE. For each sample 10,000 cells were acquired on a Facscaliber flow cytometer (BD Biosciences). The lymphocytes were initially gated (R1) on the SSC/FSC plot and all subsequent analysis was within at least this gate. On a CD2-PE versus ZAP-70 plot, the tumor population was gated (R2) (Fig. 1a). Four different analysis strategies were then employed. (1) Percentage positivity (PP) was measured (Figs. 1b and 1c) within the gate R1 + R2; a quadrant marker was placed on the isotype control (Fig. 1b) such that the cells in the lower right (LR) quadrant measured <1%. PP was read from the LR quadrant of the test plot (Fig. 1c). (2) The ratio of the geometric means of the R2 populations for test and control plots was measured (Figs. 1d and 1e). (3) The quadrant marker was placed to the left of the T and NK cell population such that cells in the upper left quadrant were <1% and the PP in the tumor population was measured as LR/(LR + LL) (Fig. 1f). (4) The ratio of the geometric mean of the CLL population to that of the T and NK cell population was measured. For both antibodies, results were analyzed by all four methods of analysis by three independent observers. Time of Analysis and Anticoagulant Analysis The effect of time of analysis after collection and choice of anticoagulant on ZAP-70 expression evaluated using the Caltag ratio were analyzed in 29 patient samples. The samples were chosen according to their previously characterized ZAP-70 status, which in all cases was concordant with the IgV H mutational status. Twenty one were ZAP-70 negative and 8 were ZAP-70 positive. Freshly isolated whole blood was collected into either preservative free heparin or EDTA vacutainers. Sample analysis on the day of collection was carried out within the first 12 h after collection and subsequently 24 and 48 h after the initial analysis.

3 ZAP-70 BY FLOW CYTOMETRY 237 FIG. 1. Showing the four different methods of analysis used. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.] IgVH Gene Mutational Status Immunoglobulin variable region genes were sequenced as previously described (3). Briefly, the variable region was amplified by PCR using a mixture of oligonucleotide 5 0 primers specific for each leader sequence of the V H families 1 7, together with a 3 0 constant region primer or a 5 0 framework 1 (FW1) consensus primer, and a 3 0 consensus J H primer. PCR products were purified (Qiagen, West Sussex, UK) and sequenced directly using an automated DNA sequencer (ABI 377/310, Applied Biosystems, Foster City, CA). Nucleotide sequences were aligned to EMBL/GenBank and V-BASE (12), using MacVector 4.0 (International Biotechnologies, New Haven, CT), or Lasergene (DNASTAR, Madison, WI) sequencing analysis software. As previously determined, a cut-off of 98% germ-line homology was taken to define the unmutated subset. Statistical Methods Correlations between different antibody results, and between flow cytometry results and IgVH gene mutational status, were measured with Spearman s correlation coefficient. The cut-offs for ZAP-70 positivity that best discriminated between mutated and unmutated IgVH gene mutational status, and the sensitivity and specificity for each method of analysis were assessed by receiver operating characteristic analysis.

4 238 BEST ET AL. FIG. 2. Interobserver differences, as found by (a) percentage method, in bands of 1% and (b) ratio method, in bands of RESULTS Interobserver Variability is Much Greater for Analysis by Percentage than Analysis by Ratio of Geometric Means For each antibody, the agreement between the three observers was assessed for analysis method (1), hereafter called percentage and method (2), hereafter called the ratio method. Because the results were not normally distributed, the agreement was assessed by grouping results, in bands of 1% for the percentage method and in bands of 0.02 for the ratio method. Results are shown in Figure 2. For both antibodies, interobserver variability was much greater for the percentage method than the ratio method. For example, for the percentage method, the number of observations that differed between the three observers by 1% was 111 (51%) for the Upstate antibody and 83(38%) for the Caltag antibody. For the ratio method, the number of observations which differed by 0.02 were 188 (87%) for the Upstate antibody and 171 (79%) for the Caltag antibody. For both antibodies, 27% of observations differed by more than 3% between the three observers by the percentage method, whereas for the ratio method only 4% (Upstate) and 6% (Caltag) of observations differed by only >0.1. For subsequent analysis, the mean result for all three observers was used. Comparison of the Two Antibodies Shows Significant Correlation Between Antibodies and Both Correlate with IgVH Gene Mutational Status Correlation between the two antibodies was assessed by taking an average of the results from the three observers. Both the percentage and the ratio methods showed significant correlation between the Caltag and Upstate antibodies: r ¼ 0.65 (p < 0.001) for the percentage method and r ¼ 0.70 (P < 0.001) for the ratio method (Spearman s correlation coefficient). Establishment of cut-offs for ZAP-70: Cut-offs for ZAP- 70 positivity for the percentage and ratio methods and both antibodies were determined by optimal concordance with IgVH gene mutational status. For percentages these were 10% for the Upstate antibody and 15% for the Caltag antibody; for ratios these were 1.3 for the Upstate antibody and 1.5 for the Caltag antibody. Sensitivity and specificity for each antibody and method of analysis is shown in Table 1: the Upstate ratio method showed the best concordance with IgVH gene mutational status, with sensitivity of 89% and specificity of 91%; the Caltag ratio method was comparable, with sensitivity of 89% and specificity of 88%; the Caltag percent- Table 1 Comparison of CLL Population to Isotype Control and T and NK Cell Population Method Cut-off (%) Sensitivity (%) Specificity (%) CLL population to isotype control Upstate % Caltag % Upstate, ratio Caltag, ratio CLL population to T and NK cell population Upstate %, T cell gate Upstate %, T cell gate Caltag %, T cell gate Caltag %, T cell gate Upstate ratio, CLL: T cell Caltag ratio, CLL: T cell

5 ZAP-70 BY FLOW CYTOMETRY 239 Table 2 Details of Discordant Cases Pt. No. Caltag % Caltag ratio Upstate % Upstate ratio Vgene % Stability Treatment Discordant result S N Caltag % S N Caltag % S N Caltag % S N Caltag ratio S N Caltag ratio S N Upstate % P Y Caltag % P Y Caltag % P Y Caltag % P Y Caltag % P Y Caltag%, Upstate ratio P Y Upstate % and ratio P Y Caltag % and ratio S N Upstate % and ratio P Y Caltag % and ratio S N Upstate % and ratio P Y Upstate % and ratio P Y ZAP-70+, mutated P Y ZAP-70+, mutated P Y ZAP-70, unmutated S N ZAP-70, unmutated S, stable; P, progressive disease. age method was clearly inferior to the Upstate percentage (70% sensitivity and 88% specificity for Caltag, 85 and 88%, respectively, for Upstate). A third method of analysis of measuring percentage ZAP-70 positivity by setting the quadrant marker on the T and NK cells rather than on an isotype control was assessed. Sensitivity and specificity of this measurement compared with IgVH gene status is shown in Table 1. With the Upstate antibody, the main effect of changing to T and NK cell gating was to increase the number of ZAP-70 negative unmutated cases (from 4 to 13 for a 10% cut-off, and to 18 with a 15% cut-off). Sensitivity and specificity were 59 and 81%, respectively, for a 10% cut-off, and 33 and 93%, respectively, for a 15% cut-off. By contrast with the Caltag antibody, changing to T and NK cell gating resulted in an increase in the number of mutated ZAP-70 positive cases from 5 to 11 if the same 15% cut-off was used, but with a 20% cut-off the mutated ZAP-70 negative cases were unchanged at 5. Sensitivity and specificity were 78 and 62%, respectively, for a 15% cut-off and 74 and 89%, respectively, for a 20% cut-off. Finally, the effect of using a ratio of the geometric mean of the CLL population to that of the T and NK cell population was assessed. Optimal cut-offs were again established by comparison with IgVH gene mutational status and were 0.18 for the Upstate and 0.2 for the Caltag antibody. Sensitivity and specificity were 79 and 81%, respectively, for the Upstate antibody and 88 and 67% for the Caltag antibody. Table 2 shows details of 21 cases in which there was any discordance between one or more of Upstate %, Upstate ratio, Caltag %, Caltag ratio, and IgVH gene mutational status. Clinical details (disease stability and treatment) are given for information but were not used in the initial assessment of which measurement was deemed to be discordant. In most cases, the discordant result was clearly defined. In patients 1 10, one ZAP-70 flow result was discordant: in 7 cases this was the Caltag %, in 2 it was the Caltag ratio, and in 1 it was the Upstate ratio. Patient 11 was discordant by 2 criteria: the Caltag % and the Upstate ratio (borderline at 1.29). In patients 12 17, one ZAP-70 antibody result was discordant by both percentage and ratio: in 2 cases it was the Caltag antibody, and in 4 cases the Upstate antibody. Patients had ZAP-70 flow results that were all concordant, but all 4 cases show ZAP-70 results that were discordant with IgVH gene mutational status. Choice of Anticoagulant and Time of Analysis Are Important in the Ratiometric Analysis of ZAP-70 Expression The time of analysis after collection and choice of anticoagulant were investigated in 29 patient samples. Analysis was carried out on samples collected into heparin or EDTA on the day of collection, and after 24 and 48 h. We observed significant variation in the Caltag ratio over the course of the 48-h time period. In the heparin-preserved samples, there was an average decrease of 13% in the ratio, compared to a 3% increase in the same samples collected into EDTA. As the majority of the patient samples analyzed in a routine laboratory setting will be tested within the first 24 h, we looked at the difference between the day of collection and the 24-h time point. Again we observed a significant difference in the Caltag ratio between the 2 anticoagulants during the first 24 h. The percent change in the ratio between the day of collection and the 24 h

6 240 BEST ET AL. FIG. 3. Graph showing the percent difference in the Caltag control ratio between the day of collection and 24-h time point. test (Fig. 3) suggests that less variation in the Caltag ratio occurs over the course of the first 24 h in samples collected into heparin rather than EDTA. DISCUSSION In most routine flow cytometry laboratories, CLL will be the most commonly diagnosed haematological malignancy and it would be an advantage if the diagnostic panel could also target antigens of prognostic significance. One such antigen is CD38 (13), which shows correlation with IgVH gene mutational status of the order of 70%, and in some (8,9,14), but not all, (15) studies has independent prognostic significance. ZAP-70 is emerging as a useful prognostic marker, particularly in Binet stage A CLL, and shows better correlation with IgVH gene mutations: series using the Upstate 2F3.2 clone show sensitivities and specificities of the order of 85% (5,6,16). This antibody, however, is unconjugated, unstable at 48C, and was not developed for flow cytometry. Moreover, our in-house method of fixation and permeabilization with paraformaldehyde and ethanol on ficolled cells is more suited to batching for research studies than for use as part of a standard panel. The 1E7.2 antibody clone by contrast is increasingly being marketed for flow cytometry: conjugated to Alexa Fluor 488, (Caltag); FITC or PE (BD Biosciences) the antibody is stable at 48C and prepared for internal staining with commonly used commercial kits. It is thus readily incorporated into a standard CLL panel. The principal problem with ZAP-70 measurement by flow cytometry is the relatively weak, homogeneous staining in the positive cases, which means that small changes in placing the marker on control cells result in large changes in the percentage of cells deemed to be positive. We therefore hypothesized that a method of analysis that used instead the ratio of the geometric means of test to isotype control populations would be more reliable, and here demonstrate that in contrast to the percentage method, it is highly reproducible between three independent observers. Moreover when assessing ZAP-70 flow results against IgVH gene mutational status, the Caltag percentage method is clearly inferior to the ratio method, and, among the 21 discordant patients, the Caltag percentage method is most commonly the one which is out of line. We included a third method of analysis, namely using the T and NK cell population as a positive control rather than using an isotype negative control, because one early article on ZAP-70 in CLL used this method (5) and some other groups have adopted it. The effect of changing to T cell gating is different for the two antibody methods: the marked increase in ZAP-70 negative, unmutated cases with the Upstate method is essentially because the intensity of the T and NK cell population is relatively higher with this method; hence, there is likely to be less overlap with a positive CLL population. The T and NK cell population stains less intensely with the Caltag method, the overlap between this and a positive CLL population is greater, and there is relatively less difference between gating using T and NK cells and gating using the isotype control. Using a ratio of the geometric mean of CLL to T and NK cell populations significantly improves concordance with IgVH genes but this is still less good than using the ratio of CLL population to an isotype control. The existence of cases that are truly discordant between ZAP-70 protein expression and IgVH gene mutational status is well described (4,6,7); in some cases for instance confirmatory evidence exists in the form of cdna array expression or quantitative RT-PCR. ZAP-70 negative, unmutated CLL cases often have clinically aggressive disease; the majority of ZAP-70 positive, mutated cases have relatively low levels of mutation, at around the 96 97% range (5,11), and many also have progressive disease. Our patients fall into this category. Several recent studies have addressed the problems of ZAP-70 protein measurement by flow cytometry in small numbers of CLL patients. Gibbs et al. (17), using a method very similar to our Caltag percentage method, found a concordance of 91% with IgVH gene mutational status. Two recent studies favor a comparison of population intensities rather than percentage values. Bakke et al. (18), in a study on 37 CLL patients without reference to IgVH gene mutational status, favor a ratiometric method (CLL to T and NK cell populations) as giving superior correlations between different commercial antibodies. Also without reference to IgVH genes is a study by Kay et al. (19) on 35 CLL patients that advocates the use of calibration beads for measuring intensity of staining of the CLL population. Further progress might be made with the ZAP-70 flow assay if different antibody clones become available: the J antibody (from BD Sciences, San Diego) for example has been proposed as having a higher signal to noise ratio than the 1E7.2 clone (20). During this analysis we have highlighted the fact that the time of analysis after collection and choice of anticoagulant are variables that also require consideration. As ZAP-70 testing is still not widely available to all routine haematology laboratories samples are often sent to other centers involving the sample spending time in transit. Our

7 observations suggest that the Caltag ratio varies significantly with time and between samples collected into heparin or EDTA. Although there is significant variability in the ratio regardless of anticoagulant this data suggests that within the first 24 h after collection, the time frame in which most samples are likely to be analyzed, heparin serves as the best anticoagulant in limiting the percent variation in the ratio. The suggestion that significant variability can occur between samples from the same patient, even in the relatively small number of samples analysed as part of this study, warrants further investigation. In summary, a flow cytometry method for ZAP-70, if it is to be widely used in conjunction with the diagnostic panel, needs to be simple in terms of cell preparation and robust in analysis. The Caltag ratio method described here, using the 1E7.2 clone with the commercial Fix and Perm kit fits these criteria well, and with sensitivity of 89% and specificity of 88% shows a very acceptable concordance with IgVH gene mutational status. LITERATURE CITED 1. Montillo M, Hamblin T, Hallek M, Montserrat E, Morra E. Chronic lymphocytic leukemia: Novel prognostic factors and their relevance for risk-adapted therapeutic strategies. Haematologica 2005;90: DamleRN,WasilT,FaisF,etal. IgVgenemutationstatusandCD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94: Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94: Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia sub-type with unmutated immunoglobulin genes, inferior clinical outcome and distinct gene expression profile. Blood 2003;101: Crespo M, Bosch F, Villamor N, et al. ZAP-70 Expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348: Orchard JA, Ibbotson RE, Davis Z, et al. ZAP-70 expression and prognosis in chronic lymphocytic leukaemia. Lancet 2004;363: ZAP-70 BY FLOW CYTOMETRY Rassenti LZ, Huynh L, Toy TL, et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004;351: Schroers R, Griesinger F, Trumper L, et al. Combined analysis of ZAP-70 and CD38 expression as a predictor of disease progression in B-cell chronic lymphocytic leukemia. Leukemia 2005;19: Del Principe MI, Del Poeta G, Venditti A, et al. Clinical significance of soluble p53 protein in B-cell chronic lymphocytic leukemia. Haematologica 2004;89: Durig J, Nuckel H, Cremer M, et al. ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia. Leukemia 2003;17: Orchard J, Ibbotson R, Best G, Parker A, Oscier D. ZAP-70 in B cell malignancies. Leuk Lymphoma 2005;46: Cook GP, Tomlinson IM. The human immunoglobulin VH repertoire. Immunol Today 1995;16: Mainou-Fowler T, Dignum HM, Proctor SJ, Summerfield GP. The prognostic value of CD38 expression and its quantification in B cell chronic lymphocytic leukemia (B-CLL). Leuk Lymphoma 2004;45: Hamblin TJ, Orchard JA, Ibbotson RE, et al. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002;99: Oscier DG, Gardiner AC, Mould SJ, et al. Multivariate analysis of prognostic factors in CLL: Clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 2002;100: Oppezzo P, Vasconcelos Y, Settegrana C, et al. The LPL/ADAM29 expression ratio is a novel prognosis indicator in chronic lymphocytic leukemia. Blood 2005;106: Gibbs G, Bromidge T, Howe D, Hopkins J, Johnson S. Comparison of flow cytometric methods for the measurement of ZAP-70 expression in a routine diagnostic laboratory. Clin Lab Haematol 2005;27: Bakke AC, Purtzer Z, Leis J, Huang J. A robust ratio metric method for analysis of Zap-70 expression in chronic lymphocytic leukemia (CLL). Cytometry Part B Clin Cytom 2006;70B: 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 Part B Clin Cytom 2006; 70B: Jorgensen J, Jones D, Rajyalakshmi L, Cooper DG. Flow cytometric detection of ZAP-70 in chronic lymphocytic leukaemia: Addition of a novel second monoclonal antibody improves correlation with IgVH mutation status. Blood 2005;106:916a. 21. Corcoran M, Parker A, Orchard J, et al. ZAP-70 methylation status is associated with ZAP-70 expression status in chronic lymphocytic leukemia. Haematologica 2005;90:

Key terms: B-cell chronic lymphocytic leukemia (B-CLL); zeta-chain associated protein 70 (ZAP-70); flow cytometry

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