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

Transcription

1 Cytometry Part B (Clinical Cytometry) 70B: (2006) Comparison of Methods for Determining Zeta-Chain Associated Protein 70 (ZAP-70) Expression in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL) Agnieszka Bojarska-Junak, 1 * Krzysztof Giannopoulos, 1 Malgorzata Kowal, 2 Anna Dmoszyńska, 2 and Jacek Roliński 1 1 Department of Clinical Immunology, Medical University of Lublin, Lublin, Poland 2 Department of Hematooncology, Medical University of Lublin, Lublin, Poland Background: Zeta-chain associated protein of 70kDa (ZAP-70) is the most promising surrogate marker for the immunoglobulin heavy chain variable region (IgV H ) mutation status in B-cell chronic lymphocytic leukemia (B-CLL). Crespo et al. proposed the detection of ZAP-70 by flow cytometry. Recently several novel monoclonal antibodies appeared on market. Methods: We compared different staining methods utilizing monoclonal antibodies (moab) against ZAP- 70: anti-zap-70 PE, clone 1E7.2, anti-zap-70 PE, clone 17A/P-ZAP70 directly stained with flourochrome as well as anti-zap-70 antibody, clone 2F3.2 stained with Zenon TM Alexa Fluor W 488 Labeling Kit. Additionally different reagents for permeabilization such as IntraPrep, FIX & PERM, Perm/Wash and 70% ethanol/paraformaldehyde were used to find the most clinically relevant and easy assay to determine ZAP-70 expression in B-CLL. Moreover we compared results of ZAP-70 expression assessed by whole blood protocol with those obtained using-peripheral mononuclear cells isolated from whole blood. Results: Anti-ZAP-70 moab clone 17A/P-ZAP70 gave elevated results for all B-CLL patients as well as healthy controls. Staining with anti-zap-70 moab clone 1E7.2 and anti-zap-70 moab clone 2F3.2 was effective in distinguishing negative and positive cells for ZAP-70 protein expression. Not statistically significant discrepancies of ZAP-70 expression were noticed between different fixation and permeabilization methods. Conclusion: Basing on results obtained during this study we can recommend use of anti-zap-70 PE, clone 1E7.2 moab utilizing whole blood protocol with commercial kits for permeabilization as an easy method that brings compatible results to the original method proposed by Crespo et al. q 2006 International Society for Analytical Cytology Key terms: B-cell chronic lymphocytic leukemia (B-CLL); zeta-chain associated protein 70 (ZAP-70); flow cytometry INTRODUCTION B-cell chronic lymphocytic leukemia (B-CLL) follows an extremely variable course with survival from months to years (1,2,3). Recently, there has been made the breakthrough in the identification of molecular and cellular markers that may predict the progression of the disease. Certain cytogenetic abnormalities such as del 11q, del 17p, chromosome, trisomy 12 or del 13q have been defined as being of prognostic value for B-CLL (4). Hamblin et al. (5) and Damle et al. (6) made an observation indicating that clinical behavior of B-CLL is related to the mutational status of immunoglobulin heavy chain variable region (IgV H ) genes. Clinically, CLL cells with unmutated IgV H genes are associated with a poorer prognosis than those with mutations. Although, there is a general agreement that the mutational status of Ig genes constitutes a very strong prognostic factor for B-CLL, routine analysis is difficult, costly and inaccessible for most clinical diagnos- Grant sponsor: State Committee for Scientific Research; Grant numbers: 2 PO5B , 2 PO5B *Correspondence to: Agnieszka Bojarska-Junak, Clinical Immunology Department, Medical University of Lublin, ul. Jaczewskiego 8, Lublin, Poland. agnieszkajunak@poczta.onet.pl Received 2 May 2006; Accepted 16 May 2006 Published online in Wiley InterScience ( com). DOI: /cyto.b q 2006 International Society for Analytical Cytology

2 294 BOJARSKA-JUNAK ET AL. tic laboratories. The identification of surrogate markers for mutational status is therefore an important purpose in B-CLL research. The expression of CD38 correlates with IgVH mutation status but tends to change during disease progression (7,8). However, other studies noted the discrepancy of CD38 expression and IgVH mutational status up to 30% (9). Considering recently published studies (8,10,11), zetachain associated protein of 70kDa (ZAP- 70) is the most promising surrogate marker of-the IgV H mutation status. ZAP-70 is a signaling molecule from Syk/ZAP-70 protein kinase family normally utilized by T and NK cell and abnormously expressed in B-CLL cells (12,13). Crespo et al. (8) proposed the method of ZAP-70 detection by a flow cytometric analysis. The ZAP-70 protein expression on B-CLL detected by flow-cytometric method, correlated with IgV H mutational status, disease progression, and survival (8) revealed even better prognostic value compared to IgV H mutation (11). Although individual reports have mentioned satisfactory results for the detection of ZAP- 70, no methodological consensus has been achieved among laboratories. In our previous publication (14) we presented a modification of the method described originally by Crespo et al. (8) for flow cytometric examination of the ZAP-70 protein expression. The crucial step of intracellular staining of the ZAP antigen followed by FITC-conjugated goat antimouse IgG secondary staining was replaced by a one-step staining method. The anti-zap-70 monoclonal antibody (moab) was labeled separately by Zenon TM Alexa Fluor Mouse IgG 2a Labeling Kit enabling separate identification of surface and cytoplasmatic molecules (14). In the current paper, we compared different clones of moab against ZAP-70 with direct and indirect staining and ZAP-70 expression utilizing whole blood protocol versus mononuclear cells isolated from peripheral blood. Additionally the use of different reagents for permeabilization was investigated. We wanted to find the most clinically relevant and easy assay to determine ZAP-70 expression in B-CLL. MATERIALS AND METHODS Patients and Samples Peripheral blood samples were obtained from 46 newly diagnosed, untreated patients with B-CLL. The diagnosis of B-CLL was made on the basis of clinical examination, as well as morphological and immunological criteria. The median age was 65 years (ranging from 46 83) including 16 female and 30 male patients. According to the Binet classification, 22 patients were at stage A, 16 patients were at stage B, 8 patients were at stage C (Table 1). All peripheral blood samples were collected into heparinized tubes or EDTA tubes (6 samples) and immediately processed. Peripheral blood from 12 healthy persons (6 female and 6 male) was also analyzed. Their median age was 60 years (range: years). The Local Ethical Committee approved this study. Reagents Anti-ZAP-70 antibody, clone 2F3.2 (mouse monoclonal IgG 2a ) was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-ZAP-70 PE, clone 1E7.2 (Isotype: IgG 1 ) was obtained from Caltag Laboratories (Burlingame, USA). Anti-ZAP-70 PE, clone 17A/P-ZAP70 (Isotype IgG 1 ) was obtained from BD Biosciences (San Diego, USA), and Zenon TM Alexa Fluor Mouse IgG 2a Labeling Kit from Molecular Probes (Eugene, Oregon, USA). Monoclonal antibodies (moab) anti-cd19 PE (Clone HIB19, Isotype IgG 1 ) and CD19 FITC (Clone HIB19, Isotype IgG 1 ) were obtained from BD PharMingen (San Diego, USA). CD5 PE-Cy5 (TC, TRI-Color 1 ) (Clone CD5-5D7, Isotype IgG 1 ) was purchased from Caltag Laboratories (Burlingame, USA). Mouse IgG 1 PE isotypic control was obtained from Caltag Laboratories. Mouse IgG 2a isotypic control was from BD Biosciences Pharmingen (San Diego, USA). IntraPrep TM was purchased from Immunotech S.A. (Marseille, France). Cytofix/Cytoperm TM solution and Perm/ Wash TM buffer were obtained from BD PharMingen (San Diego, USA). Fix & Perm 1 Cell Permeabilization Kit was purchased from Caltag Laboratories (Burlingame, USA). Lymphoprep was purchased from Nycomed (Oslo, Norway). Cell Preparation Each fresh blood sample was stained within 1 2 hours and analyzed immediately upon the completion of a staining process. Either whole blood cells were used or peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway) for 25 minutes at 400 g at room temperature. Interphase cells were collected, washed twice and resuspended in phosphate-buffered saline (PBS). Staining for Cell Surface Antigens One million of PBMC (110 6 cells in 100ml) or 100 ml of whole blood were stained with monoclonal antibodies against the cell-surface marker: CD19 PE, CD19 FITC and CD5 PE-Cy5 (20 minutes at room temperature). Following membrane staining, fixation/permeabilization procedures were performed. ZAP-70 Protein Staining For each sample, intracellular ZAP-70 protein staining was performed with the PE-conjugated anti-zap-70 (clone 1E7.2) moab from Caltag (Antibody 1), anti-zap- 70 (clone 2F3.2) antibody solution from Upstate Biotechnology labeled by Zenon TM Alexa Fluor Mouse IgG 2a Labeling Kit (Antibody 2) and PE-conjugated anti- ZAP-70 antibody (clone 17A/P-ZAP70) from BD Biosciences (Antibody 3). Each staining experiment was performed on PBMC as well as on fresh whole blood cells lysed subsequently during a permeabilization step. Four methodes of fixation and permeabilization were used in the study.

3 COMPARISON OF METHODS FOR DETERMINING ZAP-70 EXPRESSION IN PATIENTS WITH B-CLL Zenon Complex Formation. For ZAP-70 immunocytochemical reaction, the concentrations of 0.5 mg, 1 mg and 2 mg of anti-zap-70 moab (clone 2F3.2) per cells were tested. The best results were obtained with 1 mg moab, and this concentration was used in further 295 experiments. Five ml of Zenon mouse IgG labeling reagent (Component A) was mixed with 1mg of the anti- ZAP-70 antibody (clone 2F3.2) solution or appropriate isotypic control. The mixture was incubated for 5 minutes at room temperature. Then 5 ml of the Zenon Table 1 Main biologic and clinical characteristics of the B-CLL patients. The percentage of ZAP-70 positive CD19 + /CD5 + cells obtained with anti-zap-70 antibody, clone 2F3.2 labeled by Zenon Alexa Fluor 488 and with anti- ZAP-70 PE clone 1E7.2. If more than 20% of all B-CLL cells were positive based on the the cursor position, the patients were considered positive for ZAP-70 expression 1A Patient No# Antibody 1 Zenon Alexa Fluor 488 Clone 2F3.2 [%] Antibody 2 Anti-ZAP-70 PE Clone 1E7.2 [%] Antibody 3 Anti ZAP-70 PE Clone 17A/P-ZAP-70 [%] Age Sex Binet stage M A M A M A M B M B F A F A M B F A M C M A M A M A M A F B F A M A M C F A M B M B M B F C M C M C F B M A F A M C M A M B M C M B F A F B M B M B M B M A M B F C M B F A F A F A F A ZAP-70 positive patients n ¼ 22 (47.82%) n ¼ 25 (54.34%) n ¼ 14 (100%) Median Min-Max p value/the Wilcoxon non-parametric test/ p ¼ The Spearman rank correlation coefficient R ¼ p ¼

4 296 BOJARSKA-JUNAK ET AL. Table 1 Main biologic and clinical characteristics of the B-CLL patients. The percentage of ZAP-70 positive CD19 + /CD5 + cells obtained with anti-zap-70 antibody, clone 2F3.2 labeled by Zenon Alexa Fluor 488 and with anti- ZAP-70 PE clone 1E7.2. If more than 20% of all B-CLL cells were positive based on the the cursor position, the patients were considered positive for ZAP-70 expression (continued) 1B Healthy Persons Antibody 1 Zenon Alexa Fluor 488 Clone 2F3.2 [%] Antibody 2 Anti-ZAP-70 PE Clone 1E7.2 [%] Antibody 3 Anti ZAP-70 PE Clone 17A/P-ZAP-70 [%] Age Sex M F M M M F F M M F F F Median 2.1 1, Min-Max p value/the Wilcoxon non-parametric test/ p ¼ The Spearman rank correlation coefficient R ¼ p ¼ blocking reagent (Component B) was added to the reaction mixture. The solution was incubated for additional 5 minutes and applied to the cell sample during intracellular staining. Fixation/Permeablization Methods and Intracellular Staining IntraPrep. Following membrane staining, the cells were resuspended in 100 ml of Reagent 1 (fixation) for 15 minutes at room temperature, and then washed in PBS. After washing, permeabilization was induced with Reagent 2 and simultaneously remaining erythrocytes were lysed. The cells were incubated in 100 ml of Reagent 2 and with one of the three anti-zap-70 moab (anti-zap- 70 PE moab clone 1E7.2, anti-zap-70 PE moab clone 17A/P-ZAP70 or anti-zap-70 moab clone 2F3.2, labeled by Zenon TM Alexa Fluor Mouse IgG 2a Labeling Kit) or appropriate isotypic control for 30 minutes. Prolonged permeabilization/incubation time was chosen to allow optimal labeling without altered morphology or cell loss in comparison with a 15-min incubation as recommended by the manufacturer. Perm/Wash. Following membrane staining, the cells were fixed in 200 ml of Cytofix/Cytoperm solution for 15 minutes at 48C. Fixed cells were then permeabilized by washing twice in the Perm/Wash buffer. Cells were incubated for 30 minutes in 100 ml of the Perm/Wash buffer containing PE- conjugated anti-zap-70 antibody from Caltag, anti-zap-70 PE from BD Bioscience or anti-zap-70 antibody solution labeled by Zenon TM Alexa Fluor Mouse IgG 2a Labeling Kit, or appropriate negative control. FIX & PERM. Following the cell surface markers staining, 100ml of fixation solution (Reagent A) was added. Tubes were incubated for 15 minutes in the dark at room temperature. After washing, the cells were incubated (30 minutes at room temperature) with 100ml of permeabilization medium (Reagent B) and the appropriate volume of each anti-zap-70 antibody or corresponding isotypic control. 70% ethanol/paraformaldehyde. As an alternative, a noncommercial method was used. Following membrane staining, the cells were fixed in cold 1% paraformaldehyde solution in PBS for 15 minutes at room temperature and permeabilized with 70% ethanol for at least 1 hour at 208C. After fixation and permeabilization, the cells were washed with PBS. After centrifugation, the supernatant was discarded. Next, either isotypic control or one of the three ZAP-70 antibodies was added to the appropriate sample tubes and incubated for 30 minutes in the dark at room temperature. Flow Cytometric Analysis After intracellular staining, samples were washed twice with PBS and promptly analyzed by flow cytometry using FIG. 1. The dot plots show representative data from one B-CLL patient, illustrating the method for the identification of CD19 + /CD5 + leukaemic cells expressing ZAP-70 following three color staining. Lymphocyte population was gated (R1) using FSC and SSC plot. Next, the R1 gated events were analysed for CD19 + /CD5 + staining and positive cells were selected (region R2 and R3) (1a, 2a). We used dot plots of CD19 PE versus CD5 TC (1a) or CD19 FITC versus CD5 TC (2a). Final dot plots: CD19 PE/isotype control (1b) CD19 PE/ZAP-70 Alexa Fluor (1c), CD19 FITC/isotype control (2b) and CD19 FITC/ZAP-70 PE (2c) were established by combined gating of events using R1 and R2. The numbers in the upper right quadrant on the dot plots 1c and 2c represent the percentage of CD19+/CD5+/ZAP-70+ cells. The dot plots 1d, 1e, 2d and 2e show ZAP-70 expression after all lymphocyte gatings (R1). Upper left quadrants of dot plots 1e and 2e show ZAP-70 expression by CD19- lymphocytes (generally T and NK cells), which provided a useful positive control for antibody activity. [Color figure can be viewed in the online issue, which is available at

5 COMPARISON OF METHODS FOR DETERMINING ZAP-70 EXPRESSION IN PATIENTS WITH B-CLL 297 FIG. 1.

6 298 BOJARSKA-JUNAK ET AL. a Becton Dickinson FACSCalibur instrument equipped with 488-nm argon laser. Five data parameters were acquired and stored: linear forward and side scatter (FSC, SSC), green-fluorescence (FL1), orange-fluorescence (FL2) and red-fluorescence (FL3). An acquisition gate was established based on FSC and SSC that included mononuclear cells and excluded dead cells and debris events were acquired and analyzed using CellQuest Software. The percentage of positive cells was measured from a cutoff set using isotype-matched nonspecific control antibody. Statistical Analysis Statistical comparisons were done for paired samples. The Wilcoxon non-parametric test was used to analyze the statistical significance of the results. The Spearman rank correlation coefficient was used for correlation testing. We used Statistica 5.0 PL software for all statistical procedures. Differences were considered as statistically significant when the p-value was < RESULTS Comparison of Different moab Against ZAP-70 In our first assessment, for each sample, intracellular ZAP-70 protein staining was performed with PE-conjugated anti-zap-70 antibody clone 1E7.2 (Antibody 1) and anti-zap-70 antibody solution clone 2F3.2, labeled by Zenon TM Alexa Fluor Mouse IgG 2a Labeling Kit (Antibody 2). The results of the ZAP-70 staining using the two methods are shown in Tables 1A and 1B. Each method was effective in distinguishing negative and positive cells for the ZAP-70 protein expression. The sequence of analytic steps for the identification of CD19 + /CD5 + leukaemic cells expressing ZAP-70 is presented in Figure 1. In all samples, ZAP-70 was positive in CD19 cells (generally T and NK cells), which were considered as an internal standard (Figure 1). No significant differences were observed in the percentage of B-CLL cells staining with Antibody 1 ( %) or with Antibody 2 ( %). ZAP-70 PE clone 1E7.2 (Antibody 1) and ZAP-70 clone 2F3.2 labeled by Zenon procedure (Antibody 2) were compared based on the percentage of ZAP-70 positive CD19+/CD5+ cells. Likewise, the Spearman correlation test showed a direct correlation between the results obtained using both antibodies (R ¼ 0.903). Antibody 3, clone 17A/P-ZAP70, gave elevated results for all B-CLL patients (mean %, median %, range %) (Table 1A). For that antibody, the percentage of CD19+/CD5+ cells expressing ZAP-70 was always greater than 20% and discrimination between ZAP-70 positive and ZAP-70 negative cases of B- CLL patients was not possible. Likewise, in B CD19+ cells from healthy volunteers the antibody gave elevated results that were equal to CD19 cells (T-cells and NK cells) (mean %, median %, range %). Conversely, in the samples from healthy persons the proportion of CD19+ cells expressing ZAP-70 was % (median 2.1%; range %) for ZAP-70 clone 2F3.2 and % (median 1.51%; range %) for ZAP-70 clone 1E7.2 were not significantly different (Table 1B). Fixation and Permaebilization In this study, samples were fixed and permeabilized in one of four ways: IntraPrep, Cytofix/Cytoperm and Perm/ Wash, FIX&PERM and as an alternative fixation and permeabilization was performed with paraformaldehyde and ice cold ethanol. Each commercial method of fixation and permeabilzation was effective in distinguish negative and positive cells for ZAP-70 expression. Variability of ZAP-70 expression was noticed, but, the observed discrepancy was not statistically significant (Table 2). In addition, our results showed a decreased percentage of CD19 negative cells in samples treated with paraformaldehyde/ethanol when compared with samples treated by commercial methods (data not shown). Moreover, after paraformaldehyde/ethanol procedure, greater discrepancy was observed when ZAP-70 antibody clone 1E7.2 was used. Permeabilization of whole blood samples from 14 patients showed no significant difference in the percentage of B-CLL cells positive for ZAP-70 in comparison to PBMC staining (Table 3). In spite of a lower expression of ZAP-70 in whole blood, patients remained in the same group of ZAP-70 positive or ZAP-70 negative. The results of direct ZAP-70 staining of whole blood (without prior PBMC separation) obtained using IntraPrep were compatible to those obtained by Perm/Wash or FIX&PERM permeabilization. However, fixation and permeabilization method utilizing alcohol and paraformaldehyde of whole blood samples produced the results incompatible with those obtained with other fixation methods (data not shown). DISCUSSION Nowadays, many clinical trail for CLL patients aim to individualize treatment according to the prognostic factors. The prognostic factors are well defined, starting from the clinical staging systems (15,16). Other categories include the performance status, age, pattern of bone marrow involvement, serum levels of b 2 -microglobulin and soluble CD23 molecule, P53 expression, somatic mutations of the V H immunoglobulin genes and different genomic aberrations (17,18,19). However, not all of prognostic factors with proven clinical significance are easily accessible in routine laboratories. There is enthusiasm that the ZAP-70 expression assay might become available to routine cytometric laboratories (20). Several flow cytometric methods exist to detect ZAP-70 protein expression. However, no consensus has been made as to which antibody clone, type of fixation and permeabilization or even the cell preparation to be used. Recently, new clones of anti-zap-70 antibodies conjugated with different fluorochromes have appeared on the market. In our previous study (14), the Zenon labeling of ZAP-70 antibody was used. Zenon methodology was previously used for immunocytochemical detection

7 COMPARISON OF METHODS FOR DETERMINING ZAP-70 EXPRESSION IN PATIENTS WITH B-CLL 299 Table 2 The percentage of ZAP-70 positive CD19 + /CD5 + cells obtained with IntraPrep, Perm/Wash, FIX&PERM or 70% ethanol using peripheral blood mononuclear cells (anti ZAP-70 clone 1E7.2 (C) and anti-zap-70 clone 2F3.2 (Z) were used) IntraPrep [A] Cytofix/Cytoperm Perm/Wash [B] FIX & PERM [C] Paraformaldehyde and Ethanol [D] 1. Z Z Z Z Z Z Z Z C C C C C C C C C Median Min-Max p value /The Wilcoxon non-parametric test/ The Spearman rank correlation coefficient A vs. B: p ¼ A vs. C: p ¼ A vs. D p ¼ B vs. C: p ¼ B vs. D: p ¼ C vs. D: p ¼ A vs. B: R ¼1 A vs. C: R ¼ 1 A vs. D: R ¼ B vs. D: R ¼ B vs. C: R ¼ 1 C vs. D: R ¼ of caspase activation (27). In this procedure, individual cells can be sequentially analyzed for the expression of surface markers and intracellular ZAP-70 protein. In the present paper we compared three anti-zap-70 antibodies: anti-zap-70 PE clone 1E7.2 and anti-zap-70 PE clone 17A/P-ZAP70 with anti-zap-70 clone 2F3.2. We found that anti-zap-70 antibody clone 1E7.2, directly conjugated with PE, provides fully comparable results with clone 2F3.2. This method is simple and enables one to directly distinguish between surface and intracellular molecules, as secondary anti- mouse antibody is not needed and surface molecules are stained at first. Here, we reported a significant concordance with the results obtained using moab clone 2F3.2 with the Zenon labeling Kit. This also clarifies the concerns about the use of a moab conjugated with PE, since this fluorochrome requires a correct compensation with FITC, which might lower the sensivity, as FITC conjugated antibody binds highly expressed antigens (like CD19 or CD5). Recently Gibbs et al. (21) reported that clone 1E7.2 directly conjugated with the Alexa Fluor 488 flourochrome give results that significantly correlate with clone 2F3.2 from Upstate and with IgV H sequence homology (21). However, analyzing those data, individual discordance in 3 cases out of a total 33 using Fix & Perm Caltag permeabilization Kit was noted and even higher values of 15 cases using paraformaldehyde/methanol Table 3 The percentage of ZAP-70 positive CD19 + /CD5 + cells obtained from peripheral blood mononuclear cells (PBMC) or whole blood (anti ZAP-70 clone 1E7.2 (C) and anti-zap-70 clone 2F3.2 (Z) were used) PBMC Whole Blood 1.Z Z Z Z Z Z Z Median p value p ¼ C C C C C C C Median p value p ¼ TOTAL Median Min-Max p value / The Wilcoxon non-parametric test / p ¼ 0.729

8 300 BOJARSKA-JUNAK ET AL. permeabilization protocol when compared with ZAP positivity between IgV H groups (using 20% cut-off). In our report we found no such discordance regardless the permeabilization method used. Moreover, using the paraformaldehyde/ethanol permeabilization method, discordance was observed in one patient when compared with the Cytofix commercial Kit. The values tend to be lower using this method and results vary from acquisition to acquisition. Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. Fixation/permeabilization methods used for the detection of intracellular antigens often lead to the destruction of cellular morphology and surface immunoreactivity, properties useful in flow cytometry for the characterization of cells in heterogeneous populations. The paraformaldehyde/ethanol method brings incompatible results from whole blood and requires purification of peripheral blood lymphocytes prior to fixation (22,23). The selection EDTA as an anticoagulant for blood collection allows both hematologic and flow cytometric estimation using the same sample. In our opinion, the whole blood samples collected into heparinized tubes gave less discrepancies in the percentage of ZAP-70 positive leukemic cells (data not shown). This might be the reason why Gibbs et al. (21), in their recent paper, experienced some discordant results using similar clones of anti-zap-70 antibodies. Nicholson et al. (24) found that lymphocyte separation of blood collected in heparin was better than blood collected in EDTA. For the first time, we show that the results of the ZAP- 70 expression using PE-conjugated anti- ZAP-70 clone 1E7.2 are significantly compatible with data obtained utilizing anti-zap-70 clone 2F3.2 pure mouse antibody labeled by ZENON labeling Kit, showing no difference with original staining method proposed by Crespo et al. (8). Moreover, we conclude that this simple assay might be accessed from whole blood samples with Fix/Perm permeabilization reagents or other commercial permeabilization procedures. We found that whole blood method is a reliable method for ZAP-70 protein staining in flow cytometry. The whole blood lysis method is generally recommended because it does not employ density gradients centrifugation, which may lead to differential loss of specific subpopulations, assuming that all leukocyte subsets are equally tolerant to the lysis method used. In the present report, we found that density gradient centrifugation resulted in a small reduction of the CD19- cells number and an increase in the percentage of CD19+ cells compared to the fresh whole blood lysis technique (data not show). This small increase in the percentage of CD19+ B cells might be due to the loss of CD19- cells (generally T and NK cells) during the gradient centrifugation procedure. Previous reports have shown the change in the percentage of B cells, after density gradient centrifugation of normal human blood (25,26). In our study, the magnitude of the change in the median percentage was small and may hold no significance. Because density gradient centrifugation might selectively result in the loss of a lymphocyte subpopulation, the use of a whole blood technique for ZAP-70 staining is highly recommended. Furthermore, following IntraPrep, PermWash or Fix&Perm incubation, it was possible to stain for ZAP-70 during permeabilization step. The standardization of a ZAP-70 staining procedure remains a real challenge. In the diagnostic laboratory, the choice of particular technique is increasingly influenced by versatility, rapidity or cost. In this context, FIX&PERM, PermWash or IntraPrep are easy to perform and require the same amount of time to complete. However, sometimes the cost of commercial procedures may be prohibitive for routine diagnostic use. Regarding this economic aspect, we also used inexpensive paraformaldehyde/ethanol procedure recommended for detecting intracellular antigens. However, we observed some limitations such as cell loss. In conclusion, our data indicate that FIX&PERM, Perm- Wash or IntraPrep and anti-zap-70 PE clone 1E7.2 or clone 2F3.2 might be used to evaluate the expression of ZAP-70 using whole blood. The methodology of staining with clone 1E7.2 is simple, as this moab is directly conjugated with a fluorochrome. 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