An Optimized Whole Blood Method for Flow Cytometric Measurement of ZAP-70 Protein Expression in Chronic Lymphocytic Leukemia

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1 Cytometry Part B (Clinical Cytometry) 70B: (2006) An Optimized Whole Blood Method for Flow Cytometric Measurement of ZAP-70 Protein Expression in Chronic Lymphocytic Leukemia T. Vincent Shankey, 1 * Meryl Forman, 1 Paul Scibelli, 1 Jeffrey Cobb, 1 Cecilia M. Smith, 1 Rhonda Mills, 1 Karen Holdaway, 2 Elizabeth Bernal-Hoyos, 3 Mafalda Van Der Heiden, 3 Jan Popma, 4 and Mike Keeney 4 1 Beckman Coulter Incorporated, Miami, Florida 2 Beckman Coulter Pty Ltd, New South Wales, Australia 3 Douglass Hanly Moir Pathology/Sonic Healthcare, Sydney, Australia 4 London Health Sciences Centre, London, Ontario, Canada Background: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. Methods: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4 17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-zap-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. Results: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. Conclusions: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory. q 2006 International Society for Analytical Cytology Key terms: ZAP-70; chronic lymphocytic leukemia; flow cytometry; intracellular antigen staining Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of small lymphocytes, most commonly demonstrating surface markers (CD5 þ, CD19 þ ) consistent with a subset of B-lymphocytes. It is the most common leukemia diagnosed in the US (over 8,000 new cases per year) and Europe, and in the majority of cases, presents in men over 40 years of age. In most individuals, the disease is indolent, with patients surviving for prolonged periods without definitive therapy. In the minority of cases, frequently in younger individuals, the disease rapidly progresses despite aggressive treatments. A number of markers have been proposed as useful in predicting disease course in B-cell CLL, including lymphocyte count doubling time (1), serum b 2 microglobulin *Correspondence to: T. Vincent Shankey, PhD, Advanced Technology Cntr, M/S 22-A01, Beckman Coulter, Inc., SW 147th Ave., Miami, FL 33196, USA. vincent.shankey@coulter.com Received 14 May 2006; Accepted 19 May 2006 Published online in Wiley InterScience ( com). DOI: /cyto.b q 2006 International Society for Analytical Cytology

2 260 SHANKEY ET AL. (2), serum CD23 (3), serum CD44 (4), serum thymidine kinase (5), and a number of cytogenetic markers (6). Studies originally aimed at understanding the level of maturation of B-cell CLLs demonstrated that these leukemias variably expressed restricted sets of immunoglobulin genes, in either the germ-line (indicative of relatively immature) or mutated (more mature) configuration (7). In 2001, using gene-expression arrays and clustering algorithms, Louis Staudt s group published preliminary results from the analysis of 33 CLL patients, which demonstrated that expression of several genes correlated with immunoglobulin heavy chain variable regions (IgV H ) mutational status, and that zeta associated protein, mw 70 kda (ZAP-70) was the most tightly discriminating gene between these two groups (8). The first correlation of ZAP-70 protein expression in B-cell CLL with IgV H mutational status used Western-blot analysis to demonstrate that 12/12 CLL samples with nonmutated IgV H genes expressed ZAP-70 protein, while only 1/10 CLL samples with mutated IgV H genes was positive for ZAP- 70 protein expression (9). In 2003, Staudt s group published the results of gene expression and clustering analysis of 107 B-cell CLL patients, showing tight association of ZAP-70 gene expression with germ-line IgV H configuration, and disease progression (measured by time to treatment after diagnosis) (10). The first published article, using flow cytometry to measure ZAP-70 protein expression, demonstrated that the majority of CLL patients lacking IgV H mutations expressed ZAP-70 protein, while most (not all) CLL patients with IgV H mutations expressed lower percentages of ZAP-70 positive cells (11). Since that time, a number of articles have been published comparing ZAP- 70 expression measured by flow cytometry with either IgV H mutational status, or disease progression (12 25). These studies have employed either whole blood, or more frequently isolated peripheral blood mononuclear cells (PBMC). Most studies used saponin-based permeabilization, and ZAP-70 detection using one of 2 anti- ZAP-70 antibody clones (1E7.2 or 2F3.2). At best, the separation seen in these assays between ZAP-70 positive T-cells and negative B-cells (signal-to-noise, S/N) is in the range of 6 9, and frequently appeared to be as low as 3 4. It is difficult to reproduce or standardize clinical assays with performance in this low S/N range. We have developed a whole blood assay for ZAP-70 expression that includes a unique sample fixation and permeabilization technique. In conjunction with an improved anti-zap-70 antibody conjugate, this provides a 18- to 22-fold difference in the level of ZAP-70 protein measured in normal T-cells (ZAP-70 positive) as compared to normal B-cells (ZAP-70 negative). We have employed this significantly improved flow-cytometry based assay in combination with standardized instrumentation setup and data analysis techniques to determine levels of ZAP-70 protein seen in CLL samples, and have evaluated the intra- and interlaboratory performance of this optimized assay in the analysis of CLL patient material. MATERIALS AND METHODS Whole Blood Samples Normal whole blood samples were obtained from donors following institutionally approved protocols. Whole blood was collected into EDTA containing vacutainer tubes (BD Vacutainer Systems, Franklin Lakes, NJ) and stored at room temperature until used, generally within 4 h of venipuncture, unless otherwise indicated. Whole blood samples from CLL patients obtained at all sites using local institutionally approved protocols were collected into EDTA vacutainers, and processed within 24 h unless otherwise indicated. Antibodies and Reagents EM grade, methanol-free formaldehyde was purchased as a 10% solution (Ultra Pure) from Polysciences (Warrington, PA). Formaldehyde was stored at room temperature in the dark, and used within 6 months of purchase. Triton X-100 (Surfact-Amps-X100, 10% solution packed in sealed ampoules under nitrogen) was purchased from Pierce Biotechnology (Rockford, IL) and was dissolved in PBS (calcium and magnesium free) immediately before use. Commercially available anti-zap-70 antibodies used in these studies included the following clones/conjugates: 2F3.2-FITC (Upstate Cell Signaling Solutions), 136F12- Alexa Fluor 647 (Beckman Coulter, Inc.), 1E7.2-PE (Caltag Laboratories), and 29-FITC (BD Transduction Laboratories). In addition, a custom PE conjugate of the SBZAP clone (Beckman Coulter, Inc.) was prepared by Custom Design Services, Beckman Coulter, Inc. Commercially available whole blood fixation, permeabilization, and RBC lysis kits used in these studies contained a formaldehyde fixative and either saponin-based cell permeabilization. (IntraPrep TM (Beckman Coulter, Inc), Fix&Perm TM (Caltag Laboratories)), or a methanol-based permeabilization system (Phosflow Lyse/Fix Buffer and Perm Buffer II, Becton Dickinson). All kits were used as indicated in information provided by the individual manufacturers. Antibodies to cell surface-specific markers were custom conjugates made by the Custom Design Services, Beckman Coulter, Inc. Antibodies to cell surface markers used included CD5-FITC (clone BL1a/IgG2a), CD19-PECy5 (clone J4.119/IgG1), CD3-PECy7 (clone UCHT1/IgG1), and CD56- PECy7 (clone N901/IgG1). Whole Blood Fixation and Permeabilization for Measurement of Cell Surface Markers Plus Cytoplasmic ZAP-70 The finalized technique used in this study to fix and permeabilize white blood cells and lyse red blood cells is a modification of a recently published technique used to measure the expression of phopsho-specific epitopes (26). For experiments where both cell surface and cytoplasmic (ZAP-70) antigens were stained, 100 ml of whole blood was first incubated with 10 ml of a mixture of anti-cd5-fitc, CD19-PECy5 and CD3 plus CD56-PECy7

3 at room temperature for 20 min. The concentrations of the four different cell surface (CD) antibodies were tittered and used in the mixture at final concentrations that gave optimized cell surface staining for each target epitope in this assay. Following incubation, formaldehyde was added directly to the sample to provide a final concentration of 4.0%. After incubation at room temperature for 10 min, Triton X-100 was added to provide a final concentration of 0.2% (working Triton X-100 solution was preheated to 378C prior to addition, to enhance RBC lysis). Samples were incubated at room temperature for an additional 10 min, after addition of Triton X-100. Samples were washed two times with wash buffer (PBS/ 0.1 mm EDTA/2% BSA/0.1% NaN 3, ph 7.2) before addition of anti-zap-70 antibody and incubated at room temperature for an additional 30 min. The optimal concentration of SBZAP-PE (and all anti-zap-70 antibodies) was determined by titration; the optimal antibody concentration used showed the best S/N for separation of normal T-cells and B-cells (for SBZAP-PE, 0.2 mg/100 ml). Following two washes with wash buffer, samples were resuspended in 1 ml analysis buffer (PBS/0.1% paraformaldehyde/0.1% NaN 3, ph 7.2) and analyzed. Whole blood samples (fixed and permeabilized as indicated earlier) processed for cell surface staining with single-color reagents in separate tubes were used for instrument set-up and standardization. Briefly, four tubes containing 100 ml of whole blood were first incubated separately with anti-cd45-fitc, CD45-PE, CD45-PECy5, and CD45-PECy7 at room temperature for 20 min at the optimal antibody concentrations. Following incubation, samples were fixed, permeabilized, and washed in an identical manner as samples stained for ZAP-70 plus cell surface CD expression (without ZAP-70 antibody addition). Flow Cytometry Samples were analyzed using a Beckman Coulter (Miami, FL) FC 500 flow cytometer in standard singlelaser (488 nm, 20 mw) configuration, with fluorescence emission collected using a 525 nm BP filter for FITC, a 575 nm BP filter for PE, a 675 nm BP filter for PECy5, and a 755 LP filter for PECy7. Forward scatter versus side scatter (both linear) histograms were used with a discriminator on forward scatter to remove most of the RBC debris from the acquisition, and to establish a gating region for subsequent analysis of lymphocyte subpopulations based on surface marker (CD) expression. In routine experiments, listmode files were generated containing between 25 and 50 thousand events in the lymphocyte scatter gate (established using light scatter alone). Listmode files were subsequently analyzed using CXP software (Beckman Coulter, Inc. Miami, FL). Flow cytometer instrument setup was performed using Flow-Set TM fluorospheres (Beckman Coulter, Inc) and the Autosetup application, with target channels optimized for this assay, to give maximum S/N and minimal spectral fluorescence crossover. Compensation was established using OPTIMIZED WHOLE BLOOD METHOD FOR ZAP-70 MEASUREMENT 261 the individual CD45 stained samples. The resulting matrix inversion calculations were determined for compensation coefficients, and used for the analysis of samples stained with anti-zap-70 in conjunction with cell surface markers. RESULTS Whole Blood Optimization for ZAP-70 Staining The effects of varying both time and concentration of the two key process steps, fixation by formaldehyde and lyse/permeabilization by Triton X-100, on light scatter and ZAP-70 expression were examined by a design of experiments (DOE) using either a Box-Behnken (27) or a central composite model (28) and implemented using Design-Expert TM 5 software (Stat-Ease, Inc., Minneapolis, MN). Formaldehyde concentrations from 3% to 5% and Triton X-100 concentrations from to 0.1% to 0.4% were examined in relation to incubation times of 5 15 min or min, respectively. The resulting Box Behnken models were significant for both light scatter (P ¼ ) and ZAP-70 expression (P < ). For the analysis of these experiments, the lower limits were set to enhance light scatter with an optimal ZAP- 70 S/N of >25:1. The data showed that by decreasing the concentration of Triton X-100, the concentration of formaldehyde that could be used widens. Increasing the incubation times for either formaldehyde or Triton X-100 inversely affected the amount of formaldehyde that could be used. A second round of DOE optimization used a central composite design that varied the lyse/permeabilization incubation time (5 15 min) and the concentration of Triton X-100 (from 0.05% to 0.25%, final concentration). On the basis of the previous DOE results, we set the formaldehyde concentration at 4% and the fixation time at 10 min. ZAP-70 and all surface markers were evaluated for S/N, along with RBC lysis and light scatter profiles. The significant models obtained were for ZAP-70, CD19, and CD3. Since the S/N values for CD19 and CD3 were greater than 73 and 47, respectively, we optimized for ZAP-70. Using Triton X-100 at 0.2% final concentration opened the lyse/permeabilization time window to 5 12 min provided an optimal S/N for ZAP-70. ZAP-70 Antibodies and Fixation/Permeabilization Method Comparisons A number of different anti-zap-70 antibody clones (including mouse and rabbit monoclonal antibodies) were screened to determine which clone-conjugate combination provided the best separation, or S/N for ZAP-70 positive normal peripheral blood T-cells and NK-cells from ZAP-70 negative normal B-cells. For the experiments shown in Figure 1, five different commercially available anti-zap-70 clones were evaluated using either a saponin-based permeabilization method (Intra-Prep TM Beckman Coulter, Inc.) or the optimized formaldehyde/ Triton X-100 technique. For each set of experiments, individual anti-zap-70 antibody-conjugates were run at mul-

4 262 SHANKEY ET AL. FIG. 1. Comparative performance of five directly conjugated monoclonal antibodies to ZAP-70 in normal T lymphocytes. Both FA/Saponin (A) and the optimized FA/TX100 (B) fix and perm methods were evaluated to ensure that a particular epitope was not adversely affected by one method alone. All clones tested (except for clone 29-FITC) demonstrated an approximate 2-fold increase in ZAP-70 S/N with the optimized methodology. The SBZAP-PE clone showed highest S/N using either methodology. Red: ZAP-70 events (CD19 þ ) in lymphocyte gate; Blue: ZAP-70 positive T-cells (CD3 þ ); Green: ZAP-70 positive NK-cells (CD56 þ ). tiple final antibody concentrations (from 0.0 to 2.0 mg antibody conjugate per 100 ml final reaction volume) to ensure that optimal S/N was obtained for each antibody. As shown in Figure 1, the antibodies tested showed a S/N based on MFI, comparing ZAP-70 positive T-lymphocytes to ZAP-70 negative B-lymphocytes, that ranged from 2.2 to 11.4 using the saponin-based method, while the identical whole blood donor showed values that ranged from 0 to 21.6 using the optimized formaldehyde/triton X-100 method and these same antibody conjugates. The results shown here are representative of identical experiments performed using six different normal blood donors. In general, the formaldehyde/triton X-100 method increased the S/N from 1.6- to 2.3-fold. As also shown in Figure 1, the two PE conjugates tested showed the best S/N characteristics in the saponin-based method, and both PE conjugates showed a better S/N using the formaldehyde/triton X-100 method. Different Commercially Available Blood Fixation/Permeabilization Techniques and ZAP-70 Expression To determine the impact of different commercially available whole blood fixation and permeabilization techniques on the intensity of ZAP-70 staining in normal T-cells, three different commercially available kits were used in conjunction with the anti-zap-70 antibody conjugate that showed the best S/N in preliminary testing (SBZAP-PE). Whole blood samples from individual normal donors were processed using three different commercially available kits, as per instructions provided by each manufacturer, or processed using the optimized formaldehyde/triton X-100 technique. All samples were stained using an optimal concentration of SBZAP-PE (0.2 mg antibody per 100 ml final staining volume), and all were incubated for 30 min at room temperature. As shown in Figure 2, all three commercial kits gave similar staining patterns for white blood cell populations, with S/N nearly identical for the three commercial fix and perm kits. In contrast, whole blood samples fixed and permeabilized using the optimized formaldehyde/triton X- 100 method showed a 2.0- to 2.5-fold increase in S/N as compared to the formaldehyde/saponin or formaldehyde/diethylene glycol/methanol/methods. As also shown in Figure 2 (bottom panels), the light scatter patterns (ZAP-70 versus side scatter) are similar for all fixation and permeabilization techniques used here. The results shown are representative of identical experiments performed using five different normal blood donors. Stability of ZAP-70 Protein Expression in Normal Whole Blood Samples To investigate the stability of ZAP-70 protein expression in a positive normal blood cell population (normal T-cells), whole blood samples from different normal donors (age 46 55) were studied. Samples were stored at room temperature for up to 48 h before processing. Within 2 h of

5 OPTIMIZED WHOLE BLOOD METHOD FOR ZAP-70 MEASUREMENT 263 FIG. 2. Comparison of the optimized FA/TX100 method (A, left panel) to three commercially available fix and perm kits (B, right panels) using the SBZAP-PE conjugate. The overlay plots of ZAP-70 signal within each gated lymphocyte subset (top panels) show negative expression in CD19 þ B cells (red), positive expression in CD3 þ T cells (blue) and highest expression in CD56 þ NK cells (green). The S/N for ZAP-70 expression is shown for each method using the MFI ratio for negative B cell to positive T cell populations. collection, or after 24 or 48 h of storage at room temperature, aliquots of each whole blood sample were fixed and permeabilized using the optimized formaldehyde/triton X-100 method. After staining for cytoplasmic ZAP-70 expression, samples were washed with sample buffer and resuspended in 1 ml analysis buffer. Each sample was analyzed within 1 h of processing. Samples suspended in analysis buffer were also stored at 48C in the dark, and reanalyzed after an additional 24 or 48 h of storage. FIG. 3. ZAP-70 protein stability in blood specimens through 48 h post-venipuncture, analyzed immediately (A). A decrease of 17% (S/N) is observed within the first 24 h, with an additional loss of 4% observed at 48 h in unfixed specimens. ZAP-70 protein expression decreases logarithmically over time (R 2 ¼ ). (B) Fixed samples held at (4 8)8C for 24 h before analysis, using specimens prepared at 2, 24 and 48 h showed an additional loss of S/N (R 2 ¼ 0.998).

6 264 SHANKEY ET AL. FIG. 4.(A) In normal whole blood, T, B, and NK cell lymphocytes are identified by the surface marker combinations in the two parameter histograms (top panels). ZAP-70 single parameter histograms (bottom panels in A) gated on the lymphocyte subsets give the mean fluorescence intensity (MFI) for each subset and are used to calculate the MFI ratios of T/ normal B and T/NK. These ratios are used as internal reference standards for evaluation of the ZAP-70 expression in the abnormal B cell (CD19 þ CD5 þ ) population. (B) In a whole blood sample taken from a CLL patient, lymphocytes are identified using a light scatter gate and populations identified using combinations of CD19, CD5, and CD3 þ CD56 (upper panels). ZAP-70 protein levels in CD19 þ CD5 þ CLL cells is indexed to that measured in internal negative (CD19 þ CD5 ) and positive (CD3 þ CD5 þ ) control cell populations (bottom panel). As shown in Figure 3 (left panel), whole blood samples stored at room temperature before fixation and permeabilization had a 17% decrease in ZAP-70 expression (MFI of T-cells/MFI of B-cells) after 24 h, with an additional 4% loss after the next 24 h of storage at this temperature. Analysis of individual samples following storage of postfixed (0.1% paraformaldehyde) stained samples demonstrated an additional 4% loss in ZAP-70 signal after 24 h at 48C, and a further 2% loss in signal after 48 h storage (Fig. 3, right panel). The overall regression of the

7 signal loss both before and after sample processing (R 2 ¼ 0.99 in each case) indicates that a similar decay occurs in the normal samples tested, suggesting that a similar biological (or chemical) process is involved in all normal samples tested. Light scatter profiles (both FS and SS) did not change significantly for samples stored for up to 48 h in analysis buffer (PBS containing 0.1% paraformaldehyde) as compared to freshly fixed and processed samples from the same donor. In addition, storage in analysis buffer resulted in no significant changes in fluorescence (MFI) for any of the cell surface markers used here for storage periods of up to 48 h (data not shown). OPTIMIZED WHOLE BLOOD METHOD FOR ZAP-70 MEASUREMENT 265 Whole Blood Sample ZAP-70 Analysis For the analysis of ZAP-70 expression in B-cell CLL samples, we chose to use a combination of surface markers that include CD5-FITC, CD19-PECy5, and a mixture of CD3 plus CD56 both labeled with PECy7. As demonstrated in Figure 4, this combination allows the identification of residual normal B-cells (CD19 þ CD5 ), CLL cells (CD19 þ 5 þ ), T-cells (CD3 þ CD5 þ )andnk-cells(cd56 þ CD5 ). The concentration of the individual antibody conjugates to CD markers used here was optimized to provide the best signal for each surface marker when used with the optimized formaldehyde/triton X-100 method. Results of the analysis of a normal whole blood sample (Fig. 4, top panels) demonstrates the use of the cell surface markers chosen for this assay for subsequent analysis of ZAP-70 levels in normal T (CD3 þ CD5 þ ), B (CD19 þ CD5 ), and NK (CD56 þ CD5 ) populations. Using an initial light scatter gate (FS vs. SS), surface markers are subsequently analyzed on the cells falling within the lymphocyte light scatter gate. As shown here, the target subset of CD19 þ CD5 þ cells found in normal blood donors (representing % of all lymphocytes in normal donors) (29) shows the same level of ZAP-70 protein expression as that seen in CD19 þ CD5 cells. Results of the analysis of a representative B-cell CLL sample is shown in the lower panels in Figure 4. Using an identical gating and analysis strategy as used for normal specimens, the ZAP-70 protein content in CLL cells (CD19 þ CD5 þ ) is compared to that found in the residual B-cells (CD19 þ CD5 ) and T-cells (CD3 þ CD5 þ ) (Fig. 4, bottom panel). As shown here, the separation of ZAP-70 signal in residual T-cells from that seen in residual B-cells is 25 fold (MFI CD3 þ cells/mfi CD19 þ CD5 cells), while the CLL population shows a 3- to 4-fold higher ZAP-70 level than that seen in the residual B-cell population. Using other ZAP-70 antibody clones, or suboptimal fixation/permeabilization technique, this small difference may be undetectable. ZAP-70 Expression Patterns in CLL Samples Using the optimized whole blood assay, samples from a number of CLL samples have been analyzed by two collaborating laboratories, in addition to CLL samples analyzed at Beckman Coulter. Representative data obtained from FIG. 5. Flow cytometric data from three B-cell CLL specimens all analyzed by a single participating laboratory, demonstrating the expression of ZAP-70 protein in CD19 þ /CD5 þ populations. Comparison between conventional method using quadrant analysis to generate percentage positives for each subset (left panels) versus approach evaluating the relative MFI of the ZAP-70 expression in the CLL population to internal negative and positive control cell populations (right panels). one participating lab (E.B-H. and M.V.D.H.) is shown in Figure 5. Three different CLL samples, one negative for ZAP-70 (top panels), one with low levels of ZAP-70 (middle panels), and one with elevated levels of ZAP-70 (bottom panels) are shown. The results of quadrant analysis of CD19 þ vs. ZAP-70 expression of cells in the lymphocyte gates are shown in the left panels in Figure 5. One of the techniques that has been proposed to measure ZAP-70 expression is to index the CLL ZAP-70 expression using normal B-cells (15). Using this type of index (here, using internal CD19 þ CD5 cells), setting quadrants to make 99% of the internal B-cells ZAP-70 negative, these three specimens are 0.8% (sample A), 29.5% (B), and 89.9% (C) positive for ZAP-70 expression (Table 1). Indexing the ZAP-70 level measured in these CLL samples to internal B-cells provides an index of 1.1 (sample A), 2.1 (B), and 5.8 (C) (Table 1). An alternative method has been proposed by Bakke et al. (20) indexing the MFI of CLL cells to internal T-cells. Using this type of approach, the CLL cells in these three specimens have an index of 0.05 (sample A), 0.18 (B), and 0.31 (C) (Table 1). We propose

8 266 SHANKEY ET AL. Table 1 Data from Three B-cell CLL Specimens (A, B, and C) Analyzed in One Laboratory, Measuring the Expression of ZAP-70 Protein in CD19þ/CD5þ Populations (A) ZAP-70 Negative (B) ZAP-70 Low (C) ZAP-70 High ZAP-70 MFI T-cells (CD3 þ CD5 þ ) CD5 B-cells CLL cells (CD5 þ CD19 þ ) NK-cells MFI Ratios T/CD5 B-cells NK/T CLL/CD5 B-cells CLL/T-cells CLL Z index a Quadrant analysis (ZAP-70 % positive) CD5þB Comparison between conventional analysis method using quadrant analysis to generate percentage positives for each subset versus approach evaluating the relative MFI of the ZAP- 70 expression in the CLL population to internal control populations. See Figure 6 for data. a CLL Z index ¼ (CLL MFI B-cell MFI)/(T-cell MFI B-cell MFI) 100. small (total variation less than 10%), similar to the variation shown in Figure 5 (and Table 1) for the results from another collaborating laboratory. Another point shown here is that only one of these eight samples had insufficient numbers of internal CD19 þ CD5 B-cells to use as a negative ZAP-70 control (defined as fewer than 20 CD19 þ CD5 cells). The minimum number of internal negative control cells that are needed, and their distribution (CV) need robust statistical validation with sufficient numbers of CLL samples. At present, we recommend for samples showing few CD19 þ CD5 cells, that a minimum of 100,000 lymphocytes are collected in listmode files. In our experience using this recommendation, ~10% of the CLL samples that were analyzed thus far lack sufficient cells to use as an internal ZAP-70 negative control. For samples lacking sufficient numbers of internal negative control cells, we propose the use of only internal positive cells, or the addition of a here a more sensitive and reproducible method to index the ZAP-70 level in the CLL cells using both the internal negative (CD19 þ CD5 B-cells) and positive (CD3 þ CD5 þ T- cells) control cell populations, using a CLL Z Index, where: CLL Z Index ¼ ðcll MFI B-cell MFIÞ ðt-cell MFI B-cell MFIÞ 100: The advantages of this approach are that it provides internal controls for both positive and negative ZAP-70 expression, and by using MFI values, normalizes the ZAP- 70 expression in each sample to a relative value scale with the end values set by these internal controls. Using this approach, sample A has a CLL Z Index of 0.3 (identical to the internal negative control), B a Z Index of 6.0, and C a Z index of 27.0 (Table 1). In the event that the MFI of the CLL population is less than that of the CD19 þ CD5 cells (due to statistical fluctuations) we propose that the CLL Z index default to zero. Intralaboratory Reproducibility of ZAP-70 Expression To demonstrate the reproducibility of the optimized whole blood assay within a single laboratory using CLL samples from different patients, results from one collaborating laboratory (M.K. and J.P.) are presented in Figure 6. Shown here are the results of the analysis of eight different CLL samples, processed and analyzed on three different days over a 1-month time period. As shown here, the variation in MFI for T-cells and for residual CD19 þ CD5 B-cells was FIG. 6. Intralaboratory variability of optimized whole blood assay of CLL samples. Eight different CLL samples were prepared and analyzed on three different days over a 1 month period by one participating laboratory. CD19 þ CD5 B-cells (green), CD19 þ CD5 þ CLL cells (blue), CD3 þ CD5 þ T-cells (pink), CD56 þ CD5 NK-cells (purple) are shown in overlay plots. Reproducibility of assay is shown by relatively small changes in the positions (MFI) for the internal ZAP-70 negative (red line) and positive (T-cells) (blue line) control populations for different cell preparations.

9 negative control cell or bead population, or the use of the MFI value for the negative population obtained from other samples run at the same time. The last approach requires reproducible MFI values for the negative population, which we have demonstrated in these limited studies. Further testing must be employed to determine which approach is best for samples lacking internal negative cells. Interlaboratory Reproducibility of ZAP-70 Expression To date, over 40 CLL samples from different patients have been analyzed in three different laboratories, all using identical reagents, instrumentation, and set-up protocols. The extent of interlaboratory variability in the data is demonstrated in Figure 7, showing two representative results from each laboratory. The results shown here are strikingly similar to those shown for intralaboratory variability (Fig. 6). Here, the total variation in MFI for T-cells and for residual CD19 þ CD5 B-cells was still <20%, and again, only 10% of the individual CLL samples analyzed lacked sufficient CD19 þ CD5 B-cells to act as an internal negative control for ZAP-70 expression. Another point illustrated here is the sample to sample variability in the ZAP-70 ratio of internal NK (CD56 þ CD5 ) cells to T-cells FIG. 7. Interlaboratory variability of optimized whole blood assay of CLL samples. Six different CLL samples were prepared and analyzed in three different laboratories, showing similar relative positions (and MFI values) for internal ZAP-70 negative and positive control populations. OPTIMIZED WHOLE BLOOD METHOD FOR ZAP-70 MEASUREMENT 267 (CD3 þ CD5 þ ) in CLL samples, suggesting variable levels of ZAP-70 expression in NK, but not in T-cells. DISCUSSION Prognostic markers for CLL have gained considerable attention with ZAP-70 proposed as a marker to predict disease course in CLL. The first published flow cytometry study to measure ZAP-70 protein expression in CLL (11) employed saponin-based permeabilization and an indirect anti-zap-70 staining, which resulted in a poor separation between the ZAP-70 signal in negative versus positive (internal TþNK cells) populations (S/N about 4/1). Most of the subsequently reported studies measuring ZAP-70 have used a combination of quadrant analyses (frequently set using isotype controls) and an arbitrary threshold value for ZAP-70 expression (11,15), beyond which samples are considered positive. The results of the study reported here have established two important issues for the development of a clinically useful assay. First, the optimized cell fixation and permeabilization technique used in conjunction with a unique anti-zap-70-pe conjugate provides a significant improvement in the assay performance as compared with the results shown in previously published studies. Second, by using a carefully standardized assay, including instrumentation set-up, we have demonstrated significant assay reproducibility both within and between laboratories. The choice of antibodies used to measure ZAP-70 protein in clinical samples is important. For cell surface markers, CD5 should be included in order to differentiate ZAP-70 expression in CD19 þ 5 þ cells from that in other cell populations, particularly CD19 þ CD5 cells. Without CD5, the ZAP-70 levels measured in CLL cells will be mixed with varying percentages of other cell populations, broadening the CV of the CLL specific ZAP-70 measurement, and likely shifting the MFI value. In our experience, it is also important to optimize the concentrations of anti- CD antibodies used in the cell surface staining mix. This can have a significant impact on the amount of compensation required in the assay. Regarding the anti-zap-70 used, the antibody providing the best separation (S/N) between positive and negative populations should provide the most robust assay. In our method development experiments, we used a number of different anti-zap-70 clones, which target different epitopes in different parts of the ZAP-70 molecule. Our results show that the S/N characteristics of all (but one) of the clones tested using normal blood was higher using the formaldehyde/triton X-100 technique. Since the technique, which included an alcohol permeabilization step, showed no improvement in S/N, the different epitopes tested here do not appear to require epitope unmasking (26). Previously published studies have utilized a number of different approaches to index or measure ZAP-70 levels in the CLL population. A top down approach, using TþNK cells as an internal positive control population was used in the first published study (11). However, this study did not specify the exact cutoff-point used to mark the bound-

10 268 SHANKEY ET AL. ary for the internal ZAP-70 positive population. Two studies have subsequently demonstrated that relatively small changes in the cursor settings used to define ZAP-70 positive levels can have a profound impact on the assay results (20,30), a problem exacerbated for ZAP-70 assays having poor separation between positive and negative populations (20). Another approach has used a bottom up analysis, setting the ZAP-70 negative boundary using normal B-cells (15). By setting this boundary to make 99.9% of all normal B-cells negative for ZAP-70 expression, the authors were able to set a dichotomous cutoff-point to separate progressors from nonprogressors, and more important, were able to demonstrate that this approach provided a better predictor for disease course than IgV H mutational status in these same patients. Other approaches have used added beads to calculate MESF values for CLL cells (25), or used a ratio metric method (20) comparing the MFI of internal TþNK to that of the CLL cells (similar to one of the approaches shown in Table 1 [CLL/T]), or have used isotype controls to set a negative (background) staining level for the CLL cells (14,24). Isotype controls (or similar negative staining approaches) fail to adequately control for nonspecific binding of the specific antibody used in the assay to the target cell population. The MESF approach offers a useful methodology; the only drawback is the added cost to each test, which we argue is unnecessary with the use of internal control cell populations. In contrast to all of these approaches, the strategy taken in our assay, utilizing both internal positive and negative control cell populations, in conjunction with improved assay methodologies (fixation/permeabilization technique, anti- ZAP-70 antibody conjugate, and highly standardized instrumentation set-up), together provide a more robust and reproducible approach to measure ZAP-70 expression levels in CLL samples. We propose the use of internal CD19 þ CD5 cells in CLL samples as a ZAP-70 negative control population. While the issue of whether these cells or normal peripheral blood B-cells express ZAP-70 is somewhat controversial (31), this issue is irrelevant for this assay. The CD19 þ CD5 cells in CLL samples may express little or no ZAP-70 protein. In either case, flow cytometry is very poor at resolving small differences in expression, particularly at low expression levels. The measured level of ZAP- 70 in these cells must be reproducible, both within and between laboratories, a concept validated in our studies. One issue that our studies has shown is that ~10% of the CLL samples studied thus far lack sufficient CD19 þ CD5 cells to serve as a negative control population. For these types of samples, either an internal T-cell dependent measurement will have to be employed (20), or a negative control cell or bead population will have to be added to the sample, or the MFI value obtained from the negative population from other samples could be used. Further testing must be employed to determine which of these is best for samples lacking internal negative control cells. The one critical issue in any clinical assay is its ability to make useful measurements. As discussed earlier, a number of different methods have been proposed to measure ZAP- 70 levels using flow cytometry, including our proposal to use internal positive and negative control cell populations (CLL Z Index). Whatever the measurement technique applied or the assay methodology used, that entire approach must be employed to establish specific cutoff-points points for disease progression. To make this or any ZAP-70 methodology useful to the clinical community, methodology dependent cutoff-points must be established then validated by interlaboratory studies. At present, the use of cutoffpoints established in one laboratory by another laboratory (that might be using different analytes and/or gating and analysis methods) represents a significant pitfall. If these assays are not appropriately standardized and validated, we run the risk of publishing papers on a marker that in the end will not prove useful to the clinician treating CLL patients. 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