Flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia: correlation with immunocytochemistry and Western blot analysis.
Objective.--To describe our FC method for detecting ZAP-70 in CLL and assess whether this assay is useful in estimating the ZAP-70 protein level in CLL cells.
Design.--ZAP-70 expression was assessed by FC in 45 consecutive newly diagnosed CLL patients, and the results were correlated with those of immunocytochemistry and Western blot analysis.
Results.--With >25% ZAP-70-positive B cells as the cutoff, the FC results had a perfect concordance with those of immunocytochemistry (39/39, 100%) and Western blot analysis (7/7, 100%). The use of autofluorescence controls was found to be superior to other alternatives. Overall, 19 (42%) of 45 cases were ZAP-70 positive in our series. Since only 7 cases (16%) had >20% to 30% ZAP-70-positive B cells, the cutoff of >25% readily separated CLL into positive and negative groups in most cases. ZAP-70 positivity was significantly associated with atypical morphology but not other laboratory parameters evaluated.
Conclusions.--With proper specimen processing and the use of directly fluorescence-conjugated anti-ZAP-70 antibody, one can readily incorporate ZAP-70 into the routine FC study panel for CLL. Our data suggest that FC is a rapid and useful method to estimate the ZAP-70 protein expression level in CLL.
Chronic lymphocytic leukemia (CLL) is a B-cell chronic lymphoproliferative disorder characterized by proliferation of small B cells expressing the CD5 antigen. (1,2) Although the disease is indolent in many patients, who require no immediate therapeutic intervention, a subset of CLL patients has evidence of disease progression (ie, advancing clinical stage) shortly after the initial diagnosis. (3) Previous studies have attempted to establish prognostic markers that identify patients with aggressive disease; features that have been associated with aggressiveness in CLL include chromosomal abnormalities (ie, trisomy 12 and del11q23), CD38 expression, loss of p53, and the absence of somatic mutations in the variable regions of the immunoglobulin heavy chain (IgH) gene. (4-7) More recent studies have shown that the zeta-associated protein of 70 kd (ZAP-70) is aberrantly expressed in a subset of CLL, and its expression correlates with an unmutated status of the IgH gene and predicts worse clinical outcome in early-stage CLL patients. (8-11)
ZAP-70 is a cytoplasmic tyrosine kinase of the Syk family; it normally plays an important role in mediating activation signals for the T-cell receptor. (12) ZAP-70 is expressed at a low level in normal B cells; little is known with regard to its function in normal B cells, although previous studies showed that ZAP-70 can reconstitute Syk-deficient B cells, indicating that ZAP-70 may be associated with enhanced signal transduction via the B-cell receptor complex, similar to its function in T cells. In keeping with this concept, expression of ZAP-70 in CLL has been associated with increased B-cell receptor signaling. (13)
In view of the prognostic significance of ZAP-70 in CLL, its detection has become important in the initial workup of newly diagnosed CLL cases. Flow cytometry (FC) is probably the method of choice since most cases of CLL are diagnosed by immunophenotyping using FC, and it would be logical to incorporate ZAP-70 assessment into the immunophenotyping study panel used to diagnose CLL. We identified 5 publications (9-11,14,15) that have described FC methods and protocols to detect ZAP-70, including the first published article, by Crespo et al, (10) describing the clinical significance of ZAP-70. Three of these 5 studies used the same commercially available, indirectly conjugated anti-ZAP-70 antibody. Nevertheless, for clinical diagnostic laboratories, it would be more suitable and efficient to assess ZAP-70 using directly conjugated anti-ZAP-70 antibodies. In this regard, we identified only 2 studies reporting the use of an anti-ZAP-70 antibody that is directly conjugated to the dye. (11,15)
In this report, we describe our FC method to detect ZAP-70 that uses a commercially available, directly Alexa 647-conjugated anti-ZAP-70 antibody. We aimed to determine whether the FC method is useful in readily distinguishing CLL cases with relatively high ZAP-70 levels from those with relatively low ZAP-70 levels. To achieve this objective, we correlated the FC results with those of immunocytochemistry and Western blot analysis, 2 other semiquantitative protein assays that can be performed in a routine clinical laboratory. Lastly, we assessed whether ZAP-70 is associated with other pathologic parameters, such as atypical morphology and/or unusual CLL immunophenotype (ie, positivity for CD22, CD79b, and FMC7).
MATERIALS AND METHODS
Peripheral blood samples in sodium heparin were collected from 45 consecutive patients presenting with absolute lymphocytosis (>5.0 x [10.sup.3]/[micro]L). All of these patients were subsequently confirmed to have CLL according to the criteria described by the World Health Organization (2) based on the morphologic and immunophenotypic features analyzed by FC. All samples were received and analyzed at the Cross Cancer Institute, Edmonton, Alberta, between November 2004 and April 2005. This study has been reviewed and approved by the institutional ethics committee.
Antibodies and Flow Cytometry
Anti-ZAP-70 (clone 136F12) rabbit monoclonal antibody (Cell Signaling, Beverly, Mass) directly conjugated with Alexa 647 was used for FC. Other antibodies used in the immunophenotyping of CLL included fluorescein isothiocyanate-conjugated anti-IgG1, -[lambda], -CD20, -CD22, and -FMC7; phycoerythrin-conjugated anti-IgG2a, -[kappa], -CD5, -CD79b, -CD23, and -CD3; peridinin chlorophyll protein-conjugated anti-IgG1 and -CD45; and allophycocyanin-conjugated anti-IgG1, -CD19, and -CD3 (BD Biosciences, Mississauga, Ontario). A flow cytometer FACSCalibur (BD Biosciences) was used, and CaliBRITE 3 and CaliBRITE allophycocyanin beads (BD Biosciences) were used to validate the instrument performance. Flow-set fluorospheres and the allophycocyanin (675/ 633) setup kit (Beckman-Coulter, Mississauga, Ontario) were run daily to standardize the FACSCalibur.
For ZAP-70 staining, 2 tubes on each peripheral blood sample were initially stained using fluorescein isothiocyanate-conjugated anti-CD20 (B cells), phycoerythrin-conjugated anti-CD3 (T cells), and peridinin chlorophyll protein-conjugated anti-CD45 for 15 minutes at room temperature in the dark. Subsequently, these cells were fixed using Intraprep Reagent 1 (Beckman-Coulter) for 20 minutes at room temperature. The cells were washed with 1x phosphate-buffered saline ([PBS], pH = 7.2), and the supernatant was aspirated without disturbing the cell pellet. Permeation was achieved by adding Intraprep Reagent 2 (Beckman-Coulter) to the cell samples and incubating for 10 minutes at room temperature. After washing with PBS three times, 10 [micro]L of Alexa 647-conjugated anti-ZAP-70 antibody was added to one tube (test sample) but not the other tube (negative control). Both tubes were incubated and mixed for 30 minutes at room temperature. Cell samples were washed once with PBS and resuspended in PBS for flow cytometric analysis using the FACSCalibur and Cellquest Pro software (Becton Dickinson). CD45 versus SSC gating strategy was used to isolate the lymphoid cell population. ZAP-70 expression was calculated as the percentage of CD20-positive cells expressing ZAP-70 above the baseline threshold ([less than or equal to] 2%) set by the autofluorescence controls. CD22, FMC7, and CD79a were considered positive when >20% of CD20-positive cells expressed the antigen.
Peripheral blood samples from 39 of the 45 patients were available for immunocytochemical analysis. Mononuclear cells were separated using Ficoll-Paque (Amersham, Uppsala, Sweden), washed in PBS (pH = 7.2), and subjected to cytospin slide preparation. All cytospin preparations were made on the same day the specimens were received in the laboratory. The slides were air dried overnight and fixed in acetone for 15 minutes. Anti-ZAP-70 monoclonal antibody (clone 2F3.2, Upstate, Charlottesville, Va), 1 in 50 dilution, was applied to the slides for 30 minutes at room temperature in a humidified chamber. After washing with PBS, the slides were incubated with anti-mouse IgG labeled to a polymer with peroxidase enzyme (EnVision Plus, Dako-Cytomation, Carpinteria, Calif) for 30 min at room temperature. After another washing with PBS, cytospins were incubated with 3,3'-diaminobenzidine/[H.sub.2][O.sub.2] (DAB; Dako) for color development, and hematoxylin was used as a counterstain. For each case, a score of 0, 1, or 2 was given by 2 independent observers (G.W.S., R.L.). Granulocytes and monocytes were excluded from scoring, as were intensely staining lymphocytes (presumed to be T cells). The following scoring criteria were used: 0 = no definitive staining in the majority (ie, >50%) of lymphocytes, 1 = definite but weak staining in the majority of lymphocytes, 2 = strong staining in the majority of lymphocytes. Evaluation of the immunostaining was performed without knowing the FC or Western blot analysis results. Cases with discrepancies in scoring were reviewed by both observers on a multiheaded microscope. Atypical CLL cell morphology was defined as >15% of CLL cells with cleaved nuclear contours and/or plasmacytoid features. (1)
Cell Sorting and Western Blot Analysis
Peripheral blood samples from 7 patients were available for cell sorting and Western blot analysis. Whole blood (heparin or ethylene-diaminetetraacetic acid) was purified over Ficoll Paque PLUS to isolate peripheral blood mononuclear cells. The peripheral blood mononuclear cells were retrieved, washed with 1x PBS, and stained with fluorescein isothiocyanate-conjugated anti-CD19 and phycoerythrin-conjugated anti-CD5, along with matched isotype antibodies. The double-positive (CD19-positive/ CD5-positive) cells were high-speed sorted using an EPICS ALTRA (Beckman-Coulter) and collected into fetal bovine serum. Purities of >95% were achieved on reanalysis of the sorted population. The cells were washed with 1x PBS and centrifuged at 1500 rpm for 10 min. The supernatant was aspirated and the cell pellet was frozen at -80[degrees]C until further testing. Western blot analysis of sorted CD5-positive/CD19-positive lymphocytes was performed using standard techniques, as described previously. (16) For each case, a score of 0, 1, or 2 was assigned by 2 independent observers based on the chemiluminescence intensity (0 = no definitive band, 1 = faint band, 2 = definitive and strong band).
Statistical differences between the 2 groups (ZAP-70-positive group versus ZAP-70-negative group) were determined by the Student t test or Fisher exact test (2-tailed) where appropriate.
We first determined the ZAP-70 expression in the CD3-positive T cells and CD20-positive B cells from 10 healthy individuals. Using our autofluorescence controls, a median of 58% of CD3-positive cells were positive for ZAP-70. All 10 individuals had <20% of CD20-positive B cells that were ZAP-70 positive, and we observed a median of 14% of normal CD20-positive cells that were ZAP-70 positive in these healthy individuals.
The study group comprised 45 consecutive, newly diagnosed CLL patients. As summarized in Table 1, there were 28 men and 17 women, with a median age of 67 years (range, 38-88 years). Their lymphocyte count ranged from 4.6 to 40.0 x [10.sup.3]/[micro]L.
By FC, a wide range of ZAP-70 positivity was obtained (1%-90%). To establish the optimal cutoff for ZAP-70 positivity, we correlated the FC results with those obtained from immunocytochemistry and Western blot analysis. As summarized in Table 2, we found that >25% of ZAP-70 positivity within the CD20-positive cell population correlated the best with results from the other 2 methods. Of the total 45 cases evaluated by FC (illustrated in Figure 1, a through f), 39 (87%) cases were assessed by immunocytochemistry (Figure 2, a through c). The concordance between the 2 observers in scoring the immunocytochemically stained slides was 92% (36/39 cases). All 23 cases scored 0 by immunocytochemistry were [less than or equal to] 25% ZAP-70-positive cells by FC. In contrast, all 16 cases scored 1 or 2 by immunocytochemistry were >25% ZAP-70-positive cells by FC. Using Western blot analysis of CD5-positive/ CD19-positive cells sorted by FC, we evaluated 7 cases, as illustrated in Figure 3 and summarized in Table 2. The concordance between the 2 observers in scoring the Western blot analysis results was 100%. All 3 cases scored as 0 or 1 by Western blot had [less than or equal to] 25% ZAP-70-positive cells by FC and had a score of 0 by immunocytochemistry. In contrast, all 4 cases scored as 2 by Western blot analysis had >25% ZAP-70-positive cells by FC and had a score of 1 or 2 by immunocytochemistry. The use of other percentages of ZAP-70-positive cells as the cutoff resulted in increased discrepancies among results obtained with the 3 methods.
[FIGURES 1-3 OMITTED]
While we used the isotype antibody as the negative control to set the baseline threshold for detecting ZAP-70, one alternative approach would be to use the lowest expression level of ZAP-70 in benign T cells, as used by 3 other studies. (9,10,14) As illustrated in Figure 1, a through f, this approach was limited by 2 factors. First, the lowest level of ZAP-70 in T cells was not constant, as evidenced by comparing case 1 (Figure 1, a and d) and case 3 (Figure 1, c and f). The second limitation is exemplified by case 2 (Figure 1, b and e), in which there was no well-defined T-cell population detectable. One other alternative would be to apply a constant baseline threshold to all cases, but we found that the use of this alternative method resulted in higher discordance in the results generated by FC, immunocytochemistry, and Western blot analysis.
Using >25% ZAP-70 positivity as a cutoff, 19 cases (42%) were considered ZAP-70 positive. Figure 4, a, illustrates the raw FC data and Figure 4, b, illustrates the grouping of CLL patients based on 0% to 20%, >20% to 30%, or >30% ZAP-70-positive cells. Most of the 45 cases examined by FC had a percentage of ZAP-70-positive cells falling into the [less than or equal to] 20% or >30% categories; only 7 cases (16%) had a percentage of ZAP-70-positive cells falling into the >20% to 30% range. Thus, most cases (84%) had a percentage of ZAP-70-positive B cells that was relatively remote from the 25% cutoff, and CLL cases can be readily separated into 2 distinct subsets-ZAP-70 positive or ZAP-70 negative.
[FIGURE 4 OMITTED]
We also determined the correlation of ZAP-70 positivity with various clinical and pathologic parameters. As summarized in Table 1, atypical morphologic features, as defined by >15% of CLL cells with cleaved nuclear contours and/or plasmacytoid features, (1) were found only in the ZAP-70-positive group (P = .001). The determination of atypical features was made at the time of the initial morphologic examination of the peripheral blood smear, without knowledge of any FC data. No significant differences were found between ZAP-70 positivity and other parameters, including the absolute lymphocyte count at presentation, patient age at presentation, or the presence of an unusual CLL immunophenotype (including expression of FMC7, CD22, or CD79b).
Chronic lymphocytic leukemia is a hematologic malignancy 2with a variable clinical course. Recent reports suggest that a number of prognostic markers may be helpful in stratifying patients into different risk groups. Currently, the strongest predictor of aggressive disease is the absence of somatic hypermutation within the variable regions of the IgH gene. Nevertheless, the ability to directly detect these changes is not readily available in most clinical laboratories. Recently, it has been shown that ZAP-70 serves as a surrogate marker for the IgH mutational status in CLL. (10) ZAP-70 by itself also has been reported as an independent prognostic marker for CLL patients. (9) Thus, detection of ZAP-70 has become an important part of the diagnostic workup of CLL. In the literature, there are only a small number of studies that have reported the methods for detecting ZAP-70 in CLL. As discussed, we identified 5 reports that have described FC methods; the use of directly conjugated anti-ZAP-70 antibodies, which is more suitable for clinical laboratories, was described in only 2 reports.
In this report, we describe our experience with the ZAP-70 detection assay in CLL. To determine whether FC detection of ZAP-70 is truly useful in distinguishing CLL cases with high ZAP-70 expression from those with low ZAP-70 expression, we correlated our FC data with those obtained using immunocytochemistry and Western blot analysis. By FC, we found that ZAP-70 expression is relatively weak in the vast majority of cases, represented by relatively small shifts above the baseline threshold set by the autofluorescence control. Thus, compared with the expression of most of the other markers commonly used to diagnose CLL (such as CD20, CD23, and FMC7), detection of ZAP-70 is overall not a robust assay. However, we found that a cutoff of >25% by FC correlated well with results obtained by immunocytochemistry and Western blot analysis. These findings support the concept that the relatively small shifts in the ZAP-70 expression level detectable by FC truly reflect higher levels of ZAP-70 protein expression. Thus, our findings suggest that the use of the direct fluorescence-conjugated anti-ZAP-70 antibody is a rapid and useful method to estimate the ZAP-70 protein level in CLL, and this method can be readily incorporated into the routine FC study panel. Of note, the overall frequency of ZAP-70-positive cases in our series (ie, 19/45 [42%]) is comparable to the results reported by Rassenti et al (11) (36% with >30% ZAP-70-positive B cells), and Crespo et al (10) and Durig et al (9) (57% and 51%, respectively, with >20% ZAP-70-positive B cells).
Several technical aspects of this assay deserve further mention. The first point is related to the use of a commercially available kit for cell fixation and permeabilization. During the optimization testing, we altered the manufacturer's recommended methodology to achieve optimal staining results. These changes included an increase in the fixation time (Intraprep Reagent 1) from 15 minutes to 20 minutes, an increase in the permeabilization time (Intraprep Reagent 2) from 5 minutes to 10 minutes, an introduction of 3 washes with PBS between permeabilization and staining, and a prolongation of the incubation time with anti-ZAP antibody from 15 minutes to 30 minutes. In our experience, these modifications are critical to the success of ZAP-70 staining; a dramatic decrease in staining was observed when the time durations for these steps were shortened. The second point is related to the use of antibody dilution. We performed dilution studies and we found that 10 [micro] L (rather than 20 [micro]L as recommended by the manufacturer) also produced excellent results. The third point is related to the setting of the baseline threshold. As described above, we attempted several approaches, including the use of the autofluorescence level, the lowest ZAP-70 expression level of benign T cells and natural killer cells, and a constant baseline threshold. We found that the use of the autofluorescence background level is superior to the other methods. The use of the ZAP-70 level in T cells and natural killer cells is problematic because ZAP-70 is known to be upregulated in these cells in some CLL patients. (17) We also found that in cases where CLL cells predominate, the relatively small number of T cells and natural killer cells made the definition of the cutoff difficult to establish. Lastly, we have evaluated 2 other commercially available, directly conjugated anti-ZAP-70 antibodies, with one from Santa Cruz (clone G4, sc-17760; Santa Cruz, Calif) and one from BD Biotechnology (clone 29; Mississauga, Ontario), and we found that our fixation and permeabilization protocols worked well with the antibody from BD Biotechnology but not with that from Santa Cruz. Nevertheless, further testing is required to fully compare the reactivities of these antibodies.
The finding that only 16% of the cases had >20% to 30% ZAP-70-positive cells suggests that the distinction between ZAP-70-negative and ZAP-70-positive cases can be readily achieved in most cases; the percentages of ZAP-70-positive cells are likely to be sufficiently remote from the "cutoff" value of 25%. Cases with >20% to 30% ZAP-70-positive cells are perceivably problematic, since any random errors occurring during fixation, staining, and/or gating may likely lead to different conclusions regarding the ZAP-70 expression status. Fortunately, only a minority of cases are in this "gray zone." These cases were also relatively uncommon in one other previous study, as Rassenti et al (11) found only 10% of their cases possessing >20% to 30% ZAP-70-positive cells. Currently, in our laboratory, we report the result as "borderline positive" when the ZAP-70-positive cells are >25% to 30%. Long-term follow-up data will be needed to verify the clinical behavior of these borderline cases.
Although immunocytochemistry was primarily used for correlation, our findings suggest that the use of immunocytochemistry applied to cytospin preparations may be a feasible alternative to FC analysis. The advantages of using immunocytochemistry are that it is relatively inexpensive and the assay can be batched to better utilize the laboratory resources. With our once-a-week immunocytochemistry staining schedule, we found no appreciable differences in the quality of the immunocytochemical stain that may be related to the age of cytospin preparations. However, immunocytochemistry applied to cytospin preparations carries certain limitations. In our study, strict criteria (as described above) were required to properly score each case. Difficulties may be encountered when the proportion of leukemic cells is relatively small (ie, <50% of the total white cells), although this scenario is relatively infrequent, and there were only 3 (of 39) cases in our series with leukemic cells <50%.
In this study, we assessed ZAP-70 expression in the CD20-positive cell population by FC. In view of the relatively weak expression of CD20 in CLL cells, CD19 theoretically would be a better choice for setting up the B-cell gate. After the submission of this manuscript, we performed a parallel study in which we compared the proportions of ZAP-70-positive cells in 22 CLL cases using either the CD20-gating strategy or the CD19-gating strategy. Of these 22 cases tested, we identified only 1 case in which discrepancy occurred. The relative rarity of the discrepancy is probably due to the fact that CLL is the predominant cell population in the blood in most patients, who were all newly diagnosed in this study. We also determined whether removal of CD3-positive cells by gating will lead to significantly different results. When we reanalyzed our data derived from the 45 cases described in this report, we found 2 cases in which the conclusions were different as a result of the exclusion of CD3-positive cells by gating. Of note, in both of these 2 cases, the proportion of ZAP-70-positive B cells was in the range of >20% to 30%. As discussed above, the chance of inaccurately labeling the ZAP-70 status for these 2 cases is intrinsically high. With the advent and introduction of more sophisticated flow cytometry instruments in the near future, we believe that there are ample opportunities to improve the gating strategy for this assay.
In conclusion, we found that, with the use of directly fluorescence-conjugated anti-ZAP-70 antibody, one can readily incorporate this marker into the routine FC study panel for CLL. We found that ZAP-70 expression, as defined by >25% positive B cells, correlated with the results from immunocytochemistry and Western blots, and these findings support that ZAP-70 positivity detected by FC genuinely reflects a higher level of ZAP-70 protein expression in these cells. Because of the relatively small number of samples, future studies using newer FC instruments, larger sample size, and better gating strategies are warranted to improve the reliability of this assay.
Accepted for publication July 7, 2006.
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Graham W. Slack, MD; Juanita Wizniak, BSc; Laith Dabbagh, MSc; Xinzhe Shi, MD; Pascal Gelebart, PhD; Raymond Lai, MD, PhD
From the Department of Laboratory Medicine and Pathology, University of Alberta (Drs Slack, Shi, Gelebart, and Lai); and the Department of Laboratory Medicine and Pathology, Cross Cancer Institute (Ms Wizniak, Mr Dabbagh, and Drs Gelebart and Lai), Edmonton, Alberta.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Raymond Lai, MD, PhD, Cross Cancer Institute and University of Alberta Department of Laboratory Medicine and Pathology, 11560 University Ave, Edmonton, Alberta, Canada T6G 1Z2 (e-mail: firstname.lastname@example.org).
Table 1. Clinical and Pathologic Characteristics of 45 Newly Diagnosed Chronic Lymphocytic Leukemia (CLL) Patients Based on ZAP-70 Expression ZAP-70 Positive, n = 19 Age, y 67 [+ or -] 15 Sex 68% male Absolute lymphocyte count at presentation, /[micro]L 17.7 [+ or -] 16.8 x [10.sup.3] Platelet count, /[micro]L 196 [+ or -] 86 x [10.sup.3] Hemoglobin, g/dL 13.5 [+ or -] 3.1 Atypical morphology, No./total (%) 7/19 (37) Unusual CLL immunophenotype CD22 positive, No./total (%) 3/19 (16) FMC7 positive, No./total (%) 0/19 (0) CD79b positive, No./total (%) 7/19 (37) ZAP-70 Negative, n = 26 Age, y 65 [+ or -] 12 Sex 60% male Absolute lymphocyte count at presentation, /[micro]L 16.4 [+ or -] 11.0 x [10.sup.3] Platelet count, /[micro]L 168 [+ or -] 71 x [10.sup.3] Hemoglobin, g/dL 13.1 [+ or -] 3.4 Atypical morphology, No./total (%) 0 Unusual CLL immunophenotype CD22 positive, No./total (%) 4/26 (15) FMC7 positive, No./total (%) 2/26 (8) CD79b positive, No./total (%) 12/26 (46) P Value Age, y .32 Sex .75 Absolute lymphocyte count at presentation, /[micro]L .38 Platelet count, /[micro]L .14 Hemoglobin, g/dL .35 Atypical morphology, No./total (%) .001 Unusual CLL immunophenotype CD22 positive, No./total (%) >.99 FMC7 positive, No./total (%) .50 CD79b positive, No./total (%) .56 Table 2. Correlation of ZAP-70 Expression Detected by Flow Cytometry, Immunocytochemistry (IC), and Western Blot (WB) Analysis No. of Studied Cases Flow Cytometry Flow Cytometry ZAP-70 [less than Score ZAP-70 > 25% or equal to] 25% IC, 0 0 23 IC, 1 6 0 IC, 2 10 0 16 23 Total 39 WB, 0 or 1 0 3 WB, 2 4 0 4 3 Total 7