Evaluation of a multicolor, single tube technique for the determination of lymphocyte subpopulations

To evaluate the fully automated FACSCantoTM software, we compared lymphocyte subpopulation counts obtained using a 3-color FACSCaliburTM-CELLQuest and a 6-color FACSCantoTMFACSCanto software techniques. High correlations were observed between data obtained with these techniques. Our study indicates that FACSCanto clinical software is accurate and sensitive in single platform lymphocyte immunophenotyping. Key terms Lymphocyte immunophenotyping, FACSCanto software, multicolor analysis. Recently, flow cytometry has become the principal technique for the diagnosis and monitoring of cellular immunodeficiencies (Helbert et al., 2000). New instruments, methodologies and reagents have been developed to improve accuracy, precision and standardization in lymphocyte subpopulations count (LSc) (Storie et al., 2004). Clinical laboratories are now routinely using single platform assays with a lyse-no wash methodology which reduces inter-laboratory variability (Gratama et al., 2002) but requires a complex analysis with a multiple gating strategy (Lambert et al., 2006; Ashman et al., 2007). The recent availability of new multicolor flow cytometers has allowed to perform absolute LSc in a single tube, including B lymphocytes and natural killer (NK) cells, with a reduction in cost and time. In 2004, the 6-color flow cytometer FACSCantoTM provided with FACSCanto and FACSDivaTM softwares (BD, Biosciences, San Josè, CA) was approved by the Food and Drug Administration (FDA) for in vitro LSc. Lambert et al. evaluated the performance of this cytometer using a lyse-no wash, single platform technique and a manual gating analysis. Similarly, Ashman et al. compared the performance of FACSDiva and FACSCanto software using a lyse/wash double platform technique. They concluded that FACSDiva software should be preferred for 6-color LSc, as it offers a better manual gating performance. The aim of our study was to evaluate the performance of the completely auto-gating new FACSCanto clinical software using the 6-color single tube reagent (TBNKTM) in a lyse-no wash and single platform technique (6-ST). We compared LSc obtained with this approach with those obtained with 3-color multiple tube technique (3-MT) and FACSCaliburTM, routinely used in our clinical diagnostic procedures. AC CE PT ED on O cber 3, 2017 by gest httpcvi.asm .rg/ D ow nladed fom LSc were measured in EDTA whole blood samples of 40 consecutive subjects referred to our laboratory (14 adults; 22 children; 4 neonates) and 10 adult healthy blood donors. Patients included 17 HIV infections, 6 kidney transplant recipients and 17 undiagnosed cases. Samples were processed within 8 hours of blood draw, using in parallel the 3-MT (Schenker et al., 1993) with the following monoclonal antibodies: CD3 FITC (clone SK7), CD4 PE (clone SK3), CD8 PE (clone RPA-T8), CD3/CD16+CD56 FITC/PE bundle, CD19 PE (clone SJ25C1) and CD45 PerCP (clone 2D1); and the 6-ST (trucount tubeTM) with the TBNKTM kit that combines CD3 FITC (clone SK7), CD16 PE (clone B73.1), CD56 PE (clone NCAM16.2), CD45 PerCP-Cy-5.5 (clone 2D1), CD4 PE-Cy7 (clone SK3), CD19 APC (clone SJ25C1) and CD8 APC-Cy7 (clone SK1), according to manufacturer’s recommendations. Full blood counts were performed for all samples using a Sysmex XE 2100TM hematology analyzer. Tubes of 3-MT were acquired both with FACSCaliburTM (CELLQuestTM software v. 3.3; fig.1 panel A) and with FACSCanto (FACSDivaTM software v. 4.0.2; fig. 1 panel B). Data analysis employed CD45/SSC gating on lymphocytes and then dot plot evaluations of two antigen coexpression on CD45+ lymphocyte events (CD3/CD4; CD3/CD8; CD19; CD3/CD16+56. Each absolute LSc was determined multiplying percentages obtained in the analysis by the hematology analyzer lymphocyte counts. Samples processed with 6-ST were acquired and analyzed with FACSCantoTM and FACSCanto clinical software (v 2.0; fig.1 panel C). This software automatically calculates the absolute LSc using the internal reference beads contained in truCount tube. In order to exclude operator variability (Schnizlein-Bick et al., 2000) all the analyses were performed by the same operator. Correlations of LSc obtained with 3-MT and 6-ST were assessed using Pearson correlation coefficient and linear regression analysis. Wilcoxon signed rank test was used to assess differences between the two methods. All statistical analyses were performed with GraphPad PrismTM 4.0 statistical software. Instruments’ calibrations were highly stable over a period of three months. The automatic compensation setting included in FACSCanto software passed in all performances with CVs always below 20%. Excellent correlations (r > 0.94) were observed between all LSc obtained with 3-MT and 6-ST and between 3-MT performed with both CELLQuestTM and FACSDivaTM software (r > 0.98). As reported in table I, 6-ST produced higher and statistically different counts in all LSc as compared to 3-MT. AC CE PT E on O cber 3, 2017 by gest httpcvi.asm .rg/ D ow nladed fom The 6-ST lymphosums (sum of percentages of CD3+, CD19+ and NK cells) were significantly higher than those obtained with 3-MT (99.5 ± 0.32% vs. 94.1 ± 3.7%; p < 0.0001). No difference was found between 3-MT lymphosums obtained with CELLQuestTM and FACSDivaTM software (94.1 ± 3.7% vs. 93.7 ± 3.7%; p=0.084). We also recalculated LSc using the 6-ST percentages and the hematology analyzer lymphocyte counts, in a double platform approach (Table II). Only CD3+/CD4+, CD3+/CD8+ and CD19+ cell counts remained statistically different using 6-ST and 3-MT performed with FACSCantoTM flow cytometer. FACSCanto files were also reanalyzed with FACSDiva and results were not statistically different from those obtained with FACSCanto software (data not shown). In our study we evaluated the performance of similar methodology and instrumentation used by Lambert et al. but employing the new fully automated FACSCanto clinical software for data analysis, in parallel with our internal reference method. FACSCanto software, although evaluated by Ashman et al. less accurate than FACSDiva software in LSc, seems to be precise and sensitive as FACSDiva software, when we employed it in a lyse-no wash, single platform assay using TBNK reagent. Differently from FACSDiva, FACSCanto software automatically compensates all 6 fluorescences and creates all gates needed to evaluate lymphocyte subsets. Higher LSc obtained with 6-ST could be due to different platform approaches, instrument sensitivity and brighter fluorochromes used in the TBNKTM reagent. Since we did not find any statistical variation comparing data obtained using only a double platform technique, we could exclude that differences were due to the platform approach used. At the same time, a minimally different instrument sensitivity, related only to CD19+ cell counts, was found when 3-MT was performed on either FACSCalibur or FACSCanto flow cytometers. A unique manual gating strategy employed by a single operator for both software (CELLQuest and FACSDiva) probably contributed to obtain similar counts. Higher CD4+, CD8+ and CD19+ cell counts found with the 6-ST seem to be related to the introduction of brighter fluorochromes in TBNK reagent, i.e. CD4 PECy7, CD8 APC and CD19 APC-Cy7, instead of PE conjugated antibodies in 3-MT. It is important to notice that, in spite of these differences in LSc, no modifications in clinical reports were required. TBNK reagent in combination with FACSCanto software allows LSc in shorter time and with a smaller blood sample, a crucial requirement in testing newborns or pediatrics, as in our setting. The higher lymphosum obtained with 6-ST supports a higher accuracy in characterizing a larger amount of lymphocytes. Certainly, the use of complex instruments with multicolor analysis in which every fluorochrome has to be accurately compensated, especially in a lyse-no wash technique, could be problematic for a junior operator. The implementation of new fully automated software can overcome these problems. AC CE PT ED on O cber 3, 2017 by gest httpcvi.asm .rg/ D ow nladed fom In conclusion, our study indicates that FACSCanto clinical software is accurate, sensitive and easy to apply for LSc using the TBNK reagent and a single platform approach in clinical routine setting.