Dual-platform Flow Cytometry Research Articles

A-075 Development and Validation of Image-based Cell Counter Microscanner C3 for CD34-positive Hematopoietic Stem Cell Enumeration

Abstract Background Accurate measurement of CD34-positive hematopoietic stem cells (HSCs) is critical for successful peripheral blood stem cell transplantation (PBSCT). Flow cytometry is by far the gold standard for measuring CD34-positive stem cells. However, its complicated procedure requires time, cost and highly-trained operators, thus the necessity of simpler and cheaper alternative method is emerging. We have developed a new image-based cell counter, Microscanner C3 (MSC3, Biozentech, South Korea) as an alternative method for enumeration of CD34-positive HSCs and evaluated its performance. Methods 100 µl of apheresis product was stained with CD45-BB515 and CD34-PE antibodies (Becton Dickinson, USA) at room temperature in the dark. Red blood cell lysis with 500 µl of OptiLyze C solution (Beckman Coulter, USA) was followed by centrifugation. After the removal of supernatants, the sample was resuspended with 500 µL PBS, which then infused to filter paper imbedded BZ-1 microchip (Biozentech, South Korea). Thirty-fields of the loaded microchip was auto-scanned by MSC3 and its dedicated software analyzed the images and yielded CD34-positive cell fraction out of CD45-positive cells as percentage. Results of thirty-six samples were compared to that of FacsLyric (Beckton Dickinson, USA) flow cytometry and accuracy and imprecision (pentaplicate measurements for three samples), repeatability (two experimenters performed triplicate measurements for three samples each), linearity (triplicate measurements for five levels), limit of detection (hexaplicate measurements for five levels) and specificity (using whole blood specimen from three donors with monoclonal gammopathy, thrombocytosis or monocytosis) was evaluated. Results CV% values of inaccuracy, imprecision and repeatability did not exceed 15%. The method was found to be linear across the measuring range. LOD was evaluated to be 7 cells/µL and no background interference was detected. The difference between the measured fraction of MSC3 and FacsLyric was within allowable limit, with an R-value greater than 0.98. Conclusion We developed a reliable method for quantifying CD34-positive HSCs based on image analysis. The limitation involved in MSC3 applications in CD34-positive HSCs is that, like dual-platform flow cytometry, the results should be combined with the results of conventional hematology analyzer in order to define absolute HSC numbers. However, with its simpler and faster procedures, MSC3 exhibited a great potential to be used as an alternative to conventional flow cytometry in CD34-positive HSCs enumeration and other applications as well.

Normal Values of T, B and NK Lymphocyte Subpopulations in Peripheral Blood of Healthy Cuban Adults.

INTRODUCTION Quantification of lymphocyte subpopulations is useful for evaluating immune response in states of health and disease, including immunodeficiencies, autoimmunity, infections and cancer. Studies have found that concentrations and proportions of different cell subpopulations vary with geographic location, age, sex and ethnicity. Knowing the normal values of these cells and their variation in healthy populations will contribute to improved clinical practice and scientific research. OBJECTIVE Estimate normal absolute concentrations and percentages of the most abundant lymphocyte subpopulations in peripheral blood and their relation to sex and age. METHODS A cross-sectional analysis was conducted in 129 healthy adults, 61 men and 68 women aged 18-80 years; 89 aged <50 years and 40 ≥50 years. We included individuals who agreed to participate by written informed consent. Exclusion criteria were chronic disease, or use of tobacco, alcohol or medications that can alter immune system cell numbers and functions. Through dual platform flow cytometry, we determined absolute and percentage values for T lymphocyte subsets CD3+, CD3+/CD4+T, CD3+/CD8+T, CD19+ B cells and CD3-/CD56+ natural killer cells in peripheral blood, using an 8-color flow cytometer. We estimated medians and the 2.5 and 97.5 percentiles and calculated the Pearson correlation coefficient to evaluate associations. Significance tests were also used to compare groups. The significance threshold was p = 0.05 in all cases. RESULTS Ranges of absolute values and percentages (%) were: total lymphocytes: 1200-3475 cells/μL (20.2-49.3); CD3+ T cells: 880-2623 cells/μL (56.5-84.7); CD3+/CD4+ T cells: 479-1792 cells/μL (30.3-55.7); CD3+/CD8+ T cells: 248-1101 cells/μL (13.2-42.9); CD19+ B cells: 114-1491 cells/μL (5.4-49.5); CD3-/CD56+ natural killer cells: 70-652 cells/μL (3.7-28.0); and the CD4+:CD8+ index: 0.80-3.92. Absolute numbers--but not percentages--of lymphocytes and CD3+ T cells were higher in those <50 years (p = 0.025 and 0.020, respectively). Absolute values and relative percentages of CD3+/CD8+ and relative values of CD3+/ CD4+ T cells were significantly higher in the younger subgroup (p = 0.004 and p = 0.047). Age was not associated significantly with B lymphocytes or natural killer cells. Absolute and relative values ​​of CD3+/CD4+ T lymphocytes were significantly higher in women (p = 0.009 and 0.036, respectively). CONCLUSIONS. Absolute numbers of total lymphocytes and T and CD3+/CD8+ T lymphocytes are higher in younger individuals. In percentage values, CD3+/CD4+ T lymphocytes are lower in older persons. Absolute and percentage values ​​of CD3+/CD4+ T phenotype are higher in women. These differences justify adjusting clinical analyses to different values ​​by age and sex. KEYWORDS T lymphocytes, B lymphocytes, normal values, flow cytometry, age, sex, Cuba.

Effects of Ageing on the Immune System: Infants to Elderly.

Physiological ageing is accompanied by decline in immune system function and immune alteration during ageing increases susceptibility to infections. We retrospectively analysed the data for complete blood count (CBC) and lymphocyte subsets from infant to elderly age groups to determine changes during ageing. Data from dual-platform flow cytometry and CBC were analysed to determine the percentage (%) and absolute cell counts (Abs) of peripheral blood lymphocyte subsets (CD3, CD4, CD8, CD19 and CD56+16+ cells) in infants (1 month to 1 year), children (1 year to 6 years), adolescents (12 years to 18 years), adults (21 years to 50) and elderly (70 years to 92 years). Differences in plasma cytokine levels in adults and elderly were also analysed using Randox system. Comparisons among age groups from infants through adults revealed progressive declines in the percentage of total lymphocytes and absolute numbers of T and B cells. The NK cells declined from infancy to adulthood but increased in elderly participants. The percentages of T cells increased with age from infant to adulthood and then declined. Pro-inflammatory cytokines, TNF-α and IL-6, were higher in elderly people compared to adults. The elderly group had significantly higher levels of monocyte chemoattractant protein-1 (MCP-1) and lower levels of epidermal growth factor (EGF) compared to adults. Our findings confirm and extend earlier reports on age-related changes in lymphocyte subpopulations and data generated from this study is useful for clinicians and researchers, patient management in various age groups for the interpretation of disease-related changes, as well as therapy-dependent alterations.

Age- and sex-related reference intervals of lymphocyte subsets in healthy ethnic Han Chinese children.

Immunophenotyping of blood lymphocytes has become an important tool in the diagnosis of immunologic and hematologic disorders such as immunodeficiencies, lymphoproliferative and autoimmune diseases. Lymphocyte subsets include total T-cells (CD3(+)), TH (T helper, CD3(+) CD4(+)), TC (cytotoxic T cells, CD3(+) CD8(+)), B-cells (CD3(-) CD19(+)), and NK-cells (CD3(-) CD16(+) CD56(+)). Specific lymphocyte subset reference intervals should be locally established for meaningful comparison and to obtain an accurate interpretation of the results. Reference intervals of lymphocyte subsets for Chinese children are scarce. We performed dual-platform flow cytometry to determine the reference intervals of the percentages and absolute counts of lymphocyte subsets, including total T-cells, TH cells, TC cells, B-cells, and NK-cells in 1,027 ethnic Han children aged 4 months to 7 years in Henan, China. The children were divided into seven age groups. The percentages and absolute counts differed significantly with age, with the percentages of TH cells and B cells and the CD4/CD8 ratio peaking during the first year, while the percentages of total T cells, TC cells, and NK cells were obviously increased with age; girls showed a trend toward having a higher percentage of TH cells and a higher CD4/CD8 ratio than boys. The absolute counts of lymphocyte subsets peaked during first year and then decreased steadily with age. The reference intervals of lymphocyte subsets among children from China differed from the reported values in Hong Kong, the United States, Cameroon, and Italy. The differences observed could be due to genetic and environmental factors, coupled with the methodology used. The reference intervals of lymphocyte subsets could be used as initial national reference ranges in guidelines for children aged 4 months to 7 years.

Determination of lymphocyte subset reference ranges in peripheral blood of healthy adults by a dual-platform flow cytometry method

Flow cytometry has emerged as a useful screening approach to evaluate whether specific cell populations are present or absent. Previous studies have shown different reference ranges in several countries. The aim of this study was to determine reference ranges of lymphocyte subsets in peripheral blood by flow cytometric method in Brazilian adults. In this study, relative and absolute reference ranges of lymphocyte subsets were: CD3+: 51.3–83.5%, 718–2494cells/μl; CD4+: 24.4–54.2%, 456–1492cells/μl; CD8+: 12.8–40.2%, 272–1144cells/μl; CD4+CD8+: double-positive 0.01–3.6%, 2–88cells/μl; TCR γδ: 1.0–15.9%, 19–345cells/μl; CD3+CD4−CD8−: 1.2–13.3%, 28–292cells/μl; TCR αβ+: 44.3–77.0%, 855–2384cells/μl; CD4/CD8 ratio: 0.68–3.61; CD19+: 6.3–20.8%, 112–622cells/μl; mature NK cells: 3.1–27.4%, 70–745cells/μl; immature NK cells: 0.08–1.1%, 1–23cells/μl; total NK cells: 3.7–28.5%, 82–760cells/μl; and NKT cells: 0.9–21.4%, 18–488cells/μl. Comparison with other studies showed differences among some of them. This suggests that there are differences among lymphocyte subsets in the worldwide population and also it is important to determine reference ranges in different populations in order to better assess and monitor patients.

Reference ranges of lymphocyte subsets in healthy adults and adolescents with special mention of T cell maturation subsets in adults of South Florida

BackgroundAnalysis of peripheral blood lymphocyte subsets has become an essential tool in the evaluation of outcome of diagnostic and research related questions in immunological and pathological conditions. Periodic evaluation and establishment of normal lymphocyte reference ranges are required in clinical and research settings of various immunodeficiency disorders for evaluation of the significance of observations. It is also important that age and gender specific lymphocyte subset reference ranges should be locally established for meaningful comparison and accurate result interpretation as age plays a significant role in the development of immune system. MethodsWe performed dual platform flow cytometry to determine reference ranges for lymphocyte subsets (CD3, CD4, CD8, CD19 [B cells] and CD16+CD56+ [Natural Killer – NK cells]) in 50 adolescents (age range: 12–18) and 100 adults (age range: 21–67) along with T cell maturation, activation and co-stimulatory molecules in healthy multiracial adult population of South Florida. ResultsThe lymphocyte reference ranges percentages [absolute counts – Abs, cells/μl] unadjusted for gender differences for adolescents are: CD3: 49–83 [939–2959]; CD4: 27–53 [467–1563]; CD8: 16–40 [259–1262]; CD19+ B cells: 8–31 [169–1297] and CD16+CD56+ NK cells: 3–30 [59–1178] and for adults are: CD3: 65–88 [983–3572]; CD4: 26–62 [491–2000]; CD8: 14–44 [314–2,087]; CD19+ B cells: 2–27 [64–800] and CD16+CD56+ NK cells: 2–27 [27–693]. The ranges for CD4:CD8 ratio for adolescents and adults are 0.7–2.6 and 0.6–4.4, respectively. Gender based analysis of relative percentages of lymphocyte subsets showed no significant differences between adult and adolescent males and females. The mean CD4:CD8 ratio was significantly higher in adult females than males (P=0.04) and in adolescents this difference was not significant between genders. The mean CD3 and CD4 T cell percentages were higher and CD19 cell percentages were lower in adults compared to adolescents (P<0.0001). Absolute lymphocyte counts showed a positive correlation with the absolute counts of CD3+, CD4+, CD8+, CD19+, CD16+CD56+, CD45RO+ and CD45RA+ cells (all correlations with P<0.0001 except CD45RO [P=0.01] and CD45RA [P=0.03]). ConclusionThe reference values of peripheral blood lymphocyte subsets were analyzed in healthy adolescent and adult population of South Florida. This study indicates the need for periodic evaluation and establishment of lymphocyte reference ranges for patient population served based on gender and age since these could influence immune status and treatment outcome.

Comparison of interlaboratory variation in absolute T‐cell counts by single‐platform and optimized dual‐platform methods

Previous studies have reported that the adoption of a single-platform flow cytometry cell counting method resulted in lower interlaboratory variation in absolute T cell counts as compared to predicate dual-platform flow cytometry methods which incorporate independent automated lymphocyte counts (Schnizlein-Bick et al., Clin Diagn Lab Immunol 2000;7:336-343; Reimann et al., Clin Diagn Lab Immunol 2000;7:344-351). In the present study, we asked whether use of a single-platform method could reduce variation in absolute cell counts across the laboratories in the Multicenter AIDS Cohort Study (MACS) (n = 4), as suggested by the studies cited. Identical study samples were shipped overnight to the MACS laboratories either by the National Institute of Allergy and Infectious Diseases, Division of AIDS Immunology Quality Assessment (NIAID- IQA) proficiency-testing program (n = 14), or by the Los Angeles site of the MACS (n = 10). For each sample, two tubes of blood were received; one was used for an automated complete blood count and differential, and the other for flow cytometry. The latter was performed using both our current dual-platform method (three-color CD45 gating and automated hematology) and the single-platform method (with TruCOUNT beads to generate the absolute counts). The median percent coefficients of variation (%CVs) for the dual-platform and single-platform methods were 6.6 and 9.9, respectively, for CD4 T cell counts, and 5.9 and 8.5, respectively, for CD8 T cell counts (n = 24). These differences were not statistically significant. The differences in absolute T-cell counts between the MACS sites and the median of all laboratories participating in the NIAID-IQA were smaller for the dual-platform than for single-platform absolute count method. In contrast to previous reports, we did not observe lower interlaboratory variation across the MACS sites for single-platform absolute lymphocyte subset counting relative to dual-platform methods. This result may be at least partly explained by the lower interlaboratory variation with the optimized dual-platform method in this study relative to the previous reports.

Absolute CD4+ cell count using a plastic microchip and a microscopic cell counter

We have designed and evaluated the performance of a simple, rapid, and affordable method for counting CD4(+) T-cells with the use of plastic microchips. This new system is an adaptation of a "no-lyse, no-wash," volumetric single platform assay, and absolute CD4(+) counts are determined with the use of a microscopic scanning cell counter. To assess the CD4(+) count test precision and linearity of the system, measured CD4(+) counts were compared with two other reference assays (single and dual platform flow cytometry) with the use of 123 clinical samples including samples obtained from 35 HIV-infected patients, and artificially diluted samples. A correlation between the results from the use of the new method and from the use of the two other reference assays was r = 0.98 for the clinical samples. A dilution test of the new method demonstrated a linearity of r >or= 0.99, with coefficients of variation <or=7.6% for all concentration levels. Our findings suggest that the new CD4(+) counting device can be potentially be applied for other diagnostic procedures that measure quantities of characteristic antigens or other materials on cells.

An Immunomagnetic Single-Platform Image Cytometer for Cell Enumeration Based on Antibody Specificity

Simplification of cell enumeration technologies is necessary, especially for resource-poor countries, where reliable and affordable enumeration systems are greatly needed. In this paper, an immunomagnetic single-platform image cytometer (SP ICM) for cell enumeration based on antibody specificity is reported. A chamber/magnet assembly was designed such that the immunomagnetically labeled, acridine orange-stained cells in a blood sample moved to the surface of the chamber, where a fluorescent image was captured and analyzed for cell enumeration. The system was evaluated by applying one kind of antibody to count leukocytes and one kind for each leukocyte subpopulation: CD45 for leukocytes, CD3 for T lymphocytes, and CD19 for B lymphocytes. Excellent precision and linearity were achieved. Moreover, these cell counts, each from blood specimens of 42 to 52 randomly selected patients, were compared with those obtained by SP (TruCount) and dual-platform (DP) flow cytometry (FCM) technologies. The cell counts obtained by our system were in between those obtained from the TruCount and DP FCM methods; and good correlations were achieved (R > or = 0.95). For CD4(+) counts, as we expected, the cell count by our system was significantly higher than the CD4(+) T-lymphocyte counts obtained by SP and DP FCM methods. Immunophenotyping of the immunomagnetically selected CD4(+) cells showed that, besides CD4(+) T lymphocytes, a proportion of the CD4(+) dim monocytes was also selected. Our system is a simple immunomagnetic SP ICM, which can potentially be used for enumeration of CD3(+) CD4(+) T lymphocytes in resource-poor countries if an additional CD3 immunofluorescent label is applied.

Comparison of two alternative methods for CD4+ T‐cell determination (Coulter manual CD4 count and CyFlow) against standard dual platform flow cytometry in Uganda

In this study we evaluated alternative CD4(+) T-cell counting methods in clients of a PMTCT Programme in rural Uganda. The Coulter Manual CD4 Count method for CD4(+) T-cell enumeration (Cyto-Spheres) and an automated method (volumetric, single-platform flow cytometry; CyFlow) were compared with a standard, dual-platform flow cytometry protocol (DPFC, FACScan). Correlation and precision of agreement were higher for the CyFlow method (r = 0.929 and eta = 0.08) when compared to DPFC than for the Cyto-Spheres method (r = 0.725 and eta = 0.3). Multiple linear regression analysis showed that CD4(+) cell counts by the CyFlow method were a stronger predictor for results of DPFC than those of the Cyto-Spheres method (r(2) = 0.864 and r(2) = 0.552, respectively). When compared to DPFC the CyFlow method generated higher CD4(+) cell counts than the Cyto-Spheres method, as expressed by a higher median and mean difference (+70 and +90 cells for CyFlow, +28 and -1.4 cells for Cyto-Spheres). Both, the manual Cyto-Spheres method and the CyFlow method can be used for the enumeration of CD4(+) cells in resource-limited settings. Under supervised conditions, the CyFlow method produced results more consistent with the reference method than the Cyto-Spheres method.

Comparison of capillary based microflurometric assay for CD4+ T cell count estimation with dual platform Flow cytometry

The CD4+ T cell count estimation is an important monitoring tool for HIV disease progression and efficacy of anti-retroviral treatment (ART). Due to availability of ART at low cost in developing countries, quest for reliable cost effective alternative methods for CD4+ T cell count estimation has gained importance. A simple capillary-based microflurometric assay (EasyCD4 System, Guava Technology) was compared with the conventional flow cytometric assay for estimation of CD4+ T cell counts in 79 HIV infected individuals. CD4+ T cell count estimation by both the assays showed strong correlation (r = 0.938, p < 0.001, 95% CI 0.90 to 0.96). The Bland Altman plot analysis showed that the limits of variation were within agreeable limits of ± 2SD (-161 to 129 cells/mm3). The Easy CD4 assay showed 100% sensitivity for estimating the CD4+ T cell counts < 200 cells/mm3 and < 350 cells/mm3 and 97% sensitivity to estimate CD4+ T cell count < 500 cells/mm3. The specificity ranged from 82 to 100%. The Kappa factor ranged from 0.735 for the CD4+ T cell counts < 350 cells/mm3 to 0.771 for < 500 cells/mm3 CD4+ T cell counts. The system works with a simple protocol, is easy to maintain and has low running cost. The system is compact and generates minimum amount of waste. Hence the EasyCD4 System could be applied for estimation of CD4+ T cell counts in resource poor settings.