Abstract Tumor-infiltrating lymphocytes (TILs) are naturally occurring immune cells residing in the solid tumor microenvironment. Some of these TILs are tumor specific and may possess cytotoxic anti-tumor activity. The harvesting, selection, expansion, and reinfusion of these cells represent a current therapeutic approach that may prove to be safe and effective. We developed an immunophenotyping panel to study the tumor microenvironment in human lung cancer specimens. This is a broad panel that includes blood and epithelial cell markers. Twenty lung cancer samples were collected following institutional protocol and dissociated into single cells within 24 hours of resection. Cells were then stained and data was acquired on a 3 laser Cytek™ Aurora™ spectral flow cytometer. The frequencies of cell populations were measured. Increased percentages of intermediate and non-classical monocyte subsets were observed. PD-L1 was detected on neoplastic epithelial cells and EMT (epithelial-mesenchymal transition) cells. Interestingly, a population of CD45 and EpCAM double positive cells was detected in some samples. These cells expressed both epithelial and macrophage markers, suggesting a macrophage lineage origin with abnormal expression. This streamlined single cell sample preparation and multiparametric panel provide an efficient method for the analysis of the tumor microenvironment. Expansion of the sorted cytotoxic TILs could potentially be reinfused into a patient for cancer treatment.

A white blood cell (WBC) differential is routinely performed on automated hematology analyzers as a general health check to evaluate immune status of patients. The recent advances in high parameter flow cytometry, especially full spectral flow cytometry, enable the analysis of more comprehensive leukocyte subsets to meet the increased demands in clinical research. We designed a 15-color, single-tube panel to identify and enumerate all major subsets of leukocytes on a Cytek full spectral flow cytometer. Human peripheral blood collected in K2EDTA, Heparin, ACD and Cyto-chex blood tubes were stained with 15 cFluor ®conjugated mAbs with a Lyse No Wash method and analyzed on a Cytek spectral flow cytometer. The performance of this assay was evaluated side by side with BD Multitest™ 6-color TBNK assay on BD FACSCanto II system on 13 healthy donor samples. Assay precision was examined on 12 donor samples in triplicates on three consecutive days. This 15-color panel identifies more than 20 leukocyte subpopulations: including neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, monocyte subsets, T cell subsets, B cells, NK cell subsets. The results are comparable to that from BD 6-Color TBNK assay with R 2=0.961, 0.986, 0.940, 0.954, and 0.930 for CD3+, CD4+, CD8+ T, B and NK cells respectively. The correlations for all subsets are statistically significant with P < 0.001. The coefficient of variations (CV) of intra assay precision for all cell populations of interest are less than 25%. Cytek’s single-tube 15-color pan leukocyte panel demonstrated an effective, high sensitivity flow cytometry approach that can be used for monitoring immune cell subsets in peripheral blood in discovery and translational research.

Flow cytometry is valuable for identifying and monitoring B cell subsets in patients with humoral immune disorders and patients treated with immunotherapies targeting B cells. We have designed a 13-color cFluor ®B Cell Monitoring panel that identifies plasmablasts/plasma cells (CD19 +CD20 −CD27 +CD38 ++), naïve B cells (CD27 −IgD +), IgG class switched memory (CD27 +IgG +) and unswitched memory (CD27 +IgD +and/or IgM +). Additionally, markers for CD4+ and CD8+ T cells are included for a general status of lymphocytes along with granulocyte (CD15) and monocyte (CD14) markers to allow for cleaner lymphocyte gating. The panel was tested on healthy PBMCs and peripheral blood samples collected in EDTA, Cyto-Chex BCT, or heparin tubes. The cell populations of interest were well identified in the PBMCs and blood samples collected in the different types of blood tubes. The resolution of each marker in the panel was comparable to the single-color staining. Evaluation of assay reproducibility showed that the coefficient of variance (CV) for the B cell subsets of interest were <15% in triplicate runs of 10 samples. Also, blood sample stability and post staining stability were evaluated over time. In conclusion, Cytek’s 13-color cFluor ®B Cell Monitoring panel was shown to be effective in identifying and enumerating B cell subsets. This may be a useful tool for monitoring B cell subsets of patients with humoral immune disorders or patients treated with immunotherapies targeting B cells in translational research.

The concept of myeloid derived suppressive cells (MDSCs) has emerged in recent years as a group of myeloid cells that pocess potent immunosuppressive activity. These cells expand under pathologic conditions and suppress the immune functions of T cells, dendritic cells, macrophages, and natural killer cells. They have been shown to be involved in the pathological states of cancer, autoimmune diseases, and infections, such as COVID-19. MDSCs are subset into PMN (polymorphonuclear)- and M (monocytic)-MDSCs. Phenotypic markers of MDSCs have been elucidated in recent years. Here Cytek® developed a 14-color, 16-marker, single-tube flow cytometric assay to analyzed MDSCs. The panel includes markers to exclude lymphoid lineage cells, and markers to identify PMN-MDSC and M-MDSC populations, such as CD15, CD14, CD16, CD66b, CD11b and HLA-DR. In addition, PMN- and M-MDSC specific expression markers such as Lox-1, CXCR-1, DR5 are also included. Fresh whole blood samples were stained with erythrocyte lyse wash method and the samples were analyzed on a Cytek® Aurora or 3-laser Northern Light TMinstrument. The panel performance was evaluated on blood samples collected in EDTA, Heparin, ACD, and Cytochex blood tubes as well as peripheral blood mononuclear cell (PBMC) samples. Assay precision was assessed by testing on 12 donor samples in triplicates on three consecutive days. Targeted cell populations are well identified in peripheral blood and PBMC samples with good resolution and assay precision with %CV less than 25% for all cell populations of interests. This 14-color 16-marker preoptimized flow cytometric assay can be used in clinical research and drug discovery in cancer, autoimmune diseases, infections and other inflammatory diseases.

Abstract Objective Multiparameter flow cytometry is widely and routinely used in acute myeloid leukemia (AML) diagnosis and the residual disease detection and monitoring. We have developed a single-tube, 20-color panel for AML analysis that uses the Cytek® spectral flow cytometer with increased reagent and sample efficiency. Methods Blood and bone marrow samples were stained with the 20-color panel, acquired, and analyzed using SpectroFlo® software. The resolution of each marker was compared between single stain versus the 20-color full stain on the same samples. The analytical precision for cell populations of interest and antibody cocktail stability were assessed. The limit of detection (LOD) and lower limit of quantification (LLOQ) of the 20-color assay were determined using limiting dilution method. Results The resolution of each marker in the fully stained panel was comparable to that in the single-color stained samples. Normal and aberrant myeloid cell populations were clearly identified and the coefficient of variation (CV) of cell percentage for defined populations in replicate runs were all less than 25%. The antibody cocktail was stable in the fridge for at least 14 days. Our data showed that the single-tube, 20-color assay easily achieved 0.01% on residual aberrant cells in AML, higher than the minimum required detection sensitivity of 0.1%. Conclusions Cytek’s single-tube, 20-color panel demonstrated an effective, high sensitivity flow cytometry approach that can be used for AML testing, including identifying and characterizing normal and aberrant cells, immunophenotypic classification and minimal residual disease evaluation in translation research.

Abstract With many cell surface markers expressed at very low levels the ability to identify (and sort) biologically relevant sub-populations by flow cytometry is often limited by the availability of fluorescent reagents sufficiently bright to resolve these markers. In the past year multiple new fluorochromes have been developed which have significantly improved brightness compared to previously available reagents. In particular, BD Horizon™ PE-CF594 and BD Brilliant Violet™ 421 reagents can be 2-5 times brighter than equivalent PE reagents which have typically been the brightest available. These brighter reagents, when used in multicolor analyses, provide multiple benefits including full resolution of small, dim sub-populations resulting in more accurate enumeration and enrichment via sorting. Data will be presented demonstrating this improved resolution using model systems including human T memory and Treg cell sub-populations and murine CD34+ bone marrow cells.