Isolation of tumor-infiltrating lymphocytes from preserved human tumor tissue specimens for downstream characterization
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Both normal and malignant plasma cell (PC) populations can be identified using modern flow cytometry (FC) technique in multiple myeloma (MM) patients. Expression of CD19 and CD56 markers is heterogenous on bone marrow PCs of healthy individuals. Little is known about immunophenotypically aberrant (CD19-/CD56+) PCs subpopulation of healthy people.Using six color FC, we analyzed PCs in BM samples of 11 healthy donors (HD) and compared their immunophenotypic properties with clonal PC populations from MM patients.Both immunophenotypically normal (CD19+/CD56-) and aberrant (CD19-/CD56+) PC populations could be detected in 10 of 11 HDs' BM samples and constituted the median of 60.3% (37.3-72.3) and 9.6% (0-35.7) of BM PCs, respectively. CD19, CD56, CD38, CD45, and CD20 marker expression characteristics were of little value discriminating clonal PCs of MM patients from immunophenotypically aberrant PCs of healthy donors.Our findings suggest that aberrant immunophenotype is common in BM PCs of healthy people. Improvements in FC methodology to separate normal and malignant PCs remain an open area for future investigations.
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This article provides an overview of the role of flow cytometry in the diagnosis, prognosis, and follow‐up of T and NK‐cell lymphoproliferative disorders. For each category, we will briefly discuss the immunophenotypic features of normal T and NK cells, and address technical issues in flow cytometry, the approach to diagnosis in various contexts, pitfalls in interpretation, and its use in follow‐up and post‐therapy management. In addition to reviewing the diagnostic, prognostic, and therapeutic utility of flow cytometric immunophenotyping in several of specific T and NK cell entities, we will also cover some of the new immunophenotypic markers. Furthermore, we will touch upon incorporation of flow cytometry in the final diagnosis, including correlation with other ancillary tests. © 2019 International Clinical Cytometry Society
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Immunophenotyping of peripheral blood leukocytes (PBL) is a very well documented application of Slide Based Cytometry (SBC). As for any other assay it is of highest importance to ensure that all cells which are relevant for an analysis are recognized. Unlike assays for cultured cells which have homogenous morphology immunophenotyping of PBLs is performed on cells with heterogeneous size and shape. Therefore, triggering on parameters related to cell morphology might lead to an incomplete analysis of just a subset of cells especially in pathological conditions. Several dyes stain DNA specifically in a wide variety of emission spectra. Many of them show some influence of the chromatin condensation and organization on the staining intensity. DNA dyes therefore can be used to differentiate between cell types having the same ploidy. This can be exploited for immunophenotyping since some dyes therefore can partially replace antibody staining. The concept of using DNA dyes in the setting of immunostaining has the following advantages: (1) nuclear staining provides a stable and easy triggering signal that guarantees both, that neither cells are excluded nor that debris or polluting particles are included into the analysis; (2) some DNA dyes differentiate between mononuclear and polymorphonuclear cells. A disadvantage of DNA dyes is that mostly cells have to be permeabilized. Because of this only one set of immunophenotypic markers can be stained, cells are fixed and permeabilized, and then nuclei are stained with the appropriate DNA dye. In the study we demonstrate the use of the most commonly available DNA dyes (7-AAD, To-Pro, To-To, PI etc.) in their applicability in immunophenotyping. An overview of spectral properties, fluorescence spill-over and optimal combinations with surface antigen staining will be shown. As in general for SBC only very small sample volumes are needed. This allows to serially analyze PBL in clinical settings that up to now could not be studied in detail such as in the critical ill patient, during major surgery, and in new-borns and infants.
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Abstract Aim Slide‐based cytometry (SBC) allows to “ask a cell a second time.” We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs). Methods PBLs primarily stained for CD‐markers and DNA were immobilized on a glass slide and analyzed by laser scanning cytometry. Then, iterative restaining was applied for a second and a third analysis. Based on the cells' fixed location, analyses were merged on a single‐cell level. Results We analyzed six virtual immunostainings by “recycling” the PE‐channel for four CD‐markers. Conclusion Iterative restaining might prove to be a pivotal tool for n‐color immunophenotyping exclusive to SBC concepts. © 2006 International Society for Analytical Cytology
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Immunophenotyping using high dimensional flow cytometry is a central component of human immune system multi-omic studies. We present four high parameter flow cytometry panels for deep immunophenotyping of human peripheral blood mononuclear cells (PBMC). This set of four 25+ color panels include 64 cell surface markers to resolve broad immune compartment populations, as well as activation and memory of specific T, B, natural killer (NK), and myeloid lineages. Common lineage bridging markers are integrated into each panel to allow for inter-panel quality control through major lineage frequency verification. These panels were developed using a five laser BD Symphony A5 conventional cytometer and successfully transferred to a five laser Cytek Aurora spectral cytometer capable of acquiring the panels. Nine representative PBMC samples were stained with the four phenotyping panels and acquired on both instruments to evaluate population frequency and visual staining patterns for gating between the systems. Both instruments produced comparable high quality flow cytometry data and supported our decision to acquire samples on the spectral cytometer moving forward. This modular set of panels and instrument performance metrics provide guidelines for designing flow cytometry experiments suitable for longitudinal or cross-sectional immune profiling.
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Abstract Background Immunophenotyping and enumeration of plasma cells (PCs) by flow cytometry are deemed to be prognostically significant. However, PCs enumeration by flow cytometry is challenging owing to discrepancy with morphology and PCs loss during sample processing. Enumeration and differentiation of abnormal plasma cells (APCs) and normal plasma cells (NPCs) is difficult because abnormal antigen expression can be seen in subsets of NPCs. This is particularly true when a limited panel of antibodies are relied upon. Aims and purpose To study the immunophenotypic profile of newly diagnosed multiple myeloma (MM) cases by flow cytometry and evaluate the sensitivities and specificities of individual antigens and combinations. Methods We studied immunophenotype of PCs in newly diagnosed MM cases (n = 48) and control cases (n = 10) by a 6-color, 3-tube flow cytometry panel. The sensitivities and specificities of antigens in MM were evaluated and compared with control cases. Results Majority of MM cases (n = 43) had < 3% NPCs. CD19 was the most sensitive (100%) and CD81 was the most specific marker (100%) for differentiating APCs from NPCs. CD38 MFI came out as a useful marker for APCs identification. In combination, CD19 and CD81 had a higher sensitivity and specificity to detect APCs. Conclusion NPCs may show aberrant antigen expression. A combination of multiple markers including CD81 and CD38 MFI should be used for accurate APC detection.
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