These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

IntroductionAn increased or disturbed activation and aggregation of platelets plays a major role in the pathophysiology of thrombosis and haemostasis and is related to cardiovascular disease processes. In addition to qualitative disturbances of platelet function, changes in thrombopoiesis or an increased elimination of platelets, (e. g., in autoimmune thrombocytopenia), are also of major clinical relevance. Flow cytometry is increasingly used for the specific characterisation of phenotypic alterations of platelets which are related to cellular activation, haemostatic function and to maturation of precursor cells. These new techniques also allow the study of the in vitro response of platelets to stimuli and the modification thereof under platelet-targeted therapy as well as the characterisation of platelet-specific antibodies. In this protocol, specific flow cytometric techniques for platelet analysis are recommended based on a description of the current state of flow cytometric methodology. These recommendations are an attempt to promote the use of these new techniques which are at present broadly evaluated for diagnostic purposes. Furthermore, the definition of the still open questions primarily related to the technical details of the method should help to promote the multi-center evaluation of procedures with the goal to finally develop standardized operation procedures as the basis of interlaboratory reproducibility when applied to diagnostic testing.

Cell DNA content measurements, including determination of tumor ploidy and S-phase fraction, have been performed on a wide variety of human tumors for > 20 years using flow cytometry. During this time many publications have discussed the clinical utility of cell DNA content measurements. A major impediment to the more widespread application of cytometric DNA content measurements has been the lack of agreement among these studies. Whereas some discrepancies can be attributed to poorly designed studies lacking sufficient follow-up or significant numbers of patients, in many cases the discrepancies are due to technical factors in flow or image cytometry.The terminology used to describe the results of flow cytometry studies is often confusing and not universally applied. Although a convention for nomenclature for all DNA cytometry was recommended in 1984 (l), the guidelines suggested are frequently not used in published studies. Cytometric studies should not use cytogenetic terminology (hypodiploid, peritetriploid, etc.), where no direct measurement of changes in the number or composition of individual chromosomes has been made. Rather, the terms DNA diploid and DNA aneuploid should be used, with identification of the degree of DNA content abnormality given by the use of the DNA index, or DI (ratio of mean or mode of sample G,/G, population divided by mean or mode of diploid reference cells).One critical aspect of the clinical applications of these measurements is the use of standardized procedures to prepare and analyze clinical samples and to analyze and interpret cytometry data. A number of studies have demonstrated significant intralaboratory, as well as interlaboratory variation in the results of DNA content analyses (2-5) and in the interpretation of flow cytometric data (6). The purpose of this work is to help increase the reliability and reproducibility of DNA content flow cytometry by pointing out important technical considerations for cytometry, to provide guidelines that logically follow from these considerations, and to provide a framework for the development of standards and standardization of DNA content flow cytometry. Although a number of reported studies have utilized image cytometry to provide DNA content analysis, the technical differences between image and flow cytometry suggest that guidelines for the clinical application of image cytometry be developed independently.In reviewing the published literature, it is clear that many studies fail to provide sufficient information to judge critically the quality of cytometric measurement. It is recommended that all publications using DNA content cytometry provide details of the technique used to isolate and prepare cells, data that indicate that the sample used for cytometry contains representative tumor material, details concerning the techniques used to stain cells or nuclei (dye concentration, enzyme concentrations in units, cell concentrations), and details of the techniques used to analyze DNA content histograms (debris and aggregation correction ...

Cell DNA content measurements, including determination of tumor ploidy and S-phase fraction, have been performed on a wide variety of human tumors for > 20 years using flow cytometry. During this time many publications have discussed the clinical utility of cell DNA content measurements. A major impediment to the more widespread application of cytometric DNA content measurements has been the lack of agreement among these studies. Whereas some discrepancies can be attributed to poorly designed studies lacking sufficient follow-up or significant numbers of patients, in many cases the discrepancies are due to technical factors in flow or image cytometry.The terminology used to describe the results of flow cytometry studies is often confusing and not universally applied. Although a convention for nomenclature for all DNA cytometry was recommended in 1984 (l), the guidelines suggested are frequently not used in published studies. Cytometric studies should not use cytogenetic terminology (hypodiploid, peritetriploid, etc.), where no direct measurement of changes in the number or composition of individual chromosomes has been made. Rather, the terms DNA diploid and DNA aneuploid should be used, with identification of the degree of DNA content abnormality given by the use of the DNA index, or DI (ratio of mean or mode of sample G,/G, population divided by mean or mode of diploid reference cells).One critical aspect of the clinical applications of these measurements is the use of standardized procedures to prepare and analyze clinical samples and to analyze and interpret cytometry data. A number of studies have demonstrated significant intralaboratory, as well as interlaboratory variation in the results of DNA content analyses (2-5) and in the interpretation of flow cytometric data (6). The purpose of this work is to help increase the reliability and reproducibility of DNA content flow cytometry by pointing out important technical considerations for cytometry, to provide guidelines that logically follow from these considerations, and to provide a framework for the development of standards and standardization of DNA content flow cytometry. Although a number of reported studies have utilized image cytometry to provide DNA content analysis, the technical differences between image and flow cytometry suggest that guidelines for the clinical application of image cytometry be developed independently.In reviewing the published literature, it is clear that many studies fail to provide sufficient information to judge critically the quality of cytometric measurement. It is recommended that all publications using DNA content cytometry provide details of the technique used to isolate and prepare cells, data that indicate that the sample used for cytometry contains representative tumor material, details concerning the techniques used to stain cells or nuclei (dye concentration, enzyme concentrations in units, cell concentrations), and details of the techniques used to analyze DNA content histograms (debris and aggregation correction t...