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Nature Reviews. Immunology Feb 2012The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their... (Review)
Review
The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their comprehensive elucidation has been termed the 'Human Immunology Project'. The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts. Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease. A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis. In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project.
Topics: Animals; Flow Cytometry; Humans; Immunophenotyping; Leukocytes, Mononuclear; Mice; Reference Standards
PubMed: 22343568
DOI: 10.1038/nri3158 -
Blood Apr 2008Flow cytometric immunophenotyping remains an indispensable tool for the diagnosis, classification, staging, and monitoring of hematologic neoplasms. The last 10 years... (Review)
Review
Flow cytometric immunophenotyping remains an indispensable tool for the diagnosis, classification, staging, and monitoring of hematologic neoplasms. The last 10 years have seen advances in flow cytometry instrumentation and availability of an expanded range of antibodies and fluorochromes that have improved our ability to identify different normal cell populations and recognize phenotypic aberrancies, even when present in a small proportion of the cells analyzed. Phenotypically abnormal populations have been documented in many hematologic neoplasms, including lymphoma, chronic lymphoid leukemias, plasma cell neoplasms, acute leukemia, paroxysmal nocturnal hemoglobinuria, mast cell disease, myelodysplastic syndromes, and myeloproliferative disorders. The past decade has also seen refinement of the criteria used to identify distinct disease entities with widespread adoption of the 2001 World Health Organization (WHO) classification. This classification endorses a multiparametric approach to diagnosis and outlines the morphologic, immunophenotypic, and genotypic features characteristic of each disease entity. When should flow cytometric immunophenotyping be applied? The recent Bethesda International Consensus Conference on flow cytometric immunophenotypic analysis of hematolymphoid neoplasms made recommendations on the medical indications for flow cytometric testing. This review discusses how flow cytometric testing is currently applied in these clinical situations and how the information obtained can be used to direct other testing.
Topics: Flow Cytometry; Hematologic Neoplasms; Hemoglobinuria, Paroxysmal; Humans; Immunophenotyping; Plasma Cells
PubMed: 18198345
DOI: 10.1182/blood-2007-11-120535 -
The Journal of Pathology Jan 2014The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine... (Review)
Review
The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).
Topics: Biomarkers, Tumor; Humans; Immunophenotyping; Neoplasm Staging; Neoplasms; Predictive Value of Tests; Tumor Microenvironment
PubMed: 24122236
DOI: 10.1002/path.4287 -
Cytometry. Part B, Clinical Cytometry Jul 2022Prompt diagnosis of acute promyelocytic leukemia (APL) is critical for patient care. In this study, we aimed to characterize the immunophenotype of APL and explore...
BACKGROUND
Prompt diagnosis of acute promyelocytic leukemia (APL) is critical for patient care. In this study, we aimed to characterize the immunophenotype of APL and explore immunophenotypic difference between APL and its mimics using flow cytometric analysis.
METHODS
Eighty-five cases were collected, including 47 APL, 26 NPM1-mutated acute myeloid leukemia (AML) and 12 KMT2A-rearranged AML with an APL-like immunophenotype. Immunophenotypes were analyzed using flow cytometric analysis.
RESULTS
APL showed four distinct patterns (designated a-d) based on CD45/SSC plots. Blasts in patterns a-c showed high side scatter, whereas blasts in pattern d had low side scatter and were located in the traditional blast gate. Compared with patterns a-c, pattern d of APL (APL-D) was more often positive for CD2 (p = 0.0005) and CD34 (p = 0.0002) in blasts. All NPM1-mutated AML and KMT2A-rearranged AML cases with an APL-like immunophenotype had blasts in the traditional blast gate on CD45/SSC, mimicking APL-D. In comparison, uniform CD13 and positive CD64 were seen in 100% (n = 13) APL-D cases and in only 2 of 26 (8%) NPM1-mutated AML cases (p < 0.0001). In addition, APL-D cases were more likely to be positive for CD2 and/or CD34 than NPM1-mutated AML (p < 0.0001 and p = 0.0007, respectively). In comparison with APL-D, KMT2A-rearranged AML cases were less often positive for myeloperoxidase (MPO) (p = 0.001), with none being strongly positive. Similar to NPM1-mutated AML and different from APL-D, KMT2A-rearranged AML cases were rarely positive for CD34 and all negative for CD2.
CONCLUSIONS
APL and its immunophenotypic mimics share some immunophenotypic similarities but can be distinguished by CD2, CD13, CD34, CD64, and MPO.
Topics: Antigens, CD34; Diagnosis, Differential; Flow Cytometry; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Nuclear Proteins
PubMed: 35716019
DOI: 10.1002/cyto.b.22085 -
Cytometry. Part B, Clinical Cytometry Jan 2018Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between...
Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.
Topics: Acute Disease; Child; Consensus; Flow Cytometry; Humans; Immunophenotyping; Phenotype; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 28187514
DOI: 10.1002/cyto.b.21518 -
Cytometry. Part B, Clinical Cytometry May 2021Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay...
Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms.
BACKGROUND
Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells.
METHODS
Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1.
RESULTS
We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets.
CONCLUSIONS
Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Body Fluids; Female; Flow Cytometry; Humans; Immunophenotyping; Lymphoma, T-Cell; Male; Middle Aged; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes; Young Adult
PubMed: 32333725
DOI: 10.1002/cyto.b.21881 -
Cytometry. Part a : the Journal of the... Sep 2021
Topics: Flow Cytometry; Immunophenotyping
PubMed: 34374486
DOI: 10.1002/cyto.a.24494 -
Leukemia Sep 2012Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated... (Comparative Study)
Comparative Study
Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.
Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Case-Control Studies; Europe; Flow Cytometry; Hematologic Neoplasms; Humans; Immunophenotyping; Leukocytes; Prognosis
PubMed: 22552007
DOI: 10.1038/leu.2012.120 -
Cytometry. Part B, Clinical Cytometry Jan 2018The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the...
Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project.
The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5-20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for "required" diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. "Recommended" markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as "required" for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus "recommended" panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society.
Topics: Biomarkers, Tumor; Flow Cytometry; Humans; Immunophenotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Pilot Projects; Reproducibility of Results; Retrospective Studies
PubMed: 29024461
DOI: 10.1002/cyto.b.21595 -
Advanced Science (Weinheim,... May 2023The effectiveness of neoadjuvant immune checkpoint inhibitor (ICI) therapy is confirmed in clinical trials; however, the patients suitable for receiving this therapy...
The effectiveness of neoadjuvant immune checkpoint inhibitor (ICI) therapy is confirmed in clinical trials; however, the patients suitable for receiving this therapy remain unspecified. Previous studies have demonstrated that the tumor microenvironment (TME) dominates immunotherapy; therefore, an effective TME classification strategy is required. In this study, five crucial immunophenotype-related molecules (WARS, UBE2L6, GZMB, BATF2, and LAG-3) in the TME are determined in five public gastric cancer (GC) datasets (n = 1426) and an in-house sequencing dataset (n = 79). Based on this, a GC immunophenotypic score (IPS) is constructed using the least absolute shrinkage and selection operator (LASSO) Cox, and randomSurvivalForest. IPS is characterized as immune-activated, and IPS is immune-silenced. Data from seven centers (n = 1144) indicate that the IPS is a robust and independent biomarker for GC and superior to the AJCC stage. Furthermore, patients with an IPS and a combined positive score of ≥5 are likely to benefit from neoadjuvant anti-PD-1 therapy. In summary, the IPS can be a useful quantitative tool for immunophenotyping to improve clinical outcomes and provide a practical reference for implementing neoadjuvant ICI therapy for patients with GC.
Topics: Humans; Neoadjuvant Therapy; Stomach Neoplasms; Immunophenotyping; Prognosis; Immunotherapy; Tumor Microenvironment
PubMed: 36998102
DOI: 10.1002/advs.202207417