-
Iranian Journal of Allergy, Asthma, and... Feb 2015The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. Flow cytometric analysis provides a rapid qualitative and quantitative... (Review)
Review
The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. Flow cytometric analysis provides a rapid qualitative and quantitative description of multiple characteristics of individual cells. For example, it is possible to detect the cell size and granularity, aspects of DNA and RNA content and the presence of cell surface and nuclear markers which are used to characterize the phenotype of single cells. Flow cytometry has been used for the immunophenotyping of a variety of specimens including whole blood, bone marrow, serous cavity fluids, (cerebrospinal fluid) CSF, urine and all types of body fluids. The technique has also been applied to human bronchoalveolar lavage (BAL) fluid, peritoneal fluids and blood. In this review, we describe the current status of the application of flow cytometry as a diagnostic tool in various lung diseases. We focus on the analysis of BAL cell composition in chronic obstructive lung disease (COPD), asthma, lung cancer, sarcoidosis, tuberculosis and idiopathic eosinophilic pneumonia (IEP).
Topics: Bronchoalveolar Lavage Fluid; Flow Cytometry; Humans; Immunophenotyping; Inflammation; Lung Diseases
PubMed: 25530134
DOI: No ID Found -
Annals of Hematology Mar 2022Acute myeloid leukemia (AML) is a highly heterogeneous disease showing dynamic clonal evolution patterns over time. Various subclones may be present simultaneously and...
Acute myeloid leukemia (AML) is a highly heterogeneous disease showing dynamic clonal evolution patterns over time. Various subclones may be present simultaneously and subclones may show a different expansion pattern and respond differently to applied therapies. It is already clear that immunophenotyping and genetic analyses may yield overlapping, but also complementary information. Detailed information on the genetic make-up of immunophenotypically defined subclones is however scarce. We performed error-corrected sequencing for 27 myeloid leukemia driver genes in 86, FACS-sorted immunophenotypically characterized normal and aberrant subfractions in 10 AML patients. We identified three main scenarios. In the first group of patients, the two techniques were equally well characterizing the malignancy. In the second group, most of the isolated populations did not express aberrant immunophenotypes but still harbored several genetic aberrancies, indicating that the information obtained only by immunophenotyping would be incomplete. Vice versa, one patient was identified in which genetic mutations were found only in a small fraction of the immunophenotypically defined malignant populations, indicating that the genetic analysis gave an incomplete picture of the disease. We conclude that currently, characterization of leukemic cells in AML by molecular and immunophenotypic techniques is complementary, and infer that both techniques should be used in parallel in order to obtain the most complete view on the disease.
Topics: Clonal Evolution; Gene Expression Regulation, Leukemic; Genetic Variation; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Mutation
PubMed: 35024892
DOI: 10.1007/s00277-021-04747-x -
Current Topics in Microbiology and... 2014A comprehensive study of the cellular components of the immune system requires both deep and broad immunophenotyping of numerous cell populations in an efficient and... (Review)
Review
A comprehensive study of the cellular components of the immune system requires both deep and broad immunophenotyping of numerous cell populations in an efficient and practical manner. In this chapter, we describe the technical aspects of studying the human immunome using high-dimensional (15 color) fluorescence-based immunophenotyping. We focus on the technical aspects of polychromatic flow cytometry and the initial stages in developing a panel for comprehensive leukocyte immunophenotyping (CLIP). We also briefly discuss how this panel is being used and the challenges of encyclopedic analysis of these rich data sets.
Topics: Animals; Biomarkers; Flow Cytometry; Fluorescent Dyes; Humans; Immunophenotyping; Leukocytes
PubMed: 23975032
DOI: 10.1007/82_2013_336 -
Current Oncology Reports Aug 2022Recent advances in the small field of the rare mixed phenotype acute leukemias (MPAL) are presented focusing on a better understanding of their pathophysiology and... (Review)
Review
PURPOSE OF REVIEW
Recent advances in the small field of the rare mixed phenotype acute leukemias (MPAL) are presented focusing on a better understanding of their pathophysiology and search for better therapeutic approaches.
RECENT FINDINGS
Three aspects of respective classification, therapy, and immunophenotype of MPAL are reviewed. New proposals have been made to segregate MPAL subtypes based on their genomic landscape. In parallel, it was found that a large array of therapeutic approaches has been tested in the past few years with increasingly good results. Finally, we explored the use of unsupervised flow cytometry analysis to dissect subtle variations in markers expression to better characterize the variegating aspect of MPALs. Genomic and immunophenotypic aspects more clearly link MPAL subtypes with bona fide acute myeloblastic of lymphoblastic leukemias. This is likely to impact therapeutic strategies, towards a better management and outcome.
Topics: Hematopoietic Stem Cell Transplantation; Humans; Immunophenotyping; Phenotype; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 35380407
DOI: 10.1007/s11912-022-01252-w -
Acta Cytologica 2016According to the World Health Organization (WHO), the new classification of lymphomas is mainly based on morphological, immunophenotypical, and molecular criteria.... (Review)
Review
According to the World Health Organization (WHO), the new classification of lymphomas is mainly based on morphological, immunophenotypical, and molecular criteria. Consequently, this new approach has led from the substantial role that architecture played in the past to a secondary panel highlighting the role of fine-needle biopsy (FNB). Applied together with other ancillary techniques, such as flow cytometry (FC), FNB is a potential tool for the diagnosis of lymphomas, and enlarged lymph nodes represent an excellent target for the implementation of this technique. Despite the difficulties inherent in this technology, which might pose problems in differential diagnosis, in the majority of cases this joint work allows an accurate diagnosis of malignancy and even correct subcharacterization in routine lymphomas. Additionally, in selected cases, other molecular techniques like FISH and PCR can also be performed on FNB specimens, helping in the characterization and diagnosis of lymphomas. In this review, we discuss the basic aspects of the combination of FNB cytology and FC in the diagnosis and subclassification of lymphomas. The preanalytical phase is extensively discussed. The advantages, disadvantages, and technical limitations of this joint work are addressed in general and in terms of the accurate subclassification of lymphomas.
Topics: Biopsy, Fine-Needle; Cytodiagnosis; Diagnosis, Differential; Flow Cytometry; Humans; Immunophenotyping; Lymph Nodes; Lymphoma; Neoplasms
PubMed: 27639613
DOI: 10.1159/000448679 -
Scientific Reports Dec 2022The pathogenesis of coronavirus disease 2019 (COVID-19) is not fully elucidated. COVID-19 is due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which...
The pathogenesis of coronavirus disease 2019 (COVID-19) is not fully elucidated. COVID-19 is due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes severe illness and death in some people by causing immune dysregulation and blood T cell depletion. Increased numbers of myeloid-derived suppressor cells (MDSCs) play a diverse role in the pathogenesis of many infections and cancers but their function in COVID-19 remains unclear. To evaluate the function of MDSCs in relation with the severity of COVID-19. 26 PCR-confirmed COVID-19 patients including 12 moderate and 14 severe patients along with 11 healthy age- and sex-matched controls were enrolled. 10 ml whole blood was harvested for cell isolation, immunophenotyping and stimulation. The immunophenotype of MDSCs by flow cytometry and T cells proliferation in the presence of MDSCs was evaluated. Serum TGF-β was assessed by ELISA. High percentages of M-MDSCs in males and of P-MDSCs in female patients were found in severe and moderate affected patients. Isolated MDSCs of COVID-19 patients suppressed the proliferation and intracellular levels of IFN-γ in T cells despite significant suppression of T regulatory cells but up-regulation of precursor regulatory T cells. Serum analysis shows increased levels of TGF-β in severe patients compared to moderate and control subjects (HC) (P = 0.003, P < 0.0001, respectively). The frequency of MDSCs in blood shows higher frequency among both moderate and severe patients and may be considered as a predictive factor for disease severity. MDSCs may suppress T cell proliferation by releasing TGF-β.
Topics: Male; Humans; Female; Myeloid-Derived Suppressor Cells; COVID-19; Immunophenotyping; SARS-CoV-2; Transforming Growth Factor beta
PubMed: 36581679
DOI: 10.1038/s41598-022-26943-z -
Clinics in Laboratory Medicine Sep 2007Flow cytometry-based immunophenotyping assays have become increasingly multiparametric, concomitantly analyzing multiple cellular parameters. To maximize the quality of... (Review)
Review
Flow cytometry-based immunophenotyping assays have become increasingly multiparametric, concomitantly analyzing multiple cellular parameters. To maximize the quality of the information obtained, antibody conjugate panels need to be developed with care, including requisite controls at every step. Such an optimization procedure for multicolor immunophenotyping assays is time consuming, but the value of having a reliable antibody conjugate panel that provides for sensitive detection of all molecules of interest justifies this time investment. This article outlines important considerations and procedures to undertake for the successful design and development of multicolor flow cytometry panels.
Topics: Antibodies, Monoclonal; Flow Cytometry; Fluorescent Dyes; Humans; Immunophenotyping
PubMed: 17658403
DOI: 10.1016/j.cll.2007.05.002 -
Journal of Clinical Pathology Jul 2001In the past decade, cellular immunophenotyping has become a new discipline in diagnostic haematology and immunology, and is invaluable in the rapid diagnosis of... (Review)
Review
In the past decade, cellular immunophenotyping has become a new discipline in diagnostic haematology and immunology, and is invaluable in the rapid diagnosis of leukaemia and monitoring disease progression in human immunodeficiency virus infected individuals. The introduction of bench top flow cytometers has meant that immunophenotyping is now also used for the quantitation of CD34(+) peripheral blood stem cells (PBSCs) to ensure the correct timing and adequacy of haematopoietic progenitor cell harvests. Furthermore, flow cytometry has become an important tool for the counting of leucocytes in blood components after leucocyte depletion. Because this new discipline is now such a major diagnostic and prognostic tool in the clinical arena, its use must be subject to both internal and external quality control. Such a requirement was first recognised as early as 1986 when an Inter-Regional Quality Assessment Scheme (IRQAS) was initiated for laboratories that undertook the immunocytochemical diagnosis of leukaemia using the alkaline phosphates anti-alkaline phosphatase technique. This programme began with around 25 UK laboratories. In 1990, after the introduction of two more programmes (one for leukaemia diagnosis using UV microscopy and latterly flow cytometry, and one for the enumeration of CD4(+) T cells) the IRQAS achieved UK National External Quality Assessment Scheme (UK NEQAS) status and changed its title to UK NEQAS for Leucocyte Immunophenotyping. In the past decade the once small IRQAS programme has evolved into the largest international scheme of its kind, providing EQA to over 650 laboratories world wide for leukaemia immunophenotyping, lymphocyte subset analysis, PBSCs, and more recently low level leucocyte counting. Over the years, this EQA programme has highlighted important problems, such as the inappropriate use of fluorochromes and antibody titre, and the identification of effective gating strategies, all of which have contributed directly to the high interlaboratory variations seen in cellular immunophenotyping. Furthermore, particularly in absolute counting of lymphocyte subsets, PBSCs, and the enumeration of low numbers of leucocytes, UK NEQAS for Leucocyte Immunophenotyping programmes have been instrumental in highlighting the differences that occur between single and dual platform flow cytometric technologies. As a result of these findings, UK NEQAS for Leucocyte Immunophenotyping has helped to reduce the variation seen on an interlaboratory basis and enabled greater standardisation both in the UK and internationally. These advances have been attributable to the development, by UK NEQAS for Leucocyte Immunophenotyping, of a unique whole blood stabilising process that ensures the retention of the physical characteristics (both light scatter and antigenic profile) required of cells to ensure successful cellular immunophenotyping. This major technological advancement has enabled the distribution of specimens for EQA purposes on a global scale that have minimal matrix effect and behave in a manner identical to fresh blood for several months after stabilisation.
Topics: Antigens, CD34; CD4 Lymphocyte Count; Forecasting; Hematopoietic Stem Cells; Humans; Immunophenotyping; Leukemia; Leukocyte Count; Leukocytes
PubMed: 11429420
DOI: 10.1136/jcp.54.7.508 -
Current Protocols in Cytometry Apr 2010This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in... (Review)
Review
This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and "mass action law" characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units.
Topics: Antigens; Cell Membrane; Cell Nucleus; Chromatin; DNA; Flow Cytometry; Fluorescent Dyes; Humans; Immunophenotyping; Neoplasms; Paraffin; Ploidies; Reproducibility of Results
PubMed: 20373495
DOI: 10.1002/0471142956.cy0702s52 -
Best Practice & Research. Clinical... Dec 2021Acute leukemias of ambiguous lineage (ALAL), including mixed phenotype acute leukemia (MPAL) and related entities such as early T-cell precursor acute leukemia... (Review)
Review
Acute leukemias of ambiguous lineage (ALAL), including mixed phenotype acute leukemia (MPAL) and related entities such as early T-cell precursor acute leukemia (ETP-ALL), remain diagnostic and clinical challenges due to limited understanding of pathogenesis, reliance of immunophenotyping to classify disease, and the lack of a rational approach to guide selection of appropriate therapy. Recent studies utilizing genomic sequencing and complementary approaches have provided key insights that are changing the way in which such leukemias are classified, and potentially, treated. Several recurrent genomic alterations define leukemias that straddle immunophenotypic entities, such as ZNF384-rearranged childhood B-ALL and B/myeloid MPAL, and BCL11B-rearranged T/myeloid MPAL, ETP-ALL and AML. In contrast, some cases of MPAL represent canonical ALL/AML entities exhibiting lineage aberrancy. For many cases of ALAL, experimental approaches indicate lineage aberrancy arises from acquisition of a founding genetic alteration into a hematopoietic stem or progenitor cell. Determination of optimal therapeutic approach requires genomic characterization of uniformly treated ALAL patients in prospective studies, but several approaches, including kinase inhibitors and BH3 mimetics may be efficacious in subsets of ALAL.
Topics: Child; Genomics; Humans; Immunophenotyping; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prospective Studies; Repressor Proteins; Tumor Suppressor Proteins
PubMed: 34865701
DOI: 10.1016/j.beha.2021.101329