Use of CD45 gating in three and four‐color flow cytometric immunophenotyping: Guideline from the national institute of allergy and infectious diseases, division of AIDS

Abstract

Since the publication of the "Three-color supplement to the NIAID/DAIDS Guideline for Flow Cytometric Immunophenotyping" in 1996 (1), significant scientific and technological advances in the development and production of reagents, instrumentation, and software have increased the use of multicolor flow cytometry in both research and clinical laboratories. With the increased adoption of three and four-color flow cytometry as the preferred methodology in determining patients' CD4 and CD8 T-cell counts, it has become apparent that a gating strategy that integrates the bright CD45 cells reduces interlaboratory and intralaboratory variability. Traditionally, a lymphocyte gate for immunophenotyping is derived from a bivariate frequency distribution histogram that includes 90°side scatter (SSC) and forward light scatter (FSC) frequency patterns. This type of histogram configuration is called a homogenous gating protocol (2). The advantage of the combination of bright CD45 fluorescence and light scatter, a heterogeneous gating protocol, was first reported in 1993 (3). Over the past few years, it has been determined that this alternative approach provides a more reproducible and accurate lymphocyte gate (4 -6). This heterogeneous method will be referred to as the CD45 gating method.The purpose of this article is to update the 1996 NIAID/ DAIDS Guideline and include the use of the CD45 gating method to minimize measurement variability with multicolor flow cytometry for the enumeration of T-cell subsets. Some information is provided about the advantages of four-color flow cytometry and the use of single-platform bead-based technology for determining absolute and percentage of lymphocytes. The addition of integrated fluorosphere counting provides a single-platform protocol that facilitates the simultaneous determination of both absolute and percentage of lymphocyte subsets. The specifications and recommendations were developed for use in laboratories supporting clinical trials and epidemiolog-

Flow cytometric lymphocyte subset enumeration: 10 years of external quality assessment in the Benelux countries

“…This observation confirms previous studies (22,29,38,43,44). Single-platform assays bypass the ''denominator issue,'' that is, the need to express the percentage of lymphocytes as a proportion of either leukocytes, total nucleated cells, or total events exceeding the FSC threshold.…”

“…Our recommendations provided with the debriefings and the plenary meetings reflected international recommendations (9,(36)(37)(38)(39)(40)48). As a result, the use of ''CD45-SSC'' gating increased at the expense of ''CD45-14 backgating,'' and ''lyse-no-wash'' techniques were implemented by the majority of participants instead of ''lyse and wash'' techniques.…”

“…Counterstaining of the T cells for CD4 and/or CD8 allows their further characterization (34,35). These gating strategies were adopted subsequently in the CDC (36), NIAID-DAIDS (37,38), and British Committee for Standards in Haematology (BCSH) (39,40) guidelines.…”

Age‐ and sex‐related reference intervals of lymphocyte subsets in healthy ethnic Han Chinese children

“…The use of the CD45 gating method could minimize measurement variability with multicolor flow cytometry for the enumeration of T-cell subsets (33). Flow cytometry analysis was performed with a standardized 4-color FACSCalibur analyzer (BD Biosciences, San Jose, CA).…”

Comparative Study of “Klim-Test” Monoclonal Antibodies for Phenotyping of Peripheral Blood Cells With Monoclonals From Foreign Producers

“…1А, B). Гейт R1 нарисо-ван вокруг CD45-положительных клеток с низ-ким боковым светорассеянием (лимфоциты) [7,12]. Распределяя клетки из гейта R1 по наличию рецепторов CD4 (рис.…”