Three-color supplement to the NIAID DAIDS guideline for flow cytometric immunophenotyping

Nicholson, Janet K. A.; Kidd, Pamela; Mandy, Francis; Livnat, Daniella; Kagan, Jonathan

doi:10.1002/(sici)1097-0320(19960915)26:3<227::aid-cyto8>3.0.co;2-b

Cited by 61 publications

(54 citation statements)

References 4 publications

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“…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.…”

Section: Fig 5 Variation Of Cd4þ T-cell (Panelmentioning

confidence: 86%

“…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.…”

Section: Flow Cytometric Lymphocyte Subset Enumerationmentioning

confidence: 99%

“…As for gating, the ''CD45-SSC'' strategy stood out by a positive bias for CD3 þ and CD4 þ T-cell counts relative to the ''CD45-14 backgating'' strategy. Currently, the view is widely held that ''CD45-SSC'' gating of lymphocytes is more robust than ''CD45-14 backgating'' (34,37,39); the risk of the latter method is that a too restricted FSC-SSC gate is set on lymphocytes to avoid contamination by monocytes, which then would result in an underestimation of the % CD3 þ or CD4 þ T cells (52). Furthermore, the ''dual-anchor'' gating strategy was associated with a negative bias toward NK cells.…”

Section: Fig 5 Variation Of Cd4þ T-cell (Panelmentioning

confidence: 99%

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

Levering

Wieringen

Kraan

et al. 2007

Cytometry Part B Clinical

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A biannual external quality assessment (EQA) scheme for flow cytometric lymphocyte immunophenotyping is operational in the Benelux countries since 1996. We studied the effects of the methods used on assay outcome, and whether or not this EQA exercise was effective in reducing between-laboratory variation. Eighty test samples were distributed in 20 biannual send-outs. Per send-out, 50-71 participants were requested to enumerate CD3 + , CD4 + , and CD8 + T cells, B cells, and NK cells, and to provide methodological details. Participants received written debriefings with personalized recommendations after each send-out. For this report, data were analyzed using robust multivariate regression. Five variables were associated with significant positive or negative bias of absolute lymphocyte subset counts: (i) platform methodology (i.e., singleplatform assays yielded lower CD4 + and CD8 + T-cell counts than did dual-platform assays); (ii) sample preparation technique (i.e., assays based on mononuclear cells isolation yielded lower T-cell counts than those based on red cell lysis); (iii) gating strategies based on CD45 and sideward scatter gating of lymphocytes yielded higher CD4 + T-cell counts than those based on ''backgating'' of lymphocytes guided by CD45 and CD14); (iv) stabilized samples were generally associated with higher lymphocyte subset counts than nonstabilized samples; and (v) laboratory. Platform methodology, sample stabilization, and laboratory also affected assay variability. With time, assay variability tended to decline; this trend was significant for B-cell counts only. In addition, significant bias and variability of results, independent of the variables tested for in this analysis, were also associated with individual laboratories. In spite of our recommendations, participants tended to standardize their techniques mainly with respect to sample preparation and gating strategies, but less with absolute counting techniques. Failure to fully standardize protocols may have led to only modest reductions in variability of results between laboratories. q 2007 Clinical Cytometry Society

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Section: Fig 5 Variation Of Cd4þ T-cell (Panelmentioning

confidence: 86%

Section: Flow Cytometric Lymphocyte Subset Enumerationmentioning

confidence: 99%

Section: Fig 5 Variation Of Cd4þ T-cell (Panelmentioning

confidence: 99%

See 1 more Smart Citation

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

Levering

Wieringen

Kraan

et al. 2007

Cytometry Part B Clinical

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show abstract

“…Fresh whole blood was transported to the three laboratories at ambient temperature and processed within 30 h of collection. Enumeration of lymphocyte phenotypes was performed according to standard methods (7,22), which are used in the consensus protocol of the AIDS Clinical Trial Group. Specific lymphocyte subsets were identified by three-color flow cytometry using combinations of monoclonal antibodies to lymphocyte surface antigens conjugated to fluorochromes fluorescein isothiocyanate, phycoerythrin, and energy coupled dye or peridinin chlorophyll protein.…”

Section: Methodsmentioning

confidence: 99%

Association of Selected Phenotypic Markers of Lymphocyte Activation and Differentiation with Perinatal Human Immunodeficiency Virus Transmission and Infant Infection

Lambert

Moye

Plaeger

et al. 2005

Clin Vaccine Immunol

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“…The monoclonal antibody combinations used include CD3/CD4/CD45 as well as CD3/CD8/CD45 to determine CD4 + T cells and CD8 + T cells, respectively. Using low side scatter and gating on "bright" CD45 for each tube helped ensure inclusion of only lymphocytes and eliminated the need to correct for gate purity [17]. A complete blood count, including WBC count, with automated differential was measured annually at these same visits.…”

Section: Measurement Of White Blood Cells Lymphocytes Cd4 and Cd8mentioning

confidence: 99%

Obesity and immune cell counts in women

et al. 2007

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Obesity is common in women and is associated with a number of adverse health outcomes including cardiovascular disease, infectious diseases, and cancer. We explore the relationship between obesity and immune cell counts in women in a longitudinal study of 322 women from 1999 through 2003 enrolled as HIV-negative comparators in the Women's Interagency HIV Study. Body mass index (BMI, kg/m(2)) was categorized as normal weight (BMI 18.5-24.9), overweight (BMI 25-29.9), obese (BMI 30-34.9), and morbidly obese (BMI >/=35). CD4 and CD8 counts and percents and total lymphocyte and white blood cell (WBC) counts were measured annually using standardized techniques. A mixed model repeated measures analysis was performed using an autoregressive correlation matrix. At the index visit, 61% of women were African American; mean age was 35 years, and median BMI was 29 kg/m(2). Immunologic parameters were in the reference range (median CD4 count, 995 cells/mm(3); CD8 count, 488 cells/mm(3); total lymphocyte count, 206 cells/mm(3); median WBC, 6 x 10(3) cells/mm(3)). In multivariate analyses, being overweight, obese, or morbidly obese were independently associated with higher CD4, total lymphocyte, and WBC counts than being normal weight; morbid obesity was associated with a higher CD8 count. The strongest associations between body weight and immune cell counts were demonstrated in the morbidly obese. Increasing body weight is associated with higher CD4, CD8, total lymphocyte, and WBC counts in women. Investigation into the impact of obesity on immune function and long-term adverse outcomes is needed.

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Three-color supplement to the NIAID DAIDS guideline for flow cytometric immunophenotyping

Abstract: Health and Welfare Canada, Ottawa, Ontario (F.M.); and Division of AIDS, NIAID, NIH, Bethesda, Maryland (D.L., J.K.)'Section nurnberingsystern isdesigned to maintain continuity with the 1993 "NIAID DAIDS Guidelinesfor Flow Cytornetric Immunotyping" (1).

Cited by 61 publications

References 4 publications

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

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

Association of Selected Phenotypic Markers of Lymphocyte Activation and Differentiation with Perinatal Human Immunodeficiency Virus Transmission and Infant Infection

Obesity and immune cell counts in women

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