Cytofluorometric methods for assessing absolute numbers of cell subsets in blood

Abstract

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)؉ individuals (CD4؉ T-lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34؉ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single-or multiple-color cell staining and logical gating strategies. These can be accomplished using single-or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources. Cytometry (Comm. Clin. Cytometry) 42:327-346, 2000.

Values for αβ and γδ T‐lymphocytes and CD4+, CD8+, and CD56+ subsets in healthy adult subjects: Assessment by age and gender

“…This single-platform approach offers fewer sources of variability. Some earlier studies, however, have found a good correlation of these different methods (12,18).…”

“…A minimum of 30,000 events were measured. Absolute counts of circulating cell subsets were calculated using the percentages obtained by flow cytometry, and the leukocyte count was obtained from the hematological analyzer, using a dual-platform counting technology (18).…”

A six‐color flow cytometric assay for the analysis of peripheral blood dendritic cells

“…This is achieved by the inclusion of fluorospheres as internal controls that allow precise cellular quantification and the adoption of a Lyse/No-wash flow cytometric protocol that prevents potential cell loss during washing steps (24). Although single-platform assays are routinely used in many clinical and research applications (25,26) their use in PBDC counting remains anedoctical (27,28). Because of the growing clinical relevance of PBDC assessment, we suggest that a single-platform approach should be largely applied to PBDC counting to improve the reliability of PBDC measurements.…”

Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers

“…16,18,19 At each step, cells were analyzed for percentage and viable absolute number of CD56 þ CD3 À , CD3 þ and by flow cytometry in single and dual platform approaches as described previously. 20,21 In case of a contaminating T-cell dose of X1.0 and o2.0 Â 10 5 /kg, the processed NK product was split into two units. In both centers, all patients received the NK-DLI unit freshly isolated at day þ 40 post HSCT; the next unit at day þ 100 was always cryopreserved.…”