Enumeration of Absolute Cell Counts Using unit 68 Immunophenotypic Techniques

At present the enumeration of absolute cell numbers is relevant in different research settings (e.g., cell culture) as well as in the clinical laboratory.

Absolute counting of cells or cell subsets by flow cytometry is an established technique in at least three major clinical settings:

1. Enumeration of residual leukocytes as part of the quality control of leukoreduced blood products;

2. Enumeration of CD4+ and CD8+ T cells in HIV disease monitoring;

3. Enumeration of CD34+ hematopoietic stem and progenitor cells.

Accordingly, counting absolute numbers of CD4+ T cells, CD34+ hematopoietic precursor cells (HPC), and residual leukocytes in whole blood by flow cytometry represents diverging clinical aspects of the same technical challenge. Most efficient procedures include immunofluorescence cell analysis linked to absolute cell enumeration, as well as to some stringent gating strategy in order to identify relevant and eliminate irrelevant cell populations with high efficiency. However, as the percentages and absolute cell-level ranges in these varied clinical settings are widely different, diverging technologies are necessary to achieve the necessary corresponding sensitivity, precision, and counting accuracy. The importance and technical requirements of CD34+ hematopoietic stem and progenitor cell enumeration are discussed in unit 6.4.

In the case of blood bank quality control, the enumeration of residual white blood cells (WBCs) in filtered blood products is a significant assay. The composite technology includes detection of cells based on cell morphology, nuclear fluorescence, and flow rate count with fluorospheres. The enumeration procedure is usually accomplished by flow cytometry. An accurate count is critical to prevent febrile reactions, microrganism transfer, and alloimmunization.

Enumeration of absolute CD4+ T lymphocyte number continues to be the hallmark laboratory test for staging HIV-infected patients. This is a critical surrogate marker for assessing immunodeficiency. The T cell subset value is an independent marker, yet it complements HIV plasma viral load data. As potent anti-HIV therapies are becoming more effective and complex, the CD4+ T cell levels for diagnostic/prognostic staging of patients and therapeutic/prophylactic intervention will continue to shift (Johnson et al., 1995; Lane, 1994). However, the utility of the CD4+ T cell count remains unchallenged and critical (Nicholson et al., 1994). The absolute and percent CD4+ T cell count is also of clinical relevance in other immunodeficiency conditions. These include solid-organ transplantation, the post-chemotherapy period, the recovery phase following bone marrow (or stem cell) transplants, therapy with purine nucleosides like 2-chlorodeoxyadenosine (cladribine) for hairy-cell leukemia, cytomegalovirus infection in immunocompromised patients, and protein-calorie malnutrition.

Initially, in the early 1980s, design limitations of flow cytometry restricted the availability of accurate CD4+ T cell count to the immunophenotypical identification and enumeration of lymphocyte frequencies (unit 6.5). This technical deficiency, at the time, appeared to be just a minor compromise. The additional information required to obtain absolute numbers was readily available from the hematology department. More recently, it has become possible to obtain absolute count with just a flow cytometer. It is now no longer necessary to combine data with a hematology workstation to generate absolute counts with flow cytometry. These two clinical methods for obtaining absolute counts are Phen°typte

Analysis

Contributed by Frank Mandy and Bruno Brando

Current Protocols in Cytometry (2000) 6.8.1-6.8.26 Copyright © 2000 by John Wiley & Sons, Inc.

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