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Phenotypic Analysis
sing fluorescently conjugated antibodies to identify specific antigens in or on cells
is a well-documented, simple, and straightforward procedure. In contrast to staining cells with various broad-range dyes, staining with antibodies (immunophenotyping)
provides high sensitivity and specificity. Three major approaches to immunophenotyping
are immunohistochemistry, fluorescence microscopy, and flow cytometry. In immunohistochemistry, which is used most commonly by clinical pathologists, cells on microscope
slides are stained with a specific antibody and counterstained with a secondary reagent
to develop a color reaction using an absorptive dye. Presence or absence of color can
then be observed with a standard light microscope. This approach has two drawbacks.
First, nonspecific binding can lead to misinterpretation, making the test somewhat subjective. Second, because only one antibody is used per slide, it is not possible to determine
precisely the coexpression of antibodies.
A second approach, which offers the best approach for evaluating multiple antigens simultaneously on large numbers of cells in a short time, is flow cytometry, presented in
this chapter. Two-, three-, and four-color assays are customary. The greatest disadvantage
is the requirement for a monodispersed cell suspension, as this means that the organization and architecture of cells within tissue is lost and that entire cell populations can
disappear during tissue processing. Because of this limitation, flow cytometric identification of antigens has had its greatest impact in hematology and immunology, where the
majority of cells are already in single-cell suspension. Several of these applications, such
as the enumeration of CD34+ hematopoietic progenitor cells in samples that will be used
for transplantation, have proved to be of great clinical utility. Basic techniques for immunophenotyping by flow cytometry, designed primarily for such single-cell material, are
presented in UNIT 6.2. Quality control parameters relevant to these procedures are discussed
in UNIT 6.1. Two other units are specifically dedicated to high-sensitivity immunofluorescence detection of membrane molecules present at low and very low concentrations using
classical (UNIT 6.3) and innovative (UNIT 6.14) multistep amplification procedures. Specific
protocols for the enumeration of CD34+ hematopoietic progenitor cells and peripheral
blood lymphocytes are described in UNIT 6.4 and UNIT 6.5, respectively. Recent reports have
also shown that flow cytometry immunophenotyping is well suited for rare-event analysis;
the immunophenotypic identification, enumeration, and characterization of human mast
cells and dendritic cells, detailed in UNIT 6.6 and UNIT 6.9, respectively, represent a clear
example of the utility of flow cytometry in this field. Apart from the analysis of normal
cells, clinically useful applications of flow cytometry to immunophenotyping have also
extended to the identification and study of pathologic leukocytes and other blood cells in
several different disease conditions. Three examples of flow immunophenotyping protocols which have proved to be of diagnostic utility are described in UNIT 6.7—measurement
of the ability of in vitro–activated T lymphocytes to express CD40 ligand for the diagnosis of X-linked hyper-IgM syndrome—, UNIT 6.11—immunophenotypic diagnosis of
PNH+ (paroxysmal nocturnal hemoglobinuria) cells–, and UNIT 6.19–ZAP-70 staining in
chronic lymphocytic leukemia. In the last decades, immunophenotypic characterization
of normal and pathological cells by multiparameter flow cytometry has evolved from the
Contributed by Alberto Orfao
Current Protocols in Cytometry (2005) 6.0.1-6.0.2
C 2005 by John Wiley & Sons, Inc.
Copyright 6.0.1
Supplement 32
study of surface to that of intracellular markers. In UNIT 6.20 several specific protocols are
described for the intracellular analysis of phosphoepitopes in immunophenotyped cell
populations by flow cytometry.
In addition to being a target for immunophenotyping, cells, and alternatively beads, can
be used as the layer for an immunological reaction devoted to the detection of soluble
antibodies. Such procedures have proved to be of great clinical utility in transplantation
for rapid screening for the presence of serum antibodies whose specificity is directed
against donor cells. UNIT 6.16 describes protocols for the flow cytometric assessment of
serum anti-HLA alloantibodies.
Platelet and red cell evaluation is another application of flow cytometry with considerable
clinical usefulness. UNIT 6.10 is dedicated to the immunophenotypic analysis of platelets and
platelet activation through the identification of platelet- and platelet activation–specific
surface markers. UNIT 6.15 specifically describes a protocol for the identification and enumeration of leukocyte-platelet aggregates as a method to evaluate platelet activation.
Finally, UNIT 6.17 details protocols for the identification and enumeration of fetal red blood
cells, F cells, and F reticulocytes.
For many of the applications of immunophenotyping, either enumeration of specific
cell subsets per unit sample volume or quantitative evaluation of the levels of antibody
bound per cell is mandatory. UNIT 6.8 is specifically dedicated to the enumeration of
absolute cell counts using flow cytometry-based immunophenotypic techniques. In UNIT
6.12, protocols for the evaluation of the levels of antigen expression per cell using flow
cytometry immunophenotyping-based approaches are described in detail.
Among the fluorochrome-conjugated proteins other than antibodies which are used to
stain cells, tetramers (and other multimers) of HLA class I and HLA class II loaded
with specific peptides have emerged as a particularly attractive tool for immunophenotypic identification, enumeration, and characterization of antigen-specific T lymphocytes.
Protocols for the phenotypic analysis of CD8+ and CD4+ T cells using fluorochromeconjugated HLA tetramers are described in UNIT 6.18.
A third approach, similar to immunohistochemistry and exhibiting some of the same
disadvantages, is the development of microscope-based imaging systems. Nonspecific
binding can be significantly reduced by using directly conjugated primary antibodies,
thereby eliminating the need for secondary reagents. Furthermore, simultaneous measurement of multiple colored fluorochromes conjugated to different antibodies makes
it possible to detect the coexpression patterns of different antigens on the same cells
and allows identification of aberrant antigen expression. This methodology, described in
Chapter 8, has the advantage over flow cytometry that morphological characteristics are
preserved. Its main limitation is the time involved in investigating large numbers of cells.
More recent instruments such as the laser scanning cytometers (LSC) have been developed which are capable of analyzing cells placed on a slide and which overcome several
of the major limitations of conventional microscopy-based systems; at the same time LSC
is particularly well-suited for the analysis of small samples which contain low numbers of
cells. UNIT 6.13 describes three- and four-color immunophenotypic assays for the analysis
of the expression of cell surface proteins in peripheral blood-derived leukocytes using
Alberto Orfao
Supplement 32
Current Protocols in Cytometry
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