Page 1283 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPtEr 92 Flow Cytometry 1245

TABLE 92.1 Selected lymphocyte Surface reporting of both percentages and absolute numbers is necessary
antigens for Immunophenotyping when immunophenotyping peripheral lymphocytes.
The objective of evaluating malignant cells is often to character-
t Cells ize the lineage and differentiation level of the abnormal cells,
Pan-T cell: CD3, CD2, CD7, CD5 rather than quantifying subpopulations. The pattern of reactivity
Major T-cell subset: CD4, CD8 combined with fluorescence intensity is often useful in identifying
Surface antigens associated with function: CD28, CD31, CD38,
CD45RA, CD45RO, CD62L, CXCR7 leukemic patterns, whereas the absolute number of cells usually
Activation antigens: CD25, CD40L, CD69, CD71, HLA-DR is not required. However, flow-cytometric detection and quantita-
tion of rare abnormal cells can be useful in evaluating for minimal
B Cells residual disease after therapy in lymphoproliferative disorders.
Pan-B cell: CD19, CD20, surface immunoglobulin
Major B-cell subset: CD5, CD21 PRACTICAL APPLICATIONS OF FLOW CYTOMETRY
Surface antigens associated with function: CD27, CD40, CD80, CD86
Activation antigens: CD23, CD25 Immunophenotyping Studies
nK Cells
Pan-natural killer (NK) cell: CD16, CD56 ClInICal rElEvanCE
NK subset: CD2, CD8, CD57 Immunophenotyping Studies
• Immunotyping studies can be used to identify cell subsets, lineage,
stage of cell differentiation, state of cell activation, and clonality.
• Lymphocyte results should be checked with T cells + B cells + natural
identify a similar cell subpopulation can serve as a useful check killer (NK) cells = 100%.
(e.g., total T cells by comparing CD3 and CD5 or CD2; total B • Immunophenotyping studies are not the equivalent of lymphocyte
function studies.
cells by comparing CD19 and CD20). In addition, the use of
specific reagents in >1 tube enables comparison between the
repeat values as a measure of consistency. The application of Most immunophenotyping studies aim to enumerate specific
internal checks should be performed by the flow operator as a cell subpopulations, evaluate for the presence or absence of
simple means of confirming the validity of the data. Insights particular surface antigens, identify the differentiation level of
regarding unusual biological findings may also be uncovered specific cells, determine cell lineage, evaluate for functional
through this type of evaluation (e.g., the presence of an increased correlates based on specific antigen expression, examine evidence
−
−
population of CD4 /CD8 double-negative T cells). 25 of cell activation, and/or establish evidence of monoclonality.
The challenge in performing immunophenotyping is to Quantification of a particular cell subpopulation can be readily
accurately identify cells with specific surface characteristics accomplished by flow cytometry together with a WBC count
(antigens). As previously noted, the capacity to discriminate cell and cell differential. The absolute lymphocyte count is used to
subpopulations is often enhanced through the directed use of generate population or subpopulation cell numbers based on
antibody combinations. The typical data generated consist of the percentages of the respective cell types generated with the
the percentage of negative versus positive cells when using one flow cytometer. The evaluation of absolute CD4 T-cell counts has
26
reagent and multiple subpopulations when using more than one formed the basis for monitoring patients with HIV infection.
+
reagent. Regardless of the experimental design, it is important The quantitation of CD34 hematopoietic stem cells (HSCs) in
to consider not only the percentage of cells within each subpopula- donor peripheral blood or bone marrow is used in many cellular
27
tion but also the absolute number of cells. This is most commonly reconstitution protocols. Subpopulation characterization can
obtained by multiplying the relevant percentage from the flow also be useful in the evaluation of patients with clinical history
28
cytometer by the absolute lymphocyte count obtained by using and laboratory findings suggestive of immune deficiency.
a white blood cell (WBC) count and differential. For example, This has taken on a higher level of significance in the setting
+
when assaying for CD4 T-cell counts, the percentage of CD4 of newborn screening for severe T-cell immune deficiency via
cells is multiplied by the peripheral absolute lymphocyte count the T-cell receptor excision circle (TREC) assay, which, when
to yield the absolute CD4 count. A potential problem with this abnormal, is typically followed by immunophenotyping to
approach is the requirement for two separate procedures (i.e., evaluate for naïve T cells based on CD45RA, CD127, or CD62L
dual platforms) to generate the final result. This introduces the expression.
possibility of additive error, based on the inherent errors of the When evaluating for the presence or absence of surface proteins
two different methods. It also has fueled a search for approaches associated with specific functional attributes, it is important to
that facilitate performing both tasks by flow cytometry (i.e., a realize that this approach does not assess the actual functional
single platform). One alternative involves the inclusion of a fixed status of cells. This point is clearly illustrated by the finding of
number of fluorescent beads (in a defined volume) in each tube normal B-cell numbers in most patients with common variable
as a reference standard to generate absolute numbers without immune deficiency despite the fact that these patients fail to
29
requiring the use of the complete blood count (CBC) and dif- produce Igs normally. However, changes in the characteristics
ferential to generate a lymphocyte count. An alternative approach of the B cells, particularly relative to memory B cells, provides
involves the use of impedance-based cell counting in the flow potential insight into different phenotypes of this disorder and
cytometer to generate an absolute lymphocyte count (dependent provides additional support for heterogeneity of patients with
on a fixed volume of sample being run) and then generating the disease. 29-31 Because of the limitations of immunophenotyping,
both percentage and absolute number of cells in each specific when evaluating the status of the immune system, it is common
population or subpopulation. Regardless of the approach, the practice to perform cell function testing in parallel.