7
FLOW CYTOMETRIC DETECTION OF B-CELL
NON-HODGKIN'S LYMPHOMA
M STETLER-STEVENSON
INTRODUCTION
Numerous studies have been conducted in the past
decade, relating patterns of antigen expression to the histologic classification
of lymphoid lesions with particular emphasis placed on differentiating
between non-Hodgkin's lymphoma and reactive lymphoid lesions. In flow cytometry,
light chain restriction has been an important indicator of B-cell neoplasia.
However, not all B-cell neoplasms express light chains, polyclonal B-cell
populations may be admixed in with monoclonal populations, and T-cell neoplasms
can not be evaluated in this manner. As our knowledge and sophistication
has increased, the field has moved far beyond simple light chain restriction
so that flow cytometric evaluation is an important diagnostic tool in the
evaluation of lymph nodes, spleen, blood and bone marrow for neoplastic
lymphoid populations. Indicators of B-cell neoplasia include light
chain restriction, absence of normally expressed antigens and presence
of antigens not normally present on mature peripheral B-cells.
LIGHT CHAIN RESTRICTION
The most definitive and useful immunophenotypic
determination in diagnosing lymphoma is light chain restriction. Immunoglobulin
light chain restriction is, with rare exceptions considered diagnostic
for B-cell neoplasia. Therefore, analysis of light chain expression
is one of the most important determinations in immunophenotyping of patient
specimens. In reactive lymphoid populations there is a mixture of kappa
and of lambda positive cells, with cells expressing kappa light chains
outnumbering cells expressing lambda light chains. Kappa expression is
also more common than lambda in lymphoid neoplasms with the exception of
hairy cell leukemia and mantle cell lymphoma. Light chains are not expressed
on the surface in plasmacytoid lesions and in early B-cell lesions. With
higher grade B-cell lymphomas there is a decreased incidence of light chain
expression. Monoclonal B-cells express a uniform quantity of surface Ig
while polyclonal B-cells have a heterogeneous surface Ig expression. This
results in a narrower distribution of staining intensity with anti light
chain reagents in monoclonal B-cells in comparison to polyclonal B-cells.
Numerous technical factors, such as antibody choice and cytophilic antibody
artifact, impact upon a laboratory's ability to perform this test. To correctly
assess clonality in biological fluids one must be able to detect small
numbers of monoclonal B-cells in the presence of polyclonal B-cells, debris
and cells coated with cytophilic antibodies. In addition, neoplastic B-cells
may express light chain epitopes not readily detected by all antibod-ies.
We have investigated methods for light chain analysis in 104 normal donors
and 375 patient specimens, comparing different anti-light chain antibodies
as well as strategies for analysis of specimens with low numbers of monoclonal
B-cells or cytophilic antibodies (5).
Polyclonal vs. Monoclonal Anti-Light Chain Reagents
There are conflicting reports concerning efficacy
of polyclonal verses monoclonal antibodies in detecting clonal B-cell populations.
Older reports indicate a preference for polyclonal antibodies in light
chain detection, indicating that monoclonal antibodies may fail to detect
some clonal B-cell populations (6,12). However, several investigators have
found similar staining with polyclonal and monoclonal anti-light chain
antibodies (1, 8, 10). We found that there was no statistically significant
difference in percent of kappa or lambda expressing B-cells as detected
by two polyclonal and one monoclonal sets of light chain reagents in nonneoplastic
normal controls. However we did find a difference in antibody performance
in neoplastic B-cell populations. The Becton Dickinsen monoclonal
Simulset light chain reagents had a higher sensitivity compared to the
polyclonal antibodies studied in detection of light chain restriction in
grossly monoclonal specimens. In these specimens, the Biosource International
polyclonal antibodies failed to demonstrate light chain restriction in
34% of cases, while the Sanofi Int. polyclonal antibodies failed in 22%.
The superior performance by the monoclonal light chain reagents used in
this study may be due to advances in monoclonal antibody technology in
general or to a serendipitous development of two superior anti-light chain
clones (5).
Cytophilic Antibody
Detection of monoclonal B-cell populations is occasionally
hampered by cytophilic antibodies. Immunoglobulin in the plasma may be
passively absorbed by Fc receptors present on cells resulting in apparent
surface light chain expression. Natural killer cells, activated T-cells,
monocytes, granulocytes, and some B-cells have Fc receptors capable of
binding secreted soluble Immunoglobulin in vivo and therefore stain artifactually
with anti-kappa and anti-lambda antibodies (7). Normal B-cells, however,
have infrequent nonspecific Ig binding (4). Investigators have found that
the masking effect of non B-cell binding of cytophilic immunoglobulin could
be eliminated by washing with PBS at 37o C prior to staining with antibodies
(8, 9, 13). However, Agrawal et al., (1) found that 1 of 51 samples could
not be interpreted because of cytophilic antibodies even though cells were
pre-incubated at 37o C for one hour prior to staining. Letwin et al., (10)
found that two color immunofluorescence using anti-CD20 or CD19 for selection
of B lymphocytes before analysis of surface light chains was useful in
eliminating the artifact introduced by non-B-cell cytophilic antibody staining.
We found that appropriate choice of antibody combinations eliminated cytophilic
antibody artifact (5). In the vast majority of cases, the method of Letwin
et al., (10), involving simple pairing of anti-light chain reagents with
CD20 or CD19 antibodies to gate out non-B-cells, removed cytophillic artifact
from the analysis gates. In only 9 (11%) of the cases was the cytophillic
antibody bound to the neoplastic B-cells. In all nine cases, simple pairing
of kappa and lambda reagents in the same tube allowed detection of monoclonality.
Low Numbers of B-Cells/Admixed Polyclonal B-Cells
Detection of monoclonal B-cells in specimens with
low numbers of B-cells or with admixed polyclonal B-Cells requires additional
manipulations. Clonal excess assays using Kolmogorov-Smirnov statistics
were initially used to detect small numbers of monoclonal B-cells. The
clonal excess test is based upon the observation that monoclonal B-cells
express a uniform quantity of surface Ig while polyclonal B-cells have
a heterogeneous surface Ig expression. This results in a narrower
distribution of staining intensity in monoclonal B-cells in comparison
to polyclonal B-cells. Using this method, populations of monoclonal B-cells
comprising from 1-5% of the specimen have been detected (2,3,11). Factors
such as intensity of antigen expression, co-expression of antigens, and
cell size can be used to target neoplastic B-cell populations for
light chain analysis and thus increase sensitivity. Neoplastic B-cells
frequently have higher or lower levels of B-cell antigen expression. In
addition, they may have a homogeneous size reflected by clustering in regions
of small or large forward scatter (e. g., small lymphocytic lymphoma or
large cell lymphoma). By using FSC and SSC versus B cell antigen
expression, the neoplastic cells can be separated from the polyclonal B-cells
based upon size and intensity of antigen expression. We call this technique
a “clonal search.” In our experience, clonal search significantly increases
the sensitivity of light chain detection. We have identified monoclonal
B-cell populations with light chain restriction in 54% of the cases in
which clonal searches were performed. Monoclonal populations were not observed
using standard light chain analysis methods in these cases. With live gating
(selective enrichment of a population) and pairing of CD19 and CD20 with
anti-light chain antibodies, less than 1% abnormal B-cells can be routinely
identified in a clonal search (5).
ANTIGEN ABSENCE
Neoplastic B-cells may demonstrate an absence of
normally expressed antigens. Approximately 25% of B-cell lymphomas fail
to exhibit surface light chain expression. This is more common in intermediate
or high grade lymphomas as well as plasmacytoid differentiation. In addition,
25% or greater of B-cell lymphomas fail to express one of the normal B-cell
antigens (CD19, CD20, CD22) (14).
INAPPROPRIATE ANTIGEN EXPRESSION
Neoplastic B-cells may contain antigens not normally
present on mature B-cells. An excellent example is the expression of CD5
on the surface of the neoplastic cells in chronic lymphocytic leukemia,
mantle cell lymphoma, and malignant lymphoma, diffuse small lymphocytic
type. CD5 is normally expressed by T-cells, pre B-cells and fetal B-cells.
It is expressed in low numbers
and at low levels in peripheral blood B-cells. Demonstration of significant
numbers of peripheral blood or lymph node B-cells with robust surface CD5
is therefore abnormal (14).
SUMMARY
Flow cytometry is a sensitive technique that, when
applied to immunophenotyping of specimens, allows detection of low numbers
of neoplastic cells among a predominately non-neoplastic population. Demonstration
of light chain restriction is the most definitive evidence of B-cell neoplasia,
although antigen absence and abnormal antigen expression it also of value.
Proper choice of antibody combinations, use of several anti-light chain
antibodies and the analysis methods used can increase the sensitivity of
the test.
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