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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3889-3896
Immunophenotypic Analysis of Peripheral Blood and Bone Marrow in the
Staging of B-Cell Malignant Lymphoma
By
Curtis A. Hanson,
Paul J. Kurtin,
Jerry A. Katzmann,
James D. Hoyer,
Chin-Yang Li,
Janice M. Hodnefield,
Cecelia H. Meyers,
Thomas M. Habermann, and
Thomas E. Witzig
From the Departments of Laboratory Medicine and Pathology and of
Internal Medicine, Mayo Clinic, Rochester, MN.
 |
ABSTRACT |
This study evaluated the contributing roles of flow cytometric
immunophenotyping of blood and bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in the staging of
B-cell malignant lymphoma. Flow immunophenotyping was performed on a
marrow specimen in 175 cases; a corresponding blood specimen was also
immunophenotyped in 135 of these cases. Morphologic marrow involvement
by lymphoma was found in 59 cases; flow immunophenotyping identified 54 cases with a monoclonal B-cell process:
morphology-positive/flow-positive (n = 49),
morphology-positive/flow-negative (n = 10),
morphology-negative/flow-positive (n = 5), and
morphology-negative/flow-negative (n = 111). The 10 morphology-positive/flow-negative cases included 5 follicular and 5 large-cell lymphomas with minimal marrow involvement. All 5 morphology-negative/flow-positive cases were from patients with large-cell lymphomas and bulky clinical disease. Because the blood contained the same B-cell clone in 2 of 2 morphology-negative/flow-positive cases studied, blood contamination of
marrow may account for these findings. Blood flow cytometric
immunophenotyping studies were positive in 32 cases; 30 had marrow
involvement by morphology and were from patients with follicular,
mantle cell, lymphoplasmacytic, small lymphocytic, or marginal zone
lymphomas. From our results, we conclude that (1) bone marrow flow
cytometric immunophenotyping is not a cost-effective replacement for
good morphologic evaluation in lymphoma staging and that (2) a positive
peripheral blood flow cytometric immunophenotyping study when performed
in low-grade lymphomas correlates with marrow involvement.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MALIGNANT LYMPHOMA is the most common
hematologic malignancy encountered in the Western world.1,2
Over the last decade there has been a continual growth in the number of
ancillary tools that can be used in the laboratory to evaluate
malignant lymphoma.3-10 This has proved critical in
providing the consistent and accurate information that is needed for
clinical decision making in these patients. The laboratory evaluation
of patients with malignant lymphoma remains centered on 4 primary
aspects: (1) recognition and diagnosis of disease; (2) appropriate
classification11; (3) providing information regarding
disease stage; and (4) providing prognostic indications that predict
the risk of death from disease.12,13
A complete and accurate staging of the patient with malignant lymphoma
is essential in determining the extent of disease, which may affect
both the prognosis and the potential therapeutic options. Staging of
patients with malignant lymphoma has become an increasingly
noninvasive, radiologic-based process.1,2,14 However,
obtaining a bone marrow (BM) biopsy has remained an important part of
evaluating these patients. BM involvement by lymphoma has traditionally
been detected by morphologic assessment of biopsy specimens. Compared
with unilateral BM biopsies, the evaluation of bilateral trephine
specimens apparently increases the rate of detection of lymphoma by
10% to 20%,15-17 although studies have not precisely
controlled for the size of the specimen examined.
Other ancillary laboratory studies are available that can be
incorporated to aid in the assessment of lymphoma. These include flow
cytometric immunophenotyping with monoclonal antibodies (MoAbs) directed against lymphoid-associated antigens, immunohistochemical staining on paraffin-embedded BM biopsy material, and molecular studies
of Ig genes/T-cell receptor genes or lymphoma-associated gene
translocations.3-10 The precise role of these latter 3 techniques over and above morphologic assessment has not been fully
established. Despite this lack of scientific evidence, there appears to
be an increasing belief that immunophenotyping studies are crucial in
the examination of a BM specimen for possible lymphoma. Most previous
studies have looked at immunophenotyping in specific disease
entities18-22 and not at the larger issue of how these modalities should be used in the global approach to the patient with
malignant lymphoma. With this as background, it was the intention of
this study to evaluate the contributing roles of flow cytometric immunophenotyping of blood and BM and immunohistochemical paraffin section staining of BM biopsies in the staging of B-cell malignant lymphoma and to determine how best to use these laboratory resources.
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MATERIALS AND METHODS |
Adult patients undergoing a BM aspiration and biopsy who had a
preceding or concurrent lymph node or other tissue biopsy that was
diagnosed as malignant lymphoma were identified for this study. Because
 and  surface Ig analysis offers an objective method for
determining B-cell clonality, only those lymphoma cases with a B-cell
immunophenotype were selected; T-cell lymphoma and Hodgkin's disease
patients were excluded. Marrow-based lymphoproliferative disorders,
such as chronic lymphocytic leukemia (CLL), Waldenstrom's macroglobulinemia, and hairy cell leukemia, were excluded, because marrow involvement is an integral part of those diseases. One hundred
seventy-five consecutive Mayo Clinic bilateral BM specimens from
patients with a diagnosis of B-cell malignant lymphoma and meeting the
restrictions given above were identified and were collected over a
7-month period. The study protocol was reviewed and approved by the
Institutional Review Board with consent obtained before the beginning
of this study.
Wright-Giemsa-stained slides of peripheral blood (PB) and BM aspirate
smears were reviewed for atypical lymphoid/lymphoma cells, independent
of immunophenotypic studies. Bilateral BM trephine biopsies were
obtained in all cases, fixed in B5 fixative, decalcified, and
paraffin-embedded. Five biopsy levels were cut for each specimen with
the first, third, and fifth levels stained with hematoxylin and eosin
(H&E) for morphologic assessment of malignant lymphoma.
Flow cytometric immunophenotyping of an EDTA, anticoagulated BM
aspirate specimen was performed in each case. A PB specimen obtained
within 2 days of the BM specimen was also immunophenotyped by flow
cytometry in 135 of the 175 cases. A standard, whole-blood/marrow assay
with erythrocyte cell lysis was used for preparing all of the BM
aspirate and PB specimens.23-25 Because the lymphomas
associated with these cases were classified on the basis of the lymph
node/tissue biopsy, the BM and PB immunophenotypic analyses were solely
centered on whether there was involvement by malignant lymphoma, ie,
whether there was a monoclonal B-cell population present. As such, all cases had, at minimum, immunophenotyping performed with MoAbs directed
against CD19 and and Ig. These antibodies were combined as
CD19-phycoerythrin (PE)/-fluorescein isothiocyanate (FITC) and
CD19-PE/-FITC (BioSource International, Camarillo, CA). The anti-
and anti- MoAb solutions were titrated for protein concentration and
/ signals.
Determination of monoclonality was based on a combination of flow
cytometric histogram evaluation and correlation with the percentage of
cellular positivity.21,26 A normal range of -to- ratios had been previously determined in the laboratory to be 0.3:1 to
3.0:1.26 Other MoAbs evaluated in the majority of cases included CD3, CD5, CD10, CD11c, CD16/56, CD20, CD22, CD23, and CD45.
Two-color or 3-color flow cytometric analysis was used depending on the
case findings, previous immunophenotyping studies, sample availability,
etc. Both forward/side light scatter and CD45/side light scatter were
used in each case as the primary gating methodologies. Further gating
was performed as necessary on either lymphoid subpopulations based on
cell size or as backgating on CD19+ B-cell staining
events.25
Immunohistochemical staining of the paraffin-embedded BM biopsies was
performed in all cases and used standard techniques that have been
previously published.27 Antibodies used included polyclonal
anti-CD3 and monoclonal anti-CD20 (Dako Corp, Carpinteria, CA).
| |
RESULTS |
Clinical.
The 175 patients included in this study consisted of 105 men and 70 women. The ages of these patients ranged from 19 to 94 years (median,
64 years of age); 1 patient was less than 20 years old and 11 patients
were greater than 80 years of age. One-hundred seven of the 175 patients were evaluated as part of the initial disease presentation
requiring a diagnostic biopsy procedure. The remaining 68 patients were
being evaluated for a variety of reasons, including restaging of
disease while in complete or partial remission, restaging for relapsed
disease, pre-BM transplantation study, pre-protocol study, or suspected
transformation to a more aggressive disease. The 175 patients were
staged as follows: stage I = 31, stage II = 38, stage III = 14, and
stage IV = 82; the remaining 10 patients could not be accurately staged
for various reasons. The international prognostic index
(IPI)12,13 showed the following: IPI of 0 = 25, IPI of 1 = 63, IPI of 2 = 48, IPI of 3 = 26, IPI of 4 = 11, and IPI of
5 = 2.
Lymph node/tissue/marrow biopsy.
Table 1 shows the various lymph node/tissue
diagnoses from the cases included in this study. Large-cell lymphoma
and follicular lymphomas accounted for the majority of diagnoses.
Morphologic assessment alone of the 175 BM biopsies demonstrated that
59 cases had involvement by malignant lymphoma. As expected, the small B-cell lymphoid neoplasms (follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, and marginal zone lymphoma) had the highest percentage of cases with marrow infiltration, whereas marrow involvement was less common in the large-cell lymphomas and the low-grade lymphomas of MALT type.
Immunophenotyping: BM aspirate.
Flow cytometric immunophenotyping of the BM aspirate identified 54 cases with a monoclonal B-cell process (Table 1): 29 with Ig and 25 with Ig light chain restriction. The percentage of monoclonal B
cells detected by immunophenotyping ranged from 0.09% to 58.6%
(median, 3.2%) of the overall marrow cell population (not just the
lymphoid gate). Thirty-seven cases had small monoclonal populations
that accounted for less than 5% of the overall BM aspirate cell
specimen; 14 demonstrated less than 1% monoclonal B cells.
The 121 BM cases with a negative flow cytometric study typically showed
a mixture of polyclonal B cells and a small population of
CD19+/Ig cells that represent normal
CD19+, precursor B cells. The median -to- ratio in
these 121 cases was 1.29:1 (range, 0.27:1 to 2.94:1), confirming the
normal range previously determined in the laboratory.
The concurrence rate of morphologic assessment and flow cytometric
immunophenotyping in evaluating for the presence of lymphoma was 91.4%
(160 of 175 BM cases). Forty-nine cases showed evidence of malignant
lymphoma by both morphologic and flow cytometric immunophenotypic
modalities, whereas 111 cases were negative for malignant lymphoma by
both methods (Table 2).
It is the remaining 15 cases that deserve additional description. Ten
cases demonstrated morphologic features of lymphoma on the BM biopsy
specimen and were confirmed by immunohistochemical staining, but had a
polyclonal B-cell immunophenotypic pattern. Subgating based on cell
size and backgating with CD19 or other antibodies failed to identify a
monoclonal or abnormal B-cell population. / ratios ranged from
0.20 to 1.8:1 in these 10 cases. Five of the 10 cases were from
patients with follicular lymphoma, predominantly small cleaved type,
and 5 were from patients with large-cell lymphoma
(Fig 1). One of the latter 5 patients with large-cell lymphoma had a discordant BM morphology with primarily a
small, cleaved cell infiltrate seen in the BM sections. In addition to
morphologic features indicative of lymphoma, all 10 cases demonstrated a prominent CD20+, lymphomatous infiltrate by
immunohistochemistry on paraffin sections of the marrow biopsies. The
extent of involvement by lymphoma in these cases was quite variable,
with 4 showing small, focal, paratrabecular patterns of infiltration; 4 showing less than 20% involvement in a random, nodular pattern; and 2 cases with single, albeit relatively large, foci. It seems reasonable to conclude that the discordance seen in these 10 cases may be ascribed
to either variation in sampling between the aspirate and biopsy, the
minimal marrow disease seen in some biopsies and not represented in the
aspirate specimen, or failure to aspirate the diagnostic cells due to
reticulin fibrosis that can be associated with a lymphomatous
infiltrate.16,28
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| Fig 1.
(A) BM biopsy showing a paratrabecular lymphoid
infiltrate of atypical lymphoid cells, diagnostic of involvement by
malignant lymphoma and confirmed by CD20+
immunohistochemical staining. A lymph node biopsy showed a follicular
lymphoma. Flow cytometric studies demonstrated polyclonal B cells with
no monoclonal B-cell population identified (H&E; original magnification × 400). (B) BM biopsy showing a nodular lymphoid infiltrate of
atypical, large lymphoid cells, diagnostic of involvement by malignant
lymphoma and confirmed by CD20+ immunohistochemical
staining. A lymph node biopsy showed a large-cell lymphoma. Flow
cytometric studies of the marrow aspirate demonstrated polyclonal B
cells with no monoclonal B-cell population identified (H&E; original
magnification ×400).
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The 5 BM morphology-negative, flow cytometric-positive cases were all
from patients with large-cell lymphomas
(Table 3). Four patients, independent of
the flow results, had localized, but bulky clinical disease: 1 with
stage I, 2 with stage II, 1 with stage III, and 1 with widespread stage
IV disease. All were treated with chemotherapy,
independent of knowing the positive flow cytometric immunophenotyping
results. No morphologic or immunohistochemical evidence of lymphoma was
seen in these 5 BM biopsy specimens (Fig 2). Substantial bilateral biopsy material was available, ranging from 2 to 6.5 cm of trephine biopsy. Interestingly, the PB contained the same
B-cell clone in 2 of 2 cases in which concomitant blood immunophenotyping was also performed. These 5 cases showed small monoclonal populations by flow cytometric analysis that ranged from
0.09% to 3% of the overall cell population analyzed, indicating that
all cases had an extremely small population of clonal cells. Unfortunately, insufficient follow-up time has accrued that would allow
clinical outcome analysis in this interesting subset of patients.
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| Fig 2.
BM biopsy showing a normocellular marrow with no
morphologic features of lymphoma, confirmed by negative
immunohistochemical staining. A lymph node biopsy showed a large-cell
lymphoma. Flow cytometric studies of the marrow aspirate demonstrated a
small population of monoclonal B cells (H&E; original magnification × 400).
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Immunophenotyping: PB.
PB immunophenotyping studies showed a monoclonal B-cell population in
32 of the 135 cases analyzed (Table 4).
Thirty of these 32 flow-positive cases had concurrent BM involvement as
assessed by morphologic and immunophenotypic findings. The 2 remaining flow-positive cases were the morphology-negative, large-cell lymphomas previously discussed. Table 4 shows the lymph node diagnoses in these
32 patients with positive blood studies. Not surprisingly, the
blood-positive cases were from patients with follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphomas, lymphoplasmacytic lymphoma, or splenic marginal zone lymphoma, all known to have a
relatively high incidence of PB
involvement.3,6,16,20,22,29,30 Although a positive PB
immunophenotyping study was usually associated with BM involvement by
lymphoma, a polyclonal blood study did not exclude the possibility of
lymphoma involving the marrow. Table 4 indicates that follicular
lymphoma, mantle cell lymphoma, lymphoplasmacytic lymphoma, and even
small lymphocytic lymphoma do not always demonstrate a monoclonal
B-cell population in the PB. The percentage of monoclonal B cells
detected in this study by immunophenotyping of the PB ranged from
0.01% to 49.2% (median, 2.1%) of the overall leukocyte population
(not the percentage of the lymphoid gate alone). Eighteen cases had
small monoclonal populations that accounted for less than 5% of the
overall blood cell specimen; 15 demonstrated less than 1% monoclonal B
cells.
Immunophenotyping: histogram evaluation.
Flow cytometric histogram evaluation was critical in determining or
monoclonality. Four patterns of / expression were seen in
these histograms. Bright surface Ig (sIg) expression was the most
obvious pattern to recognize and was found in 28 cases (Fig 3A). This was
reflected in the -to- ratio seen in these specimens (for ,
median of 24:1 and range of 9 to 189:1; for , median of 0.1:1 and
range of 0.01 to 0.2:1).
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| Fig 3.
Flow cytometric histograms showing distribution of
CD19/ and CD19/ staining. (A) Histograms showing bright
monoclonal surface Ig expression. (B) Histograms showing dim
monoclonal surface Ig expression. (C) Histograms showing a mixed monoclonal surface Ig expression and polyclonal B cells with a normal -to-
ratio. (D) Histograms showing a B-cell population with dim CD19 and no
surface Ig expression.
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Dim sIg expression, as typically seen in the small lymphocytic
lymphomas and chronic lymphocytic leukemia,4,6,21 reflects a diminished number of Ig molecules on the cell surface. This pattern
is easily recognized when comparing the histograms of and staining using the same fluorochrome (FITC in this study) conjugated to
the anti- and anti- antisera (Fig 3B). The distribution pattern
of surface Ig light chains can be analyzed statistically, but direct
histogram observation/analysis is easier and more reliable than using
ratios or statistics. As such, the / ratios were less dramatic in
these 11 cases, with 3 having ratios that were within the normal range
(for , median of 4.9:1 and range of 2.9 to 11.0:1; for , median
of 0.2:1 and range of 0.04 to 0.8:1).
The most difficult histogram interpretations were in those 14 cases
that had a mixture of polyclonal and monoclonal B cells (Fig 3C).
-to- ratios were of little value in these cases, and monoclonal
populations could only be recognized by and histogram pattern
comparisons (/ ratios: for , median of 2.9:1 and range of 1.3 to 4.4:1; for , median of 0.7:1 and range of 0.5 to 1.0:1).
A single case of surface Ig-negative lymphoma was identified (Fig 3D),
demonstrating a distinctly dim CD19 population that differed from the
usual CD19+ precursor cells. Surface Ig negativity was
subsequently confirmed in the diagnostic lymph node specimen of this
case by frozen section immunophenotyping.
Immunohistochemistry.
Immunohistochemical staining of the paraffin-embedded trephine sections
was performed in all cases with antibodies to CD3 (polyclonal) and
CD20. The immunohistochemical stains showed close correlation with the
morphologic findings with concurrence in 97.7% of cases (171 of 175;
Table 5). Three cases with morphologic features of malignant lymphoma did not show the lymphomatous infiltrate on the recut, biopsy sections, thus obviating the ability to assess interpretation. One case showed 2 tiny, distinctly paratrabecular foci
of CD20+ B cells, which are suggestive, but not definitive
for involvement by that patient's known follicular lymphoma. Lymphoid
aggregates were identified in 48 cases, but were not interpreted as
representing malignant lymphoma by the assessment of the morphologic
features of these aggregates. All of these latter 48 cases showed
either a mixture of CD20+, small B cells and
CD3+ T cells or an exclusive T-cell population.
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DISCUSSION |
The role of evaluating BM trephine biopsy sections is well-established
in the staging of malignant lymphoma. Staging for disease as determined
by marrow involvement has a clear implication for survival of patients
with malignant lymphoma and current therapeutic protocols may be
directed according to stage of disease.1,2,14 The
international prognostic index for patients with malignant lymphoma
includes BM involvement as 1 of its prognostic
factors.12,13 Determination of marrow involvement,
therefore, remains a crucial component of the workup of the patient
with malignant lymphoma.
The findings from this study lead to several observations and
conclusions. Morphologic evaluation of bilateral BM trephine biopsies
remains the gold standard in the staging process of malignant lymphoma.
An accepted corollary is that sufficient biopsy tissue needs to be
obtained and stained with a high-quality H&E stain when relying on
morphologic findings in this manner. Because morphology alone
identified all relevant cases with marrow involvement, we conclude that
routine BM flow cytometric immunophenotyping and immunohistochemical
staining for malignant lymphoma is not a cost-effective replacement for
good morphologic evaluation in lymphoma staging. These laboratory tools
do have a role in the laboratory, but should be reserved for those BM
cases with equivocal morphologic findings or marginally adequate biopsy
specimens. Too often, these ancillary studies are interpreted in
isolation from the clinical and morphologic perspectives. There needs
to be judicious use of these technologies and development of skills and
trust in traditional morphologic assessment.
The exact role of flow cytometric analysis and immunohistochemistry
over and above morphologic assessment has never been fully established.
A study published by Naughton et al31 concluded that flow
cytometric immunophenotyping had a limited role in the staging and
follow-up of patients with malignant lymphoma. Only 3 of 273 marrow
specimens showed a combination of flow immunophenotypic positivity and
negative BM morphology. Interestingly, 2 of these 3 were from patients
with large-cell lymphomas, similar to the results found in our study.
Dunphy18 compared BM morphology and flow cytometric
immunophenotyping in 188 specimens that also included many BM-based
disorders, such as CLL and hairy cell leukemia. More
than 80% of the cases in that study showed concordance between morphology and immunophenotypic studies, whereas less than 5% demonstrated immunophenotypic positivity in morphologically negative or
borderline BM cases. Fineberg et al19 also evaluated the yield of immunophenotyping in BMs for malignant lymphoma. However, all
cases in the report of Fineberg et al had morphologic evidence of BM
involvement by either lymphoma or CLL, and it was not a broad-based
study of malignant lymphoma. Most previous studies using
immunophenotypic laboratory methods have looked at specific disease
entities, such as Waldenstrom's macroglobulinemia, mantle cell
lymphoma, etc,20,22 and not at the larger issue of how these modalities should be used in the global approach to the patient
with malignant lymphoma. Coad et al10 looked at the role of
molecular gene rearrangement studies by polymerase chain reaction in BM
aspirates and biopsies from patients with malignant lymphoma; no
comparison with immunophenotyping was performed. This study showed that
43% of marrows that had morphologic evidence of lymphoma failed to
show clonality by molecular methods, again raising awareness of the
limitations of interpreting laboratory tests independent of the
morphologic findings.10
The 5 morphologically negative, flow cytometric
immunophenotypic-positive cases associated with large-cell lymphoma
raise interesting questions without obvious answers. All 5 of these cases were from patients with large-cell lymphoma, with 4 having localized, but bulky clinical disease. Biologically, are these findings
the result of distant, PB seeding by the bulky malignant lymphoma with
blood contamination of the marrow aspirate? Thus, if this phenomena did
occur, these would not represent true stage IV disease and should be
staged independently of the flow cytometric results. Alternatively, if
these cases actually have definite marrow involvement arising from
perhaps closely adjacent BM sites that were not represented in the
biopsy specimens obtained, they would represent true stage IV marrow
involvement. It is hard to fathom the latter possibility, because these
5 specimens were exceedingly generous in quantity and showed absolutely
no morphologic or immunohistochemical evidence of involvement, both
reliable means of detecting disease. Therapeutically, these 5 patients with large-cell lymphomas were treated with systemic chemotherapy, regardless of the flow findings. However, prognostically, if a positive
flow immunophenotyping study is to be equated with stage IV disease,
then the possible change in the international prognostic index if more
than 1 extranodal site is involved would imply an increased risk of
death from disease.12,13 However, insufficient follow-up
time is available in these patients from our study to determine if,
indeed, there is a long-term clinical significance to this finding.
Care must be taken in potentially changing our historic definition of
stage IV disease without appropriate clinical follow-up and study. The
real answer has to come from following a larger cohort of large-cell
lymphoma patients who have similar findings and determining their
eventual clinical outcome.
The PB flow cytometric immunophenotyping component of this study raises
some interesting and perhaps controversial perspectives. Lymphomas
known to have a relatively high rate of PB involvement include the
follicular lymphomas, mantle cell lymphoma, small lymphocytic lymphoma,
lymphoplasmacytic lymphoma, and marginal zone
lymphoma.3,6,16,20,22,29,30 The majority of patients in
this series with these diagnoses demonstrated clonal B-cell populations
in the PB and correspondingly had lymphoma involvement in the BM by
both morphology and immunophenotypic analysis. Whether a PB flow
cytometric immunophenotyping study could be used as a lymphoma
screening test remains to be determined. A negative study would
obviously not exclude marrow involvement. Such PB immunophenotyping
studies also do not appear to be routinely warranted in patients with
other types of lymphoma having very infrequent evidence of circulating
lymphoma cells. Regardless, it must be recognized that BM specimens can
still provide important information regarding the extent of involvement
by lymphoma and documentation of normal or abnormal hematopoietic activity.
The determination of / monoclonality is an inadequately described
and understood process. Relying exclusively on -to- ratios is
misleading and certainly could lead to false
interpretations.21,26,32 In this series, 16 of the 54 positive BM flow cases would have been excluded if based solely on
-to- ratios. Based on our experience, determination of /
monoclonality by flow cytometry must be primarily based on examination
of the respective CD19/ and CD19/ histograms. It is perhaps a
more costly approach to analyze 2 tubes containing these antibodies as
opposed to a single tube. However, the ability to directly compare and conjugated with the same fluorochrome is essential when trying
to determine whether a subtle shift in Ig expression represents
monoclonal expression.21,26,32 Such subtle shifts are
difficult to recognize if the 2 Ig light chains are conjugated to
different fluorochromes or titered to different protein concentrations.
Although flow cytometric analysis gives the perception of precise
objectivity due to the number of cells evaluated and the exact numbers
that are generated, indeed, the flow cytometric analytic process is a
culmination of a variety of subjective steps, including the setting of
gates, quadrants, and other parameters. This subjectiveness must be
recognized by both the clinician and laboratorian. Experience in
interpreting flow cytometric studies is a crucial component in
obtaining accurate and sensitive results, especially in the search for
relatively small populations of malignant cells.
Immunohistochemical staining of paraffin section material has become an
increasingly useful tool in the evaluation of malignant lymphoma.8,9,33,34 Changes in laboratory techniques and available antibodies have led to more comprehensive immunoperoxidase panels that can be performed in paraffin-embedded biopsies. Antibodies directed against CD10, CD20, CD23, CD43, , , bcl-2, and cyclin d1
are now available that work in paraffin sections and allow for the
majority of cases of B-cell malignant lymphoma to be immunophenotyped effectively by this more pathologist-friendly medium.35
However, the findings from our study should not encourage the routine
use of these stains in the staging of malignant lymphoma and support the conclusion that immunohistochemical staining of marrow biopsies does not add much diagnostic value to the lymphoma staging process. Lymphoid aggregates in BM biopsies that are of uncertain significance usually remain uncertain after immunohistochemical stains.
Several immunologic and molecular techniques are now available that can
be used in the clinical laboratory for aiding in the assessment of BM
staging for malignant lymphoma. The findings from this study suggest
that BM flow cytometric immunophenotyping and immunohistochemistry do
not significantly help in the diagnostic work-up of the patient with
B-cell malignant lymphoma. In our opinion, immunophenotyping of blood
or BM may be warranted in those cases that cannot be adequately
phenotyped in the diagnostic lymph node/tissue specimen, if that is
crucial for lymphoma classification. There may also be other situations
in which BM immunophenotyping may be beneficial, such as when
evaluating marginal quality BM biopsies with equivocal morphologic
features. It is imperative that we gain a thorough understanding of
these assays, of how these laboratory tests can contribute
diagnostically and clinically to our patients, and, just as
importantly, of their diagnostic and clinical limitations. As health
care finances become more limited and focused, judicial use of our
laboratory resources becomes essential.
| |
FOOTNOTES |
Submitted March 29, 1999; accepted August 2, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Curtis A. Hanson, MD, Mayo Clinic, 200 First St, SW, Rochester, MN 55905.
| |
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ABSTRACT
INTRODUCTION