A 44-year-old man presents with a 2 cm right inguinal
nodule, which has been present for 4-5 years and is stable in size. Four months prior, he underwent FNA
biopsy of a left parotid nodule at an outside institution; cytology and flow cytometry were consistent
with a benign lymph node. An FNA biopsy of the right inguinal nodule is performed (Figures A,B,C).
Case 7 - Figure A - Cellular smear shows variably sized but predominantly small lymphoid cells that appear cytologically unremarkable. A follicular dendritic cell is present in the field for size comparison (Papanicolaou, 400X).
Case 7 - Figure B - Predominantly small lymphoid cells with intermixed larger cells and a tingible-body macrophage (Diff-Quik, 400X).
Case 7 - Figure C - Higher power view of lymphoid cells. Note the presence of occasional clefted and angulated lymphoid cells, as well as numerous lymphoglandular bodies in the background (Diff-Quik, 600X).
Aspirate smears are cellular and show a mixed
lymphoid population composed of predominantly small lymphocytes and scattered intermediate sized forms.
The lymphocytes exhibit irregular nuclear contours. Rare tingible body macrophages are present. No
significant large cell component is identified.
Flow Cytometric Findings
Flow immunophenotyping shows a lambda
monotypic B-cell population, which expresses CD19, CD20, CD10 and CD23. There is no CD19/CD5
Immunophenotypes of Small B-cell Lymphomas
| ||CD5 ||CD23 ||CD10|
|SLL/CLL ||+ ||+ ||-|
|Mantle Cell ||+ ||- ||-|
|Marginal Zone ||- ||- ||-|
|Follicular ||- ||+/- ||+|
The patient underwent excision of his left parotid nodule at an outside institution prior to the right
inguinal FNA. Histologic sections showed an intraparenchymal lymph node effaced by follicles composed of
small cells. No lymphoepithelial lesions or tingible body macrophages were identified. Immunoperoxidase
stain for CD20 showed a predominance of B-cells, suggestive of lymphoma. However, the stain for bcl-2 stain (positive in follicular lymphomas – 100% grade 1, 85% grade 2, 75%
grade 3) was equivocal.  Molecular studies were performed to confirm the diagnosis of B-cell
lymphoma. These studies, which were pending at the time of the right inguinal FNA, showed a clonal
immunoglobulin gene rearrangement but no evidence of a t(14;18) translocation.
Detection of B-cell clonality by PCR
Immunoglobulin heavy chain (IgH) gene rearrangements occur early in B-cell development. Since they
are unique to each cell, these gene rearrangements can be used as a marker of clonality.
Identification of a clonal population should not
be interpreted as diagnostic of malignancy. Non-malignant clonal populations can be seen transiently in
the setting of infection or autoimmune disorders.
False positive results have also been
reported when PCR is performed on small or microdissected samples.  In addition, false
negative results can occur due to sampling error, sample handling (PCR assay inhibited by heparin), or
minimal detection limits. The results of this test need to be interpreted in the context of pathologic
and clinical findings.
VDJ (FR3) Assay:
- Uses VH-FR3 and JH primer set
- Detection limit: 10-15% clonal cells in a polyclonal background
- If negative, FR1c assay is performed
- Uses VH-FR1c primers
- Detection limit: 5% clonal cells in a polyclonal background (Stanford)
- Decreased specificity: Higher detection of non-malignant clonal populations
The patient's sample (Pt) showed the presence of a clonal IgH gene rearrangement:
Detection of t(14;18) by nested PCR
The t(14;18)(q32;q21) translocation is present in 80-85% of follicular lymphomas.
occurs when the bcl-2 gene on chromosome 18 is moved to one of the
immunoglobulin heavy chain (JH) regions on chromosome 14. The translocation brings the bcl-2 gene into the domain of the IgH enhancer, leading to overexpression of the
bcl-2 protein. Since bcl-2 blocks apoptosis
(programmed cell death), overexpression leads to decreased cell turnover.  This can result in
the accumulation of a monoclonal cell population and the development of lymphoma.
However, detection of a t(14;18) translocation is not diagnostic of follicular lymphoma. Rare cells
with t(14;18) have been found by PCR in reactive lymphoid tissue from normal individuals.
This suggests that bcl-2 translocations alone are not sufficient for the
development of follicular lymphoma.
False negative results can be due to sampling error, sample handling (PCR assay inhibited by heparin),
or minimal detection limits. In addition, not all t(14;18) breakpoints are detected.  The
current PCR assay uses primers for two breakpoint cluster regions – major breakpoint region (MBR) and
minor cluster region (mcr). If the translocation is outside of these two regions, it will not be
detected. The false negative rate is approximately 5-10%.
The patient's sample (Pt) did not show the presence of a t(14;18) translocation:
Discussion: Low Grade Follicular Lymphoma
FNA biopsy with flow cytometry is a rapid, cost effective, minimally invasive method for evaluating
lymph nodes, both in patients with new onset lymphadenopathy and in those with a history of lymphoma.
The World Health Organization (WHO) lymphoma classification system relies less on architecture and
allows for the definitive subclassification of many lymphomas based on the cytologic appearance of the
lymphoid cells and the immunophenotype.  Subsequently, flow cytometry plays an essential
role in the cytologic diagnosis of lymphoma.
It not only allows for the subclassification
of lymphomas based on the immunophenotype but can also detect the presence of a small monoclonal
population in a background of polyclonal lymphoid cells. Flow cytometry is especially important in the
diagnosis of low grade lymphomas since they can appear cytologically identical to a reactive lymph node,
including the presence of scattered tingible body macrophages. The differential diagnosis for a reactive
cytologic appearance includes reactive hyperplasia, low grade lymphoma, partial involvement by lymphoma
and Hodgkin lymphoma. While flow cytometry can distinguish between the first three entities, it is not
helpful in the diagnosis of Hodgkin lymphoma. If a diagnosis of Hodgkin lymphoma is suspected, it is
best to obtain material for immunohistochemical stains.
Cytologic material can also be submitted for cytogenetic (e.g. FISH for c-myc) or molecular diagnostic studies (e.g. PCR for B-cell clonality).
Cytogenetic studies require fresh material but molecular studies can be performed on fixed material.
Given the possibility of false positive results (see "Molecular Studies" section), these studies should
be interpreted in the context of cytologic, flow immunophenotypic and clinical findings.
In this case, the diagnosis of low grade follicular lymphoma was based on the presence of a
predominantly small lymphoid population and the flow cytometry findings of a CD10(+) lambda restricted
population of B-cells. The presence of CD10 expression is characteristic of follicular lymphomas but is
not seen in all cases. It is present in 60% of cases by immuohistochemistry and 90% of cases by flow
cytometry.  However, it should also be kept in mind that CD10 expression can be seen with
Burkitt's lymphoma, some diffuse large B-cell lymphomas, precursor B- or T-lymphoblastic
lymphoma/leukemia, and normal cortical thymocytes. Therefore it is important to correlate the presence
of CD10 expression with the cytologic appearance of the lymphoid cells and the clinical presentation.
By the WHO Classification, follicular lymphomas are divided into three grades based on the proportion
of small and large cells – grade 1, predominantly small cleaved; grade 2, mixed small cleaved and large
cell; grade 3, follicular large cell. However, only two grades are clinically significant. Low-grade
(grades 1 and 2) follicular lymphomas are indolent while high-grade (grade 3) follicular lymphomas are
aggressive and require treatment. Based on aspirate smears, it is often possible to distinguish between
a low-grade and high-grade follicular lymphoma but, as in tissue, there is less reproducibility with the
use of three grades.  Two different methods for grading follicular lymphomas are generally
used but there are no well-established criteria for grading based on cytologic material. The two methods
parallel those used in tissue sections – visual estimation of the percentage of large cells or a count of
At Stanford, the visual estimation method is used. In cases
where the cytologic appearance is not clearly consistent with a low grade follicular lymphoma or where
the clinical presentation is discordant with the cytologic findings, an excisional biopsy is recommended
for further evaluation.
In patients with a history of lymphoma, FNA biopsy is often used to evaluate for transformation of a
low grade lymphoma to a high grade lymphoma. In cases where the aspirate smears show predominantly small
cells or sheets of large cells, the diagnosis is clear-cut. However, for the in-between cases, there are
not well-established criteria for diagnosing transformation based on a cytologic specimen. Various
studies have looked at the utility of proliferation markers (PCNA or Ki-67), transformed lymphocyte count
and analysis of cell size by flow cytometry in the diagnosis of transformation.
methods appear to show some correlation with grade but more studies are needed.
Triaging specimens for evaluation of lymphoid lesions:
|For each aspirate, place a small drop on a slide and make two smears (one air-dried, one fixed).|
|Rinse the remainder of the specimen in a small amount (~5 mL) of RPMI (holding media).|
|Repeat aspirations until RPMI appears cloudy and small particles are seen when the fluid is agitated. This can be obtained with a minimum of two to three passes but, depending on technique, may require more.|
|Perform immediate evaluation of aspirate smear to confirm lymphoid nature of specimen and send specimen for flow cytometry.|
|If background is suggestive of Hodgkin lymphoma, use material in RPMI for immunohistochemical stains.|
|If specimen is insufficient for a full panel, perform limited flow cytometry panel (e.g. two tubes – CD19/CD5, kappa/lambda – can diagnose B-cell lymphomas by the presence of light chain restriction and CD19 expression; CD5 expression can allow further sub-classification)|
|If possible, obtain additional material for cell block, in case the flow cytometry results indicate a non-hematolymphoid process or are inconclusive.|
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