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Pathology | Getting a Second
Pathology Evaluation |
How Lymphomas are Diagnosed (a concise overview)
Adapted from www.cancer.org
Examination of all biopsy samples by pathologist
The pathologist looks at the appearance, size, and shape of the cells and how the cells are arranged.
(sometimes called morphology)
Biopsy sample is treated with antibodies that attach only to specific molecules on the cell surface. These antibodies cause color changes, which can be seen under a
microscope, helping to identify different types of lymphoma and if
the cells represent other diseases.
Flow Cytometry to help determine the exact type of lymphoma,
or exclude lymphoma
This test also looks for certain molecules on the outside surface of cells
by which antibodies (protein molecules) stick, helping to identify what types of cells they are.
This test can look at many more cells than immunohistochemistry.
Flow cytometry is most commonly used test for immunophenotyping -- classifying lymphoma cells according to the substances (antigens) on their surfaces. Different types of lymphocytes have different antigens
(distinct protein molecules) on their surface. These antigens may also change as each cell
matures - goes from one stage of development to another.
Flow cytometry can help determine whether lymph node swelling is due to
lymphoma, some other cancer, or a non-cancerous disease (such as
reactive hyperplasia - normal immune cells reacting appropriately to
Cytogenetic tests help identify the type of lymphoma at
Evaluation of the chromosomes in the lymphoma cells for
abnormalities, such as translocations (wrong order), too many
chromosomes (additions), too few chromosomes (deletions).
Molecular Genetic Studies
Tests of the DNA of lymphoma cells
to detect abnormalities in cells that can't be seen with a
Fluorescent in situ hybridization (FISH) iook for specific changes in
FISH can find most translocations that are too small to be seen with usual cytogenetic testing. It uses special fluorescent dyes that only attach to specific parts of chromosomes.
FISH can be used to look for specific changes in chromosomes. It can be used on regular blood or bone marrow samples.
Polymerase Chain Reaction (PCR) can also find translocations too small to be seen under a microscope,
even if there are very few lymphoma cells present in a sample.
Can also detect certain genes that have been "turned on" and are contributing to the lymphoma cells' abnormal growth.
Other Lab Tests
While blood tests are not used to diagnose lymphoma, they can be helpful in
monitoring for changes in an advanced lymphoma, or to look for
abnormalities that warrant further testing.
For example, if the blood counts are low, it might indicate that the lymphoma is growing in the bone marrow and
restricting blood cell production. Increasing or decreasing Levels
of lactate dehydrogenase or LDH,
can provide indications of disease direction or changes in growth
Other blood tests can help detect liver or kidney problems caused by the spread of lymphoma cells or due to the side effects of
Blood tests can also help determine if treatment is needed to correct blood levels of certain
minerals, or to make sure blood is clotting properly.
technical: Best Practice in Lymphoma Diagnosis and
British Committee for Standards in
Royal College of Pathologists
Best to avoid blood thinners prior to having a surgical
procedures, such as aspirin, fish oil, Vitamin E.
Talk to your doctor about medications and supplements you are taking.
"If sufficient material is available, it is advantageous to snap
freeze a small portion of the lymph node in liquid nitrogen.
Frozen section immunohistochemistry and DNA gene rearrangement studies
can be performed on this material." Bccancer
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biopsy of suspected lymphoma
tissue (typically a lymph node), and subsequent evaluation
by a trained pathologist is the only way to
definitively diagnose lymphoma. It may take a week or
more to get the results,
and this time of waiting often causes considerable anxiety for patient and family. The
delay is not an indication that a lymphoma was found.
When surgical resection
(removal) of a lymph node is not possible, a Fine
Needle Aspiration or Large
Needle/Core Biopsy may be performed, but each of these
procedures have diagnostic limitations. Of the the two, an
image-guided core biopsy is more reliable.
The Biopsy Report: A Patient's Guide - By Edward O.
Uthman, MD. Diplomate, American Board of Pathology Biopsy
Core Biopsy - Image Guided
Effectiveness and Safety of Image-Directed Biopsies
(free login req.)
from Southern Medical Journal
utility of computed tomography-guided core needle biopsy in the
diagnostic re-evaluation of patients with lymphoproliferative
disorders and suspected disease progression. Ann Oncol. 2003
Fine Needle Aspiration (FNA)
fine-needle aspiration cytology and flow cytometry in the diagnosis
and subclassification of primary and recurrent lymphoma
Nancy A. Young M.D.1,*, Tahseen I.
Al-Saleem M.D.1, Hormoz Ehya M.D.1, Mitchell R. Smith M.D.,
Ph.D.2Article first published online: 10 NOV 2000
In conclusion, FNA cytology combined with FC (flow cytometry)
obviates the need for tissue biopsy in many instances, particularly
when the pathologists have experience diagnosing hematologic
malignancies and correlate the findings with the FC results and the
Biopsy may be necessary when monoclonality cannot be proven in a
clinically suspicious lymph node or when lymphoma grade or subtype
cannot be determined.
Value and limitations of fine-needle aspiration
cytology in diagnosis and classification of lymphomas: A review. Diagn
Cytopathol. 1999 Oct;21(4):240-9. Review. PMID:
10495316 | Related
Lymphomas have successfully been classified by FNA cytology
following the prevalent histologic classifications. The success rate
of FNA cytology ranges from 80%-90% in diagnosis of NHL and from
67.5%-86% in its subtyping.
The cytodiagnosis of Hodgkin's disease (HD) depends upon demonstration
of Reed-Sternberg cells or Hodgkin's cells amongst appropriate
reactive cell components. The diagnostic accuracy of FNA cytology for
HD has also been invariably high (>85%). Yet, the role of cytology
in primary diagnosis, subclassification and management of patients
with lymphoma remains controversial. The differential diagnostic
problems for NHL include a group of small round cell tumors,
nonlymphoid acute leukemias and HD.
Reservations have been expressed regarding the efficacy of cytology in
separating florid reactive hyperplasia from low-grade malignant
The reported cytodiagnostic accuracy for follicular lymphomas and
nodular sclerosis type of HD is less compared to other subtypes of NHL
and HD respectively since nodular pattern and sclerosis are strict
histologic criteria which can not be appreciated in cytologic
preparations. Entities like atypical lymphoproliferative disorders,
peripheral T-cell lymphomas and Ki-1 positive anaplastic large cell
lymphomas pose diagnostic challenges to cytologists.
Despite these limitations, FNA cytology remains the first line of
investigations (screening test) used in cases of lymphadenopathy.
Besides initial diagnosis of lymphoma, it helps in detection of
residual disease, recurrences and progression of low-grade to
high-grade lymphoma, and helps in staging the disease.
About Fine Needle Aspiration and Large
Needle/Core Biopsy OncologyChannel
"because of small sample sizes and lack of information
about lymph node structure, FNA often is inadequate for the
initial diagnosis of HD or NHL. In such cases, larger tissue
samples are obtained by surgical biopsy." OncologyChannel
Fine-Needle Aspiration in Non-Hodgkin Lymphoma:
Evaluation of Cell Size by Cytomorphology and Flow Cytometry from
American Journal of Clinical Pathology - Posted 08/06/2002 Medscape
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Image-guided core-needle biopsy (IGNB)
core-needle biopsy in patients with suspected or recurrent lymphomas
It is now commonly admitted that the diagnosis of recurrence of lymphoma
can be assessed by image-guided needle biopsy (IGNB). However, the means
of obtaining tissue for the original diagnosis of lymphoma is often
surgery. The aim of this study was to compare the accuracy of IGNB at the
time of diagnosis and at the time of recurrence or progression.
The authors performed 212 IGNBs on 194 patients who eventually had a
diagnosis of lymphoma. One hundred three IGNBs were obtained at original
diagnosis and 109 at recurrence or progression. Large-cutting core-biopsy
needles, ranging in size from 20 gauge to 14 gauge, were used.
Immunohistochemistry studies were performed in all lymphoma cases.
A diagnosis of lymphoma with subtyping was obtained in 88% of all cases,
in 85% at initial diagnosis, and in 89% at follow-up. Therapy was
initiated on the basis of IGNB in 93% of all cases, in 91% at initial
diagnosis, and in 94% at follow-up. Benign complications occurred in 7.5%
of cases and did not require specific treatment. IGNB was equally
effective for making a specific diagnosis of lymphoma and initiating
therapy at the time of original diagnosis and at follow-up.
The authors recommend that IGNB be performed as the initial procedure for
the diagnosis of lymphoma in the absence of peripheral lymph nodes, either
at presentation or at recurrence. Cancer 2000;89:647-52.
Recommendations for the Reporting of Lymphoid Neoplasms
Medscape (free login req.)
A checklist for pathologists and surgeons on how to evaluate and store lymphoid tissue at biopsy.
Why do we need to have this handy? When we ask informed questions, we are more likely
to get informed care. Note that it also includes guidance on how to snap
freeze the tissue.
Clinical utility of computed tomography-guided core
needle biopsy in the diagnostic re-evaluation of patients with
lymphoproliferative disorders and suspected disease progression
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Staining Cytotoxicity (DiSC) may be useful to determine what
treatment agents will not work on your particular tumor cells.
Flow Cytometry Helps to identify the type of
An analogy might help.
Like people, lymphocytes have a life
span which includes stages of maturation where the appearance changes
as do the behaviors and roles in the body as the cells mature.
The type of cell and it's level of
maturation is identified by markers - called clusters of
differentiation (CD), similar to how people develop features at
different ages, (breasts, gray hair, etc)
Specific groups of markers help distinguish
between close cousin lymphocytes (follicular, Marginal zone, t-cell,
mantle cell, diffuse large b-cell, Hodgkins ...)
The cells are so very small (one
billion in a 1 cm lesion) that these differentiation markers
cannot be distinguished under a microscope but by "seeing"
which fluorescently tagged antibodies
stick to the cells.
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Flow cytometry is a way of
"measuring the number of cells in a sample, the percentage of
live cells in a sample, and certain characteristics of cells, such as
size, shape, and the presence of tumor markers (such as Clusters of
Differentiation - CD) on the cell surface."
"By virtue of its ability to
evaluate not only surface but also cytoplasmic and nuclear antigens,
flow cytometry continues to enjoy widespread use in various capacities
in lymphoma evaluation and treatment. Additional roles for flow
cytometry are likely to be invented in the future and should provide
distinctive uses in the molecular era." 4
"It's performed on cells in liquid suspension (i.e. blood, bone
marrow, body fluids or tissue cell suspensions) that have been
incubated with fluorescently tagged antibodies directed against
specific cell surface proteins." ~ Univ. of Washington.
It is "a highly
complex process utilized by clinical laboratories to examine blood,
body fluids, bone marrow, and tissues. The technology of flow
cytometry and the discovery of a method to produce monoclonal
antibodies have made possible the clinical use of flow cytometry for
the identification of cell populations. This technique measures
multiple characteristics within the cell nucleus or cytoplasm (e.g.,
cell size, internal structure, antigens, DNA, ploidy, and cell cycle
analysis) that help distinguish one cell type from another."
Flow Cytometry Procedure Manual (Specimen
Flow cytometry in lymphoma diagnosis and
Best Pract Res Clin Haematol. 2003 Dec;16(4):583-97.
Flow cytometric analysis of lymphomas: current
status and usefulness.
Arch Pathol Lab Med. 2006 Dec;130(12):1850-8. Review.
Flow Cytometric Analysis of Leukemia and Lymphoma - The
Follicular lymphoma differential
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Interphase fluorescence in situ
hybridization (FISH) is an alternative to conventional chromosome
analysis of chronic lymphocytic leukemia (CLL) cells.2
The FISH panel is performed for CLL
prognosis-specific genomic abnormalities as follows: ATM, Rb-1,
D13S25, Trisomy 12, p53 1
Chromosome Analysis, Chronic Lymphocytic
Leukemia (CLL) Panel by FISH
Chronic Lymphocytic Leukemia FISH Panel: Impact
on Diagnosis Medscape
Antigens - that which is capable of inducing a specific immune response
Morphology - appearance and structure
Autolysis - spontaneous rupture of
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Histology is the study of cells and tissue on the microscopic level.
Procedures and tests that follow are conducted on
tissue obtained from a biopsy.
Provides for a rapid provisional diagnosis and the
identification of antigens and or enzymes which maybe lost during
subsequent fixation and processing schedules. See source for
Preserve the tissue and cells without without
distorting or dissolving cellular constituents.
Paraffin embedded tissue are cut to a thickness of
3µm. Sections are floated on a warm water bath, then picked up onto
microscope slides and allowed to drain. Sections for staining are
prepared. See source for details:
Embedding tissues in resin offers several advantages
over frozen or paraffin sections, such as improved morphology and
Antigens - that which is capable of inducing a specific immune response
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The demonstration of antigens in tissue sections or smears by the
use of specific immunological (antibody-antigen) interactions
culminating in the attachment of a visible marker to the antigen. See
source for details.
Reveals the kinds of
surface molecules that are present on cells (typically immune cells),
such as CD20, CD22.
CD stand for clusters of
differentiation, which show the developmental stage of the cell and
the cell type. CD20, for example, is expressed only on mature
b-cells, but not t-cells.
puncture / Spinal Tap
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malignant cells in the
cerebrospinal fluid (CSF), which circulates around the brain and spinal cord.
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mediastinumis is the space that
separates the 2 lungs and contains the heart, thymus, esophagus,
trachea, the large blood vessels, and lymph nodes. A mediastinoscopy
is a procedure in which a lighted instrument (mediastinoscope) is
inserted through a neck incision to visually examine the structures in
the top of the chest cavity." Source - MedlinePlus
Diagnostics - LymphoChip
"Ultimately, this effort will guide patients towards
therapies that are tailored for their particular diseases and will
identify new molecular targets for therapeutic development."
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began a study of gene expression in lymphoid malignancies by
constructing a specialized DNA microarray, termed the "Lymphochip",
that is enriched in genes which are selectively expressed in
lymphocytes and genes which regulate lymphocyte function (1)."
LymphoChip promises to:
help identify new molecular targets
predict clinical response to
therapy and long-term outcome.
generate gene expression profiles
of lymphoma and leukemia.
help correlate response to
treatment with gene expression and thus better match patient to
treatment in the future.
show how treatment affects gene
Application of tissue microarray technology to
the study of non-Hodgkin's and Hodgkin's lymphoma. Hum Pathol.
2002 Oct;33(10):968-974. PMID: 12395368 - PubMed
Rita M. Braziel, Margaret A. Shipp, Andrew L. Feldman, Virginia
Espina, Mary Winters, Elaine S. Jaffe, Emanuel F. Petricoin III and Lance A. Liotta
PCR testing for
Minimal Residual Disease (MRD)
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Polymerase Chain Reaction (PCR)
analyses can be used to detect
small amounts of lymphoma cells in the bone marrow and
peripheral blood of patients with lymphoma. The analysis is
limited to the compartment tested (blood, bone marrow).
PCR to detect a gene
t(14;18) in blood or marrow
PCR testing can detect very small amounts of genetic material in
a biospecimen sample, such as a gene translocation t(14:18)
which produces pro-survival protein, BCL-2, in many lymphomas. When the PCR test is negative (a good result),
it means that there may be no abnormal cells in
the blood or marrow, which might be described as a
molecular remission or
minimal residual disease
A standard bone marrow test is like taking a sample of a pool
with a scoop net in the dark.
The test is only conclusive if leaves are found, because the sample
may have missed leaves floating elsewhere in the larger pool.
PCR testing is
like examining the same sample for tiny fragments of substances that exist only in leaves. Thus, a negative
finding with PCR testing provides greater
confidence that the bone marrow is clear of the abnormal cells.
The clinical significance of a molecular remission is still not clear, but achieving this kind or response has been associated
with longer duration of response among the participants in some
PCR tests are generally given after a complete
response to treatment
has been determined using CT and
a bone marrow biopsy, mainly in clinical trials.
A limitation of the test is that
it can only show the status of the compartment tested: the blood or marrow. A conversion to negative appears to be a good prognostic indicator, but not
definitively, because the test can't determine the t:14:18 status in
lymph nodes and other areas.
"The t(14;18) translocation is characteristic of B-cell
lymphomas, occurring in up to 90% of follicular lymphomas. It is also found in 20% to 30% of diffuse large
B-cell lymphomas ..."
analytical sensitivity of 1 tumor
cell in 100,000
||Monitoring Lymphoma with PCR
||Molecular monitoring of low
grade non-Hodgkin's lymphoma by gene amplification, 1991,
Abstract: Molecular monitoring by the polymerase chain reaction
(PCR) was used to detect and follow minimal disease in working
formulation category B and C on non-Hodgkin's lymphoma.
Rearrangement of the bcl-2 gene served as the target for gene
amplification. Thirty patients were studied. Bone marrow
histology was compared to PCR analysis of bone marrow aspirate
and blood. PCR upstaged disease status in approximately 50% of
Results are shown from a patient whose disease was followed with
PCR during chemotherapy from initial remission to relapse. We
conclude that PCR of bone marrow and blood can be used to
upstage disease status in low grade lymphoma and PCR of blood
may be used to monitor response to treatment with obvious
patient benefit. The general approach of molecular monitoring
provides a means for appraising therapies in the setting of
||All advanced stage
non-Hodgkin's lymphomas with a polymerase chain reaction
amplifiable breakpoint of bcl-2 have residual cells containing
the bcl-2 rearrangement at evaluation and after treatment.
Abstract: Polymerase chain reaction (PCR) of bcl-2
provides an extremely sensitive method to detect minimal disease
in approximately 50% of patients with non-Hodgkin's lymphomas
(NHL). In an attempt to determine the clinical usefulness of
this technique, we examined the bone marrow (BM) of 152 patients
with advanced-stage NHL at the time of evaluation and after
induction or salvage chemotherapy before autologous BM
transplantation. The BM proved to be an accessible and
reproducible tissue source to determine PCR positivity because
all of the 102 patients examined had the same PCR-amplifiable
breakpoint in their BM and lymph node. At the time of
evaluation, PCR analysis in advanced-stage NHL patients added
little additional information to morphologic analysis because
each technique identified BM infiltration in approximately 70%
of patients. PCR was significantly more useful in determining BM
infiltration after induction or salvage therapy. At that time,
approximately 50% of patients had morphologically normal BM,
whereas PCR analysis remained positive in 100% of those with an
amplifiable breakpoint. These observations were confirmed in a
clinical trial attempting to induce remission in previously
untreated low-grade advanced-stage NHL patients. In this series,
PCR was positive in all patients after treatment although the BM
was histologically uninvolved in 50% of cases, showing that
conventional therapy did not eradicate bcl-2-positive cells.
||Detection of occult lymphoma in the peripheral blood and bone
marrow of patients with untreated early-stage and advanced-stage
NL Berinstein, MD Reis, BY Ngan, CA Sawka, HH Jamal and B Kuzniar
Department of Medicine, Immunology, University of Toronto,
What is PCR Testing?
"Sometimes referred to as "molecular
photocopying," PCR can characterize, analyze,
and synthesize any specific piece of DNA or RNA. ... Medical
research and clinical medicine are profiting from PCR mainly in two areas: detection of
infectious disease organisms, and detection of variations and mutations in genes, especially human
Quantitative Real-Time PCR
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|"Splenectomy, with an
acceptable surgical risk, has the potential to establish the diagnosis
of NHL in patients with splenomegaly (enlargement of the spleen)
without lymphadenopathy (enlarged lymph nodes) and negative bone
Moreover, splenectomy has the capacity to modify the
disease course in patients with NHL complicated by AIHA or
Source: Splenectomy in patients with malignant
non-Hodgkin's lymphoma. Eur J Haematol. 2000 Mar;64(3):145-50. PMID:
Serum Free Light Chain Analysis
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|A test for Monoclonal Gamopathy - excess
monoclonal immunoglobulin in serum or urine, which indicates excessive