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Tests > Diagnostic Tests

Last Update: 03/24/2017

  Diagnostics | Imaging | Labs | Other Tests


How Lymphomas are Diagnosed (overview)



Fine Needle Aspiration (FNA) 


Image-guided Core Needle Biopsy

TIP  Try to set up the biopsy at a major research center. 
When setting up your appointment or when requesting this of
your oncologist, say that you want the extra tissue to be
donated for lymphoma research. 

This may also lead to a priority appointment ...
and it will increase chance that the most advanced
diagnostic tests will be used on the sample --
helping to inform your care and potentially helping
to identify trials you can consider in future.


Bone marrow biopsy


DiSC Assay


Flow Cytometry


FISH panel




Immunocytochemistry & immunophenotyping


Lumbar Puncture




Molecular Diagnostics - LymphoChip


PCR for Minimal Residual Disease  (updated)




Serum Free Light Chain Analysis

Related Topics
Diagnosis & PathologyGetting a Second Pathology Evaluation | Prognostic indicators | Performance Standards  Diagnostic Tests | Disease Direction Indicators

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 an infection). 

Cytogenetic tests help identify the type of lymphoma at the gene-level

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 microscope.

Fluorescent in situ hybridization (FISH) iook for specific changes in chromosomes. 

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 tempo.

Other blood tests can help detect liver or kidney problems caused by the spread of lymphoma cells or due to the side effects of treatments. 

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 Reporting
British Committee for Standards in Haematology  PDF
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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A 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. 

About Biopsy  Cancer Help Org | Medline Plus | InteliHealth
The Biopsy Report: A Patient's Guide - By Edward O. Uthman, MD. Diplomate, American Board of Pathology  Biopsy Terms 

Core Biopsy - Image Guided

Effectiveness and Safety of Image-Directed Biopsies  Medscape (free login req.) from Southern Medical Journal
Clinical 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 Sep;14(9):1438-41. PMID: 12954585

Fine Needle Aspiration (FNA)

From ajcp.ascpjournals.org:
Fine-Needle Aspiration Cytology in the Diagnosis of Lymphoma The Next Step, by Linda M. Sandhaus, MD http://bit.ly/1aNXroh
Utilization of fine-needle aspiration cytology and flow cytometry in the diagnosis and subclassification of primary and recurrent lymphoma http://bit.ly/gBXctz

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 clinical history. 

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 articles

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 lymphoma. 

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 free login req.

Image-guided core-needle biopsy (IGNB)

Image-guided 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. 


Related topics:

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


Bone Marrow Biopsy

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DiSC assay (optional)

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Differential Staining Cytotoxicity (DiSC) may be useful to determine what treatment agents will not work on your particular tumor cells.

About  ACOR.org

Flow cytometry

Flow Cytometry Helps to identify the type of lymphoma

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."   NCI

"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." hgsa.com

  1. Flow Cytometry Procedure Manual (Specimen Collections)  cancer.gov
  2. Flow cytometry in lymphoma diagnosis and prognosis: useful?
    Best Pract Res Clin Haematol. 2003 Dec;16(4):583-97. PMID: 14592644
  3. Flow cytometric analysis of lymphomas: current status and usefulness.
    Arch Pathol Lab Med. 2006 Dec;130(12):1850-8. Review. PMID: 17149963
  4. Flow Cytometric  Analysis of Leukemia and Lymphoma - The Basics
  5. Follicular lymphoma differential diagnosis - Stanford


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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

  1. Chromosome Analysis, Chronic Lymphocytic Leukemia (CLL) Panel by FISH  aruplab.com
  2. Chronic Lymphocytic Leukemia FISH Panel: Impact on Diagnosis   Medscape



Some terms

Antigens - that which is capable of inducing a specific immune response 

Morphology - appearance and structure of cells

Autolysis - spontaneous rupture of cell membrane

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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.

Frozen Sections

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 details. 

Specimen Fixation
Preserve the tissue and cells without without distorting or dissolving cellular constituents.
Paraffin Sections
Paraffin embedded tissue are cut to a thickness of 3m. 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:
Resin Sections
Embedding tissues in resin offers several advantages over frozen or paraffin sections, such as improved morphology and less shrinkage.

Immunocytochemistry and Immunophenotyping


Some terms

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. 
Source: hmds.org


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. 

Lumbar puncture / Spinal Tap 
(not common)


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Checks for malignant cells in the cerebrospinal fluid (CSF), which circulates around the brain and spinal cord. 

About CancerHelp UK | Medline Plus | emedicine

Mediastinoscopy (not common)


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"The 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

About MedlinePlus 

Molecular Diagnostics - LymphoChip

Medscape *
"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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"We 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)." 
Source: Full text: nih.gov

The LymphoChip promises to:

help identify new molecular targets for treatment. 


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 expression.

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
Molecular Diagnostics 
Rita M. Braziel, Margaret A. Shipp, Andrew L. Feldman, Virginia Espina, Mary Winters, Elaine S. Jaffe, Emanuel F. Petricoin III and Lance A. Liotta  asheducationbook.org

PCR testing for
Minimal Residual Disease (MRD)



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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 marrow findings. 

Moreover, splenectomy has the capacity to modify the disease course in patients with NHL complicated by AIHA or hypersplenism." 

Source: Splenectomy in patients with malignant non-Hodgkin's lymphoma. Eur J Haematol. 2000 Mar;64(3):145-50. PMID: 10997879  PubMed

Serum Free Light Chain Analysis

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A test for Monoclonal Gamopathy - excess monoclonal immunoglobulin in serum or urine, which indicates excessive plasma cells.
bullet Serum Free Light Chain Analysis
Disclaimer:  The information on Lymphomation.org is not intended to be a substitute for 
professional medical advice or to replace your relationship with a physician.
For all medical concerns,  you should always consult your doctor. 
Patients Against Lymphoma, Copyright 2004,  All Rights Reserved.