How a Diagnosis of T Cell or NK Cell Leukemia/Lymphoma is Made: Evaluation of Biopsy Specimen

By: Lorinda Soma, M.D.

Assistant Professor

Associate Director, Hematopathology Laboratory

University of Washington

What Happens After the Sample/Biopsy/Blood is Taken?

Once the sample is taken it is sent to pathology to be evaluated and tested.  This may include various types of testing, depending on the sample and diagnosis.  Modalities that are often used by the hematopathologist are (1) the microscope to look at the cells / tissue, (2) flow cytometry or immunohistochemistry to look at the proteins expressed by the cells, (3) molecular and cytogenetic studies to look at the genetics of the lymphoma/leukemia.  We will first start with how the tissue is processed so it can be looked at under the microscope (often referred to as the morphologic, microscopic or histologic evaluation).

Morphology / Microscopic Examination

In order to look at the tissue/cells under the microscope, it must be processed and stained with dyes.  With peripheral blood, a drop of blood is placed on a glass slide and gently smeared or spread across the glass slide to form a monolayer (single cell layer).  The slide is allowed to dry, and then placed in a machine, which will fix and stain the cells. Tissue biopsies are placed in a chemical fixative immediately after removal to best preserve them in their natural state and prevent decomposition. This fixative is usually 10% neutral buffered formalin. Depending on the type of biopsy, it may further need to be cut, or “sectioned” in an appropriate manner by a pathologist or pathologist assistant. The tissue is further “processed” in a machine that additionally fixes/processes the tissue using water, ethanol and other chemicals to further prepare the tissue for embedding and staining.  Once the tissue is processed (depending on the type of tissue, may require 2 – 8 or more hours), it will be embedded in paraffin wax by a specialized technician (histotechnologists).  Once placed in the wax, and the wax solidifies, a thin slice, or “section” can be cut using a specialized machine called a microtome, by specialized technicians (histotechnologists).  This thin section (~4-8 microns, less thickness than a sheet of paper) will then be stained with dyes (most commonly hematoxylin and eosin – which impart shades of blue and pink respectively).  After the tissue has been stained, it can be evaluated by the hematopathologist under the microscope.

Marker / Protein Expression Evaluation

Evaluation of the protein a cell displays or contains can be performed by a process called flow cytometry or by using immunohistochemistry.

Flow Cytometry

Flow cytometry requires fresh tissue, peripheral blood or bone marrow for evaluation. The “flow” of flow cytometry relates to the sample being fluid and “flowing” though the machine /analyzer; and the “cytometry” relates to looking at specific cell characteristics (specific proteins/markers). The sample is processed by clinical laboratory technologists who are specialized in flow cytometry, to look for various markers / proteins that are expressed on, or in the cells of interest.  The sample is processed to become a single cell suspension in fluid, with the appropriate marker/protein identifiers (antibodies) added.  The sample fluid is evaluated by a flow cytometer, which utilizes lasers to identify the different markers identifiers (antibodies). This data is processed by specialized software, so that the data can be evaluated by the hematopathologist to determine what markers/proteins the cells express.

Immunohistochemistry

Immunohistochemistry does not require fresh tissue, and is performed on the fixed, paraffin embedded tissue that is looked at under the microscope.  It is fancy word that points out the fact that it’s using an immunologic method (antibody), histologic examination (looking at tissue under the microscope), and undergoes additional chemical reactions to identify the marker/protein of interest (allows one to see the “blue flag” – see above under “antibody” for further description of the blue flag).

Genetic Findings

Various abnormalities at the genetic level may also be studied in samples being evaluated for lymphoma.  This may be done by whole chromosome analysis (looking at all the chromosomes, where the whole chromosome can be evaluated), fluorescence in-situ hybridization study (looking for a specific genetic abnormality, at the gene level, not whole chromosome levels – also referred to as a short cut term, FISH) and molecular studies (looking for specific abnormalities within the gene itself).  As an analogy, looking at the whole chromosome would be like looking at a tree.  If you wanted to specifically look at the lower branch of the tree that would be like looking at the gene (using FISH).  If you wanted to look at the leaf on that branch and see if it is damaged, you would use molecular studies to look at a specific abnormality within that gene/ tree branch. Molecular studies can also detect if a proliferation is considered “clonal,” meaning it has originated from one cell that has multiplied over and over again to make a population of cells that are the same as the original.  Although this sometimes can happen when there is an infection or other similar reaction, lymphomas / cancers are always composed of a clonal population (although it can have more than one clone composing the cancer).

How the Hematopathologist Renders a Diagnosis

Some or all of the “tools” described above may be used by the hematopathologist to arrive at a diagnosis. The tools that are used may depend on the type of sample being evaluated and how “straight forward” the lymphoma is.  Some lymphomas seem to “read the books” and they will be stereotypical, and look/act just like the books describe them.  However, sometimes, the lymphomas will only show some characteristics of a specific type of lymphoma, or may show characteristics of more than one type of lymphoma (and even sometimes including characteristics of reactive / non-cancerous entities).  This is where the “art” of medicine comes into play.  If the lymphoma is not stereotypical, the hematopathologist will use all the tools they have to decide what the lymphoma is most like, and at times, a differential diagnosis will need to be given.  As an analogy, as one moves along the color spectrum from green to blue, there will be points in that spectrum where everyone can agree the color is either green or blue.  However, along that continuum, it can be more difficult to know if something is more green or blue, when there are characteristics of both (blue-green; green-blue; aqua; teal; turquoise).