Lymphomas are malignant neoplasms (or cancers) of "lymphoid" cells. Lymphoid cells, most of which are called lymphocytes, are white blood cells (the infection fighting cells of the body). There are two major types of lymphomas: Hodgkin's disease(also known as Hodgkin's lymphoma), characterized by the presence of special cells called Reed-Sternberg cells; and non-Hodgkin's lymphomas, which are proliferations of malignant (or cancerous) lymphocytes.

How is Lymphoma Diagnosed?
The diagnosis of malignant lymphoma may involve several techniques and several individuals. When a diagnosis of lymphoma is suspected, the primary doctor or cancer specialist will do several blood tests, a thorough physical examination (including manual examination of lymph node sites) and possibly, depending on his or her suspicion of the type of lymphoma, a biopsy (removal and examination) of bone marrow.

Depending on the findings of the physical examination and initial battery of tests, the doctor may ask for more tests or send the patient to a surgeon. There are many tests that pathologists are now able to do on a blood sample or bone marrow biopsy that may suggest a diagnosis of lymphoma. However, a tissue biopsy, usually done by a surgeon, is often important for the diagnosis of lymphoma. Lymph nodes, the glands that store white blood cells and can become swollen in infection or cancer, are often the tissue that is biopsied. At times, a biopsy of a different tissue may be required.

Correct classification of lymphoma, which can only be accurately done with a tissue biopsy, gives the clinician and patient important information with respect to treatment options and prognosis. Examination of cell appearance (known as morphology), specific cell sub-type (known as phenotype) and, occasionally, the genetic make-up (known as genotype) of a lymphoid growth (refered to as a lymphoid lesion) is often necessary for the pathologist to determine if the lesion is benign (not cancerous) or malignant. If the lesion is determined to be malignant, examination of the cells will help the pathologist correctly classify the lymphoma.

In the case of highly suspected lymphoma, a lymph node biopsy is evaluated in several different ways. Each test requires the tissue from the biopsy to be processed in a specific manner. The surgeon generally submits the freshly biopsied tissue to the pathology laboratory. The pathologist, depending on characteristics of the tissue itself (i.e. how much tissue there is, whether the cells are still alive, whether the tissue is firm or not) and the clinical suspicions of the treating doctors, will process the tissue accordingly.

Let's take a look at the different methods of diagnosis one by one.

Morphology
The "gold standard" for the diagnosis of lymphoid lesions is still morphology, i.e. microscopic examination of tissue sections. The tissue is processed and stained with special dyes, then looked at using a microscope. A portion of the tissue is fixed in a chemical solution and processed in the pathology laboratory.

The specimen is then embedded in a substance called paraffin, which becomes hard and thus, makes a tissue "block." Sections (or "slices") of the lymph node tissue are cut from the tissue block (similar to slices of bread being cut from a loaf), placed on a glass slide and stained with special tissue dyes, usually hematoxylin ("H") and eosin ("E"). This slide is then evaluated by the pathologist using a microscope.

Both architectural features (i.e. the overall structure of the lymph node or tissue) and cellular features (i.e. normal mixture of cells vs. an abnormal population) of the tissue sections (slides) are evaluated with a microscope. These features are used to help separate benign from malignant lymphoid tissue. If the pathologist decides that the biopsy looks like a lymphoma, the lesion is then classified (see Lymphoma Classification) based on morphology. However, morphologic classification is increasingly being supplemented by phenotypic and genotypic studies (see below). These other studies are important to help classify the sub-type of lymphoma as well as to prevent errors which may occur using microscopic appearance alone. For example, some benign processes and even other types of cancer (such as melanoma) may look just like a lymphoma under the microscope.

Phenotype Testing
The phenotype of lymphoid cells is determined by special laboratory tests. These tests, collectively known as cell marker analysis (or "immunophenotyping"), use a variety of antibodies to detect certain cell "markers" present on the malignant lymphocytes. These specific cell markers help to further characterize the lymphocytes, and thus the type of lymphoma.

For non-Hodgkin's lymphomas, cell marker analysis helps to determine whether the lymphoid cell population is composed of many different cells or if there is only one cell type present. This determination of "clonality" of the lymphoid proliferation is extremely important because polyclonal lymphoid cell lesions (composed of many lymphocytes of many types) are benign, while monoclonal lesions (made up of many lymphocytes of one type) are malignant.

There are two main sub-groups of lymphocytes, known as T cells and B cells. Cell marker analysis can help determine whether B cells or T cells are present. The analysis can also tell how mature the cells are. Both of these features (type of lymphocyte and stage of maturity of the cells) are important in determining the sub-type of lymphoma, which is important for treatment and prognosis. Interestingly, most lymphomas in the U.S. are B cell lymphomas.

Cell marker studies are crucial in determining the type of Non-Hodgkin's lymphoma. However, they can also be helpful in determining the presence of Hodgkin's disease. Using cell marker studies alone can be quite difficult in Hodgkin's disease, so these studies must often be used in conjunction with the cell appearance to ensure a correct diagnosis. The unique Reed-Sternberg cells (see related article on lymphoma classification) must generally be present before a diagnosis of Hodgkin's lymphoma is made.

Cell marker studies can be done using "fixed" or fresh/frozen tissue. Usually, the pathologist prefers to receive a fresh specimen and he or she can then decide the best way to process the tissue. There are two main ways to do cell marker studies. The first is called flow cytometry, which involves using cells suspended in fluid. The cells are either received already suspended in fluid (in the case of blood or bone marrow), or need to be manually separated (in the case of a tissue biopsy) and placed in a fluid solution. The cells are then stained with antibodies (to detect different cell markers) and run through a machine called a flow cytometer.

The other method for doing cell marker studies is called immunohistochemistry. This involves cutting the tissue into sections (as described above) and applying antibody stains. The pathologist then examines the slides with a microscope, to see if the tissue is positive for a particular marker..

Gene Testing
In some cases the combination of morphology and cell marker studies still will not allow one to separate benign from malignant lymphoid lesions. In these cases genotypic studies, or analysis of the genetic composition of the tumor, may be necessary. Several different methods can be used to do these studies (e.g. cytogenetic analysis, Southern blot hybridization analysis and the polymerase chain reaction). Certain genetic markers or mutations detected with genotypic studies are unique to different types of lymphomas. These studies are quite specific, and they can take a significant amount of time. Final results may not be available for up to three weeks following a lymph node biopsy.

Summary
The accurate and specific diagnosis of lymphoid proliferations is complicated and involves many diagnostic techniques. The expertise and knowledge of the pathologist with respect to morphology, phenotype and genotype of the various lymphoid lesions is crucial for diagnosis. Specialized training in a lymphoma center is usually required before a pathologist can learn the necessary techniques and their appropriate use. With this specialization, a pathologist, working closely with the primary doctor and the surgeon, can usually make an accurate and specific diagnosis with respect to lymphoid lesions.