Advanced Technology for Understanding the Immune System and the Cell Biology of Cancer with Flow Cytometry and FISH

The diagnosis of cancer is often suspected and/or confirmed  from symptoms, examination, radiologic imaging, and laboratory testing. The evaluation of suspicious cells from the pathologist can provide a diagnosis, but the nuances of the specific disease has become more focused with the advanced technology of flow cytometry.

Introduction

A specific microscopic cancer diagnosis reveals the type of cells but few of its individual characteristics.

Flow cytometry is a technological method, using blood or tissue, to provide diagnosis and analysis of cancer cell characteristics and for monitoring disease in solid tumors, and is the standard in diagnosing blood and bone marrow cancers, the leukemias and lymphomas. 

It can also assess specific immune cell populations such as T-cells, B-cells, and marrow cells, and their subsets, based on their surface markers. The information provided can reveal immune strength and health and their functional responses of activation, proliferation and cytokine production. 

By identifying cancer specific cell types and their biologic characteristics, oncologists can be guided toward therapies that will be effective and individualized to each patient. This data can also be used for comparison of pre and post treatment effectiveness and can help determine how well treatment is working or whether there is a need for change.

Testing allows thousands of cells to be placed in a fluid, collected and sorted rapidly and analyzed. They are then labeled with fluorescent dyes or proteins which attach to cell marker antigens on the cell’s surface. The cells are then subjected to optical lasers causing the signal emissions that can be detected, measured and processed by computer software to determine multiple cancer specific cellular properties simultaneously.

Cytometry also allows the identification of the immune components present, the physical and chemical characteristics of each cell subtype, levels of immune activity, growth and replication and functional qualities such as cytokine activity and response to infection.

Immunophenotyping

Immunophenotyping is an application that evaluates specific antibodies attached fluorescent dyes that attach to immune cell proteins, antigens, on white blood cells to identify whether it is healthy or malignant. It is used as a diagnostic tool for a number of leukemias and inherited immunodeficiency to identify diseases.

These antigens are called cluster of differentiation or CD numbers and their presence allows therapies to be directed against a specific cellular antigens.

Immune cells have specific CD markers.. For example T cells have CD4 and CD8 while B cells have CD 20 and 19 and natural killer cells(NK) markers are CD56 and CD161.

There are a variety of other markers that can be evaluated for various aspects of immune function and activity. Measurements can include cytokines, the ability to recognize antigens and after immune activity the measurement of long term immune memory, and levels of proliferation. The marker Ki 67 is such a marker and indicates the activity of rapidly dividing cells that is used to assess aggressiveness in breast cancer. 

Fluorescence in Situ Hybridization (FISH Test)

Alterations in the cellular DNA of cancer cells, whether genetic or from alterations in the DNA expression, often caused by toxic exposures, can change cellular pathways creating abnormal protein production that affects cancer growth and replication.

FISH is genetic testing that can recognize chromosomal DNA abnormalities by allowing direct visualization of the genes. It maps genetic material identifying mutations, missing or extra gene copies or rearrangements of different genetic areas.

To perform the test tissue or blood samples are obtained to which a fluorescent compound is bound to the cancer cell’s DNA sequences. The compound absorbs light at one wavelength but when activated emits a longer wavelength of light that when viewed against a dark background allows structures to be visualized by a pathologist under an optical fluorescent microscope. 

Identifying specific gene mutations or chromosome abnormalities can assist in cancer diagnosis and treatments. It is used in various cancers including breast, chronic lymphocytic leukemia, CLL, multiple myeloma, glioma, bladder cancer, acute and chronic myeloid leukemia and melanoma. In breast cancer it assesses HER-2 gene status and in CLL TP53 mutations, both of which can provide direction for more effective treatment options.

For prognosis, FISH can identify genetic patterns that can predict how cancer is expected to behave, as well as provide information as to how a given tumor will respond to specific therapies or whether the cells might be resistant.