Applications of Flow Cytometry

Flow Cytometry

In flow cytometry, microscopic particles are suspended in a stream of fluid and passed one-by-one by a laser beam, which allows for the multiparametric analysis of thousands of individual cells per second. Prominently used in research laboratories, flow cytometry has become increasingly routine in the diagnosis, monitoring and prognosis of disease in clinical labs due to the development of less expensive and more user-friendly devices.

With the capability to measure and investigate multiple variables in individual cells, flow cytometry is a powerful tool in disease analysis. Because of its extreme sensitivity, flow cytometry allows for the detection of disease at low levels. While commonly used to determine the presence of antigens on or within cells, flow cytometry also can be used in the analysis of DNA or RNA and other functional cell studies. What follows are some of the current clinical applications of flow cytometry.

Diagnosis of Hematologic Malignancies

Immunophenotyping is one of the most significant applications of flow cytometry. This technique is crucial to the diagnosis and monitoring of leukemias and lymphomas, as treatment of these diseases often depends on antigen variables. Based on laboratory findings, leukemias are classified into chronic and acute forms. At each stage in the development of blood cells within bone marrow, the cells carry distinctive markers classified by the cluster of differentiation. Malignancies can develop at any stage.

Leukemias and lymphomas will express a different, specific set of markers depending on the stage of development and pathway of differentiation. Using these differentiations, they are classified. The evaluation is made using a panel of antibodies, usually in three or more color combinations. The panel that is selected will depend on the initial diagnosis; the results from this screen may indicate a need for further classification.

Detection of Minimal Residual Disease

For the detection of minimal residual disease (MRD), laboratory procedures must meet the criteria of sensitivity, reproducibility, applicability and specificity. MRD is defined as “disease that is beyond detection using conventional microscopy.” Flow cytometry detects abnormal immunophenotypic features in cell populations in the study specimen and is effective in detecting the presence of malignant cells in the bone marrow and other tissues of patients with hematologic malignancies after remission. Residual malignant cells are believed to cause disease relapse in many patients.

Efficacy of Cancer Chemotherapy

One of the leading challenges in oncology is choosing the most effective chemotherapeutic agent for a patient. It is difficult to account for the variances in individual tumors. Multiple drug resistance (MDR) also plays a major role in the failure of many of the natural products used as chemotherapeutic agents. Along with immunocytochemistry and molecular techniques, flow cytometry has been established as a tool to measure the mechanisms that lead to MDR. During and after chemotherapy treatment, flow cytometry is used to confirm the bonding of an antibody and monitor tumor cell extermination.

Applications in Microbiology

Due to the need to culture microorganisms for study, classic microbiology techniques remain slow in comparison to other analytic approaches. Flow cytometry allows for single or multiple microbe detection based on peculiar cytometric parameters or through flourochromes that can be bound to specific oligonucleotides or antibodies, or be used independently. This provides for faster, reliable detection of microbes. Flow cytometry also allows for the development of quantitative, highly reproducible procedures to assess drug cytotoxicity and antimicrobial susceptibility.

HIV destroys the CD4 cells that are necessary for effective immune response. The regular counting and monitoring of these cells through the use of flow cytometry can measure the effect of treatment, disease progression, and determine what infections the patient may be susceptible to. In addition to the CD4 cell count, blood serum virus concentration is the most clinically useful parameter to evaluating treatment success.

Virus detection is also important in preventing mother-to-child transmission. Due to the high cost of viral load testing and limited available resources, that testing is often not an option in sub-Saharan African, where transmission is a chief concern. While traditional flow cytometry systems are also expensive, portable benchtop systems have been successfully used in Africa to monitor and count patients’ T cells.

Cell Function Analysis

Cell function analysis can reveal clinical information, such as the expression of surface antigens, which cannot be obtained from static cellular parameters. Such considerations are relevant in diseases of the immune system and transplantation medicine, so much of the research has focused on the functional analysis of the lymphocyte. Flow cytometry can measure essentially any event that might occur during the lymphocyte activation process.

Applications in Organ Transplantation

Through the information gathered during immunophenotypic analysis, many of the mysteries of transplantation have been answered. Flow cytometry plays an important role in the detection of biologic substances and cellular antigens, crucial for the investigation of the mechanism of action for immunosuppressive drugs and the immunobiology of graft acceptance and rejection. In solid organ transplant, flow cytometry can be useful in T cell cross-matching, post-transplantation monitoring and HLA antibody screening.

A Look Ahead

Emerging and expected clinical applications for flow cytometry include further applications in clinical microbiology, high-throughput flow cytometry for predicting drug-induced hepatotoxicity, and leveraging flow cytometry to evaluate disease phenotype and the impact of treatment with immunomodulatory therapeutics.

Stephanie Ogozaly is a former editor with ADVANCE.