Flow Cytometry as a diagnostic tool

Flow Cytometry has become an integral research tool to categorise cells. A review of its applications

Flow Cytometry is a technology that simultaneously measures multiple characteristics of single cells at a rapid rate. It is a technique for counting, examining and sorting cells and other microscopic particles suspended in a stream of fluid. It could be compared to a highly-automated and much specialised fluorescence microscope. It allows simultaneous multi-parametric analysis of the physical and/or immunological characteristics of a single cell flowing through an optical and electronic detection system. Physical characteristics such as cell size, shape, and internal complexity can be analysed and any cell component or function that can be detected by a fluorescent compound can be examined.

There are mainly two types of Flow Cytometry: analysers which allow a particular type of cell population to be identified qualitatively and quantified, are mainly used in clinical set up, however can also be used for research purposes and sorters which are a dual function systems. They can be used as analysers (identify and quantify cell population) as well as sort (collect in a tube/s) the cell population/s of interest for further research. The sorters are mainly used for high end cellular research. The flow sorted cells are yet to be used in a clinical set up as regulatory guidelines and ethical issues are yet to be addressed.

Flow Cytometry has been used for the past 20 years in India, mostly in the premier ICMR/CSIR/DBT/AIDS Research labs. The last five years have seen rapid strides in applications of Flow Cytometry in the diagnostics as well as research.

Flow Cytometry has become one crucial and well established tool in medical diagnostics and clinical research particularly in the areas of transplantation, hematology, stem cells, tumor immunology, chemotherapy and genetics due its capability to detect multifarious parameters from a single cell very accurately and rapidly. This feature also provides a very distinctive edge over other conventional methodologies, as it is non invasive, easy to design, execute and provides live cell quantification.

High end Flow Cytometry equipped with cell sorters, furthermore, help in isolating live cells (stem cells/sperm cells etc.) from a mixed pool of cells.

Flow Cytometry in Diagnostics

Flow Cytometry helps in the prognosis and diagnosis of disease and disease progression, drug therapy monitoring. Today, it has become an integral tool for research labs to categorise cells in the areas of drug discovery, molecular biology, pathology, immunology, plant biology and marine biology. This technique has the ability to increase scientific knowledge of conditions such as human immunodeficiency virus (HIV) and to monitor the immune status of those who have been diagnosed with the disease. Flow Cytometry is a technology that is mainly used in the area of haematological diseases, both in the treatment and diagnostics of leukaemia- lymphomas as well as solid tumours and plays a significant role in the area of stem cell transplantation. Flow Cytometry assumes significance as it is more reliable compared to conventional techniques as analysis can be performed faster on a large number of cells (millions) as compared to few hundreds in conventional methods , which are tedious and time consuming.

Role in HIV and Cancer

A key clinical application of Flow Cytometry is in the treatment of HIV patients. It helps in enumerating the helper T cells during the course and treatment of HIV infections. The other important clinical application of flow Cytometry is that it helps in determining the DNA content and proliferation kinetics of cells in cancers like breast cancer and other malignant diseases. This technique can also be used to quantify testicular cells of infertile patients. In many cases of cancers, the normal methods of analysis are very laborious and take painstakingly long time to complete diagnosis. Various stages of cancer can be predicted reliably using sensitive monoclonal antibodies to cancer biomarkers that can be detected using Flow Cytometry.

Transplantation

Immunological rejection remains a major barrier to successful organ transplantation. Flow Cytometry has become a useful tool for monitoring immunological responses in transplant recipients. There are three areas of clinical transplantation immunology that may benefit from this technology. First, characterizing and classifying all reactive antibodies identifies high-risk donor and recipient combinations with greater precision. Second, the ability to detect subtle changes in the cellular components of the immune system cytometrically may facilitate the differential diagnosis of rejection, infection, and iatrogenic toxicity. Finally, the ease with which Flow Cytometry determines the adequacy or inadequacy of immunosuppressive therapy through T cell receptor analysis serves to maximize the beneficial effects of engraftment. Flow Cytometry is used to determine CD34 positive stem cell counts. These stem cells are used in bone marrow and peripheral blood stem cell transplantation.

Haematology

The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Many surface proteins and glycoproteins on erythrocytes, leukocytes, and platelets have been studied in great detail. The availability of monoclonal antibodies directed against these surface proteins permits Flow Cytometry analysis of erythrocytes, leukocytes, and platelets. Antibodies against intracellular proteins such as myeloperoxidase and terminal deoxynucleotidyl transferase are also commercially available and the ever increasing number of these types of antibodies permit analysis of many intra cellular proteins.

Tumor immunology

Flow Cytometry has an extremely important role in studying cellular characters in tumor immunology and chemotherapy. Primarily, the tests are conducted for the analysis of the state and stage of progression of the disease. A wide range of surface marker antibodies are available to understand whether the patient is suffering from T-cell or B-cell malignancies. Furthermore, diagnosis is also made in the sub-classification of the lymphomas and leukemias like - AML, ALL, AMPL, CLL. Analysis of cellular populations involved in anti tumor immunity and development of cell based therapies has been possible by the use of Flow Cytometry

Genetics

On the genetics front, Flow Cytometry helps in finding the ploidy status of cells. Meaning, whether the cells have 2x or 4x of the chromosomal numbers or any other anueploid or polyploidy condition. Sorters also help in the karyotyping of chromosomes and separating these chromosomes for further studies. To cite an example - human genes coding cell surface molecules can be introduced into mouse host cells using a variety of somatic cell genetic techniques. Because these human gene products can be detected using indirect immunofluoresc-ence on viable cells, the genes themselves can be monitored and manipulated using Flow Cytometry and sorting.

Flow Cytometry Research

The research in Flow Cytometry is targeted towards developing better fluorochromes, and better diagnostic markers for more accurate information. Many of the newer studies are targeted at understanding the cell signaling mechanisms in normal and diseased states like cancers, HIV, Infectious diseases etc. Research has also helped in understanding the cellular apoptotic and proliferation mechanisms and patterns. For all these studies, many new markers have been identified and put to use in Flow Cytometric applications in healthcare.

(This write up has been contributed by Beckman Coulter)