Primer

The Flow Technique

For its contribution to a clearer picture of cell behaviour and a better understanding of how cells respond to specific therapies, Flow Cytometry has become an obvious choice for diagnosis of various diseases, says Sonal Shukla.

Flow Cytometry is a technique for counting, examining and sorting cells and other microscopic particles suspended in a stream of fluid called sheath fluid. It could be compared to a highly-automated and much specialised fluorescence microscope.

Flow Cytometry has now become an obvious choice for diagnosis, as it provides much more accurate and rapid detection capabilities compared to conventional histomorpholgical analysis- microscopy. This also becomes the primary decision criterion for many doctors, hematologists and pathologists and in some cases, oncologists too opt for Flow Cytometry. The most important aspect of Flow Cytometry is its capability to detect multifarious parameters from a single cell very accurately and rapidly. This feature provides a very distinctive edge over other conventional methodologies. High-end Flow Cytometers equipped with cell sorters, furthermore, help in isolating live cells.

Flow Cytometry has been used since 20 years in India, mostly in the premier ICMR/CSIR/DBT/National Aids Control Organisation (NACO) research labs. The last five years have seen rapid strides in the applications of the Flow Cytometers in the diagnostic fields. Flow Cytometry has become one crucial and well-established tool in medical diagnostics as well as in the clinical research particularly in the areas of transplantation, haematology, tumor immunology and chemotherapy and genetics.

In 2003, industry analysts valued the Flow Cytometry market at $650 million, and anticipated a 15 per cent annual rise to reach $1.3 billion by 2008. The clinical Flow Cytometry market in India is growing at 25 per cent CAGR.

Basics of Flow Cytometry

Flow Cytometry 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 size 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 Cytometers —analysers and sorters. The analytical Flow Cytometers are used in clinics, while sorters are often used in research laboratories. "The analysers allow quantifying of a particular type of cell population/s. Whereas, sorters not only allow to quantify, but also sort the cell population/s of interest. However, the flow sorted cells are yet to be used in a clinical set-up. Presently, the use of flow-sorted cells is limited to research and analysis as new meaningful diagnostics applications are under development stage," states Dr H Krishnamurthy, Scientific Officer 'E', National Centre for Biological Sciences, Tata Institute for Fundamental Research, Bangalore.

Flow Cytometry in Treatments

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 the 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 mainly used in the area of haematological diseases, both in the treatment and diagnostics of leukaemia—lymphomas as well as solid tumours. "On the other hand, it is significant in the area of stem cell transplantation and monitoring the treatment of the HIV-infected patients," states Martin Adelmann, Product Support Manager, Systems Biology, Beckman Coulter International S.A.

It defines the physical as well as the immunological properties of certain cell subsets in the blood by use of monoclonal antibodies and by defining the immunological pattern, cellular lineages of the immunological diseases, and after treatment, monitors the progress or failure (if any) of the treatment.

"Flow Cytometry is a powerful technology, which provides for insight into finer details of cellular constituents" - Dr Amar Dasgupta Director- Operations SRL Ranbaxy Clinical Reference Laboratories Mumbai

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 clinical cytometry is that it helps in determining the DNA content and proliferation of tumor cells of various cancers like breast cancer and other malignant diseases. This technique, used for cancer treatments, was first established in 1975 by an Indian-born US Scientist, Prof Awtar Krishan of University Of Miami, Florida. 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.

“Flow Cytometry is a powerful technology, which provides for insight into finer details of cellular constituents which are not accessible by routine laboratory technology. This power of technology can, therefore, be exploited to learn about the lineage and sub-lineage of a cell, thereby helping in correct characterisation of the cell in the diagnosis of haematological malignancies,” opines Dr Amar Dasgupta, Director- Operations SRL Ranbaxy Clinical Reference Laboratories, Mumbai.

"With the use of many sensitive monoclonal antibodies Flow Cytometry, it helps in identifying the various stages of cancer accurately and the analysis is performed on a large numbers of cells (up to 10 million cells can be analysed within seconds) as compared to few hundreds in conventional methods," explains Vimal Joshi, National Market Manager, Beckman Coulter India Pvt Ltd.

The Working of Flow Cytometer

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, characterising and classifying alloreactive 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 maximise the beneficial effects of engraftment. "Flow Cytometry is used to do CD34 stem cell counts. These stem cells are used in bone marrow and pheripheral blood stem cell transplantation," informs Dr Sumeet Gujral, Associate Professor, Department of Pathology, Tata Memorial Hospital, Mumbai.

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 cytometric analysis of erythrocytes, leukocytes, and platelets. Antibodies against intracellular proteins such as myeloperoxidase and terminal deoxynucleotidyl transferase are also commercially available and permit analysis of an increasing number of intracellular markers. Flow Cytometry is used mainly in CD4/CD8 cell counts, and in immuno phenotyping of leukemias.

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 malignanices. Furthermore, diagnosis is also made in the sub-classification of the lymphomas and leukemias like—AML, ALL, AMPL, CLL.

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 anueploidic condition, sorters also help in the karyotyping of chromosomes and further separating these chromosomes for further studies. To cite another 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 immuno fluorescence on viable cells, the genes themselves can be monitored and manipulated using Flow Cytometry and sorting.

Market at a Glance

Cell Lab Quanta

The market is divided in three segments—research, industrial, and clinical diagnostics.

Research: This segment comprises mainly of premier research institutes across India, and is dependent on Government funding. The main funding agencies are Indian Council of Medical Research (ICMR), Council of Scientific and Industrial Research (CSIR), Department of Biotechnlogy (DBT) and Indian Council of Agricultural Research (ICAR). The flow cytometers used for research are high end and include cell sorting facilities.

Industrial: Industrial segment covers the pharmaceutical and biopharmaceutical companies working in the field of drug discovery research. The studies carried out here are cyto toxicity assays, apoptosis, stem cell enumeration, reticulocyte assays. This market is slowly gathering momentum due to increasing awareness of Flow Cytometry technology and its benefits.

Clinical Diagnostics: This is the fastest growing segment. With the increased awareness about the technology and usefulness in HIV monitoring, lymphoma /leukemia, HLA typing and Cross matching, Platelet enumeration, PNH Diagnosis, reticulocyte counts, DNA analysis being the most common.

Gaining Popularity

"The reasons for popularity are the ease, accuracy and rapidity with which the tests can be performed at reasonably affordable prices" - Dr Chandrashekar S P, Managing Director, Beckman Coulter India Pvt Ltd

In most of the Indian hospitals and diagnostic centers, Flow Cytometers are becoming the choice of analytical tools in diagnosis across a wide spectrum of disease conditions. "The reasons for popularity are the ease, accuracy and rapidity with which the tests can be performed at reasonably affordable prices," states Dr Chandrashekar SP, Managing Director, Beckman Coulter India Pvt Ltd. Flow Cytometers also have a key role in hospital and medical centers world-wide, where they are widely used for diagnosis and clinical research. They have been utilised for assessing ploidy, cell cycle and surface analysis of cancers; immunophenotyping of lymphomas and leukaemias (aiming to define diagnostic and prognostic value); monitoring CD4 lymphocyte levels in the blood (cytometry is the method of choice for follow up the progression or treatement response of AIDS patients). In addition, sorting and high speed sorting of very rare populations like the stem cells are becoming increasingly important in research, clinical trials and clinical applications. There are no manufactures in India for Flow Cytometers. However, two major global players in the market having direct operations in India are Beckman Coulter and Becton-Dickinson. "Its high time Indian physists should start thinking about making an indigenous cytometers in India,"suggests Dr Krishnamurthy.

sonal.shukla@expressindia.com