Immunophenotyping using flow cytometry

Dr Sushant Agrawal

One of the most common clinical applications of the flow cytometer is in the determination of cell surface antigens “Immunophenotyping”.

What is Flow Cytometry?

Flow cytometry is the measurement (-metry) of cellular (cyto-) properties as they are moving in a fluid stream (flow), past a stationary set of detectors. Latest developments go a step further and allow cells of different subtypes to be sorted and collected for further analysis. It is capable of rapid, quantitative, multi-parameter analysis of heterogenous cell populations on a cell-by-cell basis (single cell analysis).

How does it work?

Performing flow cytometry experiments generally involve three distinct, interdependent phases. First is the pre-flow cytometry phase, which involves staining of cells with fluorescent reagents. Second is the flow cytometry phase, which involves processing the stained cells using flow cytometry instrumentation and collecting data (acquiring) for one or more measurements (which are called parameters) made on each individual cell. Finally, the analysis phase involves analysing the collected data. These three phases may all be performed by a single individual during the course of one day.

Flow cytometry facilities at Thyrocare

For the first time in India, a commercial laboratory will be having two flow cytometry instruments:

1. The BD FACSCalibur Sort 4 Colour flow cytometry System is the only instrument with four-color, dual-laser (two air-cooled argon laser), automated sample loader option, and benchtop system available today that is capable of both cell analysis and sorting. It is a fully integrated multiparameter system, designed specifically to support a wide range of clinical and research applications.

2. The BD FACScan 3 Colour flow cytometry system has a fixed single air-cooled 488nm, Argon laser and three-color cell analysis capabilities. It can analyse five parameters per cell also includes simultaneous analysis of multiple parameters (upto 7).

Presently, Thyrocare’s flow cytometry services include:

Immunophenotyping for HLA-B27: HLA-B27 is one of the multiple major histocompatibility complex (MHC) class I-specific antigens. HLA-B27 testing is clinically relevant to the evaluation of seronegative spondyloarthropathies. There is a strong association of HLA-B27 with ankylosing spondylitis (AS) as well as with several other disorders, such as Reiter’s syndrome, psoriatic arthritis, and inflammatory bowel disease.

Not only does the presence of HLA-B27 significantly aid in the diagnosis of these disorders, but those patients who are HLA-B27 negative in general have a milder disease and better prognosis than patients who are HLA-B27 positive. Therefore, HLA-B27 testing is helpful even when the results are negative.

At Thyrocare, HLA B27 estimation on flow cytometry is done using automated software, which gives the value without any manual intervention. HLA B27 software classifies a given sample as HLA B27 negative or as HLA B27 positive. This classification is based on the measured median fluorescence intensity compared to a predetermined decision-marker setting.

Lymphocyte Subset Analysis(CD3/CD4/ CD8):

The typical pattern of HIV primary infection is characterized by high levels of virus in blood followed by a progressive loss of CD4 T cells, elevation of CD8 T cells and progressive impairment of T cell functions. Between the primary infection and the final progression to AIDS, there is a period of clinical latency lasting