Flow Cytometry

Malignant cells differ from their normal counterparts in the pattern of genes expressed and consequently the proteins the cell produces. Flow cytometry is a highly effective method of identifying both normal and malignant populations according to the pattern of protein expression.

Flow cytometric analysis is only applicable to liquid samples with individual cells free in suspension. Obviously, this is already the case for samples of blood, aspirated bone marrow or other tissue fluids. However, it is often possible to prepare a cell suspension from the solid tissue biopsy providing the sample is sent to the laboratory before being placed in formalin. This adds considerably to the reliability of a diagnosis made on this type of biopsy.

Flow cytometry depends on the use of monoclonal antibodies as probes to detect the presence of a specific protein or other molecule on the cell surface or in the cytoplasm or nucleus. These antibodies are labelled with a fluorescent dye that emits light of a given colour when excited by a laser. Before analysis a sample of the patient’s cells is mixed with a number of different antibodies – typically 6 or 8 – each labelled with a different dye. The choice of antibody combinations is crucial and often several assays using different but overlapping combinations would be carried out on the same sample to obtain a detailed profile of the cells present.

The flow cytometer works by focussing the suspension into stream of individual cells which pass through a series of lasers to excite the fluorescent dyes. Detectors then measure the emissions from the dyes and hence the amount of each protein expressed on each cell. Modern instruments allow several hundred thousand cells to be analysed in a few minutes.

The output of the cytometer is a file containing data on each individual cell analysed which includes the expression of the proteins included in the assay and some physical characteristics such as cell size. This data is then analysed to identify populations of cells- normal or malignant- based on patterns of protein expression.

Modern flow cytometric analysis is an efficient and highly reliable technique for investigation of patients with suspected haematological malignancies. It is the most important single investigation in most types of leukaemia and lymphoma involving blood and marrow. Flow cytometry is particularly effective in identifying malignant cells present in complex mixtures of normal cells, which is frequently the case in the bone marrow and lymph nodes. Developments in instrumentation and reagents have increased the sensitivity of detection and flow cytometry is now used to monitor patients after treatment where very low levels of residual disease may be present. This allows more precise delivery of therapy and prediction of the outcome of treatment. The most important factor that can limit the use of flow cytometry is the quality of the sample, so placing samples in the correct bottles and timely transportation to the laboratory is crucial.