FEATURE
IMMUNOLOGY
assays that are integral to the modern management protocols in hematolymphoid malignancies. This will help in rapid diagnosis and monitoring of the disease using a single platform and technology.
BACKGROUND Multicolour flow cytometry is a natural extension of the very concept of flow cytometric immunophenotyping of hemopoietic cells in health and disease. The power of the technology allows simultaneous and/or sequential examination and analysis of a large number of immunophenotypic features of cells of interest which in turn provides a detailed picture of their complex antigenic profile. The information obtained from analysis of normal hemopoietic cells provides the basis for detection of abnormalities that occur in these cells in pre-leukemic and leukemic processes. It also allows visualization and monitoring of the entire spectrum of sequential changes in the antigenic profile hemopoietic cells that occur during the evolution of the disease in hematolymphoid malignancies. This article describes the important progresses in this field in recent times and makes forecasts about the direction in which future research in and application of this approach is likely to head.
PRINCIPLE OF MULTICOLOUR IMMUNOPHENOTYPING ‘Multicolour’ flow cytometry involves simultaneous use of three or more antibody reagents in a single tube, each reagent being tagged with a different fluorochrome so that binding of each antibody to the cell could be visualized at the same time. This approach allows detection of concurrent expression of three or more antigens by a single cell thereby providing a plethora of information about the phenotype of the cells. The number and choice of fluorochromes and of antibody reagents can be increased by increasing the number of lasers thereby further enhancing the extent and depth of the information. However, technical issues with respect to the choice of the right combination of fluorochromes per tube (e.g. interference among emission spectra of the fluorochromes) and the non-availability of antibodies of interest conjugated with appropriate
“Most advanced clinical flow cytometry laboratories have settled for 8-10 colour analysis after a lot of trial and error”
fluorochromes could be a major challenge when one increases the number of reagents per tube. Therefore, the emphasis is shifting towards judicious use of flow cytometric data obtained with the help of a limited number of antibodies.
JUSTIFICATION FOR MULTICOLOUR FLOW CYTOMETRY Conventionally, flow cytometry laboratories use 2-4 antibodies tagged with different fluorochromes per tube. Therefore, smaller the number of antibody reagents per tube, larger is the number of such tubes per sample. This involves more expense on account of reagents, manpower and consumables. This approach also fails to provide a comprehensive picture of the antigenic profile of the cells that one can obtain by simultaneously looking at multiple antigens by using larger number of reagents per tube as explained above. So subtle changes in the expression
of one or more antigens that is associated with dysplastic and neoplastic changes in these cells will be missed out when one uses a limited number of markers per tube. Often the tissues (bone marrow, CSF,
fine needle aspiartes and lymph node biopsy material) received by the laboratory for immunophenotyping are very small in quantity and are precious since they are obtained after an invasive procedure. The number of neoplastic cells too in these tissues could be small. Under these circumstances putting up multiple assay tubes for immunophenotyping would not be possible and therefore, such situations provide an ideal opportunity to perform multicolour immunophenotyping using 8-10 or more reagents per tube. The new generation of clinical flow cytometers are designed for this purpose and can handle assay tubes with 8-10 colour reagents each.
ADVANTAGES OF MULTI- COLOUR FLOWCYTOMETRY Multicolour flow cytometry can potentially provide a plethora of information about the antigenic profile of the cells of interest even if the latter are present in a very small number and in a complex mixture. Predictability of the antigenic profile of normal cells of different hemopoietic lineages and that of cells at different stages of maturation allows recognition of the slightest aberration that can happen in myelodysplastic syndromes, leukemia and
MedLab Issue 3 2011 31
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40