LITMIR.BIZ

Part 1 Purpose and Principles of Immunophenotyping

      CONTENTS

      Immunophenotyping is the process by which the pattern of expression of antigens by a population of cells is determined. The presence of a specific antigen is recognised by its binding to a labelled antibody. Antibodies can be present in a polyclonal antiserum that is raised in an animal but more often they are well characterised monoclonal antibodies produced by hybridoma technology; a hybridoma is a clone of cells created by the fusion of an antibody‐producing cell with a mouse myeloma cell. Monoclonal antibodies can be labelled with an enzyme or with a chemical, known as a fluorochrome, that under certain circumstances will fluoresce. Immunophenotyping is carried out primarily by flow cytometry or immunohistochemistry. Flow cytometric immunophenotyping is applicable to cells in peripheral blood, bone marrow, body fluids (pleural, pericardial, ascitic and cerebrospinal fluids) and fine needle aspirates. Immunohistochemistry of relevance to haematological disease is applied particularly to trephine biopsy and lymph node biopsy specimens, but also to biopsy specimens from any other tissues where infiltration by haemopoietic or lymphoid cells is suspected.

Flow Cytometric Immunophenotyping

This technique determines cell size, structure (to some extent) and antigen expression. Cells in suspension are first exposed to a combination of fluorochrome‐labelled monoclonal antibodies (or other lectins or ligands) and then pass in a focused stream through a beam of light generated by a laser. Laser‐generated light is coherent (waves of light are parallel) and monochromatic (single wave length/colour). Large multichannel instruments with multiple lasers are used to identify, count, size and otherwise characterise cells that are hydrodynamically focused and pass in a single file through a narrow orifice in a flow cell. The passing of the cell through a light beam leads to both the scattering of light and the excitation of fluorochromes so that they emit a fluorescence signal. Forward scatter (FSC) of light at a narrow angle is detected and measured and is proportional to cell size. Sideways or side scatter (SSC) of light is detected and measured and is proportional to cell granularity and complexity. Antigens expressed on the surface membrane of cells or, with modified techniques, within cells are detected. After ‘permeabilisation’, both cytoplasmic and nuclear antigens can be detected.