Immunophenotyping for Haematologists. Barbara J. Bain

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Название Immunophenotyping for Haematologists
Автор произведения Barbara J. Bain
Жанр Медицина
Серия
Издательство Медицина
Год выпуска 0
isbn 9781119606154



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property is known as fluorescence. The amount of light emitted (the number of photons) is proportional to the amount of fluorochrome bound to the cell. The mean fluorescence intensity of a population indicates the strength of expression of the relevant antigen. The emitted light passes through dichroic mirrors, that is, mirrors that reflect some wavelengths and transmit others, so that it is possible, for example, to reflect SSC for measurement and transmit fluorescence signals to another detector such as a photomultiplier tube (Figure 1.1). The detector produces an electrical signal that is proportional to the amount of incident light. Some commonly used fluorochromes are shown in Table 1.1.

      The cells that are studied must be dispersed. For peripheral blood and bone marrow aspirate specimens, it is necessary to exclude mature and immature red cells. This is most simply done by lysing red cells using an ammonium chloride solution. Otherwise red cells and their precursors will appear in scatter plots and interfere with gating leucocyte populations of interest. If assessment of immunoglobulin expression is required, there must also be a washing step to remove the plasma that contains immunoglobulin, which would neutralise the monoclonal lambda‐ or kappa‐specific antibody.

Fluorescein isothiocyanate (FITC)
Phycoerythrin (PE)
Allophycocyanine (APC)
Peridinin chlorophyll (PerCP)
Cyanine 5 (Cy5), cyanine 5.5 (Cy5.5) and cyanine 7 (Cy7)
Texas red
Pacific blue
Brilliant violet
Krome orange
Alexa Fluor 488 (AF488)
Alexa Fluor 647 (AF647)
Phycoerythrin‐Texas Red X (ECD)
Phycoerythrin‐cyanine 5 (PE‐Cy5)
Phycoerythrin‐cyanine 5.5 (PE‐Cy5.5)
Phycoerythrin‐cyanine 7 (PE‐Cy7)

      Results of immunophenotyping are usually shown as a two‐dimensional plot in which FSC, SSC and the expression of certain antigens are plotted against each other, permitting the recognition of the probable nature of a cell cluster in a particular position. It is thus possible to gate on a cellular population of interest. A gate is an electronic boundary; it can either be predetermined or drawn by the operator. There are four commonly used approaches to gating of target populations: FSC versus SSC, CD45 versus SSC, CD19 versus SSC and CD34 versus SSC.

      Forward scatter is increased in relation to increasing cell size whilst SSC is influenced by cytoplasmic granularity and nuclear complexity. It is a useful means of gating on blasts when CD34 is not expressed, for example in monoblastic leukaemias. Such plots are helpful in identifying large activated lymphocytes, an excess of small lymphocytes or monocytes and even the presence of hairy cells (see Chapter 3). Granular blasts show increased SSC and this is reflected in a shift to the right in the scatter plot. This can be an early indication of a possible acute promyelocytic leukaemia.

Haematological neoplasms