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Cells and Microscopy

Flow cytometry in blood analysis

  • 3 things to know
  • Components 01, 02 and 03

Overview

Recall questions on how cells are made to pass a laser singly, what forward- and side-scattered light reveal, fluorescent antibody labelling, reading a scatter plot, and the technique's limitations.

What you need to know

Answer each one before you reveal it.

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Key term

Why is labelling with a fluorescent antibody more specific than using a conventional stain?

Key term

What does each dot on a flow cytometry scatter plot represent?

Key term

In flow cytometry, how are cells made to pass the laser one at a time?

Questions from this subtopic

Each one with the model answer underneath.

  1. 1

    What is flow cytometry?

    Model answer

    It is a technique in which cells suspended in a fluid are carried in single file at high speed past a laser beam, and the light each cell scatters and the fluorescence it emits are detected in order to count and classify it.

  2. 2

    How are cells made to pass the laser one at a time?

    Model answer

    The sample stream is injected into the centre of a faster-flowing sheath fluid, which narrows it hydrodynamically so that the cells are forced into single file.

  3. 3

    What does the amount of forward-scattered light tell you about a cell?

    Model answer

    Forward scatter is proportional to the size of the cell, so larger cells scatter more light in the forward direction.

  4. 4

    What does side-scattered light indicate?

    Model answer

    Side scatter indicates the internal complexity or granularity of the cell, so cells with many granules or a lobed nucleus scatter more light sideways.

  5. 5

    How can a neutrophil and a lymphocyte be distinguished by scattered light alone?

    Model answer

    The neutrophil is larger, so it gives greater forward scatter, and it is far more granular with a lobed nucleus, so it gives much greater side scatter than the small, agranular lymphocyte.

  6. 6

    How are fluorescent labels used in flow cytometry?

    Model answer

    Antibodies specific to a particular surface antigen are joined to a fluorescent dye, so only cells carrying that antigen bind the label and fluoresce when the laser excites them.

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