Flow Cytometry

Question 1

What is flow cytometry?

Answer 1

A method of counting fluorescently labelled cells by hydrodynamically forcing them into a narrow beam and passing them by a laser

Question 2

How does flow cytometry allow for the typing of cells?

Answer 2

Measures forward scatter of light to determine cell size

Measures side scatter of light to determine cell granularity

Cells can be labelled with fluorochromes specific to specific molecules e.g. CD molecules, these cells will fluoresce when they pass the laser

Question 3

Explain how flow cytometry works
(7)

Answer 3

Sample is taken into red-topped EDTA tubes

The sample can then be incubated with fluorochromes

The sample then undergoes sample preparation -> red cells are lysed in lysis solution

Sample is then put onboard the analyser whereby cells are hydrodynamically focused into a narrow stream so that each cell is analysed individually

Forward scatter and side scatter is measured

Any cells bound to fluorochrome will fluoresce as they pass by the laser

Optical signals are converted into electronic signals which are used to produce a histogram

Question 4

What are fluorochrome?

Answer 4

Molecules which may be excited by energy to emit light of a longer wavelength

Question 5

How are fluorochromes used in flow cytometry?
(2)

Answer 5

They are used in two ways:
- as dyes or attached to antibodies

More than one fluorochrome can be used per sample as different ones each have different emission spectra

Question 6

Give two examples of fluorochromes

Answer 6

FITC -> fluorescein isothicyanate
PE -> phycoerythrin

Question 7

What does FITC stand for?

Answer 7

Fluorescein isothiocyanate

Question 8

Talk about the sample type used for flow cytometry
(4)

Answer 8

EDTA whole blood used

Sample incubated with fluorochrome if desired

Sample prepped -> rbcs lysed

Sample transferred to Trucount tubes

Question 9

What are Trucount tubes?
(3)

Answer 9

Tubes used to determine the absolute number of positive cells in the sample

Tubes contain a known number of fluorescent beads

Cellular events compared to bead event to calculate the absolute count

Question 10

How do we interpret flow cytometry?
(3)

Answer 10

Lenses collect light emitted and route it to optical detectors whereby optical signals are converted to proportional electronic signals which are then digitalised in the form of a dot plot (histogram/cytogram)

Forward light scatter (cell size) is plotted on X-axis

Side scatter (granularity) is plotted on Y-axis

Question 11

What is meant by gating in flow cytometry?

Answer 11

Software packages which only allow certain leucocyte subsets to be enumerated

i.e. only certain cell types (cells with certain CD molecules e.g. CD20 cells) will be counted

Question 12

What are some applications of flow?

Answer 12

Mostly used to monitor AIDS and SCID

AIDS patients -> monitor CD4 and CD8 populations -> CD4 steadily decreases as disease progresses -> relative % of CD8 is elevated as well

SCID -> monitor CD8 populations -> relative percentage of CD8 is elevated in many patients

Question 13

What cell is CD3 found on

Answer 13

T-cells

Question 14

What cell is CD4 found on

Answer 14

Helper T

Question 15

What cell is CD8 found on

Answer 15

Cytotoxic T

Question 16

What cell is CD45 found on

Answer 16

Leucocytes (all of them)

Question 17

What cell is CD19 found on

Answer 17

B lymphocytes

Question 18

What cell is CD16/56 found on?

Answer 18

Natural killer cells
CD16 also found on neutrophils

Question 19

Why might CD19 be measured?

Answer 19

Used to monitor Rituximab treatment

Question 20

How does Rituximab work?

Answer 20

Rituximab attaches itself to all the CD20 proteins it finds to mark them.
Then it triggers the cells of the immune system to pick out the marked cells and kill them.
Rituximab destroys both abnormal and normal B-cells.
Once treatment is over, the body can replace the normal B-cells.

Question 22

G

Answer 22

Y