Practical 5a: Flow Cytometry

Question 1

Define flow cytometry

Answer 1

A technique for counting, examining and sorting microscopic particles suspended in a stream of fluid

Question 2

Explain how flow cytometry works
(3)

Answer 2

Physical characteristics of particles are measured while fluorescently labelled antibodies can be used to examine protein expression

A laser beam is directed at a hydro-dynamically focused stream of fluid containing the cells

A number of detectors measure properties such as forward scatter, side scatter and fluorescence at different wavelengths

Question 3

What does flow cytometry use to fluorescently mark molecules?

Answer 3

Antibodies

Question 4

Describe the stream used in flow cytometry

Answer 4

Hydro-dynamically focused stream

Question 5

What do the detectors in flow cytometry measure?

Answer 5

Forward scatter

Side scatter

Fluorescence

Question 6

What does forward scatter of light reflect?

Answer 6

The size of the particle

Question 7

What does side scatter of light reflect

Answer 7

Complexity/granularity

Question 8

Explain in your own words how the flow cytometer can count cells
(2)

Answer 8

The hydro-dynamically focused pressure of the liquid forces the cells into single file

The cells are marked with fluorescent antibodies which can be counted as they pass by the detector

Question 9

What is the fluid in the flow cytometer called?

Answer 9

Sheath fluid

Question 10

Give an example of how the flow cytometer is used to differentiate T and B lymphocytes from a sample of lymphocytes

Answer 10

Two antibody types are used:
- one is an antibody against CD4 (helper) which is fluorescently marked with FITC
- one is an antibody against CD8 (killer) which is fluorescently marked with PE

Question 11

How many different fluorescent labels can be used in a flow cytometer?

Answer 11

Up to 17-18

Question 12

How does the flow cytometer select the correct excitation wavelengths?

Answer 12

By using a set of filters

Question 13

What scale are the dot plots created by flow cytometer in?

Answer 13

Logarithmic

Question 14

What does each dot on a scatter plot indicate?

Answer 14

Each dot represents one event passing the laser

Question 15

What CD would you use to detect wbcs?

Answer 15

Cd45

Question 16

What CD would you use to detect T cells?

Answer 16

CD3

Question 17

What CD would you use to detect B cells?

Answer 17

CD19/20

Question 18

What CD would you use to detect helper T cells?

Answer 18

CD4

Question 19

What CD would you use to detect killer T cells?

Answer 19

CD8

Question 20

What CD would you use to detect neutrophils?

Answer 20

CD15

Question 21

What CD would you use to detect monocytes?

Answer 21

CD14

Question 22

What is CGD?

Answer 22

Chronic Granulomatous Disease

Question 23

How do you diagnose CGD?
(2)

Answer 23

Patient’s cells are stained with dihydrorhodamine 123 which is non-fluorescent but when exposed to ROS it is converted into rhodamine which is fluorescent

Cells are stimulates to produce ROS and if they can they fluorescence

Question 24

What is LAD?

Answer 24

Leucocyte adhesion deficiency

Question 25

What happens in leucocyte adhesion deficiency?
(2)

Answer 25

Patient’s fail to express CD18, the B2 integrin chain

As a result, leucocyte can along the endothelial walls but cannot bind firmly and get into the inflamed tissues

Question 26

How does flow cytometry diagnose leucocyte adhesion deficiency?

Answer 26

Flow cytometry can detect the expression of CD18 on white cells

Question 27

What is XLA?

Answer 27

X-linked agammaglobulinemia

Question 28

What happens in X-linked agammaglobulinemia?
(2)

Answer 28

A condition that manifests in males who have an inherited mutated copy of Brutons’ tyrosine kinase (Btk) on the X chromosome

It results in B cells not being produced

Question 29

What mutation causes XLA?

Answer 29

A mutated copy of Brutons’ tyrosine kinase