Flow Cytometry

Question 1

Describe cytometry

Answer 1

technique for making measurements on cells, usually done via microscopy

Question 2

Describe flow cytometry

Answer 2

measurements done while cells flow past detectors

Question 3

What are the 2 main parts of flow cytometry

Answer 3

• fluidics
• optics
  however also electronics and computer interface

Question 4

Describe fluidics

Answer 4

• cells do not pass through centre of measuring point - leads to variable measurements
• hydrodynamic focusing leads to reproducible measurements

Question 5

Describe optics

Answer 5

• light scattering detected
• optical filters allow light of selected wavelengths to pass through while limiting transmission of other wavelengths

Question 6

Describe fluorescence

Answer 6

• light of one wavelength absorbed and light of a different wavelength is emitted
• emitted light always of lower energy and therefore longer wavelength than the exciting light

Question 7

What are the different types of filters?

Answer 7

• dichroic filters - reflects light
• standard band pass filters - mixed wavelengths

Question 8

Describe the graphs used to analyse flow cytometry

Answer 8

• x axis is fluorescence
• y axis is number

Question 9

Describe the disadvantages of conventional cytometry

Answer 9

• labour intensive - slow
• leads to operator fatigue
• measures a small number of cells
• qualitative or semi-quantitative
• subjective

Question 10

Describe biochemical techniques

Answer 10

• measure populations rather than single cells
• differences and distribution information lost

Question 11

What are the advantages of flow cytometry?

Answer 11

• heterogeneity
• speed
• quantitative
• multiparametric
• fluorescence activated cell sorting

Question 12

What are the applications in immunology?

Answer 12

• diagnosis/prognosis of cancer
• diagnosis and monitoring of immune diseases
• cell therapy
• transplantation

Question 13

Describe gating

Answer 13

• optimize the analysis of multiparameter experiments, gating is performed to isolate subpopulations of interest
• eliminates need to physically sort cells for further analysis
• bone marrow cells are evaluated based on SSC and CD45 expression to diagnose acute lymphoblastic leukemia

Question 14

Describe flow cytometry and HIV

Answer 14

• cluster of differentiation (CD) identification of cell surface molecules on white blood cells
• CD4+ marker for Th cells
• HIV binds CD4 on surface of a Th cell to gain entry
• no. CD3+/4+ cells in blood used to monitor progression of HIV infection