Flow Cytometry

1
Flow Cytometry

• Basic concepts

2
Flow Cytometry

• Immunophenotyping

3
Instrumentation

• Fluidic based sample delivery system
• spectrofluorometer
• light scatter photometer

4
Fluidic based sample delivery system

• single cell suspension
• sheath fluid
• flow cell

5
Flow cell

• sample core stream equals cross sectional
  diameter and should approximate cell size
• sample delivery rate determines core stream
  diameter
• dont adjust to change count rate
• adjust sample conc. to change count rate

6
Flow cell

• sensing portion
• closed flow - cells illuminated in chamber
• uses quartz capillary flow cell
• open flow - cells illuminated just as exit flow
  cell

7
Light sources

• laser
• air cooled gas and solid state most common
• Argon - 488 nm
• mercury arc lamps - occasionally used for UV

8
Flow cytometer
9
Light Scatter

• forward angle light scatter (FS)
• diffraction and refraction
• proportional to cell size (approx.)
• detector located across from laser with structure
  (beam dump) to block laser light
• collects from 0.5-1? to 10-20?

10
Light Scatter

• side scatter (SS)
• describes internal structure of cell
• detected at 90? to laser

11
Fluorescence

• much lower intensity than scatter
• requires photomultiplier tube (PMT)
• detected at 90? to laser
• each PMT detects one range of wavelengths

12
Fluorescence

• range of wavelengths
• green - 500-550 nm
• orange - 550-600 nm
• scarlet - 600-650 nm
• use filters to restrict light from reaching PMT

13
Fluorochromes

• FITC
• PE
• PerCP
• PE-Cy5
• APC

14
Filters

• absorption
• colored glass
• absorbs unwanted wavelengths
• may fluoresce
• interference
• attenuate or reflect unwanted light
• most common

15
Interference filters

• band pass - pass specific range
• notch - block specific range
• long pass - pass light above wavelength
• short pass - pass light below wavelength
• dichroic - reflect specific range pass all others

16
Photomultiplier tubes

• cathode - light sensitive metal releases
  electrons proportional to light input
• electrons go to second stage (dynode) which
  amplifies (repeated many times)
• electrical output is proportional to light input

17
Data

• for each cell FCM records FS, SS and
  fluorescence of 1 to 4 channels (colors)
• listmode data - can electronically rerun specimen
• histogram data - represents data in a graph

18
Gating

• choose parameters for inclusion in analysis
• can gate on scatter, fluorescence or both

19
Sorting

• can use FCM for sorting cells
• as cells leave flow cell the flow stream is
  broken into droplets by a piezo-electric crystal
• each cell in its own droplet
• cells of interest are given a charge
• charged cells are deflected for collection

20
Flow Cytometry

• Quality Control

21
Quality Control

• Instrument QC
• optical systems
• signal processing
• color compensation
• Biologic QC
• DNA QC

22
Optical system QC

• adjusted to give max signal with min variability
• use homogeneous plastic particles (microspheres)
  with fluorescent dyes
• monitor alignment daily
• multiple methods - all good
• use one method consistently with clear acceptable
  ranges

23
Signal processing QC

• linear - DNA - range 4-8 fold
• logarithmic - fluorescence - range 103 to 104
  fold
• check amplifier performance
• at set up
• when amplifier is replaced
• at regular intervals (3-6 months)

24
Color compensation

• fluorochromes emit over a range of wavelengths
• some emissions carry over from one PMT range into
  another
• color compensation is electronic subtraction of
  carry over
• the magnitude of carry over is proportional to
  amount of true signal

25
Color compensation
26
Two Color Compensation
27
Biologic QC

• Problem - dead cells bind antibodies
  non-specifically leading to false positives
• how many dead cells are OK?
• a level that does not affect the reliability or
  accuracy of the reported results
• generally lt 15

28
DNA QC

• in paraffin embedded tissues the location of the
  diploid peak varies by fixation, age of block and
  other features
• therefore assume the lowest peak is the diploid
  peak

29
Flow Cytometry

• Basic concepts- Part 2

30
Flow Cytometer
31
Fluorochromes
PerCP
FITC
PE
32
Gating
33
Gating
34
Color Compensation
35
Color Compensation
36
Color Compensation
37
Color Compensation
38
Under Compensation
39
Good Compensation
40
Over Compensation