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AP0609-Advanced Anylytical Techniques > Flow cytometry > Flashcards

Flow cytometry Flashcards

1
Q

What is Flow cytometry

A

Flow cytometry is a laser-based technique used to detect and analyze the chemical and physical characteristics of cells or particles

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2
Q

What are the fluidics principles that underpin flow cytometry function.

A

The fluidic system in which cells are suspended in sheath fluid is used to deliver single particles to a specific point to enable accurate measurement so they can be intersected by a LASER beam.

Laminar coaxial Flow – Principle of cells near the walls moving slower due to drag and cells in the centre moving at higher speeds which allows hydrodynamic focusing.

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3
Q

What are the laser principles that underpin flow cytometry function.

A
  1. Plasma tube contains gas under pressure which fluoresces under the application of current
  2. The light emitted is reflected along the tube
  3. When these photons strike an atom in an excited state, they release another photon of the same wavelength
  4. Light leaves the chamber through a Brewster window, through a prism, a mirror, prism, chamber then is output as a light of a single wavelength

Argon 488 is mainly used

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4
Q

What is the significance of fluorochromes within flow cytometry?

A

Flow cytometry the cells can be stained using Fluorescent Dye And/or a fluorochrome.

Fluorochrome is conjugated to an antibody of choice in an amount proportional to the quantity of the Binding e.g. DNA base sequence, protein type, surface antigen.

When the laser light hits a Fluorochrome flurecence is emmited due to the atoms being a higher energy state.

Wavelength output from Fluorochrome is always higher than the input wavelength of excitation light

Key principle – Only certain light wavelengths can influence certain fluorochromes

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5
Q

How can we use The Beer-Lambert law to calculate concentration.

A

The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance.

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6
Q

Name the two most Common Fluorochromes and why you would use either?

A

FITC - 488
* Bright - Only used if what your looking for is not seen often as it is very bright and will block other signals.
* Absorption maximum close to emission lines from both the argon laser and a mercury arc lamp

R-phycoerythrin 488
* Can be excited at 488nm so only one laser required no need to use multiple lasers
* Dim- used when cell types are common

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7
Q

What is a flow cell/ flow chamber?

A

Quartz glass cuvette where the laser beam excited the fluorochromes on cells.

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8
Q

What is the initial data you get from the laser hitting your cell of interest and how is this collected?

A

The laser hits the cell and forward scatter indicates cell size
whereas side scatter relates to the complexity or granularity of the cell.
This is measured through a Photodiode.

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9
Q

What would a Dichroic mirrors do to your light emission ?

A

Allow light of a certain wavelength to be reflected while the remaining wavelengths can pass through

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10
Q

What would a Shortpass filter do to your light emission?

A

Allow light below a specified wavelength through

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11
Q

What would a Bandpass filters do to your light emission?

A

Only allows a specified range of light wavelengths through

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12
Q

What would a Longpass filters do to your light emission?

A

Allow light above a specified wavelength through

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13
Q

What are Photo diodes, and what are they’re advantages?

A

Instruments that are used for the exact measurement of the intensity of light.

  • Great for Forward scatter
  • High efficiency for visible spectrum
  • No adjustable gain
  • Requires cooling
    Newer Technology.
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14
Q

What are Photomultiplier Tubes (PMT) and what are they’re advantages?

A

Most common detector in flow cytometry which is used to detect Light & Amplify weak signals. These are usually used to detect fluorescence.

  • High sensitivity but poor efficiency in red (>650nm)
  • Adjustable gain (sensitivity)
  • Inexpensive
  • OLDER TECHNOLOGY,
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15
Q

What is the function of a pre-amplifier in the electronics portion of flow cytometer? Explain the relevance of Compensation?

A

Smooths clean signal through Compensation.

“Compensation,” as it applies to flow cytometric analysis, refers to the process of correcting for fluorescence spill over.

This is done by running coloured beads through Cytometry with different fluorochromes, to get a true signal of these signals and subtract the associated voltages

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16
Q

What is Gating and why is it useful?

A

The ability to select a population for analysis from parameters you set for example cells with low side and forward scatter when looking for Platlets.

Cells within the gate can be analysed for other parameters

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17
Q

What is a Dako CYAN Multicolor laser and what are its advantages?

A

The CyAn ADP High-Performance Flow Cytometer that uses multiple lasers instead of 1.

 More accurate population identification
 Use smaller specimens as more parameters are available to test in one tube
 Save time and reagents as fewer tubes are required to be tested
 Capable of collecting large number of events more efficiently

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18
Q

What is the principle of – Luminex

A

Polystyrene microspheres with a range of dyes on these beads as well as ratios of dyes to test multiple conditions at once.
Each microsphere is dyed with a combination of red and infra-red fluorochromes
2 lasers are used to bisect
1 is for detecting bead and 1 for detecting reagents.
Reporting laser – bead identification – what type of bead
Microsphere ID – fluorescence from PE

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19
Q

What are the 2 methods cells can be sorted?

A

Population of interest once detected is separated by one of 2 different methods
1. Electrostatic deflection of a stream in air
2. Mechanical sorting within a flow cell

20
Q

How does Electrostatic Deflection Stream-in-air sort cells?

A
  • cells form a droplet and a negative or positive charge placed on the droplet
  • Charge is created as there is 2 plates alongside and these plates place a current creating a negative or positive charge.
  • Charged droplets deflect by electrostatic field from plates held at high voltage (+/- 3000 volts).

Cells may be close together which would mean that there is a chance another cell may tailgate ( coincidence ) - This occurs if two or more cells are detected in the time frame of droplet formation

21
Q

How can you gate to avoid tail gating (Coincidence) when cell sorting?

A

Anti-Coincidence gating can be used to prevent this:

It works by creating a time window around the particle of interest relating to droplet formation If any other partial is detected in this window then the stream is directed to waste

22
Q

How is mechanical sorting completed?

A

Grabs the cell with a mechanical arm, much slower process however better for infective processes as no droplets are created

23
Q

Outline the full process of flow cytometry.

A
  1. Sheath fluid continually circulating within the system
  2. Sample is injected and cells line up individually due to hydrodynamic focusing
  3. Argon 488 is laser intercepts cell; a photodiode records the light that can pass around the cell which gives information on the side of the cell. FORWARD SCATTER
  4. At a right angle to the collection lens, side scatter is recorded due to the laser refracting from the cell’s organelles and granules.
  5. Fluorescence detectors at set up within the machine which are capable of amplifying signals using High voltage (photomultiplier tubes)
  6. Voltage leaves optical detectors in (pulse) into electronics section which will smooth and amplify signal, then compensation, and finally analogy to digital converter.
24
Q

How could you use Flow cytometry for transplatation?

A

Flow cytometry could be used to type cells into HLA classes by binding patients serum to thus they’re antibodies to Beads then binding an Anti-Hla antibody with a flurochrome conjugated to it to that antibody and using it to detect an immune response.

25
Q

How could you use flow cytometry for Stem cell typing?

A

A flurochrome can be attached to an antibody which will bind to CD34 presenting cells or beads coated in HLA typed cells, going further we can use electrostatic separation with cell sorting to separate these Haemopoietic Stem Cells.

26
Q

Explain the principle of Flow PRA and why we would use it?

A

Flow-PRA is a flow cytometric method for both anti-HLA class I and class II antibody (Ab) detection. This utilises Luminex Ab detection.

  1. HLA antibodies bound to diffrent colour beads with set amounts of PE fluorescence.
  2. Colour is known which means that this can be gated
  3. Positive results are determined by binding of anti human IgG FITC (FL1) which shows the specific type of HLA in order to know type.
  4. LIMITED TO 8 BEADS
27
Q

Explain luminex HLA typing in detail, step by step.

A
  1. 5ul Microspheres with HLA bound to surface expressing different types of light are chosen.
  2. Add 20ul of patient serum ( contains all HLA antibodies)
  3. Agitate the beads so they do not stick
  4. Incubate at 22-25oC which is key for opsonisation
  5. In the dark so light does not bleach the spheres
  6. Wash unbound Antibodies

Beads with HLA antibodies attached
from patients serum

  1. Add 100ul of anti-human IgG labelled with PE fluorochrome
  2. Repeat steps 3-6
    Beads with HLA type from patient serum as well as a antihuman IgG-PE antibody attached to that
  3. Reporting laser detects Bead type Originally bound / colour
  4. Microphere ID laser detets – PE floruchrome
27
Q

How can we use Three Colour Lymphocyte immunofluoresence (LIFT) to determine if a cell is B cell or a T cell?

A

Principle behind this being that B cells express HLA class 2 however t cells do not.

  1. Bind Known type cells to patients Serum
  2. Then add
    Anti CD3 antibodies with a PE flurochrome bound ( t Cells)

Then add
Anti PECD19 antibodies with a PerCP flurochrome bound ( B cells)

Add Anti IgG/IgM antibodies with FITC flurochrome to show the presence of the cells.

28
Q

What is the principle of PCR-SSO - HLA Typing by Luminex?

A
  1. Amplification of HLA locus of interest with biotinlyated primers
  2. Denaturation of amplified product – separate 2 bands
  3. Hybridisation of amplified product with Oligonucleotide probes with specific DNA sequence bound to beads – DNA probe on the bead will be complimentary to HLA ( 1a)
  4. Addition of Streptavidin ( binds to biotin) /PE reporter
  5. Computer analysis of positive results leads to assignments
29
Q

Why is CD34 typing important in Cancer treatment?

A

Key for cancer radiation – when radiation kills all stem cells we need to be able to properly type stem cells to give to patients to allow them to create new cells

30
Q

How do we obtain an absolute count of stem cells.

A

Run the Luminex and gate for CD45 then gate for CD35 which would show only the homeopathic stem cells – doing this creates a % of the cells that are CD35 NOT A TRUE COUNT As there is not a constant volume.

Add beads with a known volume and calculate how many beads have gone through the point to create a volume measurement

To obtain the number of cells in a collection the absolute count is multiplied by the dilution factor, the volume (l) of the pack.

Absolute Count (cells/μl) =
Number of CD34 cells (R4) + Fluorospheres Conc
/
Number of Fluorospheres counted (R7)

31
Q

What is Transfusion Related Acute Lung Injury (TRALI) and how can we test for it?

A

Respiratory arrest due to immune response.

This can be tested for using a modification of LIFT to see Lymphocytes and granulocytes HLA 1 & 2 and HNA.

32
Q

What is Neonatal AlloImmune ThrombocytoPeinia (NAITP)

A

Babies are born with low platlets due to immunological attack

85% of cases caused by HPA 1a – Mum HPA 1bb Dad HPA 1a Baby HPA 1ab – mum recognizes 1a which leads to platlets being destroyed.

33
Q

How does Immunophenotyping work?

A

 Help the diagnosis of Leukaemia’s by the presence or absence of cell surface markers
E.g. Acute Lymphocytic Leukaemia:

34
Q

How would we use Flow cytometry to assess DNA for cancer diagnosis.

A

Malignant cells are frequently aneuploid (an abnormal number of chromosomes) and can have prognostic significance.

  1. Cell is exposed to a detergent (Triton-X), which disrupts the Membrane making it purus to dye
  2. This allows Propidium iodide to enter cell and stain DNA
  3. Then measure cell counts against PI fluorescent signal
35
Q

How can we use flow cytometry for cell cycle analysis?

A

Flow cytometry is still the method of choice for fast, accurate determination of cell cycle distributions using the above method and Propidium iodide staining and PI fluorescence.

What are all cells doing as cancer cells tend to sit in G0 Phase – due to synthesis being blocked

Alternatively they can sit in G2 as they cant undergo mitosis

36
Q

Explain Chronic granulomatous disease and what is the treatment.

A

essentailly continual infection caused by neurtophills not being able to neutrophils undergo phagocytosis fully through as they are uncapable of an oxidative burst which is sending Bleach/ free radicals through to destroy the target cells.

Phagocyte NADPH oxidase is the enzyme that is responsible for the generation of the oxidative burst and is inactivated by genetic mutations

Treat patients with phorbol myristate acetate (PMA) which activates granular sites to activate granular burst – switching on NADPH oxidase.

37
Q

What is the principle of laminar flow and hydrodynamic focusing for particle alignment.

A

laminar flow is characterized by fluid particles following smooth paths in layers, with each layer moving smoothly past the adjacent layers

Hydrodynamic focusing is based on the fluid chamber becoming increasingly more narrow allowing single cells to pass through.

38
Q

How does an Acr lamp work for Flow cytometry

A

An arc lamp is a glass envelope containing a gas or vapour at high pressure
An initial high voltage spark between 2 electrodes creates a plasma arc
Plasma arc is then maintained by the application of high current at a low voltage

39
Q

What type of dector is used to detect fluorescence?

A

PMT
Detect the presence of Fluorochromes
In flow cytometry fluorescence detectors are usually place behind filters which determine the fluorochrome they are detecting

40
Q

What is Gating?

A

The ability to select a population for analysis
Cells within the gate can be analysed for other parameters

41
Q

What are intrinsic factors that can be determined using Flow cytometry? ( no reagents added)

A

Cell size(Forward Light Scatter)
Cytoplasmic granularity(90 degree Light Scatter)
Pigment content e.g. Haemoglobin

42
Q

What are extrinsic factors that can be determined using Flow cytometry? ( reagents added)

A

Structural
DNA content
DNA base ratios
RNA content

Functional
Surface and intracellular receptors.
DNA synthesis
DNA degradation (apoptosis)
Cytoplasmic Ca++
Gene expression

43
Q

Explain Electrostatic Deflection : Stream-in-air

A

Hydrodynamic focusing in a nozzle vibrated by a transducer produces a stream breaking into droplets.

Laser interrogation and signal processing followed by sort decision

Then the stream is charged

Charged droplets deflect by electrostatic field
from plates held at high voltage (+/- 3000 volts)

44
Q

Explain Phase Gating

A

Phases are set and a system determines if a cell lies within the gate

45
Q

How would you complete HLA typing using Luminex

A

1.complete PCR-SSO to amplify HLA locus of interest using biotynilated primers
2.denature amplifed product and hybridise to specific oligonucleotides bound to beads.
3. Addition of a Streptavidin or a PE reporter to the DNA during PCR analyse the PCR signals.

46
Q

How would you analyse DNA using flow cytometry ?

A

Distrupt cell using non ionic detergent trition-X

Propidium iodine binds to stoichometically meaning the number of this compound bound directly relates the the number of DNA molecules

The flow cytometer can be set for Propidium iodine detection

This dye cannot enter alive cells without an interrupted cell membrane so can be used to see apoptosis.

AP0609-Advanced Anylytical Techniques (6 decks)

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