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Advanced Analytical Techniques > Flow Cytometry and Cell Sorting > Flashcards

Flow Cytometry and Cell Sorting Flashcards

1
Q

What is flow cytometry used for?

A

To count and analyse the size, shape and properties of individual cells within a a heterogeneous population of cells

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2
Q

What does flow cytometry measure ?

A

The simultaneous measurement of multiple physical characteristics including size/ granularity/fluorescence of a single cell

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3
Q

Measurements made on what basis?

A

Per cell - 500 to 4000 cells per second in a moving fluid stream

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4
Q

Is flow cytometry data quantitative or qualitative?

A

Quantitative

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5
Q

What are the several key components of a flow cytometer?

A

The sample, fluidics, lasers, optics, detectors and a computer system

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6
Q

What do the fluidics do in a flow cytometer ?

A

Move the sample into the flow cytometer

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7
Q

what does the fluidics rely on?

A

light/fluorochromes etc

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8
Q

How is this fluidics achieved?

A

By injecting sample (clean single cell suspension) into the centre opening the close channel through which sheath fluid is flowing

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9
Q

What does the laser do?

A

Produces a coherent, plane-polarised, intense, narrow beam of light which is Monochromatic

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10
Q

Limitations of Laser

A
  • Expensive
  • Difficult to replace
  • Require servicing
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11
Q

What do the optics do?

A

Gather the light, excitation source

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12
Q

Optics consists of what?

A

an excitation source and data collection optics

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13
Q

Optics consists of what?

A

an excitation source and data collection optics

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14
Q

What’s an arc lamp?

A

Glass bulb with 2 electrodes
glass envelope containing a gas or vapour at high pressure
An initial high voltage spark between two electrodes creates a plasma arc
Maintainedby the application of high current at a low voltage

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15
Q

Limitations of arc lamp

A
  • Prone to flicker
  • Average life span of clamps are short
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16
Q

What do the detectors do?

A

Sense the light

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17
Q

What does the computer system do?

A

Outputs the data into a form that can be analysed by the researcher

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18
Q

electronics allow for the conversion of what?

A

optical signals into electronic signals for data analysis

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19
Q

what’s the purpose of electronic data?

A

analyse and converts/records the data as computers can’t read it

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20
Q

Principle of laser

A

Password two contains gas under pressure, fluoresces under application of a current
The light emitted is reflected along the tube

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21
Q

When these protons strike an atom in an excited state, they release what?

A

another proton of the same wavelength

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22
Q

When these protons strike an atom in an excited state, they release what?

A

another proton of the same wavelength

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23
Q

A small percentage of light goes through the system, where does it go from here?

A

through the prism and sends light back in of one photon

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24
Q

Properties of laser light

A

Coherent radiation at discreet wavelengths

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25
Q

Most common laser lights

A

Diode (635nm) and argon

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26
Q

What does the laser interact with?

A

fluorochrome, only specific wavelengths can interact

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27
Q

How does fluorescence occur?

A

occurs when a molecule is excited by light of one wavelength returns to the ground state by emitting light of a longer wavelength
Light that you get out will be a longer wavelength than that that went in
light of fluorochrome higher

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28
Q

What is sheath fluid?

A

filtered isotonic saline

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29
Q

The carrier fluid is?

A

isotonic so won’t burst the cell, the transport medium has no particles

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30
Q

Dynamic of sheath fluid and sample

A

Sheath fluid always flowing when machine is on, however sample only flowing when told to

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31
Q

Interrogation point

A

Laser

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32
Q

What happens when each cell passes through the laser beam?

A

The laser beam will scatter in multiple directions

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33
Q

What’s it called when light scatters in a forward manner?

A

Forward scatter

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34
Q

How is the amount of forward scatter detected?

A

Detected by a detector on the far side of the cell from the laser

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35
Q

How is the amount of forward scatter detected?

A

Detected by a detector on the far side of the cell from the laser

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36
Q

How does the detector work?

A

converts the scattered light into a voltage pulse which is directly proportional to the amount of forward scattered light

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37
Q

Forward scatter is proportional to what?

A

The size of the cell

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38
Q

What does the computer convert the data into?

A

Histogram plot x axis: amount of forward scattered light
y axis : number of cells

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39
Q

What’s it called when light scatters in a sideways manner?

A

Side scatter

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40
Q

How is side scatter detected?

A

by a detector located perpendicular to the path of the laser beam

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41
Q

Side scatter is proportional to what?

A

the shape and internal complexity of a cell

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42
Q

Analysing the forward and side scatter data together

A

Researchers can understand a cells size, shape and complexity
And divide The heterogeneous population of cells into individual populations with varying size, shape and complexity

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43
Q

Bernoulli Effect

A

Particles flow from a High to low pressure area
Viscous drag along walls slow the fluid down
Viscosity gradient created via centre of the fluid flowing faster than that against the walls, differential core in centre due to slowing of the fluid against the wall

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44
Q

Application of fluorochromes

A

In flow cytometry the cell will bind to a fluorescent dye

or

fluorochrome conjugated with an antibody in an amount proportional to the quantity of the binding constituent (e.g. DNA, RNA, surface antigen)

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45
Q

The cells emitting fluorescence intensity is proportional to what?

A

The fluorescing cellular constituent

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46
Q

Common type of Fluorochrome

A

FITC

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47
Q

Why is FITC used if item is rare?

A

it is brighter

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48
Q

What is the absorption maximum of FITC?

A

close to emission lines from both the argon laser and a mercury arc lamp

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49
Q

What is the absorption maximum of FITC?

A

close to emission lines from both the argon laser and a mercury arc lamp

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50
Q

FITC can be excited at what wavelength?

A

488nm, so only one laser required

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51
Q

Flow cell made from what?

A

quartz glass which are transparent to all wavelengths of light
cleanable

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52
Q

Flow cytometers make measurements based on what?

A

light as the excitation source

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53
Q

What does forward/low angle scatter tell us?

A

size of the cell

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54
Q

Forward Scatter Rule

A

when laser light hits a small cell, refract little light around it, more forward scatter for a bigger cell

Forward scatter is proportional to cell size; the bigger the cell, the more light is scattered, the higher the detected signal.

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55
Q

Side scatter rule

A

Side scatter is proportional to cell complexity; the more organelles/bits inside the cytoplasm, the more light scatter, the higher the detected signal.

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56
Q

Dichroic mirrors (beam splitters)

A

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

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57
Q

Short pass filters

A

light equal to or shorter than their indicated wavelength

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58
Q

Long pass filters

A

allow light equal to or longer than specified wavelength through

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59
Q

Band pass filters

A

Only allow a specified range of light wavelengths through

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60
Q

Photodiodes

A
  • Newer techology
  • Solid state
  • Good in visible spectrum
  • Requires cooling
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61
Q

Photo Multiplier Tubes (PMT)

A
  • Detects light
  • Amplify signal so good for the detection of weak fluorescence
  • PMT tubes older and cheaper
  • Can amplify the signal that goes in
  • One photon of light goes in, 8x increase in light
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62
Q

What is a flow cytometer?

A

an instrument that is capable of simultaneous measurement of multiple physical characteristics of a single cell:
- size
- granularity
- fluorescence

the measurements are made on a per cell basis at rates typically in the order of 500 to 4000 cells per second in a moving fluid stream

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63
Q

History of flow cytometry:

A
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64
Q

what are the requirements for a flow cytometer?

A

Fluidics
Optics
Electronics

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65
Q

What do Fluidics do

A

this delivers the particles individually to a specific point that is intersected by a laser beam

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66
Q

what fluid is used in the machine?

A

sheath fluid - filtered isotonic saline

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67
Q

how is this achieved?

A

the sample is injected into the centre of an enclosed channel through which sheath fluid is flowing

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68
Q

what effects occur as a result of the fluidics?

A

the Bernoulli effect - increased speed of fluid, occurs simultaneously with a decrease in pressure or decrease in the fluids potential energy
hydrodynamic focusing - laminar coaxial flow, sheet fluid is passed around the particle causing them to form into single file

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69
Q

what effects occur as a result of the fluidics?

A

the Bernoulli effect - increased speed of fluid, occurs simultaneously with a decrease in pressure or decrease in the fluids potential energy
hydrodynamic focusing - laminar coaxial flow, sheet fluid is passed around the particle causing them to form into single file

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70
Q

what are the requirements of the fluidics system? why are they required?

A

a stream velocity of 10 m/s - for hydrodynamic focusing
- 10 micron particles will then transverse their own diameter in 1 microsecond - interrogation must be rapid as a result
- made practical with the appearance of lasers and high speed electronics

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71
Q

what does the typical fluidics system look like?

A
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72
Q

What is optics?

A

consists of an excitation source and data collection optics

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73
Q

what are the different parts the optics consists of?

A

excitation source - arc lamps and laser
fluorescence
flow cell
collection optics/detectors

74
Q

arc lamps

A
  • glass envelope containing a gas or vapour at high pressure
  • high voltage spark between 2 electrodes creates a plasma arc
  • plasma arc is then maintained by the application of a high current at a low voltage
    disadvantages:
  • prone to flicker
  • average life is short
75
Q

laser:

A

produces a coherent, plane-polarised, intense, narrow beam of monochromatic light (single wavelength)
disadvantages:
expensive

76
Q

overview of the different types of lasers:

A
76
Q

overview of the different types of lasers:

A
77
Q

what is fluorescence?

A

this occurs when a molecule is excited by light of one wavelength returns to the ground state by emitting light of a longer wavelength - there is the release of a photon; the photon released can be detected

78
Q

application of fluorochromes:

A
  • in flow cytometry the cells can be stained
  • the fluorochrome can also be conjugated with an antibody in an amount proportional to the quantity of the binding constituent
  • the cells emitted fluorescence intensity will then be proportional to the fluorescing cellular constituent
79
Q

application of fluorochromes:

A
  • in flow cytometry the cells can be stained
  • the fluorochrome can also be conjugated with an antibody in an amount proportional to the quantity of the binding constituent
  • the cells emitted fluorescence intensity will then be proportional to the fluorescing cellular constituent
80
Q

what are the different types of fluorochromes used?

A

the two most common are:

  • FITC
    • bright
    • absorption maximum close to emission lines from both the argon laser and a mercury arc lamp
  • phycoerythrin
    can be excited at 488nm so only one laser is required
81
Q

what is the flow cell made from?

A

quartz cuvette

82
Q

what does the flow cell look like?

A
83
Q

what are the main features on the cells that are measured for?

A
  • high angle scatter:
    • reflection and refraction
    • allows the cell structure to be calculated - granularity
    • able to determine what the surface of the cell looks like as well
    • more internal structures the higher the side scatter
      • dead cells have a rougher surface
  • low angle scatter:
    • diffraction
    • allow the cell size to be calculated - the bigger the cell the larger the forward scatter (FS)
  • fluorescence at longer wavelengths:
    • allows for the identification of fluorochromes
    • usually PMT
  • intrinsic and extrinsic auto fluorescence:
    this is the natural emission of light from some biological structures
84
Q

Filters and Mirrors

A

filters and mirrors - diverts the light to the detectors

85
Q

what are the different types of filters and mirrors?

A
  • dichroic mirrors:
    • allows light of certain wavelengths to be reflected while the remaining wavelengths can pass through
  • bandpass filters:
    • only allows a specified range of light wavelengths through
  • shortpass filters:
    • allow light below a specified wavelength through
  • longpass filters:
    allow light above a specified wavelength through
86
Q

Name the 2 detectors

A

Photodiodes
Photo multiplier tubes (PMT)

87
Q

What is photodiodes and what are their disadvantages

A
  • newer technology
  • high efficiency for visible spectrum
  • disadvantages:
    • no adjustable gain
    • requires cooling - fans are used
88
Q

What is a photo multiplier tubes (pmt) what are the advantages and disadvantages?

A
  • it is the most common detector used in cytometry
  • old well characterised technology
  • Advantages:
    • detect light and amplify the signal - allows for the detection of weak fluorescence
    • high sensitivity adjustable gain/sensitivity
    • inexpensive
  • disadvantage:
    poor efficiency in red (>650nm)
89
Q

what is the configuration of the cytometer?

A
90
Q

What do electronics do?

A

allow the conversion of optical signals into electronic signals for data analysis

90
Q

What is the spectral overlap?

A

the pulse processing has to take in to consideration the spectral overlap

91
Q

analysis and display:

A

most common form of display is the frequency histogram - is a direct graphical representation of the number of events for each parameter analysed

92
Q

frequency histogram:

A
93
Q

Isometric Display

A
  • this has an additional Z axis to produce a 3D graph
    • the Z axis is used to plot the frequency of the events
    • can be tilted or rotated to provide better viewing angles
94
Q

1 parameter fluorescence histogram:

A
95
Q

2 parameter histogram:

A
96
Q

2 parameter histogram:

A
97
Q

what is gating?

A
  • this is the ability to select a population for analysis
  • cells within the gate can be analysed for other parameters
98
Q

Example of gate

A
99
Q

What are the cellular parameters measured by flow cytometry?

A
  • intrinsic:
    • no reagents or probes are required
    • cell size - FS
    • cytoplasmic granularity - SS
    • pigment content - haemoglobin (can test for intracellular fluorescence)
  • extrinsic:
    • reagents are required
    • DNA content
    • DNA base ratios
    • RNA content
  • functional:
    • surface + intracellular receptors
    • DNA synthesis
    • DNA degradation - apoptosis
    • cytoplasmic Ca++
    • gene expression
100
Q

What is a cell sorter?

A
  • a cell sorter is a flow cytometer with the added ability to physically separate out a population described by a gate
  • the purified fractions can be of high purity >95%
  • the original application of sorting was the purification of morphologically similar but functionally distinct lymphocyte sub-populations
101
Q

how does cell sorting work?

A
  • electrostatic deflection of a stream in air:
    Electronic delay until cell reaches break off point. Then the stream is charges.
    Charged droplets deflect by electrostatic field from plates held at high voltage.
    Various collection devices can be attached: tubes, slides, multi-well plates.
    • coincidence:
      High sample event rates cell not fulfilling the criteria may be sorted
      Occurs if two or more cells are detected in the time frame of droplet formation
      Anti-coincidence gating works by creating a time window around the principle of interest to droplet formation. Other partial is detected in this window then the stream is directed to waste.
    • accuracy of droplet charging:
      Droplet formation is a stable process but it can be affected by sheath temperature or sheath pressure. This will mean charging pulse not being delivered to the correct droplet
      Overcome –> common to charge more than one droplet
      Decrease purity without anti-coincidence gating or decrease yield with anti-coincidence gating on
    • phase gating:
      Determines if cell is in the centre or outside quarters of the droplet window.
      If the cell is. not in the centre the system can sort 2 drops rather than 1 to ensure recovery.
      The centre only collection can be applied when maximum purity is required.
  • mechanical sorting within a flow cell:
    Hydrodynamic focusing and interrogation takes place.
102
Q

What is Dako Mo Flo ?

A

Machine that analyses and sorts cells at 70,000 cells per second.
Costs £250,000

103
Q

What is needed for multi-colour?

A
  • more lasers - increased number of fluorochromes that can be used
  • more detectors - more fluorochromes means more detectors + colours needed as a result
104
Q

What is the drawback for multicolour

A

More expensive

105
Q

Dako Cyan

A
106
Q

Why is a multi-colour flow cytometer used?

A
  • 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
107
Q

What technology is multiplex flow cytometry?

A

Luminex technology

108
Q

What does Luminex do?

A
  • allows for multiple analyses in one tube
  • utilises microspheres to which reagents can be bound
109
Q

Microspheres

A

each microsphere is dyed with a combination of red and infra-red fluorochromes

110
Q

What do microspheres allow for?

A

the definition of 100 different beads

111
Q

simple surface chemistry allows for what?

A

the coupling of antibodies, antigens, peptides, oligonucleotides or receptors

112
Q

Luminex Technology

A
  • beads are incubated with sample
  • beads are washed before addition of PE reporter
  • samples are analysed on luminex
  • luminex has 96 well plate capability so high throughout is possible
113
Q

what are the different applications of flow cytometry?

A

Transplantation
Transfusion
Immunophenotyping
DNA Analysis
Immunology

114
Q

How are HLA antibodies detected

A
  • flow methods utilise beads coated in HLA or HLA typed cells
  • can be done on flow cytometer or luminex platforms
115
Q

IgG Class Antibodies what are they

A

complement fixing antibodies can cause hyper acute rejection - clinically significant
- non complement fixing
- early rejection, long term survival may be achieved
- if there are antibodies present in historical sera but currently negative there is a 25% graft survival rate/lower chance of graft survival
- IgG against HLA class I + II have poor prognosis
- non HLA T cell antibodies may be disregarded if not HLA specific

116
Q

IgM Class Antibodies

A
  • IgM presence is associated with reasonable prognosis - if present after transplantation
  • pre-existing allo-IgM antibodies against mismatched antigens of the donor are detrimental
  • IgM antibody is associated with naive Cytotoxic T-cell (CyA sensitive)
  • patients with IgM HLA antibodies may also have IgG antibodies with the same specificity
117
Q

Flow Cross Matching of Organs

A

Known as Flow PRA

118
Q

HLA antigens are bound to beads - how many beads can be tested at one time?

A

only 8 beads can be tested at one time

119
Q

positive results are determined by what?

A

the binding of anti-human IgG FITC to the beads - this can then be analysed by fluorescence in the flow cytometer

120
Q

Luminex HLA Ab detection

A

100 beads are tested at one time - there are thousands of beads per well
- there are commercial kits available - tepnel (lifecodes) or one lambda (LABScreen)
- HLA antigens are bound to multiple microspheres
- screening tests and ID test are available

121
Q

The Principle of Luminex HLA Ab detection

A
  1. HLA antigens A1,A2,A3 (in this scenario) and patient serum containing Ab HLA A3 are incubated at 22-25 degrees C in the dark with agitation
  2. They are then washed to remove unbound antibodies
  3. Ab HLA A3 is incubated with Anti human IgG-PE
  4. Washed to remove unbound Ab
  5. The reporter laser/microsphere ID laser then interacts with the PE and signals are detected
122
Q

Explain PCR-SSO (sequence specific oligonucleotide)

A
  • amplification of HLA locus of interest with biotinlyated primers
  • denaturation of amplified product
  • hybridisation of amplified product with oligonucleotide probes bound to beads
  • addition of strepavidin/PE reporters to see if there is a positive result
123
Q

complement dependent cytotoxic test

A
124
Q

Three colour lymphocyte immunofluorescence (LIFT) uses what 3 different types of specific antibody?

A
  • Anti-CD3
  • anti-CD19
  • anti IgG - this binds to the patients antibody to see if it is present
125
Q

What is the principle of LIFT?

A
126
Q

CD34 only present on which cells?

A

on 2-4% of all normal marrow mononuclear cells

127
Q

CD34 only present on which cells?

A

on 2-4% of all normal marrow mononuclear cells

128
Q

CD34 analysis of hemopoietic stem cell transplantation (HSCT):

A

CD34 positive marrow cells rescue lethally irradiated baboon - this could be applied to humans
Flow methods utilise a mix of anti-CD34, anti CD45 and fluorospheres to generate an absolute count

129
Q

what is the principle of CD34 enumeration?

A
130
Q

what disease is associated with transfusion?

A

transfusion related acute lung injury (TRALI) - due to immune response within the lungs

131
Q

Explain TRALI

A
  • severe type of non-haemolytic transfusion reaction
  • acute respiratory distress
  • etiology is unclear but associated with antibodies to white cells
    • HLA class I + II antibodies
    • anti-granulocyte antibodies (HNA) in the donors blood
132
Q

How can TRALI be analysed?

A
  • modification of LIFT
  • there is a gate on lymphocytes and granulocytes
  • incubate with anti human IgG that has FITC bound
    analyse lymphocyte and granulocyte populations for FITC fluorescence
133
Q

what disease is seen that has neonatals having low platelet counts?

A

neonatal alloimmune thrombocytopenia (NAITP)

134
Q

why are there low platelet counts in the neonatal?

A

due to immunoglobulin attacking their platelets

135
Q

what are the different antigens present on plateletes?

A
  • ABO
  • HLA class I
  • Platelet specific (Human platelet antigen (HPA)
    • 16 documented antigen systems
    • Bi-allelic co-dominant
    • numbered 1-16 with an a form and b form (e.g. HPA-1a, HPA-1b)
    • single point mutations can produce different antigens
136
Q

NAITP overview:

A
  • similar to HDFN
  • reduced platelet count at birth
  • physical characteristics include: petichae/bruising/intracranial haemorrhage
    affects 1 in 1000 pregnancies
    can happen in first pregnancy
    anti HPA-1a causes 85% of cases
  • HLA DRB3*0101 association - 1:3 chance of forming ab
  • usually unexpected - no ante natal screening programs
  • not associated with anti-HLA
137
Q

How can NAITP be detected?

A

platelet immunofluorescence test (PIFT)

138
Q

How does PIFT work in NAITP?

A

use fathers platelets and mothers serum

139
Q

why can’t you take blood from the baby?

A

hasn’t got a lot and more risk

140
Q

Gate on platelets based on what?

A

Low forward scatter and low side scatter
analyse gated region for FITC fluorescence

141
Q

Overview of PIFT principle in NAITP

A
142
Q

Immunophenotyping what does it do?

A

helps the diagnosis of leukaemia by the presence or absence of cell surface markers

143
Q

DNA analysis

A
  • this was one of the first applications for flow cytometry
  • malignant cells are often aneuploid - they have more than one set of chromosomess
    • they can have prognostic significance for particular cancers
  • the DNA content of a tumour may be expressed as the DNA index - this is the ratio between the DNA content of a tumour cell and that of a normal diploid cell
144
Q

the method for DNA analysis

A
  • propidium iodide(PI) binds stoichiometrically (the amount seen is proportional to amount that is bound)
    • the number of molecules of probe bound is equivalent to number of molecules of DNA
    • they cant enter the cell through intact membranes - the membrane must first be disrupted by a detergent (Triton-X)
    • the cells can then be measured for amount of DNA within them from the PI fluorescent signal
145
Q

cell cycle analysis

A
  • the position the cell is within the cell cycle can be determined from the amount of DNA present in the cell
  • it is still the method for fast. accurate determination of cell cycle distributions
146
Q

Chronic granulomatous disease:

A
  • neutrophils aren’t as effective
  • the phagocytes cannot form the oxidative burst - this is required to produce free radicals needed for the immune cells to kill pathogens
    • NADPH oxidase is inactivated by genetic mutation
147
Q

Symptoms for Chronic granulomatous disease:

A
  • abscesses of the skin, tissues, and organs
  • suppurative arthritis
  • suppurative arthritis
  • osteomyelitis
  • bacteremia/fungemia
  • skin infections - cellulitis/impetigo
148
Q

How is Chronic granulomatous disease diagnosed?

A
  • incubate whole blood with phorbol myristate acetate (PMA) and DHR-123
    • PMA stimulates neutrophils to undergo the oxidative burst
    • DHR-123 is an oxygen sensitive dye that fluoresces at 535nm
  • gate on FS/SS for neutrophils
  • analyse neutrophils for FL1 fluorescence
149
Q

What sort of measurements can be taken with flow cytometry? Via what method?

A

Cell size and granularity via their laser light scattering properties as well as a wide range of different cellular parameters.

150
Q

How are these measurements achieved?

A

With the use of fluorescent probes e.g. antibodies conjugated to fluorescent dyes such as fluorescein.

151
Q

What are the major drawbacks of flow cytometry?

A

Cost and availability of an instrument.You need highly skills operators, daily maintenance programmes and comprehensive service contracts. This is not feasible for smaller laboratories or laboratories in areas of the world where the instrument cost (£60k) would be prohibited

152
Q

What is the role of Human Platelet Antigen (HPA) system in NAITP?

A

This disease affects babies with a frequency between 1/600 to 1/800.It is caused by antibodies specific for platelet antigens inherited from the father which are absent in the mother, The foetal platelets are able to cross the placenta and this results in the recognition of these paternal antigens by the mothers immune system as non-self subsequently also-reactive antibodies of IgG class are generated which cross the placenta. About 80% of NAITP cases implicate antibodies against platelet antigen HPA-1a. Commonly the father is HPA-1a/1a or 1a/1b and the mother is HPA-1b/1b and developing anti-HPA-1a antibodies. They are IgG so they can cross the placenta and enter the foetus.

153
Q

What is the principle of the screening assay for NAITP?

A

To look for an immunological reaction between anti-platelet antibodies in the maternal serum against paternal platelets. The serum from the mother has all of the antibodies. The father has the platelets that possess the antigens not present in the mother. We will also use Anti Human IgG FITC because it is the brightest flurochorme which gives us the highest sensitivity and we pick IgG because it is the only one that can cross the placenta and enter the foetus’ bloodstream.

154
Q

Why is AB serum used and a negative control?

A

Because it will not possess ABO/HLA/HPA antigens which are present in the platelets as AB donors don’t have these naturally present.

155
Q

Why do we use Anti HLA as a positive control?

A

It is easy to find and it is present in all cells (HLA-1) which will subsequently be present in platelets at high volumes.

156
Q

Why do we identify platelets by flow cytometry and how do we do it?

A

Platelets have low intercellular contents as they are very small, do not have a nuclei and have few granules. Flow cytometry can use forward scatter (to identify size) and side scatter ( to identify components and fragments) to determine the platelet and the volume. Subsequently, a count can be taken from the volume found. It is a much quicker procedure.

157
Q

Why use the median fluorescence?

A

It will not be affected by outliers or skewness as this gets removed.

158
Q

Why do we go to 3DP when calculating the Negative Cut Off?

A

Because flow cytometry can only detect fluorescent to 3DP so a better accuracy cannot be achieved as you can’t claim an accuracy greater than you cannot measure.

159
Q

Why use +/- 2SD as a Negative Cut Off?

A

This will enable us to cover 97.5% of the entire population.It ensures we can detect both weak positives and false positives and are able to rule them out through confirmatory testing.

160
Q

How can you interpret the results you get?

A

Any value that is equal or greater than your negative cut off value is a positive result. You need to look at your Md X value for positive control and make sure you get a positive result and then you need to look at your test sample Md X if it’s below the negative cut off its negative if it’s equal or above it’s positive.

161
Q

What should be done next and why?

A

Since this is a screening assay one needs to confirm results as platelets have ABO/HLA/HPA antigens on the surface. This test only detects a maternal Ab that can bind to the platelets and maternal Ab could bind to any of these Ag. However, only Anti-HPA causes NAITP. One needs to confirm with MAIPA which is the golden standard procedure. If both PIFT and MAIPA come out as positive the foetus will have NAITP. The next steps would be to council mum to have a C-section at future birth, no forceps or ventures delivery and monitor Anti-HPA titre through pregnancy. If only PIFT is positive it could be a non-immune cause due to trauma, drugs like aspiration or a bacterial infection.

162
Q

How does MAIPA work?

A

MAIPA ( Monoclonal Immunobilization of Platelet Antigen) is the confirmatory test for NAITP. It takes platelets (HPA-1a1a) and add antibody that’s complementary which keeps its original shape and then an ELISA test can take place which is when you use the patient serum. It has a long self life and accuracy as it covalently binds the bead with antigen via a dehydration reaction and keeps the shape of the antigen since sometimes covalent binding can change the shape and some ELISA won’t bind to to this change.

163
Q

What causes NAITP?

A
  • antibodies specific for platelet antigens inherited from the father of which are absent on the mother
    • the foetal platelets are able to cross the placenta, this is how the mothers immune system is able to react to the antigen
    • maternal allo-reactive IgG class antibodies will then be able to pass into the placenta and bind to the neonates platelets
164
Q

what are the symptoms in the neonate?

A
  • in most cases:
    • mild thrombocytopenia
    • relatively symptom free
    • no treatment
  • severe thrombocytopenia:
    • haemorrhagic complications
    • can be as severe as intracranial haemorrhage
      • can lead to the death in ¬10% of cases
      • neurological complications in ¬20% cases
165
Q

what antigen is the cause for 80% of cases?

A
  • HPA-1a
    • common antigen
    • only a small percentage of women HPA-1a negative
  • father is commonly HPA-1a/1a or 1a/1b and the mother is HPA-1b/1b - **producing HPA-1a Ab
166
Q

what are the typical platelet counts for NAITP?

A

2 x 105 uL-1

if higher counts may suggest a different diagnosis

167
Q

how can it be tested for?

A
  • maternal serum can be tested against the paternal platelets
    • drawing blood from the neonate can potentially exacerbate its condition
168
Q

what is the principle behind this test?

A
  • the fathers platelets are used - they posses the Ag that arent present on the mother
  • mothers serum - contains the Ab
  • anti-human IgG FITC - this detects maternal Ab if bound to the platelets
169
Q

why is AB serum used as a negative control?

A
  • AB serum should posses no naturally occurring anti-A/B ABO antibodies
  • the serum can then be screened for anti-HLA and HPA Ab’s
170
Q

why is anti HLA used as a positive control?

A
  • all HLA types are easy to source
  • there are HLA present on all platelets at high density
171
Q

what is the rational for identifying platelets by flow cytometry and how is it done?

A
  • it is able to detect the platelets off their physical properties - small, no nucleus and few granules
    • low FS and low SS
172
Q

why is median fluorescence used?

A
  • this is less affected by outliers
  • it is also better for skewed normal distribution data sets
173
Q

what limits the accuracy of the test?

A

the degree of accuracy that the flow cytometer is able to measure to

174
Q

why is mean + 2SD used as a negative cutoff for data?

A
  • his covers 95% of true negative populations
  • this ensures the detection of weak positives
175
Q

how are false positives ruled out?

A

through confirmatory testing

176
Q

how are results interpreted?

A

any result that is over the mean + 2SD threshold is a positive result

177
Q

what should be done after the flow cytometry test?

A
  • confirmatory tests need to be done
    • platelets also have ABO + HLA antigen present on them - need to ensure that there isnt an Ab for one of these antigen causing the result
      MAIPA (Monoclonal Antibody-specificImmobilization of Platelet Antigen) for confirmation
  • if there is a PIFT (positive platelet immunofluorescence assay) and positive MAIPA - then the patient has NAITP
    • PIFT and - MAIPA = non-immune cause
      • trauma?
      • drugs? aspirin - can affect thrombocyte production
      • bacterial infection - infection could be the cause of the damage
178
Q

NAITP is only from which antibody?

A

anti HPA Ab

179
Q

if NAITP what needs to be done for future births?

A
  • C-section advised - less damage to baby
  • no forceps or ventouse delivery - reduce injury to babay
  • monitor the anti-HPA titre during pregnancy
180
Q

How you calculate results?

A
  • X (mean Md) = 0.187 → 0.19 (2DP)
  • SD= 0.006
  • 2SD= 0.012 → 0.1 (2DP)
  • 2SD + X = 0.20 (2DP) (this is the upper limit of 2SD)
  • this was calculated from the negative controls so anything above the upper limit of 2SD is a positive result
    • the test sample had a fluorescence >0.2 → positive result
    • the positive control also had a fluorescence >0.2, which is expected

Advanced Analytical Techniques (8 decks)

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