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Flow Cytometry Flashcards

1
Q

What is flow cytometry?

A

A laser based technique used to detect and analyse 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

Flow cytometry fluidics focus on transporting cells in a fluid stream through a flow cell in single file.

This is achieved through hydrodynamic focusing, where the sample stream is injected into a surrounding sheath fluid, narrowing and aligning the cells so they pass one-by-one through the laser for precise analysis.

This ensures accurate detection of each cell’s properties.

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3
Q

What are the laser principles that underpin flow cytometry function?y

A

lasers produce a focused beam of light at a specific wavelength to excite fluorescent markers on cells.

The laser light is generated by stimulating atoms in a plasma tube (often argon gas) with an electric current.

Mirrors and prisms filter this light into a single wavelength, allowing precise and consistent excitation of markers, which emit fluorescence for cell analysis.

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4
Q

What is the significance of fluorochromes within flow cytometry?

A

Fluorochromes are fluorescent dyes that attach to specific molecules, such as antibodies, which bind to target markers on cells.

When exposed to a laser, fluorochromes emit light at distinct wavelengths, allowing detectors to identify and quantify these markers.

This enables precise analysis of different cell types and characteristics, making fluorochromes essential for distinguishing specific cell populations.

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

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

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6
Q

What is a flow cell/flow chamber?

A

A flow cell is a small chamber where cells pass in single file to be analysed by a laser.

It allows precise alignment for detecting scattered light and fluorescence, providing data on cell characteristics like size and marker expression.

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7
Q

Name the two most common fluorochromes and why you would use them.

A

FITC - 488:
BRIGHT - only used if what you’re looking for is not seen often as it is v bright + will block other signals

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

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

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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

laser hits cells + forward scatter indicates cell size

side scatter indicates internal complexities and granularity of the cell.

measured through a photodiode.

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9
Q

What would a Dichroic mirrors do to your light emission?

A

Dichroic mirrors selectively reflect and transmit light based on wavelength.

They separate emitted fluorescence from different markers by reflecting certain wavelengths toward detectors and allowing others to pass through.

This enables the detection of multiple fluorescent signals simultaneously, improving analysis of various markers on cells.

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10
Q

What would a shortpass filter do to your light emission?

A

allow light below a specific wavelength through

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11
Q

What would a bandpass filter do to your light emission?

A

allows only a specific range of wavelengths to pass through, isolating the fluorescence signal of interest and blocking out other emissions.

This improves detection accuracy by reducing background noise.

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12
Q

What would a longpass filter 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 their advantages?

A

instruments that convert light into an electrical current.

They help detect and measure florescence by converting the light emitted from labelled cells into an electrical signal that can be analysed for different parameters.

Advantages

  • great for FSS
  • high efficiecny for visible spectrum
  • no adjustment gain
    -requires cooling

newer tech

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14
Q

What are photomultiplier tubes (PMT) and what are their advantages?

A

v sensitive detector used to measure low levels of light.

It works by amplifying photons into a strong electrical signal.

used to detect light + amplify weak signals. used to detect fluorescence.

  • high sensitivity but poor efficiency in red (>650 nm)
    -adjustable gain (sensitivity)
    -inexpensive
    -older tech
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15
Q

What is the function of a pre-amplifier in the electronics portion of flow cytometry? Explain the relevance of compensation.

A

preamplifier function - increases the strength of the weak electrical signals from the detectors so they can be more easily processed by electronic components. They boost and condition signals for accurate processing.

compensation - corrects for fluorescence overlap, ensuring precise measurements of each fluorophores signal.

w/o compensation, overlapping signals can lead to inaccurate data, making it difficult to distinguish between different cell populations or markers

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16
Q

What is gating and why is it used?

A

the ability to focus on specific cell population for analysis. this allows researchers to analyse subsets of cells based on particular characteristics, such as, size, granularity or fluorescence intensity, without interference in from irrelevant cells or debris.

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17
Q

What is a Dako CYAN Multicolour Laser and what are its advantages?

A

High-performance FC that uses multiple lasers instead of 1.

  1. more accurate population identification
  2. use smaller specimens as more parameters are available to test in 1 tube.
  3. save time and reagents as fewer tubes are needed to be tested
  4. capable of collecting large number of events more efficiently
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18
Q

What is the principle of Luminex?

A

it is a bead-based multiplex assay that uses colour-coded microspheres coated with specific Ab to detect multiple analytes simultaneously.

it combines flow cytometry with fluorescence to measure the binding of analytes to the beads - allowing for high-throughput quantification of multiple targets in a single sample.

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19
Q

What are the 2 methods by which cells can be sorted

A

after the population of intrest is detected - it is separted by these 2 methods:

  1. electrostatic deflection of a stream in air
  2. mechanical sorting within the flow cell
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20
Q

How does electrostatic deflection stream in air sort cells? (used in FACS - Fluorescence-activated cell sorting)

A

electrostatic deflection in a stream-in-air system (used in FACS) sorts cells by following these steps:

Droplet formation: The cell-containing fluid stream is broken into tiny droplets, with each droplet ideally containing a single cell.

Charge application: When a droplet containing a cell of interest (identified by its fluorescence) is formed, a specific charge (positive or negative) is applied to that droplet.

Electric field: The charged droplets pass through an electric field created by deflection plates.

Deflection: The electric field deflects the charged droplets into different collection containers based on their charge, while uncharged droplets fall into a waste stream.

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21
Q

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

A

Threshold settings: Increase the detection threshold to exclude noise and debris, ensuring only well-defined events are counted.

Pulse width gating: Use the pulse width (time the cell passes through the laser) to differentiate between single cells and cell clumps. Wider pulses may indicate multiple cells or debris.

Doublet discrimination: Gate on forward scatter area (FSC-A) versus forward scatter height (FSC-H) or side scatter area (SSC-A) versus side scatter height (SSC-H) to separate doublets or clumped cells from singlets.

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22
Q

How is mechanical sorting completed?

A

Mechanical sorting in flow cytometry is completed using physical methods to divert cells based on their characteristics, without applying an electric charge. Here’s how it works:

Fluid stream control: Cells flow in a controlled fluid stream through the system.

Detection: Cells are analyzed for specific properties (e.g., size, fluorescence) as they pass through the laser.

Mechanical diversion: A mechanical component, such as a microscale valve, mechanical gate, or nozzle, physically redirects the fluid stream containing the desired cells into separate collection channels or wells.

23
Q

Outline the full process of flow cytometry. (1-3)

A
  1. Sample Preparation
    Cell Isolation: Cells are extracted from a biological source (e.g., blood, tissue).
    Labeling: Cells are stained with fluorescently labeled antibodies that bind to specific surface markers.
  2. Hydrodynamic Focusing
    Sheath Fluid: The sample is injected into a flow cytometer where it is surrounded by sheath fluid.
    Alignment: This sheath fluid aligns the cells into a single-file stream, ensuring that they pass through the laser one at a time.
  3. Laser Excitation
    Laser Beam: As cells flow through the laser, the fluorescent dyes are excited by the light.
    Emission: Excited dyes emit light at specific wavelengths.
24
Q

How could you use flow cytometry for transplatation?

A

FC could be used to type cells into HLA classes by binding patients’ serum to their Ab to beads then binding an Anti-HLA Ab with a fluorochrome conjugated to that Ab and using it to detect an immune response

25
Q

How could you use flow cytometry for stem cell typing?

A

fluorochrome can be attached to an Ab 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 the haemopoietic stem cell.

26
Q

Explain the principle of flow PRA.

A

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

  1. HLA ab bound to different colour beads with set amounts of PE fluorescence.
  2. Colour is known which means that is can be gated
  3. +ve results are determined by binding of anti-human IgG FITC (FL1) which shows the specfic type of HLA in order to know type

limited to 8 beads

27
Q

Explain luminex HLA typing in detail, step by step.

A
  1. Sample Preparation

Collect Blood Sample: Obtain a blood sample from the patient.
Isolate Plasma or Serum: Centrifuge the sample to separate plasma or serum containing antibodies.
2. Bead Selection

Color-Coded Microspheres: Choose a panel of Luminex microspheres, each coated with specific HLA antigens (e.g., class I and class II).

  1. Antibody Incubation
    Mix Serum with Beads: Incubate the patient serum with the Luminex beads, allowing any anti-HLA antibodies in the serum to bind to their respective antigens on the beads.
  2. Washing
    Remove Unbound Antibodies: Wash the beads to eliminate unbound antibodies, ensuring only specifically bound antibodies remain.
  3. Detection Antibody

Fluorescently Labeled Detection Antibody: Add a secondary antibody that is conjugated with a fluorescent dye. This antibody binds to the patient’s antibodies that are already attached to the beads.

  1. Final Wash
    Wash Again: Wash the beads again to remove any unbound detection antibodies.
  2. Analysis
    Flow Cytometry: Run the beads through a Luminex analyzer, which uses lasers to excite the fluorescent dyes.
    Data Collection: The analyzer measures both the color (indicating the specific HLA antigen) and the intensity of fluorescence (indicating the amount of antibody bound).
  3. Interpretation of Results
    Data Output: Analyze the resulting data to determine which HLA antigens the patient’s serum reacts against, indicating potential sensitization.
    HLA Typing: Generate an HLA typing report detailing the specific HLA alleles present in the patient.
  4. Clinical Application
    Transplant Compatibility: Use the HLA typing results to assess donor-recipient compatibility for transplantation, guiding clinical decisions.
28
Q

How can we use 3 colour lymphocyte immunofluoresence (LIFT) to determine if a cell is B cell or a T cell?

A

B cells express HLA class II - T cells do not.

  1. Bind known type cells to the patient’s serum
  2. Then add anti-CD3 Ab with a PE fluorochrome bound (T cells).

then add anti-PECD19 Ab with a PerCP fluorochrome bound (B cells)

Add anti IgG/IgM Ab with FITC flurochrome to show the presence of the cells.

29
Q

What is the principle of PCR-SSO- HLA typing by luminex?

A
  1. Amplification: PCR amplifies specific HLA gene regions.
  2. Denaturation: Denaturing of amplified product = separate 2 bands
  3. Hybridisation: Amplified DNA fragments hybridise with SSO probes bound to colour-coded microspheres, each representing specific HLA alleles.
  4. Detection: A fluorescent reporter dye binds to matched probes.
  5. Signal Reading: The Luminex reader detects fluorescence, identifying HLA alleles based on the microsphere’s unique fluorescent signature and probe match.
30
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 = create new cekks

31
Q

What is transfusion related acute 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

Neonatal alloimmune thrombocytopenia (NAITP) is a condition where maternal antibodies attack fetal platelets due to an incompatibility in platelet antigens between the mother and fetus.

This immune response lowers the platelet count in the fetus, leading to thrombocytopenia, which increases the risk of bleeding complications, such as intracranial hemorrhage, in the newborn.

33
Q

How does immunophenotyping work?

A

Immunophenotyping works by using labeled antibodies that bind to specific cell surface markers unique to certain cell types. Helps the diagnosis of leukaemias. The process involves:

Sample Staining: Cells are mixed with fluorescently labeled antibodies targeting specific markers.

Flow Cytometry: The stained cells pass through a flow cytometer, where lasers excite the fluorescent labels.

Detection: The cytometer detects fluorescence signals, identifying cell types based on marker expression patterns.

Data Analysis: The pattern and intensity of fluorescence help classify cell populations, determining their type, status, and functionality.

34
Q

How would we use flow cytometry to asses DNA for cancer diagnosis?

A

Cell Permeabilisation with Triton X-100: Triton X-100, a mild detergent, is added to the cell sample to permeabilise cell membranes, allowing PI to enter and bind DNA.

DNA Staining with Propidium Iodide (PI): PI, a fluorescent dye, binds to DNA in the permeabilised cells. PI fluorescence intensity correlates with DNA content, distinguishing cells in different cell cycle phases.

Flow Cytometry Analysis: The cells pass through a flow cytometer, where lasers excite PI, and the fluorescence signal reflects DNA content.

Cancer Assessment: Abnormal DNA content (e.g., aneuploidy or high S-phase fraction) signals potential malignancy, assisting in cancer diagnosis and tumor behavior evaluation.

35
Q

How can we use flow cytometry for cell cycle analysis?

A

Flow cytometry can analyze the cell cycle by measuring DNA content:

Sample Preparation: Cells are fixed and stained with a DNA-binding dye (e.g., propidium iodide).

Flow Cytometry Detection: Cells pass through the flow cytometer, where fluorescence intensity from the dye correlates with DNA amount.

Cell Cycle Phase Identification: DNA content allows classification into G0/G1 (one DNA copy), S-phase (DNA synthesis), and G2/M (two DNA copies). Alternatively, they can sit in G2 as they cant undergo mitosis.

Analysis: Abnormal phase distributions can indicate disrupted cell cycles, common in cancer.

36
Q

Explain chronic granulomatous disease and what is the treatment

A

In chronic granulomatous disease (CGD), the affected white blood cells are primarily phagocytes, including neutrophils and macrophages. These cells normally engulf and destroy pathogens, but in CGD, they lack the ability to produce reactive oxygen species due to NADPH oxidase deficiency, impairing their pathogen-killing function.

Treatment:

  1. Antibiotics and Antifungals: Prophylactic antibiotics and antifungals prevent infections.
  2. Interferon-gamma Therapy: Boosts immune cell function to improve infection control.
  3. Bone Marrow Transplant: The only curative option, replacing defective immune cells with healthy ones.
  4. Gene Therapy (experimental): Corrects the genetic defect in patient-derived cells.
37
Q

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

A

Laminar Flow: Fluid layers flow in parallel without mixing. In flow cytometry, a sheath fluid surrounds the sample fluid, keeping particles in a narrow, controlled stream.

Hydrodynamic Focusing: The sheath fluid applies pressure, narrowing the sample stream to a single particle width. This alignment centres particles in the flow path.

Result: Particles pass individually through the laser for accurate, single-cell or particle analysis without overlap, essential for flow cytometry precision.

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 two electrodes ionises the gas, creating a plasma arc. This plasma arc is then sustained by applying a high current at a low voltage, producing intense, stable light suitable for applications requiring bright, continuous illumination, such as flow cytometry.

39
Q

What type of dector is used to detect fluorescence?

A

photomultiplier tubes (PMTs) are commonly used to detect fluorescence.

PMTs are highly sensitive detectors that convert the light emitted by fluorescently labeled cells into electrical signals.

When fluorescence from the labeled cells hits the PMT, it amplifies the signal, enabling precise measurement of fluorescence intensity, which correlates with the quantity of labeled molecules on or within the cells.

They’re usually placed behind filters which determine the fluotochrome they are detecting.

40
Q

What is gating?

A

gating is the process of selecting specific cell populations within a sample based on their characteristics, such as size, granularity, or fluorescence intensity.

Defining Regions: Gates are drawn on flow cytometry plots to define regions that include cells of interest (e.g., lymphocytes, monocytes).

Filtering Data: Only events within these gated regions are analyzed further, allowing for focused examination of particular cell populations.

Purpose: Gating helps isolate and study specific cell subsets, making it essential for targeted analysis in complex samples.

41
Q

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

A

Cell Size: Determined by forward scatter (FSC), which correlates with cell volume.

Cell Granularity/Complexity: Determined by side scatter (SSC), which reflects internal cell structure and granularity.

Cell Morphology: Based on combined FSC and SSC patterns, giving insights into cell type (e.g., distinguishing lymphocytes from granulocytes).

Pigment conteny e.g. Hb, cholorophyll

42
Q

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

A

Structural:
surface antigens
intracellular proteins
DNA/RNA content

Functional
Apoptosis markers
enzyme activites
gene expression
DNA synthesis

43
Q

Explain electrostatic deflection: steam in air

A

electrostatic deflection is used to sort cells or particles in a stream of air by applying an electric charge to droplets containing individual cells:

Droplet Formation: Cells in a fluid stream are passed through a vibrating nozzle, breaking the stream into droplets. Each droplet can contain a single cell.

Electrostatic Charging: As each droplet forms, it receives an electric charge based on the characteristics of the cell it contains (e.g., fluorescence intensity).

Deflection Plates: Charged droplets pass through an electric field created by deflection plates. The plates exert a force on the charged droplets, causing them to deflect according to their charge polarity and magnitude.

Sorting into Collection Containers: The deflected droplets are sorted into separate containers based on their characteristics, allowing precise collection of specific cell populations for further analysis.

44
Q

What is phase gating?

A

Phase gating is a flow cytometry technique that isolates cells in different cell cycle phases (G0/G1, S, G2/M) by analyzing DNA content using DNA-binding dyes. It’s used to study cell proliferation and detect cycle abnormalities.

45
Q

How would you complete HLA typing using luminex?

A

DNA Extraction: Extract DNA from the sample.

PCR Amplification: Amplify HLA gene regions using PCR to produce target DNA fragments.

Hybridization with SSO Probes: Incubate PCR products with color-coded beads, each coated with sequence-specific oligonucleotide (SSO) probes targeting specific HLA alleles.

Binding and Detection: If the PCR product matches a probe, it hybridizes, and a fluorescent reporter dye binds to this complex.

Luminex Analysis: The Luminex system detects the unique fluorescence of each bead, identifying HLA alleles based on which SSO probes hybridized with the DNA.

PCR-SSO with Luminex enables high-throughput HLA typing for clinical applications like transplantation compatibility and immune-related disease studies.

46
Q

How would you analyse DNA using flow cytometry?

A

Sample Preparation: Collect cells and fix them to preserve structure, then treat with a detergent (e.g., Triton X-100) to permeabilize cell membranes.

DNA Staining: Add a DNA-binding fluorescent dye, such as propidium iodide (PI) or DAPI, which binds proportionally to DNA content in each cell.

Flow Cytometry Analysis: Run the stained cells through the flow cytometer. The instrument’s laser excites the fluorescent dye, and the emitted fluorescence intensity correlates with DNA content.

Data Interpretation: Analyze fluorescence intensity to determine DNA content, allowing cell cycle phase determination (G0/G1, S, G2/M) or identifying DNA content abnormalities (e.g., aneuploidy) associated with malignancies.

47
Q

Laminar coaxial flow

A

cells near the walls moving slower due to drag and cells in the centre moving at higher speeds which allows hydrodynamic focusing.

48
Q

How does luminex work?

A

Microspheres: Color-coded beads (microspheres) are coated with specific capture antibodies.

Sample binding: The sample containing target analytes is added, and the analytes bind to the antibodies on the beads.

Detection antibody: A detection antibody tagged with a fluorescent dye binds to the analyte.

Laser reading: The beads pass through a laser, which identifies bead colour (analyte type) and detects fluorescence (analyte quantity).

Data analysis: Fluorescence intensity correlates with the analyte concentration.

49
Q

What is anti-coincidence gating?

A

Anti-coincidence gating is specifically designed to prevent multiple cells (doublets or clumps) from being detected as a single event during flow cytometry, ensuring accurate sorting of individual cells. It helps minimize the chance of coincidence detection, where multiple cells might be processed as one, improving sort purity and accuracy.

50
Q

Outline the full process of flow cytometry. (4-6)

A
  1. Detection
    Photodetectors: Multiple detectors capture emitted fluorescence and scattered light.
    Types of Detection:
    Forward Scatter (FSC): Measures cell size.
    Side Scatter (SSC): Measures cell granularity and internal complexity.
    Fluorescence Detection: Measures specific fluorescent signals from labeled antibodies.
  2. Signal Processing
    Analog to Digital Conversion: Detected signals are converted to electronic signals for processing.
    Data Interpretation: Data from each cell is processed to extract information about its properties.
  3. Gating
    Data Visualization: Data is displayed using dot plots, histograms, or other graphical formats.
    Gating Strategies: Users apply gates to define populations of interest based on parameters like fluorescence intensity, size, or granularity.
51
Q

Outline the full process of flow cytometry. (7-9)

A
  1. Sorting (if applicable)
    Sorting Mechanism: If sorting is required, cells are classified based on their characteristics.
    Electrostatic Sorting (FACS): Charged droplets containing cells of interest are deflected into collection tubes.

Mechanical Sorting: Cells are physically redirected to different containers based on flow dynamics.
Collection: Sorted cells are collected into separate containers for further analysis.

  1. Data Analysis
    Software Analysis: Collected data is analyzed using specialized software for detailed interpretation.
    Statistical Analysis: Comparisons and statistical evaluations are performed to draw conclusions about the cell populations.
  2. Reporting
    Result Compilation: Findings are summarized in reports, including graphs, charts, and numerical data.
    Interpretation: Conclusions regarding the cell populations, their functions, and relevance to the study are discussed.
52
Q

Why would we use flow PRA?

A

Transplant Compatibility: Assess the risk of organ rejection by determining the level of pre-existing antibodies against donor HLA antigens.

Patient Screening: Identify sensitized patients who may require desensitization before transplantation.

Monitoring: Evaluate changes in antibody levels over time in transplant candidates or recipients.

Improving Outcomes: Inform clinical decision-making to select appropriate donors and enhance transplant success rates.

53
Q

What is the process of immunophenotyping?

A

The process involves:

Sample Staining: Cells are mixed with fluorescently labeled antibodies targeting specific markers.

Flow Cytometry: The stained cells pass through a flow cytometer, where lasers excite the fluorescent labels.

Detection: The cytometer detects fluorescence signals, identifying cell types based on marker expression patterns.

Data Analysis: The pattern and intensity of fluorescence help classify cell populations, determining their type, status, and functionality.

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