Flow Cytometry Cytometry refers to the measurement and analysis of cells and cell components, often using flow cytometry or image-based cytometry techniques to study characteristics such as size, shape, and protein expression in individual cells.

1. Flow Cytometry
Abdul KỜNTEH

2. Flow Cytometry
• The measurement (-metry) of cells (cyto-) as
they flow past a detection system.
• The particles can be almost anything-cells,
beads, bacteria, microvessicles, etc.
• The characteristics being measured are
referred to as parameters.

3. The History of Flow Cytometry
• Mid-1950’s
Wallace Coulter patents and publishes his “Coulter
Principle”
• Mid-1960’s to early 1970’s
Kamentsky at IBM’s Watson Labs
Fulwyler at Los Alamos National Laboratory
• Late 1970’s
Becton-Dickinson, Coulter, and Ortho producing 4
parameter flow cytometers

4. Principle of Flow Cytometry
• The basic principle of flow cytometry is the
passage of cells in single file in front of a laser so
they can be detected, counted and sorted.
• Cell components are fluorescently labelled and
then excited by the laser to emit light at varying
wavelengths.
• The fluorescence can then be measured to
determine the amount and type of cells present
in a sample.
• Up to thousands of particles per second can be
analysed as they pass through the liquid stream.

5. • A beam of laser light is directed at a
hydrodynamically-focused stream of fluid that
carries the cells.
• Several detectors are carefully placed around the
stream, at the point where the fluid passes
through the light beam.
• One of these detectors is in line with the light
beam and is used to measure Forward Scatter or
FSC.
• Another detector is placed perpendicular to the
stream and is used to measure Side Scatter (SSC).
• Since fluorescent labels are used to detect the
different cells or components, fluorescent
detectors are also in place.

6. Forward Angle Light Scatter
FALS Sensor
Laser

7. Laser
Fluorescence Detectors
Freq
Fluorescence
FALS Sensor
Fluorescence detector
(PMT3, PMT4 etc.)

8. PMT
PMT
PMT
PMT
Dichroic
Filters
Bandpass
Filters
Flow Cytometry Optics
Laser
1
2
3
4
Flow cell

9. Clinical Uses of Flow Cytometry
• Current
Hematology
Oncology
Immunology
Blood Banking
• Future
Clinical research translates quickly into clinical
diagnostics!

10. Use of Flow Cytometry in Hematology
• Flow cytometry is a technique used in Hematology (the
study of blood) to sort cells in accordance with their
size and complexity.
• Since its conception in 1976, flow cytometry has
acquired new functions.
• It can now sort cells by fluorescence-based approaches
to analyze a cell's viability and more.
• Applications in hematology include DNA content
analysis, leukemia and lymphoma phenotyping,
immunologic monitoring of HIV-infected individuals,
and assessment of structural and functional properties
of erythrocytes, leukocytes, and platelets.

11. Use of Flow Cytometry in Oncology
• Flow cytometry is often used to test the cells from bone
marrow, lymph nodes, and blood samples. It's very
accurate in finding out the exact type of leukemia or
lymphoma a person has.
• It can also help tell lymphomas from non-cancer diseases in
the lymph nodes.
• It uses technology to find antigens in cells that characterize
the condition to determine the type of CLL a person has.
• Medical practitioners use flow cytometry results in
conjunction with other blood tests, an individual's medical
history, and other signs and symptoms to determine
treatment options and prognosis.

12. Use of Flow Cytometry in Immunology
• Immunology covers the examination of cells,
tissues, and organs of the immune system, as well
as the study of failures in immune function.
• Important areas of study include autoimmune
disease, hypersensitivity reactions,
immunodeficiency, immunotherapy, and the role
of the immune system in transplant medicine.
• Immunophenotyping by flow cytometry is a
laboratory method that detects the presence or
absence of white blood cell (WBC) markers called
antigens.

13. • The flow cytometry analysis gives the number
of CD4 cells in a cubic millimeter of blood.
• A normal CD4 count is between 500 and
1500. If your CD4 count is below 500, it means
that your immune system may be suppressed.
• Causes may include HIV or drugs used to
suppress your immune system.

14. Use of Flow Cytometryin Blood
Banking
• Whole blood is collected from a donor into a bag
which contains an anticoagulant that mixes with
the blood as it enters the bag to prevent clotting.
• A predetermined weight of blood is collected.
After sealing, the blood is first separated into red
blood cells and platelet-rich plasma by
centrifugation.
• Flow cytometry is a common technique in blood
banking. It is used, for example, for the
enumeration of residual white blood cells in
plasma and in cellular blood products.

17. Flow Cell
Injector
Tip
Fluorescence
signals
Focused laser
beam
Sheath
fluid

18. Electronics
Discriminator/Threshold
• Analyzed events must meet a certain minimum
criteria
Photodiodes and Photomultiplier Tubes (PMTs)
• Paired with each filter and convert light into an
electrical signal
Analog to Digital Converter
• Digital signals are then saved as a raw data file
Initiation of sorting by charging and deflecting
particles

21. Instrument Standardization
Increasingly important
• Complex assays
• Performed on multiple instruments within or
between laboratories
• Patient results monitored over time
EuroFlow Consortium

22. Study Questions
1. How does a flow cytometer work?
2. What’s the difference between flow cytometry and
conventional microscopy?
3. A 32 year old woman with sickle cell disease is found at
antenatal screening to be Group O R0 r with anti-U present.
What are the possible transfusion-related complications which
might arise during this pregnancy and post delivery, and how
should they be managed?
4. What does the Forward Scatter (FWD/FSC) data represent?
Why?
5. What does the Side Scatter (SSC) data represent? Why?
6. What is an “event” in a flow cytometry data?

23. 4. A 16 year old Sierra Leonean is referred for review in the
haematology clinic with a history of 'thalassaemia' treated abroad
with blood transfusions. Her Hb is 65g/L and her spleen is
measured at 5cm below the costal margin
a) Describe how you would assess her and the appropriate
investigations that you would initiate in your first clinic.
b) Outline what relevant review and tests would be needed to
achieve a full annual review of her thalassaemia
5. You are asked to review a 28 year old female patient who is
scheduled to undergo nasal polypectomy. You are informed that
there is a history of von Willebrand disease in her family and she
shows you a registration card from when she was 6 years old on
which a VWF antigen level of 0.42 iu/ml is documented.
a) Explain what you would do to review the diagnosis and the
laboratory investigations you would perform.
b) Discuss how the findings from question a) may affect how you
advise on this patient’s management.

24. • What is the clinical implication of hemoglobin
measurement in a sample of blood?