Instrument Methodology: Flow Cytometry Created by Dominique Stevens and Emily Franklin Created for MLT 1012 (Summer Session)

1. Flow Cytometry
Dominique Stevens
Emily Franklin
MLT 1012-149579
Professor Gill
6/20/18

2. What is Flow Cytometry?
Flow cytometry is the process by which an instrument, known as
a flow cytometer, collects specific data from cells that will be
transmitted to a computer for analysis.
What do we use it for?
This method is utilized by laboratory scientists to aid in the
counting and characterization of cells.

3. How It Works:
1.) Cells are collected and placed in a suspension within a collection tube.
2.) These cells are then either stained with a fluorochrome, or treated with
antibodies that help detect the presence of cancer cells.
3.) The tube is placed into the flow chamber.
4.) As the stained cells begin to flow downward in a single-file line, a laser beam is
then projected through the assembly line.
5.) The cells begin to fluoresce as they pass through the beam, and the cytometer
detects the projection of forward and side scatter of light.
6.) The light strikes the cytometer’s internal mirrors, and bounces towards the
fluorescence detectors.
7.) The detectors sort the scatter of light by size, and then transmits collected data
to a computer for analysis.
= Key Principles

4. Illustration: Graphic demonstration of a
fluorochrome-activated cell —>

5. Fluorochrome Staining
• A fluorochrome is a dye that contains a fluorescent marker. These markers will fluoresce (light up) in
reaction to specific organelles and membranes within a cell. An antibody can also be introduced to a
cell to serve as a marker, as long as it attaches itself to the cell.
• Fluorochromes contain 2 types of spectra:
a.) Excitation
b.) Emission
• There are 5 types of fluorochrome stains:
a.) Fluorescent proteins
b.) Synthetic molecules
c.) Quantum dots
d.) Polymer dyes
e.) Tandem dyes

6. Measurement of Fluorescence
• There are 3 types of filters used, to absorb light in a cytometer:
a.) Long-pass: wavelength of light above cut-off (between 550-700nm)
b.) Short-pass: wavelength of light below cut-off
c.) Band-pass: wavelength of light in a narrow range (between 500-525nm)
• There are detectors within the cytometer, that will measure the pulse of energy,
generated as the stained cells move through the laser.
• Computer software will plot the intensity of the cell’s fluorescence.

7. Measurement of Scatter
• As cells move through the laser,
light is scattered in different
directions. Plotting light scatter
can tell us the size and
granularity of the cell.
• There are 2 types of light scatter:
A. Forward- intensity is
proportional to cell size
B. Side- intensity is
proportional to granule
size

8. What can we use it for?
• In a real-world application, flow cytometry is used as a powerful
diagnostic tool.
• Data collected by the cytometer can aid an oncologist in the classification
and diagnosis of different cancers, such as leukemia and lymphoma.
From this data, the oncologist can then devise a custom, life-saving
treatment plan for the patient.
• Flow cytometry can also be used to analyze stem cells, and make sure
they are healthy for transplantation.
• It can also be used to detect autoimmune disorders, to monitor fetal-
maternal hemoglobin levels, and even to detect immunodeficiencies, such
as HIV.

9. Leukemia Panel
• In this real-world application:
1.) A bone marrow specimen was
collected from a patient.
2.) Those cells were processed
through a cytometer.
3.) Resulting data was transmitted
to a computer.
4.) The computer then produced a
histogram, based upon the light scatter
detected, as each cell flowed through
the cytometer.
A.) The histogram to
the left, reveals that
this particular patient
has Acute Leukemia, a
form of cancer.
B.) The histogram to
the right illustrates
normal bone marrow in
a healthy patient.

10. References
Bushnell, T. (17 August 2017). Why Understanding Fluorochromes Is Important in Flow Cytometry.
Retrieved from https://expertcytometry.com.
Davidson, M. W. Retrieved from https://micro.magnet.fsu.edu.
Flow Cytometry Guide. Retrieved from https://www.creative-diagnostics.com.
Flow Cytometry Laboratory. Retrieved from https://pathology.duke.edu.
Flow Cytometry for Leukemia. Retrieved from https://www.cancercenter.com.
Halasey, S. (31 January 2016). Flow Cytometry In The Clinical Lab. Retrieved from http://www.clpmag.com.
Introduction to Flow Cytometry. Retrieved from http://www.abcam.com.
Martz, E. (2003). What Is Flow Cytometry?. Retrieved from https://www.bio.umass.edu
Robertson, S. (23 December 2014). What Is Flow Cytometry?. Retrieved from https://www.news-medical.net
Taylor, I. Forward Scatter vs. Side Scatter. Retrieved from https://www.flowjo.com.
Turgeon, M. (2016). Clinical Laboratory Science. Mosby Inc.
Worth, A. Retrieved from http://www.jenner.ac.uk.