Optical Light Scatter

and

Flow Cytometry

CHAPTER 37
MLAB 1415 HEMATOLOGY
JOANNA ELLIS, MLS(ASCP)
 Objectives

 Describe the cell anlaysis principles used in flow cytometry.

 Define hydrodynamic focusing

 List and describe the two types of light scatter.

 Identify applications of flow cytometry in the clinical laboratory.

 Define Cluster of Differentiation (CD Marker).

 Define fluorochrome and describe its use in flow cytometry.

 Define immunophenotyping.
Purpose of
Flow
Cytometry:

To detect and
measure
multiple
properties of
particles or
cells for
identification
and
quantitation.

Image from Wikipedia

Introducing the particle or cell into the Flow Cell

Flow Cell  Flow cytometry is performed on

particles in suspension such as
cells or nuclei.

 The inner column of the flow cell

consists of the liquid sample.

 The outer column of sheath fluid

controls the diameter of the
sample column so that it narrows
and isolates single particles
(hydrodynamic focusing).

 The single particles pass through

a laser beam.

 The light scatter is detected by a

photodetector.
Image from SonyInsider
 Light Scatter

The light from the laser hits the single particle and
scatters in different directions.

Forward
Scatter

LASER

Side Scatter
 Types of Light Scatter

Side light scatter: light scattered at a 90◦

angle from the particle defines internal
complexity and granularity of the particle.
 Neutrophils and eosinophils produce a great deal of
side scatter due to their cytoplasmic granules.
Forward scatter: light that continues in the
forward direction relates the particle size.
 Largecells such as monocytes and neutrophils
produce more forward scatter than nRBCs, and
normal lymphs.
 Light Scatter Scatterplot

Forward Scatter

Side Scatter
Image from Peripheral Blood Smear Evaluation
 Fluorochromes

Fluorochromes are fluorescent compounds that can be

bound by antibodies to the particles in the sample.
 Examples: FITC, PE, Propidium iodide, Acridine orange

Fluorochromes are used in:

 Immunophenotyping
 Cell differentiation and enumeration
 Reticulocyte counting
 DNA analysis
 Detection of Fluorochromes

1. Laser beam excites

fluorochrome attached
to particle at one
wavelength

Photomultiplier C
Cell Tube P
U

2. Fluorochrome 3. Light is directed to the 4. Computer

emits light at photomultiplier tube that software analyzes
different detects and quanitifies the data and generates
wavelength light. scatterplot
Immunophenotyping uses Antibodies and Antigens

Immunophenotyping is the identification of

antigens using detection antibodies.
Monoclonal antibodies are manufactured using
myeloma cells to bind with a single portion of an
antigen (foreign particle/molecule that illicits an
immune response).
Polyclonal antibodies are made by injecting
antigen into animals and are directed against
multiple portions of an antigen.
Most immunophenotyping studies detect cell surface
antigens.
 Commercial Antibodies are grouped in
Clusters of Differentiation (CD Markers)

• CD designations or CD Markers: group of antibodies

that recognize the same antigen.
 Example: Commercially available Leu-4 and OKT3 are both
designated CD3
• CD marker antibodies labeled with fluorochomes
have been developed to bind with antigens of
identifiable cells. (table 37-3, page 840 in text)
• CD designations are cell markers:
 Example: CD4+ cells are helper T-cells, CD8+ cells are
Cytotoxic T-cells
 Immunophenotyping with Flow Cytometry

 The manufactured antibody (CD marker) is

Fluorochrome
attached to a fluorochrome such as FITC and
then added to the sample. Antibody

Antigen
 If the cell has the antigen that the antibody (Ab) (target
specifically binds (target molecule), the antibody molecule)
and fluorochrome attach to the cell.
Cell
 Immunophenotyping with Flow Cytometry

 When the cell/antibody/fluorochrome

complex passes through the laser beam,
the fluorochrome is excited and fluoresces
at a measureable wavelength detected by Fluorochrome
the photomultiplier tube in the flow
cytometer. Antibody

 Computer software connected with the

Antigen
flow cytometer generate a histogram that (target
visually represent the cells present. molecule)

 NO TARGET MOLECULE NO
Cell
FLUORESCENCE
 Clinical Applications of Flow Cytometry

 Blood cell enumeration and classification

 Differentials are based on light scatter as well as fluorescent staining in some
analyzers.
 Diagnosis and classification leukemia
 Immunophenotyping of CD markers specific to certain cell populations can
identify the type of leukemia.
 Diagnosis and monitoring of HIV progression
 Immunophenotyping can determine cell deficiencies. The HIV virus infects and
destroys CD4+ cells. Immunophenotyping with Flow Cytometry can determine
how many of these cells are in circulation.
 Reticulocyte enumeration
 Fluorochromes that stain RNA directly are used to more accurately (than manual
method) count immature erythrocytes.
 DNA analysis
 Certain fluorochromes attach to DNA directly. The amount of DNA is measured
to determine if there are numerical chromosomal abnormalities
 References

 "Flow Cytometry." Wikipedia. Wikimedia Commons. Web. 17 Aug.

2010. <http://www.wikipedia.org/>. image
 McKenzie, Shirlyn B., and J. Lynne. Williams. "Chapter 37." Clinical
Laboratory Hematology. Boston: Pearson, 2010. Print.
 McManus, Christopher. "Sony Acquires ICyt And Officially Enters
Flow Cytometry Business." Sony Insider. 12 Feb. 2010. Web. 17 Aug.
2010. <http://www.sonyinsider.com/2010/02/12/sony-acquires-
icyt-and-officially-enters-flow-cytometry-business/>. Image.
 Riley, Roger, G. W. James, Sandra Sommer, and Mary Jo Martin.
"Peripheral Blood Smear Evaluation." Virginia Commonwealth
University Department of Pathology. Medical College of Virginia,
18 Sept. 1999. Web. 17 Aug. 2010.
<http://www.pathology.vcu.edu/education/PathLab/pages/hemato
path/pbs.html>. Image.