Principle and applications in flow cytometry

1. Dr. POOSAPATI RATNA KUMARI
PROFESSOR
DEPT OF MICROBIOLOGY
FLOW CYTOMETRY

2. INTRODUCTION
Flow cytometry is the science of
measuring physical and chemical
properties of live cells or other
biological particles as they pass in
a fluid, single-cell stream through
a measuring apparatus.
 Flow ~ cells in motion
 Cyto ~ cell
 Metry ~ measure
Measuring properties of cells while
in a fluid stream

3. HISTORY
 16th century - Leeuwenhoek (simple microscope) –
Father of Cytometry
 1742 - Lomonosov - dark field illumination and
performed light scatter measurements
 1934 – Moldavan - photoelectric technique for
counting cells flowing through a capillary tube, and
flow cytometry was born
 1938 - Caspersson built a crude flow cytometer to
measure cell properties in the UV and visible
regions
 1940 - Crosland and Taylor developed a blood cell
counter using the sheath flow principle, light scatter
and darkfield illumination

4.  1949 - Wallace Coulter patented the first non-
optical electronic blood cell counter
 1957 - The first Model A Coulter Counter was
introduced primarily to count erythrocytes and
leukocytes from blood
 1973 - Crissman and Steinkamp introduced
propidium iodide
 Mid 1970s - Leonard Herzenberg at Stanford
coined the term, Fluorescence Activated Cell
Sorter, or FACS

5. PURPOSE OF FLOW
CYTOMETRY
 Flow cytometry detects and measures
- a particle’s relative size,
- relative granularity or internal
complexity
- relative fluorescence intensity
 These characteristics are determined using
an optical-to-electronic coupling system that
records how the cell or particle scatters
incident laser light and emits fluorescence.

6. PRINCIPLE OF FLOW
CYTOMETER
 A flow cytometer is
made up of three main
systems:
Fluidics
Optics
Electronics

7.  Fluidic system -
transports particles in a
stream to the laser beam
for interrogation.
 Optics system
consists of lasers to
illuminate the particles in
the sample stream and
optical filters to direct the
resulting light signals to
the appropriate detectors.
 Electronics system
converts the detected light
signals into electronic
signals that can be
processed by the

8. THE FLUIDICS – FLOW
SYSTEM
 One of the fundamentals of flow cytometry is the
ability to measure the properties of individual
particles, which is managed by the fluidics system
 The fluidics system consists of FLOW CELL
(Quartz chamber)
 Central channel / core – through which the sample
is injected
 Outer sheath – contains faster flowing fluid known
as Sheath fluid (0.9% Saline), enclosing the central
core.
 As the sheath fluid moves, it creates a massive drag
effect on the narrowing central chamber.
 Under optimal conditions (laminar flow) the fluid in
the central chamber will not mix with the sheath
fluid.

9. Incoming Laser
Sample
Sheath Sheath
Sheath
Sample
Sample
Core
Stream
Low Differential High Differential
Laser Focal Point

10. OPTICS
 After the cell delivery
system, the need is to
excite the cells using a
light source.
 The light source used in a
flow cytometer is
Laser (most commonly)
and Arc lamp
 Lasers are more commonly
used as they are highly
coherent and uniform and
they are monochromatic.
 Eg. Argon laser-488nm
wavelength (blue to blue
green)

11.  Forward scatter channel (FSC) - equates to the
particle’s size and can also be used to distinguish
between cellular debris and living cells.
 Side scatter channel (SSC) provides information
about the granular content within a particle.
 Both FSC and SSC are unique for every particle, and
a combination of the two may be used to differentiate
different cell types in a heterogeneous sample.

12.  Neutrophils and
eosinophils produce a
great deal of side scatter
due to their cytoplasmic
granules.
 Large cells such as
monocytes and
neutrophils produce
more forward scatter
than normal RBCs, and
normal lymphocytes

13. Why Look at FSC v. SSC
 Since FSC ~ size and SSC ~ internal
structure, a correlated measurement
between them can allow for differentiation
of cell types in a heterogeneous cell
population
FSC
SSC
Lymphocytes
Monocytes
Granulocytes
RBCs, Debris,
Dead Cells

14. FLUORESCENT ABs
- Cells are labeled with fluorescent
antibodies directed against cell
surface molecules
- Using different color
fluorochromes allows counting of
many markers simultaneously and
allows identification of several
markers on the same cell (
Multiparameter Flow)
- In the instrument, cells pass one-
by-one past a laser to excite the
fluorochromes and there are
detectors for each type of

15. OPTICAL FILTERS
 Once the fluorescence light from a
cell has been captured by the
collection optics, the spectral
component of interest for each stain
must be separated spatially for
detection.
 Optical filters are designed such
that they absorb or reflect a
wavelength of light, while
transmitting other.
 4 types of filters are used in
flow cytometry
 Long Pass filter
 Short Pass filter
 Band Pass filter
 Dichoric filter

16. OPTICAL DETECTORS
 The two main photo detector types used in flow cytometry
are
 silicon photodiodes - to detect forward scatter
detection
 Photomultiplier tubes (PMTs) - high sensitivity and
are, therefore, assigned to side scatter and fluorescence
detection.

17. FLUOROCHROMES
 Fluorochromes are
essentially dyes,
which accept light
energy (e.g. from a
laser) at a given
wavelength and re-
emit it at a longer
wavelength.
 These two processes
are called excitation
and emission.
 The process of
emission follows
extremely rapidly,
commonly in the
order of
nanoseconds, and is

18. GATING
 An important principle of flow cytometry data
analysis is to selectively visualize the cells of
interest while eliminating results from unwanted
particles e.g. dead cells and debris. This procedure
is called GATING.
 Cells have traditionally been gated according to
physical characteristics.
 Lysed whole blood cell analysis is the most
common application of gating.

19. DETECTION OF FLUOROCHROMES
1. Laser beam excites
fluorochrome
attached to particle at
one wavelength
2. Fluorochrome
emits light at
different
wavelength
3. Light is directed to the
photomultiplier tube
that detects and
quantifies the light.
4. Computer
software analyzes
data and generates
scatterplot
Photomultiplier
Tube
C
P
U
Cell

20. ELECTRONICS
 As a particle of interest passes through the
focus, fluoresces and is detected by a photo
detector, an electrical pulse is generated and
presented to the signal processing electronics.
 The instrument is triggered when this signal
exceeds a predefined threshold level.
 Electrical pulses are digitized, the data is stored
(‘list mode data’), analysed and displayed through
a computer system.
 The end result is quantitative information about
every cell analysed
 Large numbers of cells can be processed quickly

21. ELECTRONICS

22. QUALITY CONTROL IN
FLOWCYTOMETRY
Divided into 2 parts
 Internal quality control
 External quality control

23. INTERNAL QUALITY CONTROL IN
FLOWCYTOMETRY
 Internal quality control consists of a series of
activities that are performed by the laboratory
to ensure the instrument, reagents and staff are
performing within the limits set by the
laboratory.

24.  Quality control of the instrument can be divided into
three separate procedures
 First procedure is usually carried out once or twice
a year by qualified service personnel who check
the performance of components such as the lasers,
photomultiplier tubes (pmts), optical filters, and log
and linear amplifiers.
 Second procedure is performed by the operator
with each start-up of the instrument. Using
appropriate reference microbeads with dedicated
plots and instrument settings, parameters such as
cvs and mean or median channel number can be
recorded.
 Third procedure consists of the calibration of the
fluorescence channels

25. EXTERNAL QUALITY CONTROL IN
FLOWCYTOMETRY
 Samples, either fresh or stabilized, are distributed on
a regular basis, and the laboratory is asked to
process them as they would any other sample.
 Results are submitted back to a central agency for
analysis, and a report indicating the relative
performance of each participating laboratory is
issued.
 External quality control schemes check the complete
process, from the technical side of sample
preparation (staining, data analysis, interpretation)

26. ADVANTAGES DISADVANTAGES
 High speed analyses
depending on the flow rate;
 Measures single cells and
a large number of cells;
 Simultaneous analysis
multiple parameters;
 Identifies small
populations;
 Quantification of
florescence intensities;
 Sorting of predefined cells
populations (up to
70.000/s);
 Portable equipment's.
 Very expensive and
sophisticated instruments;
 Requires management by
a highly trained specialist
and on-going
maintenance by service
engineers.
 Complex instruments are
prone to problems with
the microfluidics system
(blockages) and also
require warm-up, laser
calibration and cleaning
for each use.
 Needs single cell particle;
 Little information on intra-

27. Common specimens suitable for
flowcytometry analysis include
Peripheral blood,
Bone marrow,
Body fluids,
Cerebrospinal fluid,
Urine,
Lymph node (cells or fresh tissues),
Any fine-needle aspirates,
Fresh tissues suspicious for
hematopoietic and lymphoid disorders.

28. APPLICATIONS OF FLOW
CYTOMETRY

29. FLUORESCENCE-ACTIVATED CELL SORTING
(FACS)
 Fluorescence-
activated cell
sorting (FACS) is a
development of flow
cytometry that enables
sorting of a mixture of
cells into two or more
fractions, cell-by-cell,
utilising the scatter and
fluorescence signals of
each cell.

30. APPLICATIONS IN CLINICAL
MICROBIOLOGY
DETECTION OF BACTERIA
 Flow cytometry is a sensitive analytical technique that
can be readily applied to the enumeration of viable
bacteria in a biological sample.
 The possibility of using fluorochrome-labeled
antibodies to specific antigens render them one of the
most powerful tools in the identification of pathogens.
 Flow Cytometry in conjunction with fluorescent
antibodies has been used to detect surface antigens
in -
 Hemophilus, Salmonella, Mycobacterium, Brucella,
Mycoplasma, Pseudomonas, Bacteroides and

31. DETECTION OF FUNGI
 Surface antigens of Candida albicans can be
detected by flow cytometry in conjunction with
available antibodies.
 Using FCM and antibodies directed against yeasts
, Pierard et al identified fungal pathogens and
differentiated them from non-pathogenic ones.

32. DETECTION OF PARASITES
 FCM has been used to detect intracellular
parasites such as Plasmodium.
 The absence of DNA in erythrocytes has enabled
this intracellular parasite’s DNA to be stained
with specific fluorochromes and detected by
FCM.
 The multiparameter analysis permitted by FCM
can be used to study other characteristics, such
as parasite antigens expressed by erythrocyte or
viability state of parasitized cell.

33. DETECTION OF VIRUSES
 FCM can detect viral antigens either on the surface
of or within infected cells.
 It can rapidly detect and quantify virus infected cells
using antibodies that specifically recognise surface
or internal antigens.

34. Leukocyte Analysis
CD3
 A normal person has a significant proportion of CD3-
positive lymphocytes. In the patient with leukaemia,
staining for CD3 is absent.
CD20
 In the leukaemia patient there are a large number of
cells staining positive for CD20. In the healthy person
only a few stain positive.
HLA-DR
 The leukaemia patient is HLA-DR-positive. In the
normal person only a small number of cells stain
positive.
 Being CD3-negative, CD20-positive and HLA-DR-
positive, a clinician could diagnose with certainty that
this patient is suffering from a B cell lineage
leukaemia or lymphoma

35. DNA analysis (Malignancy)
 DNA aneuploidy generally is associated with
malignancy; often correlates with many types of
cancer such as multiple myeloma, and childhood
acute lymphoblastic leukemia (ALL).
 Flow Cytometry used to differentiate malignant
cells from their normal counterparts. The
distinction between normal and leukemic bone
marrow precursors is essential for the diagnosis
and treatment monitoring of acute lymphoblastic
leukemia.
 Although conventional cytogenetics can detect
smaller DNA content differences, flow cytometry
allows more rapid analysis of a larger number of
cells.

37. Enzymatic deficiencies
 Gaucher disease is caused by a deficiency of the
enzyme glucocerebrosidase.
 Macrophages transform into pathogenic Gaucher cells
following the phagocytosis of red blood cells (RBCs)
and subsequent accumulation of glucosylceramide.
 Flow cytometry is a tool for measuring
Bglucocerebrosidase activity in Gauchers disease and
to study the abnormalities RBCs shape.

38. IMMUNOLOGY
 DIAGNOSIS AND MONITORING OF HIV
PROGRESSION
 Immunologic monitoring of HIV-infected patients is a
mainstay of the clinical flow cytometry laboratory.
 HIV infects helper/inducer T lymphocytes via the CD4
antigen.
 As HIV disease progresses, CD4-positive T
lymphocytes decrease in total number.
 The absolute CD4 count provides a powerful laboratory
measurement for predicting, staging, and monitoring
disease progression and response to treatment in HIV-
infected individuals

39. Immunophenotyping Applications
 Flow cytometry is used in “Immunophenotyping” -
Immunophenotyping is a technique used to study the
protein expressed by cells such as erythrocytes,
leukocytes, and platelets.
 Example:
 Immunophenotyping for HLA-B27.HLA-B27 is one of the
multiple major histocompatibility complex (MHC) class I-
specific antigens. Detection of HLA-B27 (positive) may
associate the patient with several other disorders, such
as Reiter’s syndrome, psoriatic arthritis, and inflammatory
bowel disease.
 Immunophenotyping of leukemias and lymphomas by
differentiating their antigen profiles. Flow cytometry can
also be used to identify leukemias that may be resistant
to therapy .
 Flow cytometry not only is can detect the presence or

40. Genetic disease and carriage
 Flow cytometry can be useful in discriminating
heterozygous gene mutations from normal
phenotypes.
 It may also identify heterozygous family members
who are carriers of a mutation for the purpose of
genetic counseling.

41. APPLICATIONS IN HEMATOLOGY
Detection of fetal Hb
 The use of flow cytometry for the detection of fetal
cells is much more objective, reproducible, and
sensitive than the Kleihauer-Betke test .
 Fluorescently labelled antibodies to the rhesus (D)
antigen can be used, or more recently, antibodies
directed against haemoglobin F
 This method has the ability to distinguish fetal cells
from F-cells (adult red cells with small amounts of
haemoglobin F).

42. PAROXYSMAL NOCTURNAL
HEMOGLOBINURIA
 Paroxysmal nocturnal hemoglobinuria (PNH) is an
acquired clonal stem cell disorder that leads to
intravascular hemolysis with associated thrombotic and
infectious complications.
 Antibodies to CD55 and CD59 are specific for decay-
accelerating factor and membrane-inhibitor of reactive
lysis, respectively, and can be analysed by flow
cytometry to make a definitive diagnosis of PNH

43. BIOLOGICAL APPLICATIONS FOR
CELL SORTING
Protein Engineering & Development
 Screening of peptide libraries for binding
 Selecting antibody mutants
 Screening for enzymatic activity
 Screening for over-producing cells
Cell Engineering
Disease Identification/Characterization
Isolating cells to characterize them based on multiple
modalities
Nucleic acid
Protein expression
Cellular function

44. FUTURE DIRECTIONS-
NEW TESTS

45. ICP 11 (1969) Distributed by Phywe,
Göttingen The first commercial flow
cytometer PDP 11 computer
TPS 1974 - 1979,
Designed by Bob Auer
Epics II 1975,
Designed by Mack Fulwyler and Jim
Corell Delivered to NCI/NIH
Early instruments

46. Modern Instruments

47. POLYCHROMATIC FLOW CYTOMETRY
In many instances the small volume of a
sample obtained from the patient is a limiting
factor for the number of tests that can be
performed .
 Hence the need for development of
polychromatic flow cytometric tests.(upto 6
fluorochromes)
 This will reduce the requirement for large
volume of specimen ,the time needed for
sample processing and total no. of antibodies

48. APPLICATIONS IN DIAGNOSIS OF
ALLERGY
 FCM is used to assess the reaction of basophils
to allergens .
 CD63 molecules in resting basophils are located
in the membranes of intracellular granules and
are not detectable on the cell surface.
 If basophils are sensitized in vivo with allergen
specific IgE , then exposure to allergen in vitro
will result in their degranulation, and therefore
the expression of CD63 on the cell surface of
activated basophils will increase in less than
10min.

49. ANTIFUNGAL SUSCEPTIBILITY
TESTING
 One advantage of FCM AST over conventional
methods is the 4 –hour incubation time compared
with current methods that require an incubation time
of 24 to 48 hrs.
 After incubation with antifungal agent, the fungal
cells are stained with a specific fluorescent dye and
the changes in fluorescence intensity of stained cells
due to efforts of antifungal agents is measured by
FCM.
 Fungal cells become more fluorescent if they are
susceptible to the antifungal agent

50. C0NCLUSION
Flow cytometry is a powerful technique for
correlating multiple characteristics on single cells.
This qualitative and quantitative technique has made
the transition from a research tool to standard clinical
testing.
Smaller, less expensive instruments and an
increasing number of clinically useful antibodies are
creating more opportunities for routine clinical
laboratories to use flow cytometry in the diagnosis and
management of disease.