The document provides a comprehensive overview of flow cytometry, detailing its principles, components, and applications in various medical laboratories for diagnosing diseases. It explains how the technology measures cell properties by analyzing light scattering and fluorescence, allowing for detailed cellular assessments and sorting. Flow cytometry is highlighted as a crucial tool for analyzing blood cells, immunophenotyping, and identifying genetic diseases.

1. L 200 LECTURE
IMMUNOLOGIC TESTING
METHODS/FLOW CYTOMETRY

2. FLOW
CYTOMETRY

3. INTRODUCTION
AIM
• At the end of the lecture, students should
understand the meaning, principle and application
of Flow Cytometry in the diagnosis of diseases.
• SPECIFIC OBJECTIVES
• For students to understand the principle of Flow
Cytometry.

4. SPECIFIC OBJECTIVES cont.
• For students to know the cellular parameters
measurable by Flow Cytometry.
• For students to know the various components of the
Flow Cytometer and the role of each component.
• For students to know the diversity in the use of Flow
Cytometry in the medical laboratory setting.

5. FLOW CYTOMETER

7. What Is Flow Cytometry?
Flow ~ (cells) in motion
Cyto ~ cell
Metry ~ measure
Measuring the properties of cells moving in a
fluid stream.

8. The Principle of Flow Cytometry
 Flow Cytometry is the technological process that allows
for the individual measurements of cell fluorescence and
light scattering.
 - for counting and examining microscopic particles such
as cells, chromosomes etc.
 This information can be used to individually sort or
separate sub-populations of cells.
 This process is performed at rates of thousands of cells
per second.

9. HOW IS THIS ACHIEVED?
 FCM combines laser-induced fluorometry and
particle-light scattering analysis.
 FCM is incorporated with two or more fluorescence
emission detection systems.
FCM is able to measure multiple parameters such as
 cell size
total protein
 cell receptors
 membrane potential
 calcium ion concentration
 granularity

10. Mechanisms involved in FCM
 Cells in suspension flow single file past
 a focused laser where they scatter light and
emit fluorescence that is collected, filtered
 and converted to digitized values that are
stored in a file
 Which can then be read by specialized
software.
Interrogation
Fluidics
Electronics
Interpretation

11. From figure 1 (Next slide)
 Using the principle of hydrodynamic
focusing
 sample is injected into the center of the
sheath flow
 combined flow reduces the diameter
 forcing the cells into the center of the stream

12. Laser optics
Laser Beam
Flow
chamber
Sheath
Sample
Y
X
Z
Y Z
X
Cells are
presented to the
laser using
principles of
hydrodynamic
focusing
Fig . 1

13. Forward and Side Scatter
FSC
Detector
Collection
Lens
SSC
Detector
Laser Beam

14. COMPONENTS OF A FLOW CYTOMETER
• A FLOW CELL
• LASERS
• MEASURING SYSTEM
• DETECTORS
• READ-OUT DEVICE

16. A FLOW CELL
A flow cell consists of a liquid stream which
carries and arranges the cells properly.
The arrangement processes are important so
that the liquid stream can pass as a single file
through the light for a purpose of sensing.

17. LASERS
Use of lamps such as xenon or mercury
high-power water -cooled lasers like argon, krypton
and dye laser
low-power air-cooled lasers like argon will result in
light signals.

18. MEASURING SYSTEM
 An optical system and measurement of conductivity
are the most commonly used measuring system.

19. Detectors
Detectors are able to measure the brief flashes of
light emitted as cells flow past the laser beam
 -as well as fluorescence signals from light into
electrical signals
 -that can later be processed by the read-out device.

20. Types of Detectors
• There are two main types of photo detectors used
in flow Cytometry.
 Photodiodes
Used for strong signals, when saturation is a potential
problem (eg. FSC detector)
 Photomultiplier tubes (PMT)
More sensitive than a Photodiode, a PMT is used for
detecting small amounts of fluorescence emitted from
fluorochromes.

21. READ-OUT DEVICE
The results are determined by measuring the
visible and fluorescent light emissions, allowing
cell sorting based on physical, biochemical and
antigenic traits.

22. Although conventional cytogenetics can detect
smaller DNA content differences, flow
cytometry allows more rapid analysis of a
larger number of cells.
Flow Cytometry
• How is it used in
diagnosing a disease?

24. Leukocyte Analysis
 As HIV disease progresses, CD4+ T lymphocytes decrease
in total number while CD38 increases.
 The absolute CD4 count using FCM provides a powerful
laboratory measurement for predicting and monitoring
the disease progression and response to treatment in HIV
infected individuals.

25. 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 leukaemia
(ALL).
 FCM is used to differentiate malignant cells from normal
cells.
 The distinction between normal and leukaemic bone
marrow precursors is essential for the diagnosis, treatment
and monitoring of ALL.

26. Enzyme deficiencies
 Gaucher disease is caused by a deficiency of the enzyme
glucocerebrosidase.
 Macrophages transform into pathogenic Gaucher cells
following the phagocytosis of RBCs and subsequent
accumulation of glucosylceramide.
 FCM is a tool for measuring B-glucocerebrosidase activity
in Gauchers disease and to study the abnormalities in RBCs
shapes.

27. Immunophenotyping applications in Haematology
Immunophenotyping is a technique used to study the
proteins expressed by cells such as erythrocytes,
leukocytes and platelets.
Eg. 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.

28. Immunophenotyping of leukaemias and lymphomas
by differentiating their antigen profiles.
Used to identify leukaemias that may be resistant to
therapy .
 FCM does not only detect the presence or absence of
antigens but also the strength of antigen expression which
can help in diagnosis.

29. Genetic disease and carrier states
 FCM can be useful in discriminating heterozygous gene
mutations from normal phenotypes.
 Identification of heterozygous family members who are
carriers of a mutation for the purpose of genetic counseling.

30. WHICH LABS ARE USING FLOW
CYTOMETRY ?
 Haematology
 Immunology
 Tumor cell analysis
 Parasitology (Urinalysis)
 microbiology lab

31. Flow Cytometry in Haematology lab
 Blood cells used to be routinely counted manually. Now,
blood cells are routinely counted by a machine (analyzer).
 This instrument combines the principle of light excitation
and emission of fluorescent signals.
 It is widely used for counting RBC, WBC, Hb, platelet, and
other blood components employing the basic cell-
counting technologies in haematology lab.

32. Haematology analyzer

33. Flow cytometry in Immunology lab
In this lab, flow cytometry is also a primary
instrument used for diagnosis of diseases.
- capable of cell sorting.
-can separate cells by category and keep them alive
for later use.
- useful in separating blood cells such as lymphocytes.

34. Flow Cytometer in Urinalysis
This instrument performs the physical, and
microscopic examination of routine urinalysis.
In a microscopy analyzer, urine sediments can be
identified and categorized based on their size, shape,
and texture.
Lab technologists report the results using a video
screen.

35. Urine analyzer

36. Fluorescence-activated cell sorter (FACS)
 FACS is a specialized type of flow Cytometer which provides
a method for sorting a heterogeneous mixture of biological
cells into two or more containers
 -one cell at a time, based upon the specific light scattering and
fluorescent characteristics of each cell.
 It can be used to separate cells that are phenotypically
different from each other
 -to know how many of them express proteins of interest and
quantify them.

37. FACS cont.
 The process begins by placing the cells into a flask and forcing
them to enter a small nozzle one at a time pass a point of
measurement.
 The cells travel down the nozzle, which is vibrated at an optimal
frequency to produce drops at fixed distance from the nozzle.
 As the cells flow down the stream, they are scanned by a laser
beam. Some of the laser light is scattered by the cells and this is
used to count the cells.
 This scattered light can also be used to measure the size and
granularity of the cells (SSc&FSc).

38. FACS cont.
 To separate a subpopulation of cells, it tags those of interest with an
antibody linked to a fluorescent dye, eg. Fluorescein isothiocyanate
(FITC).
The antibody is bound to a protein that is uniquely expressed in the
cells you want to separate.
The laser light excites the dye, which emits a colour of light
(Fluorescence) that is detected by the photomultiplier tube, or light
detector.
By collecting the information from the light (scatter and
fluorescence) a computer can determine which cells are to be
separated and collected.

39. FACS cont.
 At the end;
 different tubes with pure subpopulations of cells
 the number of cells, in each tube and the level of
fluorescence is also recorded for each cell.
 This is displayed on the computer screen in the form of
histo and cytograms.

41. FACS cont.
 Some flow cytometers on the market have eliminated the
need for fluorescence and use only light scatter for
measurement.
 Others form images using each cell's fluorescence,
scattered light, and transmitted light.

42. Gating
 A gate in cytometry is a set of value limits (boundaries) that serve to
isolate a specific group of cytometric events from a large set.
 The data generated by flow-cytometers can be plotted in a single
dimension, to produce a histogram, or in two-dimensional dot plots or
even in three dimensions.
 The regions on these plots can be sequentially separated, based on
fluorescence intensity, by creating a series of subset extractions, termed
"gates.“
 Specific gating protocols exist for diagnostic and clinical purposes
especially in relation to haematology.
 Data accumulated using the flow cytometer can be analyzed
using software such as WinMDI.

44. FLOW CYTOMETRY
How it works (summary)
 Draw cells, with excess fluid, from test tube into
machine.
 Cells pass in single file past laser.
 Laser hits cells and light is scattered.
 Photomultiplier multiplies light intensity and a light
sensor measures the amount of light and scatter pattern.
 Based on cell characteristics (size and shape), the
computer categorizes and quantifies the cells.

45. CONCLUSION
 FCM 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 expensiveinstruments and an increasing
number of clinically useful antibodies are creating more
opportunities for routine clinical laboratories to use FCM
in the diagnosis and management of disease.

46. THANKS

47. CHEMICALS
• Saline: 8.5 – 9.5g/l NaCl (0.145- 0.154M)
• pH -7.0
• NORMAL SALINE
• A solution of sodium chloride which is physiologically
normal (i.e. one that exerts the same osmotic pressure as
human plasma).
• Theoretically, this would be represented by a solution of 8.8
g/l, although in practice a solution of 9.0 g/l is generally used.
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48. CHEMICALS
• 2.0% sodium metabisulphite
• 5.0% EDTA
• 3.2% Trisodium Citrate
• TEB BUFFER [electrophoretic buffer (pH 8.2-8.6)]
• TRIS- 10.2g
• EDTA-0.6g
• BORIC ACID- 3.2g

49. 49
LEISHMAN STAINING BUFFER
• Combine two stock solutions :
• K2HPO4 (9.5g) - 50.8mls
• NaH2PO4 · 2 H2O(9.1g) -49.2mls
Top up with distilled water to 1liter
• pH -6.8

50. CHEMICALS CONT.
PHOSPHATE-BUFFERED SALINE
• Iso-osmotic phosphate buffer
• NaH2 PO4.H20 (150mmol/l) 23.4 g/l ( solution A)
• Na2HPO4 (150mmol/l) 22.3 g/l (Solution B)
• Add equal volumes of buffer {Solution A (32ml) +Solution B (68ml)}
and 9g/l NaCl to have Phosphate-buffered saline.
• E.g. 100ml of soln (A+B) + 100ml Saline
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51. CHEMICALS CONT.
• CuSO4 SOLUTION:
• Stock Solution – 170g/l
• Working Solution (Male) – Specific Gravity – 1.054
• Working Solution (Female) – Specific Gravity – 1.052
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52. DISINFECTANT SOLUTIONS
• Solutions used to inactivate any infectious
agents that may be present in blood or other
body fluids
• e.g. Sodium hypochlorite solution (bleach,
parazone or chloros)
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53. DISINFECTANT SOLUTIONS CONT.
• Hypochlorite : 0.1% concentration is
suitable for laboratory use, while for heavy
soiling and blood spillage 1% -10% may be
prepared.
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54. DISINFECTANT SOLUTIONS
• Sufficiently and correctly prepared hypochlorite
solution must always be available in all blood banks.
• Diluted hypochlorite solutions are unstable and must
be prepared regularly to ensure that sufficient active
solution is always available.
• All hypochlorite solutions, both concentrated and
diluted, must be kept out of sunlight and ideally at a
temperature of less than +25o
C.
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