Flow cytometry is the measurement of cells in a flow system, which delivers the cells singly, past a point of measurement

1. Flow Cytometry
Principles and clinical
applications
Dr. Ankit Raiyani
Hematology Department
Sahyadri Specialty Hospital

5. • Flow cytometry (FCM)
• Immunohistochemistry (IHC)
• Immunofluroscence (IF)

7. Flow cytometry
• Flow cytometry is the measurement of
cells in a flow system, which delivers the
cells singly, past a point of measurement
• In practice, the name refers to instruments
in which light is focused at the point of
measurement.
• Typically, light scatter at two different
angles and from one to six or more
fluorescence will be measured.

9. Disadvantages of FCM
• Need for liquid cell suspension. Therefore
lack of correlation with histomorphologic
features ( tissue architecture)
• Requires viable, fresh (unfixed) material
• Requires 1,00,000 events acquired by
tubes, which often limits its use in CSF,
paucicellular lesions.

10. History
• The first impedance-based flow cytometry
device, using the Coulter principle was
developed in 1953, (Wallace H. Coulter).
• Mack Fulwyler was the inventor of the forerunner
to today's flow cytometers - particularly the cell
sorter.
• The first fluorescence-based flow cytometry
device (ICP 11) was developed in 1968 by
Wolfgang Göhde from the University of Münster,

11. • The original name of the fluorescence-based
flow cytometry technology was "pulse
cytophotometry“
• "flow cytometry", as a term was accepted in
1976
• Earlier flow cytometers were conceptualized as
cell sorters. Later on ability to sort cells was
removed as it was deemed unnecessary in
routine work
• But still name FACS (Fluorescence-activated
cell sorting) is used in BD flow cytometers.

13. Principle

16. Hydrodynamic focusing

20. Sample preparation
• Types of specimens suitable for flow
cytometry:
– Blood, bone marrow aspirate, fine needle
aspirates, body fluids.
– Solid tissue biopsy like spleen, thymus has to
be processed to form a cell suspension.

21. Sample preparation
• In clinical samples, if nucleated cells are
being studied, the red blood cells are
usually lysed either by a brief exposure to
distilled water, incubation with an
ammonium chloride solution or with a
weak detergent.
• If surface antigens are to be stained, fresh
unfixed cells are reacted with the labelled
antibodies.

22. • Take sample (100μl) in each tube
• Add monoclonal antibodies and vortex
• Incubate at room temperature in dark for 20 min
• Add RBC lysing solution (1 ml)
• Centrifuge for 5 min
• Discard supernatant
• PBS wash, discard supernatant
• Add PBS (0.5 ml) to WBC pellet.

23. • For cytoplasmic or nuclear antigens, the cells
must be fixed and permeabilised to give the
antibody access to the antigen.
• There is no standard procedure for staining intra-
cellular antigens.
• Many antigens are adversely affected by some
fixatives and consequently the optimum procedure
has to be determined for each protein under study.
• Several manufacturers sell reagents which are
mostly based on permeabilisation in detergent,
usually saponin, and fixation in formaldehyde.

25. IntraPrep
• Reagent 1:
– Fixation agent
– Formaldehide
• Reagent 2:
– Permeability agent
– Saponine

26. Fluorochromes used with
monoclonal antibodies
• Argon laser (488 nm)
• Fluorochromes: Are Molecules That Emit
Fluorescence Upon Excitation With Light
• In our set up:
– FITC (fluorescein isothiocyanate)
– PE (Phytoerythrin)
– ECD (phycoerythrin-Texas Red conjugate /energy
coupled dye )
– PC5 (phycoerythrin-cyanine5 conjugate)
– PC7 (phycoerythrin-cyanine7 conjugate)

27. Fluorochromes used with
monoclonal antibodies
• Others:
– PerCP (peridinin chlorophyll-A protein)
– 7-AAD (7-actinomycin D)
– PI (propidium iodide)
– TOT-1 (thiazole orange)

29. Gating strategy
• Can be used in single parameter and two
parameter charts.
• Commonly used gates in two parameter
scatter plots.
– FSC Vs SSC
– CD45 Vs SSC
– CD19 Vs SSC

30. Single
parameter
histogram

31. Single parameter gating

32. Two parameters scatter plot

33. Two parameters scatter plots with
gate A as source

34. Two parameters density plot

36. SSC
• High SSC: PMN (including precursors),
granular blasts
• Intermediate SSC: Monocytes, blasts,
Hairy cell leukemia cells
• Low SSC: Lymphocytes, blasts,
hematogones.

37. FSC
• High FSC: monoblasts and monocytic
cells,
• Intermediate FSC: Myeloblasts, Large
lymphoma cells (DLBCL, ALCL, etc)
• Low FSC: Lymphoblasts, lymphocytes

38. CD 45 Vs SSC
• Bright CD 45 + Low SSC: lymphocytes, mature
lymphoproliferative disorder
• Bright CD45 + High SSC: Monocytes, neoplastic
monocytes (CMML, Acute Myeloid Leukemia
M4/5), HCL, PMN in Myelodysplastic Syndrome.
• Moderate CD45 + High SSC: PMN, maturing
myeloid cells, hypergranular APML,
• Moderate CD45 + slightly increased SSC:
lymphoblasts

39. CD 45 Vs SSC
• Dim CD45 + Low SSC: hematogones,
lymphoblasts
• CD45 negative + Low SSC: plasma cells,
RBC precursors.
• CD45 negative + High SSC: extrinsic
elements (metastatic cells)

40. CD138 Vs SSC gating

41. CD19 Vs SSC

42. How to obtain absolute cell count
by flow cytometer
• While flow cytometry generally gives the
percentage of a particular sub-set of cells, some
flow cytometers precisely record the the volume
of sample analysed or deliver a fixed volume of
sample. A percentage count of a sub-population
of cells can be directly converted to an absolute
count.
• In instruments without this facility, two
approaches are used to measure the absolute
count, referred to as two platform or one
platform methods.

43. Two platform approach
• Concentration of all the cells in a sample is determined
by another method.
• e.g. for blood leucocytes, TLC is calculated by coulter
counter.
• The flow cytometer is then used to determine the
percentage of cells in a particular sub-set so that the cell
concentration of the sub-set can be calculated.
• The disadvantage of the two platform method is that
errors in the two instruments are compounded and that
two instruments are required.
• E.g.: CD4/CD8/CD25 count.

44. One platform approach
• Fixed volume of sample is spiked with a known number
of fluorescent beads.
• The brightness and light scatter of the beads is different
to that of cells so that beads and cells can be easily
distinguished in the flow cytometer.
• Counting the number of beads in the portion of the
sample analyzed allows the volume analyzed to be
calculated and hence the concentration of cells.
• The disadvantage of this method is that beads can stick
to each other and to the walls of the tube leading to an
underestimation of the bead count.
• E.g.: CD4/CD8 count, CD34 count

45. CD4/CD8
count

46. CD34 count

47. Typical Problems/Challenges
– Some markers are highly expressed, others are
expressed at low levels.
– Some dyes are much brighter than others.
– Significant emission spill over from non-primary
fluorescent reagents contributes to optical
background, which can often diminish the resolution
of dim markers (due to spread after compensation).
– Some markers may be available only in certain
colors.
– New fluorochromes are not as bright or stable as
the original ones.

48. Colour compensation
• The emission spectra of fluorescent dyes
are broad.
• For example, while fluorescein
fluorescence looks, and is, predominately
green, the spectrum contains a range of
colours from green to red.
• While the peak emission is clearly
separated for each dye, there is
considerable overlap between the dyes.

50. Spectral overlap
Ccells labelled
with FITC will
appear to have
some
phycoerythrin
fluorescence.

51. Compensation
• First the PMT (photomultiplier) voltages on
all the fluorescence channels in use are
set to display the data as required;
generally to give a good separation
between negative and positive cells.
• Cells labelled singly with each of the
fluorochromes are run and the compensation
set by inputting the percentage of the one
fluorescence signal that needs to be
subtracted from another.

52. • Software is available to carry out this
procedure automatically, once the data from
each fluorochrome has been recorded.
• Color compensation is carried out by
software and can be applied off-line after the
data has been recorded

53. • Flow-Check Fluorospheres
– Fluorospheres used to check the stability of the optical and
fluidic systems.
• Flow-Set Fluorospheres
– Fluorospheres used to standardize light scatter and
fluorescence intensity for human leukocyte applications.
• QuickCOMP 2 Kit:-
– Two single-color antibody reagents (FITC and PE) that can
be used to adjust color compensation on a flow cytometer.
• QuickCOMP 4 Kit:-
– Four single-color antibody reagents (FITC, PE, ECD, and
PC5) that can be used to adjust color compensation on a
flow cytometer.

56. • Thank You!