basics of flow cytometry

1. Moderator: Dr. Rakesh Mehar
Speaker : Dr. Aksharaditya Shukla
Department Of Pathology MGM Medical College Indore

2.  Flow ~ motion
 Cyto ~ cell
 Metry ~ measure
 Measuring properties of cells while in a
fluid stream

3.  An analytical technique in which cell
suspension obtained from any unfixed
tissue /body fluid, peripheral blood or bone
marrow are stained with fluorescently labeled
antibody and then subjected to analysis by a
instrument called as flow cytometer.

4. • In 1972 L. Herzenberg (Stanford Univ.),
developed a cell sorter that separated cells
stained with fluorescent antibodies.The
Herzenberg group coined the term Fluorescence
Activated Cell Sorter (FACS).

5. Why do we need fluorescence in flow
cytometry ?
• Many cells appear the same.
• Fluorescence enables us to mark specific
components / particles /identify & characterize
sub-population.
• Enables specific discrimination
- Live / Dead, cell cycle etc.

6.  Fluidics system.
 Optic system.
 Electronic system.
 Associated computer system.

7.  Various pumps and tubings to introduce the
sample.
 The sample so introduced is surrounded by a
pressurized stream of buffered saline called as
the sheath fluid so the cells assume a laminar
flow. So that they flow in a single file through the
light beam for sensing.
 The flow cell – Heart of flow cytometry.

8.  Laser, used to excite the florescent dye
conjugated to the antibody.
 Conveying system- to convey the emitted light
to specific detector.
 Specific detectors are photomultiplier
tubes(PMT’s)that convert the photon to
electrical impulses.

9.  Measures the electrical impulses generated by
the PMT’s and convert it to digital information.

10. Acquisition and analysis of flow cytometric data.

11. Cytometry
 Localization of
antigen is possible.
 Poor enumeration of
cell subtypes.
 Limiting number of
simultaneous
measurements.
Flow Cytometry.
 Cannot tell you where
antigen is.
 Can analyze many
cells in a short time
frame.
 Can look at numerous
parameters at once.

12. FITC
FITC
FITC
FITC
FITC
FITC
Antibodies recognize specific
molecules in the surface of
some cells.
But not others
When the cells are analyzed by flow
cytometry the cells expressing the marker for
which the antibody is specific will manifest
fluorescence. Cells lacking the marker will
not manifest fluorescence.
Antibodies are artificially
conjugated to fluorochromes.
Antibodies

13.  Sensitize mouse to antigen
 Harvest spleen B cells
 Fuse with myeloma cells
 Select hybridoma clones for antibody
production
 Label antibody with fluorochrome dye / color

14. Antigen expression:
RBCs, WBCs, Platelets, Others
CD3
Cyto CD3
Tdt

15.  No reagents or probes
required (Structural)
 Cell size(Forward Light
Scatter)
 Cytoplasmic granularity(90
degree Light Scatter)
 Photsynthetic pigments
 Reagents are required.
 Structural
 DNA content
 DNA base ratios
 RNA content
 Functional
 Surface and intracellular
receptors.
 DNA synthesis
 DNA degradation
(apoptosis)
 Cytoplasmic Ca++
 Gene expression
Intrinsic Extrinsic

16.  Peripheral Blood
 Bone Marrow Aspirate & Biopsy
 Tissue
 CSF
 Others
Different anticoagulants used are EDTA , Heparin.
Acid citrate dextose is not used as it adversely affects
the viability of cells.

17.  All the samples must be handled with care.
 After the sample is incubated with the
antibody and is washed ,but before it is
subjected to be analysed by flow cytometer, the
sample should be fixed with 1 % formaldehyde
which stabilizes the antigen antibody
interaction by creating cross linkages.

18.  The sample is introduced in the flow cell by
computer driven syringe in the centre of the
sheath fluid.
 The cells from the sample tube are injected into
the sheath stream
 Flow in a flow cell is laminar.
 Hydrodynamic focusing pushes the cells to line
up single file along their long axis.

19. Light Scattering: When light from a laser
interrogates a cell, that cell scatters light in all
directions.
Forward scatter.
Side scatter.

20.  Light that is scattered in the
forward direction (along the
same axis the laser is
traveling)
 FS is the light scattered
between 1-10 degrees.
 The intensity of this signal
has been attributed to cell
size, refractive index &
absorptive properties.

21.  The index of refraction is higher in fixed and
stained cells.
 Live cells scatter more light than dead cell at
lower angle

22. FSCFSC
DetectorDetector
Laser BeamLaser Beam
Original from Purdue University Cytometry LaboratoriesOriginal from Purdue University Cytometry Laboratories

23.  Laser light that is scattered at 90 degrees to the
axis of the laser path is detected in the Side
Scatter Channel
 The intensity of this signal is proportional to
the amount of cytosolic structure in the cell (eg.
granules, cell inclusions, etc.)

24. FSCFSC
DetectorDetector
CollectionCollection
LensLens
SSCSSC
DetectorDetector
Laser BeamLaser Beam
Original from Purdue University Cytometry LaboratoriesOriginal from Purdue University Cytometry Laboratories

25. FS SS
Size Granularity

26. SS detector - Granularity
SS detecter Granularity
FS
size
FS
size
Laser
Laser
Size and
granularity

27. Allows for differentiation of cell types in a
heterogenous cell population
FSC
SSC
Lymphocytes
Monocytes
Granulocytes
RBCs, Debris,RBCs, Debris,
Dead CellsDead Cells

28.  Flow cytometer used usually have 2 lasers the
argon and helium neon laser.
 For the florochromes to be useful ,the florescent
wavelength must be higher then the excitation
light
The difference is called as stoke shift

30.  As the laser interrogates the cell, fluorochromes
on/in the cell (intrinsic or extrinsic) may absorb
some of the light and become excited
 As these fluorochromes leave their excited
state, they release energy in the form of a
photon with a specific wavelength, longer than
the excitation wavelength.
 These photons pass through the collection lens
and are split down into specific channels with
the use of filters.

31. FSCFSC
DetectorDetector
CollectionCollection
LensLens
Laser BeamLaser Beam
FluorescenceFluorescence
Detector A, B, C, etc…Detector A, B, C, etc…
Original from Purdue University Cytometry Laboratories, Modified by James MarvinOriginal from Purdue University Cytometry Laboratories, Modified by James Marvin

32.  A way to split the light into its specific
wavelengths in order to detect them
independently. This is done with filters
 2 types of filters
 Absorption filter
 Interference filter

33.  Absorption filters:
i. Made up of colored glasses.
ii. They absorb unwanted light and pass the light of
desired wavelength.
Interference filters
Attenuate or reflect the unwanted light.
i. Long pass.
ii. Short pass.
iii. Band pass
iv. Dichroic filters.

35.  The sensor converts the photon to electrical
impulses that are proportional to no. of
photons received which in turn is proportional
to the no. of florochromes molecules bound the
the cell.
 These impulses are converted to digital
information by the converter/electronic
system. The digitalized data is stored in form
of histograms or a list mode.

36.  An important aspect of the FC is the selection
of only certain cell population for analysis by a
process called as gating.
 Used to isolate a subset of cells on a plot
 Allows the ability to look at parameters specific
to only that subset.
 Gates are rectilinear , amorphous or numeric .
 The amorphous are the most versatile .

37. .

38.  Separating cells of interest from a heterogeneous
mixture.
• 5-10 parameters could be analyzed (DNA, protein
and lipid content).
• Droplet containing cells of interest is charged.

40. PMT
PMT
PMT

41.  Florescin isothiocyanate (FITC).
 Phycoerythrin.
 Texas red.
 Allophycocyanin.
 Peridinin chlorophyll.
 Tandem florochromes.
The size of the conjugate is also important
For eg. Conjugation of phycoerythrin with IgM are
insoluble and therefore not been successful

42.  1.Immunophenotyping.
 2.Diagnosis and prognostication of
immunodeficiency.
 3. To diagnose cause of allograft rejection.
 4.Diagnosis of auto antibodies in ITP .
 5. To measure nucleic acid content.
 6. DNA ploidy study in cancer.

43.  CD1a Immature T cells (common
thymocytes), Langerhans cells
 CD2 Pan–T-cell, NK cells
 CD3 Pan–T-cell
 CD4 Helper/inducer T cells
 CD5 Pan–T-cell, subset of B cells
 CD7 Pan–T-cell, NK cells, subset of AMLs

44.  CD8 Cytotoxic/suppressor T cells
 CD9 B cells, platelets, and megakaryocytes
 CD10 Immature and germinal center B cells,
mature granulocytes
 CD11b Maturing myeloid cells (both
monocytes and granulocytes)
 CD11c Hairy cell leukemia, some marginal
zone lymphomas

45. CD38 Plasma cells (bright), B-lymphoblasts and
myeloblasts (moderate)
CD41 Glycoprotein IIb/IIIa, platelets, and
megakaryocytes
CD45 Leukocyte common antigen (nonerythroid
hematopoietic cells)
CD56 NK cells, some stem-cell disorders, some
activated T cells
CD57 Subset of cytotoxic/suppressor T cells
CD61 Platelets, and megakaryocytes
CD64 Monocytes, activated granulocytes
CD103 Hairy cell leukemia
Tdt ImmatureT cells, immature B cells

46.  CD117 Myeloid blasts, promyelocytes
proerythroblasts, mast cells
 Bcl-2 Naïve B cells, most T cells, most follicular
lymphomas
 FMC7 B-cell lymphoma other than CLL/SLL
 κ-light chain Mature B cells
 γ-light chain Mature B cells

47. Benign Hematopoeitic disorders
 HIV.
 SCID.
 CVID.
 Fetomaternal hemorrhage

48.  Determination of the numbers of CD4 +ve
lymphocytes in the peripheral blood is used to
monitor patients with HIV infections (Mandy
et al., 2002). The percentage of CD4 +ve cells
can be obtained in a single tube by staining for
CD45/CD3/CD4. A cytogram of SS versus
CD45 is used to identify the lymphocytes and a
cytogram of CD4 versus CD3 to enumerate the
CD4+ve T cells. An extended panel is used to
obtain a more complete picture of the
peripheral blood lymphocytes

49.  Since the dose of anti-D given is related to the
size of the foeto-maternal haemorrhage,
quantitation of foetal-maternal haemorrhage is
therefore important.
 Quantitation is achieved by labelling the
erythrocytes in a sample of maternal blood
with FITC-conjugated, non-agglutinating anti-
D antibodies (Nance et al., 1989). A population
of as few as 0.1% foetal cells is sufficient to
sensitise the parent so at least 500,000 cells
should be analysed to obtain a statistically
significant estimation.

50.  FCM studies are done in those cases ofAML
where the blast do not show Auer rods and are
negative for MPO and NSE.( Mo and M7).

51. FCM Approach to Acute Leukemia
Acute leukemia.
Auer rods+ Auer rods –
AML + MPO /NSE-
AML FCM

52. First line
CD 19 ,CD 22, CD 10, CD 79a –B lymphoid.
CD 3,D2, CD7- T lymphoid.
CD117, CD13, CD33- Myeloid.
CD45, Tdt –Non lineage restricted

53. Second line.
SmIg, cytoplasmic Ig- B lymphoid
CD5 ,TC-R, CD8- T lymphoid.
CD41, CD61, Glycophorin - Myeloid

55. Low S-phase fraction in low-grade lymphomas. S-phase fraction,
an index of cellular
proliferation measured by DNA flow cytometry, of less than 5%
is typically seen in low-gradelow-grade
follicular lymphomafollicular lymphoma. (Courtesy J. Davidson.)

56. DNA content in Burkitt lymphoma. A high S-phase fraction, an
index of cellular
proliferation measured by DNA flow cytometry, of greater than
20% is typically seen in Burkitt
lymphoma. (Courtesy J. Davidson.)

57. Acute myeloid leukemia with the t(15;17)(q22;q12) (acute promyelocytic leukemia). Note the characteristic loss of
HLA-DR on the leukemic promyelocytes (this specimen does retain low-level HLA-DR in a subset of the
leukemic cells, which is relatively unusual). In addition, note the minimal expression of CD34 with the associated
expression of CD117, which is a promyelocyte-like immunophenotype.

58. Acute myeloid leukemia with the t(8;21)(q22;q22). Note the relatively low-level
expression of CD33, the aberrant coexpression of CD15 on the CD34+ blasts, and the
characteristic aberrant low-level coexpression of CD19, with aberrant CD56 on a subset of
myeloid

59. A bone marrow sample from a child with acute lymphoblastic leukaemia (ALL)
undergoing treatment .Mononucleated cells were separated on a density gradient
and labelled with three antibodies associated with the phenotype of the leukaemia -

61. Hairy cell leukemia. Hairy cell leukemia has a very distinctive immunophenotype, which enables the
flow cytometric identification of even very small populations of these cells (<0.01% in some cases).
This case shows the characteristic increase in side scatter (corresponding to the increased
cytoplasm in the cells) and expression of restricted surface light chains, bright CD19, CD20, CD22,
and CD11c, and aberrant CD103 and CD25.

62. Plasma cell myeloma. This case shows the characteristic bright CD38 expression of
plasma cells (arrows), with multiple abnormalities including loss of CD45 and CD19 and aberrant
coexpression of CD56 and CD33. This case also demonstrated cytoplasmic light-chain restriction
(not shown).

64. Case
5 year old boy with fever – 1 month

66. Diagnosis
CALLA- ALL
FL, BL, DLBCL,
Hematogones

68.  Based on the clinical information provided by
the clinician specific immunophenotyping
dhould be performed.
 CD45 gating should be done as a routine in
case of leukemia.
 Most diagnostic markers for CLPD are CD 19,
CD5, CD23, CD10.
 FCM is not required for CML unless the patient
is progressing to accelerated or blast phase.

69.  Thumb rule is that weak antigens should
always be probed with a strong florochrome
 Flow cytometry integrates electronics, fluidics,
computer, optics, software, and laser technologies in
a single platform.

70. By – Dr A.Shukla