flow cytometry

1. Topic : Flow Cytometry
Presenter – Dr Shweta Khinder

2. •Flow cytometry is a standard laser-based technology that is used
in the detection and measurement of physical and chemical
characteristics of cells or particles in a heterogeneous fluid
mixture.
•The properties that can be measured by this process
include a particle’s size, granularity or internal complexity,
and fluorescence intensity.
•These characteristics are determined using an optical-to-
electronic coupling system that detects the cells based on
laser scattered by the cells.

3. The basic principle of flow cytometry is based on
the measurement of light scattered by particles,
and the fluorescence observed when these
particles are passed in a stream through a laser
beam.

5. Light Scattering
•Light scattering results when a particle deflects incident laser light.
The extent to which this happens depends on the physical
properties of a particle, namely its size and internal complexity.
•Forward-scattered light (FSC) is proportional to the cell-surface
area or size of the cell. It is a measurement of mostly diffracted light
and detects rays that are just off the axis of the incident laser beam
dispersed in the forward direction by a photodiode.

6. • Side-scattered light (SSC) indicates the cell granularity or
internal complexity of the cells. SSC is a measurement of
mostly refracted and reflected light that occurs at any interface
within the cell where there is a change in the refractive index.
•The measurements of FSC and SSC are used for the differentiation
of cell types in a heterogeneous cell population.

7. Fluorescence
•Fluorescent markers used to detect the expression of cellular
molecules such as proteins or nucleic acids in a system.
•The fluorescent compound absorbs light energy over a range of
wavelengths that is characteristic of that compound.
•This absorption of light causes an electron in the fluorescent
compound to be raised to a higher energy level.
•The excited electron quickly decays to its ground state, emitting the
excess energy in the form of fluorescence which is then collected by
detectors.

8. •In a mixed population of cells, different fluorochromes can be
used to distinguish separate subpopulations.
•The fluorescence pattern of each subpopulation, combined with
FSC and SSC data, can be used to identify which cells are
present in a sample and to count their relative percentages.
•The electronics system then converts the detected light signals
into electronic signals that can be processed by the computer.

9. A flow cytometer is made up of three main systems: fluidics,
optics system, and electronics system.
Fluidics
•The purpose of the fluidics system is to transport particles in a fluid
stream to the laser beam. To accomplish this, the sample is injected
into a stream of sheath fluid (usually a buffered saline solution) within
the flow chamber.
•The design of the flow chamber allows the sample core to be focused
in the center of the sheath fluid where the laser beam then interacts
with the particles.
•Focusing is achieved by injecting the sample suspension into the
center of a sheath liquid stream. The flow of the sheath fluid moves the
particles and restricts them to the center of the sample core.

10. Optics System
•The collection optics consist of a collection lens to collect light
emitted after the particle interacts with the laser beam and a system
of optical mirrors that divert the specified wavelengths of the
collected light to designated optical detectors.
•After a cell or particle passes through the laser light, the rays
emitted on the side and the fluorescence signals are directed to the
photomultiplier tubes (PMTs), and a photodiode collects the signals.
•To achieve the specificity of a detector for a particular fluorescent
dye, a filter is placed in front of the tubes, which allows only a narrow
range of wavelengths to reach the detector.

11. Electronics system
•The electronic system converts the signals from the detectors into
digital signals that can be read by a computer.
•Once the light signals strike one side of the photodiode, they are
converted into a relative number of electrons that are multiplied to
create a more significant electrical current.
•The electrical current moves to the amplifier and is converted to a
voltage pulse.
•The highest point of the pulse is achieved when the particle
strikes the center of the beam, in which case the maximum
amount of scatter or fluorescence is achieved.
•The Analog-to-Digital Converter (ADC) then converts the pulse to
a digital number.

12. Procedure/Steps of Flow Cytometry
The process of flow cytometry consists of the following:
Sample Preparation
•Before running in the flow cytometers, the cells under analysis
must be in a single-cell suspension.
•It is then followed by mechanical filtration should to avoid
unwanted instrument clogs and obtain higher quality flow data.
•The resulting cells are then incubated in test tubes or microtiter
plates with unlabeled or fluorescently conjugated antibodies and
analyzed through the flow cytometer machine.

13. Antibody Staining
•Once the sample is prepared, the cells are coated with
fluorochrome-conjugated antibodies specific for the surface
markers present on different cells. This can be done either by
direct, indirect, or intracellular staining.
•Indirect staining, cells are incubated with an antibody directly
conjugated to a fluorophore.
•In indirect staining, the fluorophore-conjugated secondary
antibody detects the primary antibody

14. Running Samples
•At first, control samples are run to adjust the voltages in the
detectors.
•The flow rates in the cytometer are set and the sample is run.
•The intracellular staining procedure allows direct
measurement of antigens presents inside the cell cytoplasm or
nucleus. For this, the cells are first made permeable and then
are stained with antibodies in the permeabilization buffer.

15. Types of Flow Cytometry
1.Traditional flow cytometers
•The traditional cytometers are the common cytometer using sheath
fluid for focusing the sample stream.
2.Acoustic Focusing Cytometers
•In these cytometers, ultrasonic waves are used to focus the cells for
analysis.
•This prevents sample clogging and also allows higher sample inputs.
3.Cell sorters
•Cell sorters are a category of traditional flow cytometers which allows
the user to collect samples after processing.

16. 4.Imaging flow cytometer
•Imaging cytometers are traditional cytometers combined
with fluorescence microscopy.
•Imaging cytometer allows for rapid analysis of a sample
for morphology and multi-parameter fluorescence at both a
single cell and population level.

17. Applications/Uses
Flow Cytometry is used in several fields including molecular
biology, pathology, immunology, virology, plant biology, and marine
biology. Some of the common application include:
•It is used in clinical labs for the detection of malignancy in bodily
fluids like leukemia.
•Cytometers like cell sorters can be used to separate the cells of
interest in separate collection tubes physically.

18. •It can be used for the detection of the content of DNA by using
fluorescent markers.
•Flow cytometers allow the analysis of replication cells by using
fluorescent dye for four different stages of the cell cycle.
•Acoustic flow cytometers are used in the study of multi-drug
resistant bacteria in the blood and other samples.
•The different stages of cell death, apoptosis, and necrosis can be
detected by flow cytometers based on the differences in the
morphological and biochemical changes.

19. Limitations
•This process doesn’t provide information on the intracellular
location or distribution of proteins.
•Over time, debris is aggregated, which might result in false
results.
•The pre-treatment associated with sample preparation and
staining is a time-consuming process.
•Flow cytometry is an expensive process that requires highly
qualified technicians.

22. Sample Preparation and Processing
1. Types of samples: Peripheral blood , bone marrow aspirates, body
fluids, lymph node aspirates and single cell suspensions from solid tissue
specimens.
2. Anticoagulants: EDTA used for blood and BM aspirates. Samples remain
stable for 24 hrs only and need to process immediately. Heparinized
specimens remain stable for upto 48 hours. Sample collected in acid
citrate dextrose (ACD) remain viable upto 72 hours.
3. Storage: ideally should be processed within 48 hours. Duration of
storage depends upon type of anticoagulant used. Delay in processing
can cause loss of cell viability. Specimen stored at room temp 18- 22
degree Celsius in leak proof container.

23. 3. Specimen Integrity : factor affects viability of cells are time lapse
between sample collection and delivery to lab, environmental conditions
during transport of sample to laboratory.
Grossly haemolysed samples, exposure to extremes of temperature,
presence of blood clots and samples with less than 75% cell viability are
unsatisfactory for analysis.
4. Sample Processing : volume of sample used is 0.5- 1 ml of blood or BM
aspirate .ideal conc. of nucleated cells in each tube should be 0.5-2
million cells/ml.

24. 1. Lysis of Red cells: lysing reagent like water, ammonium chloride and
hypotonic buffer is used. Red cell lysis can be performed before or
after antibody labelling.
2. Removal of lysed red cells: washing with an isotonic fluid like
phosphate buffered saline are required before staining with
antibodies to remove red blood cells as these can interfere with
immunophenotypic analysis.
3. Staining with Antibodies: Conjugation with monoclonal Ab’s can be
done before or after red cell lysis. Two types of cell antigens: cell
surface antigens eg CD19, 10, 13, 33, 117, 38 etc. and intracellular
antigens eg Tdt, cytoplasmic light chains, cyCD3, cyCD22, MPO, bcl-2
etc.Ab ‘s can be used for surface, cytoplasmic & nuclear staining.
Sample processing involves 3 major steps :

25. 4. Intracytoplasmic markers:
In AML, MPO has highest senstivity & specificity for myeloid lineage.
B- ALL – CD 19 detects B CELLS
T-ALL- CD 3 detects T cell lineage

26. 5. Membrane Permeabilization: if intracellular antigens has to be studied,
the cell membrane has to be permeabilized prior to antibody labelling.
Low concentration non ionic detergents like saponin used as
permeabilizing agents. Cases where surface & intracellular antigens need
to study simultaneously , surface markers are preferably stained first
followed by cell fixation, membrane permeabilization and then
intercellular staining .
6. Negative Controls: for each sample to analysed, one isotype control
tube is recommended. The cells are mixed with Prescence of an isotypic
control. Negative control determines the level of non specific binding &
autofluorescence.

27. Antibody Panel Selection:
One step analysis where in a large comprehensive panel of all necessary
antibodies is included initially. The number of antibodies used in panel is
enough to allow a complete characterization of the atypical cells,
including aberrant expression of surface markers.
Two step analysis
Primary panel: detects the lineage and maturity level of the neoplastic
population.
Secondary panel: uses additional antibodies to further sub-characterize
the cells.It is cost effective

28. Recommendations for flow
cytometric testing, Bethesda, 2006
Based on the clinical presentation
Cytopenias, especially bicytopenia and pancytopenia;
Elevated leukocyte count, including lymphocytosis, monocytosis, and
eosinophilia;
Presence of atypical cells or blasts in the peripheral blood, bone marrow,
or body fluids;
Plasmacytosis or monoclonal gammopathy;
Organomegaly and tissue masses

29. Advantages:
Staging a previously diagnosed hematolymphoid neoplasm
Monitoring response to treatment including detection of minimal
residual disease (MRD)
Documenting relapse or progression
Diagnosing an intercurrent hematologic malignancy, such as a therapy-
related myelodysplastic syndrome (MDS).

30. Antigens with broad expression
Pan-myeloid CD13,CD33, CD64, MPO
Pan-B-cell cyCD22, CD19, CD24, cyCD79α−β
Pan-T-cell cyCD3, CD5, CD7
Antigens associated with immaturity
Pan-lymphoid and myeloid Tdt, CD34,CD133, CD135
Pan-myeloid CD117
Antigens with lineage-specific and maturation-dependent expression
Myeloid cells CD14, CD15, CD65, Lactoferrin
Erythroid cells Glycophorin A
Platelets CD41a, CD61
B-cells CD20, cylgμ, slg, κ−λ
T-cells CD1a, CD2, sCD3, CD4, CD8
NK-cells CD16, CD56

31. Acute myelogenous leukemia: Immunophenotyping is required in following
conditions:
1.To confirm morphological findings in case of AML with Auer rods, acute
monocytic leukemia having more than 80% monocytic cells and hypergranular
promyelocytic leukemia.
2. Diagnosis of sub types AML with minimal differentiation, without maturation,
acute monoblastic leukemia and variety of AML’s.
3. Detection of Prescence of leukemia cells with aberrant phenotype .
4. To rule out presence of Blasts belonging to lymphoid lineage.
MARKERS: CD34, CD 117, CD 33, CD 13, CD 15, CD 4, CD 11b ,HLA DR&
cytoplasmic myeloperoxidase.

34. • AML with minimal myeloid differentiation : the cells are agranular &
show low FS and SS. Strongly express CD 34, HLA DR and CD 117 & CD
38 POSITIVE.
• AML without maturation: CD 45 +, HLA DR +, high proportion of them
are CD 34+ and CD 117 +. Antigens associated with mature myeloid
stages that is CD 15, CD 11b, CD 16 are not expressed. Minimal 3% blast
express MPO.
• AML with granular differentiation: strong SS and CD 45 moderately
positive whereas HLA DR is NEGATIVE.CD 13,CD 33, CD 15, CD 11b
expression are +ve . Intracellular myeloperoxidase is strong.
• Acute Myelomonocytic leukemia: Immature cell markers are negative.
Express CD 33, CD 13, CD 11 b, CD 4 and HLA DR ALSO CD 14, CD
64(Monocytic Marker).
• Megakaryocytic leukemia: CD 41, CD 61 +VE

35. Positive markers: CD 13, 33, HLA DR, CD 14, CD 64, Anti MPO
Negative markers: CD 2, 3, 7, 19, 20, 22, 79a, 117
Acute myelomonocytic leukemia

36. • low orthogonal (side) light scatter
• Express markers of immaturity such as CD34 and
CD117
• Lack markers of maturation such as CD11b, CD15,
and CD16
Myeloblasts
• Express CD34 and TdT,
• Lack surface immunoglobulin and CD20.
Immature
B-lymphoid cells
• Express CD 34, TdT or CD 1a
• Lack surface expression of CD 3.
Immature
T lymphoid cells

37. ALL AML
B-lineage T-lineage
First Line CD19,CD22, CD79a,
CD10
CD7, CD2, cyCD3 CD13, CD33, CD117, anti-MPO
TdT, HLA-DR, CD34
Second Line cymu, Smlg(surface
membrane immunoglobin)
CD1a, CD5, CD8, anti-
TCR( T cell receptor)
CD41, CD42, CD61,
anti-glycophorin A
Panel of monoclonal Abs for the
Diagnosis of Acute leukemia

39. Panel of monoclonal Abs for the diagnosis of
lymphoid disorders
B-Cell T-Cell
First Line SmIg (kappa /lambda)
CD19, CD23, FMC7
mCD79b, mCD22, CD5
CD2, CD5,
Second
Line
CD11c, CD25, CD103
CD123, CD38, CD138, CyIg
CD3, CD4, CD7, CD8
HAIRY CELL LEUKAEMIA
CD11c +
CD25+
CD103+
CD123+

40. FLOW CYTOMETRY EVALUATION OF B CELLS CD 19 + IN CLPD

41. CLL/SLL: CD 5 + , CD 23+
Follicular lymphoma: CD 10 + along with CD 19 & CD 20
Hairy cell leukemia: CD 103 + in association with CD 25,
CD 22, CD 11c, and CD 123.
Splenic lymphoma with villous lymphocytes: CD 11c and
CD 79 b + in 50% cases.
Mantle cell lymphoma:CD5+, CD 22+, cyclin D 1+
DLBCL: CD 10+ along with CD19+, CD 20+. CD 30 may be
expressed in anaplastic variant . Ki 67 is very high (90)%
Burkitt lymphoma: CD 10+, BCL 6, CD 77+, CD 43+ . As
well as express CD10 without bcl 2 , in contrast to
follicular lymphoma.

42. Mature T cell neoplasm
• Diagnosis of T cell neoplasm by
Flow cytometry is more difficult
as there is no sensitive marker for
clonality
• .CD 7 is frequently missing and
lack of CD 2, CD 5, & CD 3 are also
uncommon .
• These neoplasm has been
classified on the basis of
expression of CD 4 & CD 8.
• Paradoxically absence of T cell
antigen is helpful in diagnosing T
cell neoplasm.

43. Flowcytometry in Myelodysplastic syndrome
1.A combination of morphology, to detect multilineage dysplasia in the
bone marrow and peripheral blood, and cytogenetics, to detect
characteristic clonal abnormalities are used in establishing a diagnosis
of MDS
2. Morphology may be difficult to evaluate in some patients, either due
to hypocellularity or fibrosis of the marrow
Differentiation of MDS from aplastic anemia
Classification and prognosis
FLOW CYT0METRY IN MDS

44. • Hypogranularity of neutrophils - decreased
side scatter
• Abnormal levels of expression of antigens –
CD45, CD15, CD33,
• Expression of CD34, HLA-DR
• Aberrant expression of antigens – CD7, CD2,
CD5, CD19
• Overexpression of CD56
• An altered maturation pattern is best
demonstrated by plotting CD13 vs CD16 and
CD11b vs CD16
Immunophenotypic evaluation of myeloid
dysplasia

45. Myeloid abnormalities in MDS
demonstrated by CD45 versus
side light scatter.
(A) Healthy donor bone marrow:
normal granulocytes and
precursors in the boxed region.
(B) MDS patient bone marrow
(ungated): hypogranular
neutrophils with low side scatter
(arrow) and a discrete blast
population (oval) are
demonstrated.
(C) MDS patient bone marrow
(ungated): hypogranular
neutrophils with low side scatter
(arrow) and a discrete blast
population (oval) are
demonstrated.

46. Abnormal levels of CD71
expression;
Dissynchronous expression
of
 CD45 versus CD71;
 CD45 versus glycophorin A;
 CD71 versus glycophorin A
on nucleated red cell
precursors.
Immunophenotypic evaluation
of erythroid dysplasia

47. FCM in Platelet disorders
• Role of thrombopoiesis : analysis of platelet ribonucleic acid is
done by flow cytometry using thiazole dye.
• Measurement of reticulated platelets detected is an estimate of
rate of thrombopoiesis.
• Diagnosis of platelet ass immunoglobulins eg antiplatelets
antibodies detection in ITP and Evans syndrome.
• Diagnosis of inherited & acquired platelet disorders eg Bernard
Soulier syndrome , Glanzmann thrombasthenia .
• Detects lack of GP Ib/IX and GP IIb/IIIa on cell surface
respectively using three antibodies CD 41,CD 61 & CD 42b .

48. FCM in PNH
• Rare acquired hematological disorder characterized by
anemia, intravascular hemolysis, bone marrow hypoplasia,
as well as tendency to thrombosis and infection
• Used to detect the deficiency of GPI anchored proteins on
RBC & WBC (neutrophils).
• Increased sensitivity of erythrocytes to complement-
mediated cell lysis due to deficiency of membrane-bound
GPI anchored proteins which normally function as the
inhibitors of reactive hemolysis. GPI-anchored proteins
include: complement regulatory proteins like DAF (decay
accelerating factor, CD55), MIRL (membrane inhibitor of
reactive lysis, CD59).

49. Uniform and full expression of CD55
and CD59 antigens on all cells

50. Scattergram A shows partial loss of CD59
expression (type II )

51. Absence of CD55 and CD59 expression ( type
III )

52. During remission induction Measurement of early response to treatment
End of remission induction,
early continuation
Identification of patients at a higher risk of relapse
Throughout treatment Detection of impending relapse
Throughout treatment Identification of leukemic cells in extramedullary sites (e.g., central
nervous system
Before autologous stem cell graft Detection of contaminating leukemic cells
Before autologous stem cell graft Evaluation of the efficacy of ‘purging’’ techniques
Role of flow cytometry in detection of Minimal residual disease

53. Reticulocyte enumeration
• The flow cytometric enumeration of reticulocytes uses
fluorescent dyes that bind the residual RNA, such as
thiazole orange
• This method provides excellent discrimination between
reticulocytes and mature RBCs, with greater precision,
sensitivity, and reproducibility than the traditional
method
• The fluorescence intensity is directly proportional to
the amount of RNA and related to the immaturity of
the RBC, a reticulocyte maturity index can be given

54. • The use of flow cytometry for the detection of fetal cells is much
more objective, reproducible, and sensitive.
• Fluorescently labeled antibodies to the rhesus (D) antigen can be
used, or more recently, antibodies directed against hemoglobin F
• This method has the ability to distinguish fetal cells from F-cells
(adult red cells with small amounts of hemoglobin F).
• This intracellular approach, which uses permeabilization of the
red cell membrane and an antibody to the g chain of human
hemoglobin, is precise and sensitive
Fetomaternal Haemorrhage

55. Indications of Flow Cytometry
• Acute Leukemia – determine myeloid or lymphoid lineage
, for subtyping of AML, Immunological classification of
ALL, diagnose variants of Acute leukaemia's, detection of
MRD following chemotherapy & detection of prognostic
markers.
• Chronic lymphoproliferative Disorders- distinguish B / T
cell origin, determine subtypes, identify non Hodgkin’s
lymphoma in blood & BM, identify NK cell neoplasm ,
detect prognostic and potential therapeutic markers.

56. • MDS - Blast cells, abnormal antigen expression ,
identifying cells in PNH.
• Plasma cell Dyscrasia – BM analysis for monoclonal
gammopathies, plasmacytosis
• Red cell disorders- PNH , F cell estimation, Hereditary
spherocytosis, reticulocyte estimation.
• Platelet disorders- Primary thrombocytopenic,
Glanzmann thrombasthenia, Bernard Soulier syndrome,
Von Willebrand’s disease, storage pool defects.

57. THANK YOU