Flow cytometry is a technique that uses lasers to measure physical and chemical properties of cells as they flow in a fluid stream. A flow cytometer consists of three main systems: fluidics to transport cells to the laser, optics including lasers and detectors, and electronics to convert light signals to electronic data. Cells passing through the laser beam scatter and fluoresce light which is detected and used to measure properties like size, granularity, and presence of fluorescent markers. Flow cytometry has many applications in diagnostics including immunophenotyping of leukemias and lymphomas, detecting minimal residual disease, stem cell enumeration, and detecting autoantibodies.

1. FLOW CYTOMETRY IN
DIAGNOSTICS
PRESENTED BY-
KABERI NATH
ROLL NO- 17PBT206

2. INTRODUCTION
 ‘Flow Cytometry’ as the name suggests is a technique for cell counting and
measurement of different properties of the cell (‘cyto’= cell;
‘metry’=count/measurement).
 It is a laser based technology that measures and analyses different physical
and chemical properties of the cells/particles flowing in a stream of fluid
through a beam of light.
 An optical-to-electronic coupling system is used to record the way in
which the particle emits fluorescence and scatters incident light from the
laser.
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3. COMPONENTS OF A FLOW CYTOMETER
A flow cytometer is made up of three main systems: fluidics, optics, and
electronics.
 The fluidics system transports particles in a stream to the laser beam for
interrogation.
 The 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.
 The electronics system converts the detected light signals into electronic
signals that can be processed by the computer. For some instruments equipped
with a sorting feature, the electronics system is also capable of initiating sorting
decisions to charge and deflect particles.
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4. WORKING OF A FLOW CYTOMETER
 In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any
suspended particle or cell from 0.2–150 micrometres in size is suitable for analysis.
 The portion of the fluid stream where particles are located is called the sample core.
 When particles pass through the laser intercept, they scatter laser light. Any fluorescent
molecules present on the particle fluoresce.
 The scattered and fluorescent light is collected by appropriately positioned lenses.
 A combination of beam splitters and filters steers the scattered and fluorescent light to the
appropriate detectors.
 The detectors produce electronic signals proportional to the optical signals striking them.
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FLOW CYTOMETER DESIGN

6. CONCEPT OF SCATTERING
 Physical properties, such as size (represented by forward angle light scatter) and internal
complexity (represented by right-angle scatter) can resolve certain cell populations.
 FSC collects light at 180° from the point at which the laser beam intersects the cells, usually
on a linear scale. It is correlated with cell size, and thus can distinguish normal lymphocytes
(small), monocytes (intermediate), and neoplastic cells (generally they are large in size).
 SSC collects right-angle light at 90° and is correlated with cytoplasmic granularity and
nuclear configuration.
 The combination of both FSC and SSC can distinguish normal lymphocytes, granulocytes,
and monocytes.
 The detection of lymphocytes and monocytes provides a reliable internal control to evaluate
the size of the cells of interest.
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7. FLOW CYTOMETRY ANALYSIS
ANTIBODY
 Highly specific monoclonal antibodies are used that are produced by cloned antibody secreting
cells.
 Antibodies are based on cluster of differentiation (CD)- a protocol used for identification and
distinction of cell surface antigens.
 Using CD system we can identify cells by the presence or absence of particular surface
markers for e.g. CD3+ or CD20+ etc.
 Antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or
inside cells.
 When labelled cells are passed by a light source, the fluorescent molecules are excited to a
higher energy state. Upon returning to their resting states, the fluorochromes emit light energy
at higher wavelengths.
 The use of multiple fluorochromes, each with similar excitation wavelengths and different
emission wavelengths (or “colours”), allows several cell properties to be measured
simultaneously.
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8. 8
FLUOROCHROMES
 Fluorochromes are substances that can be excited by certain light source (such as laser) and emit
a fluorescent signal at a single wavelength.
 Fluorescent dyes can directly bind to certain cellular content, such as DNA and RNA, and allow
us to perform quantitative analysis on individual cells.
 However, in most cases fluorochromes are conjugated with monoclonal antibodies, which
specifically target cellular antigens/markers.
 E.g.-Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), PE-Texas Red, Peridininchlorophyl
protein(PerCP).
 Interestingly, although some of them can be excited by the same light source, the different
fluorochromes may emit fluorescent signals with different wavelengths/colours. Thus, multiple
fluorochromes can be simultaneously excited by a light source and detected by their emission
fluorescent signals with different wavelengths, respectively.

9. APPLICATIONS
 Flow cytometry measures multiple characteristics of individual
particles flowing in single file in a stream of fluid.
 Light scattering at different angles can distinguish differences in
size and internal complexity, whereas light emitted from
fluorescently labelled antibodies can identify a wide array of cell
surface and cytoplasmic antigens.
 This approach makes flow cytometry a powerful tool for detailed
analysis of complex populations in a short period of time.
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10. ANALYSIS OF LEUKAEMIAS AND
LYMPHOMAS
 Immunophenotyping by flow cytometry is an important tool in the diagnosis and staging of patients
with a haematological neoplasm. It is used in conjunction with classical morphology.
 In the bone marrow, normal blood cells develop from stem cells in a progressive series of
differentiations, branching off to give different lineages of cells.
 Malignancies can arise at different stages in the development of a cell. A leukaemia or lymphoma will
express a specific set of markers depending on the stage and pathway of differentiation and they are
classified accordingly.
 The initial evaluation is made with a panel of antibodies usually using them in three or more colour
combinations. The basic markers include:
B cell: CD5, CD10, CD19, CD20, CD45, Kappa, Lambda;
T cell: CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD56;
Myelomonocytic: CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD45, CD56, CD117, HLA-DR;
Plasma cell: CD19, CD38, CD45, CD56, CD138.
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11. DETECTION OF MINIMAL RESIDUAL DISEASE
 Minimal residual disease (MRD) was defined as disease beyond the limit of
morphological detection using conventional microscopy.
 Patients with acute leukaemia were considered to be in remission when bone
marrow samples contained <5% neoplastic cells.
 Flow cytometric methods can detect far lower levels of disease, which can be
important in the clinical management of leukaemia.
 The residual tumour cells are detected using immunofluorescence of surface
markers.
 A panel of at least three antibodies is used, the antibodies being selected on the
basis of the immunophenotype of the original leukaemia.
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12. STEM CELL ENUMERATION
 Haematopoietic stem cells in the bone marrow can be identified by their expression
of CD34.
 Normally, the number of such cells in bone marrow is low and is negligible in
peripheral blood.
 Autologous transplantation of circulating haematopoietic progenitors cells revealed
considerable clinical advantages in comparison with bone marrow derived
haematopoietic progenitors.
 These cells can be used to repopulate a depleted bone marrow after, for example,
high dose chemotherapy.
 Using immunofluorescence, progenitors cells can be distinguished by flow
cytometry in less than 1 h based on the expression of surface membrane molecules.
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13. SOLID ORGAN TRANSPLANTATION
T cell cross-match
 Flow cytometry can be used to crossmatch a
recipient's serum with donor lymphocytes to
detect antibodies that could interfere with
engraftment.
 Prior to organ transplantation, the organ
donor's lymphocytes are incubated with
serum from the potential recipient of the graft.
 After washing, bound immunoglobulins are
detected using an FITC-conjugated anti-
human IgG antibody. The T cells are
identified using a PE-CD3 conjugate.
Post-operative monitoring
 After the organ transplant, analysis of the
peripheral blood lymphocytes may help to
indicate early rejection and bone marrow
toxicity during immunosuppressive therapies,
and to help in the differentiation of infections
from transplant rejection.
 A variety of cell surface markers and
activation antigens can be used depending on
the clinical condition and the organ
transplanted.
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14. DETECTION OF AUTOANTIBODIES
 Autoantibodies to leucocytes, platelets and erythrocytes may be found in a
variety of autoimmune conditions and can cause anaemia, leukopenia, or
thrombocytopenia.
 They are detected by immunofluorescence in either a direct or an indirect
assay.
 In the former, anti-human Ig are used to detect Ig on the surface of the
patient’s cells.
 In the indirect assay, the reaction of antibodies in the patient’s serum with
cells from a normal person is observed.
 The procedures are similar to those used for a T cell crossmatch.
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15. HIV INFECTION
 Determination of the numbers of CD4 +ve lymphocytes in the peripheral
blood is used to monitor patients with HIV infections.
 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.
 The absolute number of CD4 +ve cells is the clinically relevant parameter
for a discussion on counting cells.
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16. FOETO-MATERNAL HAEMORRHAGE
 Foeto-maternal bleeding can sensitise a Rhesus blood group D-ve mother to D+ve blood
cells from the foetus.
 In a subsequent pregnancy, haemolytic disease of the new born child can be caused by the
destruction of Rhesus D +ve blood cells of the foetus by maternal anti-D antibodies.
 Prophylactic anti-D given to the Rhesus D -ve mother shortly after delivery of a Rhesus D
+ve child significantly reduces the incidence of anti-D sensitisation in the mother and has
led to the virtual elimination of the disease from mothers so treated.
 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.
 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.
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17. IMMUNODEFICIENCY DISEASES
Diseases resulting from primary immunodeficiencies, which are usually found in infants and
young children, can be a result of defects in T-cells, B-cells, granulocytes or monocytes.
1. X-linked agammaglobulinemia-
 Decreased serum immunoglobulins of all isotypes and decreased to absent peripheral B cells.
 As B cells are significantly reduced or absent in XLA, flow analysis focuses on monocytes
and/or platelets.
2. LRBA deficiency(lipopolysaccharide-responsive beige-like anchor)-
 Reduced immunoglobulins affecting at least two isotypes, manifest with recurrent infections,
severe pulmonary and gastrointestinal complications.
 Caused by LRBA gene mutations, assessment of LRBA protein T cells, B cells, monocytes, and
NK cells provides a rapid and easy way of determining LRBA deficiency.
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3. X-linked hyper-IgM syndrome-
 Severely impaired production of class-switched immunoglobulins with normal or elevated levels
of IgM due to impaired class switch recombination.
 Caused by mutations in the CD40LG gene, encoding the CD40 ligand protein, a member of the
tumour necrosis factor (TNF) family, expressed on activated CD4+ T cells.
 Flow cytometric analysis is performed to examine expression of CD40 ligand protein.
4. DOCK8 deficiency (dedicator of cytokinesis 8)-
 Significant eosinophilia, elevated serum IgE levels, reduced T and B cells, decreased serum IgM
levels, variably impaired IgG functional antibody responses with reduced in vitro proliferation of
activated CD8+ T cells.
 The flow cytometric assay for DOCK8 is able to identify all patients with a germ line genetic
mutation in DOCK8, making it a reliable and quick diagnostic test.
 Genetic testing is complicated by the large size of the gene.

19. PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA
 Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired disease characterised
by the development of an abnormal clone of precursor cells in the bone marrow.
 The white cells and red cells produced are dysfunctional and are susceptible to
lysis.
 Conventional laboratory tests for the diagnosis of PNH include the sugar water test
and the Ham’s acid haemolysis test. Problems associated with these tests include
stringent specimen requirements and limited specificity.
 Analysis of this clonal abnormality by flow cytometry, in general, can be
accomplished by analysis of CD55 and CD59 on red cells specific for decay-
accelerating factor and membrane-inhibitor of reactive lysis, respectively.
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20. CONTAMINATING LEUKOCYTES
The presence of contaminating leukocytes in transfused products of blood
may cause a number of adverse effects. These include:
 Non-haemolytic febrile reactions
 Graft-versus-host disease
 Cytomegalovirus transmission
 Pulmonary oedema
 Alloimmunization to HLA class I antigens
Flow cytometry offers a sensitive measure of the number of remaining
leucocytes in leucofiltered products, such as plasma and red blood cells.
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21. PLATELET COUNTING AND FUNCTION21
 Flow cytometry can be used to count platelets and also to measure their surface proteins.
 The surface protein change during activation of the platelets can be used to measure their activation
state.
 Platelets are easily activated and blood has to be taken and handled with care; for this reason, whole
blood methods are generally preferred.
 Platelet analysis using thiazol orange by flow cytometry can have application in
Identification of inherited disorders
Monitoring of anti-platelet therapy
Monitoring clinical course of disease
Monitoring platelet production in thrombocytopenia
Identification of patients at risk of thrombosis
Accurate platelet counting in thrombocytopenia
Diagnosis of heparin induced thrombocytopenia

22. THE BASOPHIL ACTIVATION TEST
 Flow cytometry can be a reliable tool for monitoring basophil activation
upon allergen challenge by detecting surface expression of
degranulation/activation markers (CD63 or CD203c).
 The basophil activation test has become a standardized tool for in
vitro diagnosis of immediate allergy.
 It is also suitable for pharmacological studies on non-purified human
basophils.
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23. DNAANALYSIS
 Dyes are available such as propidium iodide that bind DNA in a proportional and linear
fashion.
 This allows the quantitation of DNA content, enabling the identification of normal diploid cells
at rest, those that are actively synthesizing DNA, and those that are either pre-mitotic or
actually in mitosis.
 Once these phases of the cell cycle have been identified, antibody detection of cell cycle-related
proteins can be combined with DNA content determination to relate protein expression with
stages of the cell cycle.
APOPTOSIS DETECTION
 Apoptotic cells have many characteristics that can be measured by flow cytometry like cell
plasma membrane changes, changes in plasma membrane permeability, changes in
mitochondrial membrane permeability, caspase activation, and DNA cleavage.
 Determination of any one or a combination of these changes by flow cytometry allows the
identification and quantification of apoptotic cells in a mixed population.
 It can also give valuable information about the molecular pathways that cells take during cell
death.
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24. ADVANTAGES
 Fast technique.
 Allows a great deal of information to be
collected.
 With the help of computerization a large
population of cells can be studied which can
provide statistically significant information.
 Flow cytometry allows cell by cell
fluorescence study.
 The development of fluorescent DNA
probes should permit analysis at
chromosome level.
LIMITATIONS
 Since cells are used in suspension,
information concerning architecture is lost, a
particular problem when determining the
phenotype of lymphoid organs.
 Results depend on specimen collection.
 The quality of the cell labelling is affected by
the viability of cells.
 Heterogeneous populations may contain a
more fragile subpopulation which might be
damaged during cell labelling, leading to
false interpretation.
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25. RECENT DEVELOPMENTS IN FLOW
CYTOMETRY
 One of the most important progresses in flow cytometry technology is the discovery of
Quantum dots (QDs) that provide a great opportunity for highly multiplexed experiments and
improved resolution. QDs do not have an absorption spectrum unlike FITC and PE.
 Instead, they have a wide absorption spectrum and so can be excited by a range of different
wavelengths.
 They are also extremely photostable at wavelengths greater than 300nm and their different
colors can be excited simultaneously by the same source.
 Antibody conjugated QDs are suitable to stain both surface antigens and intracellular antigens.
 Spectral Flow Cytometers - high-resolution measurements of the cell and other particles
 Microfabricated Flow Cytometers-use microfluidics. used to analyze single cells in a small
population, cellular differences in gene expression or response to a drug within a population of
cells.
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26. FLOW CYTOMETRY MARKET ANALYSIS
(Flow Cytometry Market Analysis By Technology (Cell-Based, Bead-Based), By Application (Research, Industrial, Clinical),
By End-use (Commercials, Hospitals, Academics, Clinical Labs), By Product & Service, & Segment Forecasts, 2014 - 2025
Published: February 2017 )
 The global flow cytometry market size was valued at USD 3.27 billion in 2016 and is expected to
grow at a CAGR of 10.6% over the forecast period.
 Key drivers include rising prevalence of chronic diseases, which needs toxicity testing such as
cancer tests and demand for rapid, accurate, & sensitive prognosis techniques for disease validation.
 Growing awareness level amongst patients and healthcare professionals and high level of healthcare
expenditure are high-rendering drivers for flow cytometry industry growth.
 associated advantages of cell-based assays such as ease-of-use, high sensitivity & reproductively,
and advancement in reagents & software used for the analysis are other factors expected to drive the
growth.
 Increasing demand for early diagnosis and rising awareness pertaining to associated benefits with
cell-based assays are the factors attributing to its dominance over the forecast period.
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27.  The instrument segment dominated the market in 2016 owing to greater penetration coupled
with advantages such as accurate result, cost-effective nature, and user-friendliness. Reagents
and consumables, owing to their increased usage in cancer diagnostics are expected to register
lucrative growth.
 Flow cytometry has applications in research, clinical diagnosis, and industrial sectors.
Research segment dominated in 2016 owing to an increasing R&D pertaining to cancer and
other infectious diseases. Flow cytometry also has applications in various industries such as
food microbiology and plant tissue culture.
 Clinical diagnosis is expected to be the fastest growing application segment by 2025. Clinical
diagnosis market is driven by factors such as increasing demand for cost-effective disease
diagnostic tools and associated benefits of these assays in disease detection.
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28.  The commercial organization segment held the largest revenue share owing to wide
applications across various verticals such as food microbiology, blood banks, and plant cell
culture. The introduction of multiplex probes and reagents for specific applications in
diagnostics & drug discovery is expected to serve this vertical with growth opportunities by
catering to the users in research and small peripheral laboratories.
 Hospitals are expected to grow at lucrative CAGR due to increasing diagnostic tests
necessitated by rising prevalence of chronic & infectious diseases, supportive reimbursement
policies, and growing awareness amongst patients.
 Some of the key players operating in this industry are Beckman Coulter, Inc.; Becton,
Dickinson and Company; Sysmex Corporation; Agilent Technologies; Merck & Co., Inc.,
Apogee Flow Systems Ltd.; Bio-Rad Laboratories, Inc.; Thermo Fisher Scientific,
Stratedigm, Inc.; Luminex Corporation; Miltenyi Biotec; and GE Healthcare.
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29. REFERENCES
1. Qadir, M., Barcos, M., Stewart, C.C., Sait, S.N., Ford, L.A. and Baer, M.R. (2006) Routine immunophenotyping in acute leukemia: Role in lineage assignment and
reassignment. Cytometry70:329-334.
2. Craig, F.E. and Foon, K.A. (2008) Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941-3967.
3. Kern, W., Haferlach, C., Haferlach, T. and Schnittger, S. (2008) Monitoring of minimal residual disease in acute myeloid leukemia. Cancer 112:4-16.
4. Mandy, F., Janossy, G., Bergeron, M., Pilon, R. and Faucher, S. (2008) Affordable CD4 T-cell enumeration for resource-limited regions: A status report for
2008. Cytometry, 74, Suppl 1:S27-39.
5. Gratama, J.W., Orfao, A. and 14 other authors (1998) Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. Cytometry 34:128-142.
6. Bell, A., Shenton, B. and Garner, G. (1998) The flow cytometric crossmatch in solid organ transplantation. Proc. RMS 33:219-220.
7. Shanahan, T. (1997) Application of flow cytometry in transplantation medicine. Immunol. Invest.26:91-101.
8. Nance, S.J., Nelson, J.M., Arndt, P,A., Lam, H.C. and Garratty, G. (1989) Quantitation of fetal-maternal hemorrhage by flow cytometry. A simple and accurate
method. Am. J. Clin. Pathol. 91:288-92.
9. Michael Brown and Carl Wittwer(2000) Flow Cytometry: Principles and Clinical Applications in Hematology.Clinical Chemistry 46:8(B) 1221–1229
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Biotechnology ISSN: 0738-8551
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12. Radhia Boumiza, Anne Lise Debard and Guillaume Monneret “The basophil activation test by flow cytometry: recent developments in clinical studies,
standardization and emerging perspectives”. Clinical and Molecular Allergy2005
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30. THANK YOU
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