Flow cytometry is a technique that uses lasers and fluorescence to analyze physical and chemical characteristics of cells as they flow in a fluid stream. It allows simultaneous analysis of thousands of cells per second based on parameters like cell size, granularity, and detection of cell surface antigens using specific antibodies labeled with fluorochromes of different colors. Specimens suitable for analysis include blood, bone marrow, body fluids, and cell suspensions generated from tissues. Flow cytometry has various applications like immunophenotyping, DNA analysis, diagnosis of conditions like PNH, reticulated cell counting, and blood bank testing.

1. BY P.RAJGOPAL
PHARMACEUTICAL ANALYSIS
GITAM INSTITUTE OF PHARMACY
Reg No:121725201007

2. CONTENTS
• INTRODUCTION
• PRINCIPLE
• CONCEPT OF SCATTRING
• IMMUNO PHENOTYPING ANALYSIS
• ANTIBODY
• FLUOROCHROMES
• PROCESSING OF SPECIMEN FOR ANALYSIS
• APPLICATIONS OF CYTOMETRY

3. INTRODUCTION
• The concept of flow cytometry has been in existence for more
than five decades.
• Flow cytometric immunophenotyping first appeared in (FCI) first
appeared in clinical laboratories in the 1980s,in the wake of the
AIDS epidemics.
• Intially utilized to assess CD4 T-Cells,the technique was soon
applied to lymphoid and eventually myeloid and neoplasm
• Current flow cytometry have the capability of simultanously
measuring of multiple parameters of individual cells in a cell
suspension.
• Thus, a large number of cell specimens can be processed with a
quick turn around time

4. • In addition, flow cytometry is also highly sensitive and detect
immunophenotype of cells in a specimen with a thousand of
cells
The parameters analyzed by flowcytometry include
• Physical properties of cells:the size cytoplasmic granularity, and
amount of DNA contents and
• Cell antigens/markers(surface, cytoplasmic and nuclear) that
can be recognized by a specific antibodies.
By using appropriate antibody panel,flow cytometry can
be reveal
• the cell type(hematopoietic,lymphoid,or non hematopoietic)
• Cell linkage(B- and T cells natural killer cells,
myeloid,monocytic cells, neuro/neuro endocrine cells and
epithelial cells)
• Cell maturation stage( precursors vs matured cells).

5. PRINCIPLE
• Flow cytometry involves the analysis of the optical and
fluroscence
Characterstics of the single particles(eg: cells, nuclei, chromosomes,)
During their passage within narrow precisely defined liquid stream.
OR
flow cytometry is a laser- or impedance-based, biophysical
technology employed in cell counting, cell sorting, biomarker
detection and protein engineering, by suspending cells in a stream
of fluid and passing them through an electronic detection
apparatus. A flow cytometer allows simultaneous multiparametric
analysis of the physical and chemical characteristics of up to
thousands of particles per second.

6. For cell analysis the basic components of a flowcytometer includes
FLOW SYSTEM
OPTICAL
SYSTEM
ELECTRONIC
SYSTEM
COMPUTER
SYSTEM

7. Schematic diagram of flow cytometry

8. CONCEPT OF SCATTERING
• Physical properties, such as size( represented by forward
angle light scatter) and internal complexity( represented by
left- angle scatter) can resolve in certain cell populations.
•
•
•
•
•

9. • FSC collects light at 180º from the point at the point intersects the
cells, usually on a linear scale it is correlated with the cell size,
and thus can distinguish normal lympocytes (small), monocytes(
intermediate) and neoplastic cells(generally they are larger in size)
• SSC collects right angle light at 90º and is correlated with
cytoplasmic granularity and nuclear configuration
• The combination of both SSCand FSC can distinguish normal
lymphocytes , granulocytes and monocytes.
• The detection of lymphocytes and monocytes provides a reliable
internal control to evalute the size of the cell of interest.

11. IMMUNOPHENOTYPING ANALYSIS
• Requires
 Antibody
 Fluorochromes
• Antibody:
• Highly specific monoclonal antibodies are used that are
produced by cloned antibody secreting cells
• Antibody are based on the cluster differentiation (CD)- a
protocol used for differentation and distinction of cell surface
antigens.
• Using CD system we can identify cells by the presence or the
absence of particular surface marker for e.g. CD3+ or CD20-
etc...,

12. FLUOROCHROMES
• Fluorochromes are substances that can be exited by certain light
source(such as laser) and emit a fluoroscent signal at a single
wavelength.
• Fluroscent dyes can directly bind to certain cellular content, such as
DNA and DNA, and allows us to perform quantitative analysis on
individual cells.
• However, in most cases fluorochromes are conjugated with mono
clonal antibodies, which specifically target cellular antigen
/markers

13. Characterstics of fluorochromes commonly used in flow
cytometry

14. IMMUNOPHENOTYPING ANAYSIS
• Antibodies conjugated to flouroscent dyes can bind specific on cell
membrance or inside cells. When labelled cells are passed to light
source, the fluoroscent molecules are excited to higher energy state.
Upon returning to their resting states, the flurochromes emits light
light energy at higher wave lengths,the use of multiple
flurochromes,each with similar excitation wavelength and different
emission wavelength (colours), allows several properties to be
measured simulatiously.

15. Simultaneous detection of multiple cells
antigen/markers
• Multiple cell antigens(Ag)are recognised by flourochromo
conjugated specific antibodies(Ab) Because different
fluorochromes have different emission wavelength/colours, they
can be simultaneously determined detected by flow cytometer.

16. Abnormal/aberrant antigenic expression can be
grouped into four categories
• Abnormal increased or decreased level of antigenic expression
(aberrant expression)
• Gain of antigen not normally expressed in cell type
• Expression of antigens not synchornized with normal
development and maturation stage of the celltype or linkage .
• Homogenous expression of the antigen(s) by a cell poplation
that normally show more heterogenous expression.

18. Processing of specimen for flow cytometry
• Specimen suitable for flow cytometry
• Theortically, any specimen from which a single cell suspension can be
generated are suitable for flow cytometry analysis.
• However,a lack of distinct antigen / markers in the cells of the interest or
tissues limit the diagonistic valuesof flow cytometry.
• Common specimen suitables for flow cytometry include:
peripheral blood
bone marrow
body fluids
Cerebrospinal fluid
Urine
Lymph nodes( cell or fresh tissus)
Any fine needle aspiration
Fresh tissue suspension for hematopoietic and lymphoid tissues

19. SPECIMENSTORAGE
• For blood and bone marrow specimens, anticoagulants such as
EDTA, heparin, or acid citrate dextrose are needed.
Fresh tissue specimens are best transported and stored in sterile
tissue culture medium
Although specimens may be stored at room
temperature,refrigeration is prefered, particularly when there is
delay for flow cytometric analysis.
For flow cytometric analysis , single cell suspensions of the fresh
tissues can be acheived by mechanical dissolution.

20. General notes on cell preparation
• Single cell suspension are required for optimal staining of
samples for flow cytometry.
• The narrow boresof the sample injection needle and tubing on
a flow cytometry will be easily clogged by aggregated cells
and debris.
• Preparation of single cell suspensionfrom solid tissue requires
Mechanical dissociation and / or enzymatic digestion for optimal
Recovery of cells from the tissue.

21. Processing of solid tissues
• Tissue is weighed and mechanically and enzymatic disaggregated
Into a single cell suspension
• Collengenase is the most commonly used enzyme, followed by
dispase and trypsin
• Enzymatic digestion is performed in an incubator or a shaking
water bath
• Mechanical disaggeration can be accomplished with paired
scalpels or scissors.
• The process often requirescentrifugation , harvest of single cells
and redigestion of tissues fragments.
• The sample should be visually inspected at all phases of tissue
digestion

22. • In the final stage ,cell suspensions are passed through a 70 to 200
micron filter to remove aggregates
• Cell suspensions are then counted and viability is determined by a
dye exclusion assay such as trypan blue
• Sample is finally incubated with requires antibodies attached with
Fluorochrome in optimal temperature and pH and analysed by
flowcytometer

23. Application of flow cytometry
DNA CONTENT Analysis
• The measurement of cellular DNA contents by flow cytometry
uses fluoroscent dyes such as propidium iodide,that intercalate
into DNA helical structure
• The fluoroscent signals is directly proportional to the amount of
DNA in the nucleus and can identify gross gains or losses in the
DNA
• Abnormal DNA content, also known as DNA content aneuploidy
can be determined in a tumor cell population.
• DNA aneuploidy generally is associated with malignancy

25. Erythrocyte analysis
• Detection and quantification of fetal red cells in maternal blood.
The use of flow cytometry for the detection of fetal cells is much
more objective reproducible , and sensitive than the kleihauer-
betke test.

26. Diagnosis of PNH
• Conventional laboratory tests for the diagnosis of PNH
Include the sugar water test and Ham’s acid hemolysis test.
• Antibodies to CD55 and CD59 are specific for decay-accelerating
factor and membrance- inhibitor of reactive lysis,respectively,can
be analyzed by flow cytometry to make a definitive diagnosis of
PNH

27. Reticulate analysis
• Reticulate count are based on identification of residual ribosomes and
RNA in immuture non nucleated red blood cells by using supravital stain.
The flow cytometric enumeration of reticulocytes uses fluoroscent dyes
that bind the residual RNA, such as thiazole orange.
• A region has been drawn on the red blood cells in the scatter plot. The
other major cluster in the scatter plot are the platlets .
• The histogram was gated on the red blood cells and the region on it
delinate cells with high(H), medium(M) and low (L) flouresence
corresponding to increasing reticulocyte maturity.N marks nucleated red
cells

28. • In the blood bank, the flow cytometry can be used as a complementary
or replacement test for red cell immunology,including RBC- bound
immunoglobilns and red cell antigens.Flow has been used to accurately
identify and phenotype the recepients’s red cells.
• Flow cytometry is being used increasingly in the blood bank to assess
leukocyte contamination in leukocyte- reduced blood products.
Leukocyte analysis:
Perhaps the best example of simultanous analysis of multiple characterstics
by flow cytometry invloves the immonophenotyping
Of leukemias and lymphomas
Immunophenotyping by flow ctyometry (FCM) is an essential aid for
accurately diagnosing and prognosticating leukamia and lymphoma.
• WHO classification has divided non-Hodgkin lymphoma into B-cell and
T/NK cell subtypes, whichare further subclassified into precursor and
peripheral lymhomas.

29. • The ability to analyze multiple cellular characteristics,along with new
antibodies and gating strategies, has substantially enhanced the utility
of flow cytometry inthe diagnosis of leukemias and lymphomas.
• Different leukemias and lymphomas often have subtle differences in
their antigen profiles that make them ideal for analysis by flow
cytometry.

31. Diagnosis of B-Cell Lymphomas by using
specific antibodies in flow cytometer

32. Identification of lineage specific antigens for
perfect
diagnosis

33. • Immunologic monitoring of HIV-infectedpatients is a
mainstay of the clinical flow cytometry
• and provides the best possible way for enumeration of CD4+ T
lymphocytes and HIV viral load.

34. • Flow cytometry can be used for lymphomaphenotyping of
fine needle aspirates, and is apowerful adjunct to cytologic
diagnosis.
• Neutropenia may be immune or nonimmune innature.
Immune neutropenia may result fromgranulocytespecific
autoantibodies, granulocytespecificalloantibodies, or
transfusion-related anti-HLA antibodies. Flow cytometry can
readily identifyanti-neutrophil antibodies that are either bound
togranulocytes or free in plasma and confirm the originof
neutropenia, possibly eliminating the need for abone marrow
procedure.
• Functional deficiencies of leukocytes can beassessed by flow
cytometry. Assays for oxidative burst,phagocytosis,
opsonization, adhesion, and structureare available.

35. • One of the clinical example is LAD type I is caused bya genetic
deficiency of β2 -integrins, which areheterodimers of CD11 and
CD18. This deficiency leadsto a loss of neutrophil and monocyte
migration.

36. • The high sensitivity and capacity for simultaneousanalysis of
multiple characteristics make flowcytometry useful for the detection
of minimalresidual disease, especially if abnormal patterns
ofantigen expression are present.

37. Platelet analysis
• Flow cytometry is an excellent method for directanalysis of
platelet-bound antibodies, and it hasalso been shown to be of
benefit in detection of freeplasma antibodies in ITP.
• The reticulated platelet count can be quantified by flow
cytometry in order to assess the rate of thrombopoiesis. This
measurement can separateunexplained thrombocytopenias into
those withincreased destruction and those with defects in platelet
production.
• The pathogenesis and molecular defects of manyprimary
thrombocytopathies are well known andrelate to defects in
structural or functional glycoproteins,such as the abnormal
expression of gpIIb/IIIa inGlanzmann thrombasthenia and gpIb in
Bernard-Soulier disease.

38. • Flow cytometry is a rapid and useful method ofobtaining a
diagnosis.

39. Other applications
• Flow cytometry is indicated in the evaluation of serous
effusionsand CSF, including aqueous or vitreous
humor of patientswith a history of hematolymphoid
neoplasia.
• Flow cytometry assists in the differential diagnosis
betweenplasma cell myeloma and monoclonal
gammopathies ofundetermined significance by
determining the percentage ofaberrant or clonal plasma
cells of all bone marrow plasma cells.
• Flow cytometry is useful in diagnostic evaluation of
unexplainedmarrow plasmacytosis by assessing
phenotypically aberrant orclonal plasma cells and its
ability to detect other underlyingmonoclonal B-cell
process.

40. • Tissue-based lymphoid neoplasias commonly affect lymph
nodes, spleen, mucosa-associated lymphoid tissue, skin, or
nonlymphoid solid organs resulting inmasses or organomegaly.
• Flow cytometry is extremely useful in the diagnosis and
subclassification of tissue-based lymphoid
neoplasias,,organomegaly and tissue infiltrates

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