Moderator: Dr. Rakesh Mehar

Speaker : Dr. Aksharaditya Shukla

Department Of Pathology MGM Medical College Indore

Flow ~ motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a

fluid stream

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.

• 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).

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.

Fluidics system. Optic system. Electronic system. Associated computer system.

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.

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.

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

Acquisition and analysis of flow cytometric data.

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.

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

Sensitize mouse to antigen Harvest spleen B cells Fuse with myeloma cells Select hybridoma clones for antibody

production Label antibody with fluorochrome dye /

color

Antigen expression:RBCs, WBCs, Platelets, Others

CD3

Cyto CD3

Tdt

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

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.

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.

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.

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

Forward scatter.Side scatter.

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.

The index of refraction is higher in fixed and stained cells.

Live cells scatter more light than dead cell at lower angle

FSCFSCDetectorDetector

Laser BeamLaser Beam

Original from Purdue University Cytometry LaboratoriesOriginal from Purdue University Cytometry Laboratories

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.)

FSCFSCDetectorDetector

CollectionCollectionLensLens

SSCSSCDetectorDetector

Laser BeamLaser Beam

Original from Purdue University Cytometry LaboratoriesOriginal from Purdue University Cytometry Laboratories

FS SS

Size Granularity

SS detector - Granularity

SS detecter Granularity

FSsize

FSsize

Laser

Laser

Size and granularity

Allows for differentiation of cell types in a heterogenous cell population

FSC

SS

C

Lymphocytes

Monocytes

Granulocytes

RBCs, Debris,RBCs, Debris,Dead CellsDead Cells

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

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.

FSCFSCDetectorDetector

CollectionCollectionLensLens

Laser BeamLaser Beam

FluorescenceFluorescenceDetector 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

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

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.

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.

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 .

.

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.

PMT

PMT

PMT

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

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.

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

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

CD38 Plasma cells (bright), B-lymphoblasts and myeloblasts (moderate)CD41 Glycoprotein IIb/IIIa, platelets, and megakaryocytesCD45 Leukocyte common antigen (nonerythroid hematopoietic cells)CD56 NK cells, some stem-cell disorders, some activated T cellsCD57 Subset of cytotoxic/suppressor T cellsCD61 Platelets, and megakaryocytesCD64 Monocytes, activated granulocytesCD103 Hairy cell leukemiaTdt ImmatureT cells, immature B cells

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

Benign Hematopoeitic disorders HIV. SCID. CVID. Fetomaternal hemorrhage

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

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.

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).

FCM Approach to Acute Leukemia

Acute leukemia.

Auer rods+ Auer rods –

AML + MPO /NSE- AML

FCM

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

Second line.

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

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

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

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.

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

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 - CD19/CD34/CD13. .

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.

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

Case

5 year old boy with fever – 1 month

DiagnosisCALLA- ALL

FL, BL, DLBCL,

Hematogones

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.

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.

By – Dr A.Shukla