FLOW CYTOMETRY Dr. MOHAMMED H SAIEMA LDAHR KAAU FACULTY OF APPLIED MEDICAL SCIENCES MEDICAL TECHNOLOGY DEPT. 2 ND YEAR MT INSTROMINTATION EXT 21060.

FLOW CYTOMETRY

Dr. MOHAMMED H SAIEMA LDAHRKAAU

FACULTY OF APPLIED MEDICAL SCIENCESMEDICAL TECHNOLOGY DEPT.

2ND YEAR MT INSTROMINTATION

EXT 21060

OPTICS (FSC)

Forward Angle Light Scatter

FALS Sensor

Laser

Optics Side Scatter Channel (SSC)

The amount of light scattered at right angle to the incident light beam depends on the internal complexity of the particle, this known as wide angle or Side Scatter (SSC) , side scatter detected at 90, to the laser beam.

SSC)Optics (

90 Degree Light Scatter

FALS Sensor

90LS Sensor

Laser

OPTICSPROPERTIES OF FSC& SSC

PROPERTIES OF FSC& SSC

As the cell passes through the laser beam, light is scattered in all directions and that scattered in the forward direction is proportional to the square of the radius of a sphere, and so to the size of the cell or particle.

The cells may be labeled with fluorochrome-linked antibodies or stained with fluorescent membrane, cytoplasmic or nuclear dye.

What can a Flow Cytometer (FCM) tell us about a cell?

Its relative size (Forward Scatter—FSC)

Its relative granularity or internal complexity (Side Scatter—SSC)

Its relative fluorescence intensity (FL1, FL2, FL3, FL4)

Optics - Fluorescence Channels

Any fluorescent molecule present in or on the particle will absorb energy from the laser light and release the absorbed energy at longer wave length, the emitted light is collected by lenses and detectors, emitted fluorescence intensity is proportional to the amount of fluorescent compound on the particle.

Laser

Fluorescence Detectors

Freq

Fluorescence

FALS Sensor

Fluorescence detector(PMT3, PMT4 etc.)

Optical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

Band passFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

Electronics

The scattered light from particles passing the laser light is converted to digital values that stored in the computer for analysis.

Electronics

FLOW CYTOMETRY

Gating. Gating is in essence electronic window that

sets upper and lower limits on the type and amount of material that passes through.

It is used to separate a sub-population from heterogeneous population.

It permits very specific questions to be asked about a particular population.

Fluorescence and Fluorochrome.

The coupling of monoclonal antibodies with fluorescent dyes is necessary for recognition and enumeration by flow cytometer.

Each fluorochrome possesses a distinctive spectral pattern of absorption (excitation) and emission.

The property of fluorescence is that, the fluorochromes present on the cell absorb the laser light and re-emit the light at a lower energy and longer wave length.

The most popular fluorochrome used in immuno-fluorescent is fluorescein isothiocyanate (FITC), which has an absorption maximum between 450 and 550 nm.

Fluorescence and Fluorochrome.

FITC emits light detected in FL1 on the FCM. Another example of an excellent combination is

Phycoerythrin (PE) as a second fluorochrome since it can absorb light at higher wave lengths than the FITC.

PE emits light can be detected in FL2 on the FCM

APPLICATION

Applications in Clinical Laboratories Immunophenotyping (HIV) CD4 absolute counts Leukemia and lymphoma immunophenotyping Cell cycle and ploidy analysis of tumors Reticulocyte enumeration Flow cross-matching (organ transplantation) Stem cell enumeration Residual white blood cell detection (QC platelet, red blood cells)

APPLICATION

Research Laboratories Immune function studies Hematopoietic stem cells Multi-drug resistance studies (cancer) Kinetics studies (cell function) Platelet analysis

(coronary disease) Environmental sample analysis Flow and FISH

APPLICATION

Immunophenotyping Is the termed used in the identification of cells

by labeling with monoclonal antibodies identified as cluster designations (CD)

CDs is a group of antibodies that all recognize the same antigen

Immunophenotyping

Is performed by labeling the cells with red, green, and / or orange labeled monoclonal antibody.

The cells are then interrogated in the Flow Cytometry, and the number, percentage of positive cells are recorded.

CD Markers used for Primary Diagnosis

T-Cell Markers Myeloid MarkersCD 2 CD11bCD3 CD11cCD4 CD13CD5 CD14CD6 CD33CD7

CD Markers used for Primary Diagnosis

B-Cell Markers Miscellaneous MarkerCD10 sIgG HAL-DRCD19 sIgM CD34CD20 Kappa CD45CD22 Lambda TdTCD23 CIgMCD25 CCd3

TYPES OF MEASUREMENTS

Intrinsic parameters: Cell size & Granularity.

Extrinsic parameters: Surface Antigens Single or multiple surface membrane antigens

are detected with fluorescinated monoclonal antibodies examples, CD45, CD4, CD8, etc).

TYPES OF MEASUREMENTS

Multi-parametric FCM In the multi-parameter approach more than

one feature of the same cell is measured, such as three color surface and cytoplasmic staining in conjunction with the cellular DNA content

leukemia and lymphoma.Distinction of myeloid leukemia from lymphatic can be precisely determined by the analysis of surface and cytoplasmic antigens.

TYPES OF MEASUREMENTS

FCM analysis of Platelets Glanzmann’s Thrombasthenia (GPIIb/IIIa). Bernard – Soulier Syndrome (GPIb). Storage Pool Disease:

Dense Granule SPD.Gray Platelets SyndromeL.

Reticulated Platelets. Anti-platelets Antibodies. Monitoring treatment with GPIIb/IIIa

antagonist

TYPES OF MEASUREMENTS

DNA content & cell cycle analysis Aneuploidy and/or elevated S-phase fraction

have been shown to be ominous prognostic indicators in breast, colon, rectal, prostate, and bladder tumours.

GG22

MM GG00

GG11

ss

0 200 400 600 800 1000

GG00GG11

ss GG22 MM

DNA AnalysisDNA Analysis

DNA content

Count

2N2N 4N4N

Normal Cell Cycle Normal Cell Cycle

APPLICATION

APPLICATION