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Flow Cytometry Methodology (Optical)

Automated systems are now based on flow-through (also called flow cytometry) optical technologies that identify cells on the basis of light scatter properties broadly equating to the cell’s physical characteristic differences.

TECHNOLOGY

Blood cell recognition is optimized by diluting specifically to the number of cells present in the blood so as to count the number of cells appropriate for statistical accuracy.

 

A focused light beam illuminates a small area of a flow cell. Cells are rapidly injected in single file (hydrodynamic focusing) through the illuminated area (interrogation zone) where the cells intersecting the light beam scatter light in all directions in a manner that is measurable and unique to each cell type.

 

The resulting light scatter information can be collected at multiple locations incident to the light beam providing a multi-dimensional analysis of the unique light scatter properties from each cellular event. Analysis and displays of the optical light scatter measurements are provided in a two-dimensional graphical form called a scatterplot. Multiple scatterplots may be generated for complete visualization of all the cellular components including sub-populations of each cell line.

 

Figure 1 demostrates the hydrodynamic focusing and principle of optical light scatter. The focused flow stream of diluted WBC are injected into the flow cell and enter the flow cell laser intercept area. The cells scatter the laser light at different angles, yielding information about cell size, internal structure, granularity and surface morphology. The optical signals the cells generate, are detected and converted to electrical impulses which are then stored and analyzed by the computer.

Figure 1 demonstrates the hydrodynamic focusing and principle of optical light scatter.

THE FLOW CYTOMETER

The flow cytometer instrument consists of three core systems: fluidics, optics, and electronics (Figure 2). The fluidics system includes a flow cell, where the sample fluid is injected. The flow cell requires sheath fluid to carry and align the cells or particles so that they pass through a narrow channel and into the laser intercept (light beam) in a single file. This hydrodynamic focusing allows the analysis of one cell at a time by laser
interrogation.2

 

The optics system consists of various filters, light detectors, and the light source, which is usually a laser line producing a single wavelength of light at a specific frequency. This is where the particles are passed through at least one laser beam. Lasers are available at different wavelengths ranging from ultraviolet to far red and have a variable range of power levels as well (photon output/time). Interrogation by the laser beam excites any compatible fluorescent probes that are conjugated to antibodies, causing the probes to emit light (or fluoresce) at specified wavelengths. A detector in front of the light beam measures forward scatter light signals (FSC) and detectors to the side measure side scatter light signals (SSC). Fluorescence detectors measure the fluorescence signal intensity emitted from positively stained cells and particles.

Figure 2. The flow cytometer instrument consists of three core systems: fluidics, optics, and electronics.

Within the flow cytometer, all of these different light signals are split into defined wavelengths and channeled by a set of filters and mirrors so that each sensor will detect fluorescence only at a specified wavelength. These sensors are called photomultiplying tubes (PMTs). Various filters are used in the flow cytometer to direct photons of the correct wavelength to each PMT. Short pass (SP) filters allow transmission of photons below a specific wavelength while long pass (LP) filters allow transmission above a specific wavelength. Band pass (BP) filters allow transmission of photons that have wavelengths within a narrow range. Each PMT will also detect any other fluorophores emitting at a similar wavelength to the fluorophore it is detecting.2

 

These light signals are converted by the electronics system to data that can be visualized and interpreted by software. The diagram below illustrates the parts and setup of a typical flow cytometer instrument.2

CLINICAL APPLICATIONS IN HEMATOLOGY

The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Many surface proteins and glycoproteins on erythrocytes, leukocytes, and platelets have been studied in great detail. The availability of monoclonal antibodies directed against these surface proteins permits flow cytometric analysis of erythrocytes, leukocytes, and platelets.

 

 

ABBOTT OPTICAL LIGHT SCATTER TECHNOLOGY

Abbott's hematology systems employ an innovative version of optical technology: The Multi-Angle Polarized Scatter Separation (MAPSS™) technology that assures the highest level of result accuracy and precision. It uses four light scatter detectors to determine various cellular features. The application of a depolarized light detector is a unique characteristic of this method and allows for specific identification of eosinophil granulocytes.

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1. Michael Brown, Carl Wittwer, Flow Cytometry: Principles and Clinical Applications in Hematology. http://clinchem.aaccjnls.org/content/46/8/1221. (August 2000).
2. BOSTER, Flow Cytometry Fundamental Principle, How FACS Works. https://www.bosterbio.com/protocol-and-troubleshooting/flow-cytometry-principle
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