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Flow cytometry. (Product Roundup).

Flow cytometry is one of those subspecialties within the field of laboratory medicine that often elicits a "don't ask me" response from even the most veteran laboratorian. That's because it is not widely practiced within the acutecare laboratory setting, nor is it covered in much detail within clinical laboratory training programs, regardless of discipline. As a result, a discussion of flow cytometry (FC) and commercially available systems requires a little background information. This installment of Product Roundup is intended to provide a brief overview of the science behind FC, describe some of the technological differences in FC applications, and for those individuals who may be gathering information for the acquisition of such a system in the future, review some of the products offered by several of the most widely known manufacturers.

By definition, FC is a means of measuring certain physical and chemical characteristics of cells and related particles in suspension, and in some cases, using these features to separate or sort subpopulations of cells for more extensive analysis. The generic flow cytometer consists of a fluid transfer apparatus, excitation light source(s), emitted light detection mechanisms, and computer hardware and software for translating electronic signals into useful data. In terms of recognizable clinical laboratory applications, FC is used in hematology, urinalysis, immunology, and molecular pathology.

Flow cytometry procedures can be subdivided into those that allow for use of "raw" samples such as blood and urine, and those that require some type of pretreatment in order to label specific cellular components with other bio-active molecules. The modern hematology analyzer is, of course, capable of processing untreated whole (anticoagulated) blood, while the traditional flow cytometer involves use of labeled antibodies and/or biochemical dyes. Although FC is currently employed many areas of biological research, this technology will become increasingly common within the laboratories of medical centers and specialty clinics as disease-specific protocols, such as lymphocyte ratio analyses for HIV-infected patients, are refined.

Beckman Coulter offers a full line of instruments for sample preparation and analysis. The company's latest advancement is the new Cytomics FC 500 series flow cytometer, which offers five-color analysis from a single laser, streamlining the measurement of cells and allowing more powerful multiparametric acquisition. A collinear second laser is also available, providing greater versatility in reagent and fluorochrome choices. The instrument is ideal for monitoring immune subsets, immunophenotyping, activation and functional studies in a diverse range of laboratory environments. Modules for automated clinical applications, such as CD4 and progenitor cells, are in development. Beckman Coulter also manufactures the EPICS line of instruments.

The XL system features the capability to analyze up to four colors of immunofluorescence from a single air-cooled laser. Other multi-color applications include: multi-parametric DNA analysis, platelet studies, reticulocyte enumeration, and cell biology/functional studies, as well as a broad range of research applications. The instrument is self-contained and biohazard safe, and users retain the flexibility to change filter elements for versatility in research settings. The XL offers state-of-the-art Digital Signal Processing (DSP) for reliable linearity and drift-free amplification and compensation. The single laser design eliminates concerns regarding multi-beam stability, signal delay, and alignment. Compensation is achieved by a unique digital compensation matrix.

For high volume users, the XL-MCL system offers walk-away sample handling with the Multi-arousel Loader (MCL). Capable of throughputs of up to 100 samples per hour, the XL-MCL incorporates positive barcode identification and true vortex mixing prior to aspiration.

The Cytomics FC 500 Series product family is equipped with the most advanced technologies available. These instruments are capable of conducting five-color analysis from either single or dual laser excitation, and are equipped with a collinear second laser that maintains a stable alignment with a single beam path and allows greater versatility in antibody and fluorochrome choices.

BD Biosciences Immunocytometry Systems is also well-established in this market. BD Biosciences manufacturers three systems based on fluorescence-activated cell sorting (FACS) technology, including the BD FACS VantageTM SE flow cytometry system, BD FACS Calibur[TM] flow cytometry system, and BD[TM] Life Sciences Research (LSR) instrument platforms.

The BD FACSVantage[TM] SE flow cytometer is now available with the BD FACSDiVa option, which features fully digital electronics and new PC-based software. The BD FACSDiVa option can acquire up to 16 channels (12 colors), and expands the instrument's ability to utilize all standard optical pathways and acquire every fluorescent measurement possible. The new four-way sorting provide faster and more efficient results, and the enhanced software expands both acquisition and analysis tools.

The BD FAGS Calibur[TM] flow cytometry system is a four-color, dual-laser, benchtop system available capable of both cell analysis and sorting. Designed specifically to support a wide range of applications, the BD FAGS Calibur[TM] flow cytometry system is a fully integrated multiparameter system. It combines a unique dual-laser technology, an automated sample loader option, and powerful software to provide the high throughput necessary to meet productivity requirements of today's clinical laboratories.

The BD[TM] LSR offers up to six fluorescence and two scatter parameters that employ alignment-free 488/UV single-laser, or an optional 488/UV/633 three-laser system. It also features an air-cooled 8-mW, 325-nm helium-cadmium laser m addition to a 15-mW, 488-nm argon-ion laser. An optional 633 nm helium-neon (HeNe) laser is also available. The BD[TM] LSR supports a wide range of multicolor cell analysis applications with a standard 488-nm-excited dyes and UV-excited dyes such as Hoechst 33342, DAPI, and Indo-1. With the optional HeNe laser, APC can also be detected.

Gytomation offers two systems, the MoFlo and CyAn. The MoFlo is a modular, custom-configured instrument that features sort rates of 70,000 cells/second with 32 simultaneous parameters and greater than 99 percent purity. This system enables a wide range of biological applications, including detection of organelles, antigens, receptors, cytokines, and enzymes; measurement of signal transduction, apoptosis, calcium flux, and membrane potential, as well as sorting of stem cells, sperm, and other rare events. The MoFlo can also be used for fluorescence resonance energy transfer, fluorescent protein work, phage display, and other emerging protocols.

The CyAn unit offers three excitation lines, 11 parameters, complete compensation, high speed, and reliable walkup operation, all in a benchtop configuration. It offers pushbutton operation that puts even a novice user at ease; with no optical alignment necessary, the user simply places a 5mL sample tube in the tube holder and chooses the desired protocol in Summit Software.

Although CompuCyte's Laser-Scanning Cytometer, the LSC, employs considerably different technology than traditional flow cytometry, it is included in this review as means of demonstrating an alternative approach to obtain data from cellular samples. According to information obtained from the company's website, "Laser scanning cytometry ups the ante for high-content cellular analysis (by) fusing the technologies of flow cytometry and image analysis."

By directing one, two, or three coaxial laser beams to a scanning mirror, the LSC optics apply the scanned beam through the microscope objective lens onto the specimen. The specimen, which is mounted on a standard microscope slide, moves past the scanning laser beam on a motorized micro-stepping stage. As cells or other fluorescent objects pass through the scanning beam(s), the emitted photons return along the same path as the laser beam(s) and are collected in the wavelength-specific photomultiplier tubes (PMT'S). CompuCyte's system can be used for such applications as apoptosis and DNA content analysis, immunophenotyping, and tissue mapping.

J.D. Myers, a laboratory scientist and sales professional, lives in Clearwater, FL.
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Author:Myers, J.D.
Publication:Medical Laboratory Observer
Geographic Code:1USA
Date:Jul 1, 2002
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