Characteristics of Automated Testing

Laboratory automation can be separated into preanalytic, analytic, and postanalytic phases. Accuracy in each of these phases is critical to quality results. The preanalytic phase includes specimen labeling (bar coding preferred), accessioning, and tracking, along with proper test ordering.

The analytic phase involves the following areas:

Automated analyzers link each specimen to its specific test request. Any results generated must be verified (approved or reviewed) by the operator before the data are released to the patient report. Useful data for this verification process include flags, signifying results outside the reference range, critical or panic values (possibly life-threatening), values that are out of the technical range for the analyzer, and failures in other checks and balances built into the system.

The postanalytic phase includes adding to patient cumulative reports, workload recording, and networks to other systems. Quality assurance (QA) procedures, including the use of quality control (QC) solutions, are part of the analytic functions of the analyzer and its interfaced computer. The Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) regulations require the documentation of all QC data associated with any test results reported (see Chapter 7). Harmonization of analytes has been gaining momentum as an essential component of the outcomes of analysis. In the future, harmonized or normalized results may be mapped together and presented numerically and graphically to reduce data output.

Nephelometry

Nephelometry has become increasingly more popular in diagnostic laboratories and depends on the light-scattering properties of antigen-antibody complexes (Fig. 13-1).

The quantity of cloudiness or turbidity in a solution can be measured photometrically. When specific antigen-coated latex particles acting as reaction intensifiers are agglutinated by their corresponding antibody, the increased light scatter of a solution can be measured by nephelometry as the macromolecular complex form. The use of polyethylene glycol (PEG) enhances and stabilizes the precipitates, thus increasing the speed and sensitivity of the technique by controlling the particle size for optimal light angle deflection. The kinetics of this change can be determined when the photometric results are analyzed by computer.

In immunology, nephelometry is used to measure complement components, immune complexes, and the presence of a variety of antibodies (Box 13-1).

In 1917, Einstein speculated that under certain conditions, atoms or molecules could absorb light or other radiation and then be stimulated to shed this gained energy. Lasers have been developed with numerous medical and industrial applications.

The electromagnetic spectrum ranges from long radio waves to short, powerful gamma rays (Fig. 13-2). Within this spectrum is a narrow band of visible or white light, composed of red, orange, yellow, green, blue, and violet light. Laser (light amplification by stimulated emission of radiation) light ranges from the ultraviolet (UV) and infrared (IR) spectrum through all the colors of the rainbow. In contrast to other diffuse forms of radiation, laser light is concentrated. It is almost exclusively of one wavelength or color, and its parallel waves travel in one direction. Through the use of fluorescent dyes, laser light can occur in numerous wavelengths. Types of lasers include glass-filled tubes of helium and neon (most common), yttrium-aluminum-garnet (YAG; an imitation diamond), argon, and krypton.

Lasers sort the energy in atoms and molecules, concentrate it, and release it in powerful waves. In most lasers, a medium of gas, liquid, or crystal is energized by high-intensity light, an electrical discharge, or even nuclear radiation. When an atom extends beyond the orbits of its electrons or when a molecule vibrates or changes its shape, it instantly snaps back, shedding energy in the form of a photon. The photon is the basic unit of all radiation. When a photon reaches an atom of the medium, the energy exchange stimulates the emission of another photon in the same wavelength and direction. This process continues until a cascade of growing energy sweeps through the medium.

Photons travel the length of the laser and bounce off mirrors. First, a few and eventually countless photons synchronize themselves until an avalanche of light streaks between the mirrors. In some gas lasers, transparent disks referred to as Brewster windows are slanted at a precise angle, which polarizes the laser’s light. The photons, which are reflected back and forth, finally gain so much energy that they exit as a powerful beam. The power of lasers to transmit energy and information is rated in watts.

Principles of Cell Cytometry

Flow cell cytometry, developed in the 1960s, combines fluid dynamics, optics, laser science, high-speed computers, and fluorochrome-conjugated monoclonal antibodies (MAbs) that rapidly classify groups of cells in heterogeneous mixtures. The principle of flow cytometry is based on cells being stained in suspension with an appropriate fluorochrome—an immunologic reagent, a dye that stains a specific component, or some other marker with specific reactivity. Fluorescent dyes used in flow cytometry must bind or react specifically with the cellular component of interest (e.g., reticulocytes, peroxidase enzyme, DNA content). Fluorescent dyes include acridine orange and thioflavin T. Pygon is preferred for fluorescein isothiocyanate (FITC) labeling. Krypton is often used as a second laser in dual-analysis systems and serves as a better light source for compounds labeled by tetramethyl-rhodamine isothiocyanate and tetramethylcyclopropyl-rhodamine isothiocyanate.

A suspension of stained cells is pressurized using gas and transported through plastic tubing to a flow chamber within the instrument (Fig. 13-3). In the flow chamber, the specimen is injected through a needle into a stream of physiologic saline called the sheath. The sheath and specimen both exit the flow chamber through a 75-µm orifice. This laminar flow design confines the cells to the center of the saline sheath, with the cells moving in single file.

The stained cells then pass through the laser beam. The laser activates the dye and the cell fluoresces. Although the fluorescence is emitted throughout a 360-degree circle, it is usually collected by optical sensors located 90 degrees relative to the laser beam. The fluorescence information is then transmitted to a computer, which controls all decisions regarding data collection, analysis, and cell sorting.

Flow cytometry performs fluorescence analysis on single cells. The major applications of this technology are as follows:

• Identification of cells

• Cell sorting before further analysis