Automated Procedures



Automated Procedures



Learning Objectives


At the conclusion of this chapter, the reader should be able to:


• Identify and give examples of the three phases in automated testing.


• Describe the principle, advantages, and disadvantages of nephelometry.


• Discuss the analysis and clinical implications of cryoglobulins.


• Explain the principle of flow cell cytometry and cite clinical applications.


• Discuss current trends in immunoassay.


• List at least three potential benefits of automated immunoassay.


• Analyze a case study related to immunoassay.


• Correctly answer case study related multiple choice questions.


• Be prepared to participate in a discussion of critical thinking questions.


• View and discuss questions related to videos about flow cytometry.


• Correctly answer end of chapter review questions.



Key Terms




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).


image
Figure 13-1 Principle of nephelometry for the measurement of antigen-antibody reactions.
Light rays are collected in a focusing lens and can ultimately be related to the antigen or antibody concentration in a sample.

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).



Box 13-1   Immunologic Assays Performed by Nephelometry





Principle


Formation of a macromolecular complex is a fundamental prerequisite for nephelometric protein quantitation. The procedure is based on the reaction between the protein being assayed and a specific antiserum. Protein in a patient’s specimen reacts with specific nephelometric antiserum to human proteins and forms insoluble complexes. When light is passed through such a suspension, the resulting complexes of insoluble precipitants scatter incident light in solutions. The scattered light can be detected with a photodiode. The amount of scattered light is proportional to the number of insoluble complexes and can be quantitated by comparing the unknown patient values with standards of known protein concentration.


The relationship between the quantity of antigen and measuring signal at a constant antibody concentration is expressed by the Heidelberger curve. If antibodies are present to excess, a proportional relationship exists between the antigen and resulting signal. If the antigen overwhelms the quantity of antibody, the measured signal drops.


By optimizing the reaction conditions, the typical antigen-antibody reactions as characterized by the Heidelberger curve are effectively shifted in the direction of high concentration. This ensures that these high concentrations will be measured on the ascending portion of the curve. At concentrations higher than the reference curve, the instrument will transmit an out of range warning.






Advantages and Disadvantages


Nephelometry represents an automated system that is rapid, reproducible, relatively simple to operate, and common in higher volume laboratories. It has many applications in the immunology laboratory. Currently, instruments using a rate method and fixed-time approach are commercially available with tests for immunoglobulin G (IgG), IgA, IgM, C3, C4, properdin, C-reactive protein (CRP), rheumatoid factor, ceruloplasmin, α1-antitrypsin, apolipoproteins, and haptoglobins.


The disadvantages of nephelometry include high initial equipment cost and interfering substances such as microbial contamination, which may cause protein denaturation and erroneous test results. Intrinsic specimen turbidity or lipemia may exceed the preset limits. In these cases, a clearing agent may be needed before an accurate assay can be performed. In addition, low-molecular-weight immunoglobulins, monoclonal immunoglobulins, and antibovine antibodies also may produce spurious results in nephelometry.



Clinical Application: Cryoglobulins


Cryoglobulin analysis is frequently requested when patient symptoms such as pain, cyanosis, Raynaud’s phenomenon, and skin ulceration on exposure to cold temperatures are present. Cryoglobulins are proteins that precipitate or gel when cooled to 0° C (32° F) and dissolve when heated. In most cases, monoclonal cryoglobulins are IgM or IgG. Occasionally, the macroglobulin is both cryoprecipitable and capable of cold-induced anti-i–mediated agglutination of red blood cells.


Cryoglobulins with a detected monoclonal protein component normally prompt a clinical investigation to determine whether an underlying disease exists. Cryoglobulins are classified as follows:



To test for the presence of cryoglobulins, blood is collected, placed in warm water, and centrifuged at room temperature. The serum is then put into a graduated centrifuge tube and placed in a 4° C (39° F) environment for 7 days. If a gel or precipitate is observed, the tube is centrifuged and the precipitate is washed at 4° C (39° F), redissolved at 37° C (98.6° F), and evaluated by double diffusion and immunoelectrophoresis for the content of the cryoglobulin. Newer methods use nephelometry with cold treatment for analysis.



Flow Cell Cytometry


Fundamentals of Laser Technology


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.


image
Figure 13-2 The electromagnetic spectrum.
YAG, Yttrium-aluminum-garnet. (From Turgeon ML: Clinical hematology: theory and procedures, ed 5, Philadelphia, 2012, Lippincott, Williams & Wilkins.)

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.


image
Figure 13-3 Laser flow cytometry.
(Courtesy Ortho Diagnostics, Raritan, NJ.)

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:




Immunophenotyping


Monoclonal antibodies, identified by a cluster designation (CD), are used in most flow cytometry immunophenotyping (Table 13-1). Cell surface molecules recognized by monoclonal antibodies are called antigens because antibodies can be produced against them or are called markers because they identify and discriminate between (mark) different cell populations. Markers can be grouped into several categories. Some are specific for cells of a particular lineage (e.g., CD4+ lymphocytes) or maturational pathway (e.g., CD34+ progenitor stem cells); the expression of others can vary, according to the state of activation or differentiation of the same cells.


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Jun 12, 2016 | Posted by in IMMUNOLOGY | Comments Off on Automated Procedures

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