The basics of biochemistry instrumentation (Proceedings)
This session will discuss basic methodologies used by selected common biochemistry analyzers and quality assurance issues which may result in better understanding of the advantages and disadvantages of various instrument types and ultimately assist with instrument selection. Additional quality issues are discussed in the "QA Tips" and "Lab Errors" sessions.
MethodsCommon methods include photometry and electrochemistry. The origins of photometry go back to the mid 1600's when Sir Isaac Newton observed that a prism separates light into colors and that light retains its color whether it is reflected, scattered or transmitted. This is the main principle behind the majority of clinical biochemistry methods. Systems relying on the principles of light may use reagents that are liquid, reconstituted liquid, or dry. Liquid chemistry systems are considered the most traditional and are what most published reference intervals and interpretive guidelines are based upon. The spectrophotometry of liquid chemistry analyzers is similar in principle to the hemoglobin concentration measurement as part of a CBC analysis. These systems rely on a chemical reaction that occurs between the sample and one or more liquid reagents in a controlled environment. In this case, the sample is typically serum or plasma. The reaction creates a color change in the mixture and light of a specific wavelength is passed through the reaction mixture. The light that is not absorbed by the reaction mixture is transmitted to a photometer and is measured. Since other conditions, as the characteristics of the reaction/measuring chamber, remain constant, the intensity of that light is proportional to the concentration. Assays may be one of two general types: endpoint and rate. Endpoint assays require a photometer that measures absorbance at the beginning and end of the reaction. The concentration of the analyte is proportional to the magnitude of change. Because sample quality issues as lipemia, icterus or hemolysis (known as serum quality indices) can alter the light stream, systems typically incorporate a blanking system to minimize the interference. The degree to which these interferences affect results varies with the method, instrument and level of interferent. The user should be aware of if and when these interferences might occur in their chemistry system.
Rate assays involve the creation of a product or depletion of a substrate. How fast the reaction occurs over a specified time is the slope (rate) of the reaction and is proportional to the activity of the analyte, usually an enzyme. The substrate, reaction temperature and pH can affect the reaction rate and thus reference intervals may vary significantly between instruments types and methodologies.
Liquid systems tend to offer the largest test selection, have the highest amount of flexibility for customized panels, and cost-effectiveness is usually proportional to workload. In other words, the more tests performed, the lower the cost per test. A higher level of technical expertise is necessary to maintain the analyzer and the complex reagent system. Control materials must be run and evaluated regularly to insure integrity of the process, periodic calibration may be required and reagents may need manual reconstitution. Liquid systems are the most common form of instrument in large referral laboratories.
Efforts to simplify the instrumentation for users have resulted in a number of variations of this theme including removing the on-site calibration process. Reconstituted liquid systems are one such variation (VetScan® , Abaxis, Union City, California) where reagent handling and calibration requirements are simplified. Heparinized whole blood or serum is added to a rotor containing freeze-dried reagent pellets that are converted to liquid after the addition of a rotor-contained diluent. Built-in quality control materials indicate the integrity of rotor handling during shipment and storage as well as integrity of the lamp and other operational issues. Calibrations for new lot numbers are performed by the manufacturer and changes are automated via matching the barcode on each rotor to information installed with a magnetic strip on a card provided to the customer. Results are suppressed if sample quality issues interfere significantly (>10%) with the accuracy of result. Test flexibility is accomplished with selection from several predefined rotors providing test menus designed for several species and common organ assessments. A similar unit (Analyst® , Hemagen Diagnostics, Columbia, Maryland) requires dilution of the serum or plasma with the automatic diluter provided. Due to the panel arrangement, cost per test is intermediate and remains relatively constant regardless of the number of rotors used.