Materials and methodsThe precise calibration and control of temperature in analysers used in clinical chemistry is very important, especially in the assay of enzymatic activity.' 2 At present, 25°C, 30°C, and 37°C are the most commonly used temperatures in enzymatic activity assays," and it has been shown that the rate of some reactions can change as much as 10 % per degree Celsius change." The Expert Panel on Enzymes of the International Federation of Clinical Chemistry recommends that temperature be maintained to ±0·05°C.o Often however, the reaction cuvette is inaccessible, and measurement of the temperature within the cuvette requires that the instrument be dismantled. Centrifugal analysers and photometers in which microcuvettes are used fall into this category, and, with the development of systems such as these, it has been necessary to find alternative methods by which the temperature in the reaction cuvettes can be monitored.Methods based on an indicator to monitor the change in pH of a buffered solution with temperature 6 7 or on the temperature-dependent acid hydrolysis of N-o-tolyl-n-glucosylamine 8 have been reported. We describe our experiences in the monitoring of temperature with the use of 3,5-dinitrosalicylic acid (DNS) and cresol red. These reagents are readily available, inexpensive, and safe to handle and provide results that are easily interpreted. 1983; 20: 153-157 Optical methods for monitoring temperature in spectrophotometric analysers Ann Clin Biochem THOMAS D O'LEARY, JANE L BADENOCH, AND RENZE BAISFrom the Division of Clinical Chemistry, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia, Australia 5000 SUMMARY A procedure is described for monitoring the temperature in the reaction cuvettes of analytical systems that use photometers. The method employs the temperature-dependent change in absorbance of solutions of either 3,5-dinitrosalicylic acid or cresol red. The procedure is simple to perform and is especially useful in monitoring the temperature in instruments such as centrifugal analysers where the reaction cuvette is inaccessible. In some of the instruments studied, methodological changes were required to ensure that reactions were carried out at the selected temperature.

We evaluated two kinetic methods for determining total amylase activity and isoenzyme composition in serum. Stability studies of reagents for measuring total activity indicate that reagents containing 4-nitrophenyl-alpha-glucosides or enzyme-linked reagents can be stored only for seven days at 4 degrees C. Methods based on 4-nitrophenyl-alpha-glucoside substrates cannot be used if the reagent absorbance at 405 nm exceeds 2. However, in the alpha-amylase EPS method (Boehringer Mannheim) an ethylidene-protected 4-nitrophenyl-alpha-D-maltoheptaoside substrate is stable for up to 28 days after reconstitution. Further studies indicated that the Amylase-DS (Beckman) and the alpha-Amylase EPS standard curves are linear to at least six times the upper limit of the reference interval. Within-batch imprecision (CV less than 1.1%) and between-batch imprecision (CV less than 3.3%) for these two methods are comparable with those for other kinetic methods, and there is excellent correlation (r2 = 0.983) between the two methods. The reference interval, determined by use of samples from 90 healthy blood donors, is 31 to 141 U/L for the amylase-DS method, 22 to 92 U/L for the alpha-Amylase EPS method. We also used these two methods to measure amylase isoenzymes after inhibiting the salivary isoenzyme with either a lectin or a monoclonal antibody. We found the monoclonal antibody method more specific than the lectin inhibition method for determining the isoenzymes.

In the Reflotron Amylase dry-reagent carrier system (Boehringer Mannheim GmbH) a new substrate is used for determining total amylase (EC 3.2.1.1) activity:indolyl-alpha-D-maltoheptaoside. The procedure shows low imprecision (median CV less than 3.2%), and results for sera, plasma, and capillary and venous blood (y) correlate well with those of a conventional alpha-amylase method involving p-nitrophenyl (PNP)-maltoheptaoside substrate (x) (for 209 blood samples: y = 0.981x + 9.7; r = 0.994). Correlation was also excellent with a method involving maltotetraose as substrate (r = 0.987). Attachment of an indoxyl residue rather than a PNP group to the maltoheptaoside did not affect the substrate response to pancreatic or salivary isoenzyme activity. Therefore, the relative proportion of these isoenzymes did not affect the correlation between the Reflotron Amylase reagent carrier and the alpha-amylase PNP-maltoheptaoside method. With a reaction time of less than 3 min, this system is especially suitable for amylase determination in situations where a prompt result is required.

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