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Measurement of Bilirubin, Cholesterol and Creatinine in Serum and Plasma, by Solid-Phase Reflectance Spectroscopy

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Measurement of Bilirubin, Cholesterol and Creatinine in Serum and Plasma, by Solid-Phase Reflectance Spectroscopy J Clin Pathol: first published as 10.1136/jcp.36.5.598 on 1 May 1983. Downloaded from J Clin Pathol 1983;36:598-601 Measurement of bilirubin, cholesterol and creatinine in serum and plasma, by solid-phase reflectance spectroscopy JF STEVENS, W TSANG, RG NEWALL* From the Department of Chemical Pathology, Middlesex Hospital Medical School, London and *Miles Laboratories Limited, Stoke Poges, Slough SUMMARY The Seralyzer chemistries for total bilirubin, cholesterol, and creatinine have been examined in the routine hospital laboratory, and have demonstrated good correlation and preci- sion when compared to established routine methodologies. The system has proved to be reliable and easy to use. Reflectance spectroscopy is being used increasingly These contain the correct filters for the chemical in the field of clinical chemistry for the measurement reactions taking place on the strip; a programme of various analytes in blood. Its use in conjunction providing for calibration and test readings, and con- with solid-phase reagents makes the system ver- version to the appropriate concentration units; satile, and extends its applications to areas beyond defined tolerance limits for acceptance of the calib-copyright. that of clinical chemistry laboratories, who are ration procedure; display of error codes, in the event dependent on the conventional wet chemistry tech- of-system faults; and a recognition system for the niques. This is available as the SeralyzerTm system identification of the correct reagent strip for each (Ames Division, Miles Laboratories Limited). The test by means of bar codes on each strip. All tests are system can provide a "Stat" function for the provi- performed at a temperature of 37°C. Tests are per- sion of urgent results, often extending the repertoire formed either as rate or end-point reactions, in the of tests available in this way. ultraviolet or visible regions of the spectrum. The http://jcp.bmj.com/ Studies have already shown the suitability of the two-point calibration is stored in the memory, so system for the routine measurement of glucose, urea that constant recalibration is unnecessary. and uric acid' 2 and a preliminary study on the meas- urement of bilirubin has also been made.3 Recently, CHEMICAL PRINCIPLES Seralyzer methods have been developed for the Total bilirubin measurement of total bilirubin, cholesterol, and The test is based on the reaction between bilirubin creatinine, in serum. These have been studied to and a diazonium salt in the presence of dyphylline on September 29, 2021 by guest. Protected assess the performance in routine clinical use in the andp-toluene sulphonic acid, the end-product being hospital laboratory, and were found to be conve- the red-purple coloured substance azobilirubin. nient and reliable for routine clinical analysis. The test requires 30,1 of undiluted serum. After an incubation period of 75 s, the concentration of Material and methods serum total bilirubin is determined by measuring the change in reflectance at 560 nm with reference to a THE SERALYZER SYSTEM calibration curve. The Seralyzer system basically comprises a micro- processor-controlled reflectance photometer in Cholesterol association with dedicated solid-phase reagent The Seralyzer cholesterol reagent strip is based on strips, as briefly outlined previously.2 The principles the reaction in which the hydrogen peroxide, gener- of the system are described by Zipp.4 ated in the cholesterol oxidase reaction, is measured Modules are supplied for each test parameter. by the oxidative coupling of 3- Dr JF Stevens present address: St. Stephen's Hospital, Chelsea, methyl-2-benzothiazolinone hydrazone (MBTH) London, SW10 9TH. and primaquine disphosphate in the presence of Accepted for publication 22 December 1982 peroxidase, producing a coloured complex. Initially, 598 J Clin Pathol: first published as 10.1136/jcp.36.5.598 on 1 May 1983. Downloaded from Measurement of bilirubin, cholesterol and creatinine in serum and plasma 599 cholesterol esters are hydrolysed to cholesterol in Table 1 Bilirubin precision results obtained by the assay the presence of cholesterol ester hydrolase. The test oftriplicates oftwo control sera with each batch ofpatient requires 30 ,ul of ninefold diluted serum. After an samples incubation period of 135 s, the concentration of serum cholesterol is determined by measuring the Seralyzer Hitachi 706D reflectance at a wavelength of 600 nm. Control A Control B Control A Control B Noof runs 21 21 21 21 Creatinine No of assays 63 62 63 63 Mean* (mg/dl) 0-88 5-03 0-82 4-86 The Seralyzer creatinine reagent strip method is Within run based on the Benedict-Behre reaction, in which SD 0-031 0-19 0-051 0 07 CV(%) 3-5 3-8 6-2 1-4 creatinine reacts with 3,5-dinitrobenzoic acid Between run (DNBA) in an alkaline medium to form a purple- SD 0-030 0-40 0-067 0-12 coloured complex. Thirty microlitres of undiluted CV(%) 3-5 8-1 8-1 2-5 Overall specimen is required. Formation of the coloured SD 0-043 0-45 0-084 0-14 complex is monitored by the Seralyzer at 560 nm, CV(%) 4-9 8-9 102 2-9 with measurements taken at five-second intervals *To convert from mg/dl to ,umoVl, multiply by 17-1. between 15 and 30 s. (a) (b) / *l S. E *- / -. I6 copyright. C ul N_ . L- 0 :D -6 N -5 http://jcp.bmj.com/ Y=0-978x .0.100 y=1.15x -11.9 r= 0.987 r =0.903 1' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 50 100 150 200 250 300 350 400 450 Hitach 706 D bilrubin (mg/dl) Hitachi 706 D cholesterol (mg/dl) 22 on September 29, 2021 by guest. Protected (c) I9% 18 16. Fig. 1 Method comparison: scatter-plots ofSeralyzer and 14. comparative methods using clinical specimens from normal and 1* 0* diseased patients. Serum was used for the estimation of 12 (a) bilirubin and (b) cholesterol; plasma was used for the 10' (c) creatinine estimation. 8' 6 V') y= 1.208x -0-0035 r=0.989 2' 0 2 4 6 8 10 12 14 16 18 Astra 4 creatinine (mg/dl) J Clin Pathol: first published as 10.1136/jcp.36.5.598 on 1 May 1983. Downloaded from 600 Stevens, Tsang, Newall PROTOCOL Table 2; replicate precision of clinical specimens Within- and between-run precision data were demonstrated a within-run coefficient of variation of obtained using serum pools and quality control sera 6-5%. Correlation of clinical specimens with the at different analyte concentrations. Accuracy was comparative method is presented in Fig. 1, with a assessed by the analysis of clinical specimens on the correlation coefficient of 0-9. The cholesterol studies Seralyzer, relative to the routine comparative were performed on a 1/9 serum dilution. The results laboratory methods. Replicate precision of the clini- indicate very similar precision for both the Seralyzer cal specimen assays was also determined. Serum was and the comparative method at the levels studied. used for the bilirubin and cholesterol assays; plasma Comparison of the two methods using clinical and serum were used for creatinine. The bilirubin specimens demonstrated good correlation; however, test accuracy was assessed using adult specimens. the observed slope and intercept reflect the fact that For trial purposes, the modules for all three the comparative Hitachi method is known to under- chemistries were calibrated in units of mg/dl; estimate cholesterol values by approximately 7% consequently, all results are reported in these units. (JF Stevens, unpublished observations, 1982). However, SI unit modules are now available. There was very good agreement between duplicate cholesterol measurements performed on the Seral- COMPARATIVE METHODS yzer; there were no gross outliers. Turbidity, often The comparative laboratory methods used were as found in hyperlipaemic samples, and which inter- follows: feres with non-protein precipitation methods, did Total bilirubin Hitachi 706D analyser-Caffeine/ not affect the reflectance method employed on the sodium benzoate solubilisation, followed by diazot- Seralyzer. isation and the addition of alkali. This is based on the Jendrassik method.5 Creatinine Cholesterol Hitachi 706D analyser-esterase Within-run Seralyzer precision using a normal action followed by cholesterol oxidase, and subse- plasma pool can be seen from Table 3; between- and quent chromogenic reaction with overall control sera precision data are presented in 4-aminophenazone.6 copyright. Creatinine Beckman Astra 4 analyser-kinetic Table 2 Cholesterol precision results-obtained by the Jaffe reaction.' assay oftriplicates oftwo control sera with each batch of patient samples Results and discussion Seralyzer Comparative Total bilirubin Control A Control B Control A Control B http://jcp.bmj.com/ Precision data are shown in Table 1. These are Noof runs 13 13 13 13 based on triplicate measurements on two control No of assays 39 37 39 39 sera in each batch of assays of clinical specimens. A Mean* (mg/dl) 155-3 350-4 144-9 346-2 Within run graphical representation of the accuracy data, SD 5-7 10-8 2-6 3-4 together with the regression parameters, are pre- CV (%) 3-7 3-1 1-8 1.0 Between run sented in Fig. 1. The Seralyzer total bilirubin system SD 16-8 4-9 11-8 11-9 an demonstrated overall CV in the region of 5-9% CV(%) 10-8 1-4 8-1 3.5 on September 29, 2021 by guest. Protected at the two levels studied, which is perfectly accept- Overall SD 17-8 11-8 12-1 12.4 able for clinical purposes and which falls within the CV (%) 11-4 3-4 8-3 3-6 current state-of-the-art requirements. Correlation studies demonstrated excellent agreement between *To convert from mg/dl to mmolIl, multiply by 0-026.
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