Application of the technicon® RA-1000™ system for the analysis of therapeutic drugs
1
12 EVALUATION OF THE ABBOTT TIPX PROCEDURE FOR THE MEASUREMENT OF SERUM DIGOXIN CONCENTRATION. J.G. Dalton, R.W. Yatscoff, P.R. Desjardins, Clinical Chemistry, Health Sciences Centre, 700 William Ave., Winnipeg, Manitoba, R3E OZ3 Digoxin is one of the most widely-monitored therapeutic drugs, however due to its low concentration in serum the method of choice has been RIA. The nature of RIA analyses is such that "Stat" results and off-hour results are difficult to provide. The TDX procedure was evaluated as a possible replacement for our existing RIA method in the hope that it would provide an improved service capability. The within-run and between-day precision were evaluated using lyophilized quality control material with concentrations of approxi- mately 0.6, 1.7, and Z.6 lag/L. The within-run results gave CV's of 9.6%, Z.7%, and Z.1%; while the between-day results were 14.1%, 5.7%, and 3.4% respectively for the 3 levels. The method was shown to be linear over the working range of the instrument, and the lowest concentration which could be reliably differentiated from zero was 0.1 pg/L. A split-sample method comparison was done on 100 patient samples using the RIA method as the reference. This gave a regression line: TDX = 1.00275 (RIA) - 0.0349, with a correlation coeffi- cient of 0.9797 and Sy/x - 0.1616. Stability of the standard curve was evaluated using four standard solutions and no significant change was seen over a 6 week period. The results were shown to be dependent upon the concentration of protein present in the specimen. This has important implications for high results where dilution of the sample may be necessary. LM. 13 APPLICATION OF THE TECHNICON®RA-1000 SYSTEM FOR THE ANALYSIS OF THERAPEUTIC DRUGS. Herbert Rose and 3acob Levine, Technicon Instruments Corp., Tarrytown, N.Y. 10951 Automated immunoassays lor a variety ol drugs can be run on the RA-1000 system. Described here are the results of the analysis of phenobarbital and phenytoin, using a latex-particle agglutination inhibition methodology. The reagents consist ol a licoll-drug conjugate (RI) and a mono- clonal antibody covalently linked to latex particles (R2). The test is perlormed by adding sample to an aliquot ol RI, incubating t~ minutes, then adding R2. The rate ol particle agglutination inhibition is determined lrom sequential absorbance measurements at 600 nm. The assay is based on the principal that drug in the sample directly competes with the multivalent ticoll-<lrug conjugate Ior antibody combining sites, thereby inhibiting agglutination. Anal)'te Range Precision Sample Phenobarbital 0-80 ug/ml < #% ,2.0 ul Phenytoin 0-30 ug/ml < 0% 2.0 ul Comparative Analyte Method r y_f. n Phenobar tal Ames 0.96 1.05x+2.61 86 Phenytoin Ames 0.97 0.93x-0.18 75 14 ALCOHOL DIPSTICK FOR ETHANOL AND METHANOL, Bhushan M. Kaput and Yedy Israel, Clinical Institute, Addiction Research Foundation, 33 Russell Street, Toronto, Ontario. M5S 2SI Development of an Alcohol Dipstick was reported earlier by our group (i). This dipstick was based on the Alcohol Dehydrogenase(ADHl-NAD system which we found to be specific for ethanol. Methanol does not interfere with this dipstick. We have now developed a dipstick for saliva and serum which will give a positive reaction with both ethanol and methanol. This strip is based on Alcohol Oxidase(AO) as the enzyme catalyst. Paper strips are soaked in Tris Buffer containing Alcohol Oxidase and Peroxidase. The dried strips are coated with tetramethylbenzidine and urea and then attached to a plastic strip. For testing purposes this reagent strip is dipped in the serl~ or a Q-tip soaked in saliva is rolled onto the reagent pad. The colour developed after one minute is read against a graded colour chart. Linear range, (10-160mg/100ml), similar to the ADH sticks (i) was observed. Linearity of this AO system can be increased or decreased by varying the amounts of urea added to the reagent mixture. (i) Kaput B.M. and Israel Y., Clin. Chem., 29, 1178, (1983). CLINICAL BIOCHEMISTRY, VOLUME 17. JUNE 1984 The AO system gives a positive with either ethanol or methanol. This system, in conjuction with the previously developed ethanol specific ADH system (i) can be used in differential diagnosis of methanol overdose before treatment with ethanol has been instituted. METHODOLOGY 15 USE OF SPREADSHEET PROGRAMS TO ASSIST IN PROGRAM DEVELOPMENT. K.L.Masse~, Dept.of Pathology, University Hospi- tal, Saskatoon, Saskatchewan STN OXO. Spread sheet programs such as VisiCalc~, MultiPlan~ and Lotus 1,2,3® have become very popular for data analysis in- volving tabular data (columns of numbers). These can also be used to test the feasibility of programs to do more complex analysis of data, as illustrated in the following example. VisiCalc~, running on the Apple Ile®, was used to test the proposed implementation in PASCAL of the Badenhuijsen & Smit algorithm for Gaussian analysis [Proc.of Medinfo-83 Seminars (1983) 291-293]. A composite curve containing two Gaussian distributions was set up by solving, in two sets of 64 cells, the equation for a normal distribution, and then adding the two together. This was then differentiated by a) calculat- ing the fractional distribution of each cell, b) taking the logarithm of this distribution, and c) finding the slope of the log function by successive differencing. The data were then transferred to the VisiTrend/VisiPlot® Program, which solved by regression analysis the equation of the straight-line segment calculated in (c) above, and al- lowed one to calculate the mean and standard deviation (S.D.) of the original curves. Calculations could be followed at each stage by graphing the functions using the VisiPlot package. A composite curve composed of two distributions with mean = 12, S.D. = 5 and mean = 20, S.D. = 2 was synthesized. Analysis reproduced 2 curves with : mean = 12.6 20.3 S.D. = 5.06 2.15 This is a simple, cost effective way of doing many one-of- a-kind analyses. 16 ASSESSMENT OF SPECIFIC GRAVITY DETERMINED BY AMES N-MULTISTIX-SG. H. Gault, V. Whelan and L. Longerich Depts. of Lab. Medicine a~dicine, General Hospital, St. John's, Nfld., AIB 3V6. Specific gravity (SG) was determined in 204 urines by N- Multistix-SG solid phase test strip. Seventy-seven of the urines were from patients with known renal disease or were positive for protein or glucose, or had pH > 6.5 (Group I) and were assayed for osmolality, SG by urinometer and re- fractometer, and several other parameters. Intercorrel- ations were calculated. Osmolality was used as the gold standard. In Group I, correlation of osmolality with N- Multistix SG (r = 0.66) was not significantly different (p > 0.I) from that found with SG determined by refracto- meter (r = 0.72), or urinometer (r = 0.60). The correlation coefficient for 45 N-Multistix-SG values determined by two staff members was 0.91. For the 127 Group II normal urines, the correlation coefficient between Multistix-SG and SG by refractometer was 0.85. Multistix-SG avoids the errors related to large molecules such as glucose and radiographic contrast media seen with SG determined by urinometer and refractometer and was found to have comparable accuracy to these two methods when compared with osmolality, in patients with known renal disease or abnormal urinalysis. 17 TITLE: A comparison of three methods for the separation of H.D.L. Cholesterol in serum. NAMES: M. Graham, A. Skinner, C. Dymond LOCATION: Clinical Chemistry Div., Halifax Infirmary, 1335 Queen Street, Halifax, NS B3J 2H6 H.D.L. Cholesterol determination is correlated to coronary heart disease risk, However, its' clinical utility is often lessened due to difficulties and imprecision of the precipi- tation method employed. Approximately 20% of all specimens received in this laboratory for H.D.L. Cholesterol determina- tion were not evaluated due to the inability of the Manganese/ Heparin method to effect adequate separation and produce supernatant suitable for analysis. A study was performed to evaluate and compare other pre- cipitation methods. The Manganese/Heparin Method used (x), and recommended by the W.H.O. was compared to the BMC, Cat. No. 543004 Magnesium/Rhosphotungstate Method, y =-0.02 + 201
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Transcript of Application of the technicon® RA-1000™ system for the analysis of therapeutic drugs
12 EVALUATION OF THE ABBOTT TIPX PROCEDURE FOR THE MEASUREMENT OF SERUM DIGOXIN CONCENTRATION. J.G. Dalton, R.W. Yatscoff , P.R. Desjardins, Clinical Chemis t ry , Health Sciences Centre , 700 William Ave., Winnipeg, Manitoba, R3E OZ3
Digoxin is one of the most widely-moni tored therapeu t ic drugs, however due to its low concent ra t ion in serum the method of choice has been RIA. The nature of RIA analyses is such that "Stat" resul ts and off-hour resul ts are diff icult to provide. The TDX procedure was eva lua ted as a possible r e p l a c e me n t for our existing RIA method in the hope that it would provide an improved serv ice capabil i ty.
The within-run and be tween-day precision were evaluated using lyophilized quality control mate r ia l with concent ra t ions of approxi- ma te ly 0.6, 1.7, and Z.6 lag/L. The within-run results gave CV's of 9.6%, Z.7%, and Z.1%; while the be tween-day results were 14.1%, 5.7%, and 3.4% respec t ive ly for the 3 levels. The method was shown to be linear over the working range of the ins t rument , and the lowest concent ra t ion which could be rel iably d i f fe ren t ia ted from zero was 0.1 pg/L.
A spl i t -sample method comparison was done on 100 pat ient samples using the RIA method as the r e fe rence . This gave a regression line: TDX = 1.00275 (RIA) - 0.0349, with a correla t ion coeff i - cient of 0.9797 and Sy/x - 0.1616.
Stability of the standard curve was eva lua ted using four s tandard solutions and no significant change was seen over a 6 week period. The resul ts were shown to be dependent upon the concentra t ion of protein present in the specimen. This has impor tan t implications for high results where dilution of the sample may be necessary.
LM. 13 APPLICATION OF THE TECHNICON®RA-1000 SYSTEM FOR THE ANALYSIS OF THERAPEUTIC DRUGS. Herber t Rose and 3acob Levine, Technicon Ins t ruments Corp., Tarry town, N.Y. 10951
Automated immunoassays lor a var ie ty ol drugs can be run on the RA-1000 system. Described here are the results of the analysis of phenobarbital and phenytoin, using a la tex-par t ic le agglut inat ion inhibition methodology.
The reagen ts consist ol a l icoll-drug conjugate (RI) and a mono- clonal antibody covalen t ly linked to latex par t ic les (R2). The tes t is pe r lo rmed by adding sample to an aliquot ol RI, incubating t~ minutes, then adding R2. The ra te ol par t ic le agglut inat ion inhibition is de te rmined l rom sequential absorbance measu remen t s a t 600 nm.
The assay is based on the principal tha t drug in the sample di rect ly compe te s with the mul t iva lent ticoll-<lrug conjugate Ior antibody combining sites, thereby inhibiting agglut inat ion.
Anal) ' te Range Precision Sample
Phenobarbital 0-80 ug/ml < #% ,2.0 ul Phenytoin 0-30 ug/ml < 0% 2.0 ul
Compara t ive Analyte Method r y_f. n
Phenobar tal Ames 0.96 1.05x+2.61 86 Phenytoin Ames 0.97 0.93x-0.18 75
14 ALCOHOL DIPSTICK FOR ETHANOL AND METHANOL, Bhushan M. Kaput and Yedy Israel, Clinical
Institute, Addiction Research Foundation, 33 Russell Street, Toronto, Ontario. M5S 2SI
Development of an Alcohol Dipstick was reported earlier by our group (i). This dipstick was based on the Alcohol Dehydrogenase(ADHl-NAD system which we found to be specific for ethanol. Methanol does not interfere with this dipstick.
We have now developed a dipstick for saliva and serum which will give a positive reaction with both ethanol and methanol. This strip is based on Alcohol Oxidase(AO) as the enzyme catalyst.
Paper strips are soaked in Tris Buffer containing Alcohol Oxidase and Peroxidase. The dried strips are coated with tetramethylbenzidine and urea and then attached to a plastic strip.
For testing purposes this reagent strip is dipped in the serl~ or a Q-tip soaked in saliva is rolled onto the reagent pad. The colour developed after one minute is read against a graded colour chart. Linear range, (10-160mg/100ml), similar to the ADH sticks (i) was observed. Linearity of this AO system can be increased or decreased by varying the amounts of urea added to the reagent mixture.
(i) Kaput B.M. and Israel Y., Clin. Chem., 29, 1178, (1983).
CLINICAL BIOCHEMISTRY, V O L U M E 17. JUNE 1984
The AO system gives a positive with either ethanol or methanol. This system, in conjuction with the previously developed ethanol specific ADH system (i) can be used in differential diagnosis of methanol overdose before treatment with ethanol has been instituted.
METHODOLOGY
15 USE OF SPREAD SHEET PROGRAMS TO ASSIST IN PROGRAM DEVELOPMENT. K.L.Masse~, Dept.of Pathology, University Hospi- ta l , Saskatoon, Saskatchewan STN OXO.
Spread sheet programs such as VisiCalc~, MultiPlan~ and Lotus 1,2,3® have become very popular for data analysis in- volving tabular data (columns of numbers). These can also be used to test the feas ib i l i t y of programs to do more complex analysis of data, as i l lustrated in the following example.
VisiCalc~, running on the Apple Ile®, was used to test the proposed implementation in PASCAL of the Badenhuijsen & Smit algorithm for Gaussian analysis [Proc.of Medinfo-83 Seminars (1983) 291-293]. A composite curve containing two Gaussian distributions was set up by solving, in two sets of 64 cel ls, the equation for a normal distr ibution, and then adding the two together. This was then differentiated by a) calculat- ing the fractional distr ibution of each cel l , b) taking the logarithm of this distr ibution, and c) finding the slope of the log function by successive differencing.
The data were then transferred to the VisiTrend/VisiPlot® Program, which solved by regression analysis the equation of the straight- l ine segment calculated in (c) above, and al- lowed one to calculate the mean and standard deviation (S.D.) of the original curves. Calculations could be followed at each stage by graphing the functions using the VisiPlot package.
A composite curve composed of two distributions with mean = 12, S.D. = 5 and mean = 20, S.D. = 2 was synthesized.
Analysis reproduced 2 curves with : mean = 12.6 20.3 S.D. = 5.06 2.15 This is a simple, cost effective way of doing many one-of-
a-kind analyses.
16 ASSESSMENT OF SPECIFIC GRAVITY DETERMINED BY AMES N-MULTISTIX-SG. H. Gault, V. Whelan and L. Longerich Depts. of Lab. Medicine a ~ d i c i n e , General Hospital, St. John's, Nf ld . , AIB 3V6.
Specific gravi ty (SG) was determined in 204 urines by N- Mult ist ix-SG sol id phase test s t r ip . Seventy-seven of the urines were from patients with known renal disease or were posi t ive for protein or glucose, or had pH > 6.5 (Group I) and were assayed for osmolal i ty, SG by urinometer and re- fractometer, and several other parameters. In te rcor re l - ations were calculated. Osmolality was used as the gold standard. In Group I , corre lat ion of osmolal i ty with N- Mu l t i s t i x SG (r = 0.66) was not s ign i f i can t l y d i f fe ren t (p > 0 . I ) from that found with SG determined by ref racto- meter (r = 0.72), or urinometer (r = 0.60). The corre lat ion coef f i c ien t for 45 N-Multistix-SG values determined by two s ta f f members was 0.91. For the 127 Group I I normal urines, the corre lat ion coe f f i c ien t between Mult ist ix-SG and SG by refractometer was 0.85.
Mult ist ix-SG avoids the errors related to large molecules such as glucose and radiographic contrast media seen with SG determined by urinometer and refractometer and was found to have comparable accuracy to these two methods when compared with osmolal i ty, in patients with known renal disease or abnormal ur inalys is .
17 TITLE: A comparison of three methods for the
separation of H.D.L. Cholesterol in serum.
NAMES: M. Graham, A. Skinner, C. Dymond
LOCATION: Clinical Chemistry Div., Halifax Infirmary, 1335 Queen Street, Halifax,
NS B3J 2H6
H.D.L. Cholesterol determination is correlated to coronary heart disease risk, However, its' clinical utility is often
lessened due to difficulties and imprecision of the precipi-
tation method employed. Approximately 20% of all specimens
received in this laboratory for H.D.L. Cholesterol determina- tion were not evaluated due to the inability of the Manganese/
Heparin method to effect adequate separation and produce
supernatant suitable for analysis.
A study was performed to evaluate and compare other pre-
cipitation methods. The Manganese/Heparin Method used (x), and recommended by the W.H.O. was compared to the BMC, Cat.
No. 543004 Magnesium/Rhosphotungstate Method, y =-0.02 +
201
