Adaptation o f EMIT D rug Assays to a Random-Access Automated Clinical Analyzer*
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1 AN N A LS O F CLINICAL A N D LABORATORY SC IE N C E, Vol. 15, No. 1, Copyright 1985, Institute for Clinical Science, Inc. Adaptation o f EMIT D rug Assays to a Random-Access Automated Clinical Analyzer* JAMES C. BOYD, M.D., M. GERALDINE SAVORY, R.T.(CSLT), MARILYN MARGREY, M.T.(ASCP), DAVID A. H EROLD, M.D.. P h.d., JAMES R. SH IPE, P h.d. and JOHN SAVORY, P h.d. Department of Pathology, University of Virginia Medical Center, Charlottesville, VA ABSTRACT Enzym e m ultiplied im m unoassay technique (EMIT ) assays for th e ophylline, carbam azepine, phenytoin, phénobarbital, valproic acid, acetaminophen, gentamicin and tobramycin, have been adapted to the Technicon RA-1000 random access analyzer. Working reagents are stable for a period of at least one week, and calibration need only be perform ed when preparing fresh reagents. Between-day precision ranged from 2.2 to 5.5 percent and correlations with similar EMIT assays perform ed on a centrifugal analyzer w ere satisfactory. Excellent results w ere obtained on pro ficiency testing samples when analyzed for theophylline, phénobarbital, phenytoin and acetam inophen. The procedures have been adapted to em ergency testing of these four drugs. Introduction O ne of the earliest autom ated analyzers for p erform ing the enzym e m u ltiplied immunoassay technique (EMIT )f drug m easurem ents was the centrifugal analyzer.2 3 4,5 6,9 These analyzers w ere ideally suited to kinetic analyses since they could readily perform m ultipoint absorbance m easu rem en ts, m aintain high throughput, and also possessed soft * Address reprint requests to: Janies C. Boyd, M.D., University of Virginia Medical Center, Box 168, Charlottesville, VA t Syva Company. ware allowing on-line data reduction for standard curve fitting and calculation of results. A major problem with the centrifugal analyzer and other similar systems is that they m ust be operated in a batch mode and are m ost efficient w hen all of the samples for a given test are available at the same time. M ore recently, random access analyzers have been introduced where m ultiple tests can be perform ed on individual specim ens. These in stru m ents can be program m ed to handle a variety of analytical approaches which include eq u ilib riu m, zero, and firsto rd er rate m easu rem en ts. O ne of th e /85/ $00.90 Institute for Clinical Science, Inc.
2 40 BOYD, SAVORY, MARGREY, HEROLD, SHIPE, AND SAVORY m ost popular of these analyzers is the Technicon RA-1000 which possesses flexibility in sam ple and reag e n t volum e, te m p e ra tu re of o p eratio n, and two reagent capability.1,7,8 Recently, addition of software allowing EMIT m ethods to be perform ed has b een introduced The present investigation was initiated to adapt several EMIT procedures to the Technicon RA-1000 and to assess the perform ance of these assays. Materials and Methods A p p a r a t u s 1. Hardware: A random access clinical analyzer* was operated according to the m anufacturer s instructions. Some details of the instrum ent characteristics and perform ance have b een re p o rte d previously.1,7,8 2. Softw are: Program s specifically desig n ed to service a variety of im m unoassays becam e available for th e RA-1000 with the release of Version 2.0 software. U nder com puter control, the analyzer takes a series of nine absorbance readings spaced at 15 second intervals. Once the nine absorbance readings have been taken, the RA-1000 co m p u ter allows th e u se r to select a variety of options in estim ating the rate of change of absorbance. For th e E M IT assays described in the present study, the computer fits a quadratic equation to the nine absorbance readings and o u tp u ts the m ean rate of change estim ated by evaluating th e first d eriv ativ e of the q u a dratic equation at the m idpoint in tim e (referred to as the Q value). To derive a standard curve, the curve fitting option on the RA-1000 is em ployed that does a cubic regression fit to the log-logit transformed Q values of the six EM IT assay standards. The standard curve is stored by the instrum ent for use w ith subsequ en t analyses until th e assay is recalibrated. The RA-1000 software does not p resently offer th e four p a ra m e te r or five param eter logistic m odels for fitting standard curves, nor does the software support an Ro adjust option to allow adjustm en t of th e stan d ard curve based on e n try of th e rate of th e zero standard each day that the assay is perform ed. To allow the concurrent evaluation of these models in fitting the data derived from th e RA-1000, a curve fittin g softw are developed for the H ew lett-packard computer by Roche Diagnostic System sf was used. Q values for th e stan d ard s and unknowns from the RA-1000 EM IT runs w ere h a n d -e n te re d into th e H ew lett- Packard com puter and fitted by either th e four p a ra m e te r or five p a ra m ete r logistic m odels. T he algorithm w hich provided the b etter fit to a given set of data was selected. R0 rate adjustm ents were supported by the Roche software for either model. Results w ere collated for the data with and without the R0 rate adjustm ent. 3. Com parison M ethods: C om parison procedures were carried out on a centrifugal analyzer^ and used the same EMIT reagents as those to be described for the Technicon RA S a m p l e s Serum sam ples for precision and correlation studies were patient specimens subm itted for analysis to the University of Virginia M edical C en ter Toxicology Laboratory. * RA-1000, Technicon Instrum ents Inc., Tarrytown, NY t Division of Hoffman LaRoche, Nutley, NJ Í Cobas Bio, Roche Diagnostic Systems.
3 ADAPTATION OF EMIT DRUG ASSAYS 41 C o n t r o l s 1. L a boratory-p repared C ontrols: Six h u n d re d m illiliters of blood bank plasm a w ere defibrinated by adding calcium chloride. The resulting serum (zero drug concentration) was used to prepare two drug pools. Phenobarbital, carbamazepine, phenytoin, valproic acid, and theophylline w ere w eighed on an analytical balance and added to a m easured volum e of one pool to achieve concentrations approaching each drug s upper linear range (high drug concentration). The zero and high drug concentrations were then pipetted volumetrically in different proportions to yield five levels of control encompassing each drug s linear range. Similarly, five levels of controls for gentamicin and tobramycin were prepared. 2. Com m ercial Controls: Two com m ercial sources of controls w ere e m ployed: (i) O rth o B i-l evel A ssayed A nticonvulsant/antiasthmatic Control Set I, I lj (ii) Therapeutic D rug Control TDC II and III. 11 R e a g e n t s C om m ercial te st kits for all of the EMIT procedures w ere obtained from Syva Company.** F or the centrifugal analyzer com parison m ethods, the reagent dilutions and m ethod param eters were as described in th e Cobas Bio EMIT C h em istry Adaptations. Reagent A (containing antibody against the drug, glucose-6-phosphate and nicotinam ide adenine dinucleotide [NAD]) and Reagent B (containing the drug bound to glucose 6-phosphate dehydrogenase in an enzym e complex) were reconstituted with 6.0 ml of deionized w ater. T he Tris-H C l buffer was diluted to 128 ml with deionized water. Reagent A then was diluted ten-fold with buffer to provide a working reagent. The calibrators w ere p repared according to th e m anufacturer s instructions. The reagents for the Technicon RA m ethods w ere prepared as follows: 1. Stock Reagents: Reagents A and B, buffer solution and calibrators were p re p a re d and sto red according to th e m anufacturer s instructions. 2. Working Reagents: Stock Reagents A and B were diluted 1:8 with buffer. Working reagents were prepared either daily or in sufficient quantities to last for a period of seven days. a. Working Reagents Prepared Daily: Stock Reagents A and B were rem oved from the refrigerator, allowed to reach room tem perature and diluted one part with eight parts buffer. Calibration was perform ed on Day 1 and the laboratory and commercial controls were assayed. The controls were then analyzed daily for periods of betw een 12 to 17 days. b. Working Reagents Prepared in Bulk: Stock R eagents A and B w ere diluted one part with eight buffers in sufficient quantity to be used over a sevenday period. These working reagents were stored refrigerated in ground glass stoppered Erlenm eyer flasks. Each day fresh aliquots of A and B w ere poured into new reagent boats. Reagents were allowed to equilibrate to room tem perature before use and unused portions discarded. Calibration was perform ed on the first day and laboratory and commercial controls analyzed each day over th e one-w eek period. Alltech Associates, Inc., Deerfield, IL Ortho Diagnostic Systems, Rontan, NJ ICL Scientific, Fountain Valley, CA ** Palo Alto, CA P r o c e d u r e s The p a ram eters for all of th e drug assays on the Technicon RA-1000 and the
4 42 BOYD, SAVORY, MARGREY, HEROLD, SHIPE, AND SAVORY TABLE I Parameters for Drug Assays T e c h n ic o n R A C o b a s B i o stability of th e in stru m en t s initial calib ration p e rfo rm ed on day 1. An R0 adjustm ent was required for gentamicin and tobram ycin but was not found to be necessary for the o th er drugs. S a m p le ( y l ) D i l u e n t ( p i ) 0 15 R e a g e n t A ( p i ) s t. D e la y ( s e c ) R e a g e n t B ( p i ) n d D e la y ( s e c ) 15 5 I n t e r v a l ( s e c ) N u m b er o f r e a d i n g s 9 7 T e m p e r a tu r e ( c) W a v e le n g th (nm) Cobas Bio com parison in stru m e n t are shown in table I. Results and Discussion C o r r e l a t i o n S t u d i e s Fifty patient samples w ere analyzed in duplicate on both th e Cobas-Bio centrifugal analyzer and the RA-1000 analyzer for each of th e seven drugs and the results are shown in table III. The values obtained using the RA-1000 w ere generally slightly higher. The accuracy of some of the proposed RA-1000 m ethods was verified by analysis of proficiency test sam ples.* These results are shown in table IV. P r e c i s i o n S t u d i e s W hen working dilutions of reagents A and B were prepared daily, precision was found to be unacceptable for three drugs investigated. C arbam azepine, phenytoin, and p h e n o b a rb ita l gave relativ e sta n d a rd deviations of 19.1, 18.0, and 18.5 percent, respectively. This lack of precision may have resulted from dilutional erro rs since sm all b u t variable amounts of reagent w ere prepared daily as needed to minimize waste. W hen reagents A and B w ere prepared in sufficient quantity to last for a period of at least 7 days, precision was greatly improved. The results given in table II show excellent precision and reflect good A p p l ic a b il i t y t o E m e r g e n c y D r u g A n a l y s e s The use of the RA-1000 was investigated for the determ ination of acetam inophen, th eo p h y llin e, p h en y to in, and phenobarbital in em ergency situations. Unlike routine therapeutic drug m onitoring, these analyses should be available day or night with reliable rapid results and m inim al technical difficulty. W ith reagent preparation and in stru m en t calibration being perform ed weekly, warm ing reagents to room te m p e ra tu re * TDM survey samples, American Association for Clinical Chemistry, Washington, DC TABLE I I Precision Studies D rug T h e o p h y l l i n e C a rb a m a - z e p i n e P h e n y t o i n P h e n o b a r b i t a l V a l p r o i c A c id G e n ta m ic i n * T o b r a m y c in * N (N u m b er o f d a y s ) M ean ( p g /m l) IQ SD (ja g /m l) RSD (%) * T h e s e a s s a y s r e q u i r e d a d a i l y RQ r a t e a d j u s t m e n t.
5 ADAPTATION OF EMIT DRUG ASSAYS 43 TABLE I I I Correlation Studies* D ru g T h e o p h y l l i n e C a rb a m a - z e p i n e P h e n y t o i n P h é n o b a r b i t a l V a l p r o i c a c i d G e n ta m ic i n T o b r a m y c in S l o p e Y - I n t e r c e p t ( u g /m l) S. E. E r * F i f t y p a t i e n t s a m p l e s, x = c e n t r i f u g a l a n a l y z e r, y = R A required more tim e than the actual analysis and reporting of results. It was questio n ed w h e th e r pre-w arm ing w ould really be necessary since the reagents are dispensed using a h eated transfer arm assem bly. Bulk d ilutions of th e two E M IT reag en ts w ere p re p a re d (as described earlier) for each of the drugs and placed in acid-washed (25 mm I. D. x 40 mm H) w eighing b o ttles w ith ground-glass stoppers. The bottles were firmly positioned in the reagent tray by placing them in pre-cut plastic reagent boat containers. A maximum of six EM IT analyses could be assigned to each reagent tray. The entire tray stored at 4 C could be rem oved, used im m ed i ately, and returned to 4 C with no waste of reagents. Fresh dilutions of reagents could th en be com bined w ith old dilutions of the same lot num ber of reagents prior to recalibration. Twenty-five serum samples were analyzed for each drug by the p rese n t m ethod and th e centrifugal TABLE IV Results of TDM Survey Samples Phenytoin Th eophylline Acetaminophen Phénobarbital (yg/m l) ( vg/ml) (\xg/ml) 13.6 ( 13.0 ) 5.2 ( 5.2 ) (100 ) 21.3 (20.2 ) 8.2 ( 8.2 ) 11.9 ( 12.06) 23.6 ( 21.3 ) 27.1 (25.7 ) 3.3 ( 3.3 ) 11.2 ( ) 55.3 ( 51.3 ) 62.7 (61.2 ) 17.6 (17.3 ) 7.1 ( 7.5 ) 3.9 ( 4.1 ) 9. 8 ( 8.3 ) 12.6 (12.1 ) 8.1 ( 8.4 ) 332 (307 ) 7.5 ( 6.1 ) 19.8 ( 19.4 ) 12.2 ( ) 4.3 ( 4.1 ) 11.1 ( 8.1 ) ( ) ( ) 512 (5 0 7 ) RA-1000 r e s u l t f o llo w e d ( i n p a r e n t h e s e s ) b y t h e m ean r e s u l t o f a l l l a b o r a t o r i e s p a r t i c i p a t i n g i n t h e TDM s u r v e y. analyzer tech n iq u e. T he correlations w ere n o t significantly d ifferen t from those in table III (data not shown). For each drug, two calibrators and one q uality control sam ple w ere ru n daily over a period of 11 days. W ith this tim e saving procedure and m inim al reagent consum ption, the day-to-day precision was similar to that shown in table II. Regression analyses on 14 patient sam ples containing acetam inophen gave a correlation coefficient of w ith centrifugal analyzer = 1.02 (RA-1000) T he accuracy of this te c h n iq u e was assessed by analyzing as unknowns ran dom ly selected sam ples from m onthly TD M proficiency testin g program s. These samples had been stored at 20 C for periods of one to two years. In table IV are co n tain ed re p re sen ta tiv e data from th a t study w hich d em o n strates ex cellen t ag re e m e n t w ith th e m ean re su lt from 250 laboratories using th e enzym atic im m unoassay procedure. The p rese n t investigation has dem onstrated that EMIT drug assays can be adapted to the Technicon RA-1000 clinical analyzer which allows these m easurem ents to be p erfo rm ed in a random access mode. Calibration is m inimal for the tests studied and working reagents can be stored at 4 C for several days. Em ergency testing can be perform ed conveniently w ith o u t b rin g in g the reagents to room tem perature.
6 44 BOYD, SAVORY, MARGREY, HEROLD, SHIPE, AND SAVORY Acknowledgments The authors would like to acknowledge the skilled technical assistance of Mrs. Judith A. Sinn and Mr. James H. Bowden, III. References 1. B r o m b e r g, I. L., P o l l a r d, A., C h e n g, J., and R o m a s c h i n, A.: An evaluation of the Technicon RA-1000 random access analyzer. Clin. Chem. 30: , F i n l e y, P. R., W i l l i a m s. R. J., and B y e r s, J. M.: Assay of phenytoin: Adaptation of EM IT to the centrifugal analyzer. Clin. Chem. 22: , F i n l e y, P. R., W i l l i a m s, R. J., L i c h t i, D. F. et al.: A ssay o f phenobarbital: A daptation of EMIT to the centrifugal analyzer. Clin. Chem. 23: , L a c h e r, D. A., S i n n, J. A., S a v o r y, J., and W i l l s, M. R.: H om ogeneous enzym e im m u noassay for theophylline with a centrifugal analyzer (Centrifugal analyzer theophylline EMIT assay). Ann. Clin. Lab. Sci. 10: , L â c h e r, D. A., S i n n, J. A., W i l l s, M. R., and S a v o r y, J. : Rapid centrifugal analyzer enzym e immunoassays for diphenylhydantoin, phénobarbital and p rim id one. Am. J. C lin. Pathol. 74: , L â c h e r, D. A., V a l d e s, R., and S a v o r y, J.: Enzym e immunoassay of carbamazepine with a centrifugal analyzer. Clin. Chem. 25: , S c h w a r t z, M. K., S t a t l a n d, B. E., C o u g l i n, J. et al.: Chemical and clinical evaluation of the random access analyzer RA Clin. Chem. 30: , S m i t h, J., S v e r j a k, D., and T e l i c u, D.: An innovative technology for random access sampling. Clin. Chem. 28: , W e n t z, P. W., S h i p e, J. R., and S a v o r y, J.: Enzyme immunoassay o f phenobarbital with a centrifugal analyzer. Clin. Chem. 22:1207, 1976.
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