Anal. Chem. 1983, 55, 202R-214R (167) Nal-Kul, S.; Wen-Tien, C.; Fu-Sheng, W.; Shan-Shan, S . Analyst (London) 1981. 706. 1229-1233. (168) Mochlzukl, T:; Toda, Y.; Kuroda, R. Talanta 1982, 2 9 , 659-662. (169) Sarma, R. N. S.; Majumdar, M. K. J . Indian Chem. SOC. 1982, 5 9 , 790-791. (170) Narayanan, A.; Pantony, D. A. Analyst (London) 1981, 706, 1145-1 149. (171) Wang, 2.; Cheng, K. L. Talanta 1982, 2 9 , 551-556. (172) Carel, A. B.; Wlmberly, J. W. Anal. Lett. 1982, 75, 493-505. (173) Sarkay, R. C.; Das, M. S. Anal. Chim. Acta 1982, 734, 401-405. (174) Victor, A. H.; Streiow, F. W. E. Anal. Chlm. Acta 1982, 738, 285-294. (175) Perez Rulz, T.; Sanchez-Pedreno, C.; Ortuno, J. A. Analyst (London) 1982, 707, 165-189. (176) Sanz-Mendei, A.; Diaz Garcia, M. E. Ana/yst (London) 1981, 706, 1268- 1274. (177) Von Borstel, D.; Halbach, P. Fresenlus' Z . Anal. Chem. 1982, 370, 431-432. (178) Rakovskii, E. E.; Zdorova, E. P.; Kullgln, V. I.; Popova, N. N.; Flshkova, N. L.; Shvedova, N. V. Zavod. Lab. 1982, 4 6 , 11-12. C A , 9 7 : 229337r. (179) Sastrl, V. S. Talanta 1982. 2 9 , 405-406. (180) Rao, T. P.; Ramakrlshna, T. V. Analyst(London) 1982, 707, 704-707. (181) Mochlzukl, T.; Kuroda, R. Analyst (London) 1982, 707, 1255-1260. C A , 96: (182) Becker, S.; Dietze, H. J. ZfI-Mitt. 1981, 4 6 , 49-59. 1545638. (183) Rucklidge, J. C.; Gorton, M. P.; Wilson, G. C.; Klllus, L. R.; Litherland, A. E.; Elmore, D.; Gove, H. E. Can. Mlneral. 1982, 2 0 , 111-119. (184) Salto, M.; Sudo, E. Nippon Klnzoku Gakkalshi 1982, 4 6 , 691-695. C A , 9 7 : 192363t. (185) Radermacher, L.; Beske, H. E. Fresenius' Z . Anal. Chem. 1981, 309, 319-324. C A , 9 6 : 6 2 1 7 6 ~ . (186) Date, A. R.; Gray, A. L. Analyst (London) 1981, 706, 1255-1267. (187) Knab, H. J. Geochlm. Cosmochlm. Acta 1981, 4 5 , 1563-1572. (168) Jacobs, M. L. J . CoalQual. 1981, 7, 20-22, 24. (189) Van Puymbroeck, J.; Gljbels, R. Fresenlus' Z . Anal. Chem. 1981, 309, 312-315. (190) Nelson, J. H.; MacDougall, J. J.; Baglin, F. G.; Freeman, D. W.; Nadler, M.; Hendrlx, James L. Appl. Spectrosc. 1982, 3 6 , 574-576. (191) Bahgat, A. A.; Fayek, M. K. Phys. Status SolidiA 1982, 7 7 , 575-581. (192) Giulianelli, J. L.; Williamson, D. L. At. Nucl. Methods Fossil Energy Res. (Proc. Am. Nucl. SOC Conf .) 7980 1982, 443-458. (193) Inazuml, A.; Isobe, T.; Tarutanl, T. Geochem. J . 1981, 75, 325-332. (194) Maquet, M.; Cetveile, B. D.; Gouet, G. Mlner. Deposlta 1981, 76, 357-373. (195) Narasimham, N. A. Pure Appl. Chem. 1982, 5 4 , 641-846. (196) Grasserbauer, M. Angew. Chem. 1981, 9 3 , 1059-1068. C A , 96: 78948s. (197) Benton, J. L.; Klmerllng, L. C. J . Electrochem. SOC. 1982, 729, 2098-2102. (196) Ishlzaki, H.; Tu, A. T. Appl. Spectrosc. 1982, 3 6 , 587-588. (199) Mulay, L. N.; Mulay, I. L. Anal. Chem. 1982, 5 4 , 216R-227R. (200) Dolaberldze, L. D.; Kamkamidze, D. K.; Dzhallashvlll, A. G.; Alkhazlshvili, T. M. Soobshch. Akad. Nauk Gruz. SSR 1982, 705, 279-300. CAI 9 7 : 1197159.
.
(201) Varma, A. Talanta 1981, 2 8 , 705-787. (202) Neeb, R. Pure Appl. Chem. 1982, 5 4 , 847-852. (203) Barker, J. F.; Chatten, S. Chem. Geol. 1982, 3 6 , 317-323. (204) Luktuke, R. D.; Gopalaraman, C. P.; Rohatgi, V. K. Curr. Sci. 1981, 5 0 , 1055-1057. (205) Amosse, J.; Fouletier, J.; Kleitz, M. Bull. Mineral. 1982, 705, 188-1 92. (206) Mltchell, J. W. Pure Appl. Chem. 1982, 5 4 , 819-834. (207) DeLong, S. E.;Lyman, P. Chem. Geol. 1982, 35, 173-176. (208) Barnes, R. M. Trends Anal. Chem. 1981, 7 , 51-55. (209) Broekaert, J. A. C. Trends Anal. Chem. 1982, 7, 249-253. (210) IUPAC Pure Appl. Chem. 1982, 5 4 , 1565-1577. (211) Javler-Son, A. ASTM Spec. Tech. Pub/. 1981, 747, 4-20. (212) Tschoepel, P. Pure Appl. Chem. 1982, 5 4 , 913-925. (213) De Groot, A. J.; Zschuppe, K. H.; Salomons, W. Hydrobiologia 1982, 9 7 -92, 689-695. (214) Vankova, V.; Minarik, L.; Houdkova, 2. Acta Mont. 1981, 5 7 , 101-111. (215) Kahn, H. L. Ind. Res. Dev. 1982, 2 4 , 150-153. (216) Zolotov, Y. A. Nat ., Aim Methods Microchem., Proc. Int. Microchem. Symp., 8th 7960 1981, 231-256. (217) Erzlnger, J.; Puchelt, H. Errmetall 1982, 3 5 , 173-179. C A , 9 7 : 48871q. (218) Hochheimer, J. T. Preclous Met. (Proc. Int. Precious Met. Inst. Conf.) 5th 7967, 1982, 333-341. (219) Kratochvil, B.; Taylor, J. K. Chemtech 1982, 72, 564-570. (220) Khvostova, V. P.; Golovnya, S. V. Zavod. Lab. 1982, 4 6 , 3-7. C A , 9 7 : 229156f. (221) Kodymova, A. Geol. Pruzkum 1982, 2 4 , 56-57. CA 97: 4 1 9 7 1 ~ . (222) Bewers, J. M.; Windom, H. L. Mar. Chem. 1982, 7 7 , 71-86. (223) Kuz'min, N. M. Zavod. Lab. 1982, 4 6 , 11-15, C A , 96: 192392f. (224) Schwedt, G. LaborPraxls 1982, 6, 452, 454, 459-460. C A , 97: 28201t. (225) Frlglerl, P. Int. Envlron . Saf. 1982, 36-37. (226) Mltchell, J. W. J . Radioanal. Chem. 1982, 6 9 , 47-105. (227) Dubansky, A.; Straka, P. Chem. Llsty 1982, 76, 430-434, C A , 97: 48738b. (228) Lister, B. Geostandards Newsletter 1982, VI, 175-205. (229) Steger, H. F. Geostandards Newsletter 1982, VI, 249-255. (230) Kramer, U.; Puchelt, H. Geostandards Newsletter 1982, VI, 221-227. (231) Date, A. R. Anal. Proc. (London) 1982, 79, 7-12. (232) Ring, E. J.; Hansen, R. G.; Steele, T. W. Rep.-MINTEK 1981, M 2 , 31 PP. (233) Flora, L.; Matteuccl, E.; Restlvo, G.; Sandrone, R. Rend. SOC. Ita/. Mlneral. Petrol. 1981, 3 7 , 517-524, C A , 9 6 : 184617t. (234) Lange, J. Sllikaftechnlk 1981, 3 2 , 362-363. C A , 96: 186012r. (235) Dletze, H. J. ZfI-Mitt. 1981, 46, 13-47. C A , 9 8 : 154562d. (236) Potts, P. J.; Thorpe, 0. W.; Watson, J. S . Chem. Geol. 1981, 3 4 , 331-352. (237) Jaffrezlc, H.; Joron, J. L.; Treuil, M. J . Radioanal. Chem. 1982, 69, 235-238. C A , 96: 1734404. (238) Bastin, J.; Bomans, M.; Dugaln, F.; Mlchaut, C.; Pujade-Renaud, J. M.; Van Audenhove, J. Analusis 1982, 70, 253-265. C A , 97: 103444e. (239) Marchandise, P.; Olie, J. L.; Robbe, D.; Legret, M. Environ. Techno/. Lett. 1982, 3 , 157-166. C A , 9 7 : 48873s.
CIinicaI Chemistry J. E. Davis," Robert L. Soisky, Linda Giering, and Saroj Malhotra Clinical Systems Division, E.
I. do Pont de Nemours and Company, Wilmington, Delaware 19898
This review covers the nominal time period from November 1980 to November 1982. The clinical chemistry literature is so extensive that we have chosen to review only a portion covering four main topics where significant change has occurred: instrumentation, ion selective electrodes, immunoassay techniques, and analytes of clinical interest. Clinical chemistry is a mature field having evolved from the "side-room" testing once practiced by physicians. The methodology and instrumentation for today's common tests evolved rapidly during the past 2 decades. These common tests are general metabolic indicators which are perturbed by many diseases. The newer tests evolving from research in molecular biology and medicine are more disease specific. The clinical needs for diagnosis and patient management are met by tests which are (1)sensitive to the presence of disease, (2) indicative of degree or extent of disease, (3) specific for a 202 R
0003-2700/83/0355-202R$01.50/0
disease, (4) robust in methodology, (5) precise and accurate, and (6) economical. This sequence makes clear that clinical utility of a test must be established before substantial method development is warranted. Some analytes discussed in the Analytes of Clinical Interest section are early in the sequence of evolution. Analytes in the immunoassay Techniques and Ion Selective Electrodes sections are more established, while the most common tests are implicit in the section on Instrumentation. Additional information on specific topics can be found in Chemical Abstracts, Biological Abstracts, and Index Medicus. Medline and other computer literature searches are also available. In the United States the most popular journal in this field is Clinical Chemistry (Winston-Salem). Other popular journals are Clinica Chimica Acta, American Journal of Clinical Pathology, N e w England Journal of Medicine, 0 1983 American
Chemical Society
CLINICAL CHEMISTRY
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R o b r t L Solsky b a r-mh chnnw h !he Now Products RESMrch Mv)slon 01 the phom Fnxbcls Dspmnml. E. 1. du pontds Nomom 6 Co. He received M R . D . in 1980 at me state untversny 01 New York at Buffalo wnh Prol~sorG. A. Recmiiz and his B.A. In 1975 horn ImCa Cailsge. ImSca. NY. lnaw me dbsctbn ol RoteH. F. Koch.
In 1975 lnaw the dteRlDn of Colin Steel. Prior 10 ]oh- NEN. she was Msnao er of Applied Research fw me G a y m m n l Systems Divlslon 01 Bald Corporation *mere she worked on me devebpment 01 instruments and memodoloey mnpioylnp IkmewnCa spectroscopy. ersw
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Lancet, and Journal of the American Medical Association.
INSTRUMENTATION Article8 specifically discussing principles, design, and evaluation regularly ap ear in journals such as Clinical Chemistry. Anolytical (!hemsryit, Annals of Clinical Biochemistry, Clinica Chimica Acta, and the Journul of Automatic Chemistry. A new journal covers thm area specifically, the Journal of Clinical Laboratory Automotion. The great number of samples analyzed in the clinical chemistry laboratory justifies specialized instrumentation. A typical hospital-based instrument for common routine tests will process 15(tlooO tests/h although instrumente exist which can process up to 9600 tests/h. Throughput is not the only measure of need. New instruments are 'selective"; sample and reagent are consumed only on those tests which are requested. They permit urgent samples to be processed immediately without seriously disrupting routine or batch prccessing. Virtually all new clinical chemistry instruments are microprocessor controlled. However, the convenience of communication in the operators native language can lead to substantial costs. Thus, some efforts have been directed toward pictographic communication (1). A pH meter from
Beckman Instruments is an example of pictographic communications. The majority of automated instruments in the clinical chemistry lab are based on well-established photometric techniques. This is not simply a reluctance to accept new technology but a m u l t of the complex nature of the samples. There is a concern that new technologies will have yet unknown interferences that will affect the clinical interpretation. Even so, in the past decade automated instruments have replaced the flame-photometric technique by ion-selective electrodes for measuring Na and K. Fluorometry is increasingly used in immunoassay systems where hi h sensitivity is needed. Below, the automated systems are &cussed according tothe dominant measurement technique: photometry, reflectometry, fluorometry and particle counting. Photometry. A new generation of multitest instruments features 'selective" testing, using a single photometer which reads multiple cuvettes multiple times. This contrasts with prior instruments which either used a photometer per test type (channel) or completed the reading of one cuvette before beginning the next. Microprocessors have made such an a p proach practical as considerable bookkeeping is required for selecting reagents, sample size, and wavelength, collating the multiple readings, checking data integrity, and calculating results. Three recent instruments exemplify this new generation. Yet an interesting diversity exists in the mechanics of imup plementation. The DACOS (Coulter Electronics) analb 600 tests/h utilizing up to 24 reagents. Three carousels are used: one each for samples, cuvettes, and reagents. Every 6 s the cuvette carousel rotates to the next station: sample addition, reagent addition and mixing, incubation, and washing. During the stationary phase, a rotating photometer reads every cuvette to provide data for end-point and rate measurements. The Hitachi 705 (distributed by Boehringer Mannheim Diagnostics) analyzes up to 180 tests/h utilizing up to 16 reagent pairs, 6 of which are refrigerated. Carousels are used for samples and cuvettes. Reagents are placed in a linear array. The cuvette carousel is stationary for 5 s while sample addition, reagent addition, mixing, and washing take place. During the next 15 8, the carousel makes a complete revolution and f d y stops at the next station. During the rotation, every cuvette is read by the photometer, permitting end-point and rate measurements. The RA loo0 (Technicon) a n a l m up to 240 tests/h utilizing up to 14 reagents. Three carouselsare used. The cuvette carousel is stationary for a few seconds while sample and reagent additions take place. During the remainder of the 15 s cycle, the carousel oscillates to effect mixing and then rotates to allow 43 of the 100 cuvettes to be read by the photometer for end-point and rate measurements. The cycle is completed as the carousel stops at the next station. The RA 1ooO makes innovative use of an immiscible fluorocarbon fluid to eliminate cross-contamination between samples and reagents (2) by isolating the aqueous phase from the tubing walls. Air-segmented continuous-flow analysis (Technicon AutoAnalyxr) has been a mainstay of the clinical laboratory for over a decade. More recently, chromatographic theory and components have made nonsegmented continuous-flow analysis feasible. The 'flow injection" techniques (3)appear most useful when the incubation is short, while air-segmented continuous flow is most useful for long incubations. Because the continuous-flow analyzers are not 'selective", these new developments may not have much impact on the clinical chemistry laboratory. Reflectometry. There were no fundamental changes in the Kodak EKTACHEM multilayer film instrumentation using reflectometry during the past 2 years. The original descriptions are part of the Symposium on Advanced Analytical Concepts for the Clinical Laboratory (4.5). Fluorometry. Adaptations of commercial photometric instruments (Abbott VP and IL Multistat) for the measurement of fluorescence (6,7)demonstrate a 100-fold increase in sensitivity which is particularly useful for immunoassays. An immunoassay system based on fluorescencepolarization, the Abhott TDx ( 4 9 ) .uses a liquid crystal element to modulate the polarization. The degree of polarization relates to the mass of the fluorescent emitter which greatly increases ANALYTICAL CMMISTRY. VOL. 55. NO. 5. APRIL 1983
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CLINICAL CHEMISTRY
when complexed by the antibody. A new front surface fluorometric system (STRATUS, American Dade) (10) for enzyme immunoassay uses radial partition chromatography to separate free from bound label on a glass-fiber filter paper which has the antibody immobilized on it. Tabs carrying the filter paper are automaticallyinserted and removed from a carousel which is coaxial to the sample carousel. Various workstations add sample, labeled-antigen, and a combined elution-solvent/enzyme-substrate. Particle Counting. Immunoassays can also use particle counting instrumentation (11),where latex particles are agglutinated by antibodies. The size difference between the free and agglutinated particles can be easily detected. Although no instrument has been developed specifically for this purpose, the current instruments for particle counting already in the clinical laboratory make this approach potentially useful.
ION SELECTIVE ELECTRODES Potentiometric membrane sensors have features which find increasing use in biomedical analysis (I). There are several excellent reviews covering clinical applications of ion selective electrodes (2-5). Additional reviews describe catalytic substances employed for specific biocatalytic membrane electrodes (6) and how they are coupled with flow-injection analysis for clinical assays (7). A building block approach (8) describes these sensors, but this can now be extended to a "family tree" (9) concept tracing the development of bioanalysis with membrane sensors. This, coupled with basic technologies, branches out into many forms and modes of applications. Several textbooks have appeared including the construction and use of ion selective microelectrodes (10, 11),continuing work on enzyme and ion selective electrodes in medicine (12), and a description of electrodes and analysis methods for drug substances (13). New advances in the use of electrodes in biomedicine are outlined resulting from the 28th Congress of the International Union of Pure and Applied Chemistry held in August 1981 (14). The published proceedings of the 14th Annual Symposium on Advanced Analytical Concepts for the Clinical to clinical Laboratory cover electrochemical techniques applied -assays (15). The develonment of new analvtical svstems based on biocatalysts with'amperometric sen';ors (18)and a discussion of instrumental concepts for making microvoltametric electrodes for clinical use (17) have been described. Electrolytes. The measurement of serum sodium and potassium with ion selective electrodes has been the center of an increased controversy arising from the discrepancies which exist in interpretation of results when correlating direct potentiometry with flame photometry (18). This will impact on the clinician's acceptance of all direct electrode measurements in biological fluids. An excellent introduction to direct vs. indirect potentiometry is available illustrating the effects of lipids and proteins on the measured Na+ concentration (19) depending upon how the sodium is measured, in whole plasma or plasma water (20). Additional evidence implicates a degree of sodium binding to bicarbonate in the sample (21). The im ortance of this effect is addressed by a discussion of Na P/Ktanomalies measured by either direct or indirect potentiometry (22). These errors are clearly shown to be due to liquid junction and activity coefficient effects. Accurate measurement by direct potentiometry requires the proper concentration and salt be used for the liquid junction solution (23). Sodium ion binding contributes minimally to the differences seen between direct potentiometry and flame photometry (24). The sample makeup, including albumin and lipid content, which shows up as viscosity changes (25), affects the correlation between the two methods (26). This effect formed the basis for determination of the volume of macromolecules, like hemoglobin (27). The distinction between direct and indirect potentiometry and flame photometry will be resolved only when standardized methods of analysis are established. This has been initiated by a reference method for serum sodium measured by a flame atomic emission-spectroscopic method, which is being evaluated by a multiple laboratory panel (28). This is being followed by a workshop on direct-potentiometric measurements in blood to be held at the National Bureau of Standards in May 1983. This meeting is cosponsored by the National 204R
ANALYTICAL CHEMISTRY, VOL. 55, NO. 5, APRIL 1983
Table I. Enzyme Electrodes substrate
enzyme
urea urea guanine NADP+ histidine urea creatinine glutathione tyrosine
urease urease reactor guanase glutathione reductase histidine decarboxylase urease creatininase reactor glutathione reductase tyrosine decarboxylase
sensing electrode ref 69 70 71 72 73 74 75 76 77
Measurement LaboratoryCenter for Analytical Chemistry and the National Committee for Clinical Laboratory Standards. It will provide a forum for clinicians, scientists, and manufacturers of clinical instruments to discuss the status and needs of direct potentiometry in blood. The goal will be to establish a working basis for the standardization of measurment technique, nomenclature, and symbology. The topics are to include activity coefficients, residual liquid-junction effects, analyte binding, water (plasma water and water activity), and reference methods and materials. Other new developments include several examples of modified potassium electrode matrices and ionophores (29-32). Serum ionized calcium measurements are affected by protein level disturbances (33-36) while examples are given for using standard materials and methods (37-39). Total COz, bicarbonate,and carbonate sensors are described, using alternate internal sensing elements and gas-permeable membranes (40-42). New films and overlays decrease the level of bromide interference on chloride electrodes (43-45). Improvements in pH electrode design include neutral carriers, nonaqueous internal electrolyte solutions, and reference electrode considerations (46-50). Ammonia-nitrogen analysis has been improved by use of new internal sensors (51-54). Lithium electrodes can now be based on neutral carriers (55); a phosphate electrode was described based on an iron wire (56). Organic Substrates. A picrate ion selective electrode has been used to measure creatinine by monitoring the kinetics of picrate disappearance which is the basis of the traditional colorimetric method (57). This technique was also applied to the determination of albumin by monitoring the excess picrate reagent in a continuous-flow instrument (58). An amino acid analyzer which couples HPLC with a copper wire electrode (59) senses the anionic forms of amino acids to levels as low as lo4 M. Several electrodes have been described for drug assays either by direct methods or by titration, in pharmaceutical dosage forms (60-63) including: (1) amperometric sensors for screening mutagens with Salmonella (64); (2) NH3 and C 0 2 sensors to assay pyridoxal 5-phosphate (the enzymatically active form of vitamin Bs)at low levels (65, 66); (3) optically active ionophores to determine enantiomeric forms of chiral ammonium ions (67). Enzyme Electrodes. Enzyme electrodes are sometimes mistaken for electrodes which measure enzyme activities. There are electrode systems which are specifically designed to assay for enzyme activity such as the use of the NHB gas electrode for kinetic determination of trypsin activity based on the hydrolysis of a synthetic substrate (68). However, most enzyme electrodes couple a basic ion or gas-sensing electrode with a specific enzyme that acts upon selected substrates. Many concepts concerning the design and theoretical principles of enzyme electrode operation can be found in the review articles mentioned earlier. Table I lists the new enzyme electrodes recently reported. Most are based on gas-sensing electrodes and, as a result, exceedingly selective detectors can be made. The combination of high biocatalytic selectivity with the inherently high gas membrane selectivity ensure the overall specificity of the electrode in biological fluids analysis. Enzyme electrodes are often considered reagentless detectors which consume negligible quantities of substrate in the sample. A recent study suggests that enzyme-based sensors may indeed convert significant substrate amounts to release byproducts into the bulk sample (78). Variables such as sample volume, exposure time, and substrate concentration seem most important, while substrate consumption is inde-
CLINICAL CHEMISTRY
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Table 11. Bacterial and Tissue Bioelectrodes sensing electrode ref substrate bacterium/ tissue NTAa Pseudomonas serine Clostridium acidiurici tyrosine Aeromonas phenologenes L-arginine Streptococcus lactis glutamate yellow squash adenosine mouse :small intestine AMP rabbit tnuscle guanine rabbit liver a
-
Nitriloacetic acid.
fJ
NH, NH, NH; NH, CO,
79 80 81 82 83 84 85 86
NH, NH, NH, Adenosine 5’-monophosphate. -
pendent of stirring rate and temperature. Highly selective electrodes may be prepared by coupling expensive, isolated enzymes with ion selective electrodes. Those enzymes are isolated and prepared from bacteria and animal and plant tissues; therefore, preparations of such materials could also function as biocatalytic agents. Table I1 lists the most recent applications of bacterial and tissue bioelectrodes for various substrate measurements. Immunoresponsive Electrodes. In the previous decade there were but a handful of sketchy reports describing rather cumbersome immunochemical sensors. Interest has since increased in the coupling of ion selective electrodes to immunochemical reactions, as new techniques such as enzyme immunoassay, are improved and the numbers of electrodebased assays increase. By using enzyme electrode technology, appropriate biocatalytic labels have been identified and effectively used for immunoassays. A model hapten system, using adenosine deaminase and an NH3 electrode, allowed antibody measurements to 50 ng (87). In an inhibition mode, the model hapten could be mol/L. An enzyme-linked quantified between lo4 and immunoassay for humari IgG, using a fluoride electrode (88) had an enzyme label of horseradish peroxidase, which liberated fluoride ion from the p-fluoraniline substrate. A pH electrode, coupled with acetylcholiiiesterase, assayed hCG between 0.05 and 25 IU/mL (89). The recent development of a practical antibody sensor (90) used crown ethers in a PVC (poly(viny1chloride)) matrix directly con‘ugated to ai hapten that could interact with a specific antidody. The electrode operated predominantly via a “selectivity shift” mechanism where the interaction of the antibody a t the surface of the sensor altered the potential generated by common ions in the serum sample. This was further explained by coupling the protein antibody absorption to the ion-exchange processes occurring a t the membrane surface (91). Other labeled and nonlabeled immunosensors are related (92), as well as EL novel umperometric assay for creatine kinase-MB isoenzyme (93). The CK-MM species is inactivated with a specific antibody and MB is coupled through a reaction sequence with ferricyanide as a redox agent. Much of the early imrnunosensor work was stimulated by research on bilayer lipid membranes (BLM), which showed great promise for mimicking natural membrane systems, but was plagued with technical difficulties. They were highly unstable and hard to form reproducibly. A possible solution is to place the BLM on an artificial support which defines the dimensions and improves the stability. A microfiltration unit is an excellent support matrix for the BLM films (94). The response to membrane-active agents was investigated but the real utility of these supported f i i s may be in the development of sensitive immunoprobes. Ion-selective electrodes are finding greater acceptance by clinicians for biological fluids analysis. This is probably due, in part, to the greater number and variety of electrode-based electrolyte analyzers. Before these instruments can be used, trusted, and relied on as the established methodologies; performance specifications and means of standardization must become established. This can come about by first settin analytical goal standards for specific samples (95). Secont a definition of activity scales for analytes at the national and international levels is required as has been done for pH (96). Finally, the chemist or clinician must realize the capabilities
Immunoassay systems
L
Antib(odles
’
Antigens
No Label
Labeled’Antlgen
A
liarnogeneour
Heterogeneous
;abe\ed~;lbodyi.,c, Sandwlch
Labeled Second Antibody
Label
Flgure 1.
and limitations of electrochemical sensors. There are rules and procedures to follow when handling spectrophotometers and other instrumenhtion and electrodes as well. With careful consideration of error propagation (97) ion-selective electrodes can deliver impressive performance in biological fluids with a minimum of hardware and sample handling.
IMMUNOASSAY TECHNIQUES An immunoassay is an analytical procedure based on the reaction between an analyk (antigen) and its specific antibody. Since it is not possible to see antigens and antibodies react, the reaction must either form a precipitate or have a detectable label. Immunoassays for the detection and quantitation of antigens can be characterized as those using: (1)no labeled reagent, (2) labeled analytes or antigens, or (3) labeled specific antibodies (Figure I). These sytems are divided into heterogeneous assays requiring a separation step and homogeneous assays requiring no separation. Sensitivity, specificity, ease, speed, simplicity of detection, precision, and availability of instrumentation will determine which assay system (label) is the most appropriate. Figure 2 lists some clinically significant analytes and the clinical levels. Heterogeneousnonisotopic immunoassay systems c,m be as sensitive as the more familiar radioimmunoassay, but there is a problem with respect to convenience and automation of several of the asscy steps. Homogeneous assay systems are, in general, less sensitive because background signal contributed by the sample is not removed. There are many types of labels that can be used in immunoassay systems These fall into five general types including: (1)radioisotopes, (2) fluorophores, (3) enzymes, (4) particles, and (5) precipitins. This part of the review highlights some of the major developments in immunoassay technology over the last 2 yearn. Developments in immunoassay have been the subject of extensive reviews (1-ll), but only a sm,dl portion is given here. Major journals in this area include Clinical Chemistry, Journal of Immunological Methods, Clinica Chimica Acta, and Analytical Clinical Biochemistry. Radioisotopes. Radioisotopes as immunoassay markers were developed about 20 years ago and have been in clinical use ever since. Their major advantage is the ability to quantitate analytes at very low concentrations. Radioimmunoassay techniques have been employed as competitive and noncompetitive procedures utilizing a variety of isotopes and, in most cases, required separations which held back the development of automation. Recently, internal quenching agents such as bismuth oxide beads have separated bound and free radiolabels (12). These particles contain a radiation-absorbing material which shield radiation from either the antibodybound or free radioligand, eliminating centrifugation and decanting, making the radioimmunoassay homogeneous. Radioactive labelsi have serious problems associated with them. In most cases, the amounts and energy of radioisotopes used in clinical laboratories present no hazard to the user unless labeling is being carried on. There are significant problems for the manufacturer, as the label decays, new batches of the material must be made on a regular basis. This decay can cause damage to the molecules further shortening the usable shelf life of the material. The entire process of manufacture, distribution, storage, use, and disposal is becoming increasingly more regulated and costly, threatening the continued supply on a large scale. However, because such low levels of analyte can be detected, radioimmunoassay will continue to play a significant role in the clinical laboratory. Fluorophores. Fluorescence immunoassay is a rapidly expanding field for clinical medicine because of its high degree of sensitivity. Both homogeneous and heterogeneous fluorANALYTICAL CHEMISTRY, VOL. 55, NO. 5, APRIL 1983 * 205R
CLINICAL CHEMISTRY CONCENTRAT I O MOLES/LITER
THERAPEUTIC DRUGS
STEROID AND AMINO ACID HORMONES
PROTEIN/POLV:EPTIDE HORMONES
ANTl8ODlES V I R A L AG, TUMOR AG, -
CELLULAR ANTIGENS E , G , BLOOD REC:.
ETHANOL
IGG (TOTAL)
SALICYLATE ACETAMINOPHEN “AN-
GENTAMICIN
TOBRAMYCIN A E B
ANTIGENS
I GG
i
DIGOXIN
(SPECIFIC)
CORTICOSTERONE T3 ESTRADIOL
Tu (FREE) ALDOSTERONE T. (FREE)
SYPHILIS
PROLACTIN
RH ANTIGEN
RUBELLA
INSULIN PARATHYROID HMMONE HGH (GROWTH HORMONE) LH (LUTEINIZING HORMONE rTSH (THYROXINE STIM, J HORMONE)
.h;h:::p“
I
1
VASOPRESSIN
Flgure 2. Classes of clinically significant analytes as a function of concentration in the sample.
escent immunoassay systems have been developed for a variety of analytes and described in the literature (13-16). Homogeneous methods have been devised showing sensitivities in the range of mol of analyte/L compared to lo4 to mol/L for enzyme immunoassays. Background interference is the major limitation in homogeneous systems. Heterogeneous methods avoid the back round interference problems to 10-1 Fmol of analyte/L are possible. and sensitivities of Many homogeneous assay systems have been described in the literature including: decreased label fluorescence induced by antibody binding (17),fluorescent polarization (18),internal reflectance spectroscopy (19), excitation transfer (20), fluorescence protection @ I ) , and substrate-labeled immunoassay (22), but only a few are commercially available. The substrate-labeled immunoassaytechnology is the basis for a system from Ames Division, Miles Laboratories which measures therapeutic drugs in human serum (23-28). The fluorophores in this system have special chemical properties where the molecules are functional enzyme substrates and nonfluorescent precursors. The labeled precursor-substrate is hydrolyzed by enzyme catalysis, and the product is measured fluorimetrically. This technique has been used recently as a simple and reliable assay for IgG and IgM, suggesting applicability to the measurement of other serum proteins (29, 30).
Several years ago, Dandliker and co-workers (31-34) described the use of fluorescence polarization in immunoassays to measure antigens and antibodies, although most of their work described measurement of small antigens (haptens). Fluorescence polarization measures the change in the emitted polarized light when a mixture is excited with polarized light. The degree of polarization of the emitted light depends largely on the thermal rotation of the fluorescent-labeled molecule. The unbound fluorescent-labeledhapten tumbles rapidly and emits unpolarized light. When the fluorescent-labeledhapten is bound to a large antibody molecule, it forms a massive molecule that tumbles more slowly and emits light polarized in the same plane as the incident light. Therefore, the degree of polarization depends on the amount of antibody binding. A system for the measurement of therapeutic drugs using the fluorescence polarization technique (35-39) in a competitive binding assay has been commercialized by Abbott Laboratories. The tracer, a fluorescein derivative, competes for antibody binding sites with the unlabeled analyte from the clinical sample. The greater the analyte concentration in the sample, the greater the portion of tracer is unbound. A standard curve determines the relationship between the concentration of analyte in the sample and the degree of polarization measured. 206R
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The sensitivity of antigen detection by fluorescence polarization is currently in the micromol to upper nmol/L range. The technique depends on a marked increase of effective size following antibody binding, which is a major limitation. An umbelliferone derivative (40)has been used as a fluorescent label in a serum theophylline determination to avoid problems that can exist with fluorescein derivatives. Another homogeneous fluorescence-immunoassay system involves Forster energy transfer in which fluorescein (FITC) is used as a donor fluorescer and rhodamine as an acceptor or quencher. When FITC-labeled antigen and rhodaminelabeled antibody bind, energy transfer occurs resulting in a quenching of the fluorescence (41). The SYVA Advance system employs this concept by measuring the quenching which occurs when fluorescent tagged antigen is bound by quencher-labeled antibody. The fluorescent tags developed for use in this system have emissions at longer wavelengths than the common interfering blood components and thus decrease interference problems. The homogeneous assay systems do not require separation, allowing for speed and ease of automation. However, they usually suffer from lack of sensitivity for a wide variety of analytes. Several heterogeneous fluorescent immunoassays using a solid phase (42,43) involve antigen or antibody labeling with competitive or noncompetitive reactions. Both competitive and indirect-“sandwich” methods are commercially available from Bio-Rad Laboratories requiring a photon counter for quantitation (44, 45). A solid-phase heterogeneous system for tobramycin (46),available from IDT Laboratories, utilizes a specific antibody immobilized on a dipstick. It requires only one test tube and no washing steps, compared to four test tubes and two washing steps previously required for serum protein assays (47). An assay system for several analytes (48-51) mixes fluorescein-labeled antigen and antiserum covalently linked to magnetizable particles. A magnet is used after equilibrium to sediment the particles, and the supernatant, containing the free fraction and interfering substances,is aspirated away. An elution buffer is added to dissociate the bound fraction and then the fluorescence of the resulting supernatant is determined. A “sandwich”-fluorescenceimmunoassay using a flow cytometer (52), does not require the physical separation of bound-from-free label. With anti-IgG-coated micropheres and fluorescein-conjugated anti-IgG in a “sandwich”-assayformat, IgG was quantitatively measured by the ability of the flow cytometer to discriminate particle-associated from solutionassociated fluorescence.
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There are several approaches that may make fluorescence a favored technique. One involves chemical modification of the fluorescent label so that the fluorescent excitation and emission spectra are shifted away from the serum background. Another involves fluorescence of the label at some time after the short-lived werum fluorescence has disappeared (53). It has been difficult to prepare specific antibodies labeled with appropriate fluorophorea to allow exploitation of time-resolved fluorescence immunoassays, although recently a highly sensitive time-resolved fluoroimmunoassay has been developed (54). Enzyme Markers. In 1971 (55,56) enzymes were identified as markers for quantitating antigens, antibodies, or haptens. Many excellent reviews have been published describing their une as labels in immunoassay systems and the advantages and disadvantages they offer (57,58). Homogeneous enzyme immunoassays involve the measurement of enzyme activity without the need for separation. The SYVA Corp. has commercializedthe EMIT system (enzyme multiplied immuno-test) using a homogeneous enzyme immunoassay method where the activity of an enzyme-hapten conjugate is changed when bound to a specific antihapten antibody. The binding of the enzyme hapten conjugate and antibody can inhibit enzyme activity (theophylline)or increase enzyme activity (T4).This technique has had considerable impact on the clinical chemistry laboratory, especially for the measurement of drugs and hormones in biological fluids (60). The sensitivity of this type of system relates to the specific activity (turnover) of the enzyme used, the period of measurement, and the degree of activity modification. Another approach to homogeneous enzyme immunoassay involves enzyme channeling (61). This assay relies upon the observed enhancement in the overall rate of a sequence of enzymatically catalyzed reactions when the enzymes are brought into close proximity. Such an assay could take several forms. One member of an immunological pair (antigen) can be labeled with El and the second member (antibody) be labeled with E, allowing these members to interact, and enhance the rate of the overall sequence of reactions. For a multideterminant antigen, two specific antibodies could be labeled with different enzymes so that binding would permit enzyme channeling to take place. Heterogeneous enzyme immunoassays can detect and quantitate either antigens or antibodies. Immunoassays for antigen quantitation can use competitive binding or solidphase antibody or solid-phase antigen techniques, Solid-phase antigen may be employed to quantitate specific antibodies as well. The methods appear to have acceptable accuracy and reproducibility. The most familiar heterogeneous immunoassay technique is the ELISA, enzyme linked immunosorbent assay (55). This type of assay has been the topic of several reviews (62, 63) and will not be discussed here. Monji and Castro (64, 65) have described enzyme assays using D-galactosidase as a label, and agarose-amino-caproylD-galactosylamine, an insoluble pseudosubstrate inhibitor of the enzyme, to separate phases. These assays, referred to as steric hindrance enzyme immunoassays (SHEIA), begin in a homogeneous liquid phase. The enzyme-labeled hormone and antibody are incubated, after which the immobilized pseudosubstrate is added. 'The hormone-enzyme complex with antibody attached cannot bind to the pseudosubstrate because of steric hindrance, whereas free enzyme conjugate reversibly attaches to pseudosubstrate. The activity bound to the pseudosubstrate or that bound to the antibody can be measured. It is possible to achieve higher sensitivity by combining labels. An enzyme can react with many substrate molecules. If the converted substrate is easily detected, the reaction is potentially very sensitive. Harris and co-workers (66,67) have developed an "ultra sensitive enzymatic immunoassay" (USERIA) which has allowed them to detect 10-le g of choloratoxin. This assay which has also been used to detect rotavirus, utilizes a radioactive substrate (3H-AMP)for the quantitation of the specifically bound enzyme labeled antibody in a conventional ELISA method. The enzyme potentiated fluorescence-immunoassay system of American Dade involves a substrate that reacts with the bound labeled antigen to form fluorescent products. The labeled antigen can be added sequentially or competitively
with the patient sample to bind remaining unbound antibody sites on the test tabt3. The heterogeneous technology removes background interference problems but may not be suitable for a variety of tests. Recently, an enzyme-potentiated fluorescence-immunoassay was recently used to detect serum ferritin (68) using 4-methylumbelliferyl-j3-~-galactopyranoside as a substrate for the &galactosidase complex to the purified antiferritin antibody. Chemiluminescent Markers. Chemiluminescent lablels in immunoassay have been limited (69,70). However, because enzymes can be used to multiply the signal and detection of photons is relatively efficient, chemiluminescencecould play a future role in sensitive immunoassays. The major problem for chemiluminescent systems is background luminescence. Chemiluminescence occurs when the electronically excited product of an exoergic chemical reaction reverts to its ground state with the subsequent emission of a photon. The emission differs from normal fluorescence and phosphorescence since prior absorption of a photon is not required. This phenomenon is generally observed in the oxidation reactions of certain classes of organic molecules, such as luminol. The most generally applicable chemiluminescence immunoassays are those iin which the chemiluminescent molecule itself is used as a labtel. Most of the work in this area has been limited to the measurement of low molecular weight haptens. 'Recently a number of steroid assays have been developed based on haptens labeled with (aminobuty1)ethylisoluminol. These assays have shown better sensitivity than their tritium labeled counterparts (71-73). Recently (74, 75) an N-succinimidyl derivative of an aryl acridinium ester has been coupled to a monoclonal antibody to a-fetoprotein and used in a two-site luminometric assay. This system is more luminescent than luminol, can be stiimulated to produce chemiluminescence under relatively mild conditions, and correlated with a standard double antibody radioimmunoassay procedure. Chemiluminesceince has been shown to enhance the sensitivity of an enzyme-linked immunoassay for detection of viral antigens (76, 77), and serum proteins (78), and for measurement of various drugs, metabolites, and hormones (79, 80). The chemiluminescent enzyme-linked immunoassay (CELISA) effects quantitation and increased sensitivity by measuring the amount of light emitted during an enzymatically catalyzed reaction of an appropriate substrate. A bioluminescent immunoassay for measuring dinitrophenol (DNP) and trinitrotoluene (TNT) has been developed with the capability of detecting mol of antigen (81). In one system, DNP or TNT was covalently linked to firefly luciferase resulting in sensitivities in the 1-2 pmole range. If the TlVT or DNP is linked to glucose 6-phosphate dihydrogenase instead of luciferase, sensitivity is increased since the enzyme has a large turnover number and "amplification" is possible. The NADH produced is measured by using immobilized bacterial NADH-FMN (-flavin mononucleotide) oxiidoreductase and luciferase. Over the years, practically any way of reacting antigens with antibodies and meamring the result has become the basis for an immunoassay. In the future, assays based on enzymes and fluorophores will play an increasingly important role. Chemiluminescenceoffers sensitivity that may be necessary while monoclonal amtibodies offer the specificity that may ultimately be required. Monoclonal Antibodies. No matter what type of label is used in the assay system, the dominant reactant in all immunoassays for antigen is the antibody. The antibody determines the specificity and the ultimate sensitivity. Only 7 years ago Kohler and Milstein (82, 83) published work on the mouse hybridoma procedure for monoclonal antibody production and opened a new era in immunology. That Ihybridoma technology has far-reaching practical applications in diagnosis and therapy in clinical medicine (84-89). The clonal selection and relative immortality of hybridoma cell lines assure the monoclonality,monospecificity, and permanent availability of their antibody products thus liberating immunologists from the constraints and difficulties previoudy associated with preparation and use of heteroantisera. The hybridoma technology consists of fusing specifically immunized B cells, from mice to murine myeloma cells, forming hybrid cells that will grow in continuous cell culture, and produce monoclonal antibodies with specificity for a single ANALYTICAL CHEMISTRY, VOL. 55, NO. 5 , APRIL 1983
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antigen, regardless of the complexity of the immunogen. Conventional polyclonal antiserum production requires highly purified immunogens, still, subpopulations of antibodies to different portions of the immunogen are produced. This makes it difficult to find antibodies that differentiate between a drug and its metabolites or a family of closely related compounds. Unwanted antibody fractions must be absorbed out, and some of the desired antibody is removed also. Monoclonal antibodies offer a means to avoid some of these problems. Antibody-producing lymphoid cells from immunized animals have a very short life when cultured under in vitro conditions. Individual myeloma cell lines can be grown permanently in culture, but the antibody they produce does not express a predefined specificity. When both types of cells are fused, hybrids can be derived which retain the essential properties of permanent growth and production and secretion of antibody with a predefined specificity. Since the hybrid cells can be cloned, it is possible to dissect the heterogeneous response of an animal. Monoclonal antibodies are well-defined chemical reagents displaying constant immunoreactivity and specificity. The antibodies can be grown in vitro as specific hybridomas in tissue culture or grown in vivo as ascites in mice. With monoclonal antibodies, a pool of these reagents with precise performance parameters can be produced. There are some disadvantages associated with monoclonal antibodies. Identifying the perfect antibody is costly and time-consuming. The affinity of the antibody may be low. The monoclonal antibody may be over-specific and a mixture of monoclonal antibodies may be required. Despite these limitations, monoclonal antibodies are slowly replacing conventional antisera in kits for clinical testing. These antibodies will have profound and wide-ranging effects in the diagnosis, prognosis, and therapy in clinical medicine. The references represent a small portion of the published information and serve as a starting place for the interested reader. By use of the assay techniques already described, monoclonal antibodies can describe T lymphocytes, their subpopulations, and state of activation in tissue sections, exudates, and blood (90,91). Most work with these reagents has concentrated on describing circulating blood lymphocytes. Monoclonal antibodies have seen great utility for classifying leukemias and lymphomas (92-99). The immunoregulatory status of T cell subsets has been studied in relatively few infectious diseases to date (100-105). However, the status may serve as indicators for following the course of infection and the patient response. Monoclonal antibodies have been used to diagnose and monitor autoimmune disease and allergy (106-108). Monoclonal antibodies provide promising tools for exploring differences between tumor cells and their normal tissue counterparts. Once identified, unique tumor markers could have considerable clinical si nificance for in vitro diagnosis, if the tumor antigen is shef into the serum, for in vivo diagnosis of tissue by labeling the specific antibody for imaging studies, and for monitoring and treating (in vivo) tumors in patients (109-124).
ANALYTES OF CLINICAL INTEREST This section reviews progress in the clinical measurement of isoenzymes, cancer markers, coagulation factors, and therapeutic drug monitoring. This limited listing of the literature is intended to allow access to some of the key references. Isoenzymes. Recognition of the nature and occurrence of multiple forms of isoenzymes has made a significant contribution to the understanding of changes in enzyme activity in blood and to the use of their measurements in diagnosis. Enzyme measurements by mass concentrations rather than the activity have been a subject of recent interest (I). Creatine kinase and prostatic acid phosphatase are the best documented examples of enzyme assays made by both immunological (mass) and conventional enzymatic activity measurements. T o date, significant advantages in the diagnostic capability of either approach has not been demonstrated (2). Creatine Kinase Isoenzymes (0: The diagnosis of acute myocardial infarction has been improved by the measurement of isoenzymes of creatine kinase. The CK isoenzyme system is comprised of three dimeric molecular forms CK-BB (brain type), CK-MB (myocardial type), and CK-MM (muscle type). 208R
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The existence of other forms of CK isoenzymes such as CK-m (mitochondrial isoenzyme), CK-variants (macro- CK type I and 11), and CK-immunoglobulin complexes has been the subject of recent studies (3) but the clinical significance of these isoenzymes is not yet established. The measurement of creatine kinase isoenzymes has focused primarily on CK-MB isoenzyme because of its role in the detection of acute myocardial infarction (AMI). The finding of elevation of CK-MB in serum due to other causes, e.g., surgery (4), strenuous exercise (5, 6) and muscle injury (7), has not compromised the significance of this isoenzyme in the diagnosis of AMI. However, sequential determination of CK-MB within the proper time frame after AMI is reported to be most critical to its diagnostic utility (8). The diagnostic benefits of other CK-isoenzymes have been the subject of several recent publications (9-11). Prognostic value of measurement of CK-BB in various pathological conditions such as tumors, brain damage, damage to pregnant uterus, and neonatal hypoxia has been reported (10, 12). Burnam et al. (13,14) studied the diagnostic value of CK-Bi (inactive CK-B monomer) in myocardial patients. Measurement of CK-MM in dried blood samples has been recommended for neonatal Duchenne muscular disease screening (15). The ratio of CKMM, to CKMM, increases in myocardial infarction and other muscle trauma (16). However, these forms of CK have not found routine application in the clinical laboratory. Comparison of several methods for separating and quantitating CK-MB and their predictive values in diagnosing myocardial infarction has been reported (17). Techniques for fractionation of CK-isoenzymes include electrophoresis, column chromatography, radioimmunoassay, and immunoinhibition (18). Electrophoresis on agarose is currently regarded as the most specific and diagnostically efficient method for the determination of CK-MB. However, column methods (9) and immunoinhibition methods can provide rapid turnaround time (1, 19, 20). Lack of specificity continues to be the major problem with widespread acceptance of immunoinhibition methods (21). Advances in monoclonal antibody technology may increase the utility of this technique. Separation, quantitation, and clinical utility of CK-isoenzymes have been an area of active investigation, however, standard reference material has yet to be developed. Lactic Acid Dehydrogenase Isoenzymes (LD). Lactic dehydrogenase measurement is a sensitive but nonspecific marker of cell damage. In practice, the determination of its isoenzymes complements the diagnostic specificity of measurements of other enzymes such as CK, aspartate aminotransferase, and alkaline phosphatase (22). Lactic dehydrogenase has five major (LD1-LDB) electrophoretically separable units (22). In normal serum LD1 is less than LD2. After an AMI, LD, predominates causing a characteristic reversal of the LD1/LD2 ratio, commonly known as “LD flip”. This ratio has excellent specificity for myocardial infarction but generally occurs as a late event which limits its diagnostic utility. Recently attention has been directed to the determination of LD, and LD1/LD ratio (23-25) as an adjunct to the laboratory evaluation of AMI. A predictive value equivalent to ECG has been reported by use of LDl/LD and CKMB combination testing (25). The availability of an immunochemical technique (23) for specific determination of LDl makes this application promising and offers potential for automation. Alkaline Phosphatase Isoenzymes. Total alkaline phosphatase measurement is one of the most frequently performed tests for diagnosis of disease in liver and bone. Isoenzyme analysis usually is not requested in the absence of increased total activity. Lately, use of separative methods in parallel with nonseparative methods have gained emphasis in prognosis and treatment (26, 27). Quantitative fractionation of alkaline phosphatase molecular forms (placental, intestinal, liver, and bone) have not been as well accepted as those for creatine kinase and lactate dehydrogenase partially because of poor analytical methodology. Placental and intestinal alkaline phosphatase have been quantified based on their sensitivity to inhibition by l-phenylalanine (28). Placental alkaline phosphatase has been used as a tumor marker, but its clinical utility is limited due to the low prevalence of tumors which secrete the marker. Liver and bone alkaline phos-
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phatase are similarly inhibited by homoarginine and levamisole (28). They have been distinguished by their differential sensitivity to inactivation by heat or denaturation by urea (29). Use of these inactivation techniques makes the differential estimation of these isoenzymes laborious and sometimes unreliable (28, 29). Development of isoenzyme-specific assays is expected to expand the diagnostic utility of alkaline phosphatase assays. Amylase Isoenzymes. Methods for separating and measuring the isoenzymes of amylase using chromogenic substrates and specific inhibitors have received some attention in the ast 2 years (30,31). Recently, a more acidic isoenzyme has een reported from cyst fluids of serous ovarian neoplasms (32). The utility of this isoenzyme in diagnosis and monitoring ovarian cancer has not yet been established, but some evidence suggests a potential value as a tumor marker. Serum amylase is most commonly used to diagnose acute pancreatitis. Cancer Markers. A number of analytes show a high degree of tumor association, but their use as cancer markers for early cancer diagnosis remains limited. The tumor markers currently in use are careinoembryonic antigen (CEA), a-fetoprotein (AFP), prostatic acid phosphatase (PAP),human chorionic gonadotropin (hCG), estrogen and progesterone receptors, thyrocalcitonin, and prolactin. Several of these have been proposed as screening aids but none gives high predictive values (33, 34), e.g., there is high incidence of false positive results. Until assays with high sensitivity and specificity can be developed, these tests will be limited to monitoring therapy or checking relapses rather than screening. Other extensively studied areas of tumor marker research are circulating immune-complexes (35) and cell surface markers (36,37). Recently, Weinhouse (38)reviewed the role of isoenzymes in gene regulation in cancer. Several otentially useful analytes have been reported polyamines $9), sialyltransferases (40),creatine kinase-BB, lactate dehydrogenase isoenzymes (41, 42). The tumor markers that have proven their usefulness in the routine clinical laboratory are discussed below. Carcinoembryonic Antigen (CEA). CEA is an oncofetal antigen which is elevated with a number of cancers such as colon cancer (43),hepatocellular cancer (44),ovarian cancer (45, 47), and cancer of stomach, lung, pancreas, and breast (48,49). It was initially touted as a universal cancer marker and a screening aid for widespread disease. At this time, its usefulness is limited to the management of disease in selected instances (50)and as an adjunct assay to radiology, exfoliative cytologic biopsy, and endoscopy (44). CEA is currently determined by a radioimmunoassay, but an enzyme assay has become available which should facilitate the determination of this marker in the routine clinical laboratory. Prostatic Acid Phosphatase (PAP): PAP is an isoenzyme of acid phosphatase used in the diagnosis of prostatic carcinoma (51). The development of radioimmunoassaysfor PAP (52),though more sensitive, does not afford improved predictive power compared to the earlier enzyme activity assays. In controlled use, PAP has shown a false positive rate of 5-8% for prostate carcinoma (53). This limits its use as a mass screening test for early detection (54,55). The assay is largely used to confirm suspected malignancy and to monitor therapy (56). Colorimetric enzymatic assays are the most commonly used methods for PAP determinations, though RIA (57) and counterimmunoelectrophoresis (58) are gaining popularity. Serial determinations to establish a base line are important in PAP tumor m;magement. Since PAP levels can be related to tumor grade prognostic staging, improvements in assay sensitivity and specificity could result in its widespread use in screening for prostatic cancer. a-Fetoprotein (AFP). AFP is a glycoprotein that is a predictor of malignancies such as hepatocellular or germ cell carcinomas (43). Abnormal levels over 400 ng mL strongly suggest malignancy but cannot be considered iagnostic (53, 59). However, serial serum AFP assays have been useful in assessing the effects of therapy and the recurrence of tumors. AFP is also elevated with fetal neural-tube defects, but its use has been controversial due to the high incidence of false positives in prenatal diagnosis. Enzyme-linked-fluorescence immunoassay and radioimmunoassay are the common measurement techniques (60). Recently, a standard preparation has become available from the Center for Disease Control which should aid in development of more specific assays (61).
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Human Chorionic Gonadotropin (hCG). Levels of this glycoprotein hormone in serum and urine are abnormally increased in ovarian and testicular cancers (59,62,63). It is also elevated in pregnancy and its measurement is more commonly used for confirming pregnancy or detecting ectopic pregnancy. The role of hCG as a diagnostic and prognostic aid in trophoblastic cancers is well established (64). Tlhe hormonal action and fitructure of hCG are very similar to those of human luteinizing: hormone (hLH) which caused a significant amount of cross-reaction in early radioimmunoassays. Recently, specific radioimmunoassays for the p subunit of hC‘G (P-hCG) have been developed which permit discrete recognition of hCG and its differentiation from P-hLH (65). Periodic monitoring of hCG levels is a useful adjunct to follow a patient’s response to therapy. Prolactin. This is a pituitary hormone which is normally involved in lactation. Abnormally elevated serum prolactin levels have been found in patients with disorders of the hypothalmic stalk areti and pituitary or hypothalmic tumors (6648). Its measurement has clinical utility in diagnosis and management of prolactin-producing pituitary tumors. Recently, radioimmunoassays for determination of prolactin have become available (6!9). Steroid Receptors. The determination of estrogen receptclrs (ER) in breast tumors has become accepted for the prognociis and prediction of response to therapy. ER determinations are done on breast biopsy samples from patients with “breast lumps”. The presence or absence of estrogen receptors is usually reported as positive or negative results. If the breast tumor has estrogen receptors, then hormone therapy may ’be beneficial. In addition to ER status, quantitative ER levels (femtomoles/mg cytosol protein) may reflect response to endocrine therapy (70, 71). Therefore, requests for quantitative reporting are increasing. Determination of progesterone receptors (pgR) and ER increases the predictive value (71) and has been recently recommended. Newer immunodiagnostic techniques arie replacing the earlier techniques of sucrose density gradient sedimentation analysis and radioimmunoassay (72, 73). Cross-reactivityamong various receptor classes is a major problem (73)and is an area needing further development. Coagulation Factors. “Coagulation Cascade” involves tlhe ability of plasma components to selectively control the conversion of fibrinogen to fibrin by a series of biochemical steps. The coagulation process is traditionally compartmentalized into an intrinsic system where all necessary factors are present in plasma and an extrinsic system where an additional external tissue factor is required (74,75). Inadequate or inappropriate clot formation is the end result of various defects. In the latter case, heparin therapy is commonly employed to prevent thromboembolism. Measurement of the various coagulation factors is tedious, time-consuming, arid sometimes irreproducible, making standardization between laboratories and reagents difficult. The introduction of synthetic substrates has revolutionized the measurement of analytes of the “Coagulation Cascade” (76,77). New methods based on chromogenic or fluorogenic peptides have improved specificity, precision, and accuracy. Assays for antithrombin 111, plasminogen, heparin, antiplaw min, factor X, and kallikrein have been developed. Potential chromogenic measurement of other “global” analytes such as prothrombin time anid activated partial thromboplastin time also exists (77). Therapeutic Drug Monitoring. Over the past 2 years, therapeutic drug monitoring (TDM) has been a fast growing area in routine clinical laboratories. Because therapeutic efficacy and toxic side effects are related to dose levels, it is helpful to measure drug concentration in the blood. This allows dosage adjustment to achieve therapeutic effects with a minimum of toxic side effects. Such measurements are especially important when renal function is changing, since most of the drugs or their metabolites are excreted through the kidneys. Not all drugs are candidates for therapeutic drug monitoring, for example, when a wide dosage margin exists between therapeutic and toxic effects or when no relationship exists between serum drug level and the pharmacologic effect (78, 79). Plasma protein binding of some drugs affects their therapeutic efficacy since it is generally accepted that only the unbound drug is available for biological actility (80). At the ANALYTICAL CHEMISTRY, VOL. 55, NO. 5, APRIL 1983
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present time, the determination of protein binding of a drug is a time-consuming and cumbersome procedure. In selected instances, salivary drug concentrations are useful in assessing free drug levels (81). The earlier techniques of gas-liquid chromatography (GLC), high-pressure liquid chromatography (HPLC), and radioimmunoassay (RIA) for the total drug measurement are being replaced by homogeneous immunoassays in routine clinical laboratories. We can anticipate further development of new technologies and automation in the clinical laboratory. In this report we will review progress in the therapeutic drug monitoring of antiasthmatic, antiarrhythmic, anticonvulsant, psychotropic drugs, aminoglycosidic antibiotics, and antineoplastic agents. Antiasthmatic Drugs. Theophylline is a potent bronchodilator for relief of acute asthma in children and adults and for treatment of apnea in premature infants (82,83). Theophylline has a narrow therapeutic index of 10-20 pg/mL in plasma. Overdose is toxic to cardiac, gastrointestinal, and central nervous systems and can result in seizure and possibly death (83). Because the metabolism of theophylline varies considerably from patient to patient, treatment can be improved by monitoring plasma levels. Theophylline is structurally similar to caffeine and theobromine which are dietary components from coffee, tea, chocolate, and cola beverages. Therefore need for a specific analytical method is obvious. HPLC (reverse phase) has been preferred because of its high specificity and simultaneous measurement of multiple components. Immunoassays with very low caffeine cross-reactivity are replacing the earlier techniques (84-86) and offer advantages of specificity, rapidity, and simplicity. Cardioactive Drugs. Lidocaine, procainamide, quinidine, disopyramide, and digoxin are commonly used antiarrhythmic drugs. All have narrow therapeutic indices (87). Overdose can result in toxicity and death. Pharmacologically active metabolites must be measured in addition to the parent compound to avoid toxicity, e.g., determination of N-acetyl procainamide in procainamide therapy. Propranolol is another cardiac drug which can induce heart failure in toxic amounts (88). Therapeutic monitoring of the drug plasma levels helps establish adequate dosage and identify noncompliant patients. The plasma protein binding of antiarrhythmic drugs plays an important role in modulating their therapeutic effect (89). It is generally accepted that the unbound or free drug is in equilibrium with the drug concentration in the myocardium. Current techniques are not yet adapted to rapid, reproducible, and inexpensive measurements of free vs. bound drug in the plasma (90). Short half-life, cross-reactivity of metabolites, and drug-drug interaction complicate the measurement and interpretation of some of these drug levels (87, 91). GLC, HPLC, and colorimetric assays are available but their utility in the clinical setting is limited mainly due to the length of time required to obtain results. The enzyme immunoassay is currently the most specific and rapid method available for most of these drugs (90). Antiepileptic Drugs. Phenytoin (Dilantin), phenobarbital, carbamazepine, primidone, ethosuximide, and valproic acid are common antiepileptic drugs. Their effect is generally related to plasma drug concentrations as are their adverse side effects (92). Monitoring ensures patient compliance and allows adjustment for synergystic effects (93). Gas chromatography and homogeneous enzyme immunoassay (94,95) are the most widely used techniques. Administration of combinations of certain drug alters drug-protein binding. Since multiple-drug therapy is common in epileptic patients, estimation of free drug levels helps to maintain the threshold plasma concentrations. A simple ultrafiltration device has facilitated free drug level determinations in the routine clinical laboratory
contribute to the therapeutic or side effects. GLC or GC/MS (gas chromatography/mass spectrometry) are the most commonly used techniques for determination of tricyclic antidepressants. HPLC is low in sensitivity and immunoassays are currently nonspecific (99). Lithium (carbonate), used for control of mania, has a therapeutic range for blood of approximately 0.5-1.5 mmol/L (102). Pharmacokinetics of lithium and the difficulties in its estimation have been recently reviewed. Red blood cell lithium level may be of value in identifying patients with intracellular lithium toxicity and normal serum levels (103). Lithium is commonly determined by using flame or atomic absorption photometry. Aminoglycosides. Gentamicin, tobramycin, and amikacin are widely used antibacterial agents particularly for lifethreatening infections. They have a narrow therapeutic margin of efficacy over toxicity (104, 105): ototoxicity (ear) and renal impairment (105,106). Their therapeutic range does not have clear end points, which may be a function of the severity of the infection. Monitoring is helpful in regulating the dose and the dosing interval. Both trough (low point before the next dose) and peak levels are of clinical importance in regulating the dosage. A number of methods for determining drug concentration are available. These include microbiological techniques, radioimmunoassays, enzyme immunoassays, and fluorescent polarization techniques (106, 107). Microbiological techniques measure the biologically active antibiotic but are not precise. Chemical techniques are rapid and precise but may also measure biologically inactive isomers. Enzyme immunoassays and fluorescent techniques have been automated recently. A growing trend in use of these assays has been experienced in the past 2 years. Antineoplastic Drugs. Methotrexate (MTX), a folate antagonist, is widely used in the treatment of various cancers and is monitored primarily at large cancer centers. It is administered in high doses to maximize cell death of the most rapidly growing cancer cells. The high dose of MTX is followed by “citrovorum factornto rescue or salvage the normal cells (108). “Citrovorum factor” cannot salvage normal cells if rescue is delayed, nor will tumor cells be killed if serum MTX levels are too low (109). Thus, the cytotoxic action of MTX requires maintenance of drug levels above a threshold. Dosing by predictive methods may reduce some of the toxic effects such as hepatotoxicity and renal dysfunction. “Citrovorum factor” and its active metabolite are the analytes measured in addition to the MTX level. The radioimmunoassay and homogeneous enzyme immunoassay are the most commonly used techniques for MTX level determination (110). The dihydrofolate reductase inhibition assay (110) has been proposed as a more sensitive and specific criteria for the management of the drug regimen but so far has found limited application because the assay is technically demanding and the enzyme and its substrate are unstable (112, 113). Other Analytes. The following are gaining importance: apoproteins (114) in cardiovascular diseases; circulating immune complexes (115,116) in autoimmune diseases; adrenocorticotropin (ACTH) (117, 118) in pituitary disorders; calcitonin (119,120) in endocrine disorders and cancer, platelet specific proteins (121) in coagulation disorders, and allergen antibodies (122, 123) in allergy. Research in molecular biology and medicine may lead to new clinical assays: psychotropic metabolites that affect behavior (118, 124); viral markers for identifying infectious diseases (125);microbial-metabolic products for identifying fungal and bacterial infections (126);and genetic markers for prenatal and newborn screening (127).
(96).
AUTOMATION AND INSTRUMENTATION
Psychotropic Drugs. Monitoring of plasma levels of this class of drugs continues to be the subject of much controversy (97)yet there is a growing demand for these assays. Tricyclic antidepressant drugs are the most commonly requested assays. Numerous reports (98-100) suggest a therapeutic window or threshold plasma concentration for the antidepressants nortriptyline, amitriptyline, and imipramine. Noncompliance and response variability of these drugs are major problems (101). Metabolites of these drugs also have pharmacological effects. Therefore, the assay methods need to include measurements of both parent compounds and their metabolites, which 210 R
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Ferrous Analysis W. A. Straub" and J. K. Hurwitz United States Steel Corporation, Research Laboratory, Monroeville, Pennsylvania 15 146
This review is produced from a search of the literature as performed with the DIALOG capabilities of Chemical Abstracts Service. The material covered in this particular review is a continuation of past reviews by the same authors and covers the period from September 1980 to October 1982. It is obvious that the level of activity in the publication of research results by analytical chemists and spectrographers in the steel industry is significantly reduced and directly reflects the malais that affects our industry. Without being overly critical, we will have approximately 300 less literature citations in this review as compared to the last one (448).
ALUMINUM Total trace aluminum was determined spectro hotometrically in some unalloyed and low-alloyed steels a ter appropriate dissolution steps as an Eriochrome Cyanine R complex (98),by using Chromazol KS as a color-formingagent (269), as a colored complex with salicylidene-o-aminophenol-4sulfonic acid (523), and with l-pheny1-2,3-dimethylpyrazolone-5-azopyrogallol(13). Low levels of A1 have been determined in high- and lowalloy steels and ferrovanadium by atomic absorption after separation from the matrix by electrolysis (94). The determination of acid soluble aluminum by AA continues to be a very popular method in the steel industry by virtue of its rapidity (51, 76,511). Low concentrations were also determined in silicon steels by using electrothermal atomization (245). Direct injection of a dissolved steel sample into an AI-H flame has been reported (282). AA has not supplanted emission spectrochemical methods for this determination as they continue to be used for both soluble and insoluble aluminum determinations in steel (135, 304). One method involves the premelting of a selected zone on a sample surface in an inert atmosphere so as to concentrate the insoluble aluminum inclusions in the surface of the remelted zone upon which the surface is mechanically removed and subsequently analyzed spectrally for the soluble portion (76). A design for an instrument has been claimed that eliminates anomalous emission spectrometric signals from aluminum inclusions, thus permitting the determination of acid-soluble A1 with high precision (452).
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Various other procedures have been applied in a general way for the analysis of steel and related materials and include the use of lasers for localized sampling prior to spectral excitation (208,311), Grimm glow-dischargesource excitation (466),ICP emission spectrometry (15,101),an acid dissolution, ignition, and NaF fluxing prior to dc arc excitation for stainless-steel analysis (299),and the use of dc plasma techniques for iron ores, sinters, and dust analysis (369). The accuracy of XRF spectrometry for determining A1 in low-alloy and stainless steels (at
