Anal. Chem. 1987. 59. 337R-350R (28W) Voss, R. H.; Rapsomatiotis, A., J. Chromtogr., 346, 205-14 (1985). (29W) Voznakova, 2.; Popi, M.; Kovar, M., Sb. Vys. Sk. Chem.-Techno/. R a z e , Anal. Chem., Hl9, 85-96 (1984). (30W) Walton, H. F., EPA/600/1-84/026, 46 pp (1984). (31W) Wang, 2.; Cui, X.; Zhang, X.; Zhu, X., Shandong Haiyang Xueyuan Xuebao. 15, 34-41 (1985). (32W) Woodrow, J. E.; Majewski, M. S., Seiber, J. N., J. Environ Sci. Health. Pari 8, 821, 143-64 (1986). (33W) Zlatkis, A.; Ghaoui, L.; Weisner. S.; Shanfieid, H., Chromatographia, 20, 343-6 (1985).
SURFACTANTS AND DETERGENTS (1X) Ahei, M.; Giger, W., Anal. Chem., 57, 1577-83 (1985). (2X) Ahei, M.; Giger, W., Anal. Chem., 57, 2584-90 (1985). (3X) Gorenc, B.; Gorenc, D.; Keber, I.; Pihiar, B., Vestn. Slov. Kern. Drus., 33, 11-23 (1986). (4X) Hirai, Y.; Tomokuni, K., Anal. Chim. Acta, 167, 409-12 (1985). (5X) HoR, M. S.; McKerreii, E. H.; Perry, J.; Watkinson, R . J., J. Chromatogr., 362, 419-24 (1986). (6X) Kikuchi, M.; Tokai, A.; Yoshida, T., Water Res., 20, 643-50 (1986). (7X) Levsen, K.; Schneider. E.; Roeligen, F. W.; Daehiing, P.; Boerboom, A. J. H.; Kistemaker, P. G.: Mciuckey, S. A., Comm. Eur. Communities, EUR 8518, Anal. Org. Micropoliut. Water, 132-40 (1984). (EX) Osburn, Q. W., J. Am. Oil Chem. Soc., 63, 257-63 (1986). (9x1 Stephanou, E., Int. J. Environ. Anal. Chem., 20, 41-54 (1985). ( l o x ) Van Hoof, F. M.; Van Craenenbroeck, W. J.; Dewaele, J. K., Int. J. Environ. Anal. Chem., 19, 155-64 (1985). (11X) Yoshikawa, S.:Sano, H.; Harada, T., Kawasaki-shi Kogai Kenkyusho Nenpo , (12), 48-55 (1985).
PESTICIDES, HERBICIDES, AND FUNGICIDES (1Y) Aider, I . L.; Zogorski, W. J., 111, Anal. Methods Pestic. Plant Growth Regul., 13, 281-93 (1984). (2Y) Bondarev, V. S.; Talaiakina, T. N.; Spiridonov, Yu. Ya.; Shestakov, V. G.; Raskin, M. S., Khim. Sel'sk. Khoz., (5),68-9 (1986). (3Y) Brennecke. R., Pflanzenschufz-Nachr., 37, 68-93 (1984). (4Y) Brennecke, R.; Vogeier, K., Pfimzenschutz-Nachr ., 37, 46-67 (1984). (5Y) Cassista, A.; Mallet, V. N., Chromatographia, 18, 305-8 (1984). (6Y) Cessna, A. J.; Grover, R.; Kerr, L. A.; Aidred, M. L., J. Agric. Food Chem., 33, 504-7 (1985). (7Y) Day, E. W., Jr.; Decker, 0. D., Anal. Methods Pestic. Plant Growth Regul., 13, 173-82 (1984). (8Y) Deyrup, C. L.; Chang, S. M.; Weintraub, R. A,; Moye. H. A,, J . Agric. Food Chem., 33, 944-7 (1985). (9Y) Drevenkar, V.; Frobe, 2.; Stengi, B.; Tkalcevic, B., Int. J. Environ. Anal. Chern., 22, 235-50 (1985). (1OY) Drevenkar, V.; Frose, 2 . ; Stengi, B.; Tkaicevic, B., Mikrochim. Acta, 1, 143-56 (1985). (11Y) Goewie, C. E.; Hogendoorn, E. A., Sci. Total Environ., 47, 349-60 (1985).
(12Y) Hill, K. M.; Hoiloweii, R. H.; Dai Cortivo, L. A., Anal. Chem., 56, 2465-8 (1984). (13Y) Hoke, S. H.; Brueggmann, E. E.; Baxter, L. J.; Trybus, T., J. Chromatogr., 357, 429-32 (1986). (14Y) Ishiwaka, K.; Kamo, E.; Sasaki, A,; Kuzuoka, S.;Yonekura, Y.; Suzuki, K.; Ito, T., Eisel Kagaku, 31, 72-8 (1985). (15Y) Kuthan, A., VodniHospcd.: B.,35, 187-90 (1985). (16Y) Lee, H. B.; Stokker, Y. D., J. Assoc. Off. Anal. Chem., 69, 568-72 (1986). (17Y) Lores, E. M.; Moore, J. C.; Knight, J.; Forester, J.; Clark, J.; Moody, P., J. Chromatogr. Sci., 23, 124-7 (1985). (18Y) Merz, W.; Neu, H. J., Vom Wasser, 65, 189-98 (1985). (19Y) Miles, C. J.; Wallace, L. R.; Moye, H. A.. J. Assoc. Off. Anal. Chem., 69, 458-61 (1986). (2OY) Nieien, M. W. F.; Koomen, G.; Frei, R. W.; Brinkman, U. A. T.. J. Lip. Chromatogr., 8, 315-32 (1985). (21Y) Sapiets, A.; Swaine, H.; Tandy, M., J. Anal. Methods Pestic. Plant Growth Regul., 13, 33-51 (1984). (22Y) She, L. K.; Brinkman, U. A. T.; Frei, R. W., Anal. Lett., 17, 915-31 (1984). (23Y) Spittler, T. D.; Marafioti, R. A.; Lahr, L. M., J . Chromatogr., 317, 527-31 (1984). (24Y) Sundaram, K. M. S.; Feng, C.; Broks, J., J . Liq. Chromatogr., 8, 2607-24 (1965). (25Y) Swaine, H.; Tandy, M., J. Anal. Methods Pestic. Plant Growth Regul., 13, 103-20 (1984). (26Y) Tamakawa, K.; Ohgane, Y.; Aihara, Y.; Hiroshima, K.; Katoh, M.; Mishima, Y.; Seki, T., Sendai-shi Eisei Shikenshoho, 1984 (14), 261-6 (1985). (27Y) Tamakawa, K.; Ogane, Y.; Kato, M.; Mishima, Y.; Seki, T.; Tsunoda, A., EiseiKagaku, 32, 153-8 (1986). (28Y) Tsukioka, T.; Shimizu, S.; Murakami. T., Analyst (London), 110, 39-42 (1985). (29Y) Warner, J. S.; Engel, T. M.; Mondron, P. J., EPA/600/4-85/023, 45 pp (1985). (30Y) Warner. J. S.; Engei. T. M.; Mondron, P. J , EPA/600/4-85/027. 46 pp (1985). (31Y) Warner, J. S.; Engei, T. M.; Mondron, P. J., EPA/600/4-85/026, 45 pp (1985). (32Y) Warner, J. S.; Engel, T. M.; Mondron. P. J., EPA/600/4-85/021, 45 pp (1985). (33Y) Warner, J. S.; Engel. T. M.; Mondron, P. J., EPA/600/4-85-017, 60 pp (1985). (34Y) Warner, J. S.; Engei, T. M.; Mondron, P. J., EPA/600/4-65/016, 66 pp (1985). (35Y) Warner, J. S.; Engel, T. M.; Mondron, P. J., EPA/600/4-85/022, 45 pp (1985). (36Y) Warner, J. S.; Engel, T. M.; Mondron, P. J., EPA/600/4-85/024, 46 pp (1985). (37Y) West, S. D.. Anal. Methods Pestic. Plant Growth Regul., 13, 247-66 ( 1984).
Clinical Chemistry K. E. Stinshoff,*' W. Stein: W. G.
and P.F. Laska4
Medical Department, E. I . d u Pont de Nemours International S.A., 1211 Geneva, Switzerland; Medizinische Klinik, Universitat Tubingen, 7400 Tubingen, FRG; Klinik fur Innere Medizin, Universitat Lubeck, 2400 Lubeck, FRG; and Medical Department, E. I. d u Pont de Nemours & Company, Wilmington, Delaware 19898
This review covers the period from January 1, 1985, to December 31,1986. I t is a sequel to the previous survey on clinical chemistry published in 1985 in this journal (1). Again, it focuses on those areas that we believe will have the greatest impact on the routine use of the clinical laboratory and on the improvement of medical care. In addition, we have tried to include areas not well covered in the last review (e.g., enzymology and urinalysis). The review follows the same logic as the previous one and thus uses an identical partition into Instrumentation, Data Management, Immunoassays, and Analytes of Clinical Interest. This logic is dictated by practical considerations. I t does not prevent overlap. As a matter of fact, immunologic methods have developed into a topic that increasingly touches E. I. du Pont de Nemours International S.A., Geneva.
* Universitat Tubingen. Universitat Lubeck.
4E.I. du Pont de Nemours & Company, Wilmington. 0003-2700/87/0359-337R$06.50/0
all areas of clinical chemistry. We interpret this development as a sign of ongoing change in the field of clinical chemistry: Application of traditional methods and introduction of new methods continuously have to be reconsidered, and with it the organization and scope of the clinical chemical laboratory. The emphasis on testing outside the laboratory and a short paragraph on NMR in this review are indications of such new trends.
INSTRUMENTATION A N D DATA MANAGEMENT Economic pressures on routine clinical laboratories over the last two years have remained high. Demand continues to grow for systems with improved price performance, high productivity, ease-of-use, and high reliability. Several general reviews of trends have been published (2, 3 ) . Our primary focus will be on recent developments in the clinical laboratory market recognizing that other trends are pushing more and more testing nearer to the patient. 0 1987 American Chemical Society
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General Chemistry Systems. The analysis of basic metabolites, enzymes, and to some extent therapeutic drug monitoring has become a commodity business and operation. Laboratories are demanding high price-performance and getting it from many sources (4). Three areas where automation needs improvement or development are (1) highvolume basic chemistry; (2) high volume thyroid profiling, therapeutic drug monitoring, and drugs of abuse evaluation; and (3) simplified, reliable, high-performance analysis outside the laboratory, whether in the physicians office, at the bedside, or in the home, that also includes appropriate quality control options. Physician group practices and small independent laboratories have emerged as major markets of clinical chemistry instrumentation (5-7). Dry film and slide-based systems continue to play a major role in the group practice laboratory market. Two systems were recently introduced into this marketplace, each with a novel approach. The Abbott Laboratories Vision system uses centrifugal force to process whole blood through a multichambered unit dose consumable. The system can process any combination of 10 samples and tests simultaneously (8,9). The Du Pont Analyst uses disposable plastic centrifugal rotors containing all reagents to perform up to 1 2 discrete or profile tests (10-12). One of the more novel high-volume analyzers recently introduced is the Cheml from Technicon. It provides random access to tests in a “continuous flow” format capable of 720-1800 results per hour (13,14). Throughput in this instrument is a function of the pattern of assays requested. Pattern dependent throughput is typical of many of new systems such as the American Monitor Corp. Perspective systm and the Du Pont Dimension clinical chemistry system. These systems are highly optimized to solve very specific market needs for throughput and method menu with efficient, cost-effective designs. Optimal scheduling of tests through any system in general is an unsolved problem and the optimal workflow scheduling, both in the laboratory and in instruments, may be limited by the available computer power. Sensors. Perhaps the most noticeable trend in clinical sensors is the continuing replacement of flame emission photometry for sodium and potassium assays with electrodes, particularly with those based upon neutral ion carrier technology (15). The use of electrodes in clinical chemistry has been reviewed in general terms (16,17). Recent reviews with a broader focus on the overall technology are available (18-21). Ion selective electrode (ISE) systems integrating simultaneous analysis of Na+, K+, total COz, and C1 have become practical and accepted. Routine samples include serum, urine, and whole blood. The demand for lithium monitoring remains strong and it is likely that electrode-based analyzers will become commonplace. The small size, ruggedness, and total analyzer cost advantages of electrochemical detection technology are certain to have an increasing impact on clinical chemistry, particularly as the analytical devices move from the lab to the bedside and on to the home. The clinical chemistry market, however, is very conservative in adopting new analytical techniques. Like analytical chemistry in general, most sensing is, and will continue to be, optically based. Novel nonelectrode sensors have been reviewed recently (22,23).They face a long development path before coming into routine clinical use. Optical sensor technology has continued to advance deliberately as more systems are incorporating diode array detection (24). Advances in antibody and enzyme technology have made possible the quantitation of many substances formerly restricted to radioimmunoassay, such as hormones and cancer markers. Photon counting and fluorescence are often integrated in these assay systems in order to shorten the assay time and/or to lower the detection limit. Newer techniques such as time-resolved fluorescence, laser excitation, and luminescence have demonstrated their potential; but today, these approaches are employed in only a small portion of routine immunodiagnostic testing (25-27). Immunodiagnostic Systems. Immunodiagnostic methods in clinical chemistry today include assays for tumor markers, hormones, specific proteins such as ferritin, haptens such as triiodothyronine (T3), and infectious disease tests such as those for hepatitis and HIV. Competition in this market has become more intense as manufacturers vie for the opportunity to replace radioimmunoassay (RIA). New, nonradioisotopic techniques offer clinical laboratories the benefits of avoiding 338 R
ANALYTICAL CHEMISTRY, VOL. 59, NO. 12, JUNE 15. 1987
radioisotopes while increasing their productivity via automation (28,29). There is also pressure on routine laboratories to provide service more rapidly than conventional RIA allows (30). This is occurring a t a time when the medical demand for these assays is growing much faster than any other segment of clinical chemistry. Nonradioisotopic immunoassay systems have recently been reviewed (31). Rather than focus on novel technology, which abounds in this field, we will summarize some of the more recent product introductions which wil1 tend to shape the future of clinical immunoassays. Hybritech has become a key manufacturer of monoclonal antibodies and assay systems. A product recently announced by Hybritech is the Photon Elite immunoassay system. It is a fully automated, random access, rate fluorescence system with a peak throughput of 120 assays per hour after a 1-h incubation period. The reagent system is in a unit dose format where the package containing conjugate and antibody-coated magnetic beads is also the reaction vessel. Ciba-Corning has introduced the MAGIC Lite heterogeneous system in which magnetic particles are used as the solid support and the tag is chemiluminescent (32,33).Amersham International, Ltd., has introduced a coated well system using a luminescent tag that has recently been used to evaluate an AFP and a thyrotropin assay (34-36). The Amersham technology will be outlined in greater detail under Immunoassays. Ortho Diagnostic Systems, Inc., has described a novel immunofluorometric system using latex particles which incorporate a common capture antibody (37). Sclavo, Inc., is marketing the Immpulse System which employs competitive fluorometric immunoassay principles (38). A new problem created by these automated analyzers is that it is no longer simple or easy to specify system throughput. Long incubation times must be considered as well as batch processing and inventory control constraints. NMR. Nuclear magnetic resonance remains a high-cost analytical tool. Its use is limited to the largest institutions. A recent striking preliminary result was the finding that water-suppressed proton NMR of Plasma could detect the presence of malignant tumors (39). This approach examines fat particle mobility rather than any specific tumor marker. If this is proven and cost reduced it could have a significant impact on the detection and therapy of cancer. Magnetic resonance imaging (MRI) has become a promising technology in medicine but it is not considered part of clinical chemistry (40-42). Two-dimensional shift correlated NMR has found some recent application in toxicology while considerable attention is focused on 31PNMR for metabolic studies and bone evaluation (43-45). Data Management. Hospital and laboratory information systems continue to be a growth area based upon economic necessity and management advantage (46). We will focus on developments in other areas which we believe touch the analytical chemist more closely. The handy tutorials, which are a common feature of this journal, apply very well in the clinical environment (47-51). Perhaps the most striking thing about clinical laboratory data management in the last two years is the apparent growth in the number of people productively involved with personal computers. Table I references an extraordinarily wide range of application development occurring today. I t is appropriate to include references to much of the current work in chemometrics in Table I, since much of that work and its application would not be practical without the low-cost computer power currently available in the clinical laboratory. Application development in the lab has been painfully slow for many years. This was due to the high cost and complexity of programming the typical turnkey laboratory computer system. This is still basically true for most laboratory information systems (LIS). Three things have changed dramatically in the last few years, however. One is the growth in absolute numbers of personal computers in the lab. Many of these are workstation terminals or instrument data managers, but they are there. Only a few operating systems like MS-DOS and CPMM control most of these machines. Second is the growth in learning about computers in the workplace. They are a fact of life, and since much of the underlying technology has been relatively stable for several years, the learning process is finite. Third is the development of general purpose software application packages and standards which
K l u E. 81LMn k a mnaw w i m W DC agnostic Systems D i v i h of Du mt. Hs rewived hk ck. mr. MI. degree horn univnsny in Munkh. FRG. where he s i ~ o sewed 88 a research BssiSta"t wim the Maxplanck-Sociely. Frbr to joining DU Ponl he worked in the RaD and the marketing wgenizatians of Bcehringer Mannheim GmbH and m h r l n g e r lngelheim GmbH.
Wollganp Sleh rewived his ck. rer. nat. h 1976 and hi5 Dr. med. degree in 1979 from me U n M W of Tuebingen. FRG. where he presentty lectuos in clinical chemishy. He also k in charge of me clinical dlem4c.4 lab oratory at me University Hospital in Tuebicgen. HIJ research interest canters on enrymobgy. enzyme varhnts. ceratine kinare and me clinical chemical diagnosis 01 mycardial infarction.
Table 1. Recent Computer Applications applications
55, 56 57
chemometrics general laborstory & statistics wavelength optimization sensor design predictive values reference intewalls chemometrics review spreadsheets statistics
58 59
dology 01 chemiluminescence.
P r l Lnka Is an engineer wim me h g n o s tic Systems DMsbn of Du Pont. He re-
cehed his B.A. from m State Universny 01 New York. W&a at Plambwgh. Rior to joining Du pont. he w m e d as an engineer m wmputn appiicalhms and insbumentatbn systems at Path Lab.. Inc.. Rochester. NH.
60 61,62 63 64, 65. 66 67
eommunications, networks, & graphics
electronic mail 68 data representation 69, 70 contour plotting 71 PC LANs 72 integration of analysis & data management 73, 74, 75 applicaanalysis, acquisition, & control 76,77 tions lab management 78. 79, 80 barcoding 81 toxicology clinical trials 82 &
in h o u r s in 1966 and his m.D. in 1971 hm Lee48 University. U.K. I n 1972 he was awarded a Welcome Research Fellowship at SI. BarthObmew nospnai, univeversny of London. From 1975 to 1980 he m e d as clinical chemiot and research assistant wim Haspltais of the Technical and of the Ludwig$-Maxlmillan University In Munich. FRO. I n 1980 he to& charge at tha clinical laboratmy of me depnment fw internal medicine Of the MBdiCal university in L"ebeck. FRO. where he also lectures in clinical chemistry. HIS reseamh is in the lieu 01 im munology. lmmunmssays, and the metho-
references
management cost control work schedules
instruments rmm Graham Wood received his B. Sc.
general functions
RIA
NMR,spectroscopy
interfacing manual cell counting forms & reports general problems modeling
routine workflow
project management interlab proficiency qc program rules pattern intelligent workstation recognition pathogen ID hepatitis inteipre&di& pro files^ thyroid testing reporting, RBC panels expert cystic fibrasis systems sequential testing pharmacokinetics decision trees liquid chromatography quality control
83, 84 85, 86 87 88 89 90 91 92 93 94
95. 96, 97, 98 99
100. 101. 102. 103 104 105
expert system review
106. 107 108 109 110,111 112
on instrument error checking spreadsheet templates blind split sampling
113 114 115. 116 117
the trend toward more outpatient testing and the shift to satellite and physician office testing (SPOT).
IMMUNOASSAYS
have neatly eliminated the challenge of programming altogether for the average end-user. Good examples of this are spreadsheetswhich require little or no programming skill and can be made to work by anyone who understands what problem is to be solved. There are some trends in the application of technology that are playing a more important role in the laboratory. One is the increasing demand for open networks rather than proprietary local area networks (52). I t is vitally important that communication be accurate and rapid in the hospital for both medical and economic reasons. The large number of computers and networks in today's hospitals is causing a demand for standards a t all levels of interfacing-instrument to terminal, terminal to LIS, LIS to hospital information system (HIS), as well as between applications and databases (53,541. Two U.S. organizations (the ASTM and the AACC) are attempting to formulate standards in these areas now. Unfortunately, a global approach to standardization is still missing. This problem of interfacing standards is compounded by trends in the marketplace which have needs that exceed the capabilities of most installed technology. These include
Since the previous review appeared in this journal in 1985 (I), there has been remarkably little activity in the field of immunoassays as far as new techniques are concerned. In contrast, the technology transfer from the research laboratory to a marketable end product has made advances in several areas of immunoassay, notably in nonradioisotopic immunoassays and in "dry-chemistry" immunoassays. Radioimmunoassay. Despite the predicted end of the radioimmunoassay era, there are still many substances that can only be assayed, when using commercial kits, using radioisotopic labeling. Such assays are often for hormones, which are found at such low concentrations or are only assayed in low numbers, that it has not yet been possible, or feasible commercially, to replace them by alternative immunoassays. Examples of such assays include parathyrin (parathyroid hormone-PTH), calcitonin, corticotropin (ACTH), prostaglandins, and thromboxanes. In other fields of in vitro diagnosis, the assays for proteohormones such as LH have been improved in specificity and sensitivity (lower detection limit) by the use of monoclonal antibodies and solid-phase techniques (118-122). Despite the fact that these new tests are described as 'supersensitive" (123),it is better to descrihe them as tests of the second generation, as sensitivity is a relative term. These sandwich tests, which are of immunometric ANALYTICAL CHEMISTRY. VOL. 59, NO. 12, JUNE 15, 1987
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design (immunoradiometric assay-IRMA), are far superior to their radioimmunoassay counterparts, as the choice of the two monoclonal antibodies is epitope or region specific, in contrast to the multi-(non)-specificity of the normal polyclonal antisera. The trend toward solid-phase assays continues, as these techniques are easy to handle and are more robust than liquid-phase assays. Solid phases most often used include coated tubes (118), coated balls (124), and magnetizable microparticles (125-127). The latter are known commercially in the Amerlex-M (Amersham), MAIA (Seroeno), and MAGIC Lite (Ciba-Corning) immunoassay kits. Certain areas of in vitro diagnosis use almost exclusively nonradioisotopic assay methods. As an example, drug monitoring is carried out by using enzyme- or fluorescence immunoassays in over 85% of the commerical kits offered. This is possible, because of the relatively high analyte concentration, although by using advanced electronics, it has been possible to lower the limit of detection of an assay to measure such analytes as digoxin and digitoxin, which are present in nanomolar concentrations (128). Enzyme Immunoassays. Enzyme immunoassay techniques are undergoing modification, and combinations with other detection systems are now being offered commercially. One example is the Amerlite system (129) from Amersham International, where the photometric end point determination has been replaced by a chemiluminometric one, where the light-output is stable over several minutes. (For more details see under Luminescence immunoassays.) Another combination is with fluorophores of the methylumbelliferone group, which allow a fluorometric detection. Both modifications improve the sensitivity of the enzyme immunoassay greatly (130). The 3M Diagnostic Systems FAST-test for total IgE and allergen-specificIgE uses a fluorescence-enhanced enzyme immunoassay technique. The homogeneous EMIT (enzyme multiplied immunoassay technique) and ELISA (enzyme-linked immunosorbent assay) still form the basis for the majority of enzyme immunoassay kits. Immunoenzymetric assays (IEMA) (analogous to IRMA) have been developed for proteohormones (131, 132), using monoclonal antibodies and solid-phase technology, and are becoming increasingly more popular, though they are often still more complex in handling than their IRMA counterparts and have lower detection limits comparable with corresponding IRMA methods. It can be noted here, that it is often secondary as to which label is used in an assay system, when the system itself has been optimized with respect to the assay components. The advantages of a nonradioisotopic label do not seem to counterbalance the greater complexity of the assay in many cases, and it is not to be expected that enzyme immunoassays will replace their RIA counterparts until the ease of handling is comparable. With this said, the role of enzyme- (and other alternative) immunoassays will become more important-especially in countries where the use of or acquisition of radioisotopes already is or will become prohibitive-for example, in developing countries. One must also consider, that in many such countries, items and services taken for granted in highly developed communities, such as refrigerators, data-reduction systems, and constant power supplies, are not or are only partly present. This acts as a damper, as only stable assay systems which also work under adverse conditions are practicable. Many new enzyme immunoassay kits are becoming available for bacteriological and virological markers. In contrast to earlier practice, these kits have been developed as EIAs, and not first as RIAs with subsequent modification. This reflects the upgrading of the status of the enzyme immunoassay. The main drawback of many enzyme immunoassays remains the relative instability of components after reconstitution and their sensitivity to antimicrobial agents such as azide or mercurials (thiomersal). Peroxidase labels are the most affected enzymes in this category, alkaline phosphatase and galactosidase being relatively immune (131). Fluorescence Immunoassays. Conventional fluorescence has never really contended as an alternative to radioisotopic labeling because of the quench effect of serum components, which often necessitate the extraction of the sample prior to assay (133). One way of avoiding this difficulty has been the use of fluorescence polarization techniques (Abbott TDx 340R
ANALYTICAL CHEMISTRY, VOL. 59, NO. 12, JUNE 15, 1987
system) for small molecules. The physical nature of this phenomenon is that it excludes its use for large molecules, i.e., those with low Brownian movement. The fluorescence polarization immunoassay (FPIA) has been successfully introduced in the above-mentioned Abbott TDx system to monitor drug levels in biological fluids. This method uses modern electronics, combined with the “closed-system’’ approach to optimize precision. Although reagent costs are relatively high, the need for single determinations and stored standard curve allow for an economical operation of this system. The problem of interference by substances absorbing light between 350 and 500 nm, Le., where most conventional fluorogens operate, has been overcome by using rare-earth chelates-for example, europium and terbium-as labels. These have a Stokes shift over 200 nm, otherwise stated, they can be activated with blue light, and emit a red fluorescence. This coupled with the fact that the fluorescence lasts several hundred microseconds makes it possible using laser-pulsing and delayed fluorescencesamphg, to excite the sample several hundred times in a second. The signal to noise ratio is very low, and in theory, the detection limits of such assays are equal to or are better than those using radionuclide labeling. The DELFIA system from LKB uses this time-resolved fluorescence immunoassay (TRFIA) (134-136). Assays for haptens and proteohormones are available by using TRFIA. As in the case of the Abbott TDx, the LKB DELFIA is a closed system-to put it another way, the user is fully dependent upon the supplier for assays and cannot develop his own methods for these “black boxes”. Luminescence Immunoassay. Although it is almost a decade since the first chemiluminescence labeled immunoassays were published (137),their commercialization is only now being realized. This is partly due, as it is in all alternative labeled techniques, to patent problems with labels. The theoretical detection limit of Iuminescence is lower than that of radioisotopic labeling, this being partly due to the independence from radioactive decay constants. In practice, as is the case for TRFIA, the lower detection limits are similar to those in well-constructed immunoradiometric assays (138, 139). The differentiation into bio- and chemiluminescence is made on the grounds of whether an enzyme-substrate system or a chemical oxidation step generates the energy needed for the light emission. The bioluminescent systems have all the disadvantages of label instability after reconstitution, together with the ease with which the enzymes can be inactivated (140). Chemiluminescent systems have proved to be robust, labels being usable more than 3 years after their synthesis, even when they have been kept a t -20 “C in frozen form. Bioluminescent systems have a high efficiency (up to 0.9 einstein/mol has been reported in ref 129), whereas chemiluminescent systems in aqueous media have a low light output (under 0.05 einstein/mol (in ref 129)). The robust nature of the chemiluminescent systems has outweighed the high efficiency of the bioluminescent ones, so that the former have been used as signal for immunoassays. In all cases of chemiluminescence, a highly exergonic reaction is needed to produce light in the visible spectrum. Most chemiluminescent reactions are initiated with an alkaline peroxide solution, often in the present of a heme-containing compound which acts as a pseudocatalyst (141). Chemiluminescent labels include derivatives of luminol (129) and acridine (139,142, 143). The most commonly used luminol derivative is 4-aminobutyl-N-ethylisoluminol(ABEI), the derivative of acridine being sdbstituted 9-phenyl esters (acridinium esters) (139). Commercial immunoassays using both types of label have recently become available from Henning Berlin (luminol derivatives) and Ciba-Corning (acridinium esters). As stated before, a hybrid luminescence enhanced enzyme immunoassay (LEIA) has been marketed by Amersham International (144) where the amplification effect of a peroxidase has been coupled with an enhanced chemiluminescence detection system (195). Assays offered at the time of going to press include the full thyroid panel (with the exception of thyroglobulin and transthyretin), certain tumor markers, and some of the pituitary hormones. The conventional chemiluminescence of Henning-Berlin (Lumitest) ( 1 4 5 ~and ) Ciba-Corning (MAGIC Lite) is of a short-lived nature, over 95% of the light being emitted within
5 s. The LEIA from Amersham utilities an excess of luminogen together with an enhancer (e.g., 4-iodophenol), which, together with the peroxidase, gives rise to a long-lived luminescent glow (145),which is stable over several minutes. This has the advantage that no injection is needed in the luminometer to initiate the reaction. As a result 96 wells in a microtiter plate can be read and processed in under 2 min. In contrast, the measuring time for the conventional chemiluminescent labels is 2-4 s, which allows 10-12 samples per minute to be measured. A disadvantage of chemiluminescent immunoassays, more so the “conventional”type, is that it is not possible to measure a second time, should the reaction not be monitored (sticking tube, wrong injection volume or solution). The luminescence immunoassay will take its place alongside the other alternatives to RIA, especially where high sensitivity or low sample volume, e.g., neonatology, prohibits the use of other methods (146). Another interesting development in luminescent labeling has been made in the allergy field (MAST Diagnostics) where photoplate detection of total serum IgE and allergen specific IgE’s is possible by using a Polaroid-type film with enhanced luminescence techniques (147). Such methods, where a yes/no answer is good enough, are optimal for field work or for situations where the usual e uipment for quantitative measurements is not available. ther applications would appear to be bacterial and viral antibody or antigen detection assays for use in screening or epidemics. Nonlabeled Immunoassays. This group of assays includes nephelometric, turbidimetric, and particle-counting immunoassays. The first two methods have been known in clinical chemistry laboratories for many years, the latter is now making inroads into the routine laboratory. The lower detection limits of these assays have been greatly improved by the introduction of modern electronics and microprocessor control. Both nephelometry and turbidimetry are expensive inasmuch as large quantities of specific antibodies are required for each test (large at least, when compared with RIA, EIA, FIA, and LIA). These methods are also limited to molecules which can form immune precipitates, large molecules in relatively hi h concentrations, and it is for this reason that they are still fimited to the determination of certain serum proteins. The particle-counting immunoassays (PACIA) use polystyrene microparticles and depend upon the increase in particle size, produced during the antibody-antigen reaction. ACADE has recently commercialized the Impact system which in addition to proteins also measures haptens by using particle-hapten complexes (148). The lower detection limits of PACIA type assays are at least 3 decades lower than conventional turbidimetry. Problems which occur with the PACIA method include lipemic samples, and it is still necessary in some cases to pretreat the sample before measurement, especially if the sample is to be assayed in undiluted form. Because PACIA uses counting of nonagglutinated particles (0.6-1.0 ~LM), bacterial contamination of samples may give rise to false results should the cell size lie within these limits. Sample dilution above 1:400 is reported to do away with interferences
8
(148).
Dry Chemistry Immunoassays. The recent trends toward “dry-chemistry”analysis in the clinical laboratory have also found its place in immunoassays,especially where a rapid qualitative result is optimal, i.e., pregnancy testing or bacterial infection. Hybritech’s ICON ImmunoConcentration system for human chorionic gonadotropin (BhCG) is noteworthy for the large and rapid impact it has had on the pregnancy testing market (149-151). Although it is a manual, qualitative system, its speed, sensitivity, and relative ease of use have made it a market leader. The recent introduction of the TestPack system from Abbott is a novel use of an enzyme immunoassay on a dry-carrier basis. If the result is negative, a minus sign appears in blue; if positive, a blue plus sign appears. Other similar innovations have been introduced by Stada as a dipstick ovulation test (urinary LH) or pregnancy test (urinary CG) (152),which operate in a similar way to the Abbott Test-Pack system. Again, the use of such simple assays lends itself to areas where other more complicated methods are not available, or not needed, i.e., at home or in the field. Such methods lend themselves to drug screening or patient self-check, and it is
to be expected, that the development will go in this direction.
ANALYTES OF CLINICAL INTEREST The spectrum of analytes of clinical interest increases continuously. Scientific progress, medical needs, and economic interests promote the proliferation of new diagnostic parameters and new assay methods. Clinical interpretatioh of new test methods seems to lag behind, and there is little information and consult on outdated methodologies and parameters. Such consult, however, is badly needed to support decisions to discontinue tests. The analytes of clinical interest included in this review have been selected according to the following criteria: novelty and intensity of scientific discussion; novelty with regard to the previous review; usefulness for the decision process in the medical laboratory. The discussion will be incomplete by necessity, and it also may be biased toward the authors’ field of scientific interest. However, we have tried to cover those analytes included in this review to some depth. Immunology. Assay development and instrumentation as it relates to immunology have been discussed earlier in this paper. In this paragraph we would like to cover other aspects of physiological and nonphysiologicalimmunologic reactions which are of major interest to the physician involved in clinical chemistry and pathology. Antibodies as in Vivo Reagents. Whereas the in vitro use of poly- and monoclonal antibodies is well-established, in vivo application of specific antibodies for diagnostic purposes or therapy of malignancies is still in a developmental stage (153). Initially, in hematologic malignancies,passive treatment with monoclonal antibodies was tried. Later, rejection of kidney transplants and leukemic cells during autologous bone marrow transplantation were treated with monoclonals. More recent applications include treatment with antibodies labeled with cytotoxic agents (153), bacterial or plant toxins (153), and imaging. For imaging purposes, radiolabeled specific antibodies are injected which are directed against the typical structures or products of, e.g., a tumor (153-155). The antibodies will detect these structures and will be fixed on their epitopes. Subsequently, y-camera scans, emission tomography, and whole body scans are performed if indicated, which allow detection of lesions in deep tissues or tumors of early stages. This method may become useful to diagnose, stage, and monitor certain tumors. However, more information will have to be available, and problems related to in vivo dehalogenation of radiolabels and to specificity in clinical and analytical terms will have been minimized. Antibodies as Causes of Analytical Errors. The increase in analytical sensitivity, which was achieved during the last years through the application of improved methods, resulted in more precise analytical results. However, it also led to a still growing awareness of a specific type of interference which is caused by endogenous antibodies. These antibodies are characterized by their persistence, high specificity, and affinity toward their antigen (156-161). The antibodies interfere by way of several mechanisms and cause analytical as well as diagnostic confusions: (1)The antibodies directed against the analyte in question function as a kind of binding protein: they stabilize the analyte and decrease its rate of clearance from the circulation. Simultaneously they increase the analyte’s concentration or activity in the serum. Laboratory methods, in such cases, will report elevated values which do not fit the clinical symptoms. Occasionally erroneous results will be obtained if the autoantibodies in the serum and the antibodies of a given reagent compete for the analyte. Macro CK type 1 (macro CK-BB) (156, 157), macro LDH (158-160, 162), macro ASAT (163), macro AP (164),and autoantibodies directed against insulin (I&), thyroid hormones (166),and apolipoprotein B (167)have been observed to interfere via this mechanism. (2) The antibodies directed against the analyte in question decrease its activity or mask its presence: laboratory methods in this case usually will show low results and sometimes demonstrate a special kind of cold dependence. Such results have been reported recently for macro CK-BB (156, 157), macro LDH (168-1 70), and autoantibodies directed against the von Willebrand factor (171) or platelets (172-1 74). (3) The antibodies react with constituents of the reagents applied: these effects cause erroneous analytical results and have been reported for immunoassays, especially sandwich ANALYTICAL CHEMISTRY, VOL. 59,
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immunoassays. Basically, any agent capable of cross-linking the labeled antibody to the solid-phase can generate a falsepositive result. In most cases these agents are circulating antibodies directed against the specific immunoglobulin being used in the assay. Examples recently reported include antibodies directed against immunoglobulins of sheep (175),cow (175),guinea pig (175), mouse (175-177), goat (178), and rabbit (175, 177). Preventing cross-linking of the reagent antibodies by addition of nonimmune serum of the same species often helps to overcome this type of unspecificity. The prevalence of circulating antibodies directed against sheep, guinea pig, or mouse proteins has been observed in 0.5-9% of a population of apparently healthy individuals (175). Prevalence of autoantibodies directed against serum enzymes is of a comparable level. It increases with age and becomes increasingly evident with the improvement of the sensitivity of analytical methods (161, 164). Finally, interferences from extremely elevated amounts of antibodies in typical routine methods like the determination of urea (179) or creatinine (180) have been reported. Autoantibodies. Loss of immunological tolerance as a consequence of genetic, viral, hormonal, and environmental mechanisms may result in autoantibodies directed against not only blood proteins but also tissue antigens. The course of these processes, especially their initiation and perpetuation is still not well understood, and the target antigen attacked by the antibody in question often remains obscure. However, progress was significant in demonstrating a variety of human autoantibodies. Recent reports (181-189) present methodological improvements and evidence for the occurrence as well as clinical significance of autoantibodies directed against mitochondria and acetylcholinereceptor and involved in liver diseases, endocrine autoimmune diseases, rheumatic diseases, hematologic diseases, infertility, and kidney diseases. Enzymology. Investigations in enzymology mainly focused on three topics: (1)pathophysiology and pathobiochemistry of serum and intracellular enzymes; (2) improvement of current methodologies; and (3) detection and interpretation of multiple forms of enzymes. Pathophysiology and Pathobiochemistry. In a series of papers (190-195) an approach to a quantitative diagnostic enzymology was made, based on enzyme activity in blood and lymph, amount of especially structural and mitochondrial enzymes in tissues of men and laboratory animals,their release from perfused organs, and the kinetic of enzyme adjustment in the extracellular space. Enzyme release is reported to be produced by a membrane blebbing process of which elevation of intracellular Ca2+concentration is a necessary prerequisite. In the presence of ATP, active membrane blebbing is caused by contractions of the membrane-anchored cytoskeleton. In the absence of ATP, passive membrane blebbing is induced by cell swelling, provided that the cytoskeleton has been cross-linked by Ca +.As long as ADP is present, mitochondrial enzymes are released only to a negligible extent. Furthermore, recent data show the importance of enzyme transport by lymph and the relation between lymph flow and the rate of appearance of muscle and liver enzymes in blood. An example for quantitative enzymology is the attempt to quantify the release of CK-MB in order to diagnose perioperative infraction (196).
Aging of enzymes was studied by investigating erythrocyte enzymes and postsynthetic modifications of plasma enzymes. The erythrocyte loses its capability of protein synthesis shortly after entering the circulation. By exploitation of different age-related alterations of erythrocytes, it could be shown that the cellular activity of numerous enzymes declines and that, at least partially, enzymes are lost into the circulation by an outward direct vesiculation; the amount of intracellular inactivation, however, still is unknown. The determination of intracellular enzymes may result in a new understanding of pathophysiologicalrelations: increase of GTP cyclohydrolase I activity in mononuclear cells allows detection of heterozygotes of GTP cyclohydrolase I deficiency (197), the quantitative determination of CK-BB in erythrocytes and platelets shows that this parameter indicatm reexpression of the CK-BB gene in these cells as well as high proliferation rates and, therefore, may be useful to differentiate and stage hematologic disorders (198). Several other enzymes are used as markers to monitor malignant hemopathies. These are notably in342R * ANALYTICAL CHEMISTRY, VOL. 59, NO. 12, JUNE 15, 1987
tracellular forms of LDH, hexosaminidase, esterase, acid phosphatase, and thymidine kinase (199, 200). Mechanisms which cause the increase of intracellular enzyme concentration are still investigated: CK may be increased by hormonal action (201),by growth-related effects, or after malignant transformation of the cell (198, 202). Erythrocyte aldehyde dehydrogenase has been reported to be a new indicator of ethanol abuse (203); the role of cellular phopholipases in initiating the arachidonate cascade has recently been reviewed (204). The catabolism of plasma enzymes is a field of growing interest (205), and some detailed information has been obtained for CK-MM. Once released into the circulation CKMM is stepwise modified by cleavage of the C-terminal lysine of both M-subunits. The mechanisms of its final inactivation and removal from plasma, however, are still unknown. It could be shown that the stepwise modification is caused by a modifying protein, which thereby transforms the original form, CK-MM3, into CK-MM2 and finally into CK-MM1 (206). This modification process of CK-MM is accompanied by a gradual increase of the apparent activation energy of the reaction catalyzed by CK and a decrease of the respective Michaelis constants. Consequently, the ratios of modified CK-MM1 to unmodified CK-MM3 in a given serum or the energy of activation are indices of the turnover of CK-MM and of the course of, for instance, polymyositis (206-208). Attempts to use serum CK activity as a predictor of the carrier status in Duchenne dystrophy were unsuccessful, due to problems in establishing reliable cutoff limits and a substantial overlap between CK activities in the sera of carriers and noncarriers (209-211). y-Glutamyltranspeptidase (GT) is found in cells with secretory or absorptive capacity of the liver, pancreas (212), kidney, and intestine. However, numerous studies agree that the liver is the source of most of the GT activity in serum. Kidney, pancreas, and the intestines contribute little to normal serum GT activity. Its activity in apparently healthy subjects sometimes is regarded as an "indicator of health" (213) in young as well as in elderly people (214). GT extracted from liver and kidney shows considerable heterogeneity in molecular mass and charge. It consists of light and heavy protein fragments and contains-as a membrane-bound enzymehydrophilic and hydrophobic moieties. These properties, together with differences in sialylation as well as carbohydrate content are the reasons for variant molecular mass forms of 90000-120000 Da in the isolated enzyme. The polypeptide chains are reported to be identical for GT of liver and kidney, an assumption which is substantiated by the fact that antibodies do not differentiate between GT's from various organs (215,216). Serum GT, too,shows a remarkable heterogeneity, but unlike CK or LDH there are no tissue-specific characteristics. Increase of serum GT is observed after induction of synthesis, after release by, e.g., bile salts, after regurgitation of bile, and after destruction of hepatocytes (216,217). It is removed from the circulation by the liver and excreted in bile (216).
Alkaline phosphatase (AP) shares some common features with GT: both enzymes are sensitive indicators of cholestasis (218,219);they show a considerable heterogeneity in normal serum and particularly in patients' sera; and they demonstrate a tendency to react with serum constituents and to form macromolecular complexes. Serum AP may be released by liver, bone, placenta, or intestine. The placenta and a so-called placenta-like form are also found in lung, cervix, ovary, breast, thymus, and testis (220). The clinical utility of AP together with GT, LDH, ASAT, ALAT, and other liver tests has recently been reviewed (221). The use of a biochemical serum marker for prostate carcinoma centers around three clinical situations (222,223): (1) early detection of the disease in asymptomatic patients; (2) differentiation between benign hypertrophy and carcinoma in symptomatic patients; and (3) monitoring of the course of the disease and therapy control. A summary of the results obtained with various techniques for the determination of prostate acid phosphatase (PAP) in recent years indicates that (1)PAP fails to help in situation no. 1, (2) PAP may help in situation no. 2, but predictive values still have to be improved, and (3) PAP is useful in situation no. 3. Whereas diagnosis and monitoring of chronic pancreatitis and insufficiency of the pancreas by laboratory results are still
not satisfactory (224),diagnosis of acute pancreatitis can be achieved with good results. Pancreas amylase, lipase, and trypsin are sensitive and specific parameters for which reliable assays are known. Nevertheless, determinations of amylase and lipase show some limitations: Hyperamylasemiapresents not only in pancreatic disease but also in other pathologic situations like parotitis, mumps, renal insufficiency, or macroamylasemia (225-229). Urinary measurement of amylase is not specific and is no longer encouraged. Lipase determinations still suffer from complicated techniques, low precision at the discriminationlimits, and unspecificity (227,228). Diagnostic sensitivity, specificity, and reliability are significantly improved in methods recently developed (225,230-232): determination of pancreas specific amylase, determination of lipase in the presence of colipase as turbidimetric or spectrophotometric assay (233,234),or immunological determination of lipase (235) or trypsin (236). Determinations of phospholipase A2 (237,238)and other pancreatic enzymes (239) have recently been reported and their value in the diagnosis of acute pancreatitis and pancreatic cancer has been discussed. The role of cytochrome P-450 and the induction of its activity in all forms of exocrine pancreatic disease have also been reviewed (240). The basic mechanism of the inflammatory process (241-244) involve the synthesis and release of several mediators which finally lead to the so-called "inflammatory response". An initial process causes chemotaxin mediated attraction of phagocytes. A t the focal point of an inflammation neutrophil phagocytes develop three types of activities: (1)they produce more mediators to attract and activate other phagocytes; (2) they produce by way of a "metabolic brust" oxidizing agents (0, and OH radicals) and releae oxidizing enzymes like myeloperoxidases; (3) they begin phagoctosis, a process which releases proteases and glucosidases to destroy organic materials; to limit this process of destruction of tissue and proteins like fibrinogen or fibrin, activities of extracellular lysosomal proteinases are controlled by proteinase inhibitors, which are produced by the liver as acute phase proteins. Recent investigations focused on the elastase from neutrophils (PMN elastase), a member of the elastase group of enzymes (neutrophil, macrophage, pancreatic elastase) which in fact are distinct proteinase species. Its physiological inhibitor is a1-proteinaseinhibitor, previously called a-1-antitrypsin, which is able to rapidly bind and inhibit elastase released from neutrophils into the surrounding tissue. Subsequently the enzyme-inhibitor-complex enters the circulation from which it is cleared with a half-life of about 1h. The concentration of this complex in blood and its time-dependent change renders clinically valuable information about inflammatory processes, e.g., in postoperativesepticemia,in multiple trauma (242),in affections of the lungs (245-247), or in chronic joint disease (248). It furthermore allows the determination of the biocompatibility of membranes (249). Serum angiotensin converting enzyme (ACE) is a glycoprotein bound to the membranes of the endothelial cells of pulmonary capillaries. It also is found in intestinal and renal brush borders, in monocytes, and in hyperplastic prostates. ACE catalyzes the transformtion of angiotensin I to angiotensin I1 and the degradation of bradykinin (250). Increased enzyme activity has been found in sarcoidosis (251,252)and other granulomatous inflammations. It has been reported to be a useful index of the severity and course of sarcoidosis. Attempts to use this parameter to diagnose other granulomatous diseases or hypertension met with limited success (252, 253). Nevertheless, several recent papers describe methodological improvements and mechanized determinations (254-257). Methods to measure this enzyme have proliferated, presenting now a variety of units which has caused some confusion about activities and reference ranges (258, 259). Indications to determine LDH activity increasingly include, besides liver diseases, cardiac diseases and selected cases of hemolytic diseases. Determinations of the isoenzyme LDHl gain increasing importance for patients admitted late after the onset of acute myocardial infarction, for patients with concomitant skeletal muscle trauma or disease, and for patients with unstable angina pectoris. This latter group of patients shows normal LDHl values; the values nevertheless are somewhat higher than those in stable angina indicating early myocardial damage (260).
There is increased interest in determining enolase isoenzymes, particularly those containing y subunits (CY?, y-y) which have been termed neuron-specific enolases. The y monomer previously was believed to be exclusively expressed by mature neurons; recently, however, neuron specific enolase also has been detected in neuroendocrine and ganglion cells. Determination of serum and CSF enolase isoenzymes or immunocytochemical demonstration of them in tissues is reported to be clinically useful in small-cell lung cancer and neuroblastoma (261, 262). The different intracellular locations of enzymes and their compartmentation in cytoplasma and mitochondria suggest that cytosolic enzymes are more readily released following injury than mitochondrial enzymes. Mitochondrial enzymes, therefore, may be regarded as indicators of severe cell damage and necrosis. Interest recently focused on the serum activities of mitochondrial forms of CK (macro CK type 2), GLDH, and mitochondrial ASAT in chronic liver diseases and of mitochondrial ASAT as a parameter indicating acute myocardial infarction (202, 263, 264). However, results obtained for GLDH and mitochondrial ASAT show that these parameters diagnostically are not superior to cytosolic and total ASAT, ALAT, or GT activities. Methodology. Optimized and standardized assay methods have been established for the determination of most of the diagnostically important enzymes. However, the measurement of several enzymes still needs improvement. Some improvements have recently been reported. Significant progress in analytical specificity and sensitivity of the CK-MB determination was achieved by immunoassays using polyclonal or monoclonal antibodies directed against the M or B subunits of CK (265-267). Recently, the first monoclonal antibody directed against the total CK-MB molecule was isolated (268). However, these improvements in analytical performance can only partially facilitate the diagnosis of acute infarction in clinical situations. Limitations of the parameter CK-MB itself remain, e.g., its presence in blood after trauma or in skeletal muscle disease (269). Therefore, diagnosis of infarction still requires sequential sampling during the appropriate time window and recognition of a dynamic activity-time pattern. Normal-sized,postsynthetic variants of CK-MM can be quantitated by HPLC (270),isoelectrofocusing (208),and electrophoresis (207). The description of multiple forms of alkaline phosphatase (AP)has prompted the development of methodologieswhich allow distinguishingliver and bone fractions and quantitating tumor-specific AP in patients' sera. Recommended methods include isoelectric focusing (271),HPLC (272),electrophoresis on lectin-containing agarose gel (273-275) as well as immunological techniques using monoclonal antibodies (276-278). Furthermore, a monoclonal antibody has been described which allows the differentiation of liver and bone specific forms of AP (276). Improvementsin the determination of total amylase activity have been possible after development of defined substrates, particularly those bound to paranitrophenol. Tests are now available which allow mechanized determination with stable reagents and which do not suffer from interference by glucose or pyruvate. Typical substrates are 1.4-a-D-4-nitrophenylmaltoheptaoside (279),its ethyliden-protected (280) or benzyliden-protected derivatives (281), and 2-chloro-4-nitrophenyl-0-D-maltoheptaoside (282). Specific determinations of pancreatic amylase became possible by utilizing a wheat germ derived inhibitor (283) to inhibit S-type amylase, by using isoamylase-specific monoclonal antibodies (284-286) or by quantitative electrophoresis (287). Quantitative determinations of LDHl in the case of myocardial infarction require easy-to-perform assays for the application in emergency laboratories. Time-consuming electrophoretic and manual chromatographic procedures are less suited and gradually will be replaced by immunological and inhibition methods for LDHl (288, 289). Quantitative determination of all five isoenzymes in less than 30 min became possible by use of HPLC (290). Multiple Enzyme Forms. In recent years variant CK forms have been described, which occur in patients' sera and which demonstrate larger molecular masses than normal dimeric cytoplasmic isoenzymes. These macro CK's exist in two different forms (291): macro CK type 1 (Ig-linked CK) and ANALYTICAL CHEMISTRY, VOL. 59, NO. 12, JUNE 15, 1987
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macro CK type 2 (CK of mitochondrial origin). In normal individuals and hospitalized patients the presence of macro CK type 1 is characterized by persistently elevated levels of CK activity. Macro CK type 1prevalence is estimated to be about 3%. It is seen more often in the elderly and in women than in men, and-with exception of coronary heart disease (292)-shows no relation to any specific disease. Macro CK type 2, which in certain cases is associated with circulatin normal-sizeCK-BB, shows no close relation to age or sex and is seen in hospitalized patients with a prevalence of about 3.5% (293,294). It is closely related to severe illness, particularly malignancies. Various molecular forms of GT appear in serum of patients with different liver disease. A high molecular masa form (>lo6 Da) is present in highest amounts of patients with jaundice from extrahepatic obstruction. GT forms of intermediate molecular mass (250000-500000 Da) are present in the serum of most patients with liver diseases, and the presence of band I1 B among these forms allows differentiation between extrahepatic and intrahepatic jaundice (295). However, as long as knowledge of the release of GT (217), its interaction with serum constituents, and other postsynthetic modifications of the enzyme is lacking, disagreement will remain as to the significance of the various GT patterns in cholestasis (216). The patterns become even more complex in cases of malignancies when tumors release additional variants (296,297). Various forms of serum AP have been described. These include, besides isoenzymes, postsynthetically modified and complexed forms of AP. Particularly in cholestasis several electrophoretic AP bands have been observed and a number of names proposed (218). Besides isoenzymes originating from liver, bone, placenta, and intestine, macromolecular forms are of specific interest: in cholestasis a liver AP-lipoprotein-X complex is seen (219),in hepatic malignancy a membraneparticle-AP is present, and, without special relation to any disorders, a Ig-linked AP has been identified (218, 298). Ectopic or inappropriate expression of AP in tumors now is a well-recognized phenomenon. Most examples describe an isoenzyme essentially identical with placental AP (Regan isoenzyme), placental-like isoenzyme (Nagao isoenzyme) (299), and a intestinal-like isoenzyme (Kasahara isoenzyme) (299, 300). The presence of these isoenzymes is attributed to the reexpression of the respective genes in the tumor (278). Additional AP forms are detected after isoelectric focusing of serum; the clinical significance of these forms has yet to be established (271). A comprehensivereview on multiple AP forms has recently been published (301). Lipoproteins. Many studies conducted during the last years have established serum cholesterol as a major risk factor of atherosclerosis. It still is the number one cause of morbidity and mortality in the adult population of the Western World. Therefore, it comes as no surprise that the 1985 Nobel prize for medicine was awarded to J. Goldstein and M. Brown for their work on cholesterol metabolism, especially the LDL receptor hypothesis (302). Physiology and Biochemistry. Lipids like cholesterol and triglycerides, which are structural components of cell membranes, precursors of hormones, and important sources of energy, are not sufficiently polar to be dissolved by plasma and transported, in aqueous solution, to their target cells. They only can be circulated after complexation by specific proteins, the apolipoproteins (apoA-I, apoA-11, apoA-IV, apo(a), apoB-26, apoB-48, apoB-74, apoB-100, apoC-I,apoC-11, apoC-111, apoC-IV, apoC-V, apoD which sometimes is termed apoA-111, apoE, apoF, apoG, and apoH) (303-306). For most of the apolipoproteins, genetic variants and polymorphisms have been detected (303,307-311). It is now recognized that apolipoproteinsare necessary for the synthesis and catabolism of lipoproteins, important structural components of lipoproteins, cofactors or activators of certain enzymes, and lipid transfer proteins (303, 307, 312). Complexes of lipids and apolipoproteins form the five major lipoprotein particles: chylomicrons, VLDL (very low density lipoprotein), LDL (low density lipoprotein), IDL (intermediate density lipoprotein), and HDL (high density lipoprotein). They represent distinct types of particles. Their structures are constantly changing due to interactions with each other, with enzymes of plasma and arterial walls, and with lipid transfer proteins. Chylomicrons are the largest lipoprotein particles. They are synthesized in the intestine and enter the circulation via 344R
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the thoracic duct. In their core they transport dietary triglycerides and cholesterol esters (302,303,307,312-314).The polar shell around the unpolar core of the particles consists of a phospholipid membrane which also contains cholesterol, apoB-48 (304) and apoA-IV (306). During circulation the chylomicrons incorporate into their polar shell apoC-11, apoC-111, and apoE (315)from the VLDL and HDL fractions. ApoC-I1 containing chylomicrons now activate the enzyme lipoprotein lipase (LPL) which is fixed via a polysaccharide anchor to the epithelial cells of blood vessels. LPL hydrolyses and removes triglycerides from the chylomicrons and converts them into chylomicron remnants. During hydrolysis apoC-I1 and apoC-I11again are split off and transferred back to HDL. Due to loss of apoC the remnants become poorer substrates for LPL and simultaneously are more easily resorbed by the liver via the specific apoE mediated receptor (307,312,314). VLDL is synthesized by the liver. It contains a core of endogenously synthesized triglycerides and cholesterol esters and a shell containing the structural protein apoB-100. During circulation these particles incorporate and retain apoC-11, apoC-111, and, in the case of large VLDL, also apoE from plasma HDL. They now become excellent substrates for LPL and are subsequently lipolyzed. During this process the density of VLDL increases and the particles may now be differentiated into VLDL I, VLDL 11, and VLDL 111. Furthermore, parts of apoC are transferred back to HDL 2. ApoC-I11 is reported to inhibit lipoprotein lipase and thus a premature removal of VLDL by the liver. The metabolism of VLDL and the rate of its lipolysis are genetically determined by the proportion of apoC-I1 and apoC-I11 (304, 307 312,314,316). Finally, mainly small particles of VLDL (317, 318) are converted into IDL. Hepatic apoE and apoB,E receptors are involved in this conversion, the exact mechanism is still unknown (312, 317, 319). IDL has a lower triglyceride but a higher cholesterol ester content than its precursor VLDL. It is bound to the apoB,E receptor, taken up by the liver, and catabolized. An alternative pathway is its transformation into LDL through the action of hepatic lipase, transfer proteins, and mechanisms, which among other alterations cause a transfer of apoE to HDL. LDL shows less affinity to the apoB,E receptor than IDL (303, 312, 314, 315, 317). LDL is a group of particles heterogenous in size and composition. The metabolic basis and significance of this heterogenity are unknown (317 ) . Recent studies indicate that LDL not only results from VLDL degradation but also is directly secreted by the liver (317). LDL is rich in cholesterol and is assumed to transport cholesterol to those tissues which utilize cholesterol. Its structural component apoB-100 interacts with a specific LDL receptor (apo-B,E receptor) (302, 303,312,313,317) located within the clathrin-coated pits on the membranes of target cells like intimal endothelial cells, smooth muscle cells of the arteries, fibroblasts, macrophages, and hepatocytes. Receptor-mediated processes cause endocytosis of LDL which is subsequently degraded by lysomal enzymes. The resulting free cholesterol regulates the cholesterol metabolism of the cell in three ways: (1)inhibition of cholesterol synthesis by inhibition of hydroxymethylglutaryl-CoA-reductase(HMG-CoA-reductase);(2) activation of acyl-CoA-cholesterol-acyltransferase(ACAT) (320); ( 3 ) reduction of the number of receptors on the cell membrane. HDL is a very heterogenous group of particles. So far, discoidal HDL particles, HDL 2, HDL 3, and HDL, have been identified in man. They originate from several sources: the intestine, the liver, peripheral cells, and metabolism of VLDL and chylomicrons (312). HDL is secreted as a discoidal particle. It is characterized by high amounts of structural apoA-I and apoA-II(304) and small amounts of apoC-I, apoE, as well as apoA-IV which are reported to enhance the activity of lecithine-cholesterol-acyltransferase(LCAT). Discoidal HDL is rapidly converted by LCAT into the spherical plasma HDL. HDL additionally contains small amounts of apoC-I1which can activate LPL and of apoD the biological function of which is still unknown (312). When HDL 2 becomes enriched in triacylglycerols it is assumed to be converted into HDL 3 by hepatic lipase. HDL 3 is the lipoprotein of hi hest density (312,314). During circulation apoC is aquired y! VLDL and chylomicrons, and as a result the apoA-I pool in HDL is augmented. HDL 3 interacts with peripheral cells and incorporates free cholesterol from cell
membranes. By action of LCAT the cholesterol is esterified and HDL 3 is transformed into HDL 2. Additional decrease of apoA-I and an increase of apoE result in the formation of “HDL with E” (HDL,), which is reported to be taken up by the hepatic apoE receptor, thereby allowing a reverse transport of cholesterol from peripheral cells to the liver (312, 314). Reverse cholesterol transport is also mediated by plasma cholesterol ester transfer proteins which transfer cholesterol from HDL back to nascent VLDL and chylomicrons (312, 321). Other studies, however, suggest that the inverse relationship between HDL concentration and coronary heart disease is not based on an influence of HDL on body stores of cholesterol (322). This indicates that the effect of HDL on coronary heart disease is far from being fully understood, and it appears that the metabolism of cholesterol is much more complicated than it appears today. Lp(a) is a lipoprotein present in virtually all individuals (312). It contains apoB and apo(a). Lp(a) is assumed to be an LDL particle to which apo(a) is attached via disulfide bonds to apoB (323). High levels of Lp(a) appear to be correlated to an enhanced risk of atherosclerosis. Lipoprotein X and Lp(X)-like materials are complexes which are found in serum as a result of regurgitation of bile, e.g., in cholestasis. Their composition has been reported to consist of phospholipids and unesterified cholesterol (88%), of proteins, mostly apoC and albumin (6%), and of cholesterol esters, triglycerides and bile salts (6%) (324). They do not appear to be converted into spherical HDL particles by action of LCAT (312). The physiological importance of Lp(X) is not clear. It can be formed in a number of situations, especially those where the surface materials (free cholesterol, phospholipids) exceed the transport and metabolic capacities of HDL, transfer proteins, and LCAT (314). Pathology and Pathobiochemistry. Hyperbetalipoproteinemias are genetically determined disorders. They may be characterized by an overproduction of apoB (325) only. Alternatively, and as a rule, we observe familial hypercholesterolemia with elevated LDL and apoB concentrations due to increased synthesis and decreased metabolism, low HDL levels, and a high risk of atherosclerosis. This syndrome is well-explained by a deficiency of cell receptors for plasma LDL. Such a deficiency of receptors leads to a decreased rate of removal of IDL and LDL and a simultaneous increase in the rate of production of especially small LDL via VLDL (303, 313,317). In cases of hyperlipidemia VLDL may also contain apoB-48 and LDL may contain apoB-74 and apoB-26 which appear to be fragments of apoB-100 (304). Their physiological function is unknown. In cases of low numbers of LDL receptors larger LDL particles are observed which contain more cholesterol esters and add to the risk of atherosclerosis (317). Generally the risk of atherosclerosis is high due to a prolonged and direct interaction of LDL with the endothelial cells (scavenger pathway), due to an increase in cholesterol biosynthesis which results from the failure of the mechanism to regulate HMG-CoA-reductase activity, due to direct effects of LDL on platelet aggregation and proliferation of smooth muscle cells of the vessels, and due to elevated lipid peroxide levels (326, 327). Familial hypercholesterolemia is the form most often seen in patients and therefore the most frequent reason for the development of atherosclerosis and related diseases. There is no doubt that reduction in serum cholesterol concentration by only 1% results in a 2% reduction in the incidence of myocardial infarction (328-330). Tangier disease is a rare disorder characterized by a virtual absence of apoA-I, apoA-11, and HDL from serum and by low levels of LDL and LCAT. These changes are caused by a variant apoA which is rapidly catabolized. This variant apoA differs from normal apoA amino acid composition (303,312), resulting from a failure to cleave the prosegment. Clinically, Tangier patients appear to show no increased risk for atherosclerosis. Hypoalphalipoproteinemia is characterized by decreased levels of HDL, apoA-I, and increased concentrations of chylomicrons and by a significantly higher risk for coronary heart disease (303). Abetalipoproteinemia results from the absence of apoB or the presence of a defect apoB (303,312,331,332).Homozygotes have no detectable levels of chylomicrons, VLDL, or LDL in their blood, and suffer from malabsorption, neurological symptoms, retinal pigmentory degeneration, and
acanthocytosis. In a variant form chylomicrons and VLDL are present, because the underlying defect only affects synthesis of liver apoB-100, whereas synthesis of apoB-48 remains unaffected. Genetic deficiencies of apoC-I1 or LPL result in triglyceridemia. In these cases chylomicronsare metabolized at a low rate (type I of Fredrickson) (303,312,314,315,333).Genetic deficiency of hepatic lipase leads to an accumulation of IDL, triglyceride-rich LDL and HDL 2 (312, 314). Genetic deficiency of apoE has also been described (334, 335). Type I11 hyperlipidemia is associated with the polymorphism of apoE (303,307,315).ApoE receptors primarily react with the isoproteins apoE-3 and apoE-4. Patients who are homozygotic in apoE-2 therefore accumulate P-VLDL in their blood which is composed of remnants of chylomicrons and VLDL (broad beta disease) and which contains a high amount of triglycerides. Triglycerides increase results from the inhibition of LPL by the high concentration of defect apoE. Patients suffering from type I11 hyperlipidemia are at high risk for atherosclerosis. Their apoE has a high affinity to macrophages, which become overloaded with cholesterol esters and transform into foam cells. Inadequate lipolysis causes hypertriglyceridemia and increased VLDL. The risk for coronary heart disease is increased particularly in individuals with a high ratio of LDL to HDL (315, 317). The clinical significance of the lower limit for cholesterol concentrations in blood is not well-understood. In a recent paper an association between malnutrition, low serum cholesterol, and the generation of neoplasms was discussed (336). Determinations of Lipoproteins and of Cholesterol Subfractions. A variety of assays exist to determine or purify cholesterol subfractions. New modifications have recently been described and evaluated: HDL (337-344), VLDL (341), LDL (341, 345, 346), Lp(X) (347). There are three major methods to quantitate concentrations of apolipoproteins (303, 304,348,349): chromatography, isoelectric focusing and immunoassays. Problems involved are lack of standardized procedures,lack of reliable quality control materials, changing immunoreactivity due to self-association and aggregation of the particles, varying content of lipid which may mask epitopes, and different affinities of the antisera. Assays and assay modifications have been recently described for apoA-I (350-354), apoB (352,353,355-358),Lp(a) (359),apoC (353), apoC-I11(360,361).Reference ranges are not yet well defined, especially those of apolipoproteins (362, 363). In addition, the clinical significance and physiological importance of socalled protective factors like TxPA are not understood at this time (364). Measuring analytes in lipemic serum has always been a frustrating problem. A solution to this problem based on enzymatic lipolysis with the aid of lipase and a-cyclodextrin has recently been described (365). Metabolites/Drugs. Bilirubin. Hepatic bile formation and the determination of bilirubin and its derivatives are still topics of interest. Bile formation now is believed to be an osmotic process resulting from active secretion of solutes followed by passive water movement (366). Hepatic uptake mechanisms have been shown to be dependent on the transmembrane ionic and potential differences, which are regulated by Na+,K+-ATPaseand on a specific bile acid membrane transport protein. This membrane transport protein also binds substances like phalloidin, antamanide, penicillin, and other organic anions in competition to bile acids. Steps involved in intracellular metabolism of bile acids are less well-known. The importance of electrolyte transport processes and especially the role of the canalicular chloride-bicarbonate exchanger in bile formation have recently been highlighted (367). Methods used to measure bilirubin usually are based either on colorimetry of azopigments of bilirubin or on direct spectrophotometry, e.g., in specimens from newborns. These methods suffer from interferences by a variety of substances. To avoid these interferences, current assays have been modified and new methodologies developed (368-371) including assays utilizing bilirubin oxidase (372),HPLC (373,374),or the determination of albumin bound bilirubin (375, 376). Porphyrias. Many details of the biochemistry of porphyrias (377,378)are still not understood. A major difficulty is the lack of suitable assays for the determination of those enzymes which control the heme synthesis. Insufficient activity of one ANALYTICAL CHEMISTRY, VOL. 59, NO. 12, JUNE 15, 1987
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or more of these enzymes causes porphyria and the subsequent accumulation of toxic heme precursors. Porphyrias are relatively rare and mostly inherited. However, lack of knowledge of their clinical symptoms may lead to severe misdiagnoses: the symptoms may be mistaken as neurological, surgical, or psychiatric disorders. Classification of porphyrias is possible in several ways: according to the site of accumulation of the heme precursors, e.g., hepatic or erythropoietic porphyria; according to clinical symptoms, e.g., acute or nonacute porphyria; and according to the underlying enzyme defect. Typical defects (and their concomitant porphyrias in parentheses) are known for all enzymes involved in the synthesis of the heme molecule: aminolevulinic dehydratase (acute porphyria); porphobilinogen deaminase (acute intermittent porphyria); uroporphyrinogen-I11 synthetase (congenital erythropoietic porphyria); uroporphyrinogen decarboxylase (porphyria cutanea tarda (PCT), toxic porphyria, hepatoerythropoietic porphyria); coproporphyrinogen oxidase (hereditary coproporphyria, harderoporphyria); protoporphyrinogen oxidase (porphyria variegata); ferrochelatase (protoporphyria). In familial PCT inheritance of the enzyme defect is autosomal domina& the clinical expression, however, is dependent on additional factors, e.g., ethanol abuse. An interesting hypothesis proposes that sporadic PCT (379) is seen in patients homozygous for the defect in uroporphyrinogen decarboxylase which in these cases is inherited as a recessive trait. The disorder becomes manifest if the patient simultaneously is heterozygous for idiopathic hemochromatosis. The role of ethanol and estrogens as precipitating agents of this disease is well-known, the underlying mechanisms, however, still are obscure. The analytical method of choice to determine the porphyrin precursors porphobilinogen and aminolevulinic acid, and total porphyrin concentration, is ion-exchange chromatography. Determination of porphyrin patterns in urine, stool, and cells is done with best results using HPLC (380-382). The enzymes of the heme pathway can be measured, but are, at present, reserved to specialized laboratories except for the determination of aminolevulinic acid dehydratase and porphobilinogen deaminase (377). Free Drugs. During the last years the development of therapeutic drug monitoring has rapidly progressed. Once administered drugs are soon distributed between tissue and blood. In blood, drugs may exist in their free form or may be bound to serum constituents, especially serum proteins. The extent of this binding may vary from more than 99% to less than 10%. The pharmacologically active form of the drug is assumed to be the free form. Therefore, determinations of free drugs gain increasing interest, particularly for drugs which usually show a high affinity to plasma proteins (fraction of bound drug >0.80 (383-385), e.g., amitryptiline, carbamazepine, chlordiazepoxid, chlorpromazine, desipramine, diazepam, digitoxin, nortriptyline, phenytoin, propanolol, salicylic acid, tolbutamide, valproic acid, and warfarin). Typical factors influencing the binding of drugs to proteins are alterations in the concentration of binding proteins and competition from other binding substances, e.g., other drugs, free fatty acids, or hormones. However, not all of the alterations of drug binding will be of clinical concern. Free drug measurements are of special interest: in patients with progressing liver and renal diseases, where concentrations of binding proteins may fluctuate rapidly; in patients simultaneously treated with phenytoin and valproic acid or warfarin and non-steroidal antiinflammatory agents, because of possible drug interactions on the binding proteins; in acutely ill patients who are treated with cardiac active drugs (p blockers, calcium channel blockers) which bind to rapidly changing a-l-glycoprotein and lipo-proteins; and during the perinatal period. Equilibrium dialysis and ultrafiltration are the methods of choice for the measurement of free drugs. Very rapid separation of bound and free drug is achieved by ultrafiltration utilizing a pressure gradient generated by centrifugation (386). Pharmacokinetic considerations have a direct impact on drug monitoring: patient data like age, sex, weight, etc. as well as timing of the drug administration and phlebotomy are essential to relate serum concentration and treatment doses for a given drug. For theophylline, a recent monograph gives a pragmatic guidance and a concise review (387). For digoxin, digoxin-like factors have also to be taken into consideration (385, 388-390). 346R
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Urine Analysis. Discussions in the field of urine analysis mainly centered around four topics which have specifically been addressed in two recent workshops: urinalysis; urinary enzymes; proteinuria; and microalbuminuria (391, 392). Urinalysis. Urinalysis is one of the most frequent laboratory tests. As a rule, it still is done manually and therefore remains one of the most labor-intensive routine tests. Improvements of urinalysis have been proposed, and three alternatives are currently discussed: (1) Performance of an optimized quantitative manual urinalysis, which is even more labor-intensive but will give best results because standardized counting chambers, urine volumes, suspension volumes, and magnifications are used. Its main advantages are higher sensitivity and specificity in the detection of casts, proof of glomerular hematuria based on a rate of a t least 10% of acanthocytes (typical, by extrusions deformed erythrocytes), and optimal quantitation of particulate analytes (392-395). Though the procedure is labor-intensive, it may nevertheless be economic provided the prevalence of pathological specimen and/or the number of patients a t risk is high. (2) The most radical changes of the classical urinalysis result from the development of test strips with nine or more different analytical pads. Several groups of investigators showed that there will be a significant cut of cost if strip analyses are used for screening purposes to exclude all nonpathologic specimens (396). For this purpose, only clear urines of the typical color from low risk patients should be included. Discolored urines, urines from high risk patients, and urines showing positive strip results have to be examined by quantitative microscopy (397). Recently, inexpensive mechanized test strip photometers have been introduced which allow quantitation and quality control (392,398). Advantages and limitations of urine screening with test strips are still controversely discussed. Whereas the performance of the blood detection test generally is reported to be sufficiently sensitive and specific, there is some concern about the sensitivity and specificity of the albumin and leukocyte test strips. The albumin test strip of course cannot exclude proteinuria and the sensitivity of some leukocyte test strips seems to be insufficient to detect every relevant leukocyturia. Test strip screening is of highest efficiency in laboratories with a low prevalence of pathological samples. Reports discussing test strip screening and subsequent microscopy of pathological samples have recently been published (392,396, 397,399-404). (3) Attempts to reduce labor-intensity or urinalysis while retaining microscopy resulted in the development of “automated intelligent microscopy” (393, 405). The Yellow Iris is an analyzer that performs computer-assisted microscopy and utilizes dipstick analyses to automatically run complete urinalyses a t a throughput of about one to three samples per minute. Actual throughput rates vary because of particulate composition of the specimens. Papers on automated microscopy have recently been published (393, 405, 406). A definitive answer to the question of how to perform urine analysis is difficult. The prevalence of pathological samples and nephrological disorders, the quality of the test strips applied, and the protocol of sampling, storing, and handling of the urine specimens all determine the efficiency of any given method of urinalysis. Urinary Enzymes. Release of urinary enzymes such as alanine aminopeptidase (AAP), N-acetylglucosaminidase (NAG), and y-glutamyltransferase (GT) may take place prerenal, renal, and postrenal. Typical serum enzymes pass the glomerula under physiological conditions at rates depending on their molecular masses. In addition to this filtration process renal resorption and inactivation processes also determine the amount of enzyme finally found in the urine. Furthermore, the enzyme activity in urine is influenced by the stability of the enzymes under the “unphysiological conditions” of urine. Determination of urine enzyme activity generally requires an appropriate sampling (recommended: second urine in the morning), sample preparation (chromatography to remove salts and small inhibitor molecules), and the relation of enzyme activity to creatinine, sampling time or urine volume. Interpretation of urine enzyme activities seems much more difficult than that of serum enzymes. Increased values should be classified as: (1)alteration in which increased enzyme content of the kidney is paralelled by excretion (AAP, GT) without tubular injury; (2) lesion, which is paralleled by changes in proximal tubular resorption, measured by either renal glucosuria or microglobulinuria; (3)
necrosis, characterized by the appearance of parts of the cytoskeleton, intracellular enzymes, and distal nephron structures in urine. Urinary enzyme analysis is of value to monitor renal transplant rejection, to detect nephrotoxic agents, to monitor diabetic nephropathy, and to detect renal artery embolism (391, 392). Proteinuria. In cases of patients with suspected nephrological diseases and in cases of positive albuminuria, test strip regults should be quantified and the protein pattern examined (392). The objective is to differentiate between glomerular and tubular proteinuria and to distinguish selective proteinuria from nonselective or mixed proteinurias. Selective glomerular proteinuria is characterized by the presence, in urine, of albumin and transferrin and the absence of high-molecularweight proteins. It indicates minimum change nephritis. Nonselective gromerular proteinuria indicates damage of the structure of the glomerular basal membrane as it is observed in chronic glomerulopathies. Tubular proteinuria shows a resorption disturbance of the tubulus cells for low-molecular-weight proteins like albumin, a-1-microglobulin, and retinol-binding protein. Differentiation of proteinurias can be achieved by applying one- and or two-dimensional electrophoretic procedures followed y silver staining (407, 408). Single proteins are best detected by immunological techniques (392). Clinical significance of albumin dimerization in urine is currently investigated (409, 410). Quantitative determinations of total urinary proteins are still a major problem: most analytical methods are sensitive to various interferences, and an international protein standard is still lacking (392,411). Microalbuminuria. There is increasing clinical demand for parameters which allow very early detection of renal diseases. While the utility of creatinine and other kinds of clearances still is discussed controversely (412-41 7), recent interest focuses on urinary albumin. Normo- and microalbuminuria are relatively new terms to describe the small amounts of albumin which pass the tubulus system after glomerular filtration without being resorbed and finally appear in the urine. In cases with increased glomerular albumin filtration or impaired tubular resorption, an increased urinary albumin excretion is observed. In test strip negative normoalbuminuria there is an excretion rate of less than 26 mg of albumin per day, in microalbuminuria the albumin excretion rate ranges from 27 to 250 mg per day. Various methods have been developed to quantify low albumin concentrations in urine: radial immunodiffusion, rocket immunoelectrophoresis, and, very recently, fluorescein-labeled (418) as well as enzyme-labeled immunoassays (419, 420). Immunologic determination of normo- and microalbuminuria is of great value to monitor potential diabetic nephropathy: while minimal glomerular changes (microalbuminuria) are reported to be reversible, persistent proteinuria is irreversible and has to be prevented. The mechanisms affecting the glomerular filtration process are still somewhat unclear. Renal plasma flow, hydraulic pressure, and pore size as well as charge of the basement membrane of the glomerulum are reported to be part of this very complex system. In addition, a recent report documents the diagnostic value of microalbuminuria in oreeclampsia
l
(421).
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