Contributions of Analytical Chemistry
Π Kristen J. Skogerboe Department of Laboratory Medicine SB-10 University of Washington School of Medicine Seattle, WA 98195
Clinical chemistry is built on the strong foundation of analytical chemistry, and fundamental knowledge of analyti cal chemistry, medicine, and physiolo gy is essential to the success of the clin ical chemist. Because of the applied na ture of clinical chemistry, technological developments in many areas of science are eventually used in a clinical setting. The close relationship between analyt ical and clinical chemistry provides an interesting example of the impact of analytical chemistry on other scientific disciplines. Most analytical research focuses on development of state-of-the-art instru mentation and establishment of meth ods that provide accurate and precise measurements. The amount of time it takes to transfer technology from the academic research laboratory through industrial development to routine use is determined by the intent of the clini cal laboratory to provide the most clini cally useful lab tests quickly and inex pensively and to provide patients with the best possible care. Whether or not an analytical tech nique will ever be used in the clinical laboratory depends on whether it • provides valuable clinical informa tion, • is cost effective for the health care system, 0003-2700/88/A360-1271/S01.50/0 © 1988 American Chemical Society
to the Clinical Laboratory
• minimizes assay time, • is accurate over the entire physio logic range, • offers acceptable precision levels, and • is simple and reliable in routine implementation. If a method is to have clinical utility, quantitative data obtained for a chemi cal species must correlate well with some physiologic condition. A test will not be useful to the clinician unless it provides diagnostic or prognostic infor mation. Clinical testing must also be cost effective. If an assay costs too much, physicians and insurance com panies may judge that the information gained by doing the test does not justi fy the cost. Turnaround time is another
REPORT important consideration in clinical chemistry; a rapid assay may be critical in an emergency. The accuracy of clini cal assays plays an important role in implementing new technologies. A physician does not want to make a clin ical decision based on an assay that may not reflect the actual concentra tion of the measured analyte. The re quired degree of precision varies, de pending on the analyte being mea sured. Finally, reliable instrumentation is a prerequisite in the clinical lab. A mal functioning piece of equipment that yields an erroneous laboratory value can complicate the clinical assessment
of a patient. All of these criteria are reviewed carefully before a new tech nology or method is implemented in the clinical laboratory. These criteria are second nature to individuals whose careers center around laboratory medicine, but they may not be obvious to analytical chem ists who believe that a technique may have clinical potential. Because both economics and quality health care as sume important roles in clinical chem istry, much of the research and devel opment is performed by companies that manufacture clinical instrumenta tion and laboratory reagents. State-of-the-art instrumentation in clinical labs now includes automated spectrophotometers, and 40% of the tests performed in large centralized laboratories are based on ion-selective electrodes. These methods have met the requirements for commercializa tion. Which new technologies will emerge as valuable contributors to the clinical laboratory? Will lasers, optical fibers, diode arrays, and microbore chromatography become commonplace in lab medicine? This REPORT will highlight several analytical techniques that are being used in state-of-the-art clinical labs and will illustrate how oth er advances in instrumentation may contribute to clinical chemistry in the future. Biosensors for transcutaneous and on-line monitoring
The development of sensors for clinical use has been an active area of analyti cal research and the focus of several reviews (1,2). Biosensors present excit-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988 · 1271 A
ing possibilities because of their rela tive simplicity, small size, and ease of operation. The commercial viability of some of these sensors has been ques tioned, however, raising issues of biocompatibility, clinical accuracy and precision, and sensor lifetime. One positive new use of sensor tech nology is the application of biosensors to on-line monitoring in the newborn (neonatal) intensive care unit. Biosen sors are particularly valuable for neo natal monitoring because these tiny patients may have a total of only 120 mL of blood, and obtaining an adequate blood sample can be difficult and dan gerous to the patient. (The average 75kg adult has a blood volume of 5 L.) Both electrodes and optical probes are used to transcutaneously (across the skin) monitor the oxygenation of blood in a premature infant. These noninva sive methods are used at the bedside to ensure that an infant is receiving the correct amount of oxygen via a respira tor. Premature infants can die from too little oxygen and suffer lung and brain damage from too much. Because these fragile neonates require intensive care, it is desirable to monitor oxygenation continually, thus enabling early inter vention in a life-threatening respira tory crisis. Transcutaneous monitoring of the partial pressures of oxygen and carbon dioxide is accomplished by using ionselective electrodes (ISEs) attached to the skin surface. The electrode is en closed in a heated adhesive "patch" that is placed on the infant's skin. The heat of the patch (~44 °C) warms the skin, inducing blood flow to the area under the patch. The oxygen and carbon dioxide in the blood diffuse across the skin, through semiperme able membranes, and are detected by the electrode. The signal obtained by this transcutaneous method correlates well with oxygen levels measured in ar terial blood samples (3). Biosensors based on optical fibers are also used to assess oxygen status. One such instrument, called a pulse ox imeter, determines the percentage of oxyhemoglobin saturation of function al hemoglobin by measuring the ab sorption of red and near-IR light as it passes through tissue. A pulsatile arte rial bed (such as the patient's toe) is sandwiched between a pair of lightemitting diodes (~660 and 925 nm) and a photodetector. Light at each wave length passes through the toe, is col lected by an optical fiber, and is sent to the photodetector. The intensity of light reaching the detector is deter mined by the pigmentation and thick ness of the skin and the absorption of venous and arterial blood in the tissue. The absorbance of skin and venous blood is constant; that of arterial blood changes because of pulsatile blood
flow. With synchronous detection, the constant background can be subtracted from the arterial signal, and the cor rected bichromatic absorbance of light can be used to calculate the amount of oxygenated and nonoxygenated hemo globin. The success of these electrochemical and optical sensors reflects the poten tial for other probes in patient moni toring. One new direction for optical probe research may be the use of nearIR analysis, which has been clinically demonstrated by the pulse oximeter. The advantage of the near-IR spectral region, compared with visible radia tion, is the substantial throughput of near-IR radiation through tissue, which implies t h a t transcutaneous monitoring of other clinical analytes may be possible in the near-IR region. The measurement of cytochrome-c oxi dase using near-IR has been reported (4). Although transcutaneous applica tions have not yet been demonstrated, near-IR has been used for the quantita tion of serum lipids (5) and fats in hu man samples (6). Given the success of pulse oximetry, it is possible that this technology can be used to monitor lip ids, fats, and other analytes. Although transcutaneous sensors are advantageous because no blood needs to be drawn, it is certainly not possible to measure all analytes across the skin. Therefore biosensors that can be in serted into the body would be clinically useful. One interesting example of a sensor that could be used for invasive monitoring is an optical sensor that measures penicillin (7). The experi mental configuration of this probe is shown in Figure 1. Penicillin present in a sample solution is converted to penicilloic acid. The reaction is enzymatically catalyzed by penicillinase that has been immobilized on the end of a fiber-optic probe, and the amount of penicilloic acid formed is proportional to the fluorescence measured by the same fiber. The specificity provided by the enzyme system coupled with the optical fiber indicates the potential utility of probes of this type in clinical analysis. Polarization spectroscopy
Although automated absorbance spec trophotometers are still the mainstay of most clinical analyses, instrumenta tion based on other spectroscopic prin ciples is emerging. For example, fluo rescence polarization spectroscopy (FPS) is becoming a common laborato ry technique. FPS measures the anisotropy, or rotation, of a fluorophore be tween the time it absorbs light and emits that light as fluorescence. FPS was once used only in research labs to determine molecular size and other properties of a molecule in solution. Now, however, commercially available
1272 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
Fluorescence
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Penicillinase immobilized membrane Figure 1. Configuration of fiber-optic biosensor for measurement of penicil lin.
FPS instruments are used in the clini cal laboratory in several different ways. One unique application of FPS is in the area of assessing fetal lung maturi ty. Lack of lung surfactants, which en able the air sacs to expand and stay open, makes a premature infant sus ceptible to respiratory distress syn drome (RDS) and decreases the chance of survival. Physicians want to avoid delivering an infant before the lungs begin to produce adequate surfactant. Analysis of the amniotic fluid, which bathes the fetal lung and contains some of the lipophilic surfactant, is used to determine fetal lung maturity. For years, the standard method for such analyses has been a time-consuming thin-layer chromatographic procedure that measures the ratio of lecithin to sphingomylin in the amniotic fluid. A faster FPS method, however, is now often used. In this analysis, a fluorescent tag is added to the sample and attaches to the surfactant material. Because the frictional resistance of the sample de creases as more lung surfactant is present, the microviscosity of a sample coming from a fetus with mature lungs is lower than that of a sample originat ing from an immature fetus. Decreas ing FPS values correlate well with de creased probability that an infant will develop RDS (8). Thus the FPS result can be used to evaluate the lung matu rity of a fetus carried by a woman in premature labor. If the results indicate that the lungs are immature, the physi cian may put off elective delivery or decide to deliver early if the mother is at risk. A second application of FPS is in the area of therapeutic drug monitoring,
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Fluorescence polarization immunoassay and a characteristic response curve.
where FPS is coupled with immunoas say to quantitate a drug contained in a blood or urine sample (Figure 2). The sample is added to a cuvette containing antibodies specific for the analyte be ing monitored and a tracer, consisting of the drug with a fluorescent tag. Be cause any analyte present in the sam ple will compete with the tracer to bind with the antibody, there is a direct cor relation between the amount of analyte in the sample and the quantity of free tracer in solution. The free tracer ro tates more easily in solution and has a lower degree of fluorescence polariza tion than tracer bound to antibody. Therefore, as the analyte concentra tion increases, the polarization de creases, yielding a characteristic re sponse curve. FPS-based immunoas says have been developed for a number of analytes, including anticonvulsant drugs and sedatives. Because FPS only works well for small molecules with rel atively large Brownian motion, it can not be used to assay large analytes. Optical rotary dispersion (ORD) and circular dichroism (CD) also have po tential clinical laboratory use, because many molecules of clinical interest dis play optical activity (OA). OA has been exploited for the quantitation of cannabinoids by CD (9) and in laser-based ORD detection following liquid chro matography (LC) for determination of cholesterol and amino acids (10, 11). Because OA measurements provide considerable selectivity, complex sam ples may not need extraction to isolate the analyte from interfering com pounds or matrix constituents. Assays based on OA detection are not routine ly performed in the clinical lab because the instrumentation is complex and the available commercial instrumentation is limited in sensitivity. Eventually, however, the capability for selective as say of physiologic analytes and drugs where enantiomeric monitoring is criti cal may encourage the development of sensitive OA detection in clinical in strumentation.
chemiluminescence has the potential for greater sensitivity than either ra diochemical or fluorescence-based im munoassays. An assay of this type might be based on the creation of an antibody-antigen complex with a chemiluminescent label. When this complex is combined with a reagent that initiates a chemiluminescent reac tion, the amount of light emitted is re lated to the quantity of antigen present in the sample. A number of labels have been investigated, including luminol (12) and acridinium esters (13), and commercial products based on this general technology have been intro duced for a number of analytes such as thyroid and pituitary hormones. Gas-phase chemiluminescence is also being implemented for the mea surement of analytes of clinical inter est, and it is the basis of an assay of urinary nitrogen for which a commer cial instrument is available (Figure 3). An injected urine sample is carried by oxygen into a combustion furnace
Other spectroscopic techniques
Techniques such as chemilumines cence, laser and time-resolved fluores cence, and photothermal deflection are among the spectroscopic methods that have recognized clinical potential. Al though they are based on different principles, each provides either im proved sensitivity or selectivity and uses technology that is not found in every laboratory. Several of these methods are the basis for commercially available clinical instrumentation; oth ers have only demonstrated feasibility. Chemiluminescence. This tech nique is routinely used in the laborato ry, and the use of chemiluminescent labels in immunoassay has been partic ularly successful. As in FPS immunoas says, this spectroscopic label bypasses the use of radiolabels. But unlike FPS techniques, chemiluminescence is not limited to lower molecular weight ana lytes, and concentrations as low as 10"15 mol/L can be measured. Thus
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Reaction chamber
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Figure 3. Experimental arrangement for total nitrogen determination by gas-phase chemiluminescence.
1274 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
where all nitrogen in the sample is con verted to nitric oxide. Water vapor is removed via a dryer, and the nitric ox ide is mixed with ozone, creating an excited nitrogen dioxide molecule that relaxes and emits light. NO + 0 3 — NO; + 0 2 — N 0 2 + hv The emission of red and near-IR radia tion from this molecule correlates well with the amount of total nitrogen present in the sample. Because the amount of nitrogen in the urine corre lates with the breakdown of protein by the body, total nitrogen levels provide the physician with a way to assess the nutritional status of a patient. If a pa tient is using stored protein to meet metabolic needs, the nitrogen level in the urine exceeds the amount of nitro gen taken in from food. For years, the standard clinical method for determin ing total nitrogen has been the Kjeldahl method, a time-consuming test that uses caustic reagents. The chemiluminescent total nitrogen method not only correlates with the Kjeldahl meth od (14) but also is rapid and requires no reagents. Thus the convenience and time-saving factors justify this applica tion of chemiluminescence in the clini cal lab. Fluorescence. This technique is be ginning to come of age in the clinical lab. Traditionally, the application of fluorescence to the analysis of clinical specimens has often been difficult be cause of quenching and background fluorescence of native fluorophores. Spectral interferences can be mini mized by careful instrumental filter de sign and further reduced with signalprocessing techniques. Time-resolved emission techniques reduce the signal contribution caused by background emission, thus improving fluorescence applications (15). The commercial ap plication of time-resolved fluorescence to the area of fluorometric immunoas says is now underway. A representative fluorometric immu noassay involves formation of an anti body-antigen complex in which the antigen may be a drug or hormone of clinical interest present in blood or urine. A second fluorescent-labeled antibody is then attached to the anti gen. This sandwich-type complex is ex tracted from solution and subjected to fluorometric analysis. If the fluoromet ric label is conventional so that normal fluorescence is measured, this tech nique may not be more sensitive than conventional methods based on iso tope-labeled immunoassay. However, if the assay is based on the more ad vanced time-resolved fluorescence, im provements are obtained. Assays that use lanthanide chelates as fluorescent labels have demonstrat ed this potential for improvement.
These labels work well because the flu orescence lifetime of lanthanide metal chelates is ~ 1 ms, compared with decay lifetimes of only a few nanoseconds for native fluorophores. In an assay for thyroid-stimulating hormone with a europium label, the detection limit achieved by time-resolved fluorescence is 10 times better than that obtained with classical techniques (16). Further improvements in fluorescence detect ability have been sought by using laser light as an excitation source. A com mercial nitrogen laser-based, time-re solved fluorometer has been applied to the lanthanide-labeled assay of hor mones (17), where gated detection de creases the background signal and in creased laser power increases sensitiv ity. These two features combined yield improved detection limits. In addition to its application to im munoassay, laser-excited fluorescence may have other uses in the clinical lab (18). Some research has focused on the use of laser fluorometry for the assay of enzymes in biological samples. Argonion laser radiation has been used for the measurement of enzyme activity (19), where the laser system improved the detection limit of the enzyme sub strate by 25-fold relative to using a con ventional light source. A He-Cd laser has also been used in enzyme assay sys tems (20), where 1 X 10" 14 mol of the enzymatic reaction product, NADP, was detected. In applications such as these, where detectability and selectiv ity are critical considerations, the cost and unfamiliarity of lasers may be overridden by clinical need. Photothermal deflection. Another unique laser-based technology that may become important in the clinical lab is photothermal deflection spectro photometry (PTDS), one of a class of ultrasensitive spectroscopies that de termine the heat produced by the ab sorption of light. Photothermal meth ods require absorption of radiation from a focused laser beam. In one con figuration, the heat produced as the ex cited molecule undergoes radiationless decay causes a laser beam aligned par allel to the sample surface to be de flected. An aperture placed in front of a photodetector limits the amount of light reaching the detector when ab sorption occurs. Determining the de flection caused by the absorption, rath er than measuring simple absorbance, can improve detection limits by several orders of magnitude. PTDS has been used for detection in thin-layer chromatography and elec trophoresis (21). For both of these techniques it is easy to identify situa tions in which ultrasensitive PTDS de tection would provide an advantage in clinical analysis. For example, in the assessment of urinary protein by elec trophoresis, protein concentrations are
1276 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
on the order of 1 mg/dL and are diffi cult to quantitate without sample preconcentration. In certain disorders, quantitation of trace levels of proteins may aid in diagnosis or treatment, and physicians are beginning to be interest ed in how small changes in the level of urinary albumin may correlate with clinical outcome in diabetes (22). Re cently a PTDS densitometer was used in the assay of proteins separated by polyacrylamide gel electrophoresis, ex tending the detection limit to 1 ng of protein (21). Instrumentation based on the PTDS principle is not yet commer cially available, but the diagnostic util ity that has been demonstrated may justify commercial development. Chromatography in clinical analysis Gas and liquid chromatography are in tegral parts of the clinical lab. Even with the widespread use of immunoas says, chromatography is still used for analyses when antibodies either cannot be developed or are not specific enough. Chromatography is also useful for monitoring concentration rather than activity and for determining me tabolites that might be physiologically active. One area of research is the develop ment of microbore LC. The trend to ward smaller columns, and detectors designed to accommodate this type of chromatography, should eventually be appreciated by clinical chemists. Mi crobore systems are advantageous for applications in which the amount of sample is limited, such as pediatric drug monitoring. Because youngsters have smaller blood volumes, sample size is often limited. The benefit of mi crobore chromatography has been demonstrated in the analysis of chlor amphenicol, a drug given to young chil dren to fight infection (23). In this study, the amount of blood serum re quired for the assay was reduced to only 10 μι,. Microbore columns also reduce sol vent consumption. In the assay of cy closporin, a drug given to organ trans plant recipients, solvent consumption was reduced by 80% (24) by using mi crobore rather than conventional LC. In the cost-conscious clinical lab, this advantage may encourage the switch to microbore columns. Another emerging trend in clinical chromatography is the use of sophisti cated detectors that provide qualita tive as well as quantitative informa tion. The use of photodiode array (PDA) detectors has been reported; PDA detection is particularly useful in determining drugs when co-eluting, in terfering compounds jeopardize accu racy. PDA detection has also been used in the diagnosis of genetically inherited metabolic defects such as maple syrup disease (25). Here, spectra from the
PDA, obtained with ion-exchange chromatography help indentify abnor mal organic acids in urine. The future The diverse methodologies discussed here indicate that the research and de velopment efforts in analytical chemis try are being applied in the clinical lab oratory. Techniques based on biosen sors, chemiluminescence, and timeresolved fluorescence have met the criteria for clinical implementation and are routinely used. Further devel opments in spectroscopy, chromatog raphy, and electrochemistry will con tinue to contribute to the clinical lab oratory. References (1) Czaban, J. C. Anal. Chem. 1985, 57, 345 A-356 A. (2) Seitz, W. R. Anal. Chem. 1984, 56, 16 A-34 A. (3) Graham, G.; Kenny, M. A. Clin. Chem. 1980,26,629-33. (4) Ferrari, M.; Giannini, I.; Carpi, Α.; Fasella, P. Physiol. Chem. Phys. Med. NMR 1983,15,107-13. (5) Peuchant, E.; Salles, C.; Jensen, R. Anal. Chem. 1987,59,1816-19. (6) Peuchant, E.; Salles, C.; Jensen, R. Clin. Chem. 1988,34, 5-8. (7) Fuh, M. R.; Burgess, L. W.; Christian, G. D. Anal. Chem. 1988,60,433-35. (8) Tait, J. F.; Franklin, R. W.; Simpson, J. B.; Ashwood, E. R. Clin. Chem. 1986, 32, 248-54.
(9) Han, S. M.; Purdie, N. Anal. Chem. 1985,57,2068-71. (10) Yeune, E. S.; Steenhook, L. E.; Wood ruff, S. D.; Kuo, J. C. Anal. Chem. 1980, 52,1399-1402. (11) Reitsma, B. H.; Yeung, E. S. Anal. Chem. 1987,59,1059-61. (12) Kohen, F.; Kim, J. B.; Lindner, H. R.; Barnard, G. In Bioluminescence and Chemiluminescence, Basic Chemistry and Analytical Applications; DeLuca, M.; McElroy, W. D., Eds.; Academic: New York, 1981; pp. 351-56. (13) Sturgess, M. L.; Weeks, I.; Mpoko, C. N.; Laing, I.; Woodhead, J.S. Clin. Chem. 1986,32,532-35. (14) Skogerboe, K. J.; Rettmer, R. L.; Sundquist, J.; Gargett, Α.; Labbe, R. F., submitted for publication in Clin. Chem. (15) Lytle, F. E.; Kelsey, M. S. Anal. Chem. 1974,46,855-60. (16) Lawson, N.; Mike, N.; Wilson, R.; Pandov, H. Clin. Chem. 1986,32,684-86. (17) Dechaud, H.; Bador, R.; Claustrât, F.; Desuzinges, C. Clin. Chem. 1986, 32, 1323-27. (18) Sepaniak, M.J. Clin. Chem. 1985, 31, 671-78. (19) Egan, B. Z.; Lee, N. E.; Burtis, C. Α.; Kao, J.Y.; Holland, J. M. Clin Chem. 1983,29,1616-19. (20) Imasaka, T.; Zare, R. N. Anal. Chem. 1979,57,2082-85. (21) Peck, K.; Morris, M. D. Anal. Chem. 1986,56,2879-83. (22) Mogensen, C. E. Kidney International 1987,37,673-89. (23) Wong, S.H-Y.; Cudny, B.; Aziz, O.; Marzouk, N.; Sheeran, R. Clin. Chem. 1987 33 1021. (24) Anne'sley, T.; Matz, K.; Balogh, L.; Clayton, L.; Giacherio, D. Clin. Chem.
1986,32,1407-9. (25) Allen, K. R; Khan, R.; Watson, D. Clin. Chem. 1985,31,561-63.
Kristen J. Skogerboe is a senior fellow in the Department of Laboratory Medicine at the University of Wash ington in Seattle. After graduating in 1982 from Colorado State University in Fort Collins, she worked as a sum mer analytical research participant at the Procter and Gamble Company in Cincinnati, OH. She received her Ph.D. in analytical chemistry in 1987 from the Ames Laboratory at Iowa State University under the direction of E. S. Yeung. Her research interests include applications of laser spectros copy and fiber optics to chromatogra phy and unique uses of spectroscopy in clinical research.
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