FUTURE TRENDS
IN
CLINICAL CHEMISTRY Robert S. Melville Automated Clinical Laboratory Section Biomedical Engineering Program National Institute of General Medical Sciences Bethesda, Md. 20014
To practice good medicine today, the physician needs accurate, sensitive, and appropriate laboratory data which can help define the differences between sick and well people. The major task for the clinical laboratory scientist for several years to come will be to determine the best ways of providing this information to the user physicians. To do this, the clinical laboratory scientist must take full advantage of the advanced concepts and techniques developed, not only in his own field, but in other disciplines such as physics, biology, chemistry, engineering, and the medical sciences. Many of the most useful tests and techniques applied to medical problems today derive from basic ideas or concepts developed for a special scientific discipline and adapted for the clinical laboratory use only after further research and development. The clinical laboratory is located strategically at the crossroads between the basic and clinical sciences. As the practice of medicine becomes more of a science and less of an art, its excellence will depend more and more on the development of new and improved laboratory measurements, instruments, and systems. Over the past two decades, the clinical laboratory, playing a major role in this development, has called upon many different disciplines for input. These collaborative efforts have led to the emergence of the clinical laboratory sciences. In the specific field of clinical chemistry, its roots are in analytical chemistry. It is apparent that the clinical laboratory sciences have more to offer pre-
ventive medicine than is currently appreciated. For example, public health workers and physicians have long advocated the annual checkup for disease prevention and health maintenance. Recently, however, the costbenefit effectiveness of annual checkups has been challenged with the advice that annual examinations and/or certain prescribed tests be carried out only in populations with unusual risk. At some time in the future, I would expect further automation of the clinical laboratory to alleviate some of these problems, but the main questions now should be: Are we ready for automation? What do we intend to automate? Automation is expensive, and unless the results of automating a test or a group of tests will provide better data than existed prior to the changes—at a cost which reflects some benefit to the patient or can be justified on the basis of important new information— one wonders if the effort is justifiable. To try to automate a technique in the absence of well-defined goals for automation is an exercise in futility. Scientists keep insisting that we need better methods and machines to provide greater accuracy, precision, and sensitivity. What do we mean by greater? Is this a real goal or even an ideal one? Can we define the ultimate limits we need for these qualities, or will we constantly strive for more accurate data at higher and higher costs? As an analytical chemist, I say there can be no compromise with accuracy; yet, as a clinical chemist, I realize that we compromise every time we perform a test. There is, however,
594 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
a practical limit toward which we should aim—the analytical error of our methods should not exceed the normal physiological variations of the substance measured which are observed on a nonsick individual. With this degree of accuracy we could, at least, be certain that a change in value of the parameter being measured is indicative of a real physiological change in the patient and not a variation due to analytical error. One of the interesting trends in the clinical laboratory sciences is the growing recognition that certain combinations of laboratory data can be used for the early detection of disease. At the same time, clinical testing of sufficient sensitivity to detect the subtle changes which occur in blood parameters as an individual's physiology changes from what Dr. Sidney Garfield of the Kaiser Permanente Group terms well to worried-well, and finally, to sick has yet to be developed. There is room for a great deal of research and development in the clinical laboratory sciences before we arrive at a point where the "ultimate" automated instruments can be defined as to function and then developed. The chances are that 10 years hence, we will be using many new approaches to determine the state of health in patients. We keep uncovering new diseases, genetic aberrations, important new enzymes, and new ways of detecting human diseases. These, in turn, stimulate the development of newer and more effective methods for measurement of biological, chemical, and physical phenomena occurring within the body.
Report
Lest we get the idea that collaborative research between the basic sciences and the clinical laboratory is always fruitful, I would like to utter a word of caution. All too frequently, an experiment, or a piece of apparatus designed by a biological scientist with the idea of measuring a particular biological phenomenon, fails to achieve its goal because the scientist did not understand the engineering principles involved. On the other hand, the engineer may have been capable of designing a perfectly adequate piece of apparatus or of modeling a physiological system, only to have it fail miserably because the engineer did not converse with the biologist and thus failed to appreciate the complexities and interrelationships within the biological system. A notable failure was the electronic stethoscope. It sounds like a marvelous idea, and indeed it is. The only trouble with it is that the original prototypes were completely unusable by the physician. Why? Because they furnished too much information (noise). All of the body noises were picked up and tended to merge in the electronic stethoscope so that one sound is almost indistinguishable from another. Now, what are the trends for the future in clinical chemistry? Most of us, in order to think about future needs, have to start with models of what we suspect the future will be like. This type of thinking can be dangerous because frequently the models turn out to be wrong, or worse still, are taken seriously and used as a basis for action. At best, these models tend to make us think in terms of the state-
ly what the clinical chemistry laboratory would look like even 10 years from now. Nevertheless, there are ideas and concepts under development in research laboratories today which will undoubtedly have a great impact on the clinical laboratory within the next 5-10 years. I will discuss only a few of the areas of greatest promise. Immunospecific Reagents
of-the-art. For example, it was only 30 years ago that the first all-electronic digital computer ENIAC (electronic numerical integrator and computer) was installed at the University of Pennsylvania. It occupied the entire basement of the University's School of Electrical Engineering, weighed almost 30 tons, and contained more than 18 000 vacuum tubes. (It required more than 1 500 square ft of floor space.) At the time of installation, no one foresaw the full impact that electronic data processing would have on our daily lives. Even more important, no one believed that machines equivalent to or faster than ENIAC would, within a 30-year period, occupy a desk top or even fit into one's pocket. I do not believe that we are any better at precise forecasting today than we were in the case with ENIAC, and I would not presume to tell you exact-
These reagents are used for the detection and measurement of specific groups or sites on hormones, serum proteins and macromolecules, subcellular particles, viruses, bacteria, and whole cells. Today, in the clinical laboratory many of the assays using immunospecificity employ a radioactive label either on the antigen or antibody. In some instances, two or three different radioisotopes are used in the same test. The total number of radioimmunoassays done is increasing so rapidly that both production of the isotopes and disposal of radioactive wastes may, in the foreseeable future, become major problems. If alternative methods possessing the same or similar sensitivities, but without the disadvantages, can be developed, they will be a significant contribution toward a cleaner environment. Fluorescent-labeled antibodies provide the basis for one alternative. While they have been widely used for histochemical localization of antigens, attempts to use them in automated procedures have encountered a variety of problems which include not only the fundamental problems associated with any immunologic reaction but
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977 · 595 A
also the problems generally associated with fluorescence measurements. These latter include the standardization and calibration of the fluorescence measuring system, control of stray and scattered light, the problems of fading, quenching, wavelength shifting, "nonspecific" absorption, and instability of reagents—to name only a few.
proaches the separation speeds usually seen with gas chromatography. Moreover, it would appear to be particularly well suited to the separation of the biological substances encountered in the clinical laboratory without the necessity of derivatization. Other advantages are that columns are generally operated at or near room temperature and can accommodate ionic and relatively polar species directly. The variations which can be applied to chromatography are almost unlimited and range from reverse phase, exclusion, and affinity chromatography to hyperpressure (about 5 000 psi), not to mention the research and develop-
ment being carried out on resins and the uses of gradient elution. All of these variations have wide potential for the solution of difficult clinical problems. Gas Chromatography-Mass Spectrometry ( 1 )
Experts say that if these problems with fluorescence can be solved, a method will be available which will equal the radioimmunoassay method in sensitivity. This opens a large field for some interesting and productive research with practical results which can be applied to the delivery of health care. High-Performance Liquid Chromatography
Another field of promise is highperformance column liquid chromatography (HPLC). A list of the experts working in this field and their accomplishments is long and impressive. While it is true that HPLC is not extensively used in the clinical laboratory, it probably should be since it can handle larger fluid volumes and ap-
Another major development within the past few years has been the use of gas chromatography-mass spectrometry for the structural determination of molecules and for quantitative analysis of specific components in a mixture or for qualitative identification of several components. Mass spectrometers are classified according to the method of ionization employed in the source. Electron impact (EI) mass spectrometry is based upon a relatively high energy process, direct bombardment by electrons (20 eV or higher energy). Many fragments are usually formed. This is an advantage in identification or structural studies, but it is not desirable in quantitative work. Chemical ionization (CI) and atmospheric pressure ionization (API) methods are based upon ionmolecule reactions and involve relatively low energy processes. Very few ions are formed from a single organic compound, and it is often possible with API methods to establish selec-
596 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
tive ionization conditions. These circumstances are favorable for quantitative applications. There are a number of ways in which mass spectrometric analytical systems may be used. For example, it appears feasible to develop methods for identifying microorganisms by "fingerprinting" metabolic products. Another possible application lies in the development of in vivo methods for testing metabolic function through the use of stable isotope-labeled compounds. Since most applications of gas chromatography-mass spectrometry ultimately rely on the interpretation of mass spectra and therefore on knowledge of the types of reactions which are involved in the fragmentation process, mass spectrometry requires the services of competent organic chemists and physicists. In recent years, considerable progress has been made in the development of low energy methods of ionization for mass spectrometry. These techniques are potentially useful in both structural and analytical problems and are generally complementary to electron ionization. Three principal methods are now in either routine or experimental use in a number of laboratories. Chemical Ionization. Ions are formed by collision between neutral sample molecules and ions formed by ion-molecule reactions, by using a reagent gas at relatively high pressure (~1 mm Hg). Field Ionization. Ions are produced by the effect of a very high electrostatic field (up to 108 V/cm). The sample is introduced in the conventional manner, and the spectra exhibit many of the characteristics described for chemical ionization but are not broken up or ionized as completely. Field Desorption. A variation of field ionization in which ions are formed in a high electrostatic field but with the important experimental difference that low-temperature vaporization of sample molecules from the anode on which they have been coated occurs simultaneously. The present status of field desorption is that the experimental manipulations which are involved are relatively difficult and routine use must await further development. One of the uses of API mass spectrometry will be drug monitoring during chronic drug therapy. For example, epilepsy requires continuous treatment with anticonvulsant drugs. Excellent quantitative results can be obtained when l:, C-labeled compounds are used as internal reference standards. The sample consisting of a chloroform extract of serum is injected di-
rectly i n t o t h e v a p o r i z e r - s o u r c e a s sembly. T h e t e c h n i q u e is now used t o q u a n t i f y d r u g s such as d i p h e n y l h y d a n t o i n , p h é n o b a r b i t a l , a n d o t h e r s in serum. Mass spectrometric analytical m e t h o d s can be developed for n o n i n vasive t e s t i n g t h r o u g h use of saliva a n d b r e a t h s a m p l e s . T h i s relatively u n e x p l o r e d a r e a of work deserves investigation. M u l t i c o m p o n e n t analyses of selected g r o u p s of c o m p o u n d s p r e s e n t in b o d y fluids m a y prove t o b e useful b o t h in diagnosis a n d in e s t a b l i s h i n g criteria for use in p r e v e n t i v e m e d i c i n e . T h e s e analyses are usually d e s c r i b e d as " m e t a b o l i c profiles"; t h e y are b a s e d largely u p o n gas c h r o m a t o g r a p h y for separation purposes and upon mass s p e c t r o m e t r y for identification of individual components. E n v i r o n m e n t a l h a z a r d s are n o t generally c o n s i d e r e d as falling i n t o t h e general field of clinical c h e m i s t r y . N e v e r t h e l e s s , it m a y in t h e future be necessary t o i n c l u d e a n a l y t i c a l m e t h ods for toxic organic c o m p o u n d s in t h e work of h o s p i t a l l a b o r a t o r i e s . T h i s m a y include facilities for diagnosis in a c c i d e n t a l poisoning ( p a r t i c u l a r l y of y o u n g c h i l d r e n ) a n d analyses of pesticide b u r d e n s carried by h u m a n s (for example, chlorinated hydrocarbons a n d r e l a t e d c o m p o u n d s ) . M a s s spect r o m e t r y has g r e a t usefulness in work of this kind.
Cell Sorting Techniques (2) Finally, t h e t e c h n i q u e of cell sorting, which was developed in t h e m i d 1960's, h a s a d v a n c e d t o t h e p o i n t w h e r e a wide range of f u n d a m e n t a l basic a n d clinical p r o b l e m s can b e s t u d i e d on p u r e colonies of cells. T h e t e c h n i q u e itself is i n t e r e s t i n g e n o u g h
to d e s e r v e s o m e discussion since it e m p l o y s a n u m b e r of physical p h e n o m e n a : I n its essence, s u s p e n d e d cells (and t h e s e m a y b e b a c t e r i a l cells, cells from h u m a n blood, or from p r a c tically a n y o t h e r source) a r e m a d e t o flow in single file across a b e a m of light. T h e light sources m o s t f r e q u e n t ly used are t h e a r g o n - i o n laser a n d the. h e l i u m - n e o n laser. As each individual cell passes t h r o u g h t h e b e a m , m e a s u r e m e n t s are t a k e n of t h e light a b sorbed or s c a t t e r e d by it or of fluorescence from it (if t h e cells were labeled w i t h a fluorescent dye). T h e cell-sorting s y s t e m can t h u s identify from t h e passing s t r e a m those cells which m e e t t h e s e p a r a t i o n criteria e s t a b l i s h e d by t h e investigator. T o achieve sorting, t h e s t r e a m of fluid c o n t a i n i n g t h e cells in single file is b r o k e n i n t o t i n y uniform d r o p l e t s after it passes t h e light b e a m . T h i s is d o n e b y v i b r a t i n g t h e orifice from which t h e s t r e a m e m e r g e s a t u l t r a s o n ic r a t e s (10 000 H z is typical). T h e i d e a is t h a t n o d r o p l e t s h o u l d c o n t a i n m o r e t h a n one cell a n d very few cont a i n n o cells. T h e d e t e c t o r signals which were rec o r d e d as t h e cell p a s s e d t h e laser are c o n v e r t e d t o electrical p u l s e s t h a t a r e processed in such a way t h a t t h e y charge t h e d r o p l e t c o n t a i n i n g t h e cell from which t h e signals originated. T h e d r o p l e t t h e n passes t h r o u g h a n elect r i c field t h a t deflects it i n t o a separate container. Once t h e cells are s e p a r a t e d into p u r e classes, t h e variety of information which can be o b t a i n e d is i m p r e s sive. F o r e x a m p l e , a g r o u p of scientists in California h a v e isolated subclasses of T - l y m p h o c y t e s from blood (the T l y m p h o c y t e is t h e t y p e n e e d e d for cellular i m m u n i t y a n d i m m u n e regulat i o n ) . B y use of a n t i b o d i e s which h a v e b e e n tagged with a fluorescent dye, t h e cell surface a n t i g e n s can b e lab e l e d — t h e cells s e p a r a t e d a n d used to s t u d y t h e i m m u n e system. An i n t e r e s t i n g i t e m which r e c e n t l y a p p e a r e d in t h e p a p e r involved a t e s t of possible significance for t h e d e t e c tion of d i a b e t e s in h u m a n beings, b a s e d on w h e t h e r t h e h o r m o n e (ra-
diolabeled) insulin a t t a c h e s t o lymp h o c y t e s or n o t . In t h e d i a b e t i c , att a c h m e n t does n o t occur. T h i s can be used to d e t e c t i n d i v i d u a l s p r e d i s p o s e d to diabetes. I n conclusion, A l d o u s H u x l e y once said, " F a c t s d o n o t cease to exist because t h e y are i g n o r e d . " P e o p l e will c o n t i n u e t o b e c o m e ill ( a n d hopefully recover). If we, working in clinical research a r e a s , c a n p r o v i d e b e t t e r ways of d e t e c t i n g illness, p e r h a p s t h e e n t i r e processes leading t o sickness m a y one d a y be u n d e r s t o o d a n d a n o t h e r b a t t l e for survival won.
References (1) "Selected Approaches to Gas Chromatography-Mass Spectrometry in Laboratory Medicine—Report of a One Day Conference", R. S. Melville and V. F. Dobson, Eds., DHEW Publ. No. (NIH) 75-762. (2) L. B. Epstein, H. W. Kreth, and L. A. Herzenberg, Cell. Immunol., 12, 407 (1974). Presented at the 27th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1, 1976.
R o b e r t S. M e l v i l l e h a s served as Chief of t h e A u t o m a t e d Clinical L a b o r a t o r y Section in t h e Biomedical Engineering P r o g r a m , N a t i o n a l I n s t i t u t e of G e n e r a l M e d i c a l Sciences, since 1968. In his p r e s e n t position, Dr. M e l ville has p l a y e d a major role in t h e crea t i o n of a n a t i o n a l p r o g r a m for t h e s u p p o r t of research a n d t r a i n i n g in clinical laboratories. Dr. Melville received his A B degree from Clark U n i versity a n d his P h D in b i o c h e m i s t r y from t h e U n i v e r s i t y of Iowa. A founder a n d i m m e d i a t e p a s t p r e s i d e n t of t h e N a t i o n a l Registry in Clinical C h e m i s t r y , he is c r e d i t e d with being an influence in t h e provision of cred e n t i a l s for clinical c h e m i s t s a t t h e b a c c a l a u r e a t e level. In 1972 Dr. Melville received t h e J o s e p h H . Roe Award s p o n s o r e d by t h e A m e r i c a n Ins t r u m e n t Co. a n d p r e s e n t e d by t h e C a p i t a l Section of t h e AACC. H e was p r e s e n t e d with t h e A A C C ' s Fisher A w a r d in Clinical C h e m i s t r y in 1976.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977 · 597 A
