TY - JOUR
AU - Rosenfeld,, Louis
AB - Abstract This segment of history aims to inform the new, and remind the not-so-new, members of the profession about the relatively recent period that initiated the dominant role played by technological innovation in the modern investigation of disease. The 12 years from 1948 to 1960 were notable for introduction of the Vacutainer tube, electrophoresis, radioimmunoassay, and the AutoAnalyzer. Also appearing during this interval were new organizations, publications, programs, and services that established a firm foundation for the professional status of clinical chemists. It was a golden age. Except for photoelectric colorimeters, the clinical chemistry laboratories in 1948—and in many places even later—were not very different from those of 1925. The basic technology and equipment were essentially unchanged. There was lots of glassware of different kinds—pipettes, burettes, wooden racks of test tubes, funnels, filter paper, cylinders, flasks, and beakers—as well as visual colorimeters, centrifuges, water baths, an exhaust hood for evaporating organic solvents after extractions, a microscope for examining urine sediments, a double-pan analytical beam balance for weighing reagents and standard chemicals, and perhaps a pH meter. The most complicated apparatus was the Van Slyke volumetric gas device—manually operated. The emphasis was on classical chemical and biological techniques that did not require instrumentation. The unparalleled growth and wide-ranging research that began after World War II and have continued into the new century, often aided by government funding for biomedical research and development as civilian health has become a major national goal, have impacted the operations of the clinical chemistry laboratory. The years from 1948 to 1960 were especially notable for the innovative technology that produced better methods for the investigation of many diseases, in many cases leading to better treatment. New programs, organizations, publications, and services were introduced that built a firm foundation for the professional status of clinical chemists and had lasting impact on the practice of clinical chemistry. Of the many advances in clinical chemistry during those years, I have chosen what I consider the most significant. Bio-Science: The Era of Referral Chemistry Commercial laboratories for analysis of biological specimens were not uncommon at the mid-20th century. In fact, these services were offered by enterprising physicians before the turn of the century. Following World War I, there was an increase in the number of these laboratories in response to the growing number of analytical methods with clinical relevance. At mid-century, a new kind of commercial enterprise appeared—the specialized reference laboratory—and a new name was added to the clinical chemistry vocabulary. Bio-Science Laboratories (BSL)1 was started by one Navy and three Army officers who had met after World War II at Camp Detrick, Maryland while awaiting discharge to civilian life. One was a physician, the other three had doctorates in bacteriology. Pooling their finances, they decided to enter the laboratory business. The four were Richard Henry, Sam Berkman, Orville Golub, and Milton Segalove (Fig. 1 ). They opened for business in February 1948 (1). The break that gave BSL an entry into the medical community was a reproducible method for chemically measuring protein-bound iodine (PBI) of serum and, by inference, the serum thyroxine concentration for assessing thyroid status. The only other method was by measurement of the basal metabolic rate, a nonspecific and highly variable procedure. The PBI method developed by Albert Chaney at the Los Angeles County Hospital was complex and challenging and required special glassware and meticulous technique. It involved precipitation of serum proteins with their protein-bound thyroxine, washing the precipitate to remove inorganic iodine, wet digestion with chromic acid to convert thyroxine iodine to inorganic iodide, distillation of released iodine into a receiver, and measurement of that iodine by its catalytic reduction of ceric ion by arsenious acid. The diminution in the yellow color of the reaction solution was a measure of the iodine concentration. Although laborious, the method measured PBI reliably. Once the test became available to the Los Angeles medical community, business accelerated markedly. When the alkaline dry ash method of Barker et al. (2) was published in 1951, BSL quickly adapted it to mass production. Requests for other difficult analyses—so-called “reference” or “specialized” procedures—started to come in. Soon, BSL led the way with a wide range of specialty tests in chemistry, toxicology, microbiology, and immunology made available to the medical community nationwide. BSL’s contribution to the practice of laboratory medicine was the creation (or discovery) of a place in the economy, i.e., a national market for new, specialized tests. American Association of Clinical Chemists During the first two decades of the 20th century, new methods based on visual colorimetry and small volumes of specimen were a stimulus to the growth of clinical chemistry. The professional prestige of biochemists was largely advanced by their success in developing diagnostic tests for the practicing physician, and by 1940 biochemistry was able to survive on its own. Medical school biochemists gravitated to newer, exciting, and more academically prestigious lines of research. Development of clinical methods was abandoned to the clinical chemists, who found themselves no longer at the center of the biochemical sphere. Meanwhile, the methods of the clinical chemistry laboratory continued to increase in variety. Eventually, they became sufficiently complex for a new class of specialist—clinical chemist or doctoral scientist—to emerge and take charge of research and responsibility for directing the routine work in the clinical chemistry laboratory. By the late 1940s, concerned with professional identity and the perceived low image and status of the “clinical chemist” with respect to academic biochemists, clinical chemists in New York City met to plan formation of a professional association. The broadly stated objectives were to gain understanding and recognition by government, the public, and the medical establishment. On December 15, 1948, nine PhD clinical chemists from the major New York City private and municipal hospitals met at Mt. Sinai Hospital in New York City to organize a professional association of clinical chemists, locally at first, and then on a national scale. This meeting marked the founding of the American Association of Clinical Chemists (AACC; renamed American Association for Clinical Chemistry in 1976). The group was motivated by two major concerns. One was the need to upgrade the quality and accuracy of chemical analyses; the other was to review, clarify, and strengthen their professional autonomous standing as PhD chemists with medical colleagues and the public. Status and authority were confused because the city denied a license as a laboratory director to anyone without an MD degree. Aware of similar grievances elsewhere, AACC decided to contact hospital chemists throughout the United States. In 1948, the practice of medicine in general and of laboratory medicine in particular was very different from what we experience as we enter the 21st century. The war against bacteria was fought mainly with sulfonamides and penicillin, mood-altering pharmacology was almost unknown, adrenal and thyroid hypofunction were treated with endocrine organ extracts, and plasma cholesterol was newly recognized as playing some sort of role in atherosclerosis and coronary artery disease. Clinical chemistry’s contribution to the assessment of health and disease was secondary to those of bacteriology, hematology, and urinalysis. Thyroid status was evaluated by determination of the basal metabolic rate. The few hormones that could be measured, e.g., for determination of pregnancy, were bioassayed using frogs, rabbits, and mice. The only enzyme assays were for amylase, lipase, and acid and alkaline phosphatase. Analytical precision was satisfied by analysis in duplicate. By the end of 1954, there were 629 members and seven chartered local sections in the AACC: the New York, Boston, Philadelphia, Southern California, Chicago, Washington-Baltimore-Richmond, and Midwest sections. Annual meetings were held in conjunction with meetings of the American Chemical Society (ACS) until 1958, when the 10th annual meeting was held independently September 4–6, in Iowa City, Iowa. By then, with a membership of 748, the bank balance of the Association was $8752.19 (3). The new organization issued The Clinical Chemist (1949), a newsletter; formed regional sections; established a code of ethics (1952); initiated publication of Clinical Chemistry (1955), a bimonthly journal; sought licensing; and developed distinctive career patterns of training (4). Overseas the Scandinavian Journal of Clinical & Laboratory Investigation began publication in 1949 and was followed in 1956 by Clinica Chimica Acta, an international journal based in The Netherlands. The AACC launched a series of Standard (later renamed Selected Methods of Clinical Chemistry) in 1953. Eventually, the evaluation process became too slow, and the series was discontinued with volume 11 in 1986. Another series, Advances in Clinical Chemistry, begun in 1958, aimed to provide a readable account of selected important developments, their roots in allied fundamental disciplines, and their impact on the progress of medical science. Articles were written by experts who were working in the field that they described. Volume 33 appeared in 1998. An index of the series was published in 1999 as volume 34. Foreign Societies and the International Federation Even prior to the organization of the American group, professional societies of clinical chemists had begun to form in Europe. The first national society of clinical chemistry was the Société Française de Biologie Clinique, formed in 1942. The Nederlandse Vereniging voor Klinische Chemie and the Finnish Society of Clinical Chemistry were organized in 1947 and were the first to include “Clinical Chemistry” in their name. In 1953, the Association of Clinical Biochemists was founded in the United Kingdom. The Swedish Society for Clinical Chemistry was formed in 1954. At the same time that the AACC was being formed in response to professional needs of clinical chemists, a similar situation was developing in the United Kingdom. When the National Health Service was set up in the United Kingdom in 1948, there was no mechanism for deciding the gradings and salary scales of the growing number of nonmedical employees. By 1951, it was clear that there was a need for a professional body to deal with the increasing number of clinical biochemists in the United Kingdom and to provide a forum for discussions on their rapidly expanding specialty. The regional groups of clinical biochemists decided to form their own national society, which would deal exclusively with their specialty. The inaugural meeting was held at the Hammersmith Hospital, London, on March 28, 1953. Seventy-five people were present and signed the register. At the end of 1995, total membership was 2294. The Association of Clinical Biochemists owes its foundation to Earl J. King (1901–1962), professor of chemical pathology at the Postgraduate Medical School in the University of London. An eminent and highly respected scientist, he was known particularly for his work in clinical enzymology and in developing colorimetric methods of analysis. King had a PhD and a DSc degree, but no medical qualification other than an Honorary MD, a circumstance that was encouraging to the many nonmedical clinical biochemists who were struggling locally to improve their professional status (5). In 1952, King suggested that the newly forming national societies of clinical chemistry should join into an international organization under the auspices of the International Union of Pure and Applied Chemistry (IUPAC). This was accomplished on July 24, 1952, at the Second International Congress of Biochemistry in Paris by the formation of the International Association of Clinical Biochemists. The name was changed to International Federation of Clinical Chemistry (IFCC) in 1955. In the interim, the First International Congress of Clinical Chemistry was held in Amsterdam in 1954. The initial objectives of the Federation were to “Advance knowledge and promote the interests of biochemistry in its clinical (medical) aspects”. In 1967, the IFCC formally separated from IUPAC. By 1998, societies in 75 countries on six continents, and 41 corporate groups active in clinical chemistry had become members (6). Proficiency Testing Shortly after the end of World War II, directors of clinical laboratories in the Philadelphia area became alarmed over the frequency of divergent results when the same blood or serum specimen was sent to two different clinical laboratories for analysis. As a result of these discrepancies, a survey was made of the accuracy of the more common chemical measurements made in hospital laboratories throughout the state. The findings were published in 1947 by Belk and Sunderman (7). This led in 1949 to surveys in other states and a monthly proficiency testing service that mailed ampules of sera or solutions to the participating clinical laboratories throughout the country and abroad. Statistical analyses of the values reported were subsequently returned to the laboratories with a current review of methodology and a bibliography. The outcome of proficiency testing was an overall improvement in the quality of the work of the laboratories subscribing to this service. By the time the Sunderman Proficiency Testing Service was turned over to the American Society of Clinical Pathologists (ASCP) in 1985, similar services had become available from other professional societies of laboratory science and from manufacturers of diagnostic laboratory instruments. A parallel development included workshops and seminars on the newest applications in laboratory science. These were first initiated by the ASCP in 1954 in recognition of the need for continuing education for the scientific and technical staff of clinical laboratories. These education programs have become a feature of national and regional meetings of many scientific associations concerned with laboratory medicine. The Vacutainer Tube The hypodermic syringe was the first product sold by the Becton Dickinson Company. The syringes offered for sale at the time were all manufactured in Europe. The Luer syringe, produced in France, was the first all-glass hypodermic syringe. It was a marked improvement over earlier types made of hard rubber or metal and glass that wore out quickly or eroded in sterilization. Deciding to manufacture on their own, Maxwell W. Becton (1868–1951) and Fairleigh S. Dickinson, Sr. (1866–1948) purchased the American patent rights to the Luer glass syringe in 1899 (8). Improvements in the Luer syringe in 1925 with the introduction of the Yale Luer-Lok syringe virtually eliminated the danger of a needle slipping off the syringe while in use. Meanwhile, the search went on for a syringe with completely interchangeable parts. The first American-made interchangeable syringe was developed by Joseph J. Kleiner (1897–1974), who entered the surgical supply business in 1920. One product in the line of diagnostic instruments was a Japanese syringe with interchangeable parts. However, the syringe failed to maintain a vacuum in the barrel. The fault had nothing to do with interchangeability, but with the fact that these syringes were made of lime glass, which deteriorated after repeated sterilization. Kleiner decided to have the syringes made of borosilicate glass, which is much harder than lime glass, and according to his specifications. These required more time and labor to grind to an interchangeable fit and could only be produced in small lots of a dozen matched barrels and plungers. In 1935, Kleiner founded Multifit, Inc. to import these syringes from Japan and package them for sale at a competitive price, despite the additional work and cost, because of the low wage scale in Japan. World War II ended the Japanese source of these interchangeable syringes. In 1943, Kleiner joined Becton Dickinson and brought with him the rights to the Multifit syringe. This syringe represented a major technological advance, making it possible to produce syringes of a quality equal in performance and durability to the older custom-ground plunger and companion barrel, which were individually matched to be a perfect fit and were numbered, but which had to be discarded when one of the two pieces broke. With the new syringes, unbroken parts could be refitted into any intact matching part. In 1952, the company introduced Multifit syringes engineered so precisely that every syringe manufactured and distributed within each of three zones of the United States had completely interchangeable parts. Making crafted glass syringes reusable, however, did not diminish the laborious process of washing and sterilizing. Although Kleiner’s Multifit syringe had helped to deal with the problem of glass breakage, the instrument was too expensive not to reuse. The company now made a major commitment to the development of a disposable syringe and needle. Years earlier, Kleiner had approached the company about purchasing his Evacutainer blood collection tube. This device consisted of an evacuated test tube sealed with a rubber stopper, a double-pointed needle, and a holder for fitting the two together. When in use, the short-end needle is pressed against the stopper as the other end makes the venipuncture. When the stopper is pierced with the short needle, the blood pressure pushes and the vacuum pulls the blood into the tube. The tube served as both a syringe for drawing blood and as a sealed container for transporting the sample. Many blood samples could be drawn by leaving the needle in the vein and using vacuum tubes containing different anticoagulants for specific laboratory tests. In Kleiner’s early design, the stopper sometimes leaked, and over a period of time the tubes often lost their vacuum and would not work. Because of these quality issues, the company declined Kleiner’s offer. Kleiner got his idea for the Evacutainer from observing the problems associated with blood drawing in medical offices and the difficulty in obtaining small samples of blood. The procedure was slow, and spillage could occur during transfer of the blood from syringe to test tube. Sometimes more than one venipuncture was needed for additional samples for different blood tests. Eventually, the company agreed to introduce Kleiner’s tube rather than risk having a competitor produce something similar. One of the early steps taken to maintain the vacuum in the collection tube was to package them in a vacuum can similar to vacuum coffee cans. Within a short time, the product, renamed Vacutainer, became the company’s largest dollar sales item. Key to the success of the Vacutainer Tube was the growing importance of the clinical laboratory in medical diagnosis. Blood was the medium for much of the diagnosis, and the Vacutainer made its collection and handling easy and reliable. A patent for the Vacutainer blood-collecting apparatus was granted on February 1, 1949. Electrophoresis moving boundary With the growing awareness during the first half of the 20th century of the importance of proteins as structural components of protoplasm, enzymes, and hormones; in transport of oxygen and carbon dioxide; and in blood clotting, the isolation, identification, and characterization of proteins posed a challenge that was met by the invention of new physical devices for their separation. Chief among these was moving boundary electrophoresis and its successor techniques. The principle of electrophoresis (Greek: “borne by electricity”) has been known for nearly 200 years. In 1809, only 10 years after Alessandro Volta (1745–1827) had built the first galvanic cell, a Russian physicist, Ferdinand Friedrich Reuss (1778–1852), reported that when electricity was passed through glass tubing containing water and clay, colloidal clay particles moved toward the positive electrode and water moved toward the negative electrode. Further development depended on the discovery of the relationship between current and the electric field, the laws relating electricity and chemical change, and the development of chemistry and physics in general. In 1937, Arne Tiselius (1902–1971) reported his development of a new electrophoresis instrument that overcame the difficulties of convection currents and blurred boundaries in previous designs, caused by the heat-producing current during electrophoresis, and permitted quantitative analysis of a mixture of proteins in solution. His separation of horse serum into four distinct zones, albumin and three globulin components that he named alpha, beta, and gamma, demonstrated his instrument’s value as an analytical tool (9). The moving boundary technique showed that the proteins are not ill-defined lyophilic colloids but well-defined substances. The method produces concentration gradients in the solution as the proteins separate according to electric charge density of the individual protein molecules. There is only partial separation of the proteins, and only some of the slowest and fastest components can be taken out of the electrophoresis tube. Tiselius’s apparatus became commercially available in 1945, but because of its cost and size, not many were made. The moving boundary electrophoresis method was not suited for the routine clinical laboratory because of its inherent technical complexity. The method required 2 mL of plasma for each analysis, preliminary dialysis for 24 h against buffer solution, and was limited to analyzing one specimen at a time. In addition, the optical recording system required the procedure to be carried out in an air-conditioned room to prevent condensation on the lens during photography, and a dark room was needed for photographic developing. The apparatus was 19.5 feet long, and each component had to be anchored by a heavy concrete foundation to minimize vibrations. Later commercial variations were more compact and portable. Because of the need to complete a run (90 min) and then develop the photographic record of the refractive index increments of the partially separated proteins before placing the next specimen in the thermostatically controlled cooling bath, only two analyses could be completed in one working day (10). filter paper The need for absolute separations, which were not practical with the moving boundary apparatus, gave impetus to the development of electrophoresis on solid supporting medium. The first use of filter paper as a stabilizing matrix for electrophoresis appears to be that of König (11), who in 1937 determined the electric charge of a substance by its direction of migration. His report contained suggestions for staining, using ultraviolet light for locating the separated substances, and eluting segments for analysis in a test tube. The paper strip was suspended horizontally between the electrodes and was not covered. König’s report was temporarily eclipsed by the work of Tiselius the same year and attracted little attention because it appeared in Portuguese in a Brazilian journal report on a South American chemical congress. Two years later, von Klobusitzky and König (12), writing in a German journal, more fully described their experiments with paper electrophoresis and the separation of a yellow pigment from snake venom—the first application of paper electrophoresis to the separation of protein mixtures. In 1946, Consden et al. (13) separated amino acids and peptides in an electrical field on a thin slab of silica jelly in a water-cooled glass trough and first demonstrated separation into zones by staining with ninhydrin. In 1948, Wieland and Fischer (14) were the first to report the use of paper for the electrophoretic separation of amino acids and peptides. The breakthrough came in 1950, when four laboratories, in the United States, Germany, and Sweden, without knowledge of König’s work independently and almost simultaneously reported procedures for electrophoresis on paper for the separation of protein into components similar to those found by free electrophoresis. In the United States, Emmett L. Durrum’s protocol and data had been reported from an Army medical laboratory 1 year earlier, presented to the ACS meeting in San Francisco on March 29, 1949, and published in the Journal of the ACS in July 1950 (15). The simple and inexpensive paper electrophoresis method rapidly achieved popularity as a routine clinical laboratory test and led to widespread investigations of the variations of plasma proteins in disease. The advantages over the moving boundary method were speed, small sample volume, inexpensive and portable equipment, and technical ease. Durrum’s one-point suspension, freely hanging, nonhorizontal paper was popular in the United States. The designs from Germany and Sweden were in a horizontal mode. An improved version of the original Durrum cell, accommodating eight paper strips, was introduced in 1954 by the Spinco Division of Beckman Instruments (Fig. 2 ) and was soon widely accepted in the United States and abroad (16). Absolute separation and isolation of the plasma proteins by electrophoresis on paper produced discrete bands that can be visualized by staining and quantified by elution of the dye. The absorbance readings of the eluates, plotted against distances of migration, yield a pattern similar to that obtained when the intact stained strip is scanned by a densitometer. The pattern is similar to that obtained with moving boundary electrophoresis, although these analytical methods are based on different properties of the protein molecule: dye-binding and refractive index increments. cellulose acetate The empirical nature of protein staining, deviation from Beer’s law, and uneven structure of the paper stimulated the search for other support media. Cellulose acetate, introduced by Kohn (17) in England in 1957, achieved better resolution of all fractions and drastically reduced the migration time from 16 h to 20 min, as well as the sample size and scale of operation. In the United States, Grunbaum et al. (18) expanded on Kohn’s work and developed the basic features of the Microzone system, introduced by Beckman Instruments in late 1963. Agarose gel, whose scale of operation resembles cellulose acetate rather than paper, was introduced by Elevitch et al. (19) in 1966. By 1985, agarose gel had almost completely replaced cellulose acetate. Sigma and Kit Methods Another innovation that appeared in the 1950s was “kit” methodology from the Sigma Chemical Company of St. Louis, Missouri. All of the reagents for the analysis were prepackaged, ready for use, with instructions. The user provided glassware and an instrument for reading the endpoint. The company’s entry into diagnostic reagents was the result of a chance meeting between Dan Broida (1913–1981), president of Sigma, and Oliver H. Lowry, head of pharmacology at Washington University Medical School in St. Louis. Lowry had developed an assay (20) for alkaline phosphatase and needed a new source for the p-nitrophenyl phosphate substrate. The chemical was no longer available from Eastman Kodak, and Lowry asked Broida if Sigma would like to make this chemical for general use (21). Sigma, whose product line consisted of only a few biochemicals, was willing. In 1951, the company decided to market Lowry’s procedure and packaged the prepared buffer and p-nitrophenyl phosphate as a kit (Sigma Technical Bulletin No. 104). In 1955, Sigma offered a second method (Sigma Technical Bulletin No. 410) for determination of glutamic oxaloacetic transaminase (SGO) by a kinetic ultraviolet technique (22). The procedure had limited use, because few laboratories had spectrophotometers that could measure in the near-ultraviolet range (340 nm). The following year Sigma introduced a colorimetric version of this determination (Sigma Technical Bulletin No. 505) (23). This made the test more accessible because, by now, every clinical laboratory had a photoelectric colorimeter that could measure across the visible range. Although the term “kit” may have been popularized by Sigma’s application to enzyme analysis, the word itself has long been in the American lexicon, e.g., “the whole kit and caboodle”. The concept of the self-contained total package for chemical analysis also predates its use for enzyme analysis. As early as 1919, the LaMotte Chemical Products Company of Baltimore, Maryland, began to market a series of “chemical outfits” for the commonly tested constituents of blood, serum, and urine, packaged in small portable wooden chests. The outfit contained all of the reagents and the apparatus needed for the complete analysis, from preparation of a protein-free filtrate, if necessary, to a comparator block for matching the color of the test reaction with that of standard color tubes, also provided. Complete instructions accompanied each set. Perhaps the first commercially available kit method was for the determination of hemoglobin by visual comparison of a diluted blood specimen with an artificial calibrator whose color corresponded to the tint of a solution of “normal blood”. The simple apparatus was exhibited in 1878 by its designer, William Richard Gowers (1845–1915), an English neurologist (24). Diagnostic Enzymology Before the mid-1950s, the only serum enzymes with diagnostic value were amylase, lipase, and acid and alkaline phosphatase. A stimulus to diagnostic enzymology occurred in 1954 when LaDue et al. (25) demonstrated increased activity of serum glutamic oxaloacetic transaminase [aspartate aminotransferase (SGO)] after myocardial infarction. The heart muscle possesses a high concentration of transaminase, and when an infarction occurs, the damaged tissue releases the enzyme into the circulating blood, producing high serum SGO concentrations. Development of a relatively rapid spectrophotometric assay of this enzyme’s activity (22) led to improvement in the diagnosis of this illness. Although most infarctions can be diagnosed without difficulty, the SGO test provides diagnostic information in those remaining cases that can be easily confused with other pathologic conditions where the electrocardiogram is not clear. Other enzymes widely distributed in animal tissues were also shown to have diagnostic significance when serum concentrations were increased. Two of these are glutamic pyruvic transaminase [alanine aminotransferase (SGP)] and lactate dehydrogenase (LD). The liver is especially rich in SGP transaminase. Serum concentrations of this enzyme are increased in viral hepatitis and other liver diseases associated with hepatic necrosis weeks before appearance of jaundice. SGO is also present in relatively high concentrations in liver, and serum concentrations are increased following acute liver cell injury. However, the increase in SGP is greater and persists longer than that of SGO activity. This suggests that SGP might be a more specific index of liver cell damage than SGO. Furthermore, because cardiac SGP activity is low, its concentrations in serum are not appreciably altered by acute cardiac necrosis (26)(27). Despite their high activities in heart and liver tissue, neither SGO nor SGP is specific for either organ. LD activity is also increased in liver disease. However, although present at higher concentrations in other tissues, LD has appreciable activity in cardiac muscle and is used primarily for diagnosis of myocardial infarction. Like serum SGO, LD concentrations increase in a characteristic fashion after myocardial infarction (28). Five electrophoretically distinct LD enzymes, called isoenzymes, have been shown to exist. The relative amount of each isoenzyme differs in various tissues and is characteristic for that tissue (29). If the isoenzymes are numbered 1 to 5 starting at the anode, isoenzymes 1 and 2 are high in heart tissue, and isoenzyme 5 is high in liver. The LD isoenzymes released into the blood when tissue necrosis occurs alter the normal pattern to reflect the higher concentrations and relative distribution in the damaged tissue. The change in the serum pattern may be helpful in making a diagnosis. Thus, in myocardial infarction, the proportions of isoenzymes 1 and 2 increase in serum and persist longer than that usually observed for SGO. The pattern appears to be a more sensitive, specific, and lasting indicator of myocardial necrosis than total serum enzyme activity. In liver damage, there is an increase in the activity of isoenzyme 5. Many nonspecific methods—essentially group reactions based on the joint behavior and interrelationship of all the serum proteins—were introduced before the mid-20th century. In these reactions, albumin acts as a protective colloid to inhibit the tendency of a globulin fraction to be precipitated by the particular chemical reagents used. The protection may fail when the albumin is decreased or the γ-globulin is increased or changed qualitatively by a pathological process. Because this occurs most frequently in liver disorders, these procedures were often referred to erroneously as “liver function” tests. Positive results were manifested by varying degrees of opacity, coagulation, precipitation, flocculation, or turbidity. The most popular were cephalin-cholesterol flocculation (1939), thymol turbidity (1944), thymol flocculation (1946), and zinc turbidity (1947). These tests had limited usefulness in making clinical distinction between obstructive and infectious liver and biliary disease. With the introduction in the 1950s of the SGO, SGP, and LD enzyme assays, and serum protein electrophoresis on paper—as old and new were offered side by side—the last of these nonspecific tests began to fade into obscurity. Radioimmunoassay (RIA) During the mid-1950s, some unexpected findings by Solomon A. Berson (1918–1972; Fig. 3 ) and Rosalyn S. Yalow (1921– ; Fig. 4 ) led to the introduction of a new arena of analysis in clinical chemistry with the interaction of physics and biology. Even more significant than their use of γ-emitting isotopes in a clinical laboratory was their demonstration of antibodies as quantitative analytical tools. Berson and Yalow studied the metabolism of 131I-labeled insulin after intravenous administration to nondiabetic and diabetic subjects. They were surprised to find that the radioactive insulin disappeared more slowly from the plasma of patients who had received insulin, either for the treatment of diabetes or as shock therapy for schizophrenia, than from the plasma of subjects who had never received insulin. The slow rate of insulin disappearance was not attributable to the diabetes, but to the development of antibodies in response to the prior administration of exogenous insulin. These antibodies complexed with the labeled insulin acting as antigen in the plasma and prevented the insulin molecule from passing through the capillary walls and reaching its place of action. Adult-onset diabetics do not degrade insulin rapidly; they make enough but fail to utilize it efficiently (30). The antibody presumed to be present could not be detected by classic immunological techniques because at such low concentrations there was no precipitate formed. During these studies, Berson and Yalow developed a highly sensitive test tube radioisotopic method for detecting soluble antigen-antibody complexes that could determine insulin concentrations with a sensitivity 1000-fold greater than existing methods (31). The RIA of plasma insulin developed by Berson and Yalow introduced a new method of laboratory measurement, γ-ray emission from radiolabeled antigens or antibodies, and a new instrument for its detection, a gamma counter. The new technology, based on competitive interaction, permitted rapid and accurate assay of biologically important hormones, polypeptides, and drugs, which previously were measured by time-consuming chemical procedures or bioassay, if they were measured at all. Because production of antigen, antibody, and radioisotopic labels for the analysis was beyond the facilities and acceptable expense of the clinical laboratory, this new technique gave rise to a new industry, i.e., the production of packaged kits containing all of the necessary components for a successful RIA, including calibrators and controls for 100 or more tests. There also sprang up support industries feeding this new technology with accessories for the assay. Once someone shows the way, the rest comes easy. What followed was the development of novel configurations for the antigen-antibody reaction and the introduction of nonisotopic labels. Soon, previously untapped hormones succumbed to the new analytical technique of immunoassay. The clinical chemistry laboratory with its special expertise in quantitative analysis and quality control fell heir to this new technology and expanded its services. Skeggs and the AutoAnalyzer In 1957, the AutoAnalyzer became the first machine in a series of engineering marvels designed to meet specific analytical needs in the clinical chemistry laboratory. It was the beginning of major involvement of industry and commerce in the needs of the clinical chemistry laboratory beyond that of glassware, chemical reagents, laboratory furniture, and minor accessories. Until then, except for the volumetric gas apparatus for measuring bicarbonate concentration in blood plasma designed by Donald D. Van Slyke (1883–1971) in 1917, instruments finding use in the clinical chemistry laboratory had been designed primarily for applications elsewhere. Even the visual colorimeter designed and manufactured in 1854 by Jules Duboscq (1817–1886) was first used for color control in industry, e.g., to measure the amount of caramel in syrup. The first tentative step leading to automated analysis occurred in 1933, with the introduction of a commercially available photoelectric colorimeter intended for use in the industrial laboratory. What happened then was the replacement of the subjective observation of a color match on a visual colorimeter with the objective and impartial reading by an instrument. If we fast-forward a quarter of a century, we come to the start of the modern era of automated blood analysis. The story begins when Leonard T. Skeggs, Jr. (1918– ; Fig. 5 ) completed work for his PhD in biochemistry at Western Reserve University under Victor Caryl Myers (1883–1948). After his graduation in 1948, Skeggs accepted an appointment at the Veterans Hospital with responsibility for supervision of the clinical chemistry laboratory. Skeggs soon began to look for a way to deal with the error-prone repetitive steps in manual analysis and the increasing workload made worse by insufficient staffing in the laboratory. He wanted to build a machine that would complete a blood analysis from start to finish without any intervention by a technician. Working at home in his basement, Skeggs assembled a number of modules with specific tasks that constituted a continuous flow sequence of analysis. During the early 1950s, his prototype was rejected by four companies. Responses ranged from complete apathy to advice that he get a patent first. In January 1954, Skeggs’s prototype working model no. 3 was demonstrated to the Technicon Corporation, Tarrytown, New York (32). Known primarily for the AutoTechnicon—an automatic tissue processor that moved surgical specimens through the steps of fixation, dehydration, clearing, and embedding with paraffin in a timed sequence in preparation for staining—the company saw the possibilities of this totally new concept and went on to develop and produce the machine commercially. They brought the AutoAnalyzer to the market in 1957. The first model sold for $3500 and was an immediate success (Fig. 6 ). This analytical system was a radical departure from the usual laboratory instrument. Instead of performing a single task, it performed in automated sequence all of the analytical steps required to obtain a quantitative answer. It did this not by mimicking the manual movements of a technician, but by using common physical and chemical techniques in a novel, heretofore unused manner. There were three unique features of the machine in addition to continuous flow: (a) dialyzing membrane to remove proteins and provide a protein-free dialysate without centrifugation or filtration (Skeggs was involved in improving an artificial kidney machine and made the conceptual leap from a substitute for glomerular filtration in vivo to the use of dialysis in vitro); (b) first polyethylene, and later tygon tubing, with samples separated and segmented by air bubbles intended to “scrub” the tubing and prevent mixing of successive samples en route; (c) mixing coils in which columns of liquid representing discrete samples plus reagents were propelled while being continuously inverted and mixed, separated by bubbles, as the solutions moved up and down through the coils (33). Samples and reagents were introduced individually in parallel into the system at appropriate points in the continuous stream by the forward propulsive pressure of a constant-rate peristaltic pump. Heating or incubation, if necessary, was performed during continuous passage through a lengthy glass coil inside a heated bath that provided sufficient time for the reaction to take place. Photometric measurement of the exiting solution was performed by continuous monitoring of a flow-through cuvette at a given wavelength and recorded on a moving strip-chart recorder as a series of peaks at 20–40 per hour. Concurrent analysis of calibrators or reference samples provided data for a calibration curve. Later design configurations were capable of simultaneous analysis of 20 analytes per sample at the rate of 150 samples per hour. Because of the uniqueness of the AutoAnalyzer concept, Technicon set up an in-house facility to teach the proper use of this machine. During the 1-week course of hands-on instruction, each student worked on his or her own system. This innovative style of training was adopted by other manufacturers for their automated devices. Industry accepted the responsibility of instructing laboratory personnel because it recognized that the complexities of the new instrumentation could not be grasped by simply reading an instruction manual. Even before continuous-flow analysis was advancing toward the limit of its potential, it was being challenged by newly developed systems of discrete sample analysis. With samples isolated in separate containers, there was the advantage of speed and rapid changeover from one assay to another on the same instrument. The Robot Chemist was the first commercially available discrete sample analyzer. It was designed to automate wet-chemical analytical procedures by mechanically duplicating the sequential steps performed manually (34). Conceived by Research Specialties Co., Richmond, California, the machine, first marketed in 1959, was a large desk-sized structure mounted on the floor. The device was later redesigned into a bench-top unit, but it did not meet with much success or general acceptance, largely because of the complexity of its electro-mechanical and electronic components and the lack of adequate resources for servicing problems with the numerous moving parts. Furthermore, the company did not provide training sessions for customers. Production was discontinued in 1969, hastened by competition from Technicon’s advanced systems of multiple analysis. Although supplanted by the continuous-flow system of the AutoAnalyzer, it was the discrete sample concept of the Robot Chemist that was adopted by instrument designers and manufacturers and achieved the dominant position in the clinical laboratory. Conclusion Newcomers to clinical chemistry will not experience the techniques, the methods, and the instruments that were the mainstay of the laboratory before mechanization began to make inroads in the late 1950s. Nostalgia being what it is, those who did experience the laboratory of the pre-automation era tend to remember the excitement of learning new methods, developing new skills, and using different apparatus, even as they recall how primitive the old technologies often were. The work was hands-on from start to finish, with no walkaway systems. You could see what was going on. And yet, there is no denying that although the modern clinical laboratory may be a duller, less versatile, and more technical place of work, it gets far more done at far less cost and with far greater accuracy than anything that came before it. What can be learned from this review of history? Mainly, that scientific advances are not tied to the uniform passage of time, and unique analytical breakthroughs may occur when the principles of one scientific discipline interface successfully with those of another. Furthermore, we must look to the history of the past—there is nowhere else to look—to understand how we arrived at the present and to prepare for the future. If we should take history for granted, we would become orphans in time, castaways on a desert island called “the present” with no idea of where we came from or where we are going. Figure 1. Open in new tabDownload slide The founders of Bio-Science Laboratories. Left to right: Richard Henry, MD; Orville Golub, PhD; Milton Segalove, PhD; and Sam Berkman, PhD. Photo taken in February 1969, at the time of Dr. Segalove’s retirement. Figure 1. Open in new tabDownload slide The founders of Bio-Science Laboratories. Left to right: Richard Henry, MD; Orville Golub, PhD; Milton Segalove, PhD; and Sam Berkman, PhD. Photo taken in February 1969, at the time of Dr. Segalove’s retirement. Figure 2. Open in new tabDownload slide Paper electrophoresis cell and cover. Courtesy of The Beckman Heritage Center, Fullerton, CA. Figure 2. Open in new tabDownload slide Paper electrophoresis cell and cover. Courtesy of The Beckman Heritage Center, Fullerton, CA. Figure 3. Open in new tabDownload slide Solomon A. Berson. Courtesy of Mrs. Miriam Berson. Figure 3. Open in new tabDownload slide Solomon A. Berson. Courtesy of Mrs. Miriam Berson. Figure 4. Open in new tabDownload slide Rosalyn S. Yalow. Figure 4. Open in new tabDownload slide Rosalyn S. Yalow. Figure 5. Open in new tabDownload slide Leonard T. Skeggs. Figure 5. Open in new tabDownload slide Leonard T. Skeggs. Figure 6. Open in new tabDownload slide Single channel AutoAnalyzer. Courtesy of Bayer Corporation, Diagnostics Division, Tarrytown, NY. Left to right: chart-strip recorder; colorimeter (front); heating bath (rear); dialyzer with dialysis assembly submerged in water bath; proportioning pump with manifold assembly (front); sampling tray (rear); reagent bottles. Figure 6. Open in new tabDownload slide Single channel AutoAnalyzer. Courtesy of Bayer Corporation, Diagnostics Division, Tarrytown, NY. Left to right: chart-strip recorder; colorimeter (front); heating bath (rear); dialyzer with dialysis assembly submerged in water bath; proportioning pump with manifold assembly (front); sampling tray (rear); reagent bottles. " Address correspondence to: 1417 East 52nd St., Brooklyn, NY 11234. 1 " Nonstandard abbreviations: BSL, Bio-Science Laboratories; PBI, protein-bound iodine; ACS, American Chemical Society; ASCP, American Association of Clinical Pathologists; SGO, glutamic oxaloacetic transaminase; SGP, glutamic pyruvic transaminase; and LD, lactate dehydrogenase. References 1 Lee ND. A history of Bio-Science Laboratories. Clin Chem 1994 ; 40 : 149 -157. Crossref Search ADS PubMed 2 Barker SB, Humphrey MJ, Soley MH. The clinical determination of protein-bound iodine. J Clin Invest 1951 ; 30 : 55 -62. Crossref Search ADS PubMed 3 AACC organizing committee meetings. Dec. 15, 1948; Jan. 11, Feb. 1, 1949. [Also see minutes of the meeting of the AACC Executive Committee, Sept. 3, 1958.]. 4 Routh JI. Training of clinical chemists in the United States: a brief history. Clin Chem 1974 ; 20 : 1251 -1253. Crossref Search ADS 5 Broughton P, Lines J. The Association of Clinical Biochemists. The first forty years. Sherwood R, ed. London: ACB Venture Publications, 1996:9–20.. 6 Gouget B, ed. IFCC Handbook 1997–1999. Paris: Elsevier, 1998:144pp.. 7 Belk WP, Sunderman FW. A survey of the accuracy of chemical analyses in clinical laboratories. Am J Clin Pathol 1947 ; 17 : 853 -861. Crossref Search ADS PubMed 8 The Echo. Franklin Lakes, NJ: Becton Dickinson and Company, 1991(Spring);11:3–5; 1991(September);11:5–7; 1996(December);16:1.. 9 Tiselius A. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans Faraday Soc 1937 ; 33 : 524 -531. Crossref Search ADS 10 Rosenfeld L. Origins of clinical chemistry. The evolution of protein analysis 1982 : 162 -193 Academic Press New York. . 11 König P. Applicaçăo da electrophorése nos trabalhos chimicos com quantidades pequenas. In: Actas e trabalhos do terceiro congresso sul-americano de chimica. Rio de Janeiro e Sâo Paulo, 1937;2:334–6.. 12 von Klobusitzky D, König P.. Biochemische studien über die gifte der schlangengattung Bothrops. VI. Arch Exp Pathol Pharmakol 1939 ; 192 : 271 -275. Crossref Search ADS 13 Consden R, Gordon AH, Martin AJP. Ionophoresis in silica jelly. A method for the separation of amino-acids and peptides. Biochem J 1946 ; 40 : 33 -41. Crossref Search ADS 14 Wieland T, Fischer E. Über elektrophorese auf filtrierpapier. Naturwissenschaften 1948 ; 35 : 29 -30. 15 Durrum EL. A microelectrophoretic and microionophoretic technique. J Am Chem Soc 1950 ; 72 : 2943 -2948. Crossref Search ADS 16 Williams FG, Jr, Pickels EG, Durrum EL. Improved hanging-strip paper-electrophoresis technique. Science 1955 ; 121 : 829 -830. Crossref Search ADS PubMed 17 Kohn J. A cellulose acetate supporting medium for zone electrophoresis. Clin Chim Acta 1957 ; 2 : 297 -303. Crossref Search ADS PubMed 18 Grunbaum BW, Zec J, Durrum EL. Application of an improved microelectrophoresis technique and immunoelectrophoresis of the serum proteins on cellulose acetate. Microchem J 1963 ; 7 : 41 -53. Crossref Search ADS 19 Elevitch FR, Aronson SB, Feichtmeir TV, Enterline ML. Thin gel electrophoresis in agarose. Am J Clin Pathol 1966 ; 46 : 692 -697. Crossref Search ADS 20 Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 1946 ; 164 : 321 -329. PubMed 21 Berger L. Sigma Diagnostics: pioneer of kits for clinical chemistry. Clin Chem 1993 ; 39 : 902 -903. Crossref Search ADS PubMed 22 Karmen A. A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood serum. J Clin Invest 1955 ; 34 : 131 -133. PubMed 23 Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957 ; 28 : 56 -63. Crossref Search ADS PubMed 24 Gowers WR. An apparatus for the clinical estimation of haemoglobin. Trans Clin Soc London 1879 ; 12 : 64 -67. 25 LaDue JS, Wróblewski F, Karmen A. Serum glutamic oxaloacetic transaminase activity in human acute transmural myocardial infarction. Science 1954 ; 120 : 497 -499. Crossref Search ADS PubMed 26 Wróblewski F, LaDue JS. Serum glutamic pyruvic transaminase in cardiac and hepatic disease. Proc Soc Exp Biol Med 1956 ; 91 : 569 -571. Crossref Search ADS PubMed 27 Wróblewski F. The clinical significance of alterations in transaminase activities of serum and other body fluids. Adv Clin Chem 1958 ; 1 : 313 -351. Crossref Search ADS PubMed 28 Wróblewski F, LaDue JS. Lactic dehydrogenase activity in blood. Proc Soc Exp Biol Med 1955 ; 90 : 210 -213. Crossref Search ADS PubMed 29 Wróblewski F. Diagnostic dissection by isoenzymes. Pure Appl Chem 1961 ; 3 : 385 -396. Crossref Search ADS 30 Berson SA, Yalow RS, Bauman A, Rothschild MA, Newerly K. Insulin-I131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects. J Clin Invest 1956 ; 35 : 170 -190. Crossref Search ADS PubMed 31 Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest 1960 ; 39 : 1157 -1175. Crossref Search ADS PubMed 32 Lewis LA. Leonard Tucker Skeggs—a multifaceted diamond. Clin Chem 1981 ; 27 : 1465 -1468. Crossref Search ADS PubMed 33 Skeggs LT, Jr. An automatic method for colorimetric analysis. Am J Clin Pathol 1957 ; 28 : 311 -322. Crossref Search ADS PubMed 34 Crawford WC Jr. The Robot Chemist: a new approach to the automation of wet-chemistry analysis. Ann N Y Acad Sci 1968–69;153:655–9.. © 2000 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Golden Age of Clinical Chemistry: 1948–1960
JF - Clinical Chemistry
DO - 10.1093/clinchem/46.10.1705
DA - 2000-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-golden-age-of-clinical-chemistry-1948-1960-sxsD0636LY
SP - 1705
VL - 46
IS - 10
DP - DeepDyve
ER -