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Articles |
Both authors are retirees of the Eastman Kodak Co.
a Address correspondence to this author, at: PO Box 252, Pittsford, NY 14534–0252.
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Introduction |
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 Top Introduction References |
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Przybylowicz was strongly taken with some of the suggestions. He formed a team consisting of Charles Warburton, Leo Kunzelsauer, and William Fellows to explore some possibilities. The goal that emerged from these early experiments was to create dry, thin films containing all of the reagents necessary for clinical analysis by colorimetry. Reagents in a matrix of hydrophilic polymer would be coated on top of a transparent plastic base and dried. Upon applying the test sample to the film, water and the analytes would diffuse into the reagent layers, initiating the reaction sequence(s). The extent of reaction would be determined by colorimetry.
Spreading the test sample over the film proved to be a considerable
challenge: The fluid tended to form a bead on the surface of the film.
The exploratory group initially used filter paper, plastic filters, or
woven fabric to spread the fluid over the reagent layer. A better
solution was to coat the spreading layer as a slurry of solid particles
along with a binding material over the reagent layer(s); this would
become a porous solid formed in place during the drying of the coated
slurry. A preferred composition, developed by Al Goffe, was based on
cellulose acetate pigmented with titanium dioxide coated out of a
solvent–nonsolvent mixture. When dried, this mixture yielded a highly
porous structure. A schematic cross-section of an analysis slide with
such a spreading layer coated over the reagent layer is shown in Fig. 1
; Fig. 2
is a photomicrograph of the TiO2–cellulose
acetate layer. When a drop of test fluid, such as serum, is touched to
the spreading layer, it spreads rapidly until the capillary pores are
filled. Applying twice the volume of fluid covers about twice the area
(this was shown to be true for volumes of 5 to 15 µL). The spreading
layer thus provides a coverage of fluid per unit area that is nearly
independent of the volume of the applied drop. A 10-µL drop spreads
to an area of ~1 cm2
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Because a spreading layer of TiO2–cellulose acetate
is almost opaque, the colored reaction products must be quantified by
reflection densitometry. Using instruments designed by the Kodak
Research Laboratories, light of appropriate wavelength is directed
through the film base into the reagent layers at an angle of 45° and
the back-reflected light is detected at 90°, thus minimizing specular
reflection. Readings are made through a window in a small confined
volume. The film is held at a controlled temperature as the reaction
proceeds. A schematic of this setup is provided in Fig. 3
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By the end of 1971, the exploratory group demonstrated that promising results could be obtained by use of common clinical chemical reaction schemes. Indeed, they produced a glucose analysis film based on the glucose oxidase and horseradish peroxidase-catalyzed oxidation of o-dianisidine with acceptable precision. A patent covering thin-film analysis was granted in 1976 (1).
At this point, management decided to expand the effort by creating three new groups: (a) a chemical-biochemical group, under Cornelius Unruh and subsequently Pat Grisdale, which would provide reagents and chemical and biochemical analysis concepts; (b) a coating technology group, under Dick Spayd, to coat layers and to turn them into analytical test elements (small slides, 16 mm square, proved to be the best format); (c) a bioanalytical group under Henry Curme to test the performance of the analysis films and to diagnose problems as they occurred. These group leaders, or Laboratory Heads as they were officially known, were all persons with 10–20 years experience in photographic research and development at Kodak. Laboratory administration brought Roy Rand, an experienced clinical chemist, into the effort early to provide much needed clinical chemistry experience and to help direct the testing program. In addition, several young graduates, trained in various biochemical specialties, were added at this time. Typically, each analysis film was developed by a team comprising one professional from each of the three groups mentioned above.
Technical hurdles appeared everywhere. High temperatures were inconvenient because enzymes were inactivated and films dried out before reactions could go to completion. The gels formed by rehydrating hydrophilic polymers were too dense to allow large molecules, such as albumin, to penetrate them. Thus, different strategies had to be devised for the analysis of large molecules. Corrosive chemicals, such as used in the Abell–Kendall determination of cholesterol, could damage the films and the apparatus holding them. Nevertheless, the layered format with the spreading layer was early recognized to have important advantages. While the exclusion properties of the gel made analysis of large molecules difficult, other large molecules, e.g., hemoglobin (a photometric and chemical interferent), remained in the white, opaque spreading layer, minimizing its photometric detection. Sensitivity of the layered film assays to hemoglobin was found to be considerably less than in conventional spectrophotometric systems. In general, the serum proteins do not enter the gel layer because of its low porosity.
The teams were attracted to the use of enzymes as reagents, not only for their high specificity but also because enzyme-catalyzed reactions proceed rapidly at 37 °C. The o-dianisidine in the glucose element was replaced by a modified Trinder reagent (2). This reaction sequence involves the peroxidase–hydrogen peroxide catalyzed oxidation of 4-aminoantipyrene and further coupling with a phenol or a naphthol to form a dye. Mechanistic studies done in Curme's laboratory, to everyone's relief, showed that glucose oxidase and peroxidase (as well as other enzymes) showed the same activity in swollen polymer films as they did in conventional aqueous environments. Variants of the peroxidase-catalyzed oxidation of certain dye precursors (leuco dyes) worked well in half a dozen of the assays. With Kodak's expertise in the dye field, it was not difficult to find a dye precursor system that gave useful reflectance values for analytes of widely varying concentrations such as glucose (3.9–5.8 mmol/L) and uric acid (150–420 mmol/L). Adjustments to the detection reactions were necessary because all reactions were performed with undiluted serum and the researchers did not have the luxury of adjusting dilution ratios to fit the concentrations of the various analytes.
Zona Pierce and David Frank designed a spreading layer to handle large
molecules such as albumin (3). This layer consisted of
1-µm polymer spheres lightly "glued" together and to the
transparent film base with an appropriate binder. A photograph of such
a layer is shown in Fig. 4
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As the assays were developed, the teams better understood just how useful the thin-layer format could be. For example, they found that a layer containing ascorbic acid oxidase could be coated ("scavenger layer") to eliminate ascorbate interference in the uric acid assay. 2
One of the more creative uses of layer chemistry was conceived by Barbara Bruschi, another of our young chemists, who worked in the coating technology laboratory. Bruschi wanted to quantify the ammonia released by the enzyme-catalyzed hydrolysis of urea by the use of an irreversible indicator dye. To prevent interference from the inherently alkaline assay system, Bruschi overcoated the indicator with a thin layer of cellulose acetate. Being permeable to small, neutral molecules, such as NH3, but not to ions, this layered system registered ammonia with great sensitivity and was totally insensitive to ionic base (5).
When members of management saw that the teams has produced successful assays for quite different analytes—glucose, urea, and uric acid—and that the thin films had many advantages, their confidence grew further and they approved the hiring of more scientists from several disciplines. Almost all of the people were just out of graduate school or had just completed postdoctoral work. The new arrivals often made important contributions to the projects within months of their arrival. The work was exciting and morale was excellent. However, some difficult analytes still remained.
For example, the analysis of cholesterol and its esters loomed as a "show-stopper." The cholesterol team was simply unable to incorporate enough acidity in the films to achieve an Abell–Kendall-type reaction, let alone produce the swings in pH necessary to hydrolyze the cholesterol esters and then run the strongly acidic color-forming reactions. And, as mentioned earlier, there was also concern about the effects of high acidity on the physical equipment. However, a solution was found by Charles Goodhue, one of the few microbiologists employed by Kodak. Having read a literature reference to an organism that produced an enzyme, not well characterized, called cholesterol dehydrogenase, Goodhue wrote to the NIH scientist who had discovered the organism and obtained a sample. Goodhue cultured the organism and then assigned Roy Snoke, a newly hired enzymologist, the job of isolating the enzyme and determining how it could be used. Snoke's subsequent findings produced jubilation throughout the project. When a cholesterol dispersion was exposed to the cell extract, one of the products was hydrogen peroxide. This meant that the enzyme was an oxidase, not a dehydrogenase, and that colored reaction products could readily be produced by oxidative coupling, as used in the glucose and other assays. The problem of hydrolyzing the cholesterol esters was solved by Ted Esders, another of the new staff. Esders found cholesterol esterase activity in Lipase M, a commercially available preparation used in cleaning formulations. A workable scheme for purifying the enzyme was found in a few months.
Nothing having to do with cholesterol analysis proved easy: Translation of the new chemical discoveries to a working film presented new challenges. The most serious was that the hydrophobic nature of cholesterol hampered its diffusion from the spreading layer to the underlying reagent. A second obstacle was the pseudoperoxidase activity of hemoglobin, which can catalyze side reactions with hydrogen peroxide. Glen Dappen, of the coating technology laboratory, solved these problems by designing an entirely new film architecture, in which all of the enzymes and the dye precursors necessary for the analysis were incorporated in the spreading layer. In this format cholesterol does not have to diffuse, and peroxide and dye precursors can compete effectively with the hemoglobin for hydrogen peroxide. Because the titanium dioxide spreading layer was too opaque for the measurement of dye absorbance or reflectance, reactions were carried out in a barium sulfate layer, which is notably less opaque when wet (6).
Conventional analyses for triglycerides presented challenges almost as
great as those involved with cholesterol. Goodhue and Esders decided to
try an enzymatic approach and soon demonstrated that their purified
Lipase M effectively hydrolyzed the glycerol esters to glycerol. They
then applied classical methods of microbiology to screening large
numbers of organisms suspected of showing activity against glycerol or
glycerol phosphate. The search was successful and, again, results could
not have been better. Esders found that the organism
Streptococcus faecium produces the enzyme 
-glycerol
phosphate oxidase. The end product of the reaction is hydrogen
peroxide, which meant that detection could be achieved by oxidative dye
chemistry (5).
It was, of course, necessary to evaluate progress in the development of the films, which we did with hundreds, perhaps thousands, of reflectance measurements. Early in the project, 10-µL drops of test fluid were manually dispensed to the films and, after a suitable period (usually 5 min), the reflectance of the analyte reaction products was determined with a breadboard densitometer. As technology improved, it became possible to reproduce densities (analogous to absorbances) to within 1 x 10-4 density units at reflectance densities of 1.2.
To accommodate the high volume of testing required to support the program, automated breadboards had to be designed and built. This permitted the accumulation of performance statistics to support the optimization of film formulations. It also permitted the identification of and quantification of interferences.
An automated breadboard was built in the Apparatus Division under the direction of George Scherer and Clyde Glover, both veteran Kodak engineers. This machine automatically dispensed 10-µL drops, positioned the analysis slides, and placed the slides in a thermostated incubator. A reflectance photometer recorded the reflectance of each slide on each rotation as it passed an observation point, and automated data reduction converted the reflectance data to concentration units. The throughput of the instrument was impressive.
Glover and his group later moved to the Research Laboratories to design and build prototypes for later-generation instrumentation. Scherer, as Manager of Engineering and Equipment Manufacture, provided prototypes and production analyzers. By 1978, several colorimetric tests had been converted to thin-film formats, and the veil of corporate secrecy began to be removed. Descriptions of the various designs were summarized in papers by Spayd et al. (5) and Curme et al. (7).
The first of five assays had been cleared by the FDA by 1980 and were being used by a number of laboratories who agreed to participate in a market trial. Their response was enthusiastic, and management's confidence continued to grow. Many of the concepts were incorporated in the commercial line of Kodak Ektachem Analyzers (now Johnson and Johnson Vitros Analyzers), the first of which, the Kodak Ektachem 400 Analyzer, reached the market in 1980 with 12 analyses available. The discrete nature and excellent stability of the thin-film reagent format coupled within the random-access instrumentation provided a flexible alternative to the sequential multichannel analysis systems then prevalent in clinical laboratories.
Characterization of the performance of the rapidly expanding menu of thin-film assays, which required extensive testing of diverse abnormal as well as more normal patients' samples, began to occupy more and more of the researchers' time. To give these functions the needed focus, a new and independent group was formed in 1975 with Rand as Director. Known as the Evaluation and Reference Laboratory, it also took on responsibility for developing the modified reference methods required to support the calibration of the thin-film system and to evaluate accuracy.3 Field testing was supervised by the Marketing Division, where Baitsholts played a major role. Another major activity of the Evaluation and Reference Laboratory was the establishment of a Precision and Accuracy Committee to establish precision and accuracy goals for each analyte. Several prominent clinical laboratory professionals agreed to act as consultants in this area. Ed Sylvestre, a senior Kodak statistician, also greatly aided the effort; his paper on partitioning total allowable error into goals for bias, precision, and interferences should be consulted by interested persons (8).
Once the colorimetric assays were shown to be feasible, it became clear
that the utility of the system would be greatly enhanced by the ability
to analyze for the electrolytes and clinically significant enzymes.
Jack Chang, an electrochemist and laboratory head in the Analytical
Sciences Division, called attention to the increasing use of
ion-selective electrodes for measuring electrolyte concentrations in
body fluids. He believed that such electrodes might well be made in the
thin-film format and he soon proved his point. He evaporated a thin
film of silver onto a film base and treated the film with bromine,
converting the surface to silver bromide. When a drop of sodium bromide
solution was placed on this film and touched with a tiny reference
electrode, the potentiometric response was found to be instantaneous,
stable, and Nernstian. Silver–silver chloride electrodes for the
analysis of chloride were produced by a similar process. A schematic
cross-section of such an electrode is shown in Fig. 5
; bromide interference was eliminated by the use of a cellulose
acetate overcoat.
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An improved configuration consisted of two small strips of the
thin-film electrodes mounted side by side on a plastic support. A drop
of test fluid was applied to one side and reference fluid to the other.
Liquid contact between the two sides was formed through a small piece
of ion-free paper laid across the two electrodes. Fluid from each side
diffused across the filter paper until a liquid junction was formed. As
indicated by the Nernst equation, the potential difference between the
two electrodes in such a cell, with proper allowance for junction
potential, is a measure of the ratio of the chloride activities on the
two sides of the cell. Fig. 6
shows a schematic of such a slide and a picture of a production
chloride slide.
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To measure sodium, potassium, and bicarbonate, more-complex electrodes had to be developed. A reference layer was coated over the silver–silver chloride electrode and an appropriate ion-selective layer coated over the reference layer. Achieving electrodes that worked well took some excellent organic chemistry, particularly to obtain the required ionophores, some excellent electrochemistry, and much clinical systems work. The design and fabrication of a sensitive and very stable potentiometer was difficult but achieved after several years. Slide formulations and supporting instrumentation were achieved eventually, and the electrolytes could be determined with excellent precision.
By this time, a separate instrumentation laboratory, under Paul Schnipelsky's leadership, had been formed. One of this group's many accomplishments was their presentation to a surprised management of a 4-in. (~10-cm) cube box that dispensed test and reference fluids onto slides and gave a digital readout of any of the four electrolytes. Ray Jakubowicz had joined Schnipelsky in this "bootleg" operation. Its success caused management to start "thinking small." Later, the DT60, an easy-to-use desktop analyzer designed for the doctor's office and other dispersed sites, became available.
The development of assays of the NADH-coupled enzymes [aspartate and
alanine aminotransferases (AST, ALT) and lactate dehydrogenase (LDH)]
turned out to be relatively straightforward. Good comparative assays
were available and were adaptable to the thin-film format. Enzyme
activities determined with the thin-film system were shown to be
similar to those obtained with conventional clinical laboratory assays,
provided the comparison methods for AST and ALT used pyridoxal
phosphate. The instrumentation described earlier could be configured to
take reflectance readings at 340 nm and other wavelengths every few
seconds for several minutes. Algorithms were developed that would
determine the time interval over which any set of readings displayed
linear kinetic behavior and would calculate the enzyme activities based
on the data in this interval. The instrument people were able to
provide photometric quality sufficient to generate results with
precision and accuracy well above industry norms. Other enzymes
(creatine kinase, alkaline phosphatase, lipase, amylase, and
-glutamyltransferase) were determined with colorimetric rate assays
(11)(12)(13)(14)(15)(16). Kay Whitmore, soon to be President of Kodak,
recognized this achievement as outstanding and indicated this
personally to each member of the Enzyme Team.
In the work described above, the Kodak Research Laboratories had the very active and able help of the Systems Laboratory, the Reference and Evaluation Laboratory, the Apparatus Division, the Film Manufacturing Division, and the Marketing Division. Prototype equipment and slides made by the production divisions were first evaluated by the Evaluation Laboratory, then field-tested under Marketing supervision. The Eastman Kodak Ektachem 400 Analyzer was first shown in 1980 and made commercially available in 1981 for determination of 12 analytes. The system continues to evolve today, now under the ownership of Johnson and Johnson. The Vitros 950 Analyzer, introduced in 1995, now includes some immunoassays.
A last note about Przybylowicz. Stefan Augielski, president-elect of the Polish Chemical Society, heard Przybylowicz describe the thin-film system at the AACC meeting in San Francisco in 1978. The two became friends and Augielski invited Przybylowicz, who is of Polish descent, to speak to the Polish Chemical Society about this new analytical technology. Przybylowicz decided to deliver the lecture in Polish and spent his summer trying to develop some facility in the language of his forebears. He delivered the address in Poland in September 1979. While history has not recorded the quality of his Polish, 2 min into the paper he was interrupted as the audience of 1300 persons rose to their feet and gave him a standing ovation.
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Acknowledgments |
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Footnotes |
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2 Production quantity amounts of the enzyme were made possible when the young biochemist, T.W. Wu, showed that it could be isolated from the skin of the zucchini squash [4].
3 It was intended from the start that highly accurate methods be used. A detailed history of the Reference Laboratory is planned.
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References |
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TopIntroductionReferences |
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-glutamyl transferase (GGT). US
Patent 4,751,178, 1988..
The following articles in journals at HighWire Press have cited this article:
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  N. Raby, C. Bonneau, S. Gillier, J. Le, R. Granouillet, J. Frey, and A. Chamson Single Dilution for Urine Assays on the Vitros 250 or 700 Analyzers Clin. Chem., August 1, 1998; 44(8): 1746 - 1748. [Full Text] [PDF]
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