Early history of Eastman Kodak Ektachem slides and instrumentation.
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Biomedical subjects
Publications and source records attributed to R N Rand.
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At least four bilirubin fractions can be separated and identified by liquid chromatography, the least understood being the "delta" fraction (B delta), which apparently is covalently bound to albumin. To learn more about the incidence and significance of B delta, we assayed serum from 539 infants and children, both by the chromatographic method and the routine colorimetric Jendrassik-Grof method. The proportion of B delta appeared to correlate with both age and disease course. For infants younger than 28 days B delta generally was less than 2% of total bilirubin; for hyperbilirubinemic older infants and children the median B delta value was 35%. High B delta (greater than 50% of total bilirubin) in newborns was associated with intra- and extra-hepatic cholestasis, biliary cirrhosis, biliary atresia, and hepatitis. Among older infants and children, a proportion of low B delta (less than 10%) was found in hemolytic anemias, sepsis, shock, and other non-hepatic jaundice. In several cases, when low B delta was accompanied by increased conjugated bilirubin, the prognosis was very poor. Delayed clearance of B delta from the circulation together with its reactivity in direct diazo methods may interfere with interpretation of values for conjugated bilirubin as measured by classical methods.
An improved procedure for the Laurell "rocket" technique is described. Samples were electrophoresed in an agarose gel containing anti-human albumin. The gel plates were processed, the peaks stained, and peak heights used to calculate albumin concentrations. Factors affecting precision were (a) adequate heating of agarose gel before antibody is added, (b) accurate leveling of the gel surface during plate formation, (c) applied voltage during sample application, and (d) avoidance of the "edge" effect on sample placement in the gel. Multi-plate long-term precision (CV) for the method was 6.2% at a mean albumin concentration of 13 g/L and 3.0% at 37 g/L. Analytical recovery of 8 and 11 g of albumin per liter was 99 to 100%. There was negligible interference from hemoglobin and dextran as well as several common substances that bind to albumin--bilirubin and salicylate. Because of its high accuracy and good long-term precision, the method is a possible candidate reference method for serum albumin.
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In this slide, unconjugated bilirubin and its sugar conjugates interact with a cationic polymeric mordant to form spectrally enhanced complexes having similar absorptivities at approximately 400 nm. With reflection densitometry and appropriate mathematical transformation, readings at this wavelength are linearly related to bilirubin concentrations up to 260 mg/L. The slide requires 10 microL of serum, is precise (total CV less than 2% determined over 20 days for the analyte range 39-184 mg/L), gives results that correlate well with the Doumas et al. modification of the Jendrassik-Gróf method (slope 0.95, intercept 0.3, Sy . x 3.4, r = 0.991), and is relatively interference free. Also, the slide measures less loss of bilirubin after in vitro illumination of serum specimens than do diazo tests. An intermediate layer in the slide minimizes the spectral interference from hemoglobin and prevents the detection of the strongly protein-linked ("delta") bilirubin found in many jaundiced adults. The method is recommended for newborns (less than or equal to 14 days), in whom the incidence of delta bilirubin is negligible.
The transferability of the candidate Reference Method for total serum protein was tested in eight laboratories in the United States and Europe. National Bureau of Standards SRM 927 (bovine serum albumin) was used in each analytical run as the calibration standard. The mean absorptivity value obtained for this material was 0.2983 L g-1 cm-1. Four serum pools prepared at the Centers for Disease Control were analyzed on each of 15 days. Within-run variation of the protein values (expressed as CV) in the eight laboratories ranged from 0.1 to 2.5% and day-to-day (total) variation in six of the laboratories ranged from 0.4 to 1%.
Using the general concept of a dry multilayer analytical element, we can change chemical procedures and configurations to assay several blood components. In the assay of serum urea nitrogen, urease in the reagent layer catalyzes the hydrolysis of urea. A semipermeable membrane excludes aqueous base, but allows ammonia to diffuse to an underlying indicator layer. For the amylase determination, the enzyme hydrolyzes a dyed-starch substrate coated on top of the spreading layer; this produces small fragments, which diffuse to a registration layer. The increase of absorbance at 540 nm is correlated with amylase activity. Bilirubin complexes with a cationic polymer at the interface between the spreading and reagent layers. The direct reading at 460 nm allows determination of total bilirubin in the range 1 to 500 mg/liter. Tirglycerides are hydrolyzed in the spreading layer, and the resulting soluble glycerol readily diffuses into the reagent layer, where it is phosphorylated and subsequently oxidized by glycerophosphate oxidase to yield dihydroxyacetone phosphate and hydrogen peroxide. Peroxidase catalyzes production of a color commensurate with the hydrogen peroxide produced.
Dry, thin films containing all necessary reagents for clinical analysis by colorimetry have been designed. Reagents in a matrix of hydrophilic polymer are coated on top of a transparent plastic base. A white isotropically porous polymer spreading layer, 80% void volume, is coated over the reagent layer(s). In the analysis, a drop (typically 10 microliter) of undiluted serum or other fluid is touched to the spreading layer. The fluid spreads rapidly and uniformly through the pore structure, filling a void volume corresponding to the drop volume. Water and low-molecular-weight components diffuse from the spreading layer into the reagent layer(s), initiating the reaction sequence. The spreading layer acts also as a white optical diffuser for reflection densitometry. Optical reflection density is linearized through use of the function developed by Williams and Clapper [J. Opt. Soc. Am. 43, 595 (1953)] to convert reflection to transmission density. A wide variety of chemical assays are compatible with this format. As an example, for the glucose film we found coefficients of variation of 1.5% in predicting glucose concentrations in control sera during 20 days. Results for glucose concentrations in several hundred patients' sera by the present method were very cose to those obtained with the Center for Disease Control's hexokinase reference method.
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