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.

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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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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.