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.

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.

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We used two coated thin films to measure the concentrations of unconjugated, conjugated, and total bilirubin as well as bilirubin covalently bound to albumin ("delta" bilirubin) in more than 400 serum samples. We measured the unconjugated and conjugated species by determining their reflection densities at two wavelengths (400 and 460 nm) on a coating designed for the enhanced spectral measurement of bilirubin but which does not register the delta form. Total bilirubin was measured by use of a diazo-based thin film (Clin Chem 29: 37-41, 1983). We estimated the concentration of delta bilirubin by subtracting the sum of unconjugated and conjugated bilirubin from the concentration of total bilirubin. All measurements agree well with those by comparative methods, as shown by linear regression. Slopes ranged from 0.92 to 1.02, correlation coefficients from 0.935 and 0.998. Linear combinations of these values can also be used to compute other results; e.g., the sum of conjugated and delta bilirubin can be considered to be an estimate of "direct"-reacting bilirubin.