With the bromelised test cell technique, as commonly used, that is the autoanalyser manifold subject to ambient temperature with the exception of the incubation coils at 37 °C, an anomalous inherent variability has been observed in the anti-D assay of a certain proportion of antisera; these show a significant difference in the estimates of anti-D concentration obtained using the added bromelin and bromelised test cell techniques. The inherent error of assay exhibited by such antisera is partially dependent on the test cell and can be significantly improved by the use of a bromelised pool of test cells. Such a technique results in an overall reproducibility with these antisera of ±20% which should be regarded as the reproducibility for the technique as a whole, although with a proportion of anti-D antisera the estimates of antibody concentration will be obtained with greater precision.

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Three out of 28 commercial preparations of bovine serum albumin have been encountered which have an inhibitory effect on the assay of anti-Rh(o)(D) using the Technicon AutoAnalyser. The inhibitory property, which can also be demonstrated by standard manual serological techniques, appears to be directed towards the second stage of the agglutination reaction. An automated screening procedure for bovine serum albumin preparations and some properties of the inhibitor are described.

Thermodynamic and kinetic considerations, based on a standard bromelin/ methylcellulose method for anti-Rh(0)(D) quantitation using the Auto-Analyser, have shown that the proportion of antibody bound at equilibrium and its rate of attainment is dependent on the mean equilibrium constant and heterogeneity index of the particular antibody preparation. Also, upon dispersal of the deliberately induced rouleaux, a new state of equilibrium is defined, the rate of attainment of which is related to the equilibrium constant. The principle of anti-Rh(0)(D) quantitation using the Auto-Analyser depends on individual antisera behaving in an identical manner to that of the standard preparation against which they are assayed. Evidence is presented in this paper that this premise cannot be substantiated unless the test and standard antisera have the same equilibrium constant and heterogeneity index.