Biomedical subjects
Publications and source records attributed to P K Phillips.
A spectrophotometric assay of proteolytic enzyme activity is described in which the optical density obtained by a given enzyme preparation is related (2 alpha less than 0.01) to the serological activity of that preparation in a two stage technique. The assay appears to be of use for the standardisation of enzyme preparations for use in blood group serology.
After a single antigenic stimulus, not greater than 5.0 ml R2R2 red cells, anti-D was detected in 79% of D-negative volunteers increasing to 88% after subsequent spaced stimuli. The use of repeated antigenic stimuli at 2- or 4-weekly intervals to induce the immune response did not appear to increase the frequency of responders. The results obtained are compared with those of other workers and evidence is presented to suggest that the immunogenicity of red cells from different donors may have a role in determining the frequency of responders with detectable anti-D and is possibly associated with the R2 antigenic complex.
Twenty-nine Rho(D) negative male volunteers previously immunized with D-positive red cells have received series of antigenic stimuli repeated at intervals of 2 or 4 weeks. Although there is an individual variation in the magnitude of the anti-D response, the levels of anti-D achieved are, in general, higher than those obtained after stimuli spaced at intervals of several months. Moreover, certain volunteers whose plasma was not suitable for inclusion into pools for the preparation of anti-D immunoglobulin after spaced stimuli could donate regularly following the repeated stimuli. Whilst untoward clinical reactions did not occur as a result of the repeated stimuli five volunteers developed unwanted antibodies outside the Rh system and this may be related to the total dose of red cells injected.
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.
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Using the AutoAnalyzer, the percentage agglutination effected by the anti-D antisera studied showed a varied dependence on the ambient temperature over the manifold subsequent to the incubation period at 37 degrees C. This leads to assays which are a function of the ambient temperature. It is suggested that the entry of a relatively large volume of rouleaux-dispersing agent results in an elution of bound antibody to a new position of equilibrium, the shift being dependent on the particular equilibrium constant of the antibody and the rate of its attainment on the ambient temperature. A constant ambient temperature will lead to greater accuracy of anti-D assay.
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The limits of accuracy for the quantitation of anti-D using the AutoAnalyzer have been reduced from +/- 26% with manually prepared dilutions to +/- 14% using an automatic pipette, as expressed by the 95% confidence limits. The error inherent in the AutoAnalyzer was estimated to contribute +/- 10% to the overall error. Problems associated with the reproducibility of this method for anti-D quantitation have been investigated, namely, the effect of the age of a given test cell, the use of different test cells, loss of sensitivity of the machine over a given day, and the reproducibility of results obtained at different positions on the standard graph.
With eight of 32 sera significantly higher estimations of anti-D concentration were obtained when test cells premodified with bromelin were used instead of bromelin as a reagent added to the reaction manifold. With one serum the difference in estimation, using the two techniques, revealed a ten-fold discrepancy. The consequences of these findings are discussed in relation to the use of the AutoAnalyzer for the quantitation of anti-D.
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