MICROASSAY METHOD FOR ANTIBIOTIC CONCENTRATIONS IN WHOLE BLOOD BY THE USE OF PAPER DISCS

Abstract

A paper disc thin-layer plate microassay method for determination of antibiotic concentrations in the body fluid, using minute amounts of test sample preparable for the assay at bedside, was evaluated as to its usefulness and reproducibility of the results thereof.
1) It was found essential to allow blood samples be absorbed in constantly the same quantities by assay discs, since the diameters of inhibition zones produced were affected by the amount of a blood sample with which the filter paper discs were impregnated.
2) When phosphate buffers at various pH, a serum and a whole blood were used in the reference assay series, it was found that the results of assay for erythromycin and streptomycin were influenced by the pH of phosphate buffer, and the results of assay for streptomycin by the serum and whole blood. It followed that it was important to use the same ones as the reference samples as much as practicable.
3) Effect of various preserving conditions on the results of assay with heparinized whole blood samples and with (non-heparinized) whole blood samples obtained immediately prior to assay was studied. We observed practically no influence due to the sample difference upon the assay results for erythromycin, tetracycline, benzyl-penicillin and streptomycin. The findings indicated practicability of the use of heparinized whole blood as the standard sample.
4) With a view to equalization of the amounts of whole blood samples with which filter paper discs saturated, the technique for preparation of saturated assay discs was standardized as follows : Allow one end of a disc to touch the whole blood sample and wait until the whole blood soak into entire disc; then remove excess blood from the disc with a blotting paper pressed lightly on it; and use it in the assay. The amount of whole blood absorbed by the disc (8mm in diam.) was 37.65±2.34mg on the average immediately after the saturation and was 24.10±1.19mg after drying. In either case the coefficient of variation was not more than 10%.
5) The difference in whole blood content between the moist disc (immediately after saturation) and dried disc did affect the diameters of inhibition zones but it was found to be abolished when the discs were allowed for prediffusion for not less than six hours before incubation. However, when allowed for prediffusion for more than twelve hours, discs impregnated with low concentrations of chloramphenicol or benzyl-penicillin showed some reduction in the diameter of inhibition zones. Consequently, it was considered advisable to perform prediffusion for six to twelve hours, unless the chemotherapeutics to be assayed for were particularly poorly diffusible.
6) To assess the assay method as to its accuracy, ranges of experimental errors in tetracycline, benzyl-penicillin, and kanendomycin assays were determined. As a result, it was found that the range of experimental error was 78-133% (minimum) to 60-193% (maximum) when single assay plates were employed, and 86-118% (min.) to 75-146% (max.) when assays were carried out in triplicate, respectively.
7) The subject paper disc technique was applied to the thin-layer plate method using spores of B. subtilis PCI 219 as test organism. As a result, a linear relationship was confirmed to exist between the logarithmic value of drug concentration in whole blood sample and the diameter of inhibition zones in cases of assay with kanamycin, tetracycline, erythromycin, and chloramphenicol.
8) In practice, levels of the active concentration of chloramphenicol in whole blood were determined by the said assay method with reasonable accuracy in individuals receiving the agent by oral route.