The main disadvantage of serum determinations is that the low level of radioactivity present requires a long time for counting.The subjects were on normal diets, and no abnormalities were detected in the blood films.The physical variation, ie, the scatter of results due to inaccuracy of counting technique, has a standard deviation of counts per second at a level of counts per second in the serum.These figures do not include errors due to pipetting.A number of patients, however, did require repeat tests for various reasons.The degree of correlation between the two parameters has also been calculated and the established reproducibility in different individuals allows precision in the diagnosis of marginally abnormal states.Hence it is now possible to investigate reliably the vitamin B absorption of various groups of patients, for example, in those following partial gastrectomy, in those who are folate deficient, and in those with endocrinopathies associated with neurological disease.We have not investigated malabsorption states but we have examined postgastrectomy patients.Our experience of the postgastrectomy group is discussed in another paper.Proetctedbyhttp: jcpb. j.com J ClinP athol: firstpublished as. jcp. on. October. Dwlfrom The new assay correlated well with a more conventional tube assay and was not influenced by the presence of antibiotics in serum.Evaluation of assay performance showed excellent interassay and intraassay precision with quantitative <a href="http://www.targetmol.com/compound/Pamabrom">Targetmol's
Pamabrom</a> recovery of added cyanocobalamin over a wide range of additions. Traditional vitamin B microbiological assays, however, are cumberand timeconsuming, demanding specialised technical input.Another disadvantage of these assays is that the presence of antibiotics in patient sera can inhibit the growth of the assay organism giving falsely low results.Most clinical laboratories therefore use competitive protein binding assays for the routine estimation of serum vitamin B.These assays are commercially available in kit form.In the introduction of a colistin sulfate resistant strain of L leichmannii meant that assays could be completed openly without aseptic precautions and the subsequent development of a test organism cryopreservation technique further shortened the assay protocol and improved overall assay performance.We describe here simple microbiological assay for vitamin B which takes advantage of these recent developments and is performed in well microtitre plates.The assay also exploits the fact that antibiotic interference can virtually be eliminated by treating affected samples with an inactivating some a enzyme.Results were calculated either manually or using a data reduction programme. Briefly, serum was diluted in with a buffer containing sodium hydroxide, pH. After mixing, autoclaving the supernatant extracts were decanted.As a reduced volume of extract supernatant is needed to complete the microtitre plate assay it was necessary to determine if extraction by autoclaving of a lesser volume of dilute serum in a smaller tube would give a similar recovery of vitamin B.A comparison of the results obtained on sera using a small extraction volume gave virtually identical results. After boiling and cooling, mgl colistin sulphate was added.A culture of L leichmannii was started by resuspension of freeze dried organism. This growth was subcultured for two successive days by adding p to a second broth each morning and incubating overnight.This procedure was essential to ensure that the culture was well established.