Biomedical subjects
Publications and source records attributed to B Leijnse.
A modification of the flow system of the Technicon SMA 12/60 Continuous Flow Analyzer is described. This modification results in a 60% reduction of the sample volume. The sample size required for the analysis of twelve constituents in serum is thereby reduced to only 0.7 ml. This modification is simple and does not require complex hardware. The reduction in sample size also leads to a considerable cost reduction (25%).
Alkaline phosphatase activities were determined in six lots of 2-amino-2-methyl-1-propanol (AMP) and in six lots of diethanolamine (DEA) buffers without preincubation of the sample. There appeared to be differences between the lot numbers in both cases, resulting in a variation in initial activity. When serum samples are preincubated with buffer a loss of activity was observed in 4 out of the 6 AMP buffers. Four human isoenzymes showed varying inactivation during preincubation with AMP buffer. No loss of activity was observed when the preincubation was done with the six DEA buffers. These results indicate that the purity of the commercially-available buffers is quite unsatisfactory.
Standardization and quality control of measurements are of crucial importance in large medical surveys. In the C.B. Heart Project serum cholesterol analyses in the central laboratory were extensively controlled with regard to precision as well as accuracy. All measurements are carried out in side-by-side duplicate, the maximum difference allowed being 0.3 mmol/l. Bench control, aiming at maintaining linearity and precision with control limits, requires insertion of standard solutions and several serum controls into every run. Control values must fall within 2 S.D. limits from the established mean values, e.g. 4.1 +/1 0.25 mmol/l and 8.2 +/- 0.25 mmol/l. A very reliable check on the precision was obtained by blind introduction of duplicate patient specimens. Repeatability (within-run precision) as calculated each month has S.D. values of approx. 0.10--0.20 mmol/l, whereas reproducibility (long-term precision) S.D. values are 0.15--0.25 mmol/l. Accuracy is checked in the Cooperative Standardization Programme of the WHO Regional Lipid Reference Centre in Prague. This makes the C.B. Heart Progject results comparable with other standardized studies. Our bias approximates +3% and usually remains within WHO "narrow limits".
The enzyme heme synthetase, involved in the final step of the biosynthesis of heme, has been assessed in rat and human bone marrow and peripheral blood. The pH optimum of the enzyme in bone marrow appeared to be pH 7.6, whereas the Michaelis constant in human and rat bone marrow was found to be 1.6 micrometer and 0.6 micrometer, respectively. Rat reticulocytes showed approximately 100-times higher heme synthetase activities than did rat erythrocytes. By contrast, human reticulocytes did not show significantly higher activities than human erythrocytes. This difference between rat and human reticulocytes could be confirmed by in vitro experiments with intact cells in which iron uptake and heme synthesis of human and rat cells were compared. Finally, heme synthetase activity was assessed in bone marrow cells of two patients with sideroblastic anemia. In both cases the enzyme activities were found to be comparable to those in control bone marrow.
According to the hypothesis of Fletcher and Huehns, functional differences exist between both iron-binding sites of transferrin. The site designated A should mainly be involved in the delivery of iron to erythroid cells, whereas site B should donate its iron preferentially to cells involved in the absorption and storage of iron. In the present study this hypothesis could be confirmed by in vitro experiments with various cell types. Iron transferrin preincubated with rat bone marrow cells donates less iron to rat bone marrow cells, Chinese hamster fibroblasts, human fibroblasts and human lymphoblasts than freshly prepared iron transferrin equal in iron and transferrin concentraion. Rat liver parenchymal cells, however, take up more iron from preincubated than from freshly prepared iron transferrin. Obviously, site A not only donates iron preferentially to erythroid cells but also to (rapidly) dividing nonerythroid cells in culture. From experiments with iron transferrin mixtures in which radioiron was present at low or high iron saturation, it could be concluded that rat bone marrow cells take up iron equally well from monoferric as from diferric transferrin. The observed functional heterogeneity could, therefore, not be ascribed to differences between monoferric and diferric transferrin.
The interference of hemoglobin in the determination of total bilirubin with the Du Pont ACA was studied. The method seemed of limited value in our opinion especially with respect to neonatal sera. By changing the wave-length setting a rather broad plateau was found when the hemoglobin concentration was plotted against the actual bilirubin concentration. On the basis of these findings a new calibration graph was constructed and programmed into the ACA. The new method correlates well with two other micro bilirubin techniques.
Laboratory results obtained in different laboratories over lengthy periods of time usually are difficult to compare. In cooperative long-term studies where such results must be pooled, thorough standardization of methods is vital. We describe a program in which comparable plasma cholesterol and glucose analyses have been obtained, by simple methods. In the Netherlands and the Soviet Union in close collaboration with the Center for Disease Control, Atlanta, Ga., U.S.A. The two laboratories produced glucose values (direct o-toluidine reaction) within 2% of the target reference values and cholesterol results (direct Liebermann-Burchard reaction) with a consistent 6-8% positive bias over the reference method values. Intralaboratory precision was subject to preset acceptance limits. The use of common control materials, exchange of patient samples, and on-site comparison of all details of laboratory procedures are vital tools in standardization efforts. A laboratory protocol that included quality requirements and rejection criteria was developed and proved to be indispensable. The experience gained should be useful in standardizing inter-laboratory results in similar studies.
We evaluated the determination of ammonia used in the Du Pont Automatic Clinical Analyzer (aca). Correlation studies were done with a manual method, based on the same enzymatic principles as the aca and with a method involving a cation-exchange resin, followed by the Berthelot color reaction. In either case, the results agreed well with the results found with the aca. For the aca the mean analytical recovery of ammonia in plasma samples was 104%. Within-run and day-to-day precision had coefficients of variation of 6% for normal values and 1 and 2% for above-normal values. The linearity, tested with primary standards and plasma samples, was good up to 1000 mumol of NH3 per liter.
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A new automatic device has been developed for the determination of the calcium content in biological fluids. The method is based on a fluorometric titration technique, in which calcium is titrated with ethyleneglycolbis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The end-point of the titration is indicated by quenching of the fluorescence of a calcium-calcein complex, in which calcein is the fluorescein-complexone indicator. To obtain proper results we found it necessary to use a 1 molar potassium hydroxide solution as titration medium. The results are printed out directly in concentration units (mmol/l). In order to carry out these determinations, several electronic and mechanical devices, including a sample-changer, titration cabinet with built-in fluorometer, control unit and electron titrator, were developed. Together they form the new calcium automatic device of which several qualitites such as precision, recovery and correlation have been studied. The disturbing influence of bilirubin has been minimized by using an excitation wavelength of 492 nm, at which there is nearly no absorption by bilirubin. At a concentration of bilirubin of as much as 500 micronmol/l, the recovery of calcium is still about 96%.
A procedure is described to judge the quality of clinical chemistry hospital laboratories in The Netherlands. In 1974, 45% of the laboratories took part in the national control scheme. A score system of the results, independent of the standard deviation, opens a provisional possibility to strive for (dynamic) reference laboratories. The Youden plots possibly will unveil systematic method-differences. Discrepancies in results seem to be caused as much by methods/instruments applied, as by unknown factors which are under investigation now.
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The glucose concentrations in whole blood (WB) and plasma (P), both prediluted with distilled water and saline, and in their protein-free filtrates, were determined with the Hoffman ferricyanide method. In whole blood prediluted with distilled water the glucose concentration appeared to be 13% higher than in whole blood prediluted with saline. In plasma, predilution with distilled water or saline did not result in different glucose values. Plasma glucose is significantly higher than whole blood glucose independent of the method of pretreatment. A direct relationship between the two values exists, which for protein-free filtrates is represented by Glu(P) equals 1.07 Glu(WB) + 0.11. The glucose concentrations in erythrocytes were calculated and also correlated to plasma glucose values. A good correlation was found when protein-free filtrates were used. Apparently, conversion factors from whole blood to plasma glucose may be used only in cases where samples are deproteinized.
1. The haemoglobin, haematocrit, transferrin and serum iron of a group of 418 healthy medical students, aging from 18 till 25 years, were determined. The mean Hb for the male students was 9.4 +/- 0.63 mmol/l, for the female students 8.7 +/- 0.56 mmol/l. 2. The correlations between Hb, Ht, SeFe and transferrin have been investigated. A positive correlation exists between Hb and SeFe and Ht and SeFe.
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