Some effects on light scattering intensity and red blood cell size distribution histograms due to sphering.

At present most haematology blood cell analysers routinely provide red blood cell (RBC) size distribution histograms. Sophisticated improvements of the instruments have re-awakened interest in the study of size histograms. The quantitative information derived from the histograms may be applied more fruitfully if insight is available, with respect to some essential principles of sizing technology and methods for treatment of RBCs before measurement. In this study the consequences of sphering RBCs are investigated in relation to the generation of size distribution histograms by means of methods based on light scattering intensity (LSI). Sphering of RBCs results in considerably narrower histograms than upsphered RBCs. The overall signal to noise ratio increases and there is a broader gap between large platelets and microcytic RBCs. Narrower size distribution ranges will enable closer modes to be separated. Compared to unsphered RBCs, microcytic sphered RBCs yield increased LSI whereas macrocytic sphered RBCs yield decreased LSI.

Measurements on the forward light scattering intensities of monodisperse latex particles and sphered or unsphered red blood cells.

Characteristics with respect to light scattering intensity (LSI) were measured on latex spheres and on sphered and unsphered red blood cells (RBC). From the discrepancies in LSI signals from polymer spheres and RBCs it is obvious that latices cannot be used for absolute calibration of RBC sizes. Sphering of RBCs did not give rise to a closer resemblance. Monodisperse latex spheres produce extremely narrow size distribution histograms. Regarding these sharp peaks and the long-term stability, polymer spheres offer definite advantages if compared with blood controls to which preservatives were added. A shift from the RBC size distribution histogram, caused by gradually developing instrument drift, can be easily detected and corrected at an early stage.

Effects of ageing on preserved red blood cell populations as measured by light scattering.

Instability of reference materials may cause problems in interpreting results for assurance of quality in haemocytometry. The stability with time for a certain preserved blood specimen is also highly dependent on the principle of measurement. In this paper some effects of ageing on red cell populations in commercially available (Ortho) Blood Controls are evaluated. Red blood cell size histograms become narrower while mean corpuscular volumes obviously decrease with time when measurements are based on light scattering intensity. In contrast, monodisperse polystyrene spheres of different sizes show perfect stability at storage. Such artificially prepared controls should be applied to achieve optimal setting and calibration of haematology analysers.

Influence of ambient temperature on the Ektachem DT60 results for total bilirubin and total protein.

We evaluated the Kodak Ektachem DT60 analyzer with the DTE module in a two-month clinical trial before its introduction in the Netherlands. At ambient temperatures that differed from that at which the DT60 was calibrated, aberrant behavior for total bilirubin and total protein assays was observed. At subnormal temperatures the former gave higher results than expected, and the latter, lower results. We found a similar effect for total protein determined by the manual biuret reaction at different temperatures, when results were calculated from calibration at a fixed temperature. Total bilirubin assayed by the manual diazobilirubin method showed no such effect. Although the DT60 analyzer is equipped with a temperature-regulated incubator, we conclude that the manufactures's recommended room temperature range for these assays should be narrowed and the three-month calibration period adjusted according to external circumstances.

Preparation of an optically clear frozen human control serum.

A new method was developed for the preparation of a human control serum. All equipment used will be available in any clinical laboratory. An in-between storage period of 2 months at -20 degrees C for the combined serum pool proved obligatory to obtain a stable, well defined control serum. In our procedure the heavy turbidity inherent to outdated thawed serum is removed by vacuum filtration over Avicel cellulose. The resulting serum filtrate is clarified to a degree such that it can readily be passed through a filterset consisting of four filters with diminishing pore size from 8.0 to 0.22 micron. The time needed for the total filtration of 10 litres will not exceed 2 to 3 h using 90 mm diameter filters. In our experience over a period of more than 3 years, these controls stored at -20 degrees C had a stability of at least 1 year for non-enzymatic as well as most enzymatic components. Information is provided on the stability and concentration levels of components measured by immunological methods, that is, radioimmunoassay and turbidimetry. We monitor 50 parameters with the control serum described.

The determination of the complement components C1q, C4 and C3 in serum and cerebrospinal fluid by radioimmunoassay.

Non-competitive 2-site radioimmunoassays (RIA) for the determination of the complement proteins C1q, C4 and C3 in cerebrospinal fluid (CSF) are described. The quantitative results of the RIAs were the same as those obtained by other assay methods: radial immunodiffusion and turbidimetry and, in the case of C4, the haemolytic assay. The concentrations of the complement proteins in paired CSF and serum samples from a group of 60 patients were measured, as well as those of albumin and IgG. The ratios (concentration in CSF)/(concentration in serum) of the complement proteins correlated poorly with that of albumin. In contrast, the ratio of IgG was significantly correlated with that of albumin. The ratios of the complement proteins were higher than might be expected on the basis of their molecular masses. This suggests that these proteins may be synthesized within the normal central nervous system.

Radioimmunoassay for normalized thyroxine with use of Sephadex columns.

We describe a reproducible radioimmunoassay, with use of Sephadex columns, for measuring normalized thyroxine. In comparison with a competitive protein-binding procedure, the present method is more specific because antibody rather than thyroxine-binding globulin is responsible for the competition between endogeneous and tracer thyroxine. In barbital buffer, various concentrations of thyroxine binding pre-albumin and albumin had no influence on the results. Values obtained by the present method correlated well with those by the free thyroxine index and a generally accepted thyroxine normalized method.

A radioimmunoassay for thyroxine on Sephadex columns. Investigations on the influence of some buffers on the binding of thyroxine to serum proteins.

A thyroxine radioimmunoassay procedure (T4RIA) based on incubation and separation on Sephadex columns is presented. The assay is rapid and easily to perform; if necessary columns may be regenerated. The raising of antibodies against thyroxine in goats is described in detail. The specificity of the antiserum towards the coupling of several compounds related to thyroxine has been tested. Influence of some buffers on the binding of thyroxine to serum proteins has been investigated. The results of T4RIA in patient sera were in agreement with those obtained by a competitive binding method. The within-day variation was approximately 4% (coefficient of variation, C.V.); day-to-day variation was 7% C.V.

A kinetic study on the influence of the parameters in the determination of inorganic phosphate by the molybdenum blue reaction.

Parameters which influence the initial rate of formation of phosphomolybdenum blue from phosphate, molybdate, polyvinylpyrrolidone and o-phenylenediamine were investigated. From kinetic data it is concluded that polyvinylpyrrolidone not only acts as a protective colloid, but that it takes part in the reaction and a reaction mechanism is proposed dealing with the stoichiometry of polyvinylpyrrolidone. A rate procedure for the determination of inorganic phosphate in serum and urine, based on this study, is given. Deproteinization of samples is not necessary. The method is rapid requiring one minute for quantitative readings. Because of this short period there is hardly any hydrolysis of organic phosphate esters. As the rate of color development is determined blanks can be omitted. Turbid samples may be analysed with little error.