A stable human platelet-white blood cell control for the Coulter Model S-Plus II.

The preparation and evaluation of a human platelet-white blood cell control of at least 5 months' stability, as measured in the Coulter Counter Model S-Plus II, is described. In some preparations minor departures from log normal platelet (PLT) distributions were found, impeding the generation of values for mean PLT volume (MPV) and PLT distribution width (PDW). In many cases, however, excellent linearity for PLT and white blood cell (WBC) counts and low coefficients of variation for PLT and WBC counts and PDW were found. Some possible factors causing the large coefficient of variation found for MPV values are mentioned.

Laboratory preparation and evaluation of a multiparameter hemocytometry control.

A protocol for the laboratory preparation of a multiparameter hemocytometry control is given. Human platelets, stabilized by a basically simplified and inexpensive fixation procedure, are added to our previously described white and red blood cell control. Evaluation of this multiparameter control shows good precision characteristics and acceptable mechanical stability for at least 7 weeks, as measured in the Coulter counter Model S Plus-II. The control can basically contribute to the realization of the essence of internal quality control: continuous self-auditing and continuous attempts at improvement of performance.

Outdated blood and redundant buffy-coats as sources for the preparation of multiparameter controls for Coulter-type (resistive-particle) hemocytometry.

Outdated, buffy-coat depleted, CPDA-1 blood and redundant buffy-coats were used as sources for the laboratory preparation of controls for Coulter-type (resistive-particle) hemocytometry. Deteriorated white blood cells and platelets and potentially interfering microaggregates with volumes not exceeding 400 fl are shown to be virtually completely removed by centrifugation and filtration. Addition of fixed red blood cells as white blood cell substitutes and of isolated, fixed platelets enable the preparation of multiparameter controls of short-to-medium-term stability. The availability of these simple, inexpensive controls can contribute significantly to optimal internal quality control in hemocytometry.

Initial activity and inactivation of alkaline phosphatase in different lots of buffer.

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.

The difference between the glucose concentrations in plasma and whole blood.

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.

Heme synthetase activity in normal human and rat erythroid cells and in sideroblastic anemia.

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.

Serum cholesterol analysis in the C.B. Heart Project. Intra-and inter-laboratory continuous quality control.

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".

Influence of zinc ions addition to different lots of 2-amino-2-methyl-1-propanol (AMP) buffer on the alkaline phosphatase activities.

The influence of Zn2+ on the inactivation of alkaline phosphatase [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1 3.1] in serum during preincubation with 2-amino-2-methyl-1-propanol (AMP) buffers was investigated. Addition of Zn2+ to the buffer before preincubation increases the enzyme activity. An optimum Zn2+ concentration different for each lot of AMP buffer can be found, at which the enzyme activities are restored to a level equal to activities measured without preincubation. There is a relation between the inactivating properties of the different AMP buffers and the amount of Zn2+ needed to prevent this inactivation. Since Zn2+ chelating substituted diamines are held responsible for the inactivation by removing Zn2+ from the enzyme, we assume that the addition of Zn2+ to the buffer prevents this removal. As Zn2+ itself is an inhibitor of the enzyme, the addition of both too much or too little Zn2+ results in lower enzyme activities after preincubation with AMP buffer.

Experience in the Netherlands with an external quality control and scoring system for clinical chemistry laboratories.

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.

Unstable glycosylated hemoglobin in patients with diabetes mellitus.

Incubation of washed erythrocytes for 4 h at 37 degrees C in saline resulted in the disappearance of an unstable glycosylated fraction. In a reference group, its amount was very small, but its presence had a significant influence on the upper level of the reference range. In non-diabetic pregnant women, the unstable fraction was significantly higher than in the non-pregnant individuals, which resulted in a lower reference range for the stable HbA1. In diabetic patients, the average unstable HbA1 was about 10% of the "unincubated" level, with marked differences between individuals (0--30%), even in patients with a slightly elevated HbA1. Therefore it is important to determine the concentration of stable HbA1.

A simple automated method for the fluorometric titration of calcium in biological fluids,.

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%.

Polynuclear iron compounds in human transferrin preparations.

During commonly used saturation procedures of transferrin with iron compounds, both as ferri and ferrous, polynuclear iron compounds are easily formed, even when nitrilotriacetate (NTA) is used as a strong iron ligand. The presence of these nonspecific bound irons is demonstrated with Mossbauer spectroscopy and with electronic optical spectroscopy. But no evidence, however, has been found of two different iron binding sites. Because dialysis is not able to remove all polynuclear iron, an easy method with gel filtration has been developed that does remove the polynuclear iron. Some notes are made about the often used method, in transferrin biochemistry, of saturation determination, i.e. the quotient of the absorbances of 470 and 280 nm.

Alkaline phosphatase and cholestasis.

The process of cholestasis in both man and rat leads in the majority of cases to the appearance of a biliary band in the electrophoresis of alkaline phosphatase isoenzymes. In this article, the biochemical nature and mode of formation of the biliary band is discussed, with reference to its appearance in cholestasis and other hepatobiliary diseases.

Preparation and evaluation of a 7-parameter intralaboratory control blood of 4-month stability.

Sterile, fresh concentrates of human red blood cells suspended in a specially designed sterile preservation medium have a stability of all six red blood cell parameters of over four months as measured in the Coulter Model S-Plus II. Substitution under sterile conditions of the unstable white blood cells for (commercially available) fixed (human) red blood cells is a well-established method to obtain simulated white blood cell suspensions of long-term stability.

Basic principles and problems of haemocytometry.

After some brief remarks on counting chambers, references to the ICSH-recommended haemoglobin-determination are given. The microhaematocrit of normal blood is advocated as a potential routine calibration method. Comments are given on discrepancies between centrifugal and flow haemocytometry haematocrits of abnormal and artificial bloods. Flow haemocytometry instruments are classified into analogue and digital instruments or into electrical and optical instruments. Their hydrodynamic properties are discussed. The principles and problems of electrical and optical cell counting and sizing are dealt with. The importance of the refractive index and of flow-induced cell shape changes for the MCV determinations is stressed. It is argued that MCV and haematocrit values are exaggerated at both low and high values and consequently MCHC is erroneously constant. Various prevailing red cell distribution width (RDW) and platelet distribution width (PDW) definitions bring about considerable confusion. The major features of the counting and sizing of white blood cells and platelets are described.

Direct determination of total serum cholesterol by on-column gas-liquid chromatographic analysis without previous derivatisation compared with WHO-CDC reference method.

A method for the direct determination of total serum cholesterol by on-column gas-liquid chromatography is described. The French reference method developed by Gambert et al. served as a model for our method, which is fast and less laborious than the well-known CDC reference method of Cooper et al. based on the Abell-Kendall technique. The accuracy and precision of our on-column gas-liquid chromatographic method and the CDC reference method are comparable. The obvious advantage of this proposed gas-liquid chromatographic reference method is its increased analytical speed. The problem of the specificity of our method in relation to cholestanol is discussed.

Detection of aberrant observations in a background of an unknown multidimensional Gaussian distribution.

An exploratory iterative technique for the detection of aberrant observations on a background of a multidimensional Gaussian distribution is described. Its development was motivated by the analysis of a set of three measurements reflecting the acid-base metabolism in the blood of 2,402 intensive care patients. This new, three-dimensional treatment of such data yields a meaningful description. A technical evaluation of the method, using artificially generated data is also presented. It is shown that the model parameters of the underlying Gaussian distributions are determined with good accuracy and that the accuracy with which the contamination is estimated increases with increasing distance of the contaminating observations from the mean.

Graphical non-linear representation of multi-dimensional laboratory measurements in their clinical context.

A polygon-based graphical representation of laboratory test results using nonlinear scaling is described. It is argued that the nonlinearity of the scale and the use of colors in the representation facilitates interpretation of the test result in its relationship to the standard reference range and critical clinical decision levels. Preliminary results suggest that this representation may be fruitfully used to enhance the efficiency of the information transfer from the clinical chemistry laboratory to clinicians. Other applications, inside as well as outside the medical field, may easily be imagined.

A network thermodynamic model of the concentrating properties of the rabbit/rat kidney in the steady state using the electronic network simulation program SPICE.

A model for the simulation of the diluting and concentrating properties of the rabbit and rat kidney is developed. Translation of the physical model into an electronic one brings the model into a form that can be handled by the electronic network simulation program SPICE. The steady state responses of both kidneys to various inputs are calculated under certain conditions.

Experiences with a new assay for pancreas specific alpha-amylase.

The evaluation of an enzymatic colorimetric method to measure pancreas amylase activity in serum using two monoclonal antibodies that specifically inhibit salivary amylase is described. The assay is quick, easy to perform and can be automated easily. Within-run and day-to-day precision studies gave CVs of less than or equal to 1.5 p. cent and less than or equal to 2.6 p. cent respectively. The test is linear up to 2,150 U/L. Salivary amylase activities up to 20,000 U/L did not contribute more than 2 p. cent to pancreatic amylase activity present in a serum sample. A significant interference by bilirubin is found. Reference values were established for both pancreatic and total amylase activities in serum and used for interpreting the results of pancreatic amylase and total amylase in 72 randomly selected patients. Results obtained by this method agree well with a lectin inhibition method (r = 0.9886).