B. Leijnse

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.

Comparative studies on the interaction of various transferrins and rat bone marrow cells

Abstract 1. 1. The affinity of bovine transferrin for receptor sites on cell membranes of rat bone marrow cells was found to be much lower than of rat or human transferrin. 2. 2. The apparent Michaelis constant for rat transferrin and bovine transferrin appeared to be 1.25 × 10−6 and 3.2 × 10−6mol/l, respectively. 3. 3. The apparent Michaelis constant for rat transferrin was independent on its iron saturation. The binding at equilibrium of [125I]labelled rat transferrin to rat bone marrow cells varied only slightly with the iron saturation of transferrin. 4. 4. Rat diferric- and monoferric-transferrin had the same affinity for rat bone marrow cell receptors. 5. 5. The affinity of rat apotransferrin for these receptors was about one half of those of diferricand monoferric-transferrin.

Hydrophobic properties of alkaline phosphatases

The butanol extraction method of Morton (1950), a routine step in enzyme purification, is discussed with special reference to a hydrophobic form of alkaline phosphatase from human liver tissue. This form slowly precipitates from butanol-extracted liver tissue homogenates stored at 4 degrees C. Furthermore, it is lost when acetone precipitation is applied as a purification procedure. The soluble form in liver tissue is shown to have a higher relative hydrophobicity than the serum liver/bone isoenzyme. The use of sodium cholate in the isolation of the hydrophobic form produces an artefact in isoelectric focusing, which can be abolished by dialysis prior to focusing.

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.

A white blood cell control of long-term stability.

Adequate quality control (Q.C.) of white blood cell (WBC) channels of automated hematology instruments remains a major problem. Due to their extreme lability [I] and their great biological variability [2] native WBC are insuitable for most Q.C.methods as recently reviewed by Koepke and Protextor [3]. Therefore, the only pragmatic approach is using (commercial) controls containing stable WBC-substitutes [4], preferably in conjunction with the use of a computerized ‘average of normals’ Q.C. program [S]. WBC-substitutes used in commercial controls consist either of latex particles or of fixed human or avian RBC [6]. However, these substitutes of potentially long-term stability are seldom offered separately, but rather as so-called multiparameter controls (perhaps for commercial or practical reasons). The stability of these controls generally does not exceed 2 months, due to the presence of (stabilized) RBC and/or platelets. Consequently, there is a continual need to employ new multiparameter controls, which almost invariably come from different lots of material. Hence, these may differ in composition and subsequently the continuity of control might be lost. In this paper we describe the use of commercially available fixed human RBC as WBC-substitutes of long-term stability. To our knowledge, this is the first documented report on WBC quality control material exceeding 10 months’ periods of stability.

Isoelectric focusing of alkaline phosphatases in agarose gel.

An isoelectric focusing technique in agarose gel is presented which is suitable for alkaline phosphatases from both serum and tissue sources. An anomaly in the literature about isoelectric focusing of serum alkaline phosphatase from liver origin is discussed and a possible explanation is proposed. The presented technique is used to demonstrate some differences in behaviour of serum liver and bone isoenzymes towards neuraminidase treatment.

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.

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.

A simple automated method for the determination of bicarbonate in plasma or serum.

Abstract 1. 1. A simple automated method for the determination of bicarbonate concentration in plasma or serum is described. The results of the analyses appear in digital form, the numerical values correspond to the concentration in mmoles/1. Thus without further calculation the results can be printed out or fed directly into a computer. 2. 2. The method is based on a titration technique; after the measurement of the original sample pH, a standardized excess portion of acid is added to the sample and the carbon dioxide formed is expelled completely. The excess of acid is titrated with alkali until the original pH of the sample has been reached. The difference (in mmoles) between the acid added and the alkali needed for titration is equal to the amount of bicarbonate (in mmoles) present in the sample. 3. 3. In this manner a fully electronic, automated method has been developed which permits the processing of 30 samples per h.

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.