A calibrator for the determination of the erythrocyte concentration in electronic blood cell counting.

A stable calibrator, suitable for the quality control of electronic counting of erythrocytes, has been developed. The assigned concentration is determined by the cooperation of six laboratories using various types of blood cell counting instruments. The performance of the instruments was checked prior to operation. If required, corrections were applied, and deviations were taken into account. The suitability of the calibrator was tested in practice during a period of several years. The applicability of the preparation as a calibrator for use in routine practice was tested in 14 laboratories.

Haemodialysis-induced changes in red blood cell size-distribution histograms.

Treatment by haemodialysis may result in changes of the red blood cell size-distribution histograms in most subjects. After maintenance haemodialysis a statistically significant shift to the left is observed for the mean size distribution histogram in a group consisting of sixteen women. The shift is in agreement with the finding of significantly higher values for the microcyte fraction, as well as with significantly lower mean cell volume (MCV) values. No significant changes were observed for the macrocytic fractions of red blood cells.

Changes in size and zinc protoporphyrin/haemoglobin ratio in red cells of infants during the first months of life.

A longitudinal investigation of changes in red blood cell (RBC) size distribution and zinc protoporphyrin/haemoglobin (ZPP/Hb) ratio was performed. In the course of the first few weeks after birth RBC size distribution histograms show a loss of macrocytic RBCs demonstrated by a rapid decrease of the fraction of macrocytes, whereas the degree of RBC volume dispersion decreases simultaneously. Within this period a significant change in the fraction of microcytes was not observed. At about 6 months of age, a marked shift of RBC size distribution histograms towards lower volumes occurs. Concomitant with the lower MCV values, the values for the absolute distribution width at half peak height (ADW0.5) also showed an obvious decrease. At birth, ZPP/Hb ratios are about three times higher than those of adult subjects and decrease slowly during infancy. This suggests that iron deficiency is unlikely in the first months of life.

Chronic toxicity and carcinogenicity of bis(tri-n-butyltin)oxide (TBTO) in the rat.

In a 106-wk toxicity and carcinogenicity study, groups of 60 male and 60 female weanling Wistar rats were fed 0, 0.5, or 50 mg bis(tri-n-butyltin)oxide (TBTO)/kg diet. In males, feed consumption was increased in all treated groups and increased water consumption occurred at 5 and 50 mg/kg. During the second year, body weight decreased in the 50-mg/kg males, while the females in that group showed no weight gain. Excess mortality was confined to the 50-mg/kg group towards the end of the study. Haematological changes, comprising anaemia, lymphocytopenia and thrombocytosis were noted mainly at the high-dose level. Also, signs of decreased kidney function and increased plasma enzyme activities (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were noted. No effects on serum hormone concentrations (thyrotropin, follicle stimulating hormone, luteinizing hormone or insulin) were observed, except for a decrease in the free thyroxin:thyroxin ratio in both sexes at the high-dose level. Higher serum IgM and IgA levels were present at 50 mg/kg, while, in females, IgG was decreased. At 50 mg/kg, the ovaries, adrenals, spleen (females), heart (males), pituitary, liver and kidneys were increased in weight, but the thyroid weight was decreased in females. The total tin concentrations in liver and kidneys showed a dose relationship and, in general, the concentrations were similar after 1 and 2 yr. Non-neoplastic histological alterations after 1 yr consisted of a decrease in the cell height of the thyroid follicles in all dose groups, with a reduced number of psammoma bodies at 50 mg/kg, a decrease in splenic iron content at 5 (females only) and 50 mg/kg, and a slight bile-duct activation. After 2 yr, only the thyroid changes were still present. In addition, at 2 yr, vacuolation and pigmentation of the proximal tubular epithelium and nephrosis were enhanced at 50 mg/kg. The incidence of benign tumours of the pituitary was significantly elevated and enhanced at 0.5 and 50 mg/kg. At 50 mg/kg increases in pheochromocytomas in the adrenal medulla and in parathyroid adenomas (males) were noted, while adrenal cortical tumours were decreased (males). There was a low, non-dose-related incidence of pancreatic carcinoma. Other tumour rates were in line with control data. It is concluded that lifetime feeding of 50 mg TBTO/kg diet induces toxicity in various organ systems. An increase in some common tumours was found at the high dose, probably due to hormonal or immunological changes.(ABSTRACT TRUNCATED AT 400 WORDS)

Investigations on red cell size distribution histograms in subjects treated by maintenance haemodialysis.

In forty-nine subjects on maintenance haemodialysis quantitative information with respect to changes in the erythron was derived from the red blood cell size distribution histogram. Increased values for the fraction of microcytes were observed in seventeen subjects, whereas in three subjects increased fractions of macrocytes were established. Both fractions were found to be increased simultaneously in only one subject. In the case of normal mean corpuscular volume (MCV) values, the fractions of microcytes were shown to be increased in four male and three female subjects. In contrast, MCV values were increased in four male subjects while the fractions of macrocytes did not exceed the reference range. Several causes which may give rise to abnormalities in red blood cell size distribution histograms in subjects treated by haemodialysis are discussed. From the interpretation of results for serum ferritin concentration, serum iron concentration, total iron binding capacity, and iron saturation percentage it is difficult to establish unequivocally whether the iron supply to the patient is appropriate.

Investigation of red cell size-distribution histograms related to folate, vitamin B12 and iron state in the course of pregnancy.

Effects of gradually reduced stores of iron, folate or vitamin B12 on the erythron were investigated in the course of pregnancy in 23 subjects. Significant decreases in serum concentrations of the compounds above were shown at 20 weeks of gestation with a further decline later. An increased value for the microcyte fraction was established in two subjects, whereas seven other subjects showed increased values for the macrocyte fraction. As pregnancy proceeded a significant intra-individual change could be detected in the microcyte fraction in only one subject. In three of the seven above-mentioned subjects, the values measured for the macrocyte fraction demonstrated a further slight increase during the course of pregnancy. Values established for the absolute distribution width at half peak height (ADW0.5) did not reveal significant changes in the course of pregnancy. If compared with the mean cell volume (MCV) and ADW0.5 values, the microcyte and macrocyte fraction respectively may yield a sensitive and specific indication of anomalies of the erythron. Changes in successively determined haemocytometric values in several pregnant women might indicate decreased availability of nutrients for erythropoiesis. However, one may not draw definite conclusions because it is doubtful whether the condition of the stores in these circumstances will be reflected accurately by the corresponding serum concentrations.

Subacute toxicity of Zwittergent administered intramuscularly.

In order to assess the systemic toxicity of Zwittergent 3-14, a detergent used in the protein-detergent complex vaccines for its attractive immunogenic properties, a subacute toxicity study was performed. In this 4-week toxicity experiment five female and five male rats per group were injected intramuscularly with 0.25 ml of 0, 75, 750 and 7500 micrograms Zwittergent ml-1 sterile saline solution. Body weight and food intake were recorded weekly. At day 24 urine was collected for semiquantitative (pH, protein, ketone bodies, bilirubin and occult blood) and quantitative analyses (protein, creatinine and volume). At the end of the experiment blood was sampled for haematological [haemoglobin (Hb), packed cell volume (PCV), erythrocytes, leucocytes, reticulocytes and thrombocytes and differential white blood cell count] and biochemical analyses (alanine aminotransferase and aspartate aminotransferase. At necropsy brain, heart, liver, kidneys, spleen, thymus, adrenal glands, thyroid, pituitary gland, uterus, ovaries and testes were weighed. The underlined organs and the musculus quadriceps including the injection sites were examined histopathologically. Indications for systemic toxicity were noticed in the high-dose group and included occult blood in urine, elevated protein/creatinine ratio concomitantly with an increased urine volume and increased relative kidney weight indicating a slight disturbance of the kidney function. Some changes in haematological parameters (decreased PCV and increased numbers of thrombocytes eosinophils and monocytes) and a decreased glycogen content in the liver were recorded in the high dose group. These changes may be secondary to the extensive inflammatory reaction observed in the muscle of this high-dose group.(ABSTRACT TRUNCATED AT 250 WORDS)

Interference of plasma fluorophores on the red blood cell zinc protoporphyrin: haemoglobin ratio as determined on a haematofluorimeter.

Direct measurement of the zinc protoporphyrin:haemoglobin ratio (ZPP:Hb ratio) in blood samples is performed by using a haematofluorimeter. Interference by non-specific fluorophores can be eliminated by removing the plasma and making the measurement on washed red blood cells (RBCs). After re-suspending RBCs in isotonic saline, haematofluorimeter readings for the ZPP:Hb ratios revealed higher stability in the course of time whereas a good relationship was found with results obtained by application of an extraction method. Separate reference ranges were established for adult male and female subjects. After washing, the mean values calculated for ZPP:Hb ratios of subjects belonging to the reference groups demonstrated a reduction of 0.04 mumol ZPP mol Hb, corresponding with approximately 30%. In the patients' group, application of washing resulted in a variable decrease of ZPP:Hb ratios.

Investigation of red cell size distribution histograms of normal male and female subjects.

Size distribution histograms characterizing the red blood cell (RBC) populations of normal subjects are provided. Separate haemocytometric reference ranges were established for apparently healthy male and female adults by using an Ortho ELT 800 Hematology Analyzer, which is based on the principle of measuring light scattering intensity (LSI). Effects of sphering RBCs on some characteristics of size distribution histograms were also investigated. The absolute distribution width at half peak height (ADW0.5) of RBC size distribution histograms of sphered RBCs is shown to be approximately 40% lower than for unsphered RBCs. The mean RBC size distribution histogram determined for the group of female subjects was shown to be significantly shifted to the right in comparison with the mean size distribution histogram calculated for males, indicating a sex-related difference. The shift is concordant with a significantly higher fraction of microcytes in male subjects, as well as with lower mean corpuscular volume (MCV) values in males compared with females.

Screening for thalassaemia using the width of the Technicon H6000/H601 erythrocyte size histograms.

The red cell distribution width (RDW) calculated from the data of an erythrocyte size distribution (ESD) histogram generated by a Technicon H6000/H601 blood cell analyser was shown to be not suitable for distinguishing iron deficiency from thalassaemia trait and other microcytic conditions. The absolute distribution width at half maximum (ADW0.5) of these ESD histograms can be used efficiently for screening on thalassaemia trait. The ADW0.5 values appeared to be more powerful for detecting alpha thalassaemia and beta thalassaemia trait than the decision algorithms described earlier. A decreased MCV combined with a small ADW0.5 offers an indication of thalassaemia trait, the diagnosis being later confirmed by more definitive methods. By utilizing the ADW0.5, the number of subjects with thalassaemia trait, particularly those with alpha thalassaemia, detected in the south of The Netherlands was highly increased.

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.

Evaluation of red blood cell size distribution histograms: the fraction of microcytes.

A method is described for the assessment of the fraction of microcytes from the red blood cell (RBC) size distribution histogram. For quantification of the fraction of microcytes an upper threshold corresponding with a RBC volume of 70 fL is established. The reference interval for the fraction of microcytes covers a range from 0.01 to 0.09. In order to examine the relationship between the fraction of microcytes and the mean cell volume (MCV) values computer simulation studies were performed. The computer simulations are based on a Gaussian distributed reference RBC histogram as generated on a Technicon H 6000/H 601 Hematology Analyser. From our studies it is shown that determination of the fraction of microcytes yields a higher sensitivity than MCV values for detecting small populations of microcytes. In particular, measurement of the fraction of microcytes is very sensitive to minor changes in the MCV values if a normocytic RBC population combined with a microcytic RBC fraction shows a wide dispersion in their cell volumes.

Toxicity of methylmercury chloride in rats. III. Long-term toxicity study.

Four groups, each of 25 male and 25 female weanling rats, were given dietary levels of 0, 0.1, 0.5 and 2.5 ppm MeHgCl for 2 years. Observations were made on behaviour, growth, food intake, haematology, serum enzymes, urinalysis, microsomal liver enzymes, organ weights and histology with special reference to the nervous system, histochemistry of the kidneys and cerebellum and on tissue Hg concentrations. Significant findings included a slight growth reduction in females at 2.5 ppm, increased relative kidney weight at 2.5 ppm and histochemical changes in kidney enzymes at 2.5 ppm. No effect was seen on the nature or incidence of pathological lesions or tumours at any level. From the results obtained in the short-term, reproduction and long-term studies, the no-toxic effect level for rats appears to be between 0.1 and 0.5 ppm MeHgCl in the diet. Exposure of the Dutch population does not appear to present a health hazard at the moment because the mean intake of total Hg is still far below the intake deemed to be safe.

Toxicity of methylmercury chloride in rats I. Short-term study.

In the range-finding test, 6 groups of 4 male and 4 female weanling rats were given dietary levels of 0, 0.1,0.5, 2.5, 12.5 and 250 ppm methylmercury chloride (MeHgCl) for 2 weeks. Signs of central nervous system toxicity, weight loss and high mortality appeared at 250 ppm but not at lower levels. No haematological changes were observed at 0.1-12.5 ppm. The relative weights of the liver in females on 2.5 and 12.5 ppm and of the kidneys in females on 12.5 ppm were significantly increased; the effects in males were less marked. Total mercury concentration in the kidneys increased proportionally with increasing dietary levels of MeHgCl. In the short-term test, 5 groups of 15 male and 10 female weanling rats were given dietary levels of 0, 0.1, 0.5, 2.5 and 25 ppm MeHgCl for 12 weeks. Toxic signs, weight loss and restricted food intake were observed at 25 ppm starting from week 9 onwards. Haematological, serum enzyme and urinalysis changes were seen at 25 ppm. Liver microsomal enzyme activity was increased non-significantly and liver glycogen was depressed at 25 ppm. Organ weight changes were evident at 25 ppm and histological changes seen in the spleen, kidneys, brain, spinal cord and peripheral nerves were confined to the 25 ppm level. Histochemical changes in kidney enzymes occured at 2.5 and 25 ppm. Hg concentrations in blood, hair, kidneys, liver and brain were higher at 12 weeks than 6 weeks and generally increased with increasing MeHgCl level in the diet.

Toxicity of methylmercury chloride in rats. II. Reproduction study.

A reproduction study over 3 generations of rats was carried out in which groups of 20 female and 10 male rats received in the diet 0, 0.1, 0.5 and 2.5 ppm MeHgCl. The parameters studied included growth, food intake, haematology, serum and urinalysis, organ weights and reproductive performance, No effect was exerted on fertility index, lactation index or on the 21-day body weights of pups but the viability index was impaired at 2.5 ppm in the F1 and F2 generations. Weight gain reductions observed at 12 weeks for the 2.5 ppm level were not accompanied by reductions in food intake. At 6 months, Fla females on 2.5 ppm showed a reduced leucocyte count whilst P males on 0.5 and 2.5 ppm showed an increase in neutrophils and a decrease in lymphocytes. The relative weights of the kidneys, heart, spleen brain and thyroid were increased at 2.5 ppm and in some cases the increases of kidney weights were inconsistently seen at the 0.1 and 0.5 ppm levels. No significant histological changes were seen at any level. In a special 7-week study involving the F3a generation, weanling rats obtained from the four different F2a groups, each comprising 20 females and 10 males, were all transferred to diets containing 25 ppm MeHgCl. Toxicity signs were evident at 7 weeks. No evidence was obtained of increased susceptibility to the toxicity of MeHgCl in successive generations.

Haematological studies in a new variant of glucosephosphate isomerase deficiency (GPI Utrecht).

The haematological data in a 9-year-old Dutch child suffering from an unusual new variant of glucose-6-phosphate isomerase deficiency (GPI Utrecht) are discussed. Symptoms and signs differ in many respects from those generally observed in GPI deficiency, as mental retardation, drug sensitivity and increased susceptibility to infections were present. Extensive studies on mechanisms involved in drug-sensitive haemolytic anaemia did not reveal its causes. Though the defect was generalized, no disturbance in granulocyte and thrombocyte functions were detected.

Short-term toxicity of 1-naphthaleneacetic acid in rats.

In a 90-day feeding study, 4 groups of 10 male and 10 female rats received in the diet 0,200, 1000 and 5000 ppm 1-naphthaleneacetic acid (1-NAA). Growth and food intake was reduced significantly only in males on 5000 ppm. Haematological examination yielded essentially negative results except for a non-significant reduction at the 1000 and 5000 ppn levels in the leucocyte count, mainly due to a reduction in neutrophils. No significant effects were observed in the renal concentration test, urinalysis, renal histochemistry or histology of a wide range of organs at any level of 1-NAA tested. Increased relative weights of thyroid, testes, brain and liver were confined to the 5000 ppm level. The increase in relative liver weight was not accompanied by histological liver damage and was associated with elevated liver microsomal enzyme activity. The loss of glucose 6-phosphatase (G6Pase) and increase in glucose 6-phosphate dehydrogenase (G6PDH) seen histochemically in the centrilobular region of the liver in males on 5000 ppm, accompanied by glycogen depletion in the liver, could however be indicative of liver damage. On the basis of conventional criteria, a no-effect level of 1000 ppm would have been indicated by this study but in view of liver glycogen depletion at all levels tested a no-effect level was not established.