Examination of peripheral blood films using automated microscopy; evaluation of Diffmaster Octavia and Cellavision DM96.

BACKGROUND: Differential counting of peripheral blood cells is an important diagnostic tool. Yet, this technique requires highly trained staff, is labour intensive and has limited statistical reliability. A recent development in this field was the introduction of automated peripheral blood differential counting systems. These computerised systems provide an automated morphological analysis of peripheral blood films, including a preclassification of both red and white cells (RBCs and WBCs, respectively). AIMS: To investigate the ability of two automated microscopy systems to examine peripheral blood smears. METHODS: Two automated microscopy systems, the Cellavision Diffmaster Octavia (Octavia) and Cellavision DM96 (DM96), were evaluated. RESULTS: The overall preclassification accuracy values for the Octavia and the DM96 systems were 87% and 92%, respectively. Evaluation of accuracy (WBC analysis) showed good correlation for both automated systems when compared with manual differentiation. Total analysis time (including post classification) was 5.4 min/slide for the Octavia and 3.2 min/slide for the DM96 (100 WBC/slide) system. The DM96 required even less time than manual differentiation by an experienced biomedical scientist. CONCLUSIONS: The Octavia and the DM96 are automated cell analysis systems capable of morphological classification of RBCs and WBCs in peripheral blood smears. Classification accuracy depends on the type of pathological changes in the blood sample. Both systems operate most effectively in the analysis of non-pathological blood samples.

Different strategies in the laboratory diagnosis of autoimmune disease: immunofluorescence, enzyme-linked immunosorbent assay or both?

We investigated the clinical utility of different strategies for antinuclear antibodies (ANA) and antibodies to extractable nuclear antigens (ENA) testing. All requests for ANA and ENA (n = 485) in a 20-week period were tested by immunofluorescence (FANA) and immunodiffusion (strategy 1), enzyme-linked immunosorbent assay (ELISA) techniques (strategy 2) or a combination of FANA and ELISA (strategy 3). Results of strategy 1 were positive by FANA in 8% (by immunodiffusion in 2%). By ELISA, 11% of the samples tested positive. In 12% (n = 60) of the cases the two strategies did not agree. The positive predictive value (PPV) for autoimmune disease of strategy 1 was significantly higher than that for strategy 2, but after exclusion of rheumatoid arthritis this difference was abolished. In strategy 2 reagent costs were high but working time comparably shorter. With strategy 3 PPV results were not better, whereas costs and working time were higher. The most frequently occurring reasons for ANA/ENA test requests were: joint symptoms (37%), follow up (30%) or abnormal laboratory result (7%). In a survey of the clinicians 66% replied that the test result did not have any consequences, irrespective of the result or the strategy used. We conclude that FANA and immunodiffusion are superior to ELISA techniques. However, the clinical value of ANA/ENA testing is low and more selective test ordering is strongly recommended.

Iron uptake in blood-brain barrier endothelial cells cultured in iron-depleted and iron-enriched media.

Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood-brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood-brain barrier endothelial cells were grown in either iron-enriched or iron-depleted medium. Iron-enriched cells showed a reduction in surface-bound and total transferrin receptor numbers compared with iron-depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron-enriched cultures was higher, whereas the transferrin receptor externalization rate in iron-enriched cultures was lower than the rate in iron-depleted cultures. Moreover, blood-brain barrier endothelial cells cultured in iron-enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood-brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.

Regional distribution of iron, transferrin, ferritin, and oxidatively-modified proteins in young and aged Fischer 344 rat brains.

Iron dysregulation in the brain is thought to contribute to the oxidative damage seen in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. A role for iron in the oxidative stress thought to contribute to normal ageing is less certain. To better characterize the role of iron in normal ageing, the concentrations of iron, transferrin, ferritin, and protein carbonyl groups are measured in nine separate regions of Fischer 344 rats. The largest (approximately 30%) age-related increases in brain iron concentration are seen in the temporal cortex, medial septum, and cerebellum. Ferritin concentration in these same brain regions increases 50 to 250% with age, while protein carbonyl concentration is only -27 to +4%, of young rats. These results indicate that an increase in the major iron-binding protein ferritin compensates for any age-related increase in iron concentration, and suggest that the increased ferritin is cytoprotective, serving to prevent the accumulation of protein carbonyl groups (a principal product of metal-catalysed oxidation of proteins).

Transcytosis of 6.6-nm gold-labeled transferrin: an ultrastructural study in cultured porcine blood-brain barrier endothelial cells.

The mechanism and regulation of iron transport to the brain are largely unknown. The large surface area of the blood-brain barrier capillaries and the presence of transferrin receptors on the luminal plasma membranes of the blood-brain barrier endothelial cells (BBB-ECs) suggest that these cells actively participate in the transport of iron into the brain. In this paper, we describe the ultrastructural morphology of primary and first-passage cultures of BBB-ECs grown on different types of porous membranes. To investigate the mechanism of iron transport into and across the BBB-ECs, porous membrane grown first-passage cells were incubated with 6.6-nm gold-labeled transferrin and studied with electron microscopy. Results are suggestive for a transcytosis of transferrin through the BBB-ECs.

Isolation and partial characterization of two porcine spleen ferritin fractions with different electrophoretic mobility.

Ferritin isolated from porcine spleen could routinely be separated in two fractions on nondenaturating gradient gels. Both fractions could be isolated with a purity of 96% when applied to two serially linked columns, each 200 cm in length, packed respectively with Sepharose 4B and Sepharose 6B. Both fractions were similar as judged by electron microscopy. Assessed biochemically fractions were equal with respect to subunit composition, iron and phosphorus content, as well as amino acid composition (with the exception of N-acetylglucosamine). Carbohydrate analysis showed that the fraction with an apparent mass of 440 kDa (= FFL) contained 1.8% (w/w) glycans, whereas the fraction with an apparent mass of 670 kDa (= FFH) contained nearly five times as much (neutral) sugar residues (8.9%, w/w) and 10 times as much sialic acid. This difference in amount of carbohydrate side chains might explain the dissimilarity in electrophoretic mobility of the two fractions.

Quantification of different transferrin receptor pools in primary cultures of porcine blood-brain barrier endothelial cells.

Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood-brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood-brain barrier endothelial cells, we separately quantified surface-bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large "inactive" intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100-fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve-fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.

Isolation, purification and characterization of porcine serum transferrin and hemopexin.

Two techniques are described for the isolation of porcine serum transferrin and hemopexin, respectively, yielding nearly pure proteins (> 99%) as tested with crossed immunoelectrophoresis. Porcine transferrin has an estimated molecular weight of 79 kDa and porcine hemopexin a molecular weight of 62 kDa. Both purified proteins were subjected to amino acid and carbohydrate analyses. Based on carbohydrate and sialic acid analyses, it is proposed that transferrin contains one bi-antennary glycan chain, whereas hemopexin contains two bi-antennary and one tri-antennary glycan chains.

Pulmonary distribution and efficacy of exogenous surfactant in lung-lavaged rabbits are influenced by the instillation technique.

Surfactant bolus instillation has been reported to cause changes in arterial blood pressure (BP) and cerebral blood flow velocities which may increase the risk of intraventricular haemorrhage. To avoid these effects, slow tracheal infusion was evaluated as a possible alternative method of surfactant administration. Saline lung lavages were performed in 13 anesthetized and artificially ventilated adult rabbits to produce respiratory distress syndrome. Curosurf (CS, 200 mg/kg) labeled with 14C-dipalmitoyl-phosphatidylcholine (-DPPC) and/or red microspheres (RMS) was instilled into the trachea either as a single bolus (n = 8) or by infusion during 45 min via a side-channel within the wall of the tracheal tube (n = 5). An arterial cannula was placed for monitoring of blood gases and BP. To determine surfactant distribution, the lungs were cut into 60-70 pieces and radioactivity and/or the number of RMS were measured in each piece. The distribution of RMS was closely related to the distribution of 14C-DPPC (r = 0.96). Bolus instillation of CS led to a prompt and sustained increase in PaO2 (from < 10.5 to > 40 kPa within 2 min), a transient decrease in BP, and a reasonably homogeneous pulmonary surfactant distribution. Tracheal infusion of CS changed neither BP nor PaO2 during the observation period of 60 min. The pulmonary distribution of CS was extremely uneven after infusion. The distribution of exogenous surfactant and its effects on gas exchange are influenced by the instillation method. An inadequate instillation technique may add to the causes of "poor response" after surfactant replacement.

Colloidal carbon particles as a new label for rapid immunochemical test methods: quantitative computer image analysis of results.

Colloidal carbon particles can serve as label in sol particle immunoassays. The universal applicability of these particles in qualitative and (semi)quantitative immunoassays has been demonstrated. Sol particle and/or dipstick immunoassays, not yet optimized in terms of sensitivity, are discussed. The colloidal label has been used successfully in a mouse immunoglobulin isotyping kit. Human serum albumin spotted onto nitrocellulose in a concentration range of 7.8 to 1000 ng could be detected using anti-albumin antibody absorbed onto colloidal carbon particles. It was also possible to perform a competitive assay with this conjugate for a concentration range of free human serum albumin varying from 0.25 to 6.75 micrograms. The Kunitz-type trypsin inhibitor from soybean was determined by a colloidal carbon based immunoassay in a range of 2.5 to 160 ng. In this assay, free and colloidal carbon-bound inhibitor competed for binding specific antibodies spotted onto a nitrocellulose membrane. An image- and data-processing procedure has been developed that enables a rapid and simple quantification of colloidal carbon sol particle immunoassays. The average grey level of a spot is taken as a measure for quantitative purposes. This so-called Sol-particle Image Processed ImmunoAssay (SIPIA) procedure is equally well applicable to assays using other colloidal particles.

The effect of desferrioxamine on iron metabolism and lipid peroxidation in hepatocytes of C57BL/10 mice in experimental uroporphyria.

The effects of the iron chelator desferrioxamine (DFx) on liver iron accumulation, malondialdehyde (MDA) production, porphyrin accumulation and uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37) activity were investigated over a period of 14 weeks in C57BL/10 mice, made porphyric by the administration of hexachlorobenzene (HCB) and iron-dextran (Imferon, IMF) or IMF alone. In addition, we measured the amount of low molecular weight (LMW) iron in liver tissue to determine a possible correlation with MDA production. These experiments showed that combined treatment with HCB + IMF, as well as IMF alone, resulted in porphyrin accumulation, increased MDA production and reduced URO-D activity, whereas HCB alone had no effect. DFx caused a reduction in hepatic porphyrins, this reduction being more distinct in the IMF group than in the HCB + IMF group. The effect of DFx on MDA production and URO-D activity was in agreement with the results on porphyrin accumulation. LMW iron pool measurements at 11 weeks correlated well with data on MDA production in all treated groups in that period (r2 = 0.84), suggesting both variables are interdependent. In conclusion, these results suggest an important role for iron in porphyrin accumulation, probably through its catalytic role in the generation of oxygen-related free radicals, resulting in direct damage to URO-D. The effectiveness of DFx in reducing porphyrin accumulation is probably the result of a reduction in LMW iron, thus diminishing the amount of iron available for a catalytic role in the generation of oxygen-related free radicals.

Expression of individual HMW glutenin subunit genes of wheat (Triticum aestivum L.) in relation to differences in the number and type of homoeologous subunits and differences in genetic background.

The amount of individual high-molecular-weight (HMW) glutenin subunits of bread-wheat has been studied in relation to variation at homoeologous loci and in the general genetic background. The relationships between Glu-1 loci have been studied using nearisogenic lines (NILs) of the variety Sicco and in the progenies of two crosses. Substitution of the Sicco Glu-D1 allele by a null-allele resulted in higher amounts of the homoeologous subunits. The presence of a Glu-A1 nullallele did not have a noticeable effect on the amounts of homoeologous subunits. In three out of four NILs and in the sister-lines of two crosses, the amounts of HMW-subunits did not depend on the allele make-up at homoeologous loci. Only in the NIL which contains the Glu-D1 allele, encoding subunits 1Dx2.2 and 1Dy12, was the amount of homoeologous subunits lower than the amount of these subunits in Sicco. This study suggests a relation between the amount of HMW-subunits encoded by an allele and its contribution to bread-making quality. The effect of genetic background has been studied using F4 and F5 lines of two crosses. The total amounts of subunits, relative to the total amount of kernel proteins, showed a considerable variation between lines. The ratio between individual subunits did not differ between genetic backgrounds. Because this ratio is also largely independent of differences in environmental conditions, it is concluded that the relative amount of a subunit is a valuable measure for the detection of genetically-determined differences in the expression of HMW-subunit genes.