Evaluation of an automated hematologyanalyzer (CELL-DYN 4000) for counting CD4+ T helper cells at low concentrations

In human immunodeficiency virus (HIV) infection, CD4 cell counts are useful in defining the disease state, monitoring antiviral treatment, and identifying patients at risk for opportunistic infections. Counting CD4 cells typically relies on traditional immuno-flow cytometric analyzers that require opening the tube for manipulation of the blood sample. In addition to automated blood cell counting, the CELL-DYN 4000 hematology analyzer performs a completely enclosed and automated analysis of the T lymphocyte subsets. We studied the performance characteristics of this method in blood samples containing low levels of CD4+ T cells. In one set of experiments, we emulated low level CD4 counts by use of a CD4 Positive Isolation Kit to deplete the CD4+ cells from blood samples. We used the FACScan analyzer for reference counts. Measurements were made exactly 12 hr after venepuncture in samples that were stored at room temperature. In normal samples and those with low CD4+ cell counts, there was excellent correlation between the results of the CELL-DYN and FACScan methods. Using the CELL-DYN 4000 analyzer, the precision of CD4+ and CD8+ T-cell counts was high (CV = 2 to 8%). The CD4+ T-cell count was linear over a wide range (35 to 1640 cells/microl). This study shows that CD4 and CD8 T cell counts using the CELL-DYN 4000 analyzer is suitable for normal samples and also for those with low CD4+ T cell counts. The method is rapid and automated, and blood specimens remain enclosed, minimizing the biohazard of exposure to blood of HIV patients.     

A novel presenilin 1 mutation (Y154N) in a patient with early onset Alzheimer's disease with spastic paraparesis

Early onset familial Alzheimer's disease with spastic paraparesis (FAD-SP) has been associated with mutations of the presenilin 1 gene (PSEN1). We report a pedigree of FAD-SP due to a novel missense mutation of PSEN1 (Y154N). The symptoms of the proband were characterized by presenile dementia in her 40s, preceded by spastic paraparesis in her 30s, whereas the mother of the proband presented with spastic paraparesis in her 40s, followed by symptoms of dementia in her mid 60s. The mutation was found only in the proband, and not in a normal family member, normal Japanese control subjects, patients with sporadic Alzheimer's disease or patients with familial spastic paraparesis without dementia. Thus, Y154N is a novel PSEN1 mutation responsible for FAD-SP of Japanese origin.     

Expression of CD56 antigen on acute nonlymphocytic leukemia]

CD56 antigen (detected by NKH-1) is distributed on NK cells, monocytes, and ectodermal neural cells. In this study, the blasts of 29.2% of 27 patients with acute nonlymphocytic leukemia (ANLL) expressed CD56 antigen, but not CD16, CD2, or CD3 antigen. Leukemic cells isolated from 3 patients with CD56-positive ANLL did not have NK activity. There were no significant differences between CD56-positive and CD56-negative ANLL in CD13-positive cases, CD33-positive cases, and HLA-DR-positive cases. These results suggest that CD56-positive ANLL could be so-called mixed-lineage leukemia (lymphoid-associated antigen in ANLL).     

Phenotypic modulation in lipocytes in experimental liver fibrosis

The presence of a-smooth muscle actin (smA)-positive cells has recently been reported in the fibrotic liver. Lipocytes have been considered to play important roles in hepatic fibrosis. However, the relation of the a-smA-positive cells and lipocytes has not been determined. The biological implication of a-smA expression remains unknown. To study these questions, we carried out double immunofluorescent staining of a-smA and desmin (a marker for lipocytes), or a-smA and collagen, and double immunohistochemical staining of a-smA and 5-bromo-2'-deoxyuridine (BrdUrd) in carbon tetrachloride-induced fibrotic rat livers. In normal and control livers, a-smA-positive cells were not seen in the lobules, whereas scattered desmin-positive cells were present. With the development of hepatic fibrosis, a-smA was expressed only in a portion of desmin-positive cells located predominantly around collagen bundles. A number of a-smA-positive cells in the lobules were labelled with BrdUrd. These results suggest phenotypic modulation in lipocytes and differentiation of lipocytes towards myofibroblast-like cells, since a-smA is expressed with desmin in myofibroblasts in scar tissue. The expression of a-smA may be related to events of the fibrotic process, such as tissue contraction or fibrogenesis per se.     

Light and ultrastructural features of lymphoepithelial lesions of the salivary glands in Mikulicz's disease

This investigation deals with the histogenesis of the so-called 'epimyoepithelial islands' in Mikulicz's disease of the major salivary glands and is based on light and electron microscopic study in six patients. The 'epimyoepithelial islands' represent collapsed acini prior to their complete involution and disappearance, the intraductal cellular proliferation, stratification and differentiation into luminal and peripheral myoepithelial cells with partial and complete obliteration of their lumina and finally, cord-like proliferation and formation of nests of residual pluripotential cells showing squamous metaplasia and occasional myoepithelial cell differentiation. A pink, homogeneous and hyaline material on light microscopic examination is multilayered and extracellular and is in close association with the basal lamina when viewed with electron microscope.     

Early development of four Diphyllobothrium species in the final host

The early development of four Diphyllobothrium species, D. latum, D. dendriticum, D. ditremum, and D. vogeli, are described. D. latum sheds the entire larval body easily and shows a high shedding rate of 82.1% on average. On the other hand, D. dendriticum exhibits a different developmental pattern, with a low shedding rate of 8.7% in the hamster and a high shedding rate of 34.9% in the rat. D. ditremum is difficult to recover from hamsters but shows a high shedding rate of 42.9%. D. vogeli shows a constant recovery rate of 38.3% without shedding. The species specificity of these four diphyllobothriids is discussed briefly in relation to the early developmental pattern and the growth rate.     

ROUTINE LOW AND HIGH RESOLUTION TYPING OF THE HLA-DRB GENE USING THE PCR-MPH (MICROTITRE PLATE HYBRIDIZATION) METHOD

We describe HLA-DRB1 typing using polymerase chain reaction-based microtitre plate hybridization (PCR-MPH), which can process large numbers of samples. MPH typing is similar to an enzyme-linked immunosorbent assay (ELISA), in which a tandemly ligated sequence-specific oligonucleotide is immobilized on microtitre wells. The typing procedure consisted of two steps. In the first, PCR-MPH with 16 probes was performed to determine the specificities of the serological levels (DR1, DR2, DR3, DR4, DR11, DR12, DR13, DR14, DR7, DR8, DR9 and DR10) after generic amplification (‘low resolution typing’). In the second step, DR1, DR2, DR4, DR 12/8 and DR3/11/13/14 were group-specifically amplified based on the results of the first PCR-MPH, and microtitre plate hybridization proceeded in a similar manner to the first step (‘high resolution typing’). Low resolution typing was completed within 2 h after generic amplification, and the results of high resolution typing were obtained in another 3.5 h after amplification. The allelic types classified using PCR-MPH were completely concordant with those obtained by PCR- single-strand conformation polymorphism or PCR-restriction fragment length polymorphism.     

Fine structure of hepatic sinusoids and their development in human embryos and fetuses

The fine structure of the hepatic sinusoids of 81 human embryos and fetuses and their development from 5 to 12 weeks gestation were studied. At 5 weeks gestation, sinusoid-like structures and Kupffer-like cells were observed between liver cell cords. Between 6 and 8 weeks gestation the sinusoids were completely developed. Definite Kupffer cells appear at this developmental stage, when the bone marrow has not yet formed. Floating macrophages form cell aggregates in the sinusoids which contact endothelial cells and settle as Kupffer cells. Erythroblastophagia is observed in Kupffer cells and macrophages. The endothelial linings are closed, with the attenuated cell processes and intercellular junctions between the adjoining endothelial cells. No transition was observed between Kupffer cells and endothelial cells. The findings suggest that Kupffer cells in the human embryo are extrahepatic in origin and that they reach the sinusoids via the circulatory system. Ito cells, which store fat, originate from mesenchymal cells in the septum transversum.     

Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney.

A cytopathic astrovirus was isolated from pigs with acute diarrhea in an established cell line that was derived from porcine embryonic kidneys with the aid of trypsin. The virus showed a distinct cytopathic effect characterized by an enlargement of cells and the appearance of fine granules in the cytoplasm. Porcine astrovirus was shown to have an RNA genome, as determined by the effect of 5-iodo-2'-deoxyuridine on its replication, and five polypeptides with molecular masses of 13,000, 30,000, 31,000, 36,000, and 39,000 daltons; and it was shown to be stable to lipid solvents and heating at 50 degrees C for 30 min but somewhat labile to acid (pH 3.0). The buoyant density of the isolate determined in CsCl was 1.35 g/ml. Seroconversion to the virus was evident in the paired serum specimens obtained from pigs with diarrhea that were housed at the farm where the disease occurred. The neutralization test on serum specimens collected randomly from 128 adult pigs of eight herds revealed that 50 of the serum specimens were positive for antibody to porcine astrovirus, although there was considerable variation in the prevalence among herds, ranging from 0 to 83%. Hysterectomy-produced, colostrum-deprived, 4-day-old pigs developed mild diarrhea after oral exposure to porcine astrovirus propagated in the cell culture; and the virus was isolated again from diarrheal stool specimens.     

Total hematological analysis system as a screening test for hematological malignancy

Measurement of complete blood cell count and white blood cell differentiation is an essential laboratory test and the most important screening test for hematological malignancy. Recently, several automated blood cell analyzers have been developed to improve accuracy and precision. When flag messages generated in the presence of morphological abnormalities of the samples are displayed, manual revision is necessary. In our laboratory, the manual revision rate has been 35-40%. Therefore blood cell analyzers are useful in screening for abnormalities as well as greatly reducing expensive and time-consuming manual differential procedures. In addition, automated blood cell analyzers can provide several types of useful information including the leukocyte distribution scattergram. However, most such information is not utilized in the clinical field. In the future, a total hematological analysis system will be constructed so that all information provided by automated blood cell analyzer and by manual methods are available.