Preparation of Quality Control Slides and Establishment of an External Quality Assessment Program for Five Special Stains Routinely Used in Diagnostic Hematology

BACKGROUND: In general, internal/external quality control of special stains for diagnosis of hematological diseases may be unavailable in a clinical laboratory owing to the lack of an appropriate positive/negative control material. METHODS: We developed a protocol on positive/negative control materials for five special stains (iron, myeloperoxidase [MPO], periodic acid-Schiff [PAS], Sudan black B [SBB], and alpha-naphthyl acetate esterase [ANAE]) using a hematological malignant cell line. First, we compared stainability of seven cell lines (HL-60, THP-1, K562, Kasumi-1, KG-1, KO52, and NKM-1), then confirmed duration of stable stainability. A proficiency test using external quality control materials was conducted at eleven institutions, which participated voluntarily. RESULTS: HL-60 and THP-1 cell lines, which showed good stainability among the seven cancer cell lines, were selected as external quality control materials. The stainability of a prepared cell line fixed on control slides was stable for 3–4 weeks (MPO, SBB, and PAS) or 9–10 weeks (ANAE). The stainability of paraffin-embedded control material for iron stain was stable for 3 months. The results from 11 institutions were the same on iron, MPO, SBB, and ANAE. Nevertheless, two of 10 institutes showed discrepant results on PAS. CONCLUSIONS: In this study, we demonstrated that cell lines could serve as a standard quality control material for special stains. Most institutions showed representative results on special stains except for PAS. This protocol for special stain may be useful as an external or internal quality control in a haematology laboratory.

Isolation and characterization of mesenchymal stem cells derived from umbilical cord blood / 대한산부인과학회지

OBJECTIVE: The purposes of this study were to isolate and characterize mesenchymal stem cells (MSCs) with osteogenic and adipogenic potential from umbilical cord blood (UCB). METHODS: MSCs were isolated using a density gradient centrifugation and extensive subcultivation from UCB. The proliferation capability, cell cycle, cytochemical markers, and immunophenotype of these MSCs were measured. The transcript of osteoblast-specific markers, alkaline phosphatase (ALP), and calcium deposit were analyzed from MSCs cultured in osteogenic media for 1-4 weeks, and MSCs exposed in adipogenic media were stained by Oil red after 4 weeks of culture. RESULTS: More than 84% of MSCs were in the G0/G1 phase of cell cycle. MSCs were positive for periodic acid-Schiff, and alpha-naphthyl acetate esterase activity, but negative for sudan black-B, and ALP activities. MSCs expressed CD13, CD29, CD 44, CD49e, CD51, CD54, CD 90, SH2, and SH3, but did not express CD11b, CD14, CD31, CD34, CD45, CD49d, CD106, CD133, CD144, CD146, CD163, CD166, and Stro-1. When MSCs were cultured in osteogenic media for 1-4 weeks, they expressed transcripts of osteoblastic specific markers: Runx-2, ALP, and procollagen type I. During culture of 2-4 weeks, ALP activity was detected and quantification increased significantly, and the deposition of a calcified matrix became evident. Exposure of MSCs to adipogenic media resulted in morphological change stained by Oil Red O. CONCLUSION: These data indicate that UCB contains MSCs with osteogenic and adipogenic differentiation potential, and may serve as an potential source of MSCs to be utilized in cell therapy for various diseases.

Isolation and characterization of mesenchymal stem cells derived from umbilical cord blood / 대한산부인과학회지

OBJECTIVE: The purposes of this study were to isolate and characterize mesenchymal stem cells (MSCs) with osteogenic and adipogenic potential from umbilical cord blood (UCB). METHODS: MSCs were isolated using a density gradient centrifugation and extensive subcultivation from UCB. The proliferation capability, cell cycle, cytochemical markers, and immunophenotype of these MSCs were measured. The transcript of osteoblast-specific markers, alkaline phosphatase (ALP), and calcium deposit were analyzed from MSCs cultured in osteogenic media for 1-4 weeks, and MSCs exposed in adipogenic media were stained by Oil red after 4 weeks of culture. RESULTS: More than 84% of MSCs were in the G0/G1 phase of cell cycle. MSCs were positive for periodic acid-Schiff, and alpha-naphthyl acetate esterase activity, but negative for sudan black-B, and ALP activities. MSCs expressed CD13, CD29, CD 44, CD49e, CD51, CD54, CD 90, SH2, and SH3, but did not express CD11b, CD14, CD31, CD34, CD45, CD49d, CD106, CD133, CD144, CD146, CD163, CD166, and Stro-1. When MSCs were cultured in osteogenic media for 1-4 weeks, they expressed transcripts of osteoblastic specific markers: Runx-2, ALP, and procollagen type I. During culture of 2-4 weeks, ALP activity was detected and quantification increased significantly, and the deposition of a calcified matrix became evident. Exposure of MSCs to adipogenic media resulted in morphological change stained by Oil Red O. CONCLUSION: These data indicate that UCB contains MSCs with osteogenic and adipogenic differentiation potential, and may serve as an potential source of MSCs to be utilized in cell therapy for various diseases.

metabolic activity of the adult rabbit choroid plexus: an enzymatic histochemical approach

To investigate the metabolic activity of the adult rabbit choroid plexus, using succinate dehydrogenase, phosphorylase and alpha-naphthylacetate esterase as histochemical markers of, the aerobic, glycolytic, and lipolytic pathways, respectively. Coverslip-mounted choroid plexus sections of adult rabbits were stained histochemically for the above enzymes. To characterize the esterase isoform[s], sections were incubated with various esterase modifiers before identification of the esterase activity. Sections of liver and kidney [controls] were simultaneously treated as for choroid plexus sections. Strong reactivity of the choroidal epithelium for both succinate dehydrogenase and esterase was readily detectable, while phosphorylase activity was virtually absent. In contrast to the B-isoform of esterase characteristically dominated the controls, the choroidal esterase activity was attributed mainly to C-isoform. The results suggest that the energy required for CSF formation by the adult choroid plexus is derived almost exclusively from aerobic oxidation, including fat metabolism. The high esterase activity in the choroid plexus, and in particular the unique pattern of the choroidal esterase versus the esterase of the controls, were interpreted to offer a potential target for future inhibitors of the energy of fat metabolism and thereby for CSF reduction

enzyme histochemical map of the septal area of the rat

The septal area occupies a key position between the limbic telencephalon and the nuclei of the diencephalon. Its physiological role and connections have recently gained much attention. To describe the pattern of distribution of alpha-naphthyl acetate esterases, succinate dehydrogenase and cytochrome oxidase in the septal area correlate the results with the main neural connections of this area. Serial fresh frozen coronal sections from the septal area of the brains of 40 adult male rats were stained for the above-mentioned three enzymes. Alpha-naphthyl acetate esterases demonstrated the highest reactivity in the perikarya of the medial septal division. In the lateral septal division, the enzyme reactivity was particularly high in the neuropil. Succinate dehydrogenase was mainly localized in the neuropil. The lateral septal division revealed the highest reactivity. The perikarya of the medial septal division demonstrated high reactivity in contrast with the neuropil. Cytochrome oxidase localization was comparable with that of succinate dehydrogenase with only minor differences. A schematic map for each enzyme was revealed to provide detailed base-line knowledge for future perturbation and pathological studies

Effect of toxocara canis and trichinella spiralis infection on the activity of the mitochondrial kynurenine transaminase, the cytosolic kynurenine hydrolase and the lysosomal a-naphthyl acetate esterases in the liver of mice

In this study, the role of Toxocara canis and Trichinella spiralis infection in changing enzymatic activity that included in tryptophan metabolism via kynurenine pathway, and their effects on the activity of the lysosomal enzyme alpha-naphthyl acetate esterases, was considered. Toxocara canis and Trichinella spiralis produce a significant inhibitory effect on the kynurenine hydrolase. They produce a significant increase in the activity of the free lysosomal alpha-naphthyl acetate esterases in liver. Trichinella spiralis produces a significant inhibitory effect on the kynurenine transaminase. Larvae of the Toxocara canis parasite can remain alive releasing their metabolites in the surrounding tissues for years. As a result of this fact, the inhibitory effect of the Toxocara canis infection on the kynurenine hydrolase led to the suggestion that the accumulated kynurenine may be metabolized to 3-hydroxy kynurenine which is considered as a potent carcinogen. Nematode Trichinella spiralis inhibits kynurenine transaminase and kynurenine hydrolase and may withdraw the tryptophan and metabolize it to serotonin as well as nematode Ascaridia galli which is capable to synthesize 5- hydroxytryptamine from tryptophan via 5-hydroxy tryptophan. The increase in the activity of alpha-naphthyl acetate esterases after infection with both Trichinella spiralis and Toxocara canis larvae may be due to the increased fragility and rupture of lysosomes of the liver cells leading to the leakage of this lysosomal enzymes. Also, the rise in the enzyme activity indicates tissue damage which was attributed mostly to immunological nature. Detection of alpha- naphthylacetate esterases activity in urine and blood of patients infected with these tissue nematodes for long period should not be ignored, also detection and determination of the potent carcinogen 3- hydroxy kynurenine in urine of those patients is of urgent need

Acute megakaryoblastic leukemia / 영남의대학술지

Acute megakaryoblastic leukemia is a rare and rapidly fatal disease characterized by proliferation of megakaryocyte series and atypical megakaryocytes in the bone marrow. Acute megakaryoblastic leukemia is suspicious when 1) megakaryocyte in peripheral blood, mixture of large and small mononuclear megakaryoblast in the bone marrow 2) cytoplasmic budding in blast 3) myelofibrosis (dense medullary overgrowth of reticulin fibers) 4) PAS (+), ANAE (+), SBB (−), peroxidase (−) and which is confirmed by platelet peroxidase oxidation on electron microscope or monoclonal antibody. A case of acute megakaryoblastic leukemia was studied morphologically and monoclonal antibody.

Caracterizaçäo morfológica e funcional de células aderentes de baço de camundongo / Morphological and functional characterization of adherent splenic cells of mice

A separaçäo, caracterizaçäo e ensaio funcional das células inflamatórias presentes no local de lesäo têm se tornado imperiosos no estudo de diversas doenças. Através da utilizaçäo de métodos histoquímicos para esterase e fosfatase ácida, bem como do Teste de Fagocitose e da coloraçäo pelo Giemsa, realizados nas células esplénicas de dez camundongos, foi possível se caracterizar bem os componentes do Sistema Fagocítico Mononuclear e distinguir os outros tipos de células presentes, além de permitir a quantificaçäo diferencial das mesmas