Chunhwa Ihm

Evaluation of reticulocyte haemoglobin content as marker of iron deficiency and predictor of response to intravenous iron in haemodialysis patients

Because serum ferritin and transferrin saturation (TS) have a limitation in estimating iron status in haemodialysis patients, the reticulocyte haemoglobin content (CHr) has been proposed as a new tool. We investigate the accuracy of CHr in comparison with conventional tests and the relationship between changes in CHr and haemoglobin levels after therapy. We selected 140 haemodialysis patients receiving rHuEPO and intravenous iron supplementation and measured their complete blood count, CHr and iron parameters. Iron deficiency was defined as a ferritin <100 μg/l and/or a TS <20%. Hb, CHr, ferritin and TS levels were determined 1 month after therapy. Fifty‐three patients were iron deficient. CHr were distributed with 33.7 ± 1.4 pg in the iron sufficient group and with 29.9 ± 1.9 pg in the iron deficient group (P = 0.001). The cutoff value of CHr for detecting iron deficiency was <32.4 pg. In iron deficient patients, a significant correlation was found between CHr and TS. The change in CHr after therapy was significantly larger in iron‐deficient patients, and a lower baseline CHr is associated with a greater haemoglobin change. CHr is useful in screening iron status in dialysis patients, and a CHr cut‐off value of 32 pg is appropriate for the assessment of iron deficiency. Moreover, CHr may serve as a predictor of the response to anaemia treatment.

SERPINE1 Intron Polymorphisms Affecting Gene Expression Are Associated With Diffuse-Type Gastric Cancer Susceptibility

A primary inhibitor of plasminogen activators, SERPINE1 (serpin peptidase inhibitor 1, clade E, member 1, also known as plasminogen activator inhibitor type 1), is an important regulator in tumorigenesis and is highly expressed in many cancers.

Exosome cargo reflects TGF-β1-mediated epithelial-to-mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells.

It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-β1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-β1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of β-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling.

The usefulness of multiplex PCR for the identification of bacteria in joint infection

Background: The diagnosis of septic arthritis (SA) relies on synovial analysis and conventional culture. But, these methods lack of sensitivity and culture is time consuming to establish a definite diagnosis. This study evaluated a new multiplex PCR assay which entailed screening PCR for Gram typing and identification PCR for species identification using two primer mixes. Methods: A total of 80 synovial fluid samples from patients with suspected SA were collected. Culture, multiplex PCR, and 16S rRNA gene PCR were performed. Results: The analytical sensitivity of multiplex PCR assay was 101 CFU/ml for each type of bacteria. There was no cross‐reactivity with common bacterial pathogens. Bacteria were detected in 20, 25, and 26 of 80 samples for culture, multiplex PCR, and 16S rRNA gene PCR, respectively. Nineteen (95%) of 20 culture‐positivesamples and 6 (10%) of 60 culture‐negative samples were positive for the multiplex PCR. Five of six samples which were positive only from multiplex PCR were also positive in 16S rRNA gene PCR. The multiplex PCR showed 2 false‐negative in 27 true‐positive samples but no false‐positive. The sensitivity and specificity of the multiplex PCR were 92.6 and 100%, and the agreement with culture and 16S rRNA gene PCR were 91.3 and 96.3%, respectively. The time to detection for multiplex PCR was a maximum of 6 hr. Conclusion: This multiplex PCR assay offers high sensitivity and improved detection speed relative to culture. The appropriate combination of this new multiplex PCR assay with culture may contribute to the accurate and rapid diagnosis of SA. J. Clin. Lab. Anal. 24:175–181, 2010.

Rapid DNA Extraction from Dried Blood Spots on Filter Paper: Potential Applications in Biobanking

Objectives Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper. Methods The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40 μL blood was used. Results DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening. Conclusion Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage.

The M142T Mutation Causes B3 Phenotype: Three Cases and an in vitro Expression Study

The B3 phenotype is the most common B subtype in Korea. The B305 allele (425 T>C, M142T) was first reported in 2 Chinese individuals; however, it has not yet been reported in the Koreans, and the impact of the M142T mutation on the expression of the B3 phenotype has also not been studied. To resolve an ABO discrepancy between a group O neonate and her group O father and A(1)B(3) mother, blood samples from these individuals and other family members were referred to our laboratory for ABO gene analysis. The B305 allele was discovered in the neonate (B305/O01), her mother (A102/ B305), and her maternal aunt (B305/O02), while her father was typed as O01/O02. Transient transfection experiments were performed in HeLa cells using the B305 allele synthesized by site-directed mutagenesis; flow cytometric analysis revealed that this transfect expressed 35.5% of the total B antigen produced by the B101 allele transfect. For comparison, Bx01 allele transfects were also created, and they expressed 11.4% of the total B antigen expressed on the surface of B101 transfects. These experiments demonstrate that the M142T (425 T>C) mutation is responsible for the B subtype phenotype produced by the B305 allele.

Fabrication of a membrane filter with controlled pore shape and its application to cell separation and strong single cell trapping

A porous membrane filter is one of the key components for sample preparation in lab-on-a-chip applications. However, most of the membranes reported to date have only been used for size-based separation since it is difficult to provide functionality to the membrane or improve the performance of the membrane. In this work, as a method to functionalize the membrane filter, controlling the shape of the membrane pores is suggested, and a convenient and mass-producible fabrication method is provided. With the proposed method, membrane filters with round, conical and funnel shape pores were successfully fabricated, and we demonstrated that the sidewall slope of the conical shape pores could be precisely controlled. To verify that the membrane filter can be functionalized by controlled pore shape, we investigated filtration and trapping performance of the membrane filter with conical shape pores. In a filtration test of 1000 cancer cells (MCF-7, a breast cancer cell line) spiked in phosphate buffered saline (PBS) solution, 77% of the total cancer cells were retained on the membrane, and each cell from among 99.3% of the retained cells was automatically isolated in a single conical pore during the filtration process. Thanks to its engineered pore shape, trapping ability of the membrane with conical pores is dramatically improved. Microparticles trapped in the conical pores maintain their locations without any losses even at a more than 30 times faster external flow rate com-pared with those mounted on conventional cylindrical pores. Also, 78% of the cells trapped in the conical pores withstand an external flow of over 300 μl min−1 whereas only 18% of the cells trapped in the cylindrical pores remain on the membrane after 120 μl min−1 of an external flow is applied.

The p.R168Q mutation is associated with the Bw phenotype and a predicted decrease in the stability of the resulting ABO glycosyltransferase

Mutation of ABO glycosyltransferase (GT) can cause protein stability changes that can result in a weak ABO phenotype. To explain the Bw phenotype of a novel ABO*Bw allele, a protein stability of the mutant GT, which enhances the information of the three‐dimensional (3D) structural analysis, was calculated.

A pocket-sized colorimetric urine reader for telemedicine in the developing countries

We have proposed a novel handheld healthcare platform, combining a pocket-sized colorimetric reader (13.5 × 6.5 × 2.5 cm3) and commercially available 10-parameter urinalysis paper strips (glucose, protein, glucose, bilirubin, urobilinogen, ketones, nitrite, pH, specific gravity, erythrocytes, and leukocytes), capable of sending data with a smart phone (Samsung electronics co., Omnia 2) using a window mobile operating system. The reader includes a novel colorimetric multi-detection module, which consists of three-chromatic light-emitting diodes (LED), silicon photodiodes (SPDs) and a novel poly(methylmethacrylate) (PMMA) optical splitter (POS). Data reading methods using conversions of the signal data (red, blue, and green) to the hue color map or the Y model data are utilized, and a curve-fitting method for the quantification is employed. The reader is battery-powered, inexpensive, light-weighing, and very speedy in analysis. And, it was applied to analyzing of a thousand of human urine samples in the Eulji university hospital and demonstrated reliable quantification of urinary glucose and protein. The features can be used by unskilled people on-site to transfer the analyzed data to experts off-site.

Sigma-Metrics of Electrolyte Tests From a Recently Launched New-Generation Proficiency Testing Program of the Korean Association of Quality Assurance for Clinical Laboratory

Hee Jin Huh, M.D.*, Yun Mi Park, M.D.*, Seungok Lee, M.D., Chunhwa Ihm, M.D., Soyeon Seo, M.D., Sang Gon Lee, M.D., Joonseok Park, M.D., and Hae-il Park, M.D. Department of Laboratory Medicine, Dongguk University Ilsan Hospital, Goyang; Siemens Healthineers, Seoul; Department of Laboratory Medicine, Incheon St. Mary’s Hospital, The Catholic University of Korea, Incheon; Department of Laboratory Medicine, School of Medicine, Eulji University, Daejeon; Samkwang Medical Laboratories, Seoul; Green Cross Laboratories, Yongin; Hanmi Medicare Inc., Seoul; Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea