All-in-one centrifugal microfluidic device for size-selective circulating tumor cell isolation with high purity.

Circulating tumor cells (CTCs) have gained increasing attention owing to their roles in cancer recurrence and progression. Due to the rarity of CTCs in the bloodstream, an enrichment process is essential for effective target cell characterization. However, in a typical pressure-driven microfluidic system, the enrichment process generally requires complicated equipment and long processing times. Furthermore, the commonly used immunoaffinity-based positive selection method is limited, as its recovery rate relies on EpCAM expression of target CTCs, which shows heterogeneity among cell types. Here, we propose a centrifugal-force-based size-selective CTC isolation platform that can isolate and enumerate CTCs from whole blood within 30 s with high purity. The device was validated using the MCF-7 breast cancer cell line spiked in phosphate-buffered saline and whole blood, and an average capture efficiency of 61% was achieved, which is typical for size-based filtration. The capture efficiency for whole blood samples varied from 44% to 84% under various flow conditions and dilution factors. Under the optimized operating conditions, a few hundred white blood cells per 1 mL of whole blood were captured, representing a 20-fold decrease compared to those obtained using a commercialized size-based CTC isolation device. In clinical validation, normalized CTC counts varied from 10 to 60 per 7.5 mL of blood from gastric and lung cancer patients, yielding a detection rate of 50% and 38%, respectively. Overall, our CTC isolation device enables rapid and label-free isolation of CTCs with high purity, which should greatly improve downstream molecular analyses of captured CTCs.

A case of congenital central hypoventilation syndrome with PHOX2B gene mutation in a Korean neonate.

Congenital central hypoventilation syndrome (CCHS) is a life-threatening disorder with apnea and cyanosis during sleep requiring immediate endotracheal intubation during the first day of life. The PHOX2B gene has been identified as the major gene involved in CCHS. This is the first report of a Korean neonate with CCHS confirmed to have a PHOX2B mutation with expanded alleles containing 20 polyalanine repeats that is a relatively small number compared to previous cases. The patient required intermittent ventilator support during sleep only and did not suffer from any other disorders of the autonomic nerve system. He consistently needs ventilator support during sleep and remains alive. Analysis of PHOX2B gene is useful for diagnosis and appropriate therapeutic intervention of CCHS patients.

Analysis of Women's Health Online News Articles Using Topic Modeling.

OBJECTIVES: This research aimed to understand the popularity of topics in the field of women's health through analysis of online news articles which were chronologically classified and examined to determine how women's health and diseases had changed over time. METHODS: Women's health and disease news articles were collated from a popular news website between 1993 to 2015 and preprocessed using gynecological medical terminology, Korean words and nouns (excluding general nouns not related to women's healthcare topics). The resultant articles (N = 7,710) were analyzed using the Latent Dirichlet Allocation algorithm and major topics were extracted. Topic trends were analyzed by year and period for women's health. RESULTS: It was observed that most of the women's health articles were focused on "Healthcare", and 9 other topics were identified that represented a relatively small proportion in 1993-2000. In 2001-2005, most of the articles were focused on "Medical Services" and "Dietary Supplements" with some specific topics that peaked people's interest, as compared to those focused on "Healthcare" in the 1990s. It was also observed that differences in the proportion of each topic was small after 2011. CONCLUSION: Changes in topics related to women's disease were not clearly distinguished in the 1990s but this changed from 2001where articles related to "women disease" appeared as articles on the topics of various diseases.

Evaluation of the Sysmex UF-100 urine cell analyzer as a screening test to reduce the need for urine cultures for community-acquired urinary tract infection.

We evaluated the UF-100 flow cytometer (TOA Medical Electronics, Kobe, Japan) as a screening test for community-acquired urinary tract infection (UTI) to reduce the need for bacterial cultures. By comparing the test results for 330 urine samples with quantitative urine cultures, we established cutoff criteria for the UF-100. To rule out hospital-acquired UTI, all urine samples were from new patients who had not been admitted to a hospital within the previous month. Abacterial cutoff value of 3,000/microL provided the best discrimination for community-acquired UTI, with a sensitivity of 94.4% and a specificity of 73.4%compared with urine culture. It was possible to forgo 58.2% of cultures with only 4 false-negative results. With a bacterial cutoff value of 1,500/microL, the sensitivity improved to 100%, but the specificity declined to 49.8%, and only 38.5% of cultures could be avoided without any false-negative results. Screening with the UF-100 for community-acquired UTI is acceptable for routine use. It would improve the efficiency of the routine microbiology laboratory, and unnecessary antibiotic prescriptions could be reduced.

Instability of Plasma and Serum Progastrin-Releasing Peptide During Repeated Freezing and Thawing.

OBJECTIVES: Progastrin-releasing peptide (proGRP) is a promising biomarker for small cell lung cancer. However, not much is known about how sample processing and storage conditions affect the stability of proGRP. Here, we examined the effects of repeated freeze-thaw cycles on the stability of proGRP in plasma and serum. METHODS: Concentrations of proGRP were measured in plasma and serum samples exposed to two, three, or four freeze-thaw cycles and these were compared with values of corresponding samples exposed to one cycle (baseline). We also performed the area under the receiver-operating-characteristic curve (AUC) analysis to determine whether the differences of proGRP concentrations between each paired plasma and serum sample (ΔproGRP) can be used for identifying the samples that have been exposed to multiple freeze-thaw cycles. RESULTS: Concentrations of proGRP gradually decreased in both plasma and serum samples with increasing numbers of freeze-thaw cycles. Reduction rates of proGRP concentrations were greater in serum than in plasma samples and serum proGRP levels declined with statistical significance (p < 0.001) up to 10.1% after four freeze-thaw cycles. The ΔproGRP measurement showed fair accuracy (AUC = 0.741) for identifying samples that had been through four freeze-thaw cycles. The sensitivity was 82.8% and specificity was 62.1% at an optimal cut-off point of > 4.9. CONCLUSION: Our study shows that the stability of circulating proGRP is affected in both plasma and serum samples by repeated freezing and thawing. We also show that ΔproGRP could be used for identifying paired plasma and serum samples subjected to multiple freeze-thaw cycles.

Effect of Repeated Freezing and Thawing on Biomarker Stability in Plasma and Serum Samples.

OBJECTIVES: The stability of circulating proteins can be affected by repeated freezing and thawing. The aim of our study was to identify the effect of repeated freezing and thawing on the plasma and serum concentrations of eight proteins [interferon-γ, interleukin (IL)-8, IL-15, IL-17A, matrix metalloproteinase (MMP)-7, tumor necrosis factor-α, vascular endothelial growth factor (VEGF), and VEGF receptor 2 (VEGF-R2)]. METHODS: We assessed the concentration changes of these proteins in 30 plasma and serum samples subjected to three, four, or five freeze-thaw cycles, and compared these with the concentration changes in the samples that were subjected to two freeze-thaw cycles before analysis. RESULTS: Repeated freezing and thawing by up to five cycles did not modify the plasma and serum concentrations of interferon-γ, IL-8, and VEGF-R2, while levels of MMP-7, tumor necrosis factor-α, and VEGF were significantly changed in both plasma and serum samples. Moreover, MMP-7 and VEGF concentrations tended to increase with freeze-thaw cycles. They were more elevated in plasma samples (up to about 15%) than in serum samples (up to about 7%), suggesting that serum is the preferred sample type for the analysis of circulating proteins. CONCLUSION: This is the first report on the effect of repeated freezing and thawing on plasma concentrations of MMP-7 and VEGF-R2. Our findings propose that researchers should consider the number of freeze-thaw cycles to select plasma or serum samples, depending on the type of analyte.

Differential expression levels of plasma-derived miR-146b and miR-155 in papillary thyroid cancer.

BACKGROUND: Specific circulating microRNAs (miRNAs) in each organ may contribute to the diagnosis and prognosis in some cancers. miRNA from papillary thyroid cancer (PTC) may be released into the bloodstream. This study was performed to detect miRNAs in the plasma and estimate their diagnostic usefulness for discriminating between benign and malignant lesions. METHODS: Patients who underwent thyroidectomy for benign thyroid nodules or PTC were enrolled in this study. The patients were divided into three groups: benign, PTC without lymph node metastasis (LNM), and PTC with LNM. The levels of miR-146b, miR-221, miR-222, and miR-155miRNA expression in blood samples before surgery were evaluated. RESULTS: Of 89 patients enrolled in this study, 19 and 70 had benign lesions (21.3%) and PTC (78.7%), respectively. The mean levels of miR-146b and miR-155 expression were higher in the PTC group than the benign group. For discrimination between benign and PTC lesions, the area under the ROC curve (AUC) for miR-146b was 0.649 with 61.4% sensitivity and 57.9% specificity. The AUC for miR-155 was 0.695 with 74.3% sensitivity and 63.2% specificity (P<0.05). The levels of miR-146b, miR-221, and miR-222 were slightly higher in the N1 group than the N0 group. The levels of miR-146b, miR-155, and miR-222 increased in proportion to tumor size. CONCLUSIONS: miR-146b and miR-155 helped to discriminate between benign and malignant lesions. Circulating miRNA is likely a useful alternate serological marker for PTC. This preliminary study suggested that circulating miRNAs may be useful as follow-up tools as well as diagnostic tools.

Clinical relevance of high-resolution single nucleotide polymorphism array in patients with relapsed acute lymphoblastic leukemia with normal karyotype: a report of three cases.

We report three patients with normal karyotype (NK) ALL, who showed genetic aberrations as determined by high-resolution single nucleotide polymorphism array (SNP-A) analysis at both diagnosis and relapse. We evaluated the clinical relevance of the SNP-A assay for the detection of subtle changes in the size of affected genetic lesions at relapse as well as the prognostic value of the assay. In our patients, application of the SNP-A assay enabled sensitive detection of cryptic changes affecting clinically important genes in NK ALL. Therefore, this assay seems to be more advantageous compared to other conventional methods such as FISH assay, HemaVision (DNA Technology, Denmark), and conventional karyotyping for the detection of an "unstable genotype" at relapse, which may be associated with microscopic clonal evolution and poor prognosis. Further comprehensive studies are required to confirm the issues presented by our case patients in this report.

Incidence, clinical features, and prognostic impact of CALR exon 9 mutations in essential thrombocythemia and primary myelofibrosis: an experience of a single tertiary hospital in Korea.

We evaluated the incidence, clinical characteristics, and prognostic impact of calreticulin (CALR) mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. In all, 48 ET and 14 PMF patients were enrolled, and the presence of CALR mutations was analyzed by direct sequencing. Patients were classified into three subgroups according to Janus kinase 2 (JAK2) V617F and CALR mutation status, and their clinical features and prognosis were compared. CALR mutations were detected in 15 (24.2%) patients, and the incidence increased to 50.0% in 30 JAK2 V617F mutation-negative cases. These included 11 patients with three known mutations (c.1092_1143del [seven cases], c.1154_1155insTTGTC [three cases], and c.1102_1135del [one case]) and 4 patients with novel mutations. ET patients carrying CALR mutation were younger, had lower white blood cell counts, and experienced less thrombosis during follow-up than those carrying JAK2 V617F mutation, while both patient groups showed similar clinical features and prognosis. In ET patients without JAK2 V617F mutation, CALR mutation did not significantly affect clinical manifestation and prognosis. In conclusion, CALR mutation analysis could be a useful diagnostic tool for ET and PMF in 50% of the cases without JAK2 V617F mutations. The prognostic impact of CALR mutations needs further investigation.

Incidence and prognostic impact of DNMT3A mutations in Korean normal karyotype acute myeloid leukemia patients.

BACKGROUND: DNA methyltransferase 3A (DNMT3A) mutation was recently introduced as a prognostic indicator in normal karyotype (NK) AML and we evaluated the incidence and prognostic impact of DNMT3A mutations in Korean NK AML patients. METHODS: Total 67 NK AML patients diagnosed during the recent 10 years were enrolled. DNMT3A mutations were analyzed by direct sequencing and categorized into nonsynonymous variations (NSV), deleterious mutations (DM), and R882 mutation based on in silico analysis results. Clinical features and prognosis were compared with respect to DNMT3A mutation status. RESULTS: Three novel (I158M, K219V, and E177V) and two known (R736H and R882H) NSVs were identified and the latter three were predicted as DMs. DNMT3A NSVs, DMs, and R882 mutation were identified in 14.9%-17.9%, 10.3%-10.4%, and 7.5% of patients, respectively. DNMT3A mutations were frequently detected in FLT3 ITD mutated patients (P=0.054, 0.071, and 0.071 in NSV, DMs, and R882 mutation, resp.) but did not affect clinical features and prognosis significantly. CONCLUSIONS: Incidences of DNMT3A NSVs, DMs, and R882 mutation are 14.9%-17.9%, 10.3%-10.4%, and 7.5%, respectively, in Korean NK AML patients. DNMT3A mutations are associated with FLT3 ITD mutations but do not affect clinical outcome significantly in Korean NK AML patients.

Antifungal susceptibilities of bloodstream isolates of Candida species from nine hospitals in Korea: application of new antifungal breakpoints and relationship to antifungal usage.

We applied the new clinical breakpoints (CBPs) of the Clinical and Laboratory Standards Institute (CLSI) to a multicenter study to determine the antifungal susceptibility of bloodstream infection (BSI) isolates of Candida species in Korea, and determined the relationship between the frequency of antifungal-resistant Candida BSI isolates and antifungal use at hospitals. Four hundred and fifty BSI isolates of Candida species were collected over a 1-year period in 2011 from nine hospitals. The susceptibilities of the isolates to four antifungal agents were determined using the CLSI M27 broth microdilution method. By applying the species-specific CBPs, non-susceptibility to fluconazole was found in 16.4% (70/428) of isolates, comprising 2.6% resistant and 13.8% susceptible-dose dependent isolates. However, non-susceptibility to voriconazole, caspofungin, or micafungin was found in 0% (0/370), 0% (0/437), or 0.5% (2/437) of the Candida BSI isolates, respectively. Of the 450 isolates, 72 (16.0%) showed decreased susceptibility to fluconazole [minimum inhibitory concentration (MIC) ≥4 µg/ml]. The total usage of systemic antifungals varied considerably among the hospitals, ranging from 190.0 to 7.7 defined daily dose per 1,000 patient days, and fluconazole was the most commonly prescribed agent (46.3%). By Spearman's correlation analysis, fluconazole usage did not show a significant correlation with the percentage of fluconazole resistant isolates at hospitals. However, fluconazole usage was significantly correlated with the percentage of fluconazole non-susceptible isolates (r = 0.733; P = 0.025) or the percentage of isolates with decreased susceptibility to fluconazole (MIC ≥4 µg/ml) (r = 0.700; P = 0.036) at hospitals. Our work represents the first South Korean multicenter study demonstrating an association between antifungal use and antifungal resistance among BSI isolates of Candida at hospitals using the new CBPs of the CLSI.

Early detection of mycobacteria using a novel hydrogel culture method.

BACKGROUND: Early laboratory detection of Mycobacterium tuberculosis is crucial for controlling tuberculosis. We developed a hydrogel mycobacterial culture method that retains the advantages of both solid and liquid methods in terms of speed, cost, and efficiency. METHODS: Mycobacterium bovis bacillus Calmette-Guérin (BCG) suspensions and 200 acid-fast bacilli (AFB)-positive clinical specimens were inoculated in Middlebrook 7H9 liquid media (Becton-Dickinson and Company, USA) and mixed with 75 µL of 9-fluorenylmethoxycarbonyl (Fmoc)-Phe-Phe-OH hydrogel stock solution in an Eppendorf tube just before culture incubation. The mixtures were cultured at 37℃ for as long as 14 days to monitor culture status. RESULTS: The number of M. bovis BCG increased with time. For 200 AFB smear-positive specimens, 155 of 158 conventional culture-positive specimens and 4 culture-negative or contaminated specimens yielded positive cultures within 14 days. For 128 specimens positive with the liquid culture method, the time to positive culture using the hydrogel method (mean, 12.6 days; range, 7 to 14 days) was significantly shorter than that for conventional liquid culture (mean, 16.2 days; range, 6 to 31 days; P<0.0001). CONCLUSIONS: The hydrogel scaffold culture system is useful for timely, economical, and efficient detection of mycobacteria in clinical specimens.

Customer satisfaction survey with clinical laboratory and phlebotomy services at a tertiary care unit level.

We performed customer satisfaction surveys for physicians and nurses regarding clinical laboratory services, and for outpatients who used phlebotomy services at a tertiary care unit level to evaluate our clinical laboratory and phlebotomy services. Thus, we wish to share our experiences with the customer satisfaction survey for clinical laboratory and phlebotomy services. Board members of our laboratory designed a study procedure and study population, and developed two types of questionnaire. A satisfaction survey for clinical laboratory services was conducted with 370 physicians and 125 nurses by using an online or paper questionnaire. The satisfaction survey for phlebotomy services was performed with 347 outpatients who received phlebotomy services by using computer-aided interviews. Mean satisfaction scores of physicians and nurses was 58.1, while outpatients' satisfaction score was 70.5. We identified several dissatisfactions with our clinical laboratory and phlebotomy services. First, physicians and nurses were most dissatisfied with the specimen collection and delivery process. Second, physicians and nurses were dissatisfied with phlebotomy services. Third, molecular genetic and cytogenetic tests were found more expensive than other tests. This study is significant in that it describes the first reference survey that offers a survey procedure and questionnaire to assess customer satisfaction with clinical laboratory and phlebotomy services at a tertiary care unit level.

Misidentification of Candida guilliermondii as C. famata among strains isolated from blood cultures by the VITEK 2 system.

Introduction. The aim of this study was to differentiate between Candida famata and Candida guilliermondii correctly by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and gene sequencing. Methods. Twenty-eight Candida strains from blood cultures that had been identified as C. famata (N = 25), C. famata/C. guilliermondii (N = 2), and C. guilliermondii (N = 1) by the VITEK 2 system using the YST ID card were included. We identified these strains by MALDI-TOF MS and gene sequencing using the 28S rRNA and ITS genes and compared the results with those obtained by the VITEK 2 system. Results. All 28 isolates were finally identified as C. guilliermondii. Sequencing analysis of the 28S rRNA gene showed 99.80%-100% similarity with C. guilliermondii for all 28 strains. The ITS gene sequencing of the strains showed 98.34%-100% homology with C. guilliermondii. By MALDI-TOF, we could correctly identify 21 (75%) of 28 C. guilliermondii isolates. Conclusion. We should suspect misidentification when C. famata is reported by the VITEK 2 system, and we always should keep in mind the possibility of misidentification of any organism when an uncommon species is reported.

Bacteroides fragilis vertebral osteomyelitis complicated by percutaneous epidural adhesiolysis.

STUDY DESIGN: A case report of anaerobic vertebral osteomyelitis after percutaneous epidural adhesiolysis. OBJECTIVE: To present a case of Bacteroides fragilis spondylodiscitis (BFS) secondary to percutaneous epidural adhesiolysis in a 38-year-old woman without predisposing factors. SUMMARY OF BACKGROUND DATA: Most cases of BFS result from hematogenous spread from a perianal abscess or sigmoidoscopy or local spread from an adjacent infection. However, BFS due to direct inoculation after percutaneous epidural adhesiolysis has not been previously reported. METHODS: A 38-year-old woman presented with spondylodiscitis at the L4-L5 level 2 weeks after percutaneous epidural adhesiolysis. Despite empirical antibiotherapy, the spondylodiscitis and an epidural abscess became much aggravated. Open biopsy and curettage was performed, and metronidazole sensitive Bacteroides fragilis was identified by tissue culture. RESULTS: Metronidazole was administrated for 5 weeks and symptoms were completely resolved. Follow-up magnetic resonance imaging showed that the spondylodiscitis was completely cured. CONCLUSION: This is the first report to be issued regarding BFS secondary to percutaneous epidural adhesiolysis. In our case, the pathogenesis may have been direct inoculation of Bacteroides fragilis into the epidural space and disc during percutaneous epidural adhesiolysis because the procedural approach used was adjacent to the anus.

The first Korean case of Sphingobacterium spiritivorum bacteremia in a patient with acute myeloid leukemia.

Sphingobacterium spiritivorum has been rarely isolated from clinical specimens of immunocompromised patients, and there have been no case reports of S. spiritivorum infection in Korea to our knowledge. We report a case of S. spiritivorum bacteremia in a 68-yr-old woman, who was diagnosed with acute myeloid leukemia and subsequently received chemotherapy. One day after chemotherapy ended, her body temperature increased to 38.3℃. A gram-negative bacillus was isolated in aerobic blood cultures and identified as S. spiritivorum by an automated biochemical system. A 16S rRNA sequencing analysis confirmed that the isolate was S. spiritivorum. The patient received antibiotic therapy for 11 days but died of septic shock. This is the first reported case of human S. spiritivorum infection in Korea. Although human infection is rare, S. spiritivorum can be a fatal opportunistic pathogen in immunocompromised patients.

Validation of circulating miRNA biomarkers for predicting lymph node metastasis in gastric cancer.

We validated candidate biomarkers using circulating miRNAs by analyzing serum miRNA concentrations from patients with gastric cancer (GC) to predict lymph node (LN) metastasis. In a pilot study, serum levels of miR-21, miR-27a, miR-106b, miR-146a, miR-148a, miR-223, and miR-433 were compared in 10 healthy donors, 16 LN-positive patients with GC, and 15 LN-negative patients with GC. Then, we compared the level of three miRNAs (miR-21, miR-146a, and miR-148a) with the total of 79 GC patients with or without LN metastasis. In the pilot study, miR-21, miR-27a, miR-106b, miR-146a, miR-148a, and miR-223 concentrations from LN-positive patients with GC were significantly different from those of LN-negative patients with GC (P < 0.001, P = 0.003, P = 0.033, P < 0.001, P <0.001, and P = 0.017, respectively). In the validation study, levels of miR-21, miR-146a, and miR-148a increased as pN stage increased (P < 0.001, P = 0.001, and P < 0.001, respectively). Levels of the miRNAs were significantly different between pN0 and pN0 in the pT1 group (P = 0.013, P = 0.004, and P = 0.035, respectively) and among clinical stages (P = 0.001, P = 0.002, and P < 0.001, respectively). No differences in miRNA levels were observed by pT stage, Lauren's classification, sex, or age. Serum concentrations of miR-21, miR-146a, and miR-148a were closely associated with GC pN stage. These serum miRNA levels could be biomarker candidates to predict the presence of LN metastasis.

Comparison of Knowledge Levels Required for SNOMED CT Coding of Diagnosis and Operation Names in Clinical Records.

OBJECTIVES: Coding Systematized Nomenclature of Medicine, Clinical Terms (SNOMED CT) with complex and polysemy clinical terms may ask coder to have a high level of knowledge of clinical domains, but with simpler clinical terms, coding may require only simpler knowledge. However, there are few studies quantitatively showing the relation between domain knowledge and coding ability. So, we tried to show the relationship between those two areas. METHODS: We extracted diagnosis and operation names from electronic medical records of a university hospital for 500 ophthalmology and 500 neurosurgery patients. The coding process involved one ophthalmologist, one neurosurgeon, and one medical record technician who had no experience of SNOMED coding, without limitation to accessing of data for coding. The coding results and domain knowledge were compared. RESULTS: 705 and 576 diagnoses, and 500 and 629 operation names from ophthalmology and neurosurgery, were enrolled, respectively. The physicians showed higher performance in coding than in MRT for all domains; all specialist physicians showed the highest performance in domains of their own departments. All three coders showed statistically better coding rates in diagnosis than in operation names (p < 0.001). CONCLUSIONS: Performance of SNOMED coding with clinical terms is strongly related to the knowledge level of the domain and the complexity of the clinical terms. Physicians who generate clinical data can be the best potential candidates as excellent coders from the aspect of coding performance.

Progastrin-releasing peptide is a candidate marker for quality control in clinical sample processing and storage.

For clinical epidemiologic and proteomic studies, the control of preanalytic variation, including sample processing and storage, is important. We evaluated the stability of progastrin-releasing peptide (ProGRP) as a marker for the quality control of stored serum and plasma samples. The ProGRP from 23 healthy volunteers was measured serially for 8 hours at room temperature, and the results were validated with clinical samples from the biobank. A significant difference in ΔProGRP was also noted between good-quality (time delay <4 hours before storage) and poor-quality (time delay ≥4 hours before storage) specimens (mean ± SD, 0.17 ± 0.08 vs 0.36 ± 0.14; P < .001; Wilcoxon signed-rank test). With a ΔProGRP cutoff of 0.22, the sensitivity and specificity of detection of the poor-quality samples were 85.7% and 75.0%, respectively, in clinical validations. We demonstrated that ΔProGRP could be used as a marker for quality control in sample processing and storage in biobanks.