Molecular Analysis of Eight American Type Culture Collection Gonococcal Strains by Neisseria gonorrhoeae Multiantigen Sequence Typing and PorB Sequence Typing / 임상검사와정도관리

BACKGROUND: Molecular epidemiological typing of Neisseria gonorrhoeae is crucial for monitoring the spread of resistant strains. As reference strains can be used for laboratory internal quality control, we genetically characterised the American Type Culture Collection (ATCC) gonococcal strains by Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) and porB sequence typing using public multilocus sequence typing (PubMLST). METHODS: Eight ATCC gonococcal reference strains (ATCC 19424, ATCC 31426, ATCC 35541, ATCC 43069, ATCC 43070, ATCC 49226, ATCC 49926, and ATCC 49981) from Culti-Loops (Thermo Fisher Scientific, USA) were cultured. After DNA extraction, porB and tbpB were amplified and sequenced. Sequence types (STs) and allele numbers were each determined by NG-MAST (http://www.ng-mast.net) and porB sequence typing using PubMLST (http://pubmlst.org/neisseria/porB/). RESULTS: ATCC 19424 was identified as ST 266 by NG-MAST, and as Allele 946 by PubMLST. ATCC31426 was assigned a novel ST by NG-MAST, and was assigned Allele 958 with 1.2% mismatch by PubMLST. ATCC 35541 was identified as ST 12 by NG-MAST, and as Allele 624 by PubMLST. ATCC 43069 and ATCC 43070 were both identified as ST 681 by NG-MAST, and as Allele 984 by PubMLST. ATCC 49226 was identified as ST 1572 by NG-MAST, and as Allele 2110 by PubMLST. ATCC 49926 and ATCC 49981 were both identified as ST 16496 by NG-MAST, and as Allele 928 by PubMLST. CONCLUSIONS: The ST data obtained for ATCC gonococcal reference strains by NG-MAST and porB sequence typing using PubMLST can be used for quality assurance of molecular epidemiological typing in clinical microbiological laboratories.

Multiplex Assay of Second-Line Anti-Tuberculosis Drugs in Dried Blood Spots Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

As dried blood spots (DBSs) have various advantages over conventional venous blood sampling, some assays for detection of one or two anti-tuberculosis (TB) drugs in DBSs have been developed. However, there are no assays currently available for the simultaneous measurement of three or more anti-TB drugs in DBSs. In this study, we developed and evaluated a multiplex method for detecting nine anti-TB drugs including streptomycin, kanamycin, clarithromycin, cycloserine, moxifloxacin, levofloxacin, para-aminosalicylic acid, prothionamide, and linezolid in DBSs by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Seventy-nine patient samples of DBS were analyzed on the UPLC-MS/MS system. All drug concentrations were determined within 4 min, and assay performance was evaluated. All drugs were clearly separated without ion suppression. Within-run and between-run precisions were 1.7-13.0% and 5.7-17.0%, respectively, at concentrations representing low and high levels for the nine drugs. Lower limits of detection and quantification were 0.06-0.6 and 0.5-5.0 µg/mL, respectively. Linearity was acceptable at five level concentrations for each drug. Correlations between drug concentrations in plasma and DBSs by using Passing-Bablock regression and Pearson's rho (ρ, 0.798-0.989) were acceptable. In conclusion, we developed a multiplex assay to measure nine second-line anti-TB drugs in DBSs successfully. This assay provided convenient and rapid drug quantification and could have applications in drug monitoring during treatment.

Biochemical and Genetic Analysis of Seven Korean Individuals With Suspected Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by a deficiency in arylsulfatase A (ARSA). However, decreased ARSA activity is also observed in pseudodeficiency (PD). To distinguish between MLD and PD, we performed gene mutation and sulfatide analyses by using dried blood spots (DBSs) from seven Korean individuals who underwent an analysis of ARSA activity. DNA was extracted from DBSs, and PCR-direct sequencing of ARSA was performed. The cDNA obtained was analyzed to confirm a novel mutation. Of the seven subjects, three were confirmed as having MLD, one was confirmed as having MLD-PD, one was confirmed as having PD, and the remaining two were obligate heterozygotes. We verified the novel pathogenic variant c.1107+1delG by performing familial and cDNA analyses. Sulfatide concentrations in DBSs were analyzed and were quantified by using ultra-performance liquid chromatography and tandem mass spectrometry, respectively. Total sulfatide concentration was inversely correlated with ARSA activity (Spearman's coefficient of rank correlation, P=0.929, P=0.0025). The results of this mutational and biochemical study on MLD will increase our understanding of the genetic characteristics of MLD in Koreans.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Measurement of Leukocyte Arylsulfatase A Activity Using a Natural Substrate

No abstract available.

Proteomic Profiling of Serum from Patients with Tuberculosis

BACKGROUND: Effective treatment and monitoring of tuberculosis (TB) requires biomarkers that can be easily evaluated in blood samples. The aim of this study was to analyze the serum proteome of patients with TB and to identify protein biomarkers for TB. METHODS: Serum samples from 26 TB patients and 31 controls were analyzed by using nano-flow ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry in data-independent mode, and protein and peptide amounts were calculated by using a label-free quantitative approach. The generated data were analyzed by using principal component analysis and partial least squares discriminant analysis, a multivariate statistical method. RESULTS: Of more than 500 proteins identified, alpha-1-antitrypsin was the most discriminative, which was 4.4 times higher in TB patients than in controls. Peptides from alpha-1-antitrypsin and antithrombin III increased in TB patients and showed a high variable importance in the projection scores and coefficient in partial least square discriminant analysis. CONCLUSIONS: Sera from patients with TB had higher alpha-1-antitrypsin levels than sera from control participants. Alpha-1-antitrypsin levels may aid in the diagnosis of TB.

Quantification of Human Plasma-Busulfan Concentration by Liquid Chromatography-Tandem Mass Spectrometry

BACKGROUND: Busulfan, an alkylating agent administered prior to hematopoietic stem cell transplantation, has a narrow therapeutic range and wide variability in metabolism. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for rapid and accurate quantification of plasma busulfan. METHODS: Busulfan was separated and detected using an LC system containing a C18 column equipped with MS/MS. The sample was eluted with a mobile phase gradient for a total run time of 10 min. Plasma busulfan concentration was quantified against a 6-point standard curve in a multiple reaction monitoring mode at mass-to-charge (m/z) 264.1 > 151.1. Precision, recovery, matrix effect, linearity, detection capability, carryover, and stability were evaluated. The range of plasma busulfan concentration was obtained by analyzing samples from 9 children receiving busulfan. RESULTS: The coefficients of variation of within-run and within-laboratory precision were all below 5%. Recoveries were all within the range of 100-105%. Linearity was verified from 0 to 5,000 ng/mL. Limit of detection and limit of quantification were 1.56 and 25 ng/mL, respectively. Carryover rate was within allowable limits. Plasma busulfan concentration was stable for 2 weeks at -20degrees C and -80degrees C, but decreased by 25% when the plasma was stored for 24 hr at room temperature, and by <5% in 24 hr at 4degrees C. The plasma busulfan concentrations were between 347 ng/mL and 5,076 ng/mL. CONCLUSIONS: Our method using LC-MS/MS enables highly accurate, reproducible, and rapid busulfan monitoring with minimal sample preparation. The method may also enable safe and proper dosage.

Genotyping for RhD and RhCEce Antigens Using Free Circulating Nucleic Acids in Plasma and Serum / 대한수혈학회지

BACKGROUND: The Rh blood group includes several antigens, of which D, C, E, c, and e are clinically important. Although nucleic acids from whole blood can be used for Rh blood group genotyping, it is also possible to genotype free circulating fetal nucleic acids from plasma and serum. We performed Rh blood group phenotyping and genotyping using nucleic acids from whole blood and free circulating nucleic acids from plasma and serum, respectively. The results were compared. METHODS: Forty-four blood samples were phenotyped and genotyped for RhD and RhCE blood groups. Phenotyping was performed by hemagglutination assay. Further tests were performed on RhD-negative samples. Nucleic acids were extracted from whole blood, plasma, and serum. Plasma and serum were prepared after filtration and genotyped by real-time polymerase chain reaction. RESULTS: RhD blood group results showed one (2.3%) discrepant case in which the DEL phenotype appeared wild RHD genotype. Among nucleic acids, there were seven discrepant results: two from plasma and five from serum based on whole blood nucleic acids. RhCE blood group results showed three (6.8%) phenotype-genotype discordances. Among nucleic acids, seven (15.9%mpared to phenotypes. Kappa coefficients of serum were lower than those of plasma. CONCLUSION: RHD and RHCE genotype could be identified by assaying free circulating nucleic acids in plasma or serum. This study suggests that plasma is more reliable than serum as a specimen for RHD and RHCE genotyping of free circulating nucleic acids.

Evaluation of the T-KB-H and 3-HB Kits for the Measurement of Serum Ketone and beta-Hydroxybutyric Acid

BACKGROUND: Diabetes mellitus and alcohol consumption are the most common causes of ketoacidosis in adults. Recently, beta-hydroxybutyric acid (betaHBA) was reported to be a potential serum biomarker in the diagnosis and monitoring of ketoacidosis. We evaluated the performance of T-KB-H and 3-HB kits for the measurement of ketone bodies [acetoacetate (AcAc)+betaHBA] and betaHBA, respectively. METHODS: Quantitative enzymatic assays were performed using the T-KB-H and 3-HB kits (Nittobo Medical Co., Japan) and the Architect ci16200 Integrated System (Abbott Laboratories, USA). Simultaneously, the ketone body levels in these serum samples were determined by gas chromatography-mas spectrometry (GC-MS). We evaluated precision and linearity of these kits and correlation with GC-MS, and established reference intervals in children and adults. RESULTS: The coefficients of variation for the T-KB-H and 3-HB kits were less than 4.0% at analyte levels of 50, 100, and 400 micromol/L. Linearity was observed for AcAc and betaHBA over a 0-1,000 micromol/L range (R2<0.99). Results from the T-KB-H and 3-HB kits were in good agreement with those from the GC-MS analysis, with correlation coefficients of 0.94 for AcAc and 0.96 for betaHBA. Reference intervals determined for the T-KB-H kit were 9.8-270.1 micromol/L and 18.5-531.8 micromol/L in children and adults, respectively. For the 3-HB kit, the reference intervals were 6.4-234.0 micromol/L and 16.0-437.2 micromol/L in children and adults, respectively. CONCLUSIONS: The T-KB-H and 3-HB kits displayed good precision, clinically acceptable linearity, and reliable correlation with an established assay. This indicates that the kits can be used clinically for measuring serum ketone bodies.

Adoption of Donor Screening Policy in a Tissue Bank at a Tertiary Hospital / 대한수혈학회지

BACKGROUND: Tissues for transplantation can save lives or restore essential functions. According to national policies and regulations, access to suitable transplantation, as well as the level of safety, quality, efficacy of donation, and transplantation of tissues, differ significantly between countries. We reviewed a few guidelines on tissue banking from the aspect of screening tests. In addition, four-year experience with screening panels for donated bones and donors at a tertiary hospital is introduced. METHODS: Seven national and international guidelines for screening tests for donors and donated tissues were reviewed. At our institution, screening tests for donation involve two steps. At retrieval, the first screening panel, including ABO/Rh typing, unexpected antibody screening, VDRL, HBsAg, anti-HBs, anti-HBc IgM, anti-HCV, anti-HIV, and microbiological cultures was performed. The second screening panel, including the same tests, except culture studies, was performed after 90 days. From 2008 to 2011, a total of 245 retrievals of bone tissue were performed and the screening panel results were analyzed. RESULTS: Mandatory screening serologic tests for living donors can differ according to local law or regulation and/or screening for endemic diseases. At our institution, among 245 donated bones for a period of four years, 61 bone tissues were discarded due to noncompliance for the second screening (n=32), contamination or no culture study results (n=9), abnormal serologic test results (n=8), and so on. CONCLUSION: Donor screening policies for tissue banking are various according to national laws or endemic disease status. Second screening tests with consideration of the window period should be adopted.

Performance Evaluation of the CAPILLARYS 2 FLEX Piercing Analyzer for HbA1c Determination

BACKGROUND: The hemoglobin A1c (HbA1c) level is widely used to monitor glycemic control in diabetes mellitus patients, and various methods are used for its determination. The CAPILLARYS 2 FLEX Piercing (Sebia) is a fully automated, high-throughput glycohemoglobin (HbA1c) analyzer based on capillary electrophoresis. METHODS: The analytical performance of the CAPILLARYS 2 FLEX Piercing analyzer was evaluated for its precision, linearity, correlation with the Variant II Turbo (Bio-Rad Laboratories, Inc.) analyzer, and its vulnerability to interference by carbamylated hemoglobin. We also investigated its agreement with National Glycohemoglobin Standardization Program (NGSP) targets. All evaluations were performed according to CLSI guidelines EP05, EP06, and EP09. RESULTS: The coefficients of variation (CVs) for within-run and total imprecision were 1.7% and 1.8% at low concentrations and 1.2% and 1.3% at high concentrations, respectively. Linearity was excellent, with R2=0.9882 in the range of 5.13-13.83%; these results highly correlated with those produced by Variant II Turbo (R2=0.9978). The 95% confidence interval (for differences from the NGSP target) was -0.3618-0.3343%. No significant interference of carbamylated hemoglobin was noted. CONCLUSIONS: The CAPILLARYS 2 FLEX Piercing analyzer showed excellent precision and linearity. Its results correlated with those obtained by the Variant II Turbo analyzer, and were agreement with the NGSP target. Therefore, its analytical performance is satisfactory for diabetes diagnosis and treatment monitoring.

Performance Evaluation of the ADAMS A1c HA-8180 Analyzer for HbA1c / 임상검사와정도관리

BACKGROUND: Hemoglobin A1c (HbA1c) levels are widely used to monitor glycemic control in diabetes mellitus patients, and various methods are used for determining HbA1c levels. The ADAMS A1c HA-8180 (Arkray, Inc., Japan) is a fully automated HbA1c analyzer based on high-performance liquid chromatography (HPLC). METHODS: The analytical performance of the ADAMS A1c HA-8180 analyzer was evaluated on the basis of its precision, linearity, correlation with the Variant II Turbo (Bio-Rad Laboratories, USA), and agreement with the National Glycohemoglobin Standardization Program (NGSP) targets. All evaluations were performed according to Clinical and Laboratory Standard Institute (CLSI) guidelines EP05, EP06, and EP09. RESULTS: Coefficients of variation (CVs) for total precision at low and high levels were 0.99% and 1.16%, respectively. The linearity was excellent with R2 = 0.99 in the range of 4.98-15.10%. Its analytical performance was well correlated with that of Variant II Turbo (r = 0.9987). The 95% confidence interval of bias between the NGSP target and the levels measured using the ADAMS A1c HA-8180 was -0.402-0.225. CONCLUSIONS: The ADAMS A1c HA-8180 showed excellent precision, linearity, correlation with Variant II Turbo, and agreement with the NGSP target. Therefore, its analytical performance is satisfactory for diagnosis and treatment monitoring of diabetes.

Use of Tandem Mass Spectrometry for Newborn Screening of 6 Lysosomal Storage Disorders in a Korean Population / 대한진단검사의학회지

BACKGROUND: We evaluated the performance of multiplex tandem mass spectrometry (MS/MS) in newborn screening for detection of 6 lysosomal storage disorders (LSDs), namely, Niemann-Pick A/B, Krabbe, Gaucher, Fabry, and Pompe diseases and Hurler syndrome. METHODS: We revised the conditions and procedures of multiplex enzyme assay for the MS/MS analysis and determined the precision of our enzyme assay and the effects of sample amounts and incubation time on the results. We also measured the degree of correlation between the enzyme activities in the dried blood spots (DBSs) and those in the leukocytes. DBSs of 211 normal newborns and 13 newborns with various LSDs were analyzed using our revised methods. RESULTS: The intra- and inter-assay precisions were 2.9-18.7% and 8.1-18.1%, respectively. The amount of product obtained was proportional to the DBS eluate volume, but a slight flattening was observed in the product vs. sample volume curve at higher sample volumes. For each enzyme assay, the amount of product obtained increased linearly with the incubation period (range, 0-24 hr). Passing and Bablok regression analysis revealed that the enzyme activities in the DBSs and those in the leukocytes were favorably correlated. The enzyme activities measured in the DBSs were consistently lower in patients with LSDs than in normal newborns. CONCLUSIONS: The performance of our revised techniques for MS/MS detection and enzyme assays was of the generally acceptable standard. To our knowledge, this is the first report on the use of MS/MS for newborn screening of LSDs in an Asian population.

Serum Ferritin Levels for Normal Hemoglobin Blood Donors in a Korean Population / 대한수혈학회지

BACKGROUND: Serum ferritin is well known as a marker that is reflective of the iron storage status in blood. But only the hemoglobin level is generally included in the current selection criteria for blood donors. Recent studies have continuously shown that the serum ferritin levels are lower for the blood donors, and especially for women and repeat donors with a normal hemoglobin level. It has been suggested that serum ferritin should be included in the blood donor selection criteria. In this study, we examined the serum ferritin status in a Korean population, and we checked the validity of the recent related studies. METHODS: The hemoglobin and serum ferritin levels of the blood donors who visited Seoul National University Bundang Hospital were measured (total donors: 353, males: 252, females: 101). The hemoglobin levels were evaluated according to gender and the serum ferritin level, and the changes in the serum ferritin level were also checked for the repeat donors. RESULTS: The median serum ferritin level was 89.7 ng/mL (2.5~97.5%, 13.0~280.7 ng/mL). As classified by the serum ferritin levels, 9.9% of the males and 38.6% of the females showed low serum ferritin (< or =30 ng/mL) with a normal hemoglobin level. Among the 33 cases of repeat donors, 25 cases showed reduced serum ferritin changes (mean change: 17.4%). CONCLUSION: Similar to other recent studies, we found that the donors with normal hemoglobin could show a low serum ferritin level and especially the women and repeat donors in a Korean population. For the blood donors' safety, it is time to consider that the serum ferritin level should be included in the criteria for blood donor selection.