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

한민제,전선희,송상훈,박경운,김진규,송정한 대한진단검사의학회 2011 Annals of Laboratory Medicine Vol.31 No.4

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. 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.

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

한민제,송정한,준선희,송상훈,박형두,박경운 대한진단검사의학회 2015 Annals of Laboratory Medicine Vol.35 No.1

한국인에서 정상혈색소 헌혈자의 혈청 페리틴 농도

한민제,장호은,박경운,송정한,한규섭 대한수혈학회 2011 大韓輸血學會誌 Vol.22 No.2

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 (≤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.

Proteomic Profiling of Serum from Patients with Tuberculosis

송상훈,한민제,최양선,단기순,양만길,송정한,박성섭,이재호 대한진단검사의학회 2014 Annals of Laboratory Medicine Vol.34 No.5

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 discrimina- tive, which was 4.4 times higher in TB patients than in controls. Peptides from alpha-1-an- titrypsin and antithrombin III increased in TB patients and showed a high variable impor- tance 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.

CAPILLARYS 2 FLEX Piercing HbA1c 검사 성능 평가

전용범,한민제,이경훈,장호은,박경운,송정한 대한진단검사의학회 2013 Laboratory Medicine Online Vol.3 No.4

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.

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

이경훈,전선희,한민제,송상훈,박종선,이재호,박경운,송정한 대한진단검사의학회 2016 Annals of Laboratory Medicine Vol.36 No.5

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.

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

문수영,임민규,홍수지,한민제,송상훈,임경수,유경상,장인진,이지원,강형진,송정한,전용범 대한진단검사의학회 2014 Annals of Laboratory Medicine Vol.34 No.1

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 sta- bility were evaluated. The range of plasma busulfan concentration was obtained by ana- lyzing 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 -20°C and -80°C, but decreased by 25% when the plasma was stored for 24 hr at room temperature, and by <5% in 24 hr at 4°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.

혈장 및 혈청 유리 핵산을 이용한 RhD 및 RhCEce 항원의 유전자형 분석

장호은,박경운,황상미,홍윤지,한민제,박정수,송정한,한규섭 대한수혈학회 2014 大韓輸血學會誌 Vol.25 No.3

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%) cases were discrepant: one from plasma and six from serum compared 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.

T-KB-H와 3-HB 키트를 이용한 혈중 총 케톤과 베타-히드록시부틸산 측정법 평가

이경훈,전선희,이광우,한민제,송상훈,박경운,송정한 대한진단검사의학회 2014 Laboratory Medicine Online Vol.4 No.1

Background: Diabetes mellitus and alcohol consumption are the most common causes of ketoacidosis in adults. Recently, β-hydroxybutyric acid (βHBA) 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)+βHBA] and βHBA, 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 chromato-graphy-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 μmol/L. Linearity was observed for AcAc and βHBA over a 0-1,000 μmol/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 βHBA. Reference intervals determined for the T-KB-H kit were 9.8-270.1 μmol/L and 18.5-531.8 μmol/L in children and adults, respectively. For the 3-HB kit, the reference intervals were 6.4-234.0 μmol/L and 16.0-437.2 μmol/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. 배경: 성인에서 케톤산증의 가장 많은 원인은 당뇨병과 알코올의 섭취에 의한 것이다. 이러한 케톤산증을 진단 및 치료 효과의 모니터링을 하는데 혈중 베타-히드록시부틸산(βHBA)의 측정을 권장하고 있다. 본 연구는 총케톤체[아세토아세테이트(AcAc)+βHBA]와 βHBA를 각각 측정할 수 있는 T-KB-H와 3-HB 키트의 유용성을 평가하고자 하였다. 방법: 효소법을 이용한 혈중 케톤 정량은 Nittobo 사의 T-KB-H와 3-HB 키트와 Abbott사의 Architect ci16200 기기를 사용하여 측정하였으며, 동시에 가스크로마토그래피-질량분석법(GC-MS)으로 측정하였다. CLSI의 지침에 따라서 정밀도, 직선성, 기존 장비와의 상관성과 소아 및 성인의 참고범위를 설정하였다. 결과: 각 키트마다의 측정한 두가지 농도에 대한 모든 정밀도는 4.00% 미만이었다. 직선성은 0-1,000 mol/L 범위에서 우수하였다(R2<0.99). GC-MS 결과와 비교하였을 때 AcAc의 경우 상관계수가 0.94, βHBA의 경우 0.96으로 양호한 상관관계를 보였다. 그리고 각각의 키트에서 구한 참고범위는 T-KB-H 키트의 경우 소아는 9.8-270.1 mol/L, 성인은 18.5-531.8 mol/L, 3-HB 키트의 경우 소아는 6.4-234.0 mol/L, 성인은 16.0-437.2 mol/L이었다. 결론: T-KB-H와 3-HB 키트는 정밀도 및 직선성이 우수하였고 기존 방법과의 상관성이 양호하므로, 임상적으로 혈중 케톤체의 농도를 측정하는데 유용하게 사용할 수 있을 것으로 생각된다.

조직은행에서의 기증자 적합성 판정

김택수,박경운,홍윤지,황상미,송정한,한규섭,한민제 대한수혈학회 2013 大韓輸血學會誌 Vol.24 No.2

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. 배경: 이식용 조직은 생명을 구하거나 장기의필수적인 기능을 유지시켜 준다. 국가 정책 및 규정에 따라 국가간 조직 기증 및 이식의 안전 수준, 품질, 효능 등에 큰 차이가 있다. 선별 검사의측면에서 몇 개의 조직 은행 관련 지침을 검토하였고, 이에 추가하여 4년간에 걸쳐 3차 의료기관에 기증된 뼈 조직 및 기증자를 대상으로 한 혈액선별검사 경험을 제시하였다. 방법: 7개의 국내 및 국제 가이드라인을 비교하였다. 연구가 수행된 조직은행의 선별 검사에는 두 단계가 있는데, 1차 선별검사로는 ABO/Rh, 비예기 항체 선별, VDRL, HBsAg, anti-HBs, anti- HBc IgM, anti-HCV, anti-HIV 및 미생물 배양검사를 수행하였다. 2차 선별검사는 배양검사를 제외한 나머지 항목에 대해 90일 이후에 시행하였다. 2008년부터 2011년까지, 245건의 뼈 조직이기증되었으며 이에 대한 기증자 선별검사 결과를분석하였다. 결과: 생존 기증자에 대한 필수 혈액 검사 항목은 가이드라인 별로 차이를 보였고, 국내 가이드라인 간에도 차이가 존재하였다. 4년 동안 기증된 245건의 뼈 조직 중 61건이 폐기되었는데, 2차선별검사 결과가 없는 경우(n=32), 조직 오염 또는 배양 검사 미시행한 경우(n=10), 비정상적인혈액 검사 결과(n=8) 등으로 나타났다. 결론: 국내 조직 은행에 대한 기증자 선별 정책에 있어 기준이 제시되어야 할 것으로 보이며, 이런 기준에는 2차 선별검사 및 항체미형성기간을충분히 고려할 필요가 있겠다.