BACKGROUND: Vitamin D has been recently shown to play important roles in the functioning of various systems. Most of the current analytical methods for measuring vitamin D levels are based on immunoassays. We simultaneously measured the levels of 25-hydroxyvitamin D3 [ 25(OH)D3 ] and 25-hydroxyvitamin D2 [ 25(OH)D2 ] in human serum by performing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after Diels-Alder derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and evaluated the performance of our method. METHODS: After liquid-liquid extraction, samples were dried under N2 at 50degrees C for 1 hr followed by Diels-Alder derivatization with ethyl acetate containing 0.1 mg/mL PTAD. The samples were resuspended in 60 microL of methanol:10 mM ammonium formate solution (1:1, V/V). C18 UPLC column and positive ion multiple reaction monitoring transitions such as m/z 558.35-->298.1, 25(OH)D3; m/z 570.35-->298.1, 25(OH)D2; and m/z 564.35-->298.1, hexadeuterated-25(OH)D3 were used for UPLC-MS/MS. RESULTS: The within-run imprecision (CVs) for 25(OH)D3 and 25(OH)D2 were 3.5-4.0% and 3.8-4.2%, respectively, and the corresponding between-run CVs were 3.3-5.5% and 4.7-5.8%. The lower limit of quantification for 25(OH)D3 and 25(OH)D2 were 0.5 and 1.0 ng/mL, respectively. The curve for interassay calibration variability data obtained over concentrations of 0-120 ng/mL for 25(OH)D3 and 0-90 ng/mL for 25(OH)D2 was linear and reproducible [ 25(OH)D3, R2=0.993; 25(OH)D2, R2=0.998]. The total 25(OH)D levels in Koreans (average, 18.7 ng/mL) were lower than those in American Caucasians, and the percentage of people with total 25(OH)D levels under 10 ng/mL was 8.1%. CONCLUSIONS: Our method to measure 25(OH)D3 and 25(OH)D2 levels by performing UPLC-MS/MS after PTAD derivatization showed good performance as a sensitive and reproducible method for routine analysis of vitamin D status.
25-Hydroxyvitamin D 2 ; Acetates ; Calcifediol ; Calibration ; Formates ; Humans ; Immunoassay ; Liquid-Liquid Extraction ; Mass Spectrometry ; Quaternary Ammonium Compounds ; Tandem Mass Spectrometry ; Triazoles ; Vitamin D

BACKGROUND: Repeating a QC that is outside 2SD from the mean (1:2s rule) appears to be a common practice. Although this form of repeat-sampling is frowned on by many, the comparative power of the approach has not been formally evaluated. METHODS: We computed power functions mathematically and by computer simulation for 4 different 1:2s repeat-sampling strategies, as well as the 1:2s rule, the 1:3s rule, and 2 common QC multirules. RESULTS: The false-rejection rates for the repeat-sampling strategies were similarly low to those of the 1:3s QC rule. The error detection rates for the repeat-sampling strategies approached those of the 1:2s QC rule for moderate to large out-of-control error conditions. In most cases, the power of the repeat-sampling strategies was superior to the power of the QC multirules we evaluated. The increase in QC utilization rate ranged from 4% to 13% for the repeat-sampling strategies investigated. CONCLUSIONS: The repeat-sampling strategies provide an effective tactic to take advantage of the desirable properties of both the 1:2s and 1:3s QC rules. Additionally, the power of the repeat-sampling strategies compares favorably with the power of 2 common QC multirules. These improvements come with a modest increase in the average number of controls tested.
Computer Simulation ; Imidazoles ; Nitro Compounds

The HACEK group of microorganisms is responsible for approximately 3-6% of endocarditis cases and is a major cause of culture-negative endocarditis. Here, we report a case of Haemophilus parainfluenzae infective endocarditis that was diagnosed by direct PCR sequencing of 16S rRNA from resected vegetation. A healthy 26-yr-old man was admitted to the emergency room (ER) on March 27, 2011 because of intermittent high fever. The patient was prescribed cefpodoxime for 5 days at the ER. Six and 11 sets of blood cultures were performed at the ER and in a general ward, respectively, using BACTEC Plus Aerobic/F (Becton-Dickinson, USA) and Lytic Anaerobic/F Plus (BD) together. Echocardiography revealed a large vegetation at the posterior mitral valve leaflet. After performing mitral valvoplasty on hospital day (HD) 11, the vegetation tissue was cultured in thioglycolate broth, blood agar, Brucella agar, and MacConkey agar for 7 days, but no organism was grown. Direct PCR sequencing of 16S rRNA of the tissue revealed the presence of H. parainfluenzae. In the 17 sets of blood cultures, bacterial growth was detected in only 2 aerobic bottles of 5 sets taken at HD 9 after 10-day and 14-day incubation. The organism was identified as H. parainfluenzae by using the VITEK NHI card (bioMerieux, France). Direct PCR sequencing of vegetation could be useful in diagnosing bacterial pathogens in infective endocarditis patients, especially in culture-negative cases.
Agar ; Brucella ; Ceftizoxime ; Echocardiography ; Emergencies ; Endocarditis ; Fever ; Haemophilus ; Haemophilus parainfluenzae ; Humans ; Mitral Valve ; Paramyxoviridae Infections ; Patients' Rooms ; Polymerase Chain Reaction ; Sequence Analysis, RNA

Bacteria belonging to the genus Tsukamurella are aerobic, gram-positive rods that are weakly acid-fast with no apparent branching. Infections of the Tsukamurella spp. are generally caused by the use of infected medical devices such as central venous catheters. The underreporting of these infections might be attributable to the frequent misdiagnosis of Tsukamurella infections as Corynebacterium or atypical Mycobacterium spp. infections. Therefore, when gram-positive aerobic rods are observed in the blood culture of a patient with a central venous catheter, it is important to consider Tsukamurella as one of the causative organisms. Here, we report the first case of a catheter-related blood stream infection caused by Tsukamurella inchonensis in a 3-yr-old Korean girl with underlying biliary atresia who underwent hepatoportoenterostomy.
Bacteria ; Biliary Atresia ; Catheters ; Central Venous Catheters ; Corynebacterium ; Diagnostic Errors ; Gram-Positive Rods ; Humans ; Nontuberculous Mycobacteria ; Rivers ; RNA, Ribosomal, 16S

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by persistent peripheral blood neutorphilia, bone marrow hypercellularity of neutrophilic granulocyte proliferation and hepatosplenomegaly. Acquired somatic mutation, JAK2 V617F, is the only molecular marker known to have association with classic BCR-ABL1 negative MPNs. However, JAK2 V617F has been detected occasionally in other MPNs such as CNL or other disease entities. We experienced a case of CNL with JAK2 V617F mutation. The patient was diagnosed according to the 2008 WHO classification criteria, and developed AML 9 months after the diagnosis of CNL. The JAK2 V617F was detected in the bone marrow throughout the clinical course. More cases are needed to establish the role of JAK2 V617F in the pathogenesis, prognosis and disease course of CNL.
Bone Marrow ; Granulocytes ; Humans ; Leukemia, Myeloid, Acute ; Leukemia, Neutrophilic, Chronic ; Neutrophils ; Prognosis

BACKGROUND: Magnetic resonance imaging (MRI) findings of white-matter lesions are different from those of lacunar infarcts; however, both these conditions are related to cardiovascular risk factors. This study was performed to investigate the differences in the relationships of white-matter lesions and lacunar infarcts with cardiovascular risk factors and differences between the metabolic characteristics of patients with these conditions. METHODS: We included 4,255 patients who showed neurological deficits during health checkups. These individuals were classified into the following 3 groups on the basis of MRI findings: normal, white-matter lesion, and lacunar infarct. The groups were compared for age; weights; prevalence of metabolic syndrome; and levels of blood pressure, blood glucose, lipid, high sensitivity C-reactive protein, and HbA1c. RESULTS: Age, body mass index (BMI); waist circumference; levels of blood pressure, blood glucose, triglycerides and HbA1c; and prevalence of metabolic syndrome and its components were the highest in lacunar infarct group, followed by white matter lesion group, and normal group. Age and diastolic blood pressure level were related to white matter lesions, and age, systolic blood pressure level, and blood glucose level were related to lacunar infarcts. Further, the prevalence of the above-mentioned lesions increased with increase of the number of the components of metabolic syndrome. CONCLUSIONS: This study suggests that lacunar infarct is more advanced lesion than white matter lesion. Among all the cardiovascular risk factors, high blood pressure and impaired fasting blood glucose levels were significantly related to white-matter lesions and lacunar infarct.
Blood Glucose ; Blood Pressure ; Body Mass Index ; C-Reactive Protein ; Fasting ; Humans ; Hypertension ; Magnetic Resonance Imaging ; Prevalence ; Risk Factors ; Stroke, Lacunar ; Triglycerides

Rheumatoid arthritis (RA) is an autoimmune disease that results in a chronic inflammatory disorder, which principally attacks the small joints. Several autoantibodies, such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody, are known to be associated with RA. Anti-proliferating cell nuclear antigen (PCNA) antibodies are mainly observed in patients with systemic lupus erythematosus (SLE). Indeed, a high titer of these antibodies is considered highly suggestive of SLE; however, anti-PCNA antibodies also appear in other autoimmune diseases. Two previous reports described RA patients with low titers of anti-PCNA antibodies, respectively. In this report, we describe a case of an RA patient exhibiting a high titer (>1:2,560) of anti-PCNA antibodies. The 56-yr-old female patient, with no underlying disease or medication history, presented with multiple joint pain and morning stiffness that had begun 6 months prior. The erythrocyte sedimentation rate (ESR) and RF were elevated (102 mm/hr and 77 IU/mL, respectively), and C-reactive protein (CRP) was 0.8 mg/dL. While the test for anti-CCP antibodies was negative, an anti-PCNA pattern (>1:2,560) and a homogeneous pattern (1:320) were detected by autoimmune target (AIT) test. The presence of anti-PCNA antibodies was subsequently confirmed using the double immunodiffusion method. The anti-dsDNA test was also positive (1:160). X-ray imaging showed soft tissue swelling of multiple joints of both hands and wrists. According to the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria, the patient was classified as having RA. This is the first case to describe high titers anti-PCNA antibodies associated with RA.
Antibodies* ; Arthralgia ; Arthritis, Rheumatoid* ; Autoantibodies ; Autoimmune Diseases ; Blood Sedimentation ; C-Reactive Protein ; Classification ; Female ; Hand ; Humans ; Immunodiffusion ; Joints ; Lupus Erythematosus, Systemic ; Proliferating Cell Nuclear Antigen ; Rheumatic Diseases ; Rheumatoid Factor ; Wrist

Pharmacogenetics is a rapidly evolving field and the number of pharmacogenetic tests for clinical use is steadily increasing. However, incorrect or inadequate implementation of pharmacogenetic tests in clinical practice may result in a rise in medical costs and adverse outcomes in patients. This document suggests guidelines for the clinical application, interpretation, and reporting of pharmacogenetic test results based on a literature review and the collection of evidence-based expert opinions. The clinical laboratory practice guidelines encompass the clinical pharmacogenetic tests covered by public medical insurance in Korea. Technical, ethical, and regulatory issues related to clinical pharmacogenetic tests have also been addressed. In particular, this document comprises the following pharmacogenetic tests: CYP2C9 and VKORC1 for warfarin, CYP2C19 for clopidogrel, CYP2D6 for tricyclic antidepressants, codeine, tamoxifen, and atomoxetine, NAT2 for isoniazid, UGT1A1 for irinotecan, TPMT for thiopurines, EGFR for tyrosine kinase inhibitors, ERBB2 (HER2) for erb-b2 receptor tyrosine kinase 2-targeted therapy, and KRAS for anti-epidermal growth factor receptor drugs. These guidelines would help improve the usefulness of pharmacogenetic tests in routine clinical settings.
Antidepressive Agents, Tricyclic ; Atomoxetine Hydrochloride ; Clinical Laboratory Services ; Codeine ; Cytochrome P-450 CYP2C19 ; Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP2D6 ; Expert Testimony ; Genetic Testing ; Humans ; Insurance ; Isoniazid ; Korea ; Pharmacogenetics ; Protein-Tyrosine Kinases ; Tamoxifen ; Warfarin

BACKGROUND: The purpose of this study is to investigate the positive rates of screening tests for inherited metabolic disorders, set cutoff values, and report the actual status of internal quality controls in LabGenomics Clinical Laboratories by using LC-MS/MS system. METHODS: We use Agilent 1260 Infinity HPLC System (Agilent Technologies, USA) for liquid chromatography, and API 2000 (AB Sciex, Canada) for MS/MS system. We set up screening tests for 55 diseases, which include metabolic disorders of 25 amino acids, 16 organic acids, and 14 fatty acids. RESULTS: We determined the analyte cutoff values as 99.9 or 0.1 percentiles in 15,000 newborn samples. The total number of samples tested from January 2012 to September 2014 was 119,948; of these, 6,681 were repeated. Of the repeated samples, 713 were presumed to be positive in the screening tests. Repeat screening with newly obtained dried blood spot specimens was recommended for these 713 samples and 600 specimens were obtained. Thus, the recall rate was 0.5% (600/119,948) for all samples and 84.2% (600/713) for the samples presumed to be positive in the screening tests. About 70 samples, that is, 0.06% of the total samples and 11.7% of the "reobtained" samples, again tested positive; we recommended confirmatory tests for these samples. CONCLUSIONS: We have presented data on the status of neonatal screening tests for inherited metabolic disorders using LC-MS/MS, including positive rates and recall rates of screening tests, set up cutoff values and reported the actual status of internal quality controls in a clinical laboratory in Korea.
Amino Acids ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Fatty Acids ; Humans ; Infant, Newborn ; Korea* ; Mass Screening ; Neonatal Screening* ; Quality Control ; Tandem Mass Spectrometry*

BACKGROUND: The XN-series (Sysmex, Japan) is the new hematology analyzer from Sysmex, with new channels to improve the accuracy of differential leukocyte count and platelet count in the low cell count range. We evaluated the analytical performance and low white blood cell (WBC) mode of the XN-2000. METHODS: Precision, linearity, and carryover were evaluated for the analyzer. We analyzed the accordance of complete blood count (CBC), reticulocyte count, and differential leukocyte count between the XN-2000 and XE-2100 (Sysmex), using 200 samples from normal controls and patients. For 80 samples with a WBC count <1.5x10(9) cells/L, the low WBC mode was evaluated by comparing the automated count with a manual differential count as the reference. RESULTS: The coefficients of variation of precision were <5% for most CBC parameters and <10% for differential leukocyte count. All results obtained with the XN-2000 showed good correlation with those obtained with the XE-2100. The correlation coefficients (r) were >0.9800 for all CBC parameters except mean corpuscular hemoglobin concentration, mean platelet volume, and platelet distribution width, and >0.9900 for differential leukocyte count except monocytes and basophils. The low WBC mode provided accurate counts for neutrophils and lymphocytes, with r>0.9300 for samples with a WBC count of 0.1-1.5x10(9) cells/L. CONCLUSIONS: The XN-2000 showed good analytical performance and correlation with the existing model, the XE-2100. The XN-2000 provided accurate results for differential leukocyte count in samples with a WBC count of 0.1-1.5x10(9) cells/L, and reduced manual slide reviews.
Basophils ; Blood Cell Count ; Blood Platelets ; Cell Count ; Erythrocyte Indices ; Hematology* ; Humans ; Leukocyte Count* ; Leukocytes ; Lymphocytes ; Mean Platelet Volume ; Monocytes ; Neutrophils ; Platelet Count ; Reticulocyte Count