Comparison of Erythrocyte and Reticulocyte Indices for Evaluation of Iron Deficiency by Two Automated Hematologic Analyzers.

The reticulocyte hemoglobin content and hypochromic erythrocyte percentage offer advantages in evaluation of iron deficiency, especially in inflammatory conditions. The aim of this study was to evaluate the correlation of reticulocyte hemoglobin content (CHr, Ret-He) and hypochromic erythrocyte percentage (%HYPO, Hypo-He) between two automated hematologic analyzers. The CHr and %HYPO values were determined using the Advia 2021i (Siemens), while the Ret-He and Hypo-He levels were assessed using the XN-3000 (Sysmex). Data from a total of 971 cases and 834 patients were collected. For reticulocyte hemoglobin content, there was a good linear correlation between CHr and Ret-He (r = 0.857, p < 0.001). For percentage of hypochromic erythrocytes, there was a better correlation between the two measures when using a second-degree polynomial equation (Hypo-He* = 0.4818 - 0.0218 x %HYPO + 0.0069 x %HYPO2) (r = 0.786, p < 0.001).

Comparative Evaluation of Bruker Biotyper and ASTA MicroIDSys for Species Identification in a Clinical Microbiology Laboratory.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for microbial identification, because of its speed and accuracy, since its introduction to clinical microbiology laboratories. In this study, we evaluated the performance of ASTA MicroIDSys, a newly developed MALDI-TOF, and compared it with the widely used Bruker Biotyper. Microbial identification with the Bruker Biotyper system was performed by using a direct smear method and the Bruker Biotyper database (reference library version 6.0.0.0). The isolates were also tested in parallel, using the ASTA MicroIDSys system with a direct smear method and the MicroIDSys database, CoreDB v1.26. A total of 914 clinical isolates were recovered from the clinical specimens. Identical results with confidence scores (≥2.0, for the Bruker Biotyper) and acceptable scores (≥140 for the ASTA MicroIDSys) were obtained for 840 (91.9%) isolates. The minor errors were defined as misidentification at the species level, and the rate was 1.1% (9/792) for Bruker Biotyper and 1.6% (13/792) for ASTA MicroIDSys. Major errors were defined as misidentification at the genus level, and the rate was 0.3% (2/792) for both Bruker Biotyper and ASTA MicroIDSys. ASTA MicroIDSys showed reliable performance for microbial identification, which was comparable to that of the Bruker Biotyper. Therefore, ASTA MicroIDSys can be applied for the identification of microorganisms in clinical microbiology laboratories.

Mild Phenotype of Xq Deletion and 3q Duplication Associated with der(X)t(X;3)(q21.1;q22.1) without Loss of XIST Gene.

We report a 10-year-old female patient with der(X)t(X;3)(q21.1;q22.1), resulting in 3q duplication and Xq deletion. The main clinical feature was primary ovarian failure and there was no abnormal phenotype corresponding to 3q duplication syndrome. This might be explained by the XIST gene at the X-inactivation center on Xq13.2, which was not deleted in this case. The conventional karyotyping was preliminarily reported as 46,X,inv(X) (q13q26) due to the similar banding patterns of Xq13.2-q24 and 3q25.33-q29. This case highlights the value of using a chromosomal microarray as a clinical diagnostic test for individuals with developmental delay or congenital anomalies.

Multiplex LC-MS/MS for simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D3, albumin, and vitamin D-binding protein with its isoforms: One-step estimation of bioavailable vitamin D and vitamin D metabolite ratio.

Bioavailable vitamin D and vitamin D metabolite ratio (VMR) have emerged as potential novel vitamin D markers. We developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine all elements necessary for the calculation of bioavailable vitamin D and VMR, including 25-hydroxyvitamin D [25-(OH)D] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], VDBP and its isoforms, and albumin. Following separate reactions of hexane extraction and trypsin digestion, serum samples were analyzed using LC-MS/MS to measure 25-(OH)D3, 25-(OH)D2, 24,25-(OH)2D3, VDBP and its isoforms, and albumin. Analytical performances were assessed. Korean (n = 229), Arab (n = 98), White (n = 99) and Black American (n = 99) samples were analyzed. Bioavailable vitamin D and VMR were calculated. All target molecules were clearly separated and accurately quantified by LC-MS/MS. Analytical performances, including imprecision, accuracy, ion suppression, limit of quantification, linearity, and comparison with existing methods were within acceptable levels. The allele frequencies of VDBP isoforms in various races resulted similar to previously known values. The levels of bioavailable vitamin D were highest in White Americans and lowest in Black Americans. We have successfully developed a multiplex LC-MS/MS-based assay method that can simultaneously perform the measurement of all parameters needed to calculate bioavailable vitamin D and VMR. Our devised method was robust and reliable in terms of analytical performances and could be applied to routine clinical samples in the future to more accurately assess vitamin D status.

Comparative Evaluation of Allplex Respiratory Panels 1, 2, 3, and BioFire FilmArray Respiratory Panel for the Detection of Respiratory Infections.

Multiplex nucleic acid amplification assays that simultaneously detect multiple respiratory pathogens in a single nasopharyngeal swab (NPS) specimen are widely used for rapid clinical diagnostics. We evaluated Allplex Respiratory Panel (RP) 1, 2, 3, and the BioFire FilmArray RP assay for detecting respiratory pathogens from NPS specimens. In all, 181 NPS specimens obtained from patients suspected of having respiratory infections during the non-influenza season (August-December 2019) were included. The Allplex RP 1, 2, and 3 detected 154 samples positive for respiratory viruses, whereas the BioFire FilmArray detected viruses in 98 samples. Co-infection with two or more viruses was detected in 41 and 17 NPS specimens by Allplex RP and the BioFire FilmArray RP, respectively. For adenoviruses, Allplex RP 1 detected 31 specimens, compared to 34 by the BioFire FilmArray. In all, 64 NPS specimens were positive for human enterovirus (HEV) and human rhinovirus (HRV) on the Allplex RP, in contrast to 39 HEV/HRV on the BioFire FilmArray. The parainfluenza virus (PIV-1-4) detection rate differed between the two systems. Most discrepant results were observed for NPS specimens with high cycle threshold values obtained by Allplex RP. This study showed concordant performance of the Allplex RP 1, 2, 3, and the BioFire FilmArray RP for the simultaneous detection of multiple respiratory viruses.

Characterization of Chlamydia trachomatis ompA Genotypes Among Sexually Transmitted Disease Patients in Korea.

BACKGROUND: Chlamydia trachomatis is the leading cause of bacterial sexually transmitted diseases (STDs) world-wide. The aim of this study was to characterize the C. trachomatis genetic profiles of clinical isolates in the Korean population by sequence analysis of the ompA gene. METHODS: Endocervical specimens from patients who were confirmed as C. trachomatis infection by real-time PCR were used for ompA sequencing. The individual sequences (about 890 nucleotides) were determined by comparison with those from known C. trachomatis strains using the BLAST search tool. Sequence variations were analyzed by comparing them with sequences from prototype strains. RESULTS: Sequence analysis using BLAST similarity search of the ompA gene from the 106 clinical isolates revealed that the most prevalent genotype corresponded to E (n = 28, 26.2%), followed by F (n = 20, 18.9%), D (n = 16, 15.1%), J (n = 16, 15.1%), G (n = 9, 8.5%), H (n = 8, 7.5%), K (n = 5, 4.7%), B (n = 2, 1.9%), and I (n = 2, 1.9%). Detailed sequence analysis based on comparison with each prototype revealed that there are sequence differences in 44 specimens (41.5%). CONCLUSIONS: This study is the update on the distribution of C. trachomatis ompA genotypes in Korea in more than a decade. The genotypes have become more diversified, with genotypes K, B, and I being newly detected. C. trachomatis genotyping is crucial for regional and global epidemiological studies, as well as for vaccine development.

Rapid Discrimination of Methicillin-Resistant Staphylococcus aureus by MALDI-TOF MS.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious pathogen in clinical settings and early detection is critical. Here, we investigated the MRSA discrimination potential of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using 320 clinical S. aureus isolates obtained in 2005-2014 and 181 isolates obtained in 2018. We conducted polymerase chain reactions (PCR) for staphylococcal cassette chromosome mec (SCCmec) typing and MALDI-TOF MS to find specific markers for methicillin resistance. We identified 21 peaks with significant differences between MRSA and methicillin-susceptible S. aureus (MSSA), as determined by mecA and SCCmec types. Each specific peak was sufficient to discriminate MRSA. We developed two methods for simple discrimination according to these peaks. First, a decision tree for MRSA based on six MRSA-specific peaks, three MSSA-specific peaks, and two SCCmec type IV peaks showed a sensitivity of 96.5%. Second, simple discrimination based on four MRSA-specific peaks and one MSSA peak had a maximum sensitivity of 88.3%. The decision tree applied to 181 S. aureus isolates from 2018 had a sensitivity of 87.6%. In conclusion, we used specific peaks to develop sensitive MRSA identification methods. This rapid and easy MALDI-TOF MS approach can improve patient management.

A comparative evaluation of BACT/ALERT FA PLUS and FN PLUS blood culture bottles and BD BACTEC Plus Aerobic and Anaerobic blood culture bottles for antimicrobial neutralization.

The performance of BACT/ALERT FA and FN PLUS (FA PLUS and FN PLUS) blood culture bottles with the BACT/ALERT VIRTUO (bioMérieux, Inc., Durham, NC) and BD BACTEC Plus Aerobic and Anaerobic (BD Aerobic and BD Anaerobic) blood culture bottles with the BD BACTEC FX (BD Diagnostics, Sparks, MD) for antimicrobial neutralization at peak serum concentration was evaluated. The following antibiotic agents and microbial strains were used: ampicillin, cefepime, cefotaxime, gentamicin, levofloxacin, meropenem, piperacillin-tazobactam, and vancomycin; methicillin-sensitive Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Bacteroides fragilis. The detection rate of FA PLUS bottles was 69.1% (259/375) and that of BD Aerobic bottles was 75.5% (283/375) (p = 0.050). In the case of anaerobic culture, the overall detection rate of FN PLUS bottles was 77.0% (231/300) and that of BD Anaerobic bottles was 71.3% (214/300) (p = 0.113). The time to detection (TTD) from aerobic culture was 2.8 h shorter in FA PLUS bottles (12.4 h) compared to BD Aerobic bottles (15.2 h) (p < 0.001). And the TTD from anaerobic culture was 1.6 h shorter in FN PLUS bottles (18.1 h) compared to BD Anaerobic bottles (19.7 h) (p = 0.061). The FA PLUS bottles exhibited a lower detection rate compared to BD Aerobic bottles, while FN PLUS bottles showed a higher detection rate compared to BD Anaerobic bottles. The BACT/ALERT VIRTUO system exhibited shorter TTD compared to the BD BACTEC FX system for both aerobic and anaerobic cultures.

Incidence and seasonality of respiratory viruses causing acute respiratory infections in the Northern United Arab Emirates.

BACKGROUND: The data on the seasonality of respiratory viruses helps to ensure the optimal vaccination period and to monitor the possible outbreaks of variant type. OBJECTIVES: This study was designed to describe the molecular epidemiology and seasonality of acute respiratory infection (ARI)-related respiratory viruses in the United Arab Emirates (UAE). METHODS: Both upper and lower respiratory specimens were collected for the analysis from all the patients who visited the Sheikh Khalifa Specialty Hospital (SKSH) with ARI for over 2 years. The multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) test was used to detect respiratory viruses, which include human adenovirus, influenza virus (FLU) A and B, respiratory syncytial virus, parainfluenza viruses, human rhinovirus (HRV), human metapneumovirus, human enterovirus, human coronavirus, and human bocavirus. RESULTS: A total of 1,362 respiratory samples were collected from 733 (53.8%) male and 629 (46.2%) female patients with ARI who visited the SKSH between November 2015 and February 2018. The rRT-PCR test revealed an overall positivity rate of 37.2% (507/1362). The positive rate increased during winter; it was highest in December and lowest in September. FLU was the most frequently detected virus (273/1362 [20.0%]), followed by human rhinovirus (146/1362 [10.7%]). The FLU positivity rate showed two peaks, which occurred in August and December. The peak-to-low ratio for FLU was 2.26 (95% confidence interval: 1.52-3.35). CONCLUSIONS: The pattern of FLU in the UAE parallels to that of temperate countries. The trend of the small peak of FLU in the summer suggests a possibility of semi-seasonal pattern in the UAE.

Performance evaluation of enzyme immunoassay for voriconazole therapeutic drug monitoring with automated clinical chemistry analyzers.

OBJECTIVE: Voriconazole is a triazole antifungal developed for the treatment of fungal infectious disease, and the clinical utility of its therapeutic drug monitoring has been evaluated. Recently, a new assay for analyzing the serum voriconazole concentration with an automated clinical chemistry analyzer was developed. We evaluated the performance of the new assay based on standardized protocols. METHODS: The analytical performance of the assay was evaluated according to its precision, trueness by recovery, limit of quantitation, linearity, and correlation with results from liquid chromatography-tandem mass spectrometry (LC-MS/MS). The evaluation was performed with the same protocol on two different routine chemistry analyzers. All evaluations were performed according to CLSI Guidelines EP15, EP17, EP6, and EP9 [1-4]. RESULTS: Coefficients of variation for within-run and between-day imprecision were 3.2-5.1% and 1.5-3.0%, respectively, on the two different analyzers for pooled serum samples. The recovery rates were in the range of 95.4-102.2%. The limit of blank was 0.0049 µg/mL, and the limit of detection of the samples was 0.0266-0.0376 µg/mL. The percent recovery at three LoQ levels were 67.9-74.6% for 0.50 µg/mL, 75.5-80.2% for 0.60 µg/mL, and 89.9-96.6% for 0.70 µg/mL. A linear relationship was demonstrated between 0.5 µg/mL and 16.0 µg/mL (R2 =0.9995-0.9998). The assay correlated well with LC-MS/MS results (R2 =0.9739-0.9828). CONCLUSIONS: The assay showed acceptable precision, trueness, linearity, and limit of quantification, and correlated well with LC-MS/MS. Therefore, its analytical performance is satisfactory for monitoring the drug concentration of voriconazole.

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.

Performance Evaluation of the FREND Cardiac Triple Cartridge on the FREND System.

BACKGROUND: We evaluated the performance of the FREND Cardiac Triple cartridge on the FREND system in the detection of cardiac markers-myoglobin, cardiac troponin I (cTnI), and creatine kinase-MB (CK-MB). METHODS: Quantitative immunoassays were performed using the FREND system (NanoEnTek, Seoul, Korea) and its cartridge. The precision, detection limits, linearity, and correlation with the Siemens Dimension Vista 500 (Siemens Healthcare Diagnostics, Deerfield, IL, USA) were evaluated. The cutoff value for each marker was calculated in healthy individuals (men and women, n = 138 each). RESULTS: The coefficients of variation for imprecision were less than 19.0% at low and high serum concentrations. The lower limits of quantification for myoglobin, cTnI, and CK-MB were 3.11, 0.073, and 0.70 ng/mL, respectively. Acceptable linearity was achieved for each marker (R2 < 0.99). The results from the FREND system were in good agreement with those from the Siemens Dimension Vista (correlation coefficients > 0.9). The cutoff values in male and female individuals (n = 138 each) were 104.3 and 98.9 ng/mL, respectively, for myoglobin, and 4.35 and 5.37 ng/mL, respectively, for CK-MB. The cutoff value for cTnI was 0.073 ng/mL. CONCLUSIONS: The FREND Cardiac Triple cartridge exhibited good precision, clinically acceptable linearity, and reliable correlation with the Dimension Vista.

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.

Performance evaluation of SD A1cCare as a HbA1c analyzer for point-of-care testing.

OBJECTIVES: Some studies have shown that a rapid feedback of HbA1c results is helpful for controlling plasma glucose levels. Point-of-care (POC) instruments that are fast, portable, and easy to use are suitable for rapid determination of HbA1c levels. Here, we evaluated the analytical performance of a newly developed POC HbA1c analyzer, SD A1cCare (SD Biosensor, Inc.). DESIGN AND METHODS: The precision, linearity, and correlation with the Variant II Turbo instrument (Bio-Rad Laboratories, Inc.) were evaluated according to CLSI guidelines for SD A1cCare. All tests were performed according to the manufacturer instructions, and statistical analyses, including linear regression and Passing-Bablok regression, were performed. Bias from the IFCC reference targets was also evaluated with 12 duplicate specimens (n=24 in total). RESULTS: The coefficients of variation based on EP9-A2 protocol were 2.6% in SI unit and 1.8% in NGSP unit. The calibration curve was linear, with R(2)=0.9911 in the range of 23.5 to 125.1 mmol/mol in SI units (4.3% to 13.6% in NGSP units). The results of the SD A1cCare correlated with those of the Variant II Turbo (r=0.986). Deviations from IFCC targets at 30, 60, and 90 mmol/mol IFCC levels were -1.95, -1.85, and -1.74 mmol/mol, respectively. CONCLUSIONS: The SD A1cCare analyzer showed excellent precision, linearity, correlation with the Variant II Turbo analyzer, and accuracy with IFCC targets. Therefore, it may be suitable for HbA1c assays in the POC setting and in small laboratories.

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.

Ultra-performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy.

RATIONALE: Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A. Accumulated sulfatides can be detected in the urine and detection of sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for measuring sulfatides in DBSs from patients with MLD. METHODS: DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to a 96-well microplate and dried, then resuspended in methanol/propanol solution. Samples were analyzed on an UPLC system. Total running time was 4 min. Quantification was achieved by multiple reaction monitoring using a tandem mass spectrometer. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimens from MLD patients (n = 9), pseudodeficiency (PD) patient (n = 1), obligate heterozygotes (OH) (n = 2) and normal controls (n = 124). RESULTS: In negative-ion mode, sulfatides species subjected to collision-induced dissociation readily fragment to produce an intense ion at m/z 96.8 (HSO4(-)). The precisions of low and high concentration controls ranged from 5.4 to 19.9%. The sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals and the PD-OH group [mean (range), 0.07 (<0.05-0.34) and 0.13 (<0.05-0.22) µg/mL, respectively]. In contrast, the DBSs from MLD patients showed a marked increase in several molecular species of sulfatide [mean (range), 2.02 (1.18-3.89) µg/mL]. CONCLUSIONS: Simultaneous detection for sulfatides using UPLC/MS/MS can be successfully applied to DBS analysis. This method provides a fast and effective screening and monitoring tool for the diagnosis and treatment of MLD.

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 -20℃ and -80℃, but decreased by 25% when the plasma was stored for 24 hr at room temperature, and by <5% in 24 hr at 4℃. 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.

Method for simultaneous analysis of nine second-line anti-tuberculosis drugs using UPLC-MS/MS.

OBJECTIVES: Therapeutic drug monitoring (TDM) of anti-tuberculosis (TB) drugs is beneficial for patients responding slowly to treatment and those with multidrug-resistant TB. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to develop a rapid method for simultaneously measuring the blood concentrations of nine second-line anti-TB drugs: streptomycin, kanamycin, clarithromycin, cycloserine, moxifloxacin, levofloxacin, para-aminosalicylic acid, prothionamide and linezolid. METHODS: Serum samples were extracted with acidified methanol and neutralized with NaOH. A Waters Acquity HSS T3 column and gradients of ammonium formate and acetonitrile in 0.1% formic acid were used for UPLC separation. Drug concentrations were determined by multiple reaction monitoring in positive ion mode, and assay performance was evaluated. We applied this method to TDM, analysing random serum samples from 85 patients treated with second-line drugs. RESULTS: Sample preparation using acidified methanol extraction followed by neutralization yielded good recovery and ionization efficiency, with chromatographic separation achieved within 3 min per sample. Within-run and between-run precisions were 1.7%-7.5% and 1.7%-12.4%, respectively, at concentrations representing low and high levels for the nine drugs. Lower limits of detection and quantification were 0.025-0.5 and 0.25-5.0 µg/mL, respectively. Linearity was acceptable at five concentrations for each drug. No ion suppression was observed at the retention time for most compounds, except for streptomycin, kanamycin and cycloserine, which were eluted close to the void volume of the column. In a limited pilot study, all quantifiable human samples had values within the validated assay ranges. CONCLUSIONS: The performance of our MS/MS detection technique was generally acceptable. The method provided rapid, sensitive and reproducible quantification of nine second-line anti-TB drugs and should facilitate drug monitoring during treatment.

Comparison of three multiple allergen simultaneous tests: RIDA allergy screen, MAST optigen, and polycheck allergy.

We compared the performances of 3 Multiple Allergen Simultaneous Test (MAST) assays: RIDA Allergy Screen (R-Biopharm, Darmstadt, Germany), MAST Optigen allergy system (Hitachi Chemical Diagnostics, Mountain View, CA), and Polycheck Allergy (Biocheck GmbH, Munster, Germany). Forty sera that tested positive with the RIDA Allergy Screen (20 for food and 20 for inhalant panel) were subjected to MAST Optigen and Polycheck Allergy. For 26 available sera with discrepant results, 62 ImmunoCAP allergen-specific IgE tests (Pharmacia Diagnostics, Uppsala, Sweden) were performed. Percent agreements (kappa value) were 87.6% (0.59) and 91.3% (0.60) between RIDA and MAST; 89.9% (0.55) and 88.3% (0.46) between RIDA and Polycheck; and 86.8% (0.51) and 90.6% (0.61) between MAST and Polycheck. Compared with ImmunoCAP, agreements (kappa value) of inhalant and food panels were 51.7% (0.04) and 33.3% (-0.38) for RIDA; 60.7% (0.27) and 81.8% (0.59) for MAST; and 65.5% (0.26) and 45.5% (0.07) for Polycheck. The agreements between RIDA, MAST, and Polycheck and ImmunoCAP-positivity were 45.7%, 88.2%, and 28.6%, respectively, and the agreements for ImmunoCAP-negativity were 37.0%, 51.9%, and 88.9%. MAST Optigen showed better agreement with ImmunoCAP than other assays in the food panel. Better sensitivity of MAST Optigen and better specificity of Polycheck Allergy were suspected.