- Clinical immunology lab
- Therapeutic drug monitoring
- Steroid hormones
- Tumor markers
- Infecious disease serology
- Trace and toxic elements
- Clinical diagnostic protein electrophoresis
- Allergy tests
Clinical Associate Professor, Pathology
Fellowship: Medical College Of Wisconsin Office of Graduate Medical Education (2006) WI
PhD Training: University of Sydney (1996) Australia
Board Certification: American Board of Clinical Chemistry, Clinical Chemistry (2008)
Ph.D., The University of Sydney, Biochemistry (1997)
M.D., Shanghai Medical University, Medicine (1987)
Current Research and Scholarly Interests
Clinical chemistry and therapeutic drug monitoring;
adult and pediatric clinical endocrine testing;
screening, detection and follow up of multiple myeloma;
clinical utility of tandem mass spectrometry and high resolution mass spectrometry.
Chromogranin A as Blood Marker in Cancer Patients
Gastroentero-pancreatic neuroendocrine tumors (GEP-NETs) are a heterogenous group of neoplasms that arise from enterochromaffin cells of the gastrointestinal (GI) tract and pancreas. They account for 50-70% of all incident NETs. Due to the lack of symptoms in the early stage of disease and the frequency of nonspecific GI symptoms, GEP-NETs are difficult to diagnose. Identification of effective biomarkers (such as Chromogranin A) to improve GEP-NET diagnosis, as well as to assess treatment efficacy, relapse and prognosis, is important for improving outcomes for patients with GEP-NETs. The purpose of this study is to validate the performance of Brahms (BRAHMS) Chromogranin A II Kryptor (KRYPTOR) assay to monitor the course of disease in patients with well-defined GEP-NETs.
Stanford is currently not accepting patients for this trial. For more information, please contact Kathleen Hornbacker, 650-721-4108.
Steroid testing by LC tandem mass spectrometry
Case-Control Study of Individuals with Discrepant Nucleocapsid and Spike Protein SARS-CoV-2 IgG Results.
Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results.Over a two-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis.There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] versus 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] versus 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6.Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.
View details for DOI 10.1093/clinchem/hvab045
View details for PubMedID 33720347
Pre-Operative Anti-Thyroid Antibodies in Differentiated Thyroid Cancer.
Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists
To evaluate the significance of anti-thyroglobulin and anti-thyroid peroxidase antibody levels in locoregional metastatic disease in patients with well-differentiated thyroid cancer.Included patients underwent initial treatment for well-differentiated thyroid cancer at our institution between 2014 and 2018. The following variables were collected: age, sex, pre-operative thyroid stimulating hormone (TSH), thyroglobulin (Tg), anti-thyroglobulin antibody (TgAb), anti-thyroid peroxidase antibody (TPOAb); extent of surgery; T-stage; N-stage; extrathyroidal extension (ETE), extranodal extension (ENE), lymphovascular invasion (LVI), and multifocal disease. The relationships between pre-operative TPOAb, TgAb, Tg, and TSH and disease status were analyzed.405 patients were included in the study. 66.4% were female. Mean age was 52 years. Elevated TgAb was associated with the presence of lymph node metastases (LNM) in both the central and lateral neck (p<0.01), with stronger correlation with N1b compared with N1a disease (p=0.03). Presence of ETE was inversely associated with TgAb titer (p=0.03). TPOAb was associated with lower T- stage, fewer LNM, and lower likelihood of ETE (p=0.04, p=0.04, p=0.02). In multivariable analysis, TgAb≥40 IU/mL was an independently predictive factor of higher N-stage (p<0.01 for N0 v. N1 and p=0.01 for N1a v. N1b), and for ENE (p<0.01). TPOAb≥60 IU/mL was associated with lower T-stage (p=0.04 for T< 3) and absence of ETE (p=0.01).Elevated pre-operative TgAb was an independent predictor of nodal metastases and ENE, while elevated TPOAb was associated with a lower pathologic T and N stage. Pre-operative anti-thyroid antibody titers may be useful to inform disease extent and features.
View details for DOI 10.1016/j.eprac.2021.06.014
View details for PubMedID 34217894
Performance evaluation and optimized reporting workflow for HIV diagnostic screening and confirmatory tests in a low prevalence setting.
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology
2021; 145: 105020
Our institution utilizes an antigen/antibody screening test followed by a confirmatory antibody assay for preliminary positive results. Given the low prevalence for HIV infections in our institution's county, we suspect that a substantial portion of the reactive screens are false positives.We aimed to characterize the false positivity rate of the HIV screening test performed at Stanford Health Care. In parallel, we modified our reporting workflow to release both the screening and confirmatory results simultaneously to mitigate the stress of a presumptive positive test.We reviewed 45,296 eligible HIV screen specimens that underwent the Abbott ARCHITECT™ 4th generation HIV antigen/antibody combination assay between August 5, 2016 and March 16, 2021. Final sample signal/cutoff (S/CO) ratios ≥ 1 were deemed positive, which triggers a reflex order for the confirmatory Bio-Rad Geenius™ HIV 1/2 Supplemental Assay. Additional chart review was performed for positive screen cases with negative or indeterminate confirmatory results.Our institution demonstrated a 0.28% (128/45,296) positive screen rate, with 12.5% (16/128) of these samples confirmed as false positives based on a negative HIV-1 RNA test. Median S/CO ratios of true positive screens were significantly higher than those with negative or indeterminate confirmatory tests (602.27vs 2.98; p = 0.0000323). We implemented a new synchronized reporting system for positive screens, which co-releases screen and confirmatory reports without compromise in the overall turnaround time.Our study demonstrates a relatively high percentage of false positive screens. Subsequently, by providing a more complete picture up front, our new reporting pipeline may reduce anxiety of a stand-alone positive screen and optimize downstream clinical decision-making.
View details for DOI 10.1016/j.jcv.2021.105020
View details for PubMedID 34736075
Simultaneous analysis of E1 and E2 by LC-MS/MS in healthy volunteers: estimation of reference intervals and comparison with a conventional E2 immunoassay.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
2021; 1178: 122563
Monitoring estrogen levels, especially estradiol (E2), is amongst others important for determining menopausal status and guidance of breast cancer treatment. We validated a serum E2 and estrone (E1) liquid chromatography tandem-mass spectrometry assay (LC-MS/MS) suitable for quantitation in human subjects. In addition, we compared our method with an E2 immunoassay (IA) and established preliminary reference values. Validation parameters were within the predetermined acceptance criteria. Assay linearity ranges were 4-1500 pmol/L for E1 and 4-2500 pmol/L for E2. Imprecision ranged from 7.4 to 9.6%. The lower limit of quantitation for E2 (8.0 pmol/L) was 11.4 times lower than the IA. The method comparison revealed differences in E2 quantitation up to 155% between both methods. The method allowed quantitation of E1 in all healthy volunteers, while E2 could not be detected in 95% versus 40% of the post-menopausal women using IA and LC-MS/MS, respectively. Male, pre-, peri- and postmenopausal female reference values were estimated. An LC-MS/MS based method combining E1 and E2 analysis was validated with superior E2 analytical sensitivity when compared to the IA.
View details for DOI 10.1016/j.jchromb.2021.122563
View details for PubMedID 34224962
Plasma as an alternative COVID-19 diagnostic specimen in a hospitalized patient negative for SARS-CoV-2 by nasopharyngeal swab.
Diagnostic microbiology and infectious disease
2021; 100 (3): 115365
We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.
View details for DOI 10.1016/j.diagmicrobio.2021.115365
View details for PubMedID 33865070
Evaluation of SARS-CoV-2 total antibody detection via a lateral flow nanoparticle fluorescence immunoassay.
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology
2021; 139: 104818
The coronavirus disease 2019 (COVID-19) endgame may benefit from simple, accurate antibody testing to characterize seroprevalence and immunization coverage.To evaluate the performance of the lateral flow QIAreach anti-SARS-CoV-2 Total rapid nanoparticle fluorescence immunoassay compared to reference isotype-specific IgG, IgM, and IgA SARS-CoV-2 ELISA using S1 or receptor binding domain (RBD) as antigens.A diagnostic comparison study was carried out using 154 well-characterized heparin plasma samples. Agreement between assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient.Overall agreement between the QIAreach anti-SARS-CoV-2 Total and any anti-spike domain (S1 or RBD) antibody isotype was 96.0 % (95 % CI 89.8-98.8), the positive percent agreement was 97.6 % (95 % CI 91.0-99.9), the negative percent agreement was 88.2 % (95 % CI 64.4-98.0). The kappa coefficient was 0.86 (95 % CI 0.72 to 0.99).The QIAreach anti-SARS-CoV-2 Total rapid antibody test provides comparable performance to high-complexity, laboratory-based ELISA.
View details for DOI 10.1016/j.jcv.2021.104818
View details for PubMedID 33932848
Plasmapheresis as an Early Treatment for Severe Hypertriglyceridemia, Acute Pancreatitis, and Diabetic Ketoacidosis.
AACE Clinical Case Reports
View details for DOI 10.1016/j.aace.2021.03.009
- The Simultaneous Quantitation of Five Tri-azole Anti-fungal Agents from Plasma Utilizing Paper Spray-Mass Spectrometry ASMS (Reboot) Conference on Mass Spectrometry and Allied Topics ASMS. 2020
Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry.
Clinical chemistry and laboratory medicine
Background Invasive fungal disease is a life-threatening condition that can be challenging to treat due to pathogen resistance, drug toxicity, and therapeutic failure secondary to suboptimal drug concentrations. Frequent therapeutic drug monitoring (TDM) is required for some anti-fungal agents to overcome these issues. Unfortunately, TDM at the institutional level is difficult, and samples are often sent to a commercial reference laboratory for analysis. To address this gap, the first paper spray-mass spectrometry assay for the simultaneous quantitation of five triazoles was developed. Methods Calibration curves for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were created utilizing plasma-based calibrants and four stable isotopic internal standards. No sample preparation was needed. Plasma samples were spotted on a paper substrate in pre-manufactured plastic cartridges, and the dried plasma spots were analyzed directly utilizing paper spray-mass spectrometry (paper spray MS/MS). All experiments were performed on a Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer. Results The calibration curves for the five anti-fungal agents showed good linearity (R2 = 0.98-1.00). The measured assay ranges (lower limit of quantification [LLOQ]-upper limit of quantitation [ULOQ]) for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were 0.5-50 μg/mL, 0.1-10 μg/mL, 0.1-10 μg/mL, 0.1-10 μg/mL, and 0.1-10 μg/mL, respectively. The inter- and intra-day accuracy and precision were less than 25% over the respective ranges. Conclusions We developed the first rapid paper spray-MS/MS assay for simultaneous quantitation of five triazole anti-fungal agents in plasma. The method may be a powerful tool for near-point-of-care TDM aimed at improving patient care by reducing the turnaround time and for use in clinical research.
View details for DOI 10.1515/cclm-2019-0895
View details for PubMedID 31926066
Large-Scale Testing of Asymptomatic Healthcare Personnel for Severe Acute Respiratory Syndrome Coronavirus 2.
Emerging infectious diseases
2020; 27 (1)
Large-scale, 1-time testing of >12,000 asymptomatic healthcare personnel in California, USA, during April-June 2020 showed that prevalence of severe acute respiratory syndrome coronavirus 2 was low (<1%). Testing might identify asymptomatic and presymptomatic persons, including some with high viral burden, enabling prompt implementation of measures to limit nosocomial spread.
View details for DOI 10.3201/eid2701.203892
View details for PubMedID 33256889
Evaluation of Abbott Anti-SARS-CoV-2 CMIA IgG and Euroimmun ELISA IgG/IgA Assays In a Clinical Lab.
Clinica chimica acta; international journal of clinical chemistry
We evaluated the test performance focusing on specificity of a fully automated Abbott Architect anti-SARS-CoV-2 CMIA IgG and Euroimmun anti-SARS-CoV-2 ELISA IgA/IgG in human plasma.We tested positive cohort of 97 samples from COVID-19 patients or healthcare workers, collected at late time points from symptom onsets. We also included another cohort of 215 samples as negative controls, 78 of which had positive serology test results of other infectious diseases or autoimmunity. Assay specificity was assessed by using a total of 847 anonymized samples which were collected before the COVID-19 pandemic from local patient populations seeking clinical care for rheumatoid diseases, thyroid cancer, and therapeutic drug monitoring.Abbott IgG, Euroimmun IgG/IgA had high precision, demonstrated by both intra- and inter-day CVs of <2%. There was no Abbott or Euroimmun IgG assay cross reactivity in the 78 samples with positive serology of non-SARS-CoV-2 infectious diseases and positive autoimmune antibodies. The Abbott IgG has specificity of 99.6%, while Euroimmun IgG and IgA were as high as 91.5% and 71.5%, respectively.our evaluation confirmed high specificity of the Abbott IgG assay, while it was lower for Euroimmun IgG. Euroimmun IgA has suboptimal specificity which may limit its clinical use. Assay sensitivity was high for both Abbott and Euroimmun IgG assays.
View details for DOI 10.1016/j.cca.2020.09.002
View details for PubMedID 32910980
Evaluation of the Aptima HCV Quant Dx Assay using Serum and Dried Blood Spots.
Journal of clinical microbiology
HCV RNA quantitation is the primary method by which active HCV infections are identified and the response to direct acting antiviral therapy is monitored. This study describes evaluation of the Aptima HCV Quant Dx assay (Aptima HCV) performed on the Panther system. The clinical performance of Aptima HCV was compared to the COBAS AmpliPrep/COBAS TaqMan HCV Test v2.0 (CAP/CTM). Overall agreement was 84.9% (186/219) with a kappa statistic of 0.755 [Standard Error 0.037; 95% confidence interval (CI) 0.682 to 0.828]. Passing-Bablok regression of log10 IU/mL values revealed a regression line of Y = 1.163*X - 0.991 [95% CI of the slope (1.103 to 1.221) and intercept (-1.341to -0.642)]. The 95% LLOD for Aptima HCV on DBS samples was calculated to be 2.43 log10 IU/mL (267 IU/mL) [95% confidence interval, 2.31 to 2.73 log10 IU/mL (204 to 540 IU/mL)]. Comparison of Aptima HCV testing on paired dried blood spot (DBS) and serum specimens collected from patients at the time of routine blood collection for CAP/CTM demonstrated an overall agreement of 90.1% (82/91) with a kappa statistic of 0.657 (Standard Error 0.101; 95% confidence interval 0.458 to 0.855) In conclusion, Aptima HCV provides a suitable alternative for HCV RNA testing on serum and DBS.
View details for PubMedID 30760534
Cinacalcet therapy in an infant with an R185Q calcium-sensing receptor mutation causing hyperparathyroidism: a case report and review of the literature.
Journal of pediatric endocrinology & metabolism : JPEM
Background Neonatal severe hyperparathyroidism (NSHPT) is commonly treated with either parathyroidectomy or pharmacologic agents with varying efficacy and numerous side effects. Reports of using cinacalcet for NSHPT have increased, however, the effective dose for pediatric patients from the onset of symptoms through infancy has not been established. Case presentation We describe the clinical course of a newborn with a de novo R185Q mutation in the calcium-sensing receptor (CASR) gene, causing NSHPT. The infant received cinacalcet from the first days of life until 1 year of age. Conclusions Cinacalcet therapy effectively controlled the patient's serum calcium, phosphorus, and parathyroid hormone (PTH) levels without side effects.
View details for PubMedID 30730839
- Automated High Throughput LC-MS/MS Quantitation of Testosterone from Serum: Validation and Interlaboratory Comparison MSACL 2019 US Annual Conference and Exhibits 2019
An Automated, Quantitative, and Multiplexed Assay Suitable for Point-of-Care Hepatitis B Virus Diagnostics.
2019; 9 (1): 15615
Hepatitis B virus (HBV) infection has a global reach with high prevalence in resource-limited areas like China and Africa. HBV patients in these areas have limited access to the currently used, costly HBV assays, which are performed in centralized clinical laboratories using single-plexed assays with bulky and expensive instruments. We aim to overcome these limitations by developing a simple and affordable HBV diagnostic platform to allow for timelier diagnosis and intervention of HBV infection. Using giant magnetoresistive (GMR) biosensor chips, we developed an automated and multiplexed quantitative platform for the measurement of a panel of HBV serology markers, including HBV "e" antigen (HBeAg), HBV surface antigen (HBsAg), and the antibody against HBsAg (anti-HBs). Our assay platform was able to detect each HBV marker with high specificity and sensitivity (with three orders of magnitude in dynamic range for each marker). Blinded analysis of HBV patient sera showed excellent correlation between our multiplexed quantitative HBsAg results and the qualitative results obtained using FDA-approved immunoassays, as well as those obtained using quantitative, single-plexed, enzyme-linked immunosorbent assays (ELISAs). The portable, automated, multiplexed, quantitative HBV serology assay platform we designed shows great promise as a more accessible alternative for HBV screening, diagnosis, and treatment monitoring.
View details for DOI 10.1038/s41598-019-52147-z
View details for PubMedID 31666635
- Implementation of User-Defined Lamotrigine and Levetiracetam Immunoassays for Therapeutic Drug Monitoring 71st AACC Annual Scientific Meeting & Clinical Lab Expo AACC. 2019
Salivary cortisol levels by tandem mass spectrometry during high dose ACTH stimulation test for adrenal insufficiency in children.
Serum cortisol measurements after ACTH stimulation are currently used to evaluate for adrenal insufficiency in children. We aim to determine if salivary cortisol measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can confirm or replace serum cortisol during high dose ACTH stimulation test to improve test compliance and interpretation. We also aim to gain preliminary understanding of normal ranges of salivary cortisol in normal children at am, bedtime, and midnight.Children aged 6-17 years meeting study criteria and tested for adrenal insufficiency were recruited to concomitantly collect saliva and serum samples during high dose ACTH stimulation test. Normal children aged 3-18 years were recruited to collect morning, bedtime, and midnight saliva samples. Salivary cortisol was measured using LC-MS/MS while serum cortisol was determined by an immunoassay.Salivary cortisol in normal children were higher at am and lower at bedtime and midnight (p value <0.0002 and <0.007, respectively). The midnight and bedtime levels were not sufficiently different (p value 0.36). Salivary cortisol during ACTH stimulation test positively and closely correlated with serum cortisol with 100% specificity and sensitivity when 18 µg/dL for serum and 500 ng/dL for salivary cortisol were used as cutoff values respectively for adrenal sufficiency.Measurement of salivary cortisol by LC-MS/MS is less invasive, more convenient and better time controlled in busy pediatric clinic, therefore is better suited for young children to be used during high dose ACTH stimulation test to evaluate for adrenal insufficiency and to assist interpretation of test results by serum cortisol.
View details for DOI 10.1007/s12020-019-02084-8
View details for PubMedID 31535345
Comparison of Transcription-Mediated Amplification and Real-Time PCR Assays for Hepatitis B Virus DNA Quantitation in Serum.
The journal of applied laboratory medicine
2019; 4 (3): 383–90
The quantification of hepatitis B (HBV) DNA in serum is critical to identify patients requiring antiviral therapy and to monitor the response to treatment.This study describes the evaluation of the Aptima HBV Quant Dx assay (Aptima HBV) performed on the automated Panther system.Aptima HBV was linear from 1.70 to 7.70 log10 IU/mL with a commercial reference panel, as well as clinical specimens representing genotypes B and C, and total imprecision, as measured by the percentage coefficient of variation (%CV) at 2.0 log10 IU/mL was <10%. The specificity of Aptima HBV was 94.7% (126/133) and 96.6% (84/87) for serum specimens from individuals without HBV exposure and individuals with resolved HBV infection, respectively. The qualitative agreement and quantitative accuracy of Aptima HBV was compared to the COBAS AmpliPrep/COBAS TaqMan HBV Test v2.0 (CAP/CTM). Overall agreement was 90.8% (187/206) with a κ statistic of 0.708 (standard error, 0.063; 95% CI, 0.585-0.831). Passing-Bablok regression revealed a regression line of y = 0.953x + 0.075 (95% CI of the slope, 0.883-1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (Aptima - CAP/CTM) showed a slight negative bias (-0.054 log10 IU/mL, and 95% limits of agreement of -1.093 to 0.984).The Aptima HBV test affords a suitable alternative to CAP/CTM for serum virus load testing and provides a key component of the diagnostic algorithm for the global eradication of viral hepatitis.
View details for DOI 10.1373/jalm.2019.029058
View details for PubMedID 31659075
Clinical Utility of an Ultrasensitive Urinary Free Cortisol Assay by Tandem Mass Spectrometry.
24 h urinary free cortisol measurement is a clinically important first-line screening test for Cushing's syndrome (CS). Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve and validate a LC-MS/MS method to measure urinary free cortisol in both adult and pediatric patients and to characterize its clinical diagnostic performance of CS by chart review.We improved a LC-MS/MS method previously reported for urinary free cortisol to be able to measure urinary and salivary cortisol in the same batch for increased efficiency. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay's analytical performance was characterized, and a retrospective patient chart review was conducted to evaluate the assay's clinical performance in diagnosing CS.The LC-MS/MS assay demonstrated enhanced sensitivity and was linear within an analytical measurement range of 10-10,000 ng/dL. Assay accuracy was satisfactory as determined by spike and recovery studies and highly correlated with a reference LC-MS/MS method. The assay's clinical diagnostic sensitivity and specificity in detecting CS was 96% and 91%, respectively, when compared to a urinary cortisol excretion of at least 50 µg/24 h.The improved LC-MS/MS method is both sensitive and specific with enhanced analytical performance and clinical diagnostic utility to screen for CS. The clinical diagnostic sensitivity and specificity were superior based on retrospective patient chart review.
View details for PubMedID 30951757
Association of 25-hydroxyvitamin D levels and cutaneous melanoma: A nested case-control study of the Women's Health Initiative Observation Study.
Journal of the American Academy of Dermatology
2018; 79 (1): 145–47
View details for PubMedID 29908819
Interfacing Complex Laboratory Instruments during a Change to Epic Beaker.
Journal of pathology informatics
2018; 9: 24
Background: Implementing a laboratory-developed test sometimes requires incorporating an unconventional device into the laboratory information system (LIS) and customizing an interface to reduce transcription error and improve turnaround time. Such a custom interface is a necessity for complicated high-volume tests such as 25-OH Vitamin D by liquid chromatography-tandem mass spectrometry (LC-MS/MS) when there is no vendor-or LIS-supplied interface available. Here, we describe our work and experience interfacing a API 5000 LC-MS/MS instrument with our newly implemented LIS, Epic Beaker, using a combination of in-house scripting software and a middleware vendor, Data Innovations.Materials and Methods: For input interfacing, custom scripting software was developed to transcribe batched order lists generated by Epic into files usable by the instrument software, Analyst. For output interfacing, results from the LC-MS/MS system were fed to a unidirectional instrument driver made by Data Innovations and selected data were transferred to the LIS.Results: Creation and validation of a new driver by Data Innovations took approximately 6 months. The interface was adopted for 25-OH Vitamin D and testosterone testing during periods of increasing test volume (4.5-fold over 8 years and 1.25-fold over 5 years). The amount of time spent reporting 25-OH Vitamin D results decreased 82% per order resulting in a savings of 1370 technician work hours and the amount of time spent reporting testosterone results decreased 75% per order resulting in a savings of 400 technician work hours.Conclusions: A mixed model using custom scripting and curated commercial middleware serve as a durable interface solution for laboratory instrumentation such as an LC-MS/MS and are flexible to future changes in instrument software, networking protocols, and the scope of LISs and work area managers.
View details for PubMedID 30034922
- Evaluation of a High Sensitivity Estrone and Estradiol Assay by LC-MS/MS 70th AACC Annual Scientific Meeting & Clinical Lab Expo AACC. 2018
Clinical Utility of an Ultrasensitive Late Night Salivary Cortisol Assay by Tandem Mass Spectrometry.
Late night salivary cortisol measurement is a clinically important and convenient screening test for Cushing's syndrome. Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve a LC-MS/MS method to measure salivary cortisol in both adult and pediatric patients and to characterize its analytical performance by method validation and clinical performance by chart review.We improved a LC-MS/MS method originally developed for urine cortisol to measure low level salivary cortisol. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay's analytical performance was characterized and retrospective patient chart review was conducted to evaluate the assay's clinical diagnostic performance.The LC-MS/MS assay showed enhanced functional sensitivity of 10 ng/dL for salivary cortisol and was linear within an analytical measurement range of 10-10,000 ng/dL. Assay accuracy was within 84-120% as determined by recovery studies and correlation with a reference method. Data from healthy adult volunteers was compiled to establish the reference interval for late night salivary cortisol. Patient chart review determined subjects with diagnosis of Cushing's syndrome or disease, and assay's clinical diagnostic sensitivity of 100% and specificity of 92% when the cutoff value was 70 ng/dL.The improved LC-MS/MS method is sensitive and specific with enhanced analytical performance and clinical diagnostic utility for screening Cushing's syndrome. The assay may have broad clinical application due to its high sensitivity and wide dynamic range.
View details for PubMedID 29197558
- Clinical Utility of an Ultrasensitive Late Night Saliva Cortisol Assay by Tandem Mass Spectrometry MSACL 2017 US 9th Annual Conference & Exhibits 2017
Implementation of Automated Calculation of Free and Bioavailable Testosterone in Epic Beaker Laboratory Information System.
Journal of pathology informatics
2017; 8: 28
Automated calculations by laboratory information system (LIS) are efficient and accurate ways of providing calculated laboratory test results. Due to the lack of established advanced mathematical functions and equation logic in LIS software, calculations beyond simple arithmetic functions require a tedious workaround. Free and bioavailable testosterone (BT) calculations require a quadratic solver currently unavailable as ready to use the function on most commercial LIS platforms. We aimed to develop a module within the Epic Beaker LIS to enable automatic quadratic equation solving capability and real-time reporting of calculated free and BT values.We developed and implemented an advanced calculation module from the ground up using existing basic calculation programming functions in the Epic Beaker LIS. A set of calculation variables were created, and mathematical logic and functions were used to link the variables and perform the actual quadratic equation based calculations. Calculations were performed in real-time during result entry events, and calculated results populated the result components in LIS automatically.Free and BT were calculated using instrument measured results of total testosterone, sex hormone binding globulin, and/or serum albumin, by applying equations widely adopted in laboratory medicine for endocrine diseases and disorders. Calculated results in Epic Beaker LIS were then compared and confirmed by manual calculations using Microsoft Excel spreadsheets and scientific calculators to have no discrepancies.Automated calculations of free and BT were successfully implemented and validated, the first of such implementation for the Epic Beaker LIS platform, eliminating the need of offline manual calculations, potential transcription error, and with improved turnaround time. It may serve as a model to build similarly complex equations when the clinical need arises.
View details for PubMedID 28828199
View details for PubMedCentralID PMC5545775
Rapid Measurement of Cyclosporine and Sirolimus in Whole Blood by Paper Spray-Tandem Mass Spectrometry.
2016; 62 (1): 295–97
View details for PubMedID 26430072
- Comparison between two fully automated immunoassay systems for the determinations of IgA anti-tissue transglutaminase antibody in celiac disease 68th AACC Annual Scientific Meeting & Clinical Lab Expo AACC. 2016
Rapid measurement of tacrolimus in whole blood by paper spray-tandem mass spectrometry (PS-MS/MS).
Clinica chimica acta; international journal of clinical chemistry
2015; 441: 99-104
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides sensitivity and specificity for monitoring tacrolimus drug level in blood, but it requires an LC system and sample preparation, which is not amenable to random access testing typical of immunoassays. Paper spray (PS) ionization generates gas phase analyte ions directly from dried blood spots without sample preparation and LC. We evaluated a PS-MS/MS method for tacrolimus drug monitoring in a clinical diagnostic laboratory.Whole blood sample was mixed with stable isotope labeled internal standard ([(13)C, (2)H2]-FK506) and spotted onto a cartridge containing triangular shaped card paper. After drying, samples were analyzed by PS MS/MS in the selected reaction monitoring (SRM) mode, with a run time of 3min/sample.Analytical measurement range was 1.5-30ng/ml. Assay inter-day imprecision was 13%, 8%, and 5% at tacrolimus concentrations of 4.5, 10.5, and 24.5ng/ml, respectively. Accuracy was determined by pure tacrolimus solution and was confirmed by result correlation to an immunoassay (slope=1.0, intercept=-0.02; r(2)=0.99), and to a conventional LC-MS/MS method (slope=0.90, intercept=0.4; r(2)=0.94).PS-MS/MS provides accurate results for tacrolimus with rapid turnaround time amenable to random access testing protocols.
View details for DOI 10.1016/j.cca.2014.12.022
View details for PubMedID 25540885
- Fast Determination of 17-Hydroxyprogesterone by LC-MS/MS for Diagnosis of Congenital Adrenal Hyperplasia MSACL 2015 Salzburg Annual Conference & Exhibits 2015
- A Rapid Sample Preparation Method for Quantitative Analysis of Cortisol in Saliva and Urine by LC-MS/MS MSACL 2015 US Annual Conference & Exhibits 2015
- Rapid measurement of tacrolimus, cyclosporin A and sirolimus in blood by paper spray-tandem mass spectrometry (PS-MS/MS) 66th AACC Annual Meeting & Clinical Lab Expo AACC. 2014
- Assessment of Normal Salivary Cortisol Values in Children Using Mass Spectrometry The Endocrine Society's 95th Annual Meeting and Expo Endocrine Society. 2013
- Rapid Measurement of Tacrolimus in Whole Blood by Paper Spray/Tandem Mass Spectrometry 65th AACC Annual Meeting & Clinical Lab Expo AACC. 2013
Serum testosterone quantitation by liquid chromatography-tandem mass spectrometry: Interference from blood collection tubes
2012; 45 (18): 1706-1709
During the development of a testosterone assay by LC-MS/MS, we encountered significant assay interference introduced by blood collection tubes. We examined a number of commonly used blood collection tubes for the presence of interference and its impact on testosterone quantitation.A number of commonly used blood collection tubes were examined by incubation of zero, low and high testosterone concentration samples with them over time, followed by sample preparation using liquid-liquid extraction and analysis by LC-MS/MS. Source of interference was identified by separately incubating blood collection tube coating, stopper and separator gel in clean glass tubes containing zero calibrator.Significant interference was found in some blood collection tubes, with the separator gel identified as the main source. The magnitude of the interference increases over time and mainly affected one of the two testosterone mass transitions used in the quantitation, making it readily detected by the discrepant results obtained by each of the two testosterone mass transitions. We were unable to eliminate the interference by adjustment of the sample preparation procedure, and by changing LC or MS parameters. Accurate quantitation of testosterone is possible when the problematic tubes are avoided, and blood collection tubes free of interference are used instead.Significant LC-MS/MS testosterone assay interference that originated from certain type of blood collection tubes hampered testosterone analysis. Examination of blood collection tube and any other laboratory test tubes for interference should therefore be an integral part of the development and validation of any LC-MS/MS assay used in a clinical diagnostic laboratory.
View details for DOI 10.1016/j.clinbiochem.2012.08.008
View details for Web of Science ID 000312048900035
View details for PubMedID 22971570
- Development and validation of a testosterone assay using liquid chromatography tandem mass spectrometry without derivatization Ned Tijdschr Klin Chem Labgeneesk 2012; 37 (3): 229-231
- Validation of confirmatory TPPA syphilis testing in the reverse sequence algorithm 64th AACC Annual Scientific Meeting & Clinical Lab Expo AACC. 2012
Rapid blood separation is superior to fluoride for preventing in vitro reductions in measured blood glucose concentration
JOURNAL OF CLINICAL PATHOLOGY
2009; 62 (8): 752-753
To determine whether tubes containing sodium fluoride negatively bias blood glucose concentration by directly comparing glucose concentrations in paired blood samples collected in tubes containing lithium heparin (Li-Heparin) and tubes containing sodium fluoride/potassium oxalate (NaF-KOx).Paired blood samples from a group of patients (n = 1040) were collected in tubes containing Li-Heparin and tubes containing NaF-KOx at the same time. All Li-Heparin samples were centrifuged soon after collection and were kept cool in transport along with NaF-KOx samples, which were centrifuged at the receiving location after an average transport time of 4 h, but immediately before analysis. Glucose concentrations in the paired samples were determined simultaneously by an automated oxidase method.The mean glucose concentrations for NaF-KOx samples and Li-Heparin samples were 5.7 mmol/l and 6.1 mmol/l, respectively, with a mean difference of 0.39 mmol/l.Rapid separation of heparinised blood is superior to fluoride alone for abrogating glycolytic effects on blood glucose measurements in the clinical laboratory.
View details for DOI 10.1136/jcp.2008.062547
View details for Web of Science ID 000268399100017
View details for PubMedID 19638548
- T3 Uptake Methods in Clinical Chemistry edited by Hickman, P. E., Koerbin, G. Pesce Kaplan Publishers. 2009; 5th: 1115
- Thyroid Autoantibodies Methods in Clinical Chemistry edited by Hickman, P. E., Koerbin, G. Pesce Kaplan Publishers. 2009; 5th: 1158
- Thyroglobulin (Tg) Methods in Clinical Chemistry edited by Hickman, P. E., Koerbin, G. Pesce Kaplan Publishers. 2009; 5th: 1151
- Overview of Pharmacogenomics and Applications for the Modern Clinical Laboratory Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory edited by Grody, W. W., Nakamura, R. M., Kiechle, F. L., Strom, C. Academic Press. 2009; 1st: 401
- A Case of Mixed Club Drugs Abuse Tietz's Applied Laboratory Medicine edited by Scott, M. G., Gronowski, A. M., Eby, C. S. Wiley. 2007; 2nd: 557–560
- A 43-Year-Old Male with Chronic Pain Tietz's Applied Laboratory Medicine edited by Scott, M. G., Gronowski, A. M., Eby, C. S. Wiley. 2007; 2nd: 561–565
Gene expression profiles in acute myeloid leukemia with common translocations using SAGE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (4): 1030-1035
Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15(+) myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15(+) cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes/pathways for more specifically targeted therapy.
View details for DOI 10.1073/pnas.0509878103
View details for Web of Science ID 000234938300036
View details for PubMedID 16418266
- The Past, Present, and Future of Pharmacogenomics Contemporary Practice In Clinical Chemistry edited by Clarke, W., Dufour, D. AACC Press. 2006; 2nd: 477
- Pharmacogenomics as an Aspect of Molecular Autopsy for Forensic Pathology/Toxicology Pharmacogenomics and Proteomics: Enabling the Practice of Personalized Medicine AACC Press. 2006
- Clinical Toxicology-Pharmacogenomics as an Adjunct for Interpretation of Drug Toxicity. Pharmacogenomics and Proteomics: Enabling the Practice of Personalized Medicine AACC Press. 2006; 1
Detecting novel low-abundant transcripts in Drosophila
RNA-A PUBLICATION OF THE RNA SOCIETY
2005; 11 (6): 939-946
Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244,313 SAGE tags from transcripts expressed in Drosophila embryonic, larval, pupae, adult, and testicular tissue. From these SAGE tags, we identified 40,823 unique SAGE tags. Our analysis showed that 55% of the 40,823 unique SAGE tags are novel without matches in currently known Drosophila transcripts, and most of the novel SAGE tags have low copy numbers. Further analysis indicated that these novel SAGE tags represent novel low-abundant transcripts expressed from loci outside of currently annotated exons including the intergenic and intronic regions, and antisense of the currently annotated exons in the Drosophila genome. Our study reveals the presence of a significant number of novel low-abundant transcripts in Drosophila, and highlights the need to isolate these novel low-abundant transcripts for further biological studies.
View details for DOI 10.1261/rna.7239605
View details for Web of Science ID 000229554000010
View details for PubMedID 15923377
- Opioids Intoxication, Diversion And Pharmacogenomics for a Case Involving Methadone And Oxycodone Ninth International Congress of Therapeutic Drug Monitoring and Clinical Toxicology Therapeutic Drug Monitoring. 2005
- Fentanyl-related Toxicity: A Summary Of 25 Postmortem Cases Ninth International Congress of Therapeutic Drug Monitoring and Clinical Toxicology Therapeutic Drug Monitoring. 2005
- CYP450 3A4 and 3A5 Genotyping in Methadone Intoxication Cases Using Pyrosequencing Ninth International Congress of Therapeutic Drug Monitoring and Clinical Toxicology Therapeutic Drug Monitoring. 2005
- Pharmacogenomics As An Adjunct Of Molecular Autopsy - A Multi-center Study For Methadone Death Certification: Preliminary Findings Of Data Acquisition And Multiplex Genotyping cyp2d6, 2C9, 2c19, 3a4 And 3A5 By Pyrosequencing™ Ninth International Congress of Therapeutic Drug Monitoring and Clinical Toxicology Therapeutic Drug Monitoring. 2005
SAGE is far more sensitive than EST for detecting low-abundance transcripts
Isolation of low-abundance transcripts expressed in a genome remains a serious challenge in transcriptome studies. The sensitivity of the methods used for analysis has a direct impact on the efficiency of the detection. We compared the EST method and the SAGE method to determine which one is more sensitive and to what extent the sensitivity is great for the detection of low-abundance transcripts.Using the same low-abundance transcripts detected by both methods as the targeted sequences, we observed that the SAGE method is 26 times more sensitive than the EST method for the detection of low-abundance transcripts.The SAGE method is more efficient than the EST method in detecting the low-abundance transcripts.
View details for Web of Science ID 000188773400001
View details for PubMedID 14704093
Over 20% of human transcripts might form sense-antisense pairs
NUCLEIC ACIDS RESEARCH
2004; 32 (16): 4812-4820
The major challenge to identifying natural sense- antisense (SA) transcripts from public databases is how to determine the correct orientation for an expressed sequence, especially an expressed sequence tag sequence. In this study, we established a set of very stringent criteria to identify the correct orientation of each human transcript. We used these orientation-reliable transcripts to create 26 741 transcription clusters in the human genome. Our analysis shows that 22% (5880) of the human transcription clusters form SA pairs, higher than any previous estimates. Our orientation-specific RT-PCR results along with the comparison of experimental data from previous studies confirm that our SA data set is reliable. This study not only demonstrates that our criteria for the prediction of SA transcripts are efficient, but also provides additional convincing data to support the view that antisense transcription is quite pervasive in the human genome. In-depth analyses show that SA transcripts have some significant differences compared with other types of transcripts, with regard to chromosomal distribution and Gene Ontology-annotated categories of physiological roles, functions and spatial localizations of gene products.
View details for DOI 10.1093/nar/gkh818
View details for Web of Science ID 000224207500011
View details for PubMedID 15356298
- Multiple Chromosome Translocations in Leukemia: Molecular Diagnosis Detection, Screening and Quantification LabMedica International 2004; 21 (2): 10-12
Screening and quantification of multiple chromosome translocations in human leukemia
2003; 49 (7): 1066-1073
Characterization of fusion gene transcripts in leukemia that result from chromosome translocations provides valuable information regarding appropriate treatment and prognosis. However, screening for multiple fusion gene transcripts is difficult with conventional PCR and state-of-the-art real-time PCR and high-density microarrays.We developed a multiplex reverse transcription-PCR (RT-PCR) assay for screening and quantification of fusion gene transcripts in human leukemia cells. Chimeric primers were used that contained gene-specific and universal sequences. PCR amplification of fusion and control gene transcripts was achieved with use of an excess of universal primers to allow the ratio of abundance of fusion gene to endogenous or exogenous controls to be maintained throughout PCR. Multiplex RT-PCR products analyzed by an ABI 310 Genetic Analyzer were consistent with those of duplex RT-PCR (single analytical sample plus control). In addition, multiplex RT-PCR results were analyzed by an assay using an oligonucleotide microarray that contained probes for the splice-junction sequences of various fusion transcripts.The multiplex RT-PCR assay enabled screening of >10 different fusion gene transcripts in a single reaction. RT-PCR followed by analysis with the ABI Prism 310 Genetic Analyzer consistently detected 1 fusion-transcript-carrying leukemia cell in 100-10 000 cells. The assay covered a 1000-fold range. Preliminary results indicate that multiplex RT-PCR products can also be analyzed by hybridization-based microarray assay.The multiplex RT-PCR analyzed by either ABI Prism 310 Genetic Analyzer or microarray provides a sensitive and specific assay for screening of multiple fusion transcripts in leukemia, with the latter an assay that is adaptable to a high-throughput system for clinical screening.
View details for Web of Science ID 000183789600007
View details for PubMedID 12816902
Imaging vascular thrombosis with Tc-99m-labeled fibrin alpha-chain peptide
JOURNAL OF NUCLEAR MEDICINE
2000; 41 (1): 161-168
An agent that permits scintigraphic detection of chronic deep venous thrombosis (DVT) or pulmonary embolism (PE) would be a welcome addition to the armamentarium of nuclear medicine. Because fibrin is the integral part of each clot, old or fresh, we hypothesized that a 99mTc-labeled fibrin alpha-chain N-terminal peptide, Gly-Pro-Arg-Pro-Pro, that binds to the C-terminal portion of the gamma-chain of fibrin can detect DVT and PE.The peptide was modified to Gly-Pro-Arg-Pro-Pro-Aba-Gly-Gly-(D)-Ala-Gly to permit efficient binding of 99mTc (99mTc-TP 850). The stability of the peptide was examined in vitro as well as in vivo. The ability of the agent to bind to rabbit, dog, and human fibrin and to inhibit adenosine diphosphate-induced platelet aggregation was examined. Blood clearance and 3-h tissue distribution were studied. DVT was induced in 8 rabbits using a stimulating electrode and in 2 rabbits by inserting a thrombin-soaked suture. PE was induced in 6 additional rabbits by introducing tantalum-impregnated blood clots into the right atrium, and the rabbits were radiographed to locate the emboli. 99mTc-TP 850 was then injected through a lateral ear vein, and each rabbit was imaged for up to 3 h. The rabbits were then killed, the heart and lungs were dissected and radiographed and the clots were harvested so that clot-to-blood radioactivity ratios could be determined.The peptide analog permitted efficient incorporation of 99mTc, which was stable in vitro and in vivo. The blood clearance was biphasic, with an alpha phase half-life of approximately 4 min (20%) and a beta phase half-life of approximately 13 min (88%). The mean binding of 99mTc-TP 850 to human, dog, and rabbit fibrin was 46% +/- 2%, 60% +/- 3%, and 56% +/- 2.5%, respectively, and the inhibitory concentration of 50% for dog and rabbit platelet aggregation was 236 pm and 167 pm, respectively. All clots, including 24-h-old pulmonary emboli, were delineated. The radioactivity associated with clots varied from 0.01 to 0.09 %ID/g, with clot-to-blood radioactivity ratios ranging from 1.2 to 12.0. However, 48-h-old pulmonary emboli had lysed and were seen neither by radiography nor by scintigraphy.A fibrin alpha-chain, N-terminal peptide that binds to the C-terminal portion of the gamma-chain of fibrin has been modified and labeled with 99mTc. The resultant peptide is stable in vitro and in vivo; binds to human, dog, and rabbit fibrin in large quantities; and inhibits platelet aggregation. The peptide clears rapidly from the blood and delineates experimental DVT and PE in rabbits. This agent is worthy of further investigation.
View details for Web of Science ID 000084762500026
View details for PubMedID 10647619
- Imaging thromboembolism with Tc-99m-labeled thrombospondin receptor analogs TP-1201 and TP-1300. Thrombosis Research 1999; 93: 191-202
Development of an enzyme-linked immunosorbent assay with monoclonal antibody for quantification of homovanillic in human urine samples
1998; 44 (8): 1674-1679
A monoclonal antibody to homovanillic acid (HVA) was prepared by synthesis of a HVA-protein conjugate (HVA-ovalbumin) as an immunogen, immunization of mice, and the subsequent hybridization technique. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and accuracy. An indirect ELISA was developed for quantification of HVA in human urine. The assay was characterized and shown to have high specificity, with cross-reactivities to vanillylmandelic acid and normetanephrine at 0.18% and <0.1%, respectively. The assay coefficients of variation were <10% within the working range of 0.5-40 mg/L. Initial results from testing urine samples of patients with neuroblastoma and other diseases were validated by HPLC, suggesting that this ELISA method is a reliable and convenient system for quantification of HVA in urine and can be used in the mass screening of neuroblastoma in infants.
View details for Web of Science ID 000075221600012
View details for PubMedID 9702954
Metabolic effects of thiopurine derivatives against human CCRF-CEM leukaemia cells
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
1998; 30 (8): 885-895
BACKGROUND and aims. To compare the metabolic effects induced by the anticancer drugs, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 6-methylmercaptopurine riboside (MMPR), which may inhibit the de novo biosynthesis of purine nucleotides or be mis-incorporated into DNA or RNA.Leukaemia cells were grown in culture, exposed to a thiopurine and cell extracts were analyzed for NTPs, dNTPs, drug metabolites and P-Rib-PP.In leukaemia cells, 6-MP was converted to MPR-MP, thio-XMP, thio-GMP, thio-GDP and thio-GTP. Metabolites of 6-TG included thio-XMP, thio-GMP, thio-GDP and thio-GTP, while MMPR-MP was the only major metabolite of MMPR, MMPR (25 microM, 4 h) induced a 16-fold increase in P-Rib-PP and 6-MP (25 microM, 4 h) induced a delayed 5.2-fold increase. MPR-MP, thio-GMP and MMPR-MP are inhibitors of amido phosphoribosyltransferase from leukaemia cells with Ki values of 114 +/- 7.10 microM, 6.20 +/- 2.10 microM and 3.09 +/- 0.30 microM, respectively.The nucleoside-5'-monophosphate derivatives of the 3 thiopurines inhibit amido phosphoribosyltransferase in growing leukaemia cells but there is also an initial inhibition of the further conversion of IMP in the pathway. In growing cells, MMPR acts solely as an inhibitor of de novo purine biosynthesis while 6-TG and to a lesser extent, 6-MP, are converted to significant concentrations of di- and tri-phosphate derivatives which may have other mechanisms of cytotoxicity.
View details for Web of Science ID 000075542700004
View details for PubMedID 9744080