Clinical Assistant Professor, Pathology
Ph.D., The University of Sydney, Biochemistry (1997)
M.D., Shanghai Medical University, Medicine (1987)
Clinical chemistry and therapeutic drug monitoring; Screening, detection and quantification of chromosome translocations in leukemia.
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 Web of Science ID 000350085300018
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
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
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
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
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
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
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
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
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
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