Run Zhang Shi

SAGE is far more sensitive than EST for detecting low-abundance transcripts

BackgroundIsolation 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.ResultsUsing 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.ConclusionsThe SAGE method is more efficient than the EST method in detecting the low-abundance transcripts.

Serum testosterone quantitation by liquid chromatography-tandem mass spectrometry: interference from blood collection tubes.

OBJECTIVES 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. DESIGN AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Rapid Measurement of Cyclosporine and Sirolimus in Whole Blood by Paper Spray-Tandem Mass Spectrometry.

To the Editor: Paper spray (PS)1 is a recently described method for the direct mass spectrometry (MS) analysis of blood and other biological samples (1–3). Paper spray is performed by depositing a sample such as whole blood onto a paper substrate contained within a disposable cartridge; extraction and ionization occur directly from the disposable cartridge with an automated MS attachment (Fig. 1). No punching or offline extraction is required. Total analysis time, including sample extraction, is several minutes. Fig. 1. Paper spray for direct analysis of blood spots by MS. Ion chronograms from the lowest calibrator in whole blood, overlaid with representative chronograms from blank blood, are shown for cyclosporine and sirolimus. Quantification was performed with the entire area under the curve for each analyte, normalized by SIL-IS, to obtain calibration curves as shown. Because of its simplicity, paper spray could lower the barrier for implementation of MS-based assays in clinical laboratories. We previously used PS-MS/MS to monitor tacrolimus in clinical samples and showed good correlation with 2 immunoassays and an external HPLC-MS/MS method (4). Here we extend this approach by demonstrating the simultaneous quantification of cyclosporine and sirolimus by PS-MS/MS. Therapeutic drug monitoring of these drugs is important for organ transplant patient care. The method used was similar to our …

Rapid blood separation is superior to fluoride for preventing in vitro reductions in measured blood glucose concentration

Aims: 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). Methods: 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. Results: 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. Conclusion: Rapid separation of heparinised blood is superior to fluoride alone for abrogating glycolytic effects on blood glucose measurements in the clinical laboratory.

Screening and Quantification of Multiple Chromosome Translocations in Human Leukemia

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Clinical utility of an ultrasensitive late night salivary cortisol assay by tandem mass spectrometry

Background: Late night salivary cortisol measurement is a clinically important and convenient screening test for Cushings 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. Methods: 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 assays analytical performance was characterized and retrospective patient chart review was conducted to evaluate the assays clinical diagnostic performance. Results: The LC‐MS/MS assay showed enhanced functional sensitivity of 10ng/dL for salivary cortisol and was linear within an analytical measurement range of 10–10,000ng/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 Cushings syndrome or disease, and assays clinical diagnostic sensitivity of 100% and specificity of 92% when the cutoff value was 70ng/dL. Conclusions: The improved LC‐MS/MS method is sensitive and specific with enhanced analytical performance and clinical diagnostic utility for screening Cushings syndrome. The assay may have broad clinical application due to its high sensitivity and wide dynamic range. HIGHLIGHTSQuantitating late night salivary cortisol by an ultra‐sensitive LC‐MS/MS assay.Establishing reference interval for late night salivary cortisol.Retrospective patient chart review of Cushings diagnosis.Selecting cutoff value to determine clinical diagnostic sensitivity and specificity.

Interfacing complex laboratory instruments during a change to epic beaker

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.

Implementation of automated calculation of free and bioavailable testosterone in Epic Beaker laboratory information system

Background: 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. Materials and Methods: 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. Results: 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. Conclusions: 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.