Transcription free integration between an ABSciex LC MS-MS API 3200 and the iSOFT Telepath system for routine

Vitamin D Analysis
Craig Webster1, Rachel Henderson1, Kevin Jones2, Phil Goddard2 & Graham Pratt2
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Birmingham Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS CSols Ltd, Runcorn, Cheshire WA7 4QX
Reviewing and Reporting Results Once a run is completed, chromatograms of calibrants, QC material and patient specimens are reviewed in Analyst. Results are sent from Analyst to Telepath using the Links for LIMS Report Result program. The results are merged directly with the LIMS data collected earlier, and reporting rules are applied to account for the detection limit of the assay (Figure 4). A results summary table is produced by the Links for LIMS report result module, showing all results and allowing IQC checks (Figure 5).

Introduction Vitamin D is essential in calcium homeostasis and bone metabolism, as well as having roles in protection against certain cancers and in immunity. There are two forms of vitamin D vitamin D2 and vitamin D3. The major source of vitamin D is cutaneous synthesis of vitamin D3 upon exposure to UV radiation, whilst dietary sources of vitamin D2 and D3 are limited in most individuals. It is well documented that vitamin D deficiency is widespread worldwide, and is a major public health problem. The most accurate way to assess vitamin D status is to measure 25-hydroxyvitamin D (25(OH)D) in serum. There are currently many different laboratory methods to measure 25(OH)D, however mass spectrometry is increasingly being used in clinical laboratories due to its sensitivity and ability to equally measure 25(OH)D2 and 25(OH)D3. At Heart of England NHS Foundation Trust (HEFT), 25(OH)D is measured using an ABSciex 3200 LC-MS/MS instrument following liquidliquid extraction. Since 2006 the number of Vitamin D requests has grown exponentially, and now exceeds 25,000 per annum, with the mass spectrometer in almost constant use. In order to maximise efficiency and handle the increasing workload, all aspects of the process have been reviewed. HPLC conditions have been optimised to reduce the run time, and in 2010 the extraction process was automated with the use of a Tecan Evoware robot. One of the rate determining steps in delivering the overall service to clinicians and patients is the reporting of total 25(OH)D results. Prior to reviewing the process a significant amount of time was spent carrying out keyboard or manual transcriptions between different computer systems and paper. The workflow was: 1. worklist gather, collection of specimens and ordering according to worklist 2. liquid-liquid extraction using the Tecan Evoware robot 3. manual set up of batch on mass spectrometer 4. data analysis on mass spectrometer (examination and integration of peaks; report generation) 5. manual transcription of 25(OH)D2 and 25(OH)D3 results into Telepath (total 25(OH)D calculated in Telepath and reported, D2 and D3 results are visible but not reported) 6. manual checking of results and authorisation Aims The overall aim of the project was to provide an error and transcription free, more rapid workflow for the setup and the reporting of vitamin D analyses, through the use of the CSols Links for LIMS instrument integration program along with barcode labels, the Tecan Evoware robot and the ABSciex Analyst software package. More specifically the project aims were to: - develop a random access system to replace the use of worklists, enabled by the use of a barcode reader on the Tecan Evoware robot - use specimen barcode information from the Tecan Evoware robot to set up batch tables on the mass spectrometer - enable the easy, efficient and accurate transfer of results from Analyst to Telepath, via the CSols Links for LIMS integration program - apply rules to data for reporting of 25(OH)D2 and 25(OH)D3 results below the limit of detection of the assay - provide practical solutions for reviewing internal quality control (IQC) data before reporting results - provide a mechanism for verification and audit of data transfer - provide a commercially available, fully supported solution that could be adapted to other assay methods Methods CSols Links for LIMS is a modular PC software application that runs in the Windows environment and is easily installable. It mediates and controls data transfer between the LIMS package (iSOFT Telepath), the mass spectrometer (ABSciex Analyst software) and the Tecan Evoware robot. There are two main Links for LIMS application programs (Setup and Report Results programs) installed, along with supporting directories, configuration files, and a configuration utility program. The majority of user interaction is done via the Setup and Report programs, while the communication with Telepath is done by locally installed background 'driver' programs. Tecan Evoware Robot All specimens are labelled on receipt by the laboratory with a numeric barcode label and tests are requested in Telepath. The Tecan Evoware robot has a barcode label reader mounted on the robot deck, which is used to read the barcodes of the specimens placed in racks (Figure 1). A file is produced containing details such as position of calibrators, IQC material and patient specimens, the volume detected and the volume pipetted by the Tecan Evoware robot during liquid-liquid extraction of 25(OH)D.

Event D2 Low D3 High/Normal D2 Low D3 Low D2 Normal/High D3 Low D2 Normal/High D3 Normal/High

D2 conc. (nmol/L) <5 <5 >5 >5

D3 conc. (nmol/L) >5 <5 <5 >5

D2 Interpretation 0 0 D2 Result D2 Result

D3 Total Vitamin D Interpretation (nmol/L) D3 Result <5 0 D3 Result D3 Result <5 D2 Result D2 Result + D3 Result

Figure 4 Truth table showing result interpretation

Figure 5 Completed results in the Links for LIMS Report Result program aligned with the LIMS sample data

IQC Checks Links for LIMS will automatically perform IQC checks by comparing values with target values (Figure 6). Results within 2 SDs of the target value are displayed in green; results between 2 & 3 SDs in yellow, and IQCs greater than 3 SDs from the target value are highlighted in red. An IQC failure will flag sample results that follow and precede it, if required. Cumulative IQC data will be available to enable trends to be easily observed.

Figure 6 CSols AqcTools software being used to chart the current and previous IQC results
LIMS Upload The final step is the error and transcription free upload of results to Telepath in a fraction of the time taken to manually enter the results into Telepath. Within the Links for LIMS Report Result module, results are sent to Telepath in a single click. Links for LIMS enters a dialog with Telepath transferring all the results, as well as audit information such as instrument number and user identity.

Figure 1 - Tecan Evoware robot showing racks of samples being loaded prior to liquid-liquid extraction of25(OH)D.

Sample validation, Instrument setup and loading The Links for LIMS Setup program reads the Tecan plate file, verifies the samples in Telepath and extracts patient information, generating a pre-run table (Figure 2). Dilution factors can be included where appropriate. The pre-run table information is used to create an instrument run file in Analyst. There are differences between the Tecan and instrument sample ordering (row vs column orientation), therefore an inhouse template instrument run file with the correct sample positioning was used by Links for LIMS to populate a run file within Analyst with sample details (Figure 3). A unique data file name is generated per run to ensure that no data is overwritten. All the LIMS information is retained by Links for LIMS until the results are reported and then archived for future recall if necessary.

Figure 7 Links for LIMS result dialog with Telepath.

Conclusion In combination with the Tecan Evoware robot barcode reader, the use of Links for LIMS in this transcription free workflow has made a number of improvements in the routine operation of vitamin D analysis. These are: a) Significant saving of analyst time, by removing the manual transcription steps and crosschecking of results in Telepath b) Improved consistency of treatment of results and reduced error rates due to automatic transfer of results along with automatic reporting rules c) Improved monitoring of assay performance through improved IQC checks and 'at-a-glance' charts Once the system is in full routine use, Links for LIMS will be extended to other instruments and/or analytes to further improve the efficiency and quality of results delivery.

Figure 2 Example Links for LIMS pre-run setup layout

Figure 3 Analyst Batch Editor showing the run after importing

Heart of England NHS Foundation Trust BirminghamHeartlands Hospital, Bordesley Green East Birmingham B9 5SS Tel: +44 (0) 121 424 3243 Web: http://www.heartofengland.nhs.uk www.heftpathology.com E-mail: Rachel.henderson@nhs.net

CSols Ltd The Heath, Runcorn, Cheshire, WA7 4QX, UK Tel: +44 (0) 1928 513535 Fax: +44 (0) 7006 061106 Web: www.csols.com E-mail: links4lims@csols.com