Leading Edge An Alpha Laboratories Ltd. Publication 2024 - Issue 2 Boosting Efficiency in Calprotectin Testing: BA200 Standalone Analyser Provides Additional Flexibility Page 4 Also featured in this issue, case studies, news and product updates on: FIT - Faecal Immunochemical Testing Page 11 Aspergillosis Page 12 Sickle Cell Anaemia Page 14 Blood Grouping Quality Control Page 16 ACE for Sarcoidosis Page 17 Basophil Activation Testing for Allergy Page 18 Urinalysis Page 19 Discover more articles on the contents page inside.
2 Your #1 Resource for Calprotectin Testing Just Got Better To meet increasing demands for knowledge, resources and support for calprotectin testing, we have re-developed our popular website at www.calprotectin.co.uk New dedicated patient area Targeted content for healthcare professionals and patients Fresh layout design Easy to navigate pages Improved optimisation for mobile View the new website at www.calprotectin.co.uk Calprotectin .co.uk From Alpha Laboratories 2 Leading Edge
Welcome to the second edition of Leading Edge for 2024. In this edition, we are excited to bring you numerous diagnostic case studies from your peers. They delve into developments that help improve efficiency and workflow in the laboratory, new applications and reviews of established and trusted products. The standalone BioSystems BA200 random access, bench top analyser offers an excellent alternative to the main stream analysers for faecal testing, with comparable performance and efficiency. On page 4 read how the team at Maidstone and Tunbridge Wells NHS Trust have benefited from its introduction to boost their calprotectin throughput. NHS Tayside is also enjoying optimised workflows by using The Alpha Portal (TAP) to manage logistics for its FIT Kit supplies. Read Lynne Taylor’s report on page 10. At the Royal Brompton and Harefield Hospital Dr Ali Nuh has been exploring detection of Galactomannan in sputum to diagnose Aspergillosis. You can read the poster he presented at LabMedUK24 on page 12. The team from Haematology at Royal Stoke University Hospital discusses sickle cell anaemia and their use of the SICKLEDEX® kit – see page 14. Plus, we report from this year’s key clinical and laboratory conferences with posters highlighting the latest updates in calprotectin, FIT, and allergy. If you have an interesting application or research data that you would like to report on in future issues of Leading Edge, we would really love to hear from you. Please contact marketing@alphalabs.co.uk Contents Boosting Capacity & Efficiency in Calprotectin Testing A New Era at Maidstone and Tunbridge Wells NHS Trust Page 4 Gastroenterology Conference Season Highlights from Key Posters Page 6 New Testing Service Helps Diagnose Animals With Intestinal Inflammation University of Edinburgh Easter Bush School and Hospital for Small Animals Page 8 fCAL turbo® Packaging Update Page 9 The Alpha Portal Complete Solutions for FIT and Calprotectin Collection Kit Logistics Page 9 Optimising the FIT Workflow at Tayside Hospital Page 10 GP Referral Rate and Clinical Outcome of Patients According to FIT Result Judith Strachan, Consultant Clinical Scientist, NHS Tayside Page 11 Detection of Galactomannan in Sputum using a LFA Ali Nuh et al., Dept of Microbiology, Royal Brompton and Harefield Hospital Page 12 Screening for Sickling Haemoglobins Sam Johnson and Lauren Morris Royal Stoke University Hospital Page 14 Blood Group Serology (BGS) Reagents for Testing Validation and Control Page 16 Bring your testing up to date with the BÜHLMANN ACE Assay Page 17 Basophil Activation Testing The Latest Updates from EAACI 2024 Page 18 NEW Strategic Partnership to Revolutionise Urine Testing Page 19 Alpha’s Sustainability Update Page 20 www.alphalabs.co.uk © 2024 Copyright Alpha Laboratories Ltd. 3
Boosting Capacity & Efficiency in Calprotectin Testing A New Era at Maidstone and Tunbridge Wells NHS Trust Dave Parris and Yolanda Price, Blood Sciences Department, Maidstone and Tunbridge Wells NHS Trust Maidstone and Tunbridge Wells NHS Trust was established in February 2000 and is the largest acute hospital trust in the South East of England. Dave Parris and Yolanda Price from the Blood Sciences department talk about their experience evolving their calprotectin testing service: “We run the calprotectin service for the whole of Kent and Medway, so it isn’t just Maidstone as we are becoming a network. We already cover the calprotectin testing for the North and East Kent areas which includes Medway, Darrent Valley, Kent and Canterbury, Margate and Ashford, plus Maidstone and Tunbridge Wells hospitals. This means we are doing around 3,000 tests a month (~36,000 per annum) which has increased from ~32,000 per annum in 2023. The samples for analysis derive from primary care for diagnosis and also secondary care where they use the results for monitoring and for flares etc. Previously, we were using the BÜHLMANN fCAL® ELISA method on the DYNEX DS2 analyser, running 4 plates a day. Each plate would take two and a half hours to run, which meant it was becoming extremely difficult to keep up, especially if a plate failed or there was an analyser issue. Then we became behind as the assay was only run in routine hours and not at weekends or at night. This capacity constraint and the fact that the numbers requiring testing are still increasing, was the main reason for considering an alternative method. We wanted to continue using a BÜHLMANN assay because of the extraction method remaining the same. In addition the published literature indicated that there wasn’t much difference between the results obtained by the fCAL ELISA and turbo methods. Other manufacturers’ methods would have required greater resource to implement, because of the validation and communication, due to different results and cut-offs that are used. Wewere aware that the BÜHLMANN fCAL®turbo assay couldbe run on the main analysers. We have Roche c702 analysers here, but they are already almost at capacity. The blood work needs prioritising, so we didn’t feel it was a good idea to introduce the calprotectin to these lines. This is why we looked into other ways of switching to the fCAL turbo method, to increase the capacity, but without it being on the main line analysers. Introducing the BA200 The BioSystems BA200 stand alone bench top analyser seemed like the perfect solution, so this is what we evaluated, starting early 2023. The evaluation was quite straight forward, and we are lucky that we have a high sample throughput because we were able to collect samples that covered the entire range of the assay. The analyser is very user friendly and reliable - you can load the samples on the BA200 and walk away. Yolanda Price and Dave Parris with the BA200 Analyser 4 Leading Edge
Patient Sample Comparisons We ran around 300 patient sample comparisons and the usual linearity, LOQ, carry over, precision, inter and intra assay precision. With the intra assay comparison we used patient samples to cover the range of the assay (30, 100 and ~300µg/g) and the kit QCs, with results similar to those that BÜHLMANN quote. The patient comparisons were very similar using the York Care pathway cut-off values (<100, 100-250 and >250µg/g), although a few did change category (but only to the next category and not major shifts). Visit our dedicated Calprotectin website to find out more about the BioSystems BA200 www.calprotectin.co.uk/BA200 We did investigate these to see if there were colonoscopy results available and we found that the fCAL turbo was probably the better result out of the two. With the EQA we are nicely in the middle of the BÜHLMANN fCAL turbo range, which gives us confidence that the BA200 is operating the same as the main analysers with the fCAL turbo assay. Improved Efficiency The speed is the biggest benefit in moving to the BA200 as the first result is ready in 10 minutes – within 30 minutes you have 70 or so samples done, compared to 38 samples in almost 3 hours with the ELISA. To get through our daily workload of about 150 samples takes less than 2 hours and frees up the user to do something else. Also, because it is more reliable you don’t get the need for repeats. The BA200 maintenance is very easy, it has an initial warm up and blank which takes about 30 minutes and once you have learnt the software it is really straight forward. Ideally, the BA200 would be plumbed in, but that isn’t possible for us with the location the analyser is in. However, the tanks work OK and it is just a once a day check to ensure there is sufficient capacity on the water and the waste. We always ran the calprotectin service daily due to our volumes and we still do. But, with the speed of the new method, if we did decide to shut down the section for a day or so to support other areas, then we know we can catch up easily the following day. Previously, it would have been an almost impossible situation to catch-up, without doing night or weekend work. This really helps with our flexibility and the overall running of the laboratory, which is a big advantage. What Does The Future Hold? Moving forward we would like to look at giving the CALEX directly to the patients to collect and prepare their sample. But for the time being that is on hold because it may require the cutoffs adjusted, due to the improved calprotectin stability. The other thing that has been highlighted for the future is that faecal elastase can also go onto the BA200. We currently send elastase outside of the network for testing, so this has the potential to be a significant cost saving. It would also help with the patient pathway because we do get samples with requests for both calprotectin and elastase. Since they are currently tested in different locations we have to prioritise which test is completed and request a second sample for the other test. With the BÜHLMANN fPELA we can run both the calprotectin and the elastase from the same CALEX extraction. Switching from the calprotectin ELISA to the turbo method is a really good strategy, especially if you have a high volume of work. Having it on a stand alone analysers just gives you additional flexibility.” BA200 Linearity DS2 vs BA200 Comparison Mean value (µg/g) (N=20) 83.5 279.9 CV% 2.7 2.1 Inter Assay Precision Mean value (µg/g) (N=20) 36.4 83.2 (IQC) 102.6 278 (IQC) 343.9 CV% 7.1 2.5 3.8 1 0.7 Intra Assay Precision Switching from the ELISA to the turbo method is really a good move especially if you have a high volume of work. www.alphalabs.co.uk © 2024 Copyright Alpha Laboratories Ltd. 5
Gastroenterology Conference Season Highlights from Key Posters Amanda Appleton, Senior Product Manager, Alpha Laboratories Ltd. BSG In mid-June the FIT and Calprotectin Product Management team were at BSG in Birmingham, where there were lots of opportunities to engage with Gastroenterologists and Specialist IBD and endoscopy nurses. There was significant interest in the IBDoc® patient home test for calprotectin as well as the new direct to patient delivery service for this, using The Alpha Portal (TAP). It was also good to meet with the endoscopy nurses, who are often the next stage in the patient pathway, following a positive FIT or calprotectin test. LabMedUK The week before BSG, LabMedUK24 was held in Brighton. Whilst the main focus here is biochemistry, there was still a significant gastroenterology element, with talks on FIT and calprotectin, plus several relevant posters. The hot topic here was The Alpha Portal (TAP). This system enables stock management and delivery of ready made patient sample collection packs, for FIT or calprotectin, to the labs or direct to GP surgeries. It offers workflow improvements and significantly eases the logistics burden on laboratories. ECCO In February Amanda Appleton joined the BÜHLMANN team at the European Crohn’s and Colitis Organisation (ECCO) conference in Stockholm. Although this is a European conference it attracts an international attendance and one of the largest contingents is from the UK. This year there were 7223 delegates (mainly clinicians), from 99 different countries, all with a primary area of interest in Inflammatory Bowel Disease. Many interesting discussions were held, mainly on POCT and patient self-testing. GINCon The year started with GINCon - No, not a Gin conference but the 11th Gastrointestinal Nursing Conference in London in January. This is always a well-attended event with a varied and interesting programme covering current developments in IBD, assessment skills, supporting stoma patients in young adults, endoscopy as well as future developments. It was good to have conversations and help attendees understand how the products supplied by Alpha Laboratories can make their day to day easier and support the patient pathways. For more information, visit: www.calprotectin.co.uk The first half of the year is certainly gastroenterology conference season. Alpha Laboratories’ Gastroenterology Product Managers - left to right: Kayleigh Roberts, Barsa Manandhar, Amanda Appleton and Chloe Rose Kayleigh Roberts at LabMedUK 6 Leading Edge
Does extraction of faeces by the patient at home impact on the precision of faecal calprotectin analysis? Presented by Oliver Clifford-Mobley from Bristol Royal Infirmary Conclusion: Patient extraction of faeces for FC analysis is workable and does not adversely impact on result precision. Does extraction of faeces by the patient at home impact on the precision of faecal calprotectin analysis? Authors – O. Clifford-Mobley, A. Fraser, A. Hayes University Hospitals Bristol & Weston NHS Foundation Trust, Bristol, United Kingdom Our data demonstrate that CVa for duplicate patient extractions is variable, but on average of similar magnitude to CVa for faecal extraction obtained by trained lab technicians. The ELISA step itself has a lower CVa. This confirms that poor reproducibility of FC analysis is from the faecal extraction step itself; however, the identity of the person performing it makes no difference. In conclusion, patient extraction of faeces for FC analysis is workable and does not adversely impact on result precision. Discussion Calprotectin, a protein released by neutrophils during an inflammatory response, is detected in faeces of patients with inflammatory bowel diseases (IBD). Measurement of faecal calprotectin (FC) is used for screening and monitoring of IBD. Analytical methods for FC require extraction of calprotectin from faeces, typically performed in clinical laboratories by trained technicians. The introduction of simple devices for faecal extraction not requiring accurate weighing (e.g. BÜHLMANN CALEX® tube) has recently allowed patients to extract the faeces themselves. Measurement of FC suffers from poor reproducibility, even in experienced hands. It is unknown whether performing faecal extraction outside of a laboratory would worsen precision. This study attempted to answer this question. Introduction Packs containing 2x CALEX tubes, a stool-pot and instructions were distributed to 47 patients attending IBD clinic at Bristol Royal Infirmary. FC was measured by the BÜHLMANN ELISA method, with reportable range of 30-1800 µg/g. Within the lab, faecal pools are extracted with every batch for quality control (QC) purposes. The coefficient of variation (CVa=SD/mean) was calculated for duplicate patient extractions and for lab extracted faecal pool QC. Patient and lab extracted CV was compared by WilcoxonMann-Whitney test. Methods CALEX tubes were returned by 32 patients. 10 had at least one FC result outside the reportable range, such that the CVa could not be calculated. CVa for the remaining 22 duplicate patient extractions and for 23 lab extracted faecal pools over the last 2 years are shown in Figure 1. There was no statistically significant difference between patient and lab extracted CVa: median 19% and 22%, p=0.204. The spread of CVa was greater for patient extracted (0.1 – 44%) than lab extracted (5.3% – 36%). Repeat analysis of extract QC gave CVa of 7.4% and 9.5%. Results Figure 1: Box and whisker plot of CVa for duplicate patient extracts and lab extracted faecal pools. p=0.204 If you were unable to attend these meetings, here is a brief summary of the findings of some of the posters that were exhibited. Scan the QR codes to view the full posters: ECCO Comparison of clinical performance of fecal calprotectin of laboratory methods with lateral flow based POC and home tests. Presented by Christian Reinhard from BÜHLMANN Conclusion: The results presented here show that the four BÜHLMANN assays measure faecal calprotectin highly comparably and show an excellent clinical performance. This allows for the use of the methods interchangeably, depending on the needs of the patients and their care team. Feasibility study of a point of care assay for rapid determination of anti-TNFa biologics in capillary blood. Presented by Benjamin Ricken from BÜHLMANN Conclusion: The newly developed rapid Quantum Blue® assays for the determination of infliximab and adalimumab in capillary blood and EDTA whole blood are very well comparable to the analysis of either biologic in serum. This study furthermore demonstrated the assays are well suited to be used in a POC setting, such as infusion centres. Evaluation of the BÜHLMANN fCAL turbo assay on the Beckman Coulter AU5822 automated chemistry analyser. Presented by Helen Witham from Royal United Hospital, Bath Conclusion: The fCAL turbo evaluation was successful and employed into routine use in February 2024 without amendment to interpretative thresholds. This is a more robust assay with an increased measuring range (20 – 2000µg/g for turbo vs 10 -600µg/g on ELISA), improved accuracy (based on EQA results) and precision. The higher upper reportable limit on the fCAL turbo (without the need for sample dilution) was in particular recognised as a significant improvement by the gastroenterology team for monitoring and tailoring treatment of patients with known IBD. • Accuracy: A limited range EQA samples were available, we obtained results as expected from UKNEQAS reports for all 6 samples analysed. • Bias: An overall negative bias was demonstrated in patient samples run on the fCAL TURBO in comparison to results obtained on the fCAL ELISA (Figure 1). This bias was unsurprising as for unknown reasons the RUH fCAL ELISA method had a long demonstrated positive bias on the UKNEQAS EQA scheme when compared to the ALTM and method mean. • Precision: fCAL TURBO method inter-assay precision data was good and in keeping with manufacturer quoted precision. The intra-assay precision using patient samples was high in the low level patient sample but not clinically significant and likely to represent imprecision at the low end of the assay dynamic range: Evaluation of the BÜHLMANN fCAL®TURBO Assay on the Beckman Coulter AU5822 Automated Chemistry Analyser H.Witham & N.J. Pullan Pathology Department (Biochemistry), Royal United Hospital Bath NHS Trust, Combe Park, Bath, BA1 3NG helen.witham@nhs.net • Faecal samples were extracted using the CALEX® Cap extraction device (within 1-3 days of sample receipt). • fCAL BÜHLMANN TURBO method is a particle enhanced turbidimetric immunoassay (PETIA): The extracted sample is incubated with reaction buffer and mixed with polystyrene nanoparticles which are coated with calprotectin-specific antibodies. If calprotectin is available in the sample, an immunoparticle agglutination occurs. The turbidity of the sample is measured by light absorbance; an increase with calprotectinimmunoparticle complex formation is proportional to calprotectin concentration which is determined from the established calibration curve. • The fCAL TURBO evaluation was successful and employed into routine use in February 2024, without amendment to interpretation thresholds. This is a more robust assay, with an increased measuring range (20 to 2000 ug/g on TURBO vs 10 – 600 ug/g on ELISA), improved accuracy (based on EQA results) and precision. The higher upper reportable limit on the fCAL TURBO assay (without the need for sample dilution) was in particular recognised as a significant improvement by the Gastroenterology team for monitoring and tailoring treatment of patients with known IBD. • fCAL BÜHLMANN TURBO was assessed for: Inter-assay precision (manufacturer IQC run >20x over the course of the verification) Intra-assay precision (individual patient extracts run ≥10x on the same day) Accuracy (UKNEQAS EQA sample extracts (x6) run and compared to ALTM and method means) Bias(patient extracts) • Patient sample extracts (n=85) across the analytical range of the assay were run on both ELISA and TURBO methods within the same 24h period to minimise any effects of sample storage. • Faecal Calprotectin analyses is used as a tool in the assessment of intestinal mucosal inflammation and an aid to IBD disease monitoring. • Since 2021 Royal United Hospitals Bath (RUH) have employed the faecal calprotectin (fCAL) BÜHLMANN ELISA method on the Werfen DS2 analyser. • With a desire to further automate the assay and improve efficiencies, we sought to evaluate the fCAL BÜHLMANN TURBO method on the Beckman Coulter AU5822. References: Turvill J. et al. Evaluation of a faecal calprotectin care pathway for use in primary care. Primary Health Care Research & Development. 2016;17(5):428-436 BÜHLMANN fCAL® turbo Calprotectin turbidimetric assay Reagent kit package insert Inter-assay precision QC1 lot 4719 (n=29) QC2 lot 4719 (n=29) Mean (µg/g) 87 280 SD (µg/g) 2.8 7.0 CV (%) 3.2 2.5 Intra-assay precision Low patient (n=11) Intermediate patient (n=11) High patient (n=10) Mean (µg/g) 37 168 1945 SD (µg/g) 8.0 7.8 36.4 CV (%) 21.3 4.7 1.9 Results Principle of Method Background Special thanks to: • Amanda Appleton (Alpha Laboratories) • RUH Biochemistry colleagues: Tracy Hunt & Maria Saborido Figure 1 Method The difference in fCAL results was most significant at lower concentrations (-30% at <100 µg/g, -13% at 100-250 µg/g and -9% at >250 µg/g) (Figure 1). An allowable difference of ±20% in included on the difference plots below to provide allowance for inherent analytical imprecision (2 x ELISA target %CV of 10%). A mean difference of -30% at concentrations <100 µg/g was deemed clinically insignificant. Figure 2 • Clinical impact: given our intention to continue use of clinical thresholds based on the York Faecal Calprotectin Care Pathway), the verification samples results from both ELISA and TURBO fCAL methods were categorised as shown in Figure 2 (thresholds currently employed where patients have repeated fCAL tests). • This analysis highlighted that we would likely see more patients classified as IBD excluded and a reduction in patients classified as IBD unlikely and those requiring urgent referral. Conclusion Verification of the BÜHLMANN automated Elastase Assay on Abbott Alinity analysers. Presented by Alexandra Matthews from Queen Elizabeth Hospital, Birmingham Conclusion: The BÜHLMANN fPELA turbo assay for faecal elastase is acceptable for clinical use – it has acceptable precision and accuracy and will reduce turnaround time throughput the laboratory. Verification of the BÜHLMANN Automated Elastase Assay on Abbott Alinity analysers Authors – Alexandra Matthews, Jaime Weaver, Lee Briarwood, Briony Johnson and Alexander Lawson. Department of Clinical Chemistry, University Hospitals Birmingham NHS Foundation Trust, Birmingham, B15 2GW, United Kingdom. The BÜHLMANN fPELA® turbo assay for faecal elastase is acceptable for clinical use – it has acceptable precision and accuracy, and will reduce turnaround time and throughput in the laboratory. Elastase-1 is a pancreas specific enzyme that is secreted into the intestinal tract and excreted in faeces. Since it is not degraded during intestinal transit, its concentration in faeces reflects exocrine pancreatic function. Low faecal elastase-1 values are indicative pancreatic insufficiency. At University Hospitals Birmingham NHS Foundation Trust, the current method for measuring pancreatic elastase is a manual, 2-day ELISA (Schebo kit). Due to increasing workload, the aim of this project was to verify a semi-automated method for analysing faecal elastase on Abbott Alinity analysers. The BÜHLMANN faecal elastase® (fPELA) turbo test is a particle-enhanced turbidimetric immunoassay that enables relatively quick quantification of elastase in faecal extracts on clinical chemistry analysers. Accuracy, imprecision, limit of quantitation (LoQ) and linearity of the BÜHLMANN fPELA® turbo assay on the Abbott Alinity was evaluated. A sample comparison with the manual Schebo ELISA assay was also performed (n=26). Bland-Altman and Passing-Bablok statistics were used to assess these results. Stability of stool samples for elastase measurement was also assessed at room temperature and 2-8°C. Method Introduction Results Conclusion References: Whitcomb DC, Buchner AM and Forsmark CE. AGA Clinical Practice Update on the Epidemiology, Evaluation, and Management of Exocrine Pancreatic Insufficiency: Expert Review. Gastroenterology. 2023;165(5):1292-1301. BÜHLMANN fPELA turbo Abbott Alinity c Application Note fPELA Interpretation Normal Mild Severe Total Schebo Manual ELISA Interpretation Normal 9 0 0 9 Mild 1 5 2 8 Severe 0 1 8 9 Total 10 6 10 26 Sample Observed mean (ug/g) Calculated CVs Within-run Total QC1 (kit) 153.0 1.0% 1.4% QC2 (kit) 394.6 0.9% 0.9% Extraction QC (pooled patient material) 164.2 8.3% 14.8% Bias Patient comparison: When compared to the manual (weighing) Schebo pancreatic elastase-1 ELISA assay, the BÜHLMANN fPELA® assay showed an overall negative bias of -5.6%. Of the 26 patient samples analysed using both the manual ELISA and fPELA methods, 22 samples showed agreement for the interpretation of numerical results, which included all possible outcomes (severe insufficiency, mild-tomoderate insufficiency, and normal exocrine function). Four patient samples showed disagreement; however, of these, 3 were still classified as having exocrine insufficiency and therefore this would not change patient management. One patient sample was interpreted as mild-to-moderate insufficiency using the Schebo Biotech pancreatic elastase-1 ELISA kit, but normal according to the BÜHLMANN fPELA® turbo Abbott Alinity method. The ELISA result for this sample (199 µg/g) was close to the cut-off for normal exocrine function (>200 µg/g). Due to the heterogenous nature of stool samples, this was deemed to be clinically acceptable. EQA: The BÜHLMANN fPELA® assay showed an overall bias of -17.7% against the EQA ALTM. This is within the acceptable target bias of ±30% taken from the UKNEQAS EQA target. The z-scores for all samples were ≤2. LOQ: The lower limit for the BÜHLMANN fPELA® turbo method was verified by repeated analysis (n = 6) of a lowlevel sample giving a CV of 18.9% at a mean concentration of 9.9 µg/g. This is below the target CV of ≤20%. Stability: An in-house stability study showed that stool samples for faecal elastase are stable for 9 days between 2 and 8°C and for 14 days at room temperature. Precision: The precision data showed acceptable withinrun and total CVs (within the acceptance target of 15%). UKLabMed Evaluation and implementation of the BÜHLMANN fCAL turbo assay and CALEX extraction tubes on the Abbott Alinity C. Presented by Claire Paterson from Aberdeen Royal Infirmary Conclusion: As well as improved assay performance monitoring, the introduction of CALEX extraction devices has improved workflow, due to its ease of use and ability to be loaded directly onto the Alinity C. This significantly reduces staff time performing extractions. The ability to use the assay on routine clinical chemistry analysers has increased the availability of backup platforms. The department has already noticed a reduction in turnaround times, and these will improve further as the roll out of CALEX patient packs continues to all requesting areas. Evaluation and implementation of the BÜHLMANN fCAL® turbo assay and CALEX® extraction tubes on the Abbott Alinity C Authors – Claire Paterson, Julie Hawken, Fiona Brandie, Kevin Deans, Ewen Millar. Department of Clinical Biochemistry, Aberdeen Royal Infirmary, NHS Grampian The BÜHLMANN fCAL® turbo method demonstrates acceptable performance on the Alinity C using intra and inter-assay imprecision. Correlation of the two methods using patient samples did not meet the acceptance criteria. Discrepancy would be expected when comparing BÜHLMANN fCAL® method to the Alegria® due to different methodologies. In addition the sample type has great variability as calprotectin in not evenly distributed throughout the stool sample and it is not possible to ‘pick’ the exact same location for comparison3. Results obtained from EQA regression analysis showed satisfactory assay performance. Due to the higher number of Alinity C users in the EQA scheme the decision was taken to move to the BÜHLMANN fCAL® method to improve performance monitoring. As well as improved assay performance monitoring, the introduction of CALEX® extraction devices has improved workflow due to its ease of use and ability to be directly loaded onto the Alinity C, significantly reducing staff time performing extractions. The ability to use the assay on routine chemistry analysers has increased the availability of backup platforms. The department has already noticed a reduction in turnaround times and these will improve further as the roll out of CALEX patient packs continues to all requesting areas. The BÜHLMANN fCAL® turbo is an automated diagnostic test which can be utilised on a variety of chemistry platforms for quantitative determination of calprotectin in human stool1. The measurement of calprotectin can assist in distinguishing Inflammatory Bowel Disease (IBD), specifically Crohn’s disease or ulcerative colitis, from Irritable Bowel Syndrome (IBS) in patients with overlapping gastrointestinal symptoms. Calprotectin can also aid disease monitoring of patients with IBD2. The CALEX® sample extraction tubes allow for at home patient sampling and direct loading onto suitable analysers1. Intra-assay imprecision was performed using 20 replicates of 2 levels of internal quality control (IQC) material. Inter-assay impression was performed over 5 days using 5 replicates of 2 levels of IQC each day. Stool samples from 51 patients were extracted, analysed on the Alinity C (using CALEX® extraction devices) and the Alegria® (using the current extraction procedure) simultaneously and compared by regression analysis. Ten samples from the NEQAS Faecal Markers were analysed using the Alinity C and compared to the all laboratory trimmed mean (ALTM). A COGNOS search was performed on APEX to gather turnaround data for January to March 2023 (Alegria®) and January to March 2024 (BÜHLMANN fCAL® on Alinity C). The number of samples, median and average TAT were established as well as the time taken to generate 95% of results from receipt in lab to result availability. To evaluate and verify the BÜHLMANN fCAL® turbo particle enhanced turbidimetric immunoassay (PETIA) and CALEX® sample extraction tubes on the Abbott Alinity C analyser compared to the current Alegria® Calprotectin ELISA based method for the measurement of Calprotectin in human stool. Materials & Method Aim Intra and Inter-assay imprecision meet the manufacturer acceptance criteria of CV < 15% [Table 1]. Patient comparison between the Alegria and BÜHLMANN fCAL® turbo on Alinity C showed R2= 0.826 [Figure 2], acceptable criteria R2 >0.95. EQA correlation between the BÜHLMANN fCAL® turbo on Alinity C and ALTM was R2= 0.9621 [Figure 2], acceptable criteria R2 >0.95. There was a slight increase in workload between JanMar 2023 where 1417 samples were received, compared to 1456 between Jan-Mar 2024. The time from sample receipt until 95% of test results were available reduced after the switch to the BÜHLMANN fCAL® on Alinity C from 8 days 19 hours to 4 days and 22 hours in the period between Jan-March [Table 2]. Non-compliant rate also more than halved. Introduction Results Conclusions References: 1. BÜHLMANN. BÜHLMANN fCAL®: IFU Calprotectin turbidimetric assay for professional use; 2023. 2. Burri, Beglinger, Faecal calprotectin – a useful tool in the management of inflammatory bowel disease. Swiss Med Wkly; 142. 2012 3. Alpha Laboratories (2023): Calprotectin Information for patients. Available from: https://www.calprotectin.co.uk/about-calprotectin/information-for-patients/#1625652930187-dd8e44ab-4c25. Accessed April 15th 2024 Intra-assay Imprecision Inter-assay Imprecision Mean SD CV Mean SD CV µg/g µg/g (%) µg/g µg/g (%) Serum QC L 85.7 3.4 1.5 92.3 5.1 5.5 Serum QC H 287 4.2 1.5 289 3.7 1.3 Number of samples Median TAT Average TAT Time from sample receipt until 95% of test results available Noncompliant Rate Jan-Mar ‘23 1417 2 days 5 hours 3 days 20 hours 8 days 19 hours 1.3% Jan-Mar ‘24 1456 1 day 19 hours 2 days 5 hours 4 days 22 hours 0.6% Table 1: Intra and Inter-assay imprecision of the BÜHLMANN fCAL® turbo assay. Figure 1: Patient comparisons: Alegria FCal vs fCAL® turbo on Alinity C. Figure 2: EQA Correlation: fCAL® turbo on Alinity C Result v NEQAS Faecal Marker ALTM. Table 2: Turnaround times between January-March 2023 using Alegria® and JanuaryMarch 2024 using Buhlmann fCAl on the Alinity C. www.alphalabs.co.uk © 2024 Copyright Alpha Laboratories Ltd. 7
New Testing Service Helps Diagnose Animals With Intestinal Inflammation University of Edinburgh Easter Bush School and Hospital for Small Animals The University of Edinburgh’s Easter Bush Clinical Pathologists and Hospital for Small Animals’ Specialists in Internal Medicine, have validated the BÜHLMANN fCAL® turbo assay for use on their Beckman analyser. The test has now been made available by the Royal (Dick) School of Veterinary Studies to help veterinary surgeons diagnose and monitor chronic inflammatory enteropathy in their feline and canine patients. The clinical value of faecal calprotectin (fCAL) in humans has been recognised for many years, for the differentiation of Inflammatory Bowel Disease (IBD) from Irritable Bowel Syndrome (IBS) and the monitoring of IBD positive patients in the management of their disease. We have reported previously on some of the studies that have evaluated fCAL in dogs and cats and even in Jock the Silverback Gorilla at Bristol zoo. These studies demonstrate that fCAL can also assess intestinal inflammation in these animal groups, though values are much lower (cut off in the literature is around 48 µg/g) than the ones seen in IBD in people. Nevertheless, the results can still be of value diagnostically. Dogs and cats with acute and chronic diarrhoea have been found to have significantly higher fCAL levels than healthy controls. Like their human counterparts, fCAL increases in dogs with IBD (in small animals called chronic enteropathy; CE) and decreases with successful treatment. fCAL correlates with clinical severity as assessed by the CCECAI (canine chronic enteropathy clinical activity index) and with severity on histopathology. In one study, fCAL values of >15.2 µg/g were able to differentiate dogs with CE, that required immunosuppressive treatment, from others. Elevated levels were also more commonly seen in dogs with partial or no response, compared to complete/ good response to treatment, though there are issues with assay precision at lower values. If you would like any more information on the new testing service then please contact ebp.enquiries@ed.ac.uk or visit www.ed.ac.uk/vet/services/easter-bush-pathology 8 Leading Edge
The Alpha Portal – Complete Solutions for FIT and Calprotectin Collection Kit Logistics Having your kits on TAP means Traceable and efficient online ordering of FIT kits, CALEX patient packs, and IBDoc kits Improved efficiency with an end-to-end ordering process Reduced administration time Improved stock management Reduced storage requirements Direct delivery to clinic, primary care, or patient fCAL turbo® Packaging Update Amanda Appleton, Senior Product Manager, Alpha Laboratories Ltd. The packaging for the BÜHLMANN fCAL turbo® calibrator and control kits is changing from the current opaque white bottles to transparent vials with colour coded droppers and caps, as shown below: The new format will enable: The user to see what volume is left in the vial, making handing easier Colour coding makes it easier to keep the calibrators in the correct order for loading onto the analyser and prevents potential contamination with replacing the wrong lid on a vial The new vials have a more secure closure to prevent leakage during transportation The outer box is smaller, decreasing the amount of packaging waste and reducing the footprint for storage What has remained the same? Everything else is the same – the contents and total volume supplied remains unchanged. Current Calibrator Kit 1 x 6 bottles (1ml/bottle) New Calibrator Kit (B-KCAL-CASET) 1 x 6 vials (1ml/vial) Current Control Kit 3 x 2 bottles (1ml/bottle) New Control Kit (B-KCAL-CONSET) 1 x 6 vials (1ml/vial) What do I need to do? There is no change to the product codes or the pricing, so you don’t need to do anything – this is for your information only. It is estimated that this stock will begin being dispatched in approximately October 2024. If you have any questions or need further information, then please don’t hesitate to get in touch www.alphalabs.co.uk Scan the QR code to see how it works. To sign up, or for more information visit: alphalabs.co.uk/contact NEW NEW www.alphalabs.co.uk © 2024 Copyright Alpha Laboratories Ltd. 9 My workflow is greatly improved as I no longer have to wrestle with deliveries and break off from other jobs to package up parcels to send to GPs. – Lynne Taylor, Ninewells Hospital, Tayside
Optimising the FIT Workflow at Tayside Hospital Lynne Taylor, Study Coordinator, Tayside Hospital Lynne Taylor is the Study Coordinator at NHS Tayside Hospital, where she has worked for 50 years. Before introducing The Alpha Portal (TAP) for distributing sample collection kits, the FIT kitting process was very manual and time consuming. Tayside transitioned to using TAP back in November 2023, and Lynne has given us an insight into the benefits of using the portal for online ordering of FIT Kits for direct delivery to GPs. Lynne Taylor, Study Coordinator at NHS Tayside Scan the QR code to watch a video showing how The Alpha Portal works. To sign up, or for more information please visit: alphalabs.co.uk/TAPor contact: fit-kit@alphalabs.co.uk Before using TAP, how did you prepare and send patient packs out to GPs? “Before we started using the portal I could spend quite a lot of my day packaging and sending FIT kits to our users. Prior to considering sending kits out, I would have to make sure I always had plenty of stock which of course included stock control and finding storage space for the thousands of FIT tubes and envelopes we got through. I also had to manually unpack and transport all of this to and from storage. Once I received the stock, I would sit and count the instruction leaflets into bundles of 50 and rubber band them and include a with compliments slip which I also had to create and print. This was very time consuming! GPs and other users would email their requests to me, and I would then pack up the appropriate amount of supplies and send them out via our mail room to be delivered.” Roughly how long did this manual process take? “I could spend two or three hours dealing with sending out kits if I was busy and of course on the days I received a delivery then it could be much longer. Now it takes just seconds to order kits to each individual GP practice. It’s great!” Did you have any reservations about using TAP? “When we first considered the portal, I must admit to being a little apprehensive about losing control of deliveries. It was our own mail room and vans we used to deliver the kits and it was easy to find out where and at what stage the delivery was at. Any concerns I had were certainly unfounded. If there is ever a query about delivery, it is dealt with very timely and efficiently. I think there has only been one so far and it turns out to be even easier to track progress through Alpha. Customer service has been very good and there is always someone to answer your queries very quickly.” My workflow is greatly improved... I can also say that using the portal has released me to take on new tasks and responsibilities. How has being on TAP improved your workflows? “My workflow is greatly improved as I no longer have to wrestle with deliveries and break off from other jobs to package up parcels to send to GPs. I can also say that using the portal has released me to take on new tasks and responsibilities.” Are there any specific features that you like about TAP? “The portal is very clear and easy to use and the initial set up was easy, all I had to do was supply a list of GPs addresses and post codes. Considering the fact I have had no negative feedback at all from our users I think they are happy with the new system too.” 10 Leading Edge
GP Referral Rate and Clinical Outcome of Patients According to FIT Result Judith Strachan, Consultant Clinical Scientist, NHS Tayside At this year’s CRUK Early Diagnosis Conference in Birmingham, Judith Strachan presented her poster entitled: GP Referral Rate and Clinical Outcome of Patients According to FIT Result. Introduction Faecal Immunochemical Tests (FIT) use specific antibodies against intact human haemoglobin and early breakdown products to quantify the amount of blood in faecal samples (f-Hb). There is increasing and compelling evidence for the use of FIT to measure f-Hb in the triaging of patients with new bowel symptoms. All of the published studies generally show that FIT with a low cut-off has a high clinical sensitivity for colorectal cancer (CRC). Thus a high result should trigger a rapid referral for colonoscopy or similar investigations. Conversely, a low or undetectable FIT result alone has been shown to have a very high negative predictive value for CRC and other significant bowel disease. In the NHS Tayside Board area, FIT-KITS, supplied by Alpha Laboratories, along with patient instruction leaflets, have been available to GP practices since 2015. Patients are provided with a pictorial instruction leaflet and requested to return the completed FIT specimen collection device as soon as possible to the GP facility and, from there, the devices are delivered to Blood Sciences, Ninewells Hospital and Medical School, Dundee, at ambient temperature, by the routine sample collection service and, if required, stored at 4oC prior to analysis on an HM-JACKarc analyser. GPs are recommended by local and national referral guidelines to request f-Hb to guide referral of patients with any new lower GI symptoms and to use 10 µgHb/g faeces as the cut-off decision point. A FIT ≥400 µgHb/g warrants an urgent referral. Aim and Method The aim of this study was to assess referral rates and clinical outcomes of patients according to the FIT result. Patients with a colorectal bloods request who also had a FIT result between January 2022 and January 2023 were accessed via the laboratory computer system. The patient’s Community Health Index number was used to look them up on Clinical Portal, the NHS Tayside electronic reports system. From there, the Clinical Portal Communications record was accessed for each patient looking for a GP referral to Gastroenterology around the time of the FIT results. If a referral had been made, the outcome of this referral was accessed along with the results of any further investigation, usually a colonoscopy. Results The number of individual patients with both colorectal bloods and FIT result was 3993. Of these: 196 patients had a FIT ≥400 µgHb/g and 182 were referred (92.9%) 125 had a FIT 100-399 µgHb/g and 115 (92.0%) referred 107 had a FIT 50-99 µgHb/g, 102 (95.3%) referred 372 had a FIT 10-49 µgHb/g, 273 (73.4%) referred 3193 had a FIT <10 µgHb/g, 860 (24.8%) referred. In summary 78.4% of patients with FIT 10-≥400 µgHb/g and 24.8 of patients with a FIT <10 µgHb/g were referred. In the group of patients with FIT 10- >400 µgHb/g, 6.5% had CRC or high risk adenoma and 4.8% had Inflammatory Bowel Disease (IBD), whilst only 0.4% (3 patients) with FIT <10 µgHb/g had cancer/HRA and no IBD detected. Conclusions Almost 25% of patients with a FIT <10 µgHb/g were referred and had further investigation. In contrast only 78.4% of patients with a FIT result that should warrant a referral (≥10 µgHb/g) were referred. Of those with a FIT <10 who had further investigations, a very small number had evidence of CRC – in all 3, this was secondary to another pathology and not due to a Primary CRC and no cases of IBD were detected. Information and guidance should be issued to local GPs to further improve referral rates according to FIT results. For more information please visit: www.faecal-immunochemical-test.co.uk www.alphalabs.co.uk © 2024 Copyright Alpha Laboratories Ltd. 11
Detection of Galactomannan in Sputum using a LFA for the Diagnosis of Chronic and Allergic Pulmonary Aspergillosis Ali Nuh et al., Department of Microbiology, Laboratory Medicine, Royal Brompton and Harefield Hospital At this year’s British Society of Medical Microbiology annual meeting Dr Ali Nuh presented his team’s study: ‘Useofsputumtodetectgalactomannanforthediagnosis of chronic and allergic pulmonary aspergillosis- a casecontrol performance evaluation of lateral flow assay.’ Introduction Aspergillosis affects millions of patients worldwide. Laboratory testing of fungal biomarkers in respiratory samples is crucial for diagnosing aspergillosis. Although bronchoalveolar lavage (BAL) is a validated method for detecting these biomarkers, it is semi-invasive and requires expertise and expensive equipment. Therefore, there is a need for an alternative, non-invasive sample, and an easily performed method. In response to this need, we investigate the utility of IMMY Aspergillus Galactomannan Lateral Flow Assay (LFA) testing in sputum samples. Sputum is a non-invasive and easily collected alternative sample, and LFA is quick and easily performed. Methods We used sputum samples from patients referred to a cardiothoracic centre with full microbiological and radiological assessments. The study involved 32 patients with chronic pulmonary aspergillosis (CPA), 28 with allergic bronchopulmonary aspergillosis (ABPA), and 62 control patients. ABPA cases were defined by the International Society for Human and Animal Mycology criteria, while CPA cases were determined based on mycological evidence, radiological findings, and multidisciplinary team discussions following the European Society for Clinical Microbiology and Infectious Diseases guidelines. Sputum galactomannan was detected by LFA as well as enzyme-linked immunosorbent assay (PLATELIATM ASPERGILLUS Ag, BIO-RAD, UK) as per the manufacturer’s instructions. LFA strips were read by automated IMMY cube reader. Results In ABPA cases, the LFA showed a sensitivity and specificity of 89% each at the manufacturer’s cutoff of 0.5, with positive and negative predictive values of 78% and 95%, respectively. 12 Leading Edge
For CPA, the LFA gave a sensitivity of 75%, specificity of 89%, positive predictive value of 77%, and negative predictive value of 87% (table 1). The GM LFA also had a strong positive correlation and moderate concordance with the GM enzymelinked immunosorbent assay optical density index (r = 0.8, p < 0.001) (figure 1) (table 1). The receiver operating characteristic (ROC) curve analysis for ABPA indicated an area under the curve (AUC) of 0.94 (95% CI: 0.89-0.98) with an optimal cutoff of ≥ 0.5, maintaining a sensitivity and specificity of 89%. For CPA, the AUC was 0.91 (95%CI: 0.85-0.96) with sensitivity and specificity values of 75% and 93%, respectively, and an additional cutoff of 0.6 was identified (figure 2). Cube Readout Method Sensitivity (95% CI) Specificity (95% CI) Positive Predictive value (95% CI) Negative Predictive value (95% CI) Kappa (95% CI) P value CPA Versus Control LFA 75 (56-88) 89 (77-95) 77 (58-89) 87 (76-94) 0.50 (0.35-0.65) < 0.001 ELISA 100 (87-100) 64 (51-76) 59 (45-72) 100 (89-100) < 0.001 ABPA Versus Control LFA 89 (71-97) 89 (77-95) 78 (60-90) 95 (84-99) 0.56 (0.40-0.72) < 0.001 ELISA 93 (75-99) 64 (51-76) 54 (39-68) 95 (82-99) < 0.001 Table 1: Diagnostic performance of galactomannan lateral flow assay (cube-readout) and ELISA in sputum Figure 1: Scatterplot of the galactomannan (GM) lateral flow assay (LFA) optical density (OD) index and the GM enzyme-linked immunosorbent (ELISA) OD index GM LFA OD and GM ELISA OD indexes. In Pearson correlation, GM LFA was strongly positively correlated with GM ELISA OD index, r (152) = 0.8, p < 0.001. Figure 2: Receiver operating characteristics analysis curves for lateral flow assay for diagnosing chronic and allergic pulmonary aspergillosis. A) Allergic bronchopulmonary aspergillosis group versus the control group and B) Chronic pulmonary aspergillosis group versus the control. Scan the QR code to view this poster online Conclusion In conclusion, the IMMY Aspergillus Galactomannan LFA showed good diagnostic performance for CPA and ABPA using sputum samples at the 0.5 cutoff. As a non-invasive and easily collectible sample, sputum holds a significant advantage over conventional and invasive bronchoalveolar lavage fluid. The study suggests the need for further prospective research into the effectiveness of sputum galactomannan LFA. Find out more about Aspergillus Galactomannan LFA at: www.alphalabs.co.uk/ AF2003 www.alphalabs.co.uk © 2024 Copyright Alpha Laboratories Ltd. 13
Screening for Sickling Haemoglobins Using the SICKLEDEX® Solubility Test Sam Johnson (She), Senior Biomedical Scientist, Haematology and Lauren Morris, Associate Practitioner, Haematology, Royal Stoke University Hospital Sickle cell anaemia (SCA) is a lifelong disease caused by a point mutation in an individual’s beta globin genes, causing a hydrophilic amino acid on the surface of the haemoglobin molecule to be substituted for a hydrophobic amino acid. This mutation causes a qualitative disorder of haemoglobin known as sickle haemoglobin, or HbS. This genetic mutation is most common in individuals of African descent, but can also be seen in individuals of Middle Eastern, Asian, Indian and Mediterranean descent (American Society of Hematology, 2024). Under low oxygen tension, the haemoglobin molecules polymerise within the erythrocyte, distorting the shape of the erythrocyte into the characteristic sickle shape. These changes are reversible at first, with erythrocytes returning to their normal shape once re-oxygenated. However, repeated episodes of de-oxygenation and re-oxygenation leads to permanent sickling of erythrocytes. These sickled erythrocytes no longer possess the required deformability to fit through the microvasculature, delaying erythrocyte transit times and causing infarcts. Activation of the immune system and damage to erythrocytes can also cause haemolysis. During these sickle crises patients experience excruciating pain, and are at risk of tissue damage from vaso-occlusions and hypovolaemic shock from haemolysis. According to the NHS England (2023), there are at least 15,000 individuals currently in the UK with sickle cell disease (SCD). Sickle cell disease is an umbrella term for inherited haemoglobin disorders where either the HbS mutation is present on both beta globin genes, or the HbS mutation is inherited as a compound heterozygous disorder with another mutation that interacts with HbS causing a sickling disorder with a similar severity to SCA. In addition, approximately 1 in 79 babies born in the UK are carriers for sickle cell (Sickle Cell Society, unknown). Carriers are usually asymptomatic, however they can be at risk of sickle crisis in certain circumstances, for example, under anaesthetic. As they are usually asymptomatic, patients are often not aware that they carry the sickle mutation and therefore are not aware that they are at risk of sickle crisis in these circumstances. It is therefore important that patients requiring surgery from high risk ethnic groups are screened for HbS prior to admission, so that sickle carriers can be diagnosed ahead of their surgery and treated accordingly. High performance liquid chromatography (HPLC) or capillary electrophoresis (CE) are common methods of diagnosis within laboratories, however these both require specialist equipment as well as staff trained in interpretation of the results. Specialist testing can also be time consuming, with results taking a number of hours or days from receipt to reporting. This is, therefore, not ideal for patients requiring fast diagnosis. For example, patients who are admitted for emergency surgery, especially if the patient is admitted out of hours, will require a result in a matter of minutes, as opposed to hours or days. Hospitals will not necessarily have the staff and equipment available out of hours to be able to perform full testing for HbS (as well as other inherited disorders of haemoglobin). There is, therefore, a need for a quick and simple screening test to facilitate testing of patients in these emergency situations, such as the sickle solubility test. Antenatal Screening Programme In the UK all pregnant women and people are offered screening for inherited disorders of haemoglobin, including HbS, usually around 10 weeks gestation. Should a haemoglobin variant (or quantitative disorders of haemoglobin, such as thalassaemia) be detected, screening is also offered to the biological father of the baby. Due to the recessive nature of the mutation, couples where each parent carries a mutated haemoglobin gene have a 25% chance of the foetus inheriting no normal haemoglobin genes. The severity of this varies depending on the nature of the mutation. There are a number of clinically significant beta chain mutations, in addition to HbS, that are detected as part of the screening programme, including HbC, HbD Punjab, HbE, HbO Arab and beta thalassaemia (a quantitative beta chain disorder). These haemoglobin mutations do not cause a sickling disorder on their own; their significance is largely due to their interaction with HbS. For example, if HbS and HbD Punjab are inherited 14 Leading Edge
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