FIT Laboratory update
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- Adelia Lester
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1 FIT Laboratory update Sally C Benton Consultant Clinical Biochemist, Berkshire and Surrey Pathology Services (BSPS) Director, Bowel Cancer Screening Southern Hub Date: 26 th February 2019
2 February FIT laboratory challenges PRE-ANALYTICAL Stool isn t homogenous and has variable consistency Pickers from all manufacturers are different Inconsistent sampling techniques by patients Faecal Haemoglobin instability Impact of Hb variants? ANALYTICAL No assay standardisation no primary reference material/ method EQA scheme challenges No established IQC material Is a single cutoff appropriate? How to verify/ validate Fit analysers for UKAS Different methods give different results Unable to confirm if results are correct
3 Quantitative FIT systems FOB gold SentiFIT NS Prime HM-JACKarc OC Sensor PLEDIA
4 Quantitative FIT systems FOB gold SentiFIT 3 FIT systems recommended by NICE HM-JACKarc OC Sensor PLEDIA
5 Pre-analytical variation: Does the mass of sample loaded affect faecal Hb concentration? Question: Does the collar on the FIT devices stop excess sample going in to the buffer as claimed by manufacturers?
6 Does the collar on the FIT devices stop excess sample going in to the buffer? Conclusion: For all 4 manufacturer devices tested the collar appears effective in stopping excess faeces entering buffer (at amounts of faeces added) Further work: Repeat with more faeces added (ie overloading FIT devices) Look at impact of f-hb result if multiple dips of probe into faeces Piggott C, John C, Bruce H, Benton S. Does the mass of sample loaded affect faecal haemoglobin concentration using the faecal immunochemical test? Ann ClinBiochem 2018; 55(6):
7 Detection of Hb by FIT Slide from Maggie Carroll
8 Hb variants Slide from Maggie Carroll Potential impact = decreased detection of Hb = risk of false negative
9 Which Variants? Slide from Maggie Carroll Variant Abbreviation Chain affected % present in the samples tested Normal Normal α 2 β 2 HbA 2 = 2.6 Cord Blood FA α 2 γ 2 β (reduced) HbF = 62 / 78 Sickle SS α 2 β S 2 β-6(a3) Glu Val 95 / 93 Sickle trait AS α 2 β S β β 37 / 39 C AC α 2 β C 2 β-6(a3) Glu Lys 35 / 38 Sickle C CS α 2 β S β C β HbS = 50 HbC = 47 HbS = 48 HbC = 45 D-Punjab AD α 2 β D 2 β-121(gh4) Glu Gln 36 /38 E AE α 2 β E 2 β-26(b8) Glu Lys 27 / 25 Hb J-Broussais AJ α 2J β 2 α90(fg2) Lys Asn 22 Hb Q-India AQ α 2Q β2 α1 64(E13) Asp>His 17 Hb Manitoba AM α 2M β 2 α2 102(G9) Ser>Arg 9.7 β-thalassemia Major BTM α 2 β 0 2 β (reduced synthesis) HbF = 100 β-thalassemia carrier BTC α 2 β + β (reduced synthesis) HbA2 = 4.7 / 4.9
10 Do the Antibodies used in FIT systems detect Haemoglobin variants? Results: 17/ 20 Hb variants adequately detected by FIT 3/ 20 inadequately detected by FIT 1 = β -thalassaemia major (no beta-globin chain) 2 = cord blood samples (contains foetal Hb). Conclusion: Hb variants with a conformational mutation were adequately detected by FIT There is a risk of false low/ negative result in variants with a missing α or β globin chain Most of these patients would have regular transfusions so probably not a common clinical problem Carroll M, John C, Mantio D, Djedovic N, Benton S An assessment of the effect of haemoglobin variants on detection by faecal immunochemical tests. Ann Clin Biochem (6):
11 Specimen collection Hb is unstable in faeces Recommendation from all FIT suppliers and international experts is to collect samples straight in to FIT tube NOT in to a poo pot for lab to transfer in to FIT tube Laboratories have had to adapt sample collection processes Some give GP s FIT tubes to hand out Some offer a FIT postal service
12 Faecal Haemoglobin Stability Mellen et al. Evaluation of sample stability for a quantitative faecal immunochemical test and comparison of two sample collection approaches. Ann Clin Bio patients provided FIT device and remaining faeces in poo pot. 11 (8%) of patients had different interpretation
13 Assay standardisation Currently no primary reference material or method Impact different results on different platforms
14 Results from EQA material run on 4 different FIT analysers
15 IFCC FIT Working Group (International Federation of Clinical Chemistry & laboratory medicine) Chair Sally C. Benton Group Members Marieke Fasa Barcey Levy Han Mo Chiu Josep-Maria Auge Erin Symonds Petr Kocna Natasha Djedovic Judith Strachan Ingrid Zegers Shizuka Takehara Samantha Jones UK NL USA Taiwan Spain Australia Czech Republic UK UK Belgium Japan UK Corporate members Maurizio Gramegna Michael Zacherl Hideyuki Hayashi Takuo Ichiyanagi Tsuyoshi Fukuda Yasunobu Masuda Mr Yosuke Doi Dr Tetsuya Kosaka Motohito Fujimura Italy Sentinel Italy Sentinel Japan Eiken Japan Eiken Japan Kyowa Japan Kyowa Japan Alfresa Japan Alfresa Japan Wako First meeting held in Athens in June nd meeting held in Barcelona in October rd meeting held in Geel in May th meeting held in Vienna in June 2018 Terms of Reference To attempt to standardize analysis of haemoglobin in faecal samples by immunochemistry (FIT) To identify all sources of pre-analytical variation and standardise if possible To establish external quality assurance and third party internal quality control programmes To determine impact of assay interference of Hb variants and other factors
16 Assay standardisation progress Southern hub collaborating with Reference lab in Belgium and all suppliers to identify a suitable reference material There will always be slight differences but we aim to align results as much as we can between suppliers NICE FIT sub study collecting samples from patients using all 4 FIT devices to see if same/ different diagnostic accuracy with different FIT kits
17 EQA schemes update 2 schemes in the UK UKNEQAS WEQAS starting in April Multiple schemes globally (>15) Varying approaches by EQA schemes Lyophilised samples Pre-loaded FIT devices Faecal like material sent to labs to load in to devices Analytical evaluation of 5 schemes being carried out by the Southern Hub research team. 11 more schemes being contacted Challenge ensuring EQA scheme assesses analytical accuracy
18 IQC material update No established 3 rd party IQC material commercially available Labs have to use manufacturers IQC
19 What should be the reporting threshold for symptomatic FIT..? Considerations; Analytical performance characteristics Manufacturers recommendations Laboratory verification/ validation NICE guidance Diagnostic accuracy NPV and PPV/ clinical specificity and sensitivity Impact on referral/ endoscopy capacity
20 Analytical performance characteristics Fraser CG & Benton SC. Detection capability of quantitative faecal immunochemical tests for haemoglobin (FIT) and reporting of low faecal haemoglobin concentrations. Clin Chem Lab Med Limit of detection (LOD) The lowest concentration at which f-hb can be detected 95% of the time The concentration where f-hb can be reliably distinguished from a blank sample Limit of quantification (LOQ) The lowest amount of f-hb than can be reliably measured according to pre-defined analytical perfomance characteristics (CV <10%) Labs should not report a numerical value less than LOQ If f-hb is greater than LOD but less than LOQ, lab could report Hb detected at very low concentrations
21 What are labs reporting? sent on ACB mailbase for information on labs offering symptomatic FIT 10 labs in England responded 9 labs use 10ug/g as threshold 1 lab uses 7ug/g Only 2 labs had worked out LOD and LOQ of their instruments
22 NICE guidance: Evidence Base/ Diagnostic accuracy 10ug/g cut off as recommended in NICE guideline Author Year Country Patients in study cut off (ug/g) NPV PPV Sensitivity Specificity Number of cancers Mowat et al 2015 Scotland Rodriguez-Alonso et al 2015 Spain Should we be reporting Godber et al 2015 Scotland Droste et al 2011 Netherlands down to limit of McDonald et al 2012 Scotland Cubiella et al 2014 Spain 787 detection?? Cut off undetectable Hb Cancers missed Author Year Country Patients in study cut off (ug/g) NPV for Specificit CRC PPV Sensitivity y Number of cancers Cancers missed Mowat et al 2015Scotland 755 Undetectable (7ug/g) Rodriguez-Alonso et al 2015Spain 1003 Undetectable (0) Widlak et al 2016England 430 Undetectable (7ug/g)
23 Impact on endoscopy Threshold to use is a local decision Local lab, endoscopy services CCG to jointly decide Currently no published evidence to demonstrate impact of symptomatic FIT on endoscopy Lower the concentration that is reported = more people referred = pressure on endoscopy = more cancers/ precancers likely to be detected
24 February FIT laboratory current status PRE-ANALYTICAL Hb variants likely to have very minimal impact Collars on FIT tubes effective more work on-going Samples should be collected straight in to FIT devices Symptomatic FIT testing is now available across much of England ANALYTICAL Identification of a primary reference material/ method underway There are a number of EQA schemes now available. The most fit for purpose still to be confirmed Guidance is available on how to verify/ validate FIT analysers Still no established IQC material
25 February FIT laboratory current status PRE-ANALYTICAL Hb variants likely to have very minimal impact Collars on FIT tubes effective more work on-going Samples should be collected straight in to FIT devices Symptomatic FIT testing is now available across much of England ANALYTICAL Identification of a primary reference material/ method underway There are a number of EQA schemes now available. The most fit for purpose still to be confirmed Guidance is available on how to verify/ validate FIT analysers Still no established IQC material POST-ANALYTICAL What threshold should be actually be used.? LOD/ LOQ??
26 Berkshire & Surrey Pathology Services A joint venture between Frimley Park Hospital, Royal Surrey County Hospital, Royal Berkshire Hospital, Wexham Park Hospital and Ashford and St. Peter s Hospitals NHS Foundation Trusts
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