IgH/TCR Clonality Status. Performance Monitoring Cover Sheet. Final IgH Clonality Result IGH 131. Uncontrolled Copy

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1 Performance Monitoring Cover Sheet Participant No: Trial No: IgH Issue Date: 20 th December 2016 Performance Monitoring: Your Result N Consensus Clonality Result Final IgH Clonality Result IGH 131 Clonal Closing Date: 27 th January 2017 Final TCR Clonality Result TCR 132 Polyclonal (not clonal) The scoring system is a rolling scheme that will identify unsatisfactory performance or persistent unsatisfactory performance of any participant. This is in order that UK NEQAS LI can provide support and guidance where needed and ensure that the Genetics NQAAP are informed as appropriate.. Overall Classification for this Trial Running Classification Visit for further information on scoring systems. Report Issue Date: 23 Mar 2017; Distribution: IGH ; Version: v1.0.0 Page 1 of 8

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3 Trial Report Participant No: Trial No: IgH Issue Date: 20 th December 2016 Closing Date: 27 th January 2017 Two lyophilised samples were issued for this trial: IGH 131 and TCR 132. IGH 131 comprised of a cell line spiked into a buffy coat and TCR 132 comprised of pooled buffy coat. The samples were issued to 102 participants, who were asked to analyse the samples for IGH and TCR gene re-arrangements, respectively. Clinical Scenario: samples IGH 131 and TCR 132 should be considered to be taken from an 82 year old male presenting with unexplained anaemia and is found to have gastric ulceration on endoscopy. The sample is fragmented and crushed with poor morphology, but there are patchy aggregates of CD20+ lymphocytes in a background of reactive CD3 positive small T cells. Formalin fixed tissue is sent for clonality testing, given the inability to repeat the endoscopy. Clonality Results: In this trial 99 (97.1%) participants returned results. Of these, 96 participants submitted results for IGH and 96 for TCR. Please Note: Numbers in all tables may not equal the number of participants due to not all participants providing all information requested or to some participants performing combinations of techniques. Table 1: Overall Results Your Result T Final IgH 131 Clonality Result Final TCR 132 Clonality Result Polyclonal (not clonal) 3 (3.1%) 84 (87.5%) Clonal 93 (96.9%) 11 (11.5%) Pseudoclonal - - Multiple reproducible peaks (n 3) - 1 (1.0%) Report Issue Date: 23 Mar 2017; Distribution: IGH ; Version: v1.0.0 Page 3 of 8

4 N N IgH/TCR Clonality Status NB: The loci and reporting nomenclature below has been standardised based on the Euroclonality/BIOMED 2 guidelines 1. Table 2: IG Results by Locus IGH VH-JH IGH DH-JH IGK VK-JK IGK Kde IGL Your Result N N o labs performing assay(s) Polyclonal (not clonal) (3.1%) 11 (29.7%) 2 (4.0%) 37 (77.1%) 16 (84.2%) Clonal 93 (96.9%) 23 (62.2%) 45 (90.0%) 5 (10.4%) - Irregular polyclonal (not clonal) No (specific) product Multiple reproducible peaks - 1 (2.7%) - 3 (6.3%) 2 (10.5%) - 1 (2.7%) - 1 (2.1%) (4.0%) - - Pseudo-clonal Not evaluable - 1 (2.7%) 1 (2.0%) 2 (4.2%) 1 (5.3%) Table 3: TCR Results by Locus Your Result N TCR Beta V-J TCR Beta D-J TCR Gamma V-J TCR Delta N o labs performing assay(s) Polyclonal (not clonal) 53 (82.8%) 43 (74.1%) 69 (74.2%) 22 (84.6%) Clonal 1 (1.6%) 7 (12.1%) 14 (15.1%) - Irregular polyclonal (not clonal) 9 (14.1%) 6 (10.3%) 6 (6.5%) 3 (11.5%) No (specific) product - 1 (1.7%) 1 (1.1%) - Multiple reproducible peaks - 1 (1.7%) 2 (2.2%) - Pseudo-clonal Not evaluable 1 (1.6%) - 1 (1.1%) 1 (3.8%) Report Issue Date: 23 Mar 2017; Distribution: IGH ; Version: v1.0.0 Page 4 of 8

5 Method Breakdown: Table 4: Kit/Protocol Invivoscribe Identiclone (IVD)Kit In-house method (BIOMED primers) Invivoscribe (RUO)Kit In-house (not BIOMED primers) Total number of data points for all IG Loci (and as % of returned results) Total number of data points for all TCR Loci (and as % of returned results) 123 (46.8%) 126 (51.9%) 115 (43.7%) 64 (26.3%) 15 (5.7%) 36 (14.8%) 3 (1.1%) 11 (4.5%) Master Diagnostica 3 (1.1%) 5 (2.1%) Other 4 (1.5%) 1 (0.4%) Report Issue Date: 23 Mar 2017; Distribution: IGH ; Version: v1.0.0 Page 5 of 8

6 Table 5: Analysis Method Capillary Electrophoresis Acrylamide Gel Electrophoresis Total (all IG Loci) Total (all TCR Loci) 218 (80.7%) 207 (82.8%) 20 (7.4%) 21 (8.4%) Heteroduplex Analysis 14 (5.2%) 11 (4.4%) Microfluidic Electrophoresis Next Generation Sequencing 6 (2.2%) 5 (2.0%) 5 (1.9%) 2 (0.8%) Radioactive Labelling 4 (1.5%) 3 (1.2%) Agarose Gel Electrophoresis 3 (1.1%) - Other - 1 Table 6: PCR Type Total (all IG Loci) Total (all TCR Loci) Multiplex 241 (91.3%) 230 (94.3%) Single 23 (8.7%) 14 (5.7%) Report Issue Date: 23 Mar 2017; Distribution: IGH ; Version: v1.0.0 Page 6 of 8

7 Trial Summary IGH: In line with sample formulation, 93/96 (96.9%) participants who returned results reported the IgH clonality status of sample IGH 131 to be Clonal. The three participants that reported the sample as Polyclonal (not clonal) all used an in-house approach, based on BIOMED, and capillary electrophoresis. There have been no significant changes in methodology since the last trial. Trial Summary TCR: 84/96 (87.5%) participants who returned results reported the TCR clonality status of sample TCR 132 to be Polyclonal (not clonal). Eleven participants reported the sample as Clonal and 1 participant reported the sample as Multiple reproducible peaks n 3. Of the laboratories that gave a Clonal result, 5 identified a clonal rearrangement in both TCR Beta D-J and TCR Gamma V-J, and 5 identified a clonal rearrangement in TCR Gamma V-J only. Those that gave a clonal band size identified 1-3 consistent minor clone(s) in TCR Beta D-J at bp; bp and bp and a single consistent minor clone in TCR Gamma V-J at ~ bp. 8/11 used the Invivoscribe Identiclone kit, 1 used an in-house (BIOMED) approach, 1 used an in-house (not BIOMED) approach, and 1 used the Invivoscribe RUO kit. Following consultation with our Specialist Advisory Group they feel that a Polyclonal (not clonal) classification would be the most appropriate interpretation in this scenario with the Optional: more detailed molecular interpretation allowing for the detection of a minor clone of unknown significance. However, as the guidelines also allow for a minor clone to be detected within a Clonal classification: clonality detected (minor clonal product) we will consider this as a satisfactory result for this trial. However, we would warn against over-interpretation of minor clones. There have been no significant changes in methodology since the last trial. References: 1. Langerak, A. W. et al. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia 26, (2012). Report Issue Date: 23 Mar 2017; Distribution: IGH ; Version: v1.0.0 Page 7 of 8

8 Information with respect to compliance with standards BS EN ISO/IEC 17043: a) The proficiency testing provider for this programme is: UK NEQAS for Leucocyte Pegasus House, 4 th Floor Suite 463A Glossop Road Sheffield, S10 2QD United Kingdom Tel: +44 (0) , Fax: +44 (0) nicola.rose@ukneqasli.co.uk b) The coordinators of UK NEQAS LI programmes are Prof David Barnett and Mr Liam Whitby c) Person(s) authorizing this report: Prof David Barnett, Director or Mr Liam Whitby, Operations Manager of UK NEQAS LI d) Pre issue testing of samples for this programme is subcontracted, although the final decision about sample suitability lies with the EQA provider; no other activities in relation to this EQA exercise were subcontracted g) The UK NEQAS LI Confidentiality Policy can be found in the Quality Manual which is available by contacting the UK NEQAS LI office. Participant details, their results and their performance data remain confidential unless revealed to the relevant NQAAP when a UK participant is identified as having performance issues i) All EQA samples are prepared in accordance with strict Standard Operational Procedures by trained personnel proven to ensure homogeneity and stability. Where appropriate/possible EQA samples are tested prior to issue. Where the sample(s) issued is stabilised blood or platelets, pre and post stability testing will have proved sample suitability prior to issue l), n), o), r) & s) Please refer to the UK NEQAS LI website at for detailed information on each programme including the scoring systems applied to assess performance (for BS EN ISO/IEC 17043:2010 accredited programmes only). Where a scoring system refers to the consensus result this means the result reported by the majority of participants for that trial issue. Advice on the interpretation of statistical analyses and the criteria on which performance is measured is also given. Please note that where different methods/procedures are used by different groups of participants these may be displayed within your report, but the same scoring system is applied to all participants irrespective of method/procedure used m) We do not assign values against reference materials or calibrants q) Details of the programme designs as authorized by The Steering Committee and Specialist Advisory Group can be found on our website at The proposed trial issue schedule for each programme is also available t) If you would like to discuss the outcomes of this trial issue, please contact UK NEQAS LI using the contact details provided. Alternatively, if you are unhappy with your performance classification for this trial, please find the appeals procedure at Report Issue Date: 23 Mar 2017; Distribution: IGH ; Version: v1.0.0 Page 8 of 8