Bone Marrow Processing Overview Document Reference: LP-SI-HMDS_BM Processing Overview

Transcription

1 EDITION NUMBER: 1 ACTIVE DATE: 27 th August 2013 REPLACES: - LOCATION OF COPIES: AUTHOR: Date: APPROVED BY: Designation Date: 1) Electronically through Q-Pulse 2) Printed out copy in HODS reception Ulrika Johansson 27 th August 2013 Dr Joya Pawade Head of Service, Haematology Oncology s Services 27 th August 2013 CONTENTS 1. PURPOSE AND SCOPE 2. REFERENCES 3. PROCEDURE 4. APPENDICES 1. Pathways 2. Complete HODS Histology repertoire 3. Complete HODS Flow Cytometry repertoire 4. Complete HODS Molecular repertoire 5. HODS staff list with contact details Approved by: Joya Pawade Page 1 of 4 Edition No: 1

2 1. PURPOSE AND SCOPE This document describes how Bone Marrow Samples are processed in the laboratory and provides an overview of the diagnostic pathways used. It contains information for how to contact the laboratory and for how to handle out-of hours sample requests. The purpose of this document is for new clinical and science staff to quickly become familiarised with the procedure it is not intended to provide a full account on how to carry out the procedure. 2. REFERENCES SI-HMDS-REF-WHO Classification Ed4, 2008 The current world health organisation guidelines: WHO Classification Tumours of Haematopoietic and Lymphoid Tissues. 4 th Edition. WHO Press, Swizerland. Eds Swerdlow, S.H., Campo, E., Harris, N.L., Jaffe, E.S., Pileri, S.A., Stein, H., Thiele, J., Vardiman, J.W. SI-HMDS-REF-NICE IOG 2003 The national institute for clinical excellence (NICE) improved outcome guidance (IOG): NICE- IOG_Haematological Cancer Services 2003 SI-HMDS-REF-NICE IOG 2012 Additional guidance published by NICE in 2012:NICE-IOG_Haematological Cancer Services Additional Guidance 2012 FLOW-REF-Johansson et al_bjh_2014 The current British Committee for Standards in Haematology (BCSH) guidelines for multicolour flow cytometry: Johansson, U., Bloxham, D., Couzens, S., Jesson, J., Morilla, R., Erber, W., & Macey, M. (2014). Guidelines on the use of multicolour flow cytometry in the diagnosis of haematological neoplasms. British journal of haematology, 165(4), Approved by: Joya Pawade Page 2 of 4 Edition No: 1

3 3. PROCEDURE Introduction The aim is to produce an integrated Bone Marrow Report incorporating all the diagnostic techniques of aspirate cytology, bone marrow histology, flow cytometry, cytogenetics and molecular diagnostics. This is done in accordance with the 2012 NICE-IOG guidelines (gateway number 17241). This will only apply to Adult Bone Marrow Samples. An overview of the bone marrow diagnostic pathway is included in Appendix 1. Sources of Bone marrow samples 1- UHB Trust 2- Weston Super-Mare 3- Ad hock samples from other trust 4- Pre transplant samples for review- From various locations, predominately the South-west. Requesting Bone Marrow samples The bone marrow investigations (slides/aspirate, flow, molecular, cytogenetics, trephine and iron stains) are requested on ICE by clinicians. The samples (slides/aspirate, EDTA samples for flow and molecular analysis, cytogenetics sample in heparin or transport media, and trephine pot) must be sent as one single package together with a print-out of the ICE request form. Every case needs to be sent immediately to the HODS reception (BRI main building, Pathology, level 8) for booking in and processing as soon as possible. All samples should be kept at room temperature. Approved by: Joya Pawade Page 3 of 4 Edition No: 1

4 Booking in Every case will be booked in on Cellpath by laboratory staff in the SI-HMDS reception and thereby given a unique single identifying number. This includes all samples from referral centres. Labels with patient s name and case request number will be attached to the request form and to all individual specimens. The request form will indicate which samples have been taken: Slides/aspirate, flow, molecular diagnostics, cytogenetic, trephine and/or trial samples. Unlabelled specimens will not be processed. If samples have been requested but not received, SI-HMDS reception staff will note this on the request form and immunophenotyping report form. The originating clinic or SpR on laboratory rotation will be contacted to ensure that no sample has been lost in transport. Iron stains are requested by duty haematologist after slide/aspirate interpretation. The request for iron stain is made on the Iron stain request sheet in the flow cytometry laboratory (LF-FLOW-Iron stain record, located on the white board next to the flow lab reception desk). Bone Marrow Trephine The trephine biopsy will be sent immediately to Histopathology, level 9, for processing. Methods are all documented on Q-pulse and can be found by a search for HIS documents. The s and antisera used are listed in appendix 2. Bone Marrow Slides/Aspirate The slides, including any iron stains requested, are stained in the laboratory and taken into the morphology room for interpretation by haematologist. Specimen from referral centres may have slides analysed locally however, all slides are reported independently by UHB HODS as part of the integrated bone marrow report. Flow Cytometry The samples need to be processed and analysed within 24 to 48 hours for optimum results. Samples arriving after Monday Thursday will be processed the following morning; If a sample arrives after on a Friday it will be processed on Monday morning. Methods are all documented on Q-pulse and can be found by a search for FLOW documents. Tests offered are attached in appendix 3. Molecular s The relevant samples identified by SI-HMDS staff are analysed immediately. The RNA based assays need urgent processing for extraction of RNA. The molecular tests currently offered are attached in appendix 4. Methods are all documented on Q-pulse and can be found by a search for MOL documents. All bone marrow specimens are stored at +4 o C for 3-4 months to make possible subsequent DNA analysis. Therefore requests for further DNA based molecular tests can be done at a later stage by the team. Should this be required, tests are requested by direct contact with SI-HMDS staff. Reflex tests form flow to molecular section are requested on internal request forms (LF-SI-HMDS-Int Mol Req), sample and form are handed directly to molecular staff or placed in the molecular sample fridge (room 60 fridge). Approved by: Joya Pawade Page 4 of 4 Edition No: 1

5 Cytogenetics All samples requiring cytogenetics testing should be dealt with immediately for best results. These samples will be forwarded to Bristol Genetics Lab, Cytogenetics, Southmead Hospital, by HODS staff. Tests offered are karyotyping, and FISH for the most common and relevant SI- HMDS related abnormalities. A complete list of tests offered is awaited form the cytogentics lab at the time of writing this document. Transports leave at 0900 or 1400hrs every week day. If necessary samples can be stored at 4 o C over night. Urgent Samples arriving after 1400hrs on a Friday will need a courier (arranged by HODS staff). Samples stored by CG lab that requires to be analysed can be activated for analysis by contacting the laboratory by DutyScientistHaemato-Oncology@nbt.nhs.uk. Trial Samples Trial samples are booked in on ICE request and forwarded to the relevant trial centre / laboratory via the BHOC Trials Unit. Trials staff must be informed in advance of the procedure to allow collection and transport arrangements for the trial sample. After Hours Monday Thursday If a sample arrives after 5pm on Monday Thursday it will be dealt with the following morning. Samples will be left at room temperature and send to level 8 in the SI-HMDS reception After Hours Friday and Weekends If a sample arrives after 5pm on a Friday or at a Weekend, it will be dealt with on Monday morning. Urgent samples For all such samples the SI-HMDS reception should be informed and appropriate members of SI-HMDS team contacted SI-HMDS reception: Flow Ulrika Johansson extension Histology Dr Joya Pawade extension Molecular Tim Clench extension Approved by: Joya Pawade Page 5 of 4 Edition No: 1

6 Appendix 1 Pathways Disease FC IHC Molecular FISH Karyotyping CML sample CML FU samples MPN MDS New AML off Trial With patients >65 years to receive only supportive care it may suffice to just confirm diagnosis. AML off Trial FU samples Myeloid progenitor Myeloid progenitor If? Mastocytosis: Mast cell Myeloid progenitor MDS Acute leukaemia Myeloid progenitor MRD where available, post each chemotherapy course MPN MPN MPN MDS MDS MDS Qualitative and quantitative BCR-ABL on blood Quantitative BCR-ABL on blood Monitoring 3 monthly JAK-2 V617F, JAK-2 exon12 MPL515 MPL Baltimore BCR-ABL FIP1L1-PDGFRa (Salisbury) Familial ET: EPOR If? Mastocytosis: KIT D816V JAK-2 if thrombocytosis SRSF2 if CMML suspected Familial MDS/AML: RUNX1 mutations Qualitative PML-Rara, t(8;21), inv16, Flt-3 NPM-1 MRD where available t(9;22) t(9;22) Until negative or suspected relapse Not unless target suspected or identified by karyotyping Not unless target suspected or identified by karyotyping If? APML: t(15;17) If? Monomyeloid with eosinophilia: inv 16 If? With maturation: t(8;21) identified at Dx: Until negative or suspected relapse Yes Yes Yes Yes identified at Dx: Until negative or suspected relapse Approved by: Joya Pawade Page 6 of 4 Edition No: 1

7 Disease FC IHC Molecular FISH Karyotyping AML 17 samples AML 17 FU samples APML FU samples Acute leukaemia Also at NBT Lymphoid Malignancies CLL/SLL MCL FL HCL Myeloid progenitor MRD where available Also at NBT Until cytogenetic remission and at suspected relapse B-LPD Clonal B cell Absolute clonal B cell count from PB MRD analysis on FU PB samples B-LPD Clonal B cell B-LPD Clonal B cell B-LPD Clonal B cell MDS MDS MDS Low grade b cell lymphoma Low grade b cell lymphoma Low grade b cell lymphoma Flt-3 (also at Cardiff) NPM-1 Trial will determine other suitable markers and centre informed. identified at Dx. Also, if identified at Dx: WT-1 and CBF to Manchester and NPM-1 to London. Quantitaive PML-Rara (at Guy s Hospital) IgVh (<60 years) NOTCH1 Sox-11 BRAF V600E If? APML: t(15;17) If? Monomyeloid with eosinophilia: inv 16 If? With maturation: t(8;21) identified at Dx: Until negative or suspected relapse t(15;17) post each course until negative Trisomy 12, Del 13q, Del 11q, del 17p P53 t(11;14) t(14;14) if diagnostic uncertainty Yes Also Sample storage locally identified at Dx: Until negative or suspected relapse Approved by: Joya Pawade Page 7 of 4 Edition No: 1

8 Disease FC IHC Molecular FISH Karyotyping NHL HG NHL HL LPD Clonal B/T cell LPD KI-67 Clonal B/T cell Low grade B cell lymphoma High grade B cell lymphoma Hodgkin Lymphoma s If suspect: TCR/IgH clonality t(8;14) if Burkitt s needs excluding Burkitt s Lymphoma LPD TdT/Partial AL High grade B cell lymphoma t(8;14) if infiltration ALL sample Acute leukaemia BCR-ABL suspected or if identified by karyotyping t(9;22), t(11;23), TEL/AML-1 t(8;14) if Burkitt s needs excluding Yes ALL FU samples MRD identified identified ALL Trial sample Acute leukaemia Also at NBT BCR-ABL Yes ALL Trial FU samples MRD Also at NBT Myeloma/PCD sample Myeloma/PCD FU samples Plasma cell Panel Plasma cell Panel CD138, CD20, CD3, CD56 CD138, CD20, CD3, CD56 <50-60 years: Del 13q, t(11;14), t(4;14), del 17p Approved by: Joya Pawade Page 8 of 4 Edition No: 1

9 Appendix 2 HODS Histology Repertoire Reactive vs Lymphoma CD20 CD3 Bcl-2 CD23 Ki67 CD30 EBV High grade lymphoma- T CD3 CD5 CD4 CD8 CD30 CD10 Ki67 EBV CD21 ALK-1 Granzyme -B TIA-1?PD1-if AILD Low grade B cell lymphoma CD20 CD3 CD5 CD10 MUM-1 CD21 CD23 Bcl-2 Bcl-6 Ki67 Cyclin d1 Cytokeratin- MALT sites CD138 Kappa and lambda light chain depending on plasma cell compartment High grade Lymphoma, mostly - B CD20 CD3 CD5 CD10 CD23 BCL-2 BCL-6 Ki67 MUM-1 EBV above 50 or immunocompromised Approved by: Joya Pawade Page 9 of 4 Edition No: 1

10 Hodgkin lymphoma CD30 CD15 CD20 CD3 EBV PAX-5 MUM-1 KI67 EMA CD23 ALK-1 (YOUNG PATIENT) Thymoma Pan CK EMA CK 20 CD3 Tdt CD1a CD20 Ki67 Reticulin NODULAR HISTIOCYTE AND LYMPHOCYTE PREDOMINANT HODGKIN LYMPHOMA/ Lymphocyte rich Hodgkin lymphoma CD20 CD30 CD15 CD23 PD-1 BCL-2 CD21 EMA EBV BOB Oct-01 CD3 Myelodysplasia CD34 CD117 CD14 Glycophorin CD61 For Myeloproliferative Neoplasm + MPO Approved by: Joya Pawade Page 10 of 4 Edition No: 1

11 Appendix 3 HODS Flow Cytometry Repertoire Panel LPD ( Initial Screen ) Extended LPD: B-LPD Extended LPD: T-LPD Plasma cell AL Initial screen to establish lineage AL: B-ALL AL: T-ALL AL: AML/MDS Non-haematopoietic Antigens Tested CD3, CD4, CD8, CD56, CD57, CD19, CD20, CD5, CD10, Kappa, Lambda, CD45 FMC7, CD79b, CD23, CD22, CD81, CD43, CD200. For HCL: CD103, CD123, CD11c, CD25 For? HG-NHL: Ki-67 If suspicious of Burkitt s/al: TdT, CD34 CD45RA, CD45RO, CD2, CD5, CD7, CD16, CD25, CD26, CD30, CD52*, TCRab, TCRgd. If suspicious of T-LBL: CD34, TdT. CD138, CD38, CD19, CD56, CD45, ckappa, clambda MPO, CD3, ccd3, CD19, ccd79a, CD7, CD34, CD45. CD10, TdT, cigm, kappa, lambda, CD25, CD20, CD22. For MRD: CD81, CD9, CD13, CD33, CD38, CD45, CD24. TdT, CD99, CD2, CD5, CD4, CD8, CD10, CD13, CD56. (for MRD and Dx) CD117, CD13, CD33, CD11b, CD16, CD2, CD7, CD19, CD56, CD14, CD64, CD36, CD105, GPA, CD71. If? Monocytic: ILT-3, CD65 If? PDCN: CD123, CD303,CD4, CD11c If? Megakaryocytic: CD41, CD42b, CD61 CD45, CD117, CD99, CD56 An initial screen to establish if abnormal lymphocytes are present in a diagnostic sample. For CLL MRD: PB is used. Antibody combination to be used for FU samples identified by PB screen at Dx/pre-treatment For MRD: Antibody combination to be used for FU samples identified at Dx. For MRD: Antibody combination to be used for FU samples identified at Dx. For MRD: Antibody combination to be used for FU samples identified at Dx. Approved by: Joya Pawade Page 11 of 4 Edition No: 1

12 Appendix 4. HODS Molecular Repertoire Name H/A Disease Relevance Technique TPMT gene mutations MPL-Baltimore JAK-2 (V617F) Hereditary Hereditary Acquired Purine-based drug sensitivity Thrombocytosis (nonmalignant) Myeloproliferative diseases JAK-2 (Exon12) Acquired Primary Polycythaemia MPL 515 mutations KIT D816V mutation SOX11 BRAF V600E etc BCRABL1 (p210 & p190) BCRABL1 (p210 & p190) ABL kinase domain mutations t(15;17) (PML- RARα) t(8;21) (RUNX1- RUNX1T1) Inv16 (CBFβ - MYH11) Acquired Primary Myelofibrosis/ET Acquired Systemic Mastocytosis N/A Acquired Overexpressed in Mantle Cell Lymphoma Hairy Cell Leukaemia and solid tumours /Prognostic Restriction enzyme digest and sequencing LightCycler probe based melting curve LightCycler probe based melting curve High Resolution melt curve/sequencing LightCycler probe based melting curve Allele-specific Blocking PCR/RT-PCR RT-QPCR High Resolution melt curve/pyrosequencing Acquired CML/ALL Nested PCR Acquired Acquired CML/ALL CML/ALL Treatment monitoring Treatment monitoring RT-QPCR PCR/Sequencing Acquired APML Nested PCR Acquired CBF AML Nested PCR Acquired CBF AML Nested PCR NPM1 Acquired AML Prognostic Sequencing NPM1 Acquired AML Treatment monitoring RT-QPCR FLT3 Acquired AML Prognostic PCR/Sequencing IgH clonality N/A Lymphoma PCR/UHG generation TCRγ/TCRβ clonality N/A Lymphoma PCR/UHG generation IgV H N/A CLL Prognostic PCR/Sequencing Approved by: Joya Pawade Page 12 of 4 Edition No: 1

13 KIT exon8 and 17 mutations Acquired CBF AML Prognostic PCR/Sequencing + as KIT D816V RUNX1 mutations Hereditary Familial MDS/AML PCR/Sequencing EPOR mutations Hereditary Familial ET PCR/Sequencing SRSF2 mutations Acquired CMML /Prognostic PCR/Sequencing/High Resolution Melt curve NOTCH1 Acquired CLL/SLL Prognostic PCR/Sequencing/AS-PCR Other tests: Factor 5 Leiden Hereditary Thrombophilia Prothrombin gene mutation Heamachromatosis genetics HHCS KRAS codons 12/13 and 61 PDGFRα Hereditary Thrombophilia Hereditary Haemochromatosis Hereditary Hyperferritinaemia with cataracts syndrome Acquired Colorectal cancer Prognostic Acquired Gastro-intestinal stromal tumours Prognostic LightCycler probe based melting curve LightCycler probe based melting curve LightCycler probe based melting curve High Resolution melt curve/sequencing High Resolution melt curve/pyrosequencing PCR/Sequencing Approved by: Joya Pawade Page 13 of 4 Edition No: 1

14 Appendix 6. HODS Laboratory Contact Information SI-HMDS Lead SI-HMDS Laboratory Manager Haemato-Oncology Lead Haematopathology Lead Molecular Haematology Lead Flow Cytometry Lead Dr Joya Pawade Elizabeth Worsam Dr Lisa Lowry Dr Joya Pawade Tim Clench Ulrika Johansson Telephone Dr Lowry Dr Pawade Specimen notification and general queries Flow Cytometry queries & results Molecular Laboratory queries & results Cytogenetics Laboratory Fax Approved by: Joya Pawade Page 14 of 4 Edition No: 1