bone marrow processing overview - swcn · bone marrow processing overview document reference:...

University Hospitals Bristol NHS Foundation Trust Directorate of Laboratory Medicine

Bone Marrow Processing Overview

Document Reference: LP-SI-HMDS_BM Processing Overview Haematology Department

Approved by: Joya Pawade of 4 Edition No: 1 NOT VALID UNLESS PRINTED ON CONTROLLED DOCUMENT PAPER

EDITION NUMBER: 1

ACTIVE DATE: 27th August 2013

REPLACES: -

LOCATION OF COPIES: 1) Electronically through Q-Pulse 2) Printed out copy in HODS reception

AUTHOR: Date:

Ulrika Johansson 27th August 2013

APPROVED BY: Designation Date:

Dr Joya Pawade Head of Service, Haematology Oncology Diagnostics Services 27th August 2013

CONTENTS 1. PURPOSE AND SCOPE 2. REFERENCES 3. PROCEDURE 4. APPENDICES

1. Diagnostic Pathways 2. Complete HODS Histology repertoire 3. Complete HODS Flow Cytometry repertoire 4. Complete HODS Molecular repertoire 5. HODS staff list with contact details





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), 455-488.





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.





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 panels 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 15.30 Monday–Thursday will be processed the following morning; If a sample arrives after 15.30 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 Diagnostics

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 +4oC 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).





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 4oC 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 email: [email protected]. 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: 0117 342 2596 Flow – Ulrika Johansson extension 22596 Histology – Dr Joya Pawade extension 22869 Molecular – Tim Clench extension 22596

mailto:[email protected]





Appendix 1 – Diagnostic Pathways

Disease FC IHC Molecular FISH Karyotyping

CML Diagnostic sample

Myeloid progenitor quantification

MPN panel

Qualitative and quantitative BCR-ABL on blood

t(9;22) Yes

CML FU samples

MPN panel

Quantitative BCR-ABL on blood Monitoring 3 monthly

t(9;22) Until negative or suspected relapse

MPN

Myeloid progenitor quantification If ? Mastocytosis: Mast cell panel

MPN panel

JAK-2 V617F, JAK-2 exon12 MPL515 MPL Baltimore BCR-ABL FIP1L1-PDGFRa (Salisbury) Familial ET: EPOR If ? Mastocytosis: KIT D816V

Not unless target suspected or identified by karyotyping

Yes

MDS

Myeloid progenitor quantification MDS panel

MDS panel

JAK-2 if thrombocytosis SRSF2 if CMML suspected Familial MDS/AML: RUNX1 mutations

Not unless target suspected or identified by karyotyping

Yes

New AML– off Trial With patients >65 years to receive only supportive care it may suffice to just confirm diagnosis.

Acute leukaemia panel

MDS panel

Qualitative PML-Rara, t(8;21), inv16, Flt-3 NPM-1

If ? APML: t(15;17) If ? Monomyeloid with eosinophilia: inv 16 If ? With maturation: t(8;21)

Yes

AML – off Trial FU samples

Myeloid progenitor quantification MRD where available, post each chemotherapy course

MDS panel MRD where available

If target identified at Dx: Until negative or suspected relapse







AML 17 Diagnostic samples

Acute leukaemia panel Also at NBT

MDS panel

Flt-3 (also at Cardiff) NPM-1 Trial will determine other suitable markers and centre informed.

If ? APML: t(15;17) If ? Monomyeloid with eosinophilia: inv 16 If ? With maturation: t(8;21)

Yes Also Sample storage locally

AML 17 FU samples

Myeloid progenitor quantification MRD where available Also at NBT

MDS panel

If target identified at Dx. Also, if identified at Dx: WT-1 and CBF to Manchester and NPM-1 to London.



APML FU samples

Until cytogenetic remission and at suspected relapse

MDS panel Quantitaive PML-Rara (at Guy’s Hospital)

t(15;17) post each course until negative

Lymphoid Malignancies

CLL/SLL

B-LPD panel Clonal B cell quantification Absolute clonal B cell count from PB MRD analysis on FU PB samples

Low grade b cell lymphoma panel

IgVh (<60 years) NOTCH1

Trisomy 12, Del 13q, Del 11q, del 17p P53

MCL B-LPD panel Clonal B cell quantification


Sox-11 t(11;14)

FL B-LPD panel Clonal B cell quantification


t(14;14) if diagnostic uncertainty

HCL B-LPD panel Clonal B cell quantification

BRAF V600E






NHL LPD panel Clonal B/T cell quantification

Low grade B cell lymphoma panel

If suspect: TCR/IgH clonality

HG NHL

LPD panel KI-67 Clonal B/T cell quantification

High grade B cell lymphoma panel

t(8;14) if Burkitt’s needs excluding

HL Hodgkin Lymphoma panels

Burkitt’s Lymphoma

LPD panel TdT/Partial AL panel

High grade B cell lymphoma panel

t(8;14) if infiltration

ALL Diagnostic sample

Acute leukaemia panel

BCR-ABL

If target 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 If target identified

If target identified

ALL –Trial Diagnostic sample

Acute leukaemia panel Also at NBT

BCR-ABL Yes

ALL –Trial FU samples

MRD Also at NBT

Myeloma/PCD Diagnostic sample

Plasma cell Panel

CD138, CD20, CD3, CD56

<50-60 years: Del 13q, t(11;14), t(4;14), del 17p

Myeloma/PCD FU samples

Plasma cell Panel

CD138, CD20, CD3, CD56





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





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





Appendix 3–HODS Flow Cytometry Repertoire

Panel Antigens Tested

LPD (“Initial Screen”) CD3, CD4, CD8, CD56, CD57, CD19, CD20, CD5, CD10, Kappa, Lambda, CD45

An initial screen to establish if abnormal lymphocytes are present in a diagnostic sample.

Extended LPD: B-LPD

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

For CLL MRD: PB is used. Antibody combination to be used for FU samples identified by PB screen at Dx/pre-treatment

Extended LPD: T-LPD

CD45RA, CD45RO, CD2, CD5, CD7, CD16, CD25, CD26, CD30, CD52*, TCRab, TCRgd. If suspicious of T-LBL: CD34, TdT.

Plasma cell CD138, CD38, CD19, CD56, CD45, cKappa, cLambda

AL Initial screen to establish lineage

MPO, CD3, cCD3, CD19, cCD79a, CD7, CD34, CD45.

AL: B-ALL

CD10, TdT, cIgM, kappa, lambda, CD25, CD20, CD22. For MRD: CD81, CD9, CD13, CD33, CD38, CD45, CD24.

For MRD: Antibody combination to be used for FU samples identified at Dx.

AL: T-ALL TdT, CD99, CD2, CD5, CD4, CD8, CD10, CD13, CD56. (for MRD and Dx)


AL: AML/MDS

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


Non-haematopoietic CD45, CD117, CD99, CD56





Appendix 4. HODS Molecular Repertoire

Name H/A Disease Relevance Technique

TPMT gene mutations

Hereditary Purine-based drug sensitivity

Restriction enzyme digest and sequencing

MPL-Baltimore Hereditary Thrombocytosis (non-malignant)

Diagnostic LightCycler probe based melting curve

JAK-2 (V617F) Acquired Myeloproliferative diseases


JAK-2 (Exon12) Acquired Primary Polycythaemia Diagnostic High Resolution melt curve/sequencing

MPL 515 mutations

Acquired Primary Myelofibrosis/ET


KIT D816V mutation

Acquired Systemic Mastocytosis Diagnostic Allele-specific Blocking PCR/RT-PCR

SOX11 N/A Overexpressed in Mantle Cell Lymphoma

Diagnostic RT-QPCR

BRAF V600E etc Acquired Hairy Cell Leukaemia and solid tumours

Diagnostic/Prognostic High Resolution melt curve/pyrosequencing

BCRABL1 (p210 & p190)

Acquired CML/ALL Diagnostic Nested PCR

BCRABL1 (p210 & p190)

Acquired CML/ALL Treatment monitoring

RT-QPCR

ABL kinase domain mutations

Acquired CML/ALL Treatment monitoring

PCR/Sequencing

t(15;17) (PML-RARα)

Acquired APML Diagnostic Nested PCR

t(8;21) (RUNX1-RUNX1T1)

Acquired CBF AML Diagnostic Nested PCR

Inv16 (CBFβ - MYH11)

Acquired CBF AML Diagnostic 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 Diagnostic PCR/UHG generation

TCRγ/TCRβ clonality

N/A Lymphoma Diagnostic PCR/UHG generation

IgVH N/A CLL Prognostic PCR/Sequencing





KIT exon8 and 17 mutations

Acquired CBF AML Prognostic PCR/Sequencing + as KIT D816V

RUNX1 mutations Hereditary Familial MDS/AML Diagnostic PCR/Sequencing

EPOR mutations Hereditary Familial ET Diagnostic PCR/Sequencing

SRSF2 mutations Acquired CMML Diagnostic/Prognostic PCR/Sequencing/High Resolution Melt curve

NOTCH1 Acquired CLL/SLL Prognostic PCR/Sequencing/AS-PCR

Other tests:

Factor 5 Leiden Hereditary Thrombophilia Diagnostic LightCycler probe based melting curve

Prothrombin gene mutation

Hereditary Thrombophilia Diagnostic LightCycler probe based melting curve

Heamachromatosis genetics

Hereditary Haemochromatosis Diagnostic LightCycler probe based melting curve

HHCS Hereditary Hyperferritinaemia with cataracts syndrome

Diagnostic High Resolution melt curve/sequencing

KRAS codons 12/13 and 61

Acquired Colorectal cancer Prognostic High Resolution melt curve/pyrosequencing

PDGFRα Acquired Gastro-intestinal stromal tumours

Prognostic PCR/Sequencing





Appendix 6. HODS Laboratory Contact Information

SI-HMDS Lead Dr Joya Pawade SI-HMDS Laboratory Manager Elizabeth Worsam Haemato-Oncology Lead Dr Lisa Lowry Haematopathology Lead Dr Joya Pawade Molecular Haematology Lead Tim Clench Flow Cytometry Lead Ulrika Johansson Telephone

07714 325 424 Dr Lowry 0117 342 2564 Dr Pawade 0117 342 2596 Specimen notification and general queries 0117 342 2596 Flow Cytometry queries & results 0117 342 2595 Molecular Laboratory queries & results 0117 323 5570 Cytogenetics Laboratory Fax

0117-342 2591

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