Molecular Pathology

Pathology

Contacts:

Sharon Forrest Immuncytochemistry & Molecular Pathology Services Manager Tel: 0151 706 4485 Email: sharon.forrest@LiverpoolFT.nhs.uk Steven Forrest Molecular Pathology Team Leader Tel: 0151 706 4485 Email: steven.forrest@LiverpoolFT.nhs.uk Professor Sarah Coupland Consultant Histopathologist Email: s.e.coupland@liverpool.ac.uk Dr Helen Kalirai Liverpool Ocular Oncology Molecular Pathology Service Email: h.kalarai@liverpool.ac.uk Sophie Thornton Liverpool Ocular Oncology Molecular Pathology Service Email: sophie.thornton@LiverpoolFT.nhs.uk

Location of Laboratory: the Molecular service is delivered from laboratories based on 4th Floor CSSB and the 3rd floor of the William Henry Duncan Building.

Services offered by the laboratory: the Molecular Pathology service offers a comprehensive test repertoire to support its clinical service activity. The service offers PCR for TCR/IgH gene rearrangement studies in haematological neoplasms and coeliac disease and RT-PCR for EGFR and BRAF gene mutation detection in non-small cell lung cancer and melanoma, respectively. In conjunction with the Directorate’s Immunocytochemistry service, a range of fluorescence in situ hybridisation (FISH) and chromogenic in situ hybridisation (CISH) protocols are also offered as diagnostic and therapeutic predictive aids for the investigation of breast cancer, non-small cell lung cancer, lymphoma and oropharyngeal carcinoma specimens.

Multiplex Ligation dependant Probe Amplification (MLPA) and micro-satellite (MSA) analyses for prognostication in ocular oncology are also offered and these services are delivered by the Liverpool Ocular Oncology Molecular Pathology Service. In 1999, the Liverpool Ocular Oncology Centre became the first centre to perform genetic typing of uveal melanoma as a prognostic service. This team provides a genetic testing service for tumours occurring in the eye; two ocular tumour types are examined: conjunctival melanoma and uveal melanoma. Using neural networks, the Liverpool Ocular Oncology Molecular Pathology Service has developed an on-line prognostic tool, which analyses all major risk factors and generates a personalised survival curve for individual patient management. One Step Nucleic Acid Amplification (OSNA) is also offered to facilitate the detection of micro and macro-metastases in sentinel lymph node samples from patients with breast cancer; further information about this service can be obtained by accessing the Histology Service sections of the User Handbook.

Laboratory opening hours: Monday to Friday 08:00-17:30.

Sample types and volumes: in situ hybridisation techniques require formalin fixed paraffin embedded (FFPE) tissue sections cut at a thickness of 4microns and mounted onto APES coated slides. It is recommended that sections for FISH and CISH analyses are mounted onto Superfrost Plus sialinized slides.

Requests for EGFR mutation testing should be sent with a Haematoxylin and Eosin (H&E) stained slide representative of the lesion, a formalin-fixed paraffin embedded tissue or cell block and a copy of the corresponding pathology report. This procedure requires at least 5% tumour infiltration and over 20 tumour cells in total.

PCR requests for investigation of TCR/IgH gene rearrangement can be performed using the following sample types: formalin-fixed paraffin embedded tissue or cell blocks, whole blood (EDTA) and ocular fluids.

BRAF mutation analysis requires FFPE tumour material with the corresponding haematoxylin and eosin stained slide. Alternatively, a copy of the corresponding pathology report is required. This procedure requires 5% tumour infiltration and over 20 tumour cells in total.

Requests for MLPA require fresh, snap frozen or FFPE uveal melanoma material with the corresponding H&E stained slide from the P0 block. This procedure requires at least 100ng DNA at a concentration of 20ng/µl.

MSA requires fresh, frozen or FFPE uveal melanoma material together with a matched whole blood sample (100µl). May-Grünwald Giemsa stained cytospin or other histological slide or copy of the corresponding pathology report. This procedure requires at least 20ng DNA.

Special precautions: sections for ISH should be cut onto APES slides and heated overnight; a temperature range of 37-50°C is acceptable. The Superfrost Plus sialinized slide type is the recommended option when preparing slides for in situ hybridisation (FISH and CISH) techniques.

It is important to ensure that fresh formalin is used for the fixation of samples for molecular testing; use of fresh formalin is required to reduce the effect of polymerisation.

Fresh tissue for molecular analysis must be transported rapidly to the laboratory on ice.

Turnaround times (TATs):
for ISH analyses TATs are 14 working days.
EGFR testing TATs: 14 working days.
PCR ICT/IgH analyses: 20 working days.
BRAF testing: 14 working days.
MLPA analysis: 20 working days.
MSA testing: 20 working days.

Instructions for completion of request forms: refer to the Liverpool Clinical Laboratories Minimum Dataset Policy (MDS) for Laboratory Investigations for guidance. Link

Instructions for transportation of samples: slides submitted for molecular review should be labelled with the sample’s unique laboratory number and patient surname and transported to the laboratory in sealed slide mailer boxes. FFPE tissue or cell blocks should be wrapped in bubble wrap or absorbent tissue to protect the block during transportation. Slides and blocks should then be placed into a jiffy bag together with all paperwork required for the analysis, i.e. request form and copy of histology report and posted to the relevant laboratory in accordance with the postal regulations in place for the transport of pathological specimens.

Blood samples, fresh tissue and ocular fluid samples for molecular analyses should be transported to the lab in sample containers labelled in accordance with the guidelines outlined in the MDS Policy.

Patient consent: where applicable e.g. MLPA and MSA prognostication testing, all patients must be consented.

Lab criteria for accepting/ rejecting samples: criteria for sample acceptance or rejection is outlined in the MDS Policy; please see this Policy document for advice. Link

Factors known to significantly affect performance of examination or interpretation of results: both fresh and formalin-fixed tissue samples can be submitted to the lab for genetic analyses. If submitting fresh tissue, the sample must be transported promptly to the laboratory on ice. Fixed tissue specimens require prompt, adequate fixation. Tissue should be placed into an adequate volume of fixative (10X the volume of the sample) as soon as possible after excision to protect against the deleterious effects of cold ischemia on molecular protocols (alteration of levels of gene expression at the RNA and protein level).

It is recommended that sections for ISH analyses are mounted onto Superfrost Plus sialinized slides. Artefacts associated with the use of other adhesive slide types can impede interpretation.

DNA quality is compromised by the use of acid fixatives and decalcification reagents and this must be borne in mind when submitting samples for molecular analyses.

Gene expression and mutation status can be altered by certain therapeutic agents, i.e. chemotherapy therefore previous treatments should be documented on the sample request form.

Availability of clinical advice when ordering examinations and on interpretation of examination results: technical advice can be obtained by contacting the Molecular Services Lead or a member of the Molecular Pathology Services team by e-mail (see above for contact details) or by telephone on 0151 706 4485.

Technical advice for MLPA and MSA analyses can be obtained by contacting Professor Sarah Coupland, Dr Helen Kalirai or Miss Sophie Thornton by e-mail (see above for contact details).

Clinical advice on examination results can be obtained by contacting the relevant member of the Consultant Pathologist team.

Protection of personal information: the laboratory complies with the mandates set out in the Data Protection Act and Caldicott regulations. Trust and local policies are also in place to assure the protection of personal information.

Complaints procedure: if you wish to raise any concerns regarding the service, please contact the Immunocytochemistry and Molecular Services Manager, Sharon Forrest, on 0151 706 4485 or by e-mail at sharon.forrest@LiverpoolFT.nhs.uk. Alternatively concerns can be raised by contacting the cellular Pathology Clinical Director Dr Vijay Sharma via email vijay.sharma@LiverpoolFT.nhs.uk or the Quality Practitioner Jane Harrison-Williams 0151 706 2000 ext 11093 or email jane.harrison-williams@LiverpoolFT.nhs.uk
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