Requests for Molecular Pathology services can be made by disease type or individual test as required.  For all non-NHS Lothian requests, an H&E stained section, corresponding formalin-fixed paraffin-embedded (FFPE) block and copy of the pathology report should be sent to the Molecular Pathology laboratory along with a completed request form, following local protocols for transport of pathology samples.

Internal requests (from within NHS Lothian) can be made using the request form, via email to molecular.pathology@nhslothian.scot.nhs.uk, or by discussion with an NHS Lothian pathologist.

All requests for EGFR mutation analysis of cell-free tumour DNA (cfDNA) specimens must be accompanied by the appropriate request form

MolPath EGFR cfDNA request form v4.pdf

.

The Molecular Pathology service is funded by NSD Scotland, meaning that all tests are provided free of charge for requests received from within NHS Scotland. 

Specimen Requirements

Molecular Pathology analysis is routinely performed on DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue samples.  Blocks should represent clinically relevant tumour (typically the most recent sample available or a metastasis) and contain a proportion of tumour cells sufficient for the required analyses.  

EGFR mutation analysis may also be performed using cfDNA extracted from blood.  Blood for cfDNA analysis must be collected using Roche Cell-Free DNA Collection Tubes.  Tubes, request forms and instructions for collection are available on request.

DPYD mutation analysis is performed on DNA extracted from blood specimens.  A minimum of 2ml of blood should be collected in an EDTA tube.  Blood specimens should be kept at room temperature and delivered to the laboratory with 48 hours of collection.

Factors known to affect Molecular Pathology testing

  • Tumour tissues, particularly small biopsies, may yield inadequate quantities of DNA for molecular analysis.  Samples determined to be inadequate during morphological assessment will be reported as insufficient.  Owing to the increasing number of tests that need to be performed using a limited amount of tissue, it is strongly recommended that specimens consisting of multiple core biopsies be embedded in separate blocks and that multiple blocks be submitted for testing.  The most appropriate block(s) for the required assays will be identified prior to testing.
  • DNA from non-neoplastic cells within a sample may dilute the analyte of interest beyond the level of detection of some molecular diagnostic assays.
  • The quality of tissue fixation, including ischemic time, duration of fixation, sample size (i.e. penetration of formalin) and processing protocols can affect the quality of DNA isolated from FFPE tissues, and therefore the results of molecular testing
  • Some tissue components including melanin, collagen, haemoglobin and myoglobin are known to inhibit PCR reactions if present in high concentration, which may lead to a higher than expected test failure rate in some specific sample types.
  • Acid decalcification of tissues (e.g. using formic acid or hydrochloric acid-based decalcifying agents) is known to degrade the DNA within FFPE samples making subsequent molecular analysis impossible.
  • The use of the plasma-thrombin technique for the preparation of cell blocks from cytology specimens can introduce DNA contamination into a sample and is strongly discouraged unless the plasma has been DNase treated
  • Certain pigmented tissue additives used to aid visualisation of core needle biopsies and dyes used to mark specimen resection margins may fluoresce under the wavelengths of light required during FISH analysis, making interpretation difficult or impossible
  • Blood collection tubes for cfDNA analysis must not be refrigerated.
  • PD-L1 immunohistochemistry has not been fully validated for use on cytology specimens.  Biopsy material is preferred, however FNA specimens may be used if they are received fixed in formalin.  Cytology specimens fixed in CytoLyt are not suitable for PD-L1 immunohistochemistry.

If analysis is required of a specimen type not described above, please contact a member of the Molecular Pathology team prior to requesting. 

Mandatory Data Set

Referring clinicians are requested to comply with NHS Lothian policy on mandatory data set requirements for patient identification on laboratory requests.  The following information must be legible on the request form:

  • Patient identifier number (CHI or NHS number)
  • Surname
  • Forename
  • Date of Birth
  • Gender
  • Location/Requesting Clinician

If the CHI/NHS number is not available, the data set must include first line of the patient’s address and postcode. 

Tissue blocks sent from external laboratories must be labeled with a unique identifier. Accompanying slides must be labeled with the unique identifier and patient surname. 

Failure to comply with this policy will result in the sample being rejected or the result being delayed.  Samples without minimum data, or for which data on the sample and accompanying documents do not match, will not be accepted and will be returned to the sender.  

Consent, Storage and Authorisation

In accordance with the requirements of the Human Tissue (Scotland) Act 2006, it is the responsibility of the referring clinician to ensure that appropriate informed consent has been obtained before any testing is undertaken.  The laboratory must be informed of any restrictions to this consent (e.g. storage of samples).

Unless otherwise informed, the laboratory assumes that all appropriate consent has been obtained from the patient for the tests requested and for storage of the derived DNA or RNA (cDNA) for future use both in assisting further testing (if required) and in the development of future diagnostic tests.  If in doubt, contact a member of the Molecular Pathology team to discuss. 

Specimen Transportation

It is the responsibility of those dispatching specimens to the laboratory to ensure that these samples are sent in accordance with any national guidelines and/or local policies for the packaging, labelling and transport of biological material.

The Health and Safety Executive guidance, ‘Biological Agents: Managing the risks in laboratories and healthcare premises’, recommends materials such as blood, tissue, excreta, secreta etc collected from humans be considered, as a minimum, Category B infectious substances.

The following packaging, labelling and transport requirements are applicable for samples from otherwise healthy individuals, or where there is no reason to suspect that they are suffering from a severe infectious disease, and are derived from Packing Instruction P650 of the European Agreement concerning the International Carriage of Dangerous Goods by Road (ADR).  Such specimens are not subject to ADR provided the specimen is carried in a packaging which will prevent any leakage and which is marked with the words "EXEMPT HUMAN SPECIMEN".

The packaging is deemed to comply with the above requirements if it meets the following conditions:

The packaging should consist of three components: leak-proof primary receptacle(s), a leak-proof secondary packaging, and an outer packaging of adequate strength for its capacity, mass and intended use, and with at least one surface having minimum dimensions of 100 mm × 100 mm. 

For liquids, absorbent material in sufficient quantity to absorb the entire contents should be placed between the primary receptacle(s) and the secondary packaging so that, during carriage, any release or leak of a liquid substance will not reach the outer packaging and will not compromise the integrity of the cushioning material.

When multiple fragile primary receptacles are placed in a single secondary packaging, they must be either individually wrapped or separated to prevent contact between them.

Packages should be clearly labelled with the delivery address and sender details. Labels must be durable and legible.