Protocol for liver sampling

The tissue samples used in the present work have been collected according to a protocol that I have specifically designed for sampling livers removed at transplantation. This protocol has the approval of the Ethics Committee of the Royal Free Hospital. Informed consent is obtained from each patient, before tissue samples are retrieved, and stored. This protocol consists of the collection of fresh tissue samples for storage and research purposes from nodular lesions in cirrhotic liver with a detailed record of the topography and the macroscopic appearance of each nodule. Tissue samples are also taken from each nodule for routine histological analysis of paraffin sections. These pathological data are recorded in a specifically designed computer database. This database has the formal approval of the Royal Free Hospital Ethics Committee.

The importance of obtaining tissue for research on hepatocellular carcinoma has been stressed in the recently published EASL consensus document on clinical management of Hepatocellular carcinoma (Bruix et al. 2001). In the discussion of the role of tissue diagnosis in the surveillance of cirrhotic patients, the EASL panel has stressed that "tumour biopsies at early stages may constitute a unique research tool to validate, in the clinical setting, the findings of experimental studies", and anticipated the future role of molecular profiling of the disease. If the possibility of obtaining biopsy samples for research purposes should be seriously considered despite the potential risks of biopsy, all the more reason that tissue easily obtainable from livers removed at transplantation should not be wasted. This protocol has been specifically designed in order to avoid compromising the routine diagnostic requirements.

2.2.1 Sampling of fresh liver

Livers are received fresh after transplantation and examined in a cryostat room under an extraction hood. The location of every distinct nodular lesion evident on external examination is recorded on a pro-forma diagram specifying diameter, colour, presence of necrosis and site (i.e. slice number from top to bottom, lobe, mid-upper/lower third, anterior/posterior, if near to gall bladder, hilum, vena cava, falciform ligament or other recognizable anatomic structures, sub capsular or intraparenchymal). This pro-forma diagram is then used as reference, at the cut-up bench after fixation.

The capsular surface is inked before any sample is taken. The middle of the tumour is sampled provided this is taken without interfering with the surgical or capsular resection margins. For example, a single cut is made with a stout bladed large knife ("brain cutting" 12 inch knife) across the tumour on the transverse plane (i.e. plane similar to a CT scan cut). One half is not interfered with further, and is kept for routine examination. Tissue may be taken from the other half for special fixation and freezing. Each lesion sampled is labelled with progressive numbers.

If no focal lesions are evident on external examination, there is no clinical diagnosis of liver tumour, or clinical details indicate a tumour without specifying the locations, then multiple neat and complete slices of uniform thickness (up to 1 cm thick) are cut in the transverse plane (i.e. plane similar to a CT scan cut) and any distinct nodule identified recorded, described and sampled as above.

The remainder of the liver is then placed in formalin for between 24-48 hours depending on size, degree of fibrosis etc and then sampled for routine histology.

Tissue taken from non-neoplastic liver, as well as from gall bladder or other adjacent tissue (e.g. fibro-adipose tissue of the falciform ligament) is also snap frozen. A few centimetres of the upper extreme of the left lobe can be removed without compromising the sampling.

Snap freezing is achieved placing the piece of fresh tissue onto a square piece of cork covered by OCT, which is then used to cover the entire specimen. This is placed in cooled isopentane for a few seconds, then in liquid nitrogen and ultimately for storage at -80°C, in dedicated, numerated and mapped storage racks.

2.2.2 Liver sampling after fixation

If abnormal nodules are found at tissue sampling, these are described in a diagram pro-forma so that they can be easily identified. Each slice of liver is numbered and photographed from top to bottom using a digital camera with close-ups on each nodule including an adjacent label bearing the corresponding paraffin block number for permanent record. Images are later transferred onto Adobe Photoshop and saved on JPEG format (Figure 2.1).

The following features of the specimen are described: external and cut surface appearance of the background liver, presence of other structures, each distinct nodular lesion as above specifying also clearance from the hilar vascular and biliary surgical ends including distance in mm. Lymph nodes are searched for in the hilar tissue.

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Figure 2.1. Example of digital photography during gross examination of a cirrhotic liver removed at transplantation. Left, low power view of slices cut on a transverse plane. Right close-up views of two slices containing tumours.

Samples are taken from each nodule from which fresh samples were taken plus all further nodules that become evident after fixation. The same numbers are allocated to the routine diagnostic histopathology samples taken from the same nodules after fixation and recorded on the macro description. If multiple samples from one lesion are taken, these are treated the same way as "extra blocks" in ordinary specimens and the allocated number recorded on the diagram and the macro description. For example:

(1)4 nodules are identified in the fresh liver. They measure respectively 30, 10, 6 and 5 mm diameter and are labelled A l, A2, A3 and A4.

(2) At surgical cut-up 3 blocks are taken from nodule "Al", 1 block is taken from nodule "A2", 1 block is taken from nodule "A3" and 1 block is taken from nodule “A4”. The blocks taken from nodule "A l" will be labelled A l , A5 & A6.

Usually, these "extra blocks" are taken from the nodule itself, adjacent satellite smaller nodules, tumour and capsular surface, tumour and adjacent vascular (e.g. vena cava, hepatic veins) or other (e.g. gall bladder) structures which may be useful for staging purposes. Blocks of hilar structures are also useful as they represent a resection margin. Lymph nodes from the hilar tissue are sampled.

If no abnormal nodules were found at tissue sampling, but become evident after fixation, the same method of sampling as above is used. In this case, as no fresh tissue has been taken from these nodules, there is no need to match paraffin and fresh samples and the method of block labelling is up to individual preference. However, it is advisable, for practice and consistency, to maintain the same system as in section 1 (i.e. allocating a number to each nodule and considering further samples as “extra blocks”).

If no nodules are found, samples are taken as per protocol described by Demetris et al (Demetris et al. 1987).

The time necessary for sampling of fresh tissue ranges from 10 minutes when no nodules are present to approximately 30 minutes when up to 8 nodules are found. The gross examination and sampling of formalin fixed livers, is of about 30 minutes including digital photography, and may extend to 45 minutes when a large number of nodules (more than a dozen) are identified. This protocol may appear time consuming, but allows the retrieval of tissue, pathological data and digital images at later stages, fairly quickly.