CHAPTER 2 Laboratory Techniques

Processing of the specimen

As soon as a needle biopsy specimen is obtained from the patient it should be expelled gently into fixative or onto a piece of glass, card or wood. Filter paper is less suitable because fibres tend to adhere to the tissue and may interfere with sectioning. The specimen must be treated with great care and excessive manipulation should be rigorously avoided; distortion of the specimen by rough handling at this stage may seriously interfere with accurate diagnosis, because diagnosis often depends on subtle criteria. At this stage, minute pieces can be put into an appropriate fixative for electron microscopy (Ch. 17), preferably by an operator experienced in this technique, and samples taken for chemical analysis or freezing. Frozen sections may be needed for demonstration of lipids. If porphyria is suspected, a very small amount of the unfixed tissue can be examined under ultraviolet light or with a suitable quartz halogen source, either whole or smeared onto a glass slide.

Tissue for paraffin embedding should be transferred to a fixative as soon as possible. When transit to the laboratory is likely to involve much movement, it is helpful to fill the container to the brim with fixative. Buffered formalin and formol saline are both suitable for routine fixation, which is accomplished after 3 h at room temperature or less at higher temperatures (Table 2.1). Operative wedge biopsies and larger specimens need longer fixation. Fixatives other than formalin are successfully used in some centres; handbooks of laboratory technique should be consulted for optimum times and conditions for each fixative.

Table 2.1 Sample tissue schedules for liver biopsies

Minute fragments can be hand-processed more quickly than larger pieces and this also avoids undue shrinkage and hardening. Automated vacuum embedding allows the time of processing of needle specimens to be drastically reduced, as shown in Table 2.1; the ultra-rapid method by which a good section can be produced in about 2 h has become important because of the need for rapid decisions on treatment in patients who have undergone liver transplantation. Frozen sections, occasionally needed for a decision at surgery, can be cut by a standard method using a cryostat. They are sometimes adequate for diagnosis of obvious lesions such as neoplasms, but are unsuitable for recognition of subtle changes, and can even be dangerously misleading.

The exact number of sections routinely cut from a block varies widely from one laboratory to another. In Scheuer’s former laboratory at the Royal Free Hospital in London, ten or more consecutive sections 3–5 µm thick are cut from each block and alternate sections used for the staining procedures outlined in the next paragraphs. The remaining sections are stored. Step sections are used when discrete lesions such as granulomas or tumour deposits are suspected or for identification of bile ducts when duct paucity is suspected. Serial or near-serial sections are helpful when utilising multiple immunohistochemical stains.

Choice of stains

The stains routinely applied to liver biopsies vary according to local custom. The minimum advised is haematoxylin and eosin (H&E) and a reliable method for connective tissue. The author prefers a silver preparation for reticulin as the principal method for showing connective tissue for reasons discussed below, but trichrome stains also have important applications and can reveal changes not easily seen in a reticulin, such as the pericellular fibrosis of steatohepatitis. Routine staining for iron enables the biopsy to be used to screen for iron storage disease and the periodic acid–Schiff stain after diastase digestion (DPAS or PASD) provides a relatively crude, but practicable screening procedure for α₁-antitrypsin deficiency as well as showing activated macrophages and bile-duct basement membranes. Stains for copper-associated protein, elastic fibres and hepatitis B surface antigen are useful and arguably essential additions to the routine list. Some pathologists like to see two H&E-stained sections, one from the beginning and the other from the end of a series of consecutive sections. Other methods are used as required for particular purposes. The extent to which ‘special’ stains form part of the routine set must be decided by each pathologist.

A reticulin preparation is important for accurate assessment of structural changes. Without it, thin layers of connective tissue and hence cirrhosis may be missed, as may foci of well-differentiated hepatocellular carcinoma in which the reticulin structure is often highly abnormal (see Fig. 11.12). Counterstaining is sometimes used, but is apt to distract rather than help, bearing in mind that the chief function of the reticulin preparation is to provide a sensitive low-power indicator of structural changes.

Stains for collagen such as chromotrope–aniline blue (CAB) are important for the detection of new collagen formation, especially in alcoholic steatohepatitis and its imitators (see Ch. 7). Collagen staining is therefore advised for any biopsy showing substantial steatosis. It also helps to show blocked veins within scars; these are easily missed on H&E staining. It is therefore wise to use a trichrome stain when vascular disease is suspected.

A stain for elastic fibres such as the orcein stain, Victoria blue or elastic–Van Gieson is also useful to identify blocked vessels. The stains often enable the pathologist to distinguish between recent collapse and old fibrosis, since only the latter is positive (see Ch. 6). Again, this distinction may be very difficult to make on H&E and even with the help of stains for collagen and reticulin. The orcein and Victoria blue also show copper-associated protein and hepatitis B surface material.

Staining for iron by Perls’ or other similar method enables not only iron but also bile, lipofuscin and other pigments to be evaluated, as discussed in Chapter 3. Counterstaining should be light to avoid obscuring small amounts of pigment.

Staining of glycogen by means of the PAS method or Best’s carmine demonstrates the extent of any liver-cell loss, and shows focal areas devoid of hepatocytes such as granulomas. Glycoproteins may be demonstrated by the PAS method after digestion with diastase to remove glycogen. This stain serves to accentuate hypertrophied macrophages, such as Kupffer cells filled with ceroid pigment after an acute hepatitis or episode of cholestasis. Alpha₁-antitrypsin bodies stain strongly, but the stain is not sufficiently sensitive to enable all examples of α₁-antitrypsin deficiency to be detected.

Staining for copper is mainly used in suspected Wilson’s disease, although, as explained in Chapter 14, it is not always helpful and may even be negative. The rhodanine method is preferred because it is easy to distinguish the orange-red colour of copper from bile, a distinction which is occasionally difficult with rubeanic acid. In Wilson’s disease, there is variable correlation between the presence of stainable copper and staining for copper-associated protein. In chronic cholestasis, however, the two usually correspond.

Other non-immunological methods useful on occasion include the Ziehl–Neelsen stain for mycobacteria and for the ova of Schistosoma mansoni. Specific staining for bilirubin is rarely necessary, but conjugated bilirubin stains a bright green colour by the Van Gieson method (Fig. 4.3). Amyloid is stained by the usual techniques.

For immunohistochemical staining, standard techniques are applied. Among antibodies that are helpful in everyday practice are those against components of the hepatitis B virus, the delta agent, cytomegalovirus and α₁-antitrypsin. Neoplasms of doubtful histogenesis or differentiation are investigated by appropriate panels of antibodies, as in any other organ. In hepatocellular carcinoma, bile canaliculi between tumour cells may stain with a polyclonal anti-CEA (carcinoembryonic antigen) antibody, cross-reacting with a canalicular antigen. Assessment of bile-duct loss may require staining of cytokeratins 7 and 19, characteristic of bile-duct rather than liver-cell cytoplasm and of the ductular reaction (see Ch. 4). The application of immunohistochemistry as well as of other modern techniques is discussed in more detail in Chapter 17.

Most of the staining methods mentioned above are used routinely in many laboratories, and can be found in the books listed under General reading at the end of this chapter. A selection of methods is given below (see Box 2.1).

Box 2.1 Staining methods