Histological techniques

Microscopic assessment of histological specimens by pathologists is almost the last step in a lengthy and laborious process. The stepwise procedure is explained below:

  • Allocation of a unique number to the incoming specimen (with barcode labels for transport containers and request forms) for the purpose of clear assignment of cassettes, slides and paraffin blocks with the report
  • Recording of patients’ clinical details and case data in the computer system
  • Macroscopic inspection with measurements and description of the tissue; for surgical specimens that cannot be completely embedded, sections of all diagnostically relevant parts (by the pathologists)
  • Embedding the specimens in cassettes
  • Dehydration of the specimens in a series of alcohol solutions of increasing concentration as a prerequisite for subsequent impregnation with liquid paraffin
  • Embedding the specimens in paraffin blocks
  • Sectioning of the blocks and mounting of the 2–5 micrometre paraffin sections on slides
  • Dewaxing and staining of the sections (mandatory haematoxylin and eosin staining and, depending on the location and clinical issue, additional special stains where necessary)
  • Sealing the stained sections using cover slips or film
  • Linking the slides and the request forms and transferring the case to the pathologist for microscopic analysis and reporting
  • Writing, validating and sending of the report
  • Electronic archiving of the report and the accompanying request form
  • Physical archiving of sections and blocks

The embedding process requires about 10 hours for most specimens and is generally done overnight in automated tissue processing machines. This explains why histological diagnoses can generally only be made over the course of the business day after receiving the specimen. Larger surgical specimens might take a day longer to allow proper fixation before cut up (generally formalin fixation over at least 24 hours). The priority is optimal tissue fixation to prevent artefacts which could compromise the microscopic evaluation of the specimen (e.g. distance of a tumour from the resection margin).

For so-called cito cases, the material is initially processed as described above. For the steps following the dehydration of the material, these specimens undergo preferential and accelerated processing so that the histological results are generally available at about 10 am on the business day after the samples have been received and the results can be forwarded by telephone or fax.

An even shorter processing time is possible using rapid embedding. Only specimens derived from needle or forceps biopsies that have been well fixed and have arrived at our lab before 12 pm can be considered for this process. Only these specimens can undergo dehydration in a short programme that lasts just a few hours and the entire process can be completed, including forwarding of the report, by late afternoon.

It is obvious that the rather complex process for fast-tracking specimens should be reserved for very urgent cases in which the histological diagnosis has immediate consequences on short-term therapeutic measures. This applies even more to frozen sections. In this case a microscopic diagnosis is made during an operation and has an enormous impact on the surgical management. The most common indication for frozen sections is intraoperative evaluation as to whether a lesion is benign or malignant or whether or not resection margins are involved by tumour. Instead of the lengthy fixation required for the paraffin sectioning technique, the tissue samples are rapidly deep frozen and sections are produced by a special microtome (cryostat). The cryostat sections are stained in a shortened procedure which generally enables the diagnosis to be made within 15 to 20 minutes of receiving the specimen and communicated to the surgeon by telephone.

Frozen sections intrinsically do not have the same technical quality as paraffin sections that are handled by a more gentle tissue processing procedure. For this very reason, the remaining tissue not used for the frozen section is processed by the normal embedding procedure described above. The final diagnosis is made after assessing the definite sections and, if necessary, only after additional special stains and possibly even immunohistochemical examinations.

By default, all histological sections are stained with haematoxylin and eosin (H&E). Depending on the source of the tissue samples and any specific considerations, additional special stains (e.g. PAS, alcian blue, Giemsa, van Gieson, elastica, Gomori, Ziehl-Neelsen, Congo red) are used which can help to visualise certain tissue structures as well as secretory products, deposits of substances, pigments or microorganisms, thus making them diagnostically useful. Since many diagnoses rely on using further investigations a broad range of special stains must be kept in stock.

The same applies to immunohistochemistry, another technique used to supplement conventional histological diagnostics. Antigens present in the tissue (e.g. structural or functional proteins, hormone receptors, growth factors, viral proteins) are labelled using specific monoclonal or polyclonal antibodies and detected using a coupled colour reaction. Immunohistochemical characterisation of a certain cell population can, for example, provide information about the histogenesis and proliferative activity of tumours that cannot be clearly classified using conventional staining techniques only. In other cases immunohistochemistry provides information for the prognosis or the effectiveness of certain therapeutic procedures. Apart from tumour pathology, immunohistochemistry is a helpful tool for diagnosing immune-mediated diseases and also to detect infectious pathogens. 

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