5 The gross room/surgical cut-up
The dissection and preparation of any specimen for histological/microscopic analysis involves more than simply the tissue processing and section cutting. Whilst the dissection and laboratory area are often perceived as the key elements of the department, it must be clearly understood that there are many other steps that follow specimen receipt. Some are specific to tissue selection and handling, and others are clearly support role in type. It is also implicit that a good laboratory will be adequately staffed by appropriately trained scientific/medical and support staff (secretarial, medical laboratory assistants, administration, etc.), as they interface at multiple levels with pathological sample handling. Indeed, a poorly staffed department will perform weakly – at best.
The histopathology department is rich in hazards (e.g. biological, chemical, radiation). These are also risks reflecting the range of materials used to store, process and analyze tissues. These may be toxic, flammable, allergic, carcinogenic, electrical, etc. Furthermore, the presence of sharp cutting implements, complex machinery and the movement of the specimens around the laboratory needs staff to be fully trained and aware of all these potential hazards. Every laboratory should have accessible and clear standard operating procedures (SOPs, see Chapter 1), of which many will reflect national/international guidelines (websites 1–3). Ongoing safety education is required and caution should be employed at every step of specimen handling for safe laboratory practice (websites 2 and 4).
A separate room is required for specimen reception, acting as the interface between hospital staff (or other visitors) and the pathological laboratory. Appropriate benching and good lighting must be available, along with good ventilation, safety equipment, disinfectants, absorption granules and protective clothing. In the event of specimen spillage (i.e., body fluids, fixative leakage or other mishap), the immediate response by staff will limit any potential local health risk, and also prevent risk to other laboratory personnel.
The key point of this room is to receive samples safely and securely. The specimen should be confirmed in terms of the identity and to be assigned a unique laboratory specimen identifier – usually a complex number. Correlation of the specimen against the clinical request form is mandatory, along with checking of appropriate clinical details mentioned against the specimen. Corroborative data in the form of the hospital number/registration index, national patient identifier number, the full name, date of birth and address are also valid ways of verifying the identity of any specimen. Multiple sources of cross reference are advocated, and if there is any doubt with regard to the probity of specimen then it should not be passed onwards until the clinician concerned has confirmed the appropriate details and probity of the sample.
In many situations the two-person rule is best followed, with two independent laboratory practitioners verifying the various details of the specimen – at all the different stages of examination. Confirmation of a minimum of three unique identifiers (as detailed above) is advisable. Once validated and identified, the case can be passed to the dissection room for examination, specimen description and block sampling.
The usual method of specimen identification is simply the year (expressed usually in two digits) with a sequential numbering system starting with one (1) and proceeding up to the final specimen of each year. There may be a check digit, usually in the form of a letter applied, but this simple system allows surgical pathology samples to be processed with ease and to be correlated against paraffin blocks, photographs and other tests (see below). Thus, case 2345 L/12 is the two thousand, three hundred and forty-fifth sample of the year 2012. The letter suffix (L) is a computer check datum to verify that the numerical data is valid.
Particular attention must be paid to cases with unusual names, or contrastingly, very common names. Names which have a variety of different spellings and any specimens that have incomplete information should be very carefully considered before being accepted.
In some cases multiple specimens from a single patient may be received on the same day for analysis. Some laboratories prefer to annotate each sample with a separate number. However, a single laboratory number may suffice, but with sub-parts of the specimen being separately designated (e.g., sample A, sample B, etc.). Within this framework, if multiple blocks were taken from a sub-part of the specimen then these can be designated with individual numbers/letters in a similar ascending fashion. Thus, a gastrectomy sample with lymph node groups and the spleen could have one case number, multiple sub-part specimens and multiple blocks that can be correlated against the surgeon’s operative dissection. For example, using the number described above, the spleen in this case could be being designated 2345 L/12.C.2 (C. indicating the sub-part of the third sample = spleen; and the block number = 2).
Barcodes can be used where appropriate facilities exist, but in general terms many laboratories still have paper request forms that will accompany the specimen as it passes through the laboratory and towards final report emission.
The ideal layout of this room is a matter of debate, varying between different laboratories and pathologists’ needs. There are multiple different design solutions existing around the general principles of a histology laboratory (Rosai 2004, Cook 2006, websites 2, 4), but it is imperative that the dissection area must have good lighting, good ventilation, non-absorbent wipe-clean surfaces, appropriate protective clothing for the laboratory personnel, gloves and other equipment (photography, tissue macerators, disposal bins). The dissection room should be a comfortable environment permitting undisturbed work by the pathologist and support technical staff. Given that the range of specimens received in most laboratories is wide, the technical staff will have to be familiar with the various requirements of different specimens that guide their subsequent handling and pathological preparation.
It is a matter of preference whether the operators within this environment sit or stand, and ideally both options should be available. Modern dissection areas often have integrated dissection desks, enclosed fluid/fixative feeds and laminar down-draft ventilation (website 5) in order to protect both the dissector and support staff from formalin vapor. All tools and materials should be ergonomically accessible. The room should have good natural and/or electric lighting (Fig. 5.1).
(Grateful thanks are expressed to Dr Caroline Verbeke and Mr Jonathan Sheriff for their assistance and consent for the illustration.)
Prior to fixation it may be relevant to reserve some tissue from the specimens for microbiology assessment (being placed into appropriate culture media) and/or electron microscopy (requiring glutaraldehyde fixation). Fresh tissue can be taken for DNA extraction, cytogenetics and molecular pathology techniques. Other specialized tests (e.g., mass spectroscopy) may also require tissue retention before standard formalin fixation. Some samples need fixation and then decalcification in EDTA (see section later in the book).
Some specimens are only examined by means of macroscopic assessment, possibly with photography and other physical techniques. Examples include various mechanical/prosthetic implants, metal bodies, bullets, gallstones and medical devices. These must be dealt with according to the needs of the request/case. It should be noted that some specimens may require retention for a prolonged period of time – as in cases of forensic/criminal investigation.
This dissection/blocking/grossing/cut-up facility must have an appropriate storage area immediately to hand. This allows clearance of examined samples promptly, without the dissecting area becoming cluttered.
The individual choice of dissecting tools will reflect the type of specimen being considered (Fig. 5.2). However, a range of cutting blades is advised, enabling the dissector to deal with small specimens through to complex and large resections. Very large knives are particularly useful for obtaining full transverse sections of organs (lungs, liver, etc.). The smaller blades are useful for precise trimming of tissues. However, before any knife is put to the specimen, it is emphasized that the tissue specimen should be well fixed. Forceps and absorbent cloths should be available. The blocks taken (vi) should not completely fill the cassette (Fig. 5.3) as this would impede processing fluid access to the tissue. Thus, tissue cassettes are generally made of plastic and conform to a variety of size standards across the developed world nowadays. Most standard blocks allow a sample of about 20 × 20 × 3 mm thick tissue to be contained and processed. There is variation in cassette size that does allow larger blocks to be selected (Fig. 5.2). This is particularly useful for histological examination of large surgical resections where the global geography of the specimen is needed for analysis. Examples could include rectal cancer resection, radical prostatectomy and autopsy lung tissue for industrial disease. However, some general rules can be developed to specimen handling/sampling.
Figure 5.2 Cut-up/grossing tools. A range of small and large bladed tools are advocated along with forceps, ruler and a fluid-resistant dissecting surface. An appropriate measure and access to photography are needed. Varying sizes of cassette (centrally) are available, in a range of colors and sizes, to permit handling of varying amounts of sample and also to indicate handling issues that follow tissue processing.