The gross room/surgical cut-up including sample handling

Introduction

The initial dissection and preparation of any specimen for histological/microscopic analysis involves more than simply the transcribed macroscopic description and sampling of the specimen. Whilst the dissection and laboratory area are often perceived as the two key elements of the department, it must be clearly understood that there are many steps which follow specimen receipt, interfacing with the dissection room, that directly affect case handling. Some are specific to tissue selection and handling and others are clearly support roles.

It should be self-evident that a good laboratory will be adequately staffed by appropriately trained scientific/medical and support staff (secretarial, medical laboratory assistants, administration, etc.), and these staff interface at multiple levels with pathological sample handling. It has been demonstrated on many occasions that a poorly staffed department, whether in personnel and/or training, will perform suboptimally, to the detriment of sample analysis and therefore patient care.

Safety first and last

The histopathology department is rich in hazards, e.g. infection/biological, chemical and radiation. There are also various risks reflecting the range of materials used to store, process and analyze tissues. These may be toxic, flammable, allergenic, carcinogenic and electrical. The presence of sharp cutting implements, complex machinery and the movement of the specimens around the laboratory heightens all of these risks. Staff need to be fully trained to be aware of all of these potential hazards and capable of operating safely in this environment. Every laboratory should have accessible and clear standard operating procedures (SOPs, see Chapter 1 ), many of which will reflect national/international guidelines ( ). Ongoing safety education as part of continuing professional development is required, and caution should be employed at every step of specimen handling for safe laboratory practice ( ).

Specimen reception

A separate room is required for specimen reception which acts as the interface between non-laboratory hospital staff, other visitors and the pathological laboratory. The area must be equipped with appropriate easily cleaned benching, adequate lighting, good ventilation, safety equipment, disinfectants, absorption granules and protective clothing. In the event of specimen spillage, e.g. body fluids, fixative leakage or other mishap, the immediate response by staff in this area will limit any potential local health risk and prevent risk to other laboratory personnel.

The key point of this room is to receive samples safely and securely. Any new specimen should have its identity confirmed and assigned a unique laboratory specimen identifier, usually a complex number. Mapping of the specimen identifier 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 valid ways of verifying the identity of any specimen. Multiple sources of cross reference are always advocated, and if there is any doubt with regard to the probity of a specimen then it should not be passed onwards until the clinician concerned has confirmed all the appropriate details.

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, usually labeled with a self-adhesive bar or quick response (QR) code label, can be passed to the dissection room for examination, specimen description and block sampling.

The usual numerical method of specimen identification is simply the year, expressed 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, often 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 2345L/17 is the two thousand, three hundred and forty-fifth sample of the year 2017. The letter L is a computer check datum to verify that the numerical data is valid.

Particular attention must be paid to cases with unusual or common names. Names which have a variety of different spellings and any specimens which have incomplete information should be carefully considered before being accepted. Those with imprecise verification data and poorly handwritten data should not be 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 are taken from a sub-part of the specimen then these can be designated with individual numbers or letters in a similar ascending fashion. Thus, a gastrectomy sample with two separate lymph node groups and the spleen could have one case number, 2345L/17, multiple sub-part specimens (A, B, C) and multiple blocks (1, 2, 3, etc.) which can be correlated against the surgeon’s operative dissection. Using the number described above, e.g. the spleen in this case could be designated 2345L/17. C.2 (C. indicating the sub-part of the third sample = spleen and the block number = 2). At this stage the sample may now confidently be passed into the dissection room.

Barcodes and QR codes can be used to assist material handling within the laboratory, but in general terms many laboratories still have paper request forms which will accompany the specimen as it passes throughout the laboratory and towards final report production (see Chapter 11 , Automation in the histology department). The departmental computer system can be set to track specimen movement through the laboratory from its receipt to the final histopathologist report authorization.

Surgical cut-up/specimen dissection/grossing

The ideal layout of this room is a matter of debate, varying between different laboratories and pathologists’ needs. Numerous design solutions exist around the general principles of a histology laboratory ( , ), but it is imperative that the dissection area must have good electrical or natural lighting, good ventilation and non-absorbent wipe-clean surfaces. Within the area there must also be appropriate protective clothing for the laboratory personnel including gloves and other equipment, e.g. photography, tissue macerators and disposal bins.

The dissection room should be a comfortable environment permitting undisturbed work by the pathologist and support technical staff ( Fig. 5.1 ). 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 which 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 to suit individual staff members. Modern dissection areas often have integrated dissection desks, enclosed fluid/fixative feeds and laminar down-draft ventilation ( ) protecting all the staff from formalin vapor and hazardous fluids. All tools and materials should be ergonomically accessible.

Thinking before dissection

Prior to fixation it may be relevant to reserve some fresh tissue from the specimens for microbiology assessment by placing into appropriate culture media and/or electron microscopy which requires glutaraldehyde fixation. Fresh tissue may be taken for frozen section immediate analysis. Unfixed tissue can also be taken for DNA extraction, cytogenetics and molecular pathology techniques. The latter is becoming increasingly common and important in the arena of personalized therapy by reflecting the tumor genotype and characteristics. Other specialized tests, e.g. enzyme assay and mass spectroscopy may also require tissue retention before standard formalin fixation.

Some specimens are only examined macroscopically, possibly with photography and other physical techniques. Examples include various mechanical/prosthetic implants, foreign bodies, bullets, gallstones and medical devices. These must be dealt with according to the needs of the specific request/case. It should be noted that some specimens may require retention for a prolonged period of time, e.g. in forensic/criminal investigation cases.

At this point, if no preliminary sampling is performed, the specimen is usually fixed, by immersion into formalin. This process fixes (see Chapter 4 ) the sample, allowing storage for a prolonged period without tissue degradation. Some samples need fixation and then decalcification in ethylenediaminetetraacetic acid (EDTA) (see Bone, Chapter 17 ).

Case handling

This dissection/blocking/grossing/cut-up facility must have an appropriate storage area immediately to hand allowing clearance of already-examined samples promptly, preventing 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 sharp cutting blades is advised, enabling the dissector to deal with small specimens through to complex and large resections. Long knives are particularly useful for obtaining full transverse sections of whole organs, e.g. lungs and liver. The smaller blades are useful for precise trimming of tissues. The blade must be sharp if one is going to confidently sample the tissue appropriately to produce blocks of the correct thickness and shape. However, before any knife is put to the specimen, it is emphasized that the tissue specimen must be well fixed.

Forceps and absorbent cloths should be available adjacent to the work area. The blocks of tissue taken should not completely fill the cassette as this impedes the later processing fluid accessing all of the tissue ( Fig. 5.3 ). Tissues are normally put into standard tissue cassettes which are usually made of plastic and now conform to a variety of size standards across the developed world. Most standard blocks allow a sample of about 20 × 20 × 3 mm thick tissue to be contained and processed adequately. There is variation in cassette size which 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, e.g. complex rectal cancer resections, radical prostatectomy and autopsy lung tissue for industrial disease.

However, some general rules can be developed to specimen handling and sampling. The specimens should be analyzed with only one pot open at any one time. The request and specimen identity should be checked, ideally by two persons, the dissector and their assistant. The sample should be described in terms of the nature, shape, size and also any defining characteristics. This means that small biopsies, e.g. endoscopic mucosal samples, may simply be afforded a simple descriptor in the form of the number of pieces and the size (SI units, usually mm) of the largest piece of tissue. An example could be ‘three pieces of brown tissue, the largest 3 mm diameter’.

Medium and large specimens ( Fig. 5.4 ) need more detail and a careful description of the various anatomical components, together with identification of macroscopic landmarks, orientation markers/sutures and any lesion(s) if relevant. The background tissues, beyond the lesion under consideration, also require description.

The sampling of any large case/resection ( Fig. 5.5 ) should follow local and national guidelines in order to provide the relevant information for the subsequent clinical management of the patient ( ).

The macroscopic description is usually dictated for subsequent secretarial transcription, or occasionally can be simply written down for typing later. Canned or proforma reports may be of value to standardize the approach to samples, but those in the dissection suite must be capable of some adaptation since no two cases are identical.

Once the case sampling has been finished, any excess material should be kept generally for 1-3 months as further sampling may be needed. Storage should be close to the dissection area on sturdy shelving with good ventilation and easy access ( Fig. 5.6 ). The shelves should not be at high level

If one knows beforehand of additional tests which will be automatically required on a specimen, e.g. a renal transplant biopsy (requires multiple levels, ancillary histochemical stains and immunohistochemistry tests), then different color cassettes or markers can be used in order to designate standardized additional actions which should follow as an automatic laboratory consequence ( Fig. 5.2 ). The different colored cassettes may also indicate the types of sample contained, as well as the urgency of any specimen.

Following dissection, the residual tissues must be stored in a ventilated secure archive format, and waste materials must be disposed of according to local/national health and safety regulations ( ).