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 (websites 1-3). 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 (websites 2 and 4).
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 (Rosai, 2011;
Lester, 2010; Cook, 2006, websites 2, 4), 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 (website 5) 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 x 20 x 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 (website 6).
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 (websites 4, 5, 8).
Photography
Photographing the macroscopic specimen, whole or during the dissection, is particularly important in cases of
complex surgical excision, e.g. Wertheim's hysterectomy, pneumonectomy and localization samples. It may also
be of use in later analysis/ case discussion (Fig. 5.5). Digital photography with hand-held basic devices has been a
major bonus to the laboratory superceding film-based photography. The opportunity to enhance the patient
record has greatly improved all pathology disciplines and allowed retrospective case reviews. Specialist and
professional photography may still be required however for cases which may be used in visual teaching
presentation, journal/book publication or in a medico-legal situation. Consideration of the potential to recognize
a patient's sample should be made, with some guidance existing on the subject (website 7).
Specimen dissection plans
Small samples
Small biopsy samples rarely need dissection and can be processed as they present i.e. whole, embedded and then
sectioned. In some cases, orientation of the specimen in the block can be facilitated by means of a dissecting
microscope or magnifying lens, e.g. when considering morphological abnormalities of small bowel biopsies.
However, the majority of small biopsies can be adequately examined at multiple levels allowing the pathologist
to mentally reconstruct the three-dimensional quality of the tissue during microscopic examination.
These small samples may benefit from being placed in a nylon bag, between metal disks with fine mesh, or
wrapped in paper in order to prevent the samples falling through the cassette perforations and being lost. Eosin
can be used as a marker for small samples in order to highlight them on the background of paper, embedding
bench or equivalent. It is recommended that a count of the small tissue biopsy fragments is always taken at the
description/ grossing stage in order to verify that all the tissue has survived processing prior to section cutting.
Core biopsies
These are treated in a similar manner to small biopsies, although their embedding requires being laid out in
longitudinal fashion so that the plane of section cuts along the majority of the tissue. At this stage, larger cores
with diameters of 4-5 mm or greater may benefit if divided into two halves along the long axis, increasing the
tissue available to review histologically. However, this risks tissue damage and can be time-consuming and
difficult. The alternative solution may be to simply provide multiple levels with retention of tissue in between the
levels for further analysis. Multiple cores, even if in the same specimen pot, often require each core to be placed in
an individual cassette in order to prevent tissue loss during 'trimming-in' after processing.
Skin biopsies
These include simple punch biopsies (handled akin to cores) and shave biopsies which should be mounted on
edge in order to provide an adequate view of the epidermis, dermis and subcuticular layers. A marker item
placed into the cassette, e.g. plastic bead or colored paper will alert such samples to embedding personnel.
Alternatively, some laboratories use cheese paste to help maintain specimen orientation (Tripathi et al., 2008). The
protein in the paste helps hold the tissue orientation during processing (Fig. 5.7).
Skin samples may include the more complex intermediate and large specimens. These are required for
removal of large defined lesions and radical skin cancer resections which may include deep soft and/ or bony
tissues.
The intermediate and larger samples of skin are often presented as an ovoid or ellipse piece of skin and
subcutis, usually with a central lesion (Fig. 5.4). These must be described in terms of the width and breadth of the
specimen together with a depth. After the fixed tissue is sectioned, usually at 3 mm intervals along the specimen
the lesion characteristics, e.g. nodule, ulcer or papule, color and the distance to the margins should be recorded.
Photography may be of value. Specimens of these skin resections are often best managed in serial transverse
section, with Indian ink or another dye being applied to different surfaces in order to confirm the orientation or
boundaries of the specimen (Fig. 5.3). Markers can be placed into cassettes in order to confirm pieces of tissue
with orientation markers, although it is generally found that specimens start with small apical transverse sections
through to the broadest point across the waist of the specimen and then taper off towards the other end.
Large resections of skin with soft tissues may require photography and then targeted block sampling, as it is
unreasonable to embed and section all of it. This should also allow the appropriate assessment of any tumor or
lesion with deep and lateral margins. Multiple blocks of the pathology within the specimen are normally taken in
order that any disease variations within the lesion will be identified. These samples, and indeed their smaller
counterparts, should be blocked to permit reporting against national and/or international standards.
Bowel specimens
Apart from endoscopic gastro-intestinal biopsies, bowel samples are generally medium and large tissue
resections which can originate from anywhere along the length of the gastrointestinal tract, e.g. esophagectomy,
gastrectomy and partial colectomy. They are best sampled with multiple (3 or
more) blocks of any lesion in relation to the adjacent mucosa, wall and serosal aspect of the tissues (Fig. 5.8),
although large geographic ('jumbo') blocks can be employed. The margins often need inking and the background
tissues including resection margins are often included as part of the relevant dissection protocol (Allen, 2013;
Allen & Cameron, 2012, website 6). Particular attention is paid to the lymph nodes which can either be manually
dissected in groups, or be identified from fat-clearance protocols (Prabhudesai et al., 2005) as described below. It
is vital that the nodes are assessed in terms of their proximity to the lesion along with their different 'level' stages.
Many cases require consideration of the high tie, i.e. the most proximal lymph node in the resection (or
equivalent).
Fat clearance
Finding lymph nodes within a large amount of fatty mesenteric or soft tissue can be particularly problematic
when trying to isolate individual small nodes. The ability to remove the adipose substrate from any specimen will
lead to an enhanced rate of
node detection and thereby sampling. This will raise the accuracy of tumor staging and patient management. One
aspect of the histological tissue handling in the cut-up room allows such node identification (Prabhudesai et al.,
2005). In this process, the fatty tissue is usually sliced into 5 mm fragments and placed into large cassettes which
increases the access of solvents to the specimen. Fat removal occurs as part of the processing of tissues, but the
blocks of fatty parenchyma are normally removed from the processing chamber before the tissues are
impregnated finally with wax. At this stage the lymph nodes can be readily palpated and identified by
transillumination of the tissue sample from below (Fig. 5.9). The sampled nodes can then be placed back in the
tissue processor in a smaller cassette, with normal embedding, sectioning and staining to follow.
Lung tissues
Aside from small bronchi, transbronchial and pleural biopsies, the lung samples commonly received for histology
are localized, wedge biopsies or lobectomy and pneumonectomy specimens. There are also occasional radical
resections which can include, e.g. pleuro-pneumonectomy, lobectomy and the chest wall. The background pleura
and lung must be evaluated along with any lesions as required in standard proforma sampling protocols (Allen,
2013; Allen & Cameron, 2012, website 6). In general terms, multiple blocks for any tumor (4 or more) along with
sampling of the pleural/mediastinal/bronchial margins are needed. Nodes are often presented in groups
separately, although careful dissection of the hilar tissues should allow further node harvest from these tissues
(Fig. 5.5).
Gynecological samples
Common samples include fragments of endometrium removed by curette or equivalent and small punch
biopsies. These are generally embedded whole and processed in one cassette. More complex samples, e.g. cone
biopsies from the cervix, need appropriate inking of margins and orientation, often in a serial block fashion across
the specimen with photography. This allows the three-dimensional assessment of dysplasia or invasive neoplasia
in relation to the various surgical margins. Uterine samples are usually sampled in terms of the cervix if included,
endometrium and myometrial tissues together with some representative sampling of common benign lesions, e.g.
fibroids (leiomyomas). Sampling will be guided by local practice together with national guidelines (Allen, 2013;
Allen & Cameron, 2012, website 6). Dysplastic and malignant lesions often require multiple blocks including
resection margins and careful examination of related lymph nodes which are usually presented, and therefore
blocked, separately. Specific tissues such as tubes and ovaries should follow similar standard guidelines in terms
of the sampling pattern, number of blocks and related tissue samples. It is emphasized that pluripotential
differentiation of tumors within the female genital tract requires multiple blocks of a tumor to be taken for full
analysis.
Breast resections
These are also common surgical resections, usually with the need for inked margins in relation to the orientation
of the specimen. Multiple blocks of the tumor are usually required. The background tissues should also be
assessed at multiple points and the lymph nodes, if present, are often examined in a tiered or grouped fashion.
This allows the tumor spread to be assessed by identifying the size of the nodes involved and the furthest node
from the primary which has been affected. Fat clearance may be required to capture all the nodes in the axillary
tail.
Soft tissue resections
These vary but should be examined with multiple blocks of tissue, background parenchyma and the margins.
Some experts advocate 1 block for every 10 mm diameter of tumor up to 10 blocks, although more may be
required on occasions. Careful slicing and examination of the specimens macroscopically will allow sampling and
consideration of all the peripheral boundaries. Furthermore, given the pervasive nature of soft tissue tumors this
widespread sampling is usually required. Tumor sampling before fixation for molecular and/or genetic analysis
may be required.
Other samples
This chapter is not designed to be able to discuss all possible resections and specimen subtypes and the groups
above are illustrative only. Several of the references below will assist the reader with a fuller range of dissection
approaches, alongside publications from governing bodies and national organizations which have produced
protocols for the analysis of specimens (Allen, 2013; Allen & Cameron, 2012, website 6).
References
Allen, D. C. (2013). Histopathology reporting. Guidelines for surgical cancer (3rd ed.). London: Springer.
Allen, D. C., & Cameron, R. I. (Eds.). (2012).
Histopathology specimens: clinical, pathological and laboratory aspects (2nd ed.). London: Springer.
Cook, D. J. (2006). Some aspects of the organization of a histology laboratory. In Cellular pathology (2nd ed.). (pp. 357-
367). Scion, Bloxham: Oxfordshire.
Prabhudesai, A. G., Dalton, R., Kumar, D., & Finlayson, A. G. (2005). Mechanized one-day fat clearance method to
increase the lymph node yield in rectal cancer specimens. British Journal of Biomedical Science, 62, 120-123.
Rosai, J. (2011). Gross techniques in surgical pathology. Rosai and Ackerman's surgical pathology (10th ed.). Edinburgh: Mosby,
25-35.
Lester, S. C. (2010). Manual of surgical pathology (3rd ed.). Elsevier-Saunders.
Tripathi, M., Sethuraman, C., Lindley, R., & Ali, R.
B. (2008). Comparison of use of cream cheese and agar gel for orientation of skin biopsies. XXIX Symposium of the ISDP,
Graz, Austria, October 2-4, 2008. American Journal of Dermatopathology, 30, 514-533.
Websites
1. Clinical Pathology Accreditation (UK) Ltd. https:// www.ukas.com/services/accreditation-services/ clinical-p
athology-accreditation/.
2. Institute of Biomedical Science. Good professional practice for biomedical scientists. http://www. ibms.org.
3. National Pathology Accreditation Advisory Council. Requirements for Pathology Laboratories.
https://www.health.gov.au/internet/main/ publishing.nsf/Content/21B7F24866EF1DEEC
A257C2A001EC403/$File/Reqmts%20for%20 Medical7o20Path7o20Services.pdf.
4. Health and Safety Executive. http://www.hse. gov.uk.
5. Ventilation in the histopathology laboratory. http: //www.afosgroup.com/news/Ventilation%
20in°720the°720Histopathology°720Laboratory. pdf.
6. Datasets and tissue pathways. Royal College of Pathologists. http://www.rcpath.org.
7. Making and using visual and audio recordings of patients - guidance for doctors. https://
 www.bma.org.uk/advice/employment/ ethics/confidentiality-and-health-records/ visual-and-audio-recordings-
of-patients.
8. The Human Tissue Act 2004. (2008). (Ethical Approval, Exceptions from Licensing and Supply of Information about
Transplants) (Amendment) Regulations. http://www.legislation.gov. uk/uksi/2008/3067/pdfs/uksi_20083067_
en.pdf.