Surgical pathology and the tissue diagnosis
Surgical pathology turns a tissue specimen into a diagnosis through gross examination, sampling, processing and microscopic interpretation. The specimen, the clinical question and the quality of preparation determine what can be established and how much work is needed.
The questions tissue examination answers
Surgical pathology examines tissue and organs removed by biopsy or surgery to diagnose, rule out or evaluate disease, or provide documentation. Almost all tissue removed during surgery undergoes pathological examination. Light microscopy of cell and tissue morphology anchors this work, supplemented for some tissues by electron microscopy, flow cytometry and molecular diagnostics.
The determination the public associates with surgical pathology — benign or malignant, after a breast or prostate biopsy — is only one part of the practice. Vast categories of benign, premalignant and malignant disease exist, and the pathologist is trained to evaluate subtle features within each and to supply information that guides treatment.
Within benign disease the assessment is often of the presence, type and severity of inflammation in biopsies from the gastrointestinal, respiratory and genitourinary tracts. Examination may also detect infection by bacteria, fungi or parasites, or the cellular changes a virus produces; ulceration, scarring, necrosis and other signs of tissue reaction are detectable in the same way. Specific patterns of reaction point beyond themselves, so that certain types of inflammation coupled with necrosis prompt a search for tuberculosis or fungal disease. Documenting that a sample is normal is itself a contribution when a patient has unexplained symptoms and diseases must be ruled out, and unsuspected malignancy is occasionally found in an organ removed for what was assumed to be benign disease.
Premalignant disease is a third distinct question. Certain benign disorders of the breast carry a significantly increased risk of future malignancy while others do not, so the pathologist's evaluation can help identify patients who need close clinical follow-up. Biopsies from the bladder, colon, cervix, endometrium, bronchus, prostate and skin can show a spectrum of dysplastic change that may precede malignancy, and the assessment of the severity and extent of that change informs what follow-up or treatment would prevent or forestall it. Where malignancy is present, each organ system has its own characteristic neoplasms, some separated only by subtle distinctions, and surgeons and oncologists depend on the pathologist to establish type and extent — depth of invasion, involvement of blood vessels and lymphatic channels, metastasis to lymph nodes and other organs, and the relationship of the cancer to the surgical margins.
Gross examination and slide preparation
Specimens may include brain, nerve, heart, blood vessel or muscle biopsies; whole gallbladders, ovaries, uteri, kidneys, prostates, spleens or lymph nodes; breast or prostate material ranging from needle biopsies to whole organs; bowel, stomach, lung or bone segments; and lower limbs removed for gangrene. Small biopsies commonly come from the oesophagus, stomach, duodenum, colon, liver, bronchus, prostate, endometrium, cervix, bladder, kidney, thyroid or oral mucosa; skin specimens range from minute biopsies to larger resections.
Evaluation has two integrally linked halves, gross and microscopic, each contributing an essential part of the assessment. Gross examination means evaluating the specimen with the naked eye, taking measurements including weight where appropriate, and describing abnormalities. Except for minute specimens under about 2 mm in thickness, dissection is needed both to assess the specimen and to prepare tissue for microscopy. Because most microscopic examination is performed on a very limited portion of the whole, deciding which portions go forward for histopathologic study is a crucial part of the macroscopic evaluation, and clinical information such as the suspected diagnosis and the reason for the biopsy or surgery guides it from the outset.
What counts as a relevant macroscopic finding varies widely by specimen. Specimens are generally serially sectioned and examined for variation in texture, consistency and colour, but a gallbladder removed for gallstones is documented chiefly by the number, size and description of the stones, while a colon segment removed for cancer needs the cancer described and also the incidental polyps or ulceration unrelated to it. An incidental finding may be clinically unsuspected cancer, for example in an appendix removed for acute appendicitis. Ink is sometimes placed at the margins or other areas of interest so they can be identified during microscopy. The dissector's controlling obligation is to capture the features on which diagnosis depends: a diagnosis cannot be made from microscopic features if the lesion stayed behind in the gross specimen. Clues that sampling missed something do appear later — a macroscopically benign-looking area containing microscopic cancer, for instance — so gross specimens are kept available for re-examination for usually weeks to months. They are eventually discarded, after which the tissue used for microscopy is the only permanent record of what was removed from the patient.
Representative portions are cut with a scalpel into sections routinely no larger than 2.0 by 2.5 cm, ideally less, and 2 to 3 mm thick. Sectioning is greatly helped by fixation, and the ideal is to place the specimen in preservative immediately on removal from the body, although the need for certain special studies, such as microbiologic cultures, precludes immediate fixation. Preservatives, commonly formalin, prevent autolysis (digestion of cells or tissues by their own enzymes), inhibit bacterial overgrowth, stabilise cellular structure, create the conditions for staining and firm the tissue for sectioning; fixative penetrates smaller specimens more easily, so large ones such as bowel segments may be opened and left in preservative overnight. The sections are transferred to plastic cassettes and placed on a tissue processor, which dehydrates the tissue and infiltrates it with paraffin, routinely overnight, although very small specimens can be prepared in a shorter time. Any short cut or technical problem at this stage can make the microscopic appearance difficult or impossible to interpret. Histotechnologists then embed each portion in a paraffin block and use a microtome to cut sections usually 4 to 6 μm thick, mount them on glass slides and stain them. A microtome holds and advances a tissue block in increments of 1 to 10 μm against a special knife blade. Both the millimetre-thick scalpel slices and the micrometre-thick microtome slices are commonly called sections, so the term alone does not distinguish the two preparations.
The routine stain is a combination of hematoxylin and eosin. Hematoxylin, a basic dye, stains acidic structures such as nuclei blue or purple; eosin, an acidic dye, gives basic structures such as cytoplasm a red or pink colour, and the pair lets the pathologist distinguish regions of cells and assess cellular detail. Most processed tissue can be evaluated adequately with hematoxylin and eosin alone, and numerous other stains highlight microorganisms or particular tissue types where it cannot. Immunohistochemistry uses antibodies to highlight specific cellular and tissue features and can help classify cancers that conventional stains delineate poorly.
Microscopic interpretation
Histopathologic findings are microscopic morphological alterations in diseased or abnormal tissue.
One of the first steps in a pathologist's training is learning the microscopic appearance of normal tissue, because the body's cell and tissue types are numerous and extremely varied. Epithelial cells of the oesophagus can be distinguished from those of the stomach, which differ from small bowel, which differ again from colon — so that cells entirely normal for the intestine represent an abnormality when they appear in the stomach. Recognising normal tissue in a normal location, and subtle departures from it, is therefore itself diagnostic work.
Familiarity with normal appearances is only a fraction of the skill. Cells and tissues undergo an effectively unlimited range of alterations, and although the difference between benign and malignant is usually obvious to a trained pathologist, morphology does not always reflect biologic behaviour. A common diagnostic difficulty arises when benign cellular alterations mimic malignancy: one hallmark of malignant cells is increased intensity of nuclear staining, hyperchromasia, together with a disproportionate increase in nuclear size relative to cytoplasm — and benign cells reacting to inflammation or other insult can show similar changes. Premalignant dysplastic change can also resemble malignancy closely enough that severe dysplasia and early cancer are morphologically indistinguishable, which is a recognised source of diagnostic difficulty in sites such as the cervix.
General histopathologic features of malignant neoplasms vary considerably between cases; morphological rules do not invariably establish malignancy.
| Feature | Findings |
|---|---|
| Tissue architecture | Distortion or obscuring of normal architecture |
| Growth patterns | Crowding and disorganisation |
| Invasion | Growth into adjacent structures or tissues, blood vessels or lymphatics |
| Necrosis | Tissue death |
| Cellular morphology | Pleomorphism (variation in size and shape); diminished differentiation or developmental immaturity |
| Nuclear atypia | Enlargement relative to cytoplasm; pleomorphism; irregular borders; altered internal appearance; hyperchromasia; coarse chromatin; prominent nucleoli, which are associated with protein synthesis |
| Mitotic figures | Increased numbers; aberrant appearance |
Looking at glass slides through a microscope is comparable to viewing a football field through a magnifying glass, and even a minute specimen may yield hundreds of microscopic sections which do not all show the same changes. Surgical pathology requires data collection and assimilation, discernment, judgement and decision making.
Differences between specimens
Time, labour and expertise depend on the size and complexity of the specimen, the nature of the pathologic process, and the specific information the pathologist has to determine and convey. At one end are specimens from sterilisation procedures — small segments of paired Fallopian tubes or vasa deferentia — where disease is rarely found and the point of examination is microscopic documentation that the surgeon resected the intended structures; inadvertent resection of something else is rare but could have serious consequences, and both halves of the evaluation are quick. Many small biopsies from the gastrointestinal tract, bronchi, bladder and prostate are handled quickly at the gross table, many of them small enough to go forward without dissection. Slightly larger specimens, such as colonic polyps, require scalpel sectioning but are generally still easily examined, measured and described at the gross table.
Microscopic evaluation of a small specimen can nonetheless be more complicated than its size suggests, because the number of microtome-cut sections is a separate decision. Several sections are routinely prepared from a single prostate needle biopsy, taken from both superficial and deep levels of the paraffin block so that all clinically significant alterations present are available for evaluation. Where the original slides are equivocal, the pathologist may request further sections from deeper in the block: a colon biopsy that cannot separate benign reactive change from malignant or dysplastic change, especially where only scant, superficial, fragmented tissue is available, may on deeper sections show unequivocal malignancy, or show inflammation or ulceration sufficient to suggest that the alterations are reactive. Patterns of inflammation suggesting infectious agents similarly prompt special stains and additional slide preparation. Each of these prolongs the evaluation, and the extra time may be what produces a definitive diagnosis.
Large specimens carry their own requirements. A colon segment removed for cancer needs the size, location and growth characteristics of the tumour, the depth of invasion into surrounding tissue and the distance from the resection margins determined and recorded, and the fat outside the bowel wall inspected carefully for lymph nodes to establish whether the cancer has metastasised. The disease present determines how much tissue must be sampled: a uterus removed for leiomyomas requires sampling of the tumours both to document them and to rule out the similar-appearing malignant leiomyosarcoma, which is quick where the tumours are few, small and characteristic in appearance and progressively more demanding as they grow larger and more numerous. More thorough sampling is required where gross inspection shows signs of malignancy such as irregular tumour borders encroaching on surrounding tissue, or the softening and sponginess of necrosis sometimes caused by a neoplasm outgrowing its blood supply. A uterus removed for endometrial cancer needs the location and extent of the cancer, the depth of invasion into the uterine wall, and whether cancer is present outside the uterus, at the external surface or in other removed organs such as ovaries and fallopian tubes. Fat removed at surgery must also be checked for lymph nodes to assess metastasis. Microscopy confirms or dispels gross impressions and identifies the subtype of malignancy and features such as invasion of blood vessels or lymphatic channels.
A breast lumpectomy specimen can be labour-intensive at both stages even when small. The surgeon's aim is to remove the cancer with a rim of noncancerous tissue around it, and the pathologist must evaluate the histopathologic features and growth characteristics of the cancer and also establish whether the margins are free of tumour. The whole external surface of the specimen is the margin, as the peel of a grapefruit would be — except that the margin of a lumpectomy is soft pliable fatty tissue that does not hold the shape a grapefruit peel does, which is what makes the assessment difficult. Margins nearest the tumour are sampled more thoroughly and distant ones more sparsely, and the orientation the surgeon conveyed must be maintained throughout, because if cancer extends to a margin the surgeon has to know which margin of the operative wound to re-excise.
Intraoperative consultation
Microscopic examination is sometimes needed during a procedure while the patient remains anaesthetised. Reasons for intraoperative consultation include the following.
- Establish the presence and nature of a lesion.
- Determine the adequacy of surgical margins.
- Establish whether tissue contains diagnosable material or additional sampling is indicated.
Working at that speed requires a cryostat to freeze the tissue, because freezing hardens it enough for microtome sectioning without waiting for fixation and paraffin embedding. A cryostat is a cabinet in which tissue is rapidly frozen, commonly to −20 °C, for cutting sections usually 4 to 6 μm thick. Diagnoses are often rendered from the resulting frozen sections, but confirmation generally awaits permanent sections, prepared from fixed, paraffin-embedded tissue: freezing preserves cellular structure less well than fixation does, the morphologic detail is inferior, and frozen tissue is more prone to artefact and distortion.
The pathology team
A surgical pathology diagnosis is produced by a team. Pathologists often have assistants who organise and record the procedures around gross examination, and some have highly trained assistants who perform gross evaluation under supervision. Histotechnologists do the microtome sectioning and slide preparation. Medical transcriptionists type the reports of findings and diagnoses. Other clerical and technical staff cover the chain from specimen receipt and log-in to distribution of the final report, and fellow pathologists consult on difficult cases. Pathology depends on clinical colleagues sharing clinical impressions and pertinent patient histories, and the accuracy of a diagnosis rests partly on what those colleagues supplied.