Anatomic Pathology | Lab Tests Online

What is anatomic pathology?

Anatomic (or anatomical) pathology is the branch of medicine that studies the effect of disease on the structure of body organs, both as a whole (grossly) and microscopically. The primary role of anatomic pathology is to identify abnormalities that can help to diagnose disease and manage treatment. Although one of the frequent uses of anatomic pathology is to help identify and manage various types of tumors or cancers, it is also valuable in evaluating other conditions, including kidney and liver diseases, autoimmune disorders, and infections, for example. In fact, in most hospitals, all tissue removed during surgery must be examined by a pathologist.

Anatomic pathology is somewhat different from clinical pathology (or laboratory medicine), which deals with the measurement of chemical constituents of blood and other body fluids (clinical chemistry), analysis of blood cells (hematology), and identification of microorganisms (microbiology), to name a few examples. While most of the tests described on this site would be categorized as clinical pathology, many are used in conjunction with anatomic pathology procedures. In fact, technical advances are blurring the distinctions between the two in many areas. Overlaps include, for example, flow cytometry, cytogenetics and molecular pathology, which can be performed on both tissue samples and blood or body fluid samples. Therefore, some knowledge of this branch of medicine may help you better understand the tests that your or a family member's health practitioner may consider in diagnosing, monitoring, and treating a condition.

There are two main subdivisions within anatomic pathology:

Histopathology, which involves examination of intact tissue under the microscope. This is often aided by the use of special staining techniques and other associated tests, such as using antibodies to identify different components of the tissue.
Cytopathology (cytology), which is the examination of single cells or small groups of cells. A common cytology test is the cervical Pap smear. Specimens are processed by physician assistants and technologists and then examined under a microscope by a physician pathologist. The pathologist gives the definitive diagnosis to the health practitioner, who then decides on the most appropriate mode of treatment and patient management.

Anatomic pathologists are also involved in performing post-mortem examinations (autopsies). An autopsy may be performed after a person has died of an illness that could not, for whatever reason, be properly or fully diagnosed before death. The physician will seek consent from the family to have an autopsy performed. If the cause of death is suspicious or related to an illegal activity, the autopsy will be performed by a forensic pathologist. Consent from next of kin is not required for such medico-legal autopsies. (For more on this, see our information page on Forensic Pathology and Autopsies.)

Histopathology: Biopsies and Examination of Tissues

Histopathology involves the examination of sampled tissues under the microscope. These may be small pieces of tissue obtained from a part of the body using a technique called biopsy or samples taken from whole organs or parts of organs removed during surgery.

Most biopsies are small samplings of the area of the body in which disease is suspected. These are called "incisional" biopsies and additional surgery or treatment may be recommended after the diagnosis is made.

Other biopsies may include the entire affected area, such as a skin mole. These are called "excisional" biopsies and examination of the adjacent, uninvolved margins helps to verify that the affected area has been completely removed.

The pathology laboratory also receives large whole organs, or parts of organs, removed during surgery, such as a uterus after a hysterectomy, the large bowel after a colectomy, or an amputation of an arm or leg. These specimens are examined as a whole (grossly) for size, shape, color, and/or presence of any external abnormalities. Then, smaller samples are taken for definitive microscopic evaluation.

Tissue Preparation

Formalin-fixed, paraffin-embedded (FFPE)

Biopsies and samples of tissue removed from organs are usually placed in formalin (diluted formaldehyde), which "fixes" the tissue by cross-linking proteins. This preserves the cellular architecture and also allows the tissue to survive the processing that comes next. Other types of fixatives may be used depending on the type of specimen or the cellular characteristics that need to be enhanced.

The tissue goes through various chemical steps (dehydration and dissolving of fat) in preparation for embedding into a paraffin (wax) block. The paraffin blocks are placed on a special machine that uses an extremely sharp knife (a microtome) to shave very thin pieces of tissue of about 5 µm (micrometers, or about 0.0002 inches) in thickness. The thin pieces are placed on a glass slide and stained with special reagents to highlight key aspects of the tissue.

Hematoxylin and eosin, also known as H&E, is the most widely used stain. It is a combination of a basic stain (hematoxylin) and an acidic stain (eosin). This reacts with acidic and basic cellular components on the slide to give, respectively, purple and pink colors to the tissues.
Frozen sections

When time is crucial (for instance, when a surgeon needs an answer while performing surgery), the pathologist will bypass the fixation, processing, and embedding in paraffin steps and perform a frozen section. The tissue is surrounded by a fluid containing polyethylene glycol and placed on a chilled metal block inside of a refrigerated device called a cryostat. Once the fluid has frozen, a laboratorian uses the microtome to thinly slice (section) the block. The thin slice is placed onto a glass slide, stained, and examined. The procedure usually takes 10-20 minutes. However, freezing of the tissue can result in some distortion of cells and some staining artifact. This is why frozen sections are often preliminary, with a final diagnosis based on the routine processing of tissue as noted above.
Touch preps

One additional method that can be done intra-operatively instead of freezing the tissue is making imprint smears (touch preps). The tissue is cut and pressed or smeared onto a glass slide, then stained and examined under the microscope. This process leaves individual cells from the tissue on the slide and avoids freezing artifacts. Imprint smears further reduce the time taken to examine tissue and are most often used to examine lymph nodes.

Special Techniques

Special staining

Pathologists use different special stains in addition to the routine hematoxylin and eosin (H&E) stain. These may highlight fat, different tissue fibers, mucus, microorganisms such as bacteria or fungi, proteins, or other biochemical substances that might be useful in identifying key elements that are characteristic of certain diseases.
Immunohistochemistry

Special stains can provide useful information but are somewhat limited in their ability to provide a definitive answer, whereas immunohistochemical stains are more specific in what they stain. This technique takes advantage of the unique properties of antibodies that have been developed to recognize specific components on or within cells. The antibodies are bound to certain stains or dyes that are readily visible when observed under a microscope. This allows a pathologist to select those antibodies designed to identify key cellular elements or tissue types that have been associated with certain diseases and assist in obtaining a final diagnosis.
Electron microscopy

Some medical situations require a higher level of microscopy than that provided by standard light microscopy. Examples include types of kidney disease (glomerulonephritis), pulmonary diseases (asbestosis), or aggressive cancers that lose their normal proteins. In these cases, a very powerful type of microscope called an electron microscope may be used. The electron microscope uses high voltage to create a wide beam of electrons that are directed at a specially stained sample. The electrons are either absorbed or scattered, creating a black and white image. It can magnify up to two million times, whereas the maximum power of a conventional light microscope is only one to two thousand times.
Genetic testing and other novel techniques
Recent advances have allowed pathologists to utilize formalin-fixed, paraffin-embedded (FFPE) tissue in a number of innovative ways. Specific areas of tissue can be removed (by micro-dissection) and the chemical constituents analyzed by mass spectrometry or other techniques.

Most often, genetic testing, which involves the study of chromosomes and their DNA, is done in the clinical pathology laboratory, but sometimes may be overseen by the anatomic pathology laboratory when tissue samples are employed. It can be a valuable tool as certain diseases may be caused by specific genetic abnormalities.

For example, sections of a chromosome may switch places (translocation) or may be missing (deletion). These chromosomal translocations and deletions can be detected using fluorescent in situ hybridization (FISH). DNA and RNA isolated from FFPE tissue can also be used to identify specific mutations or screened for abnormalities.

Because DNA and RNA from FFPE tissue are fragmented by tissue preparation (formalin fixation) and unprotected storage, mutation analysis is increasingly being performed using so-called "next generation" sequencing techniques in which millions of reactions are performed simultaneously on the short stretches of DNA, followed by the application of computer algorithms to determine the genome.

Most of these molecular techniques are currently utilized in order to identify mutations that help to guide therapy of malignant tumors, such as:

HER2/neu—breast cancer

EGFR—adenocarcinoma of the lung

KRAS—colorectal cancer

BRCA 1 & 2—breast cancer and ovarian cancer

BRAF—skin melanomas

JAK2—myeloproliferative neoplasms

For a general discussion of these tests, see the article on Genetic Tests for Targeted Cancer Therapy.

Cytopathology

Cytology is the study of individual cells and cytopathology is the study of individual cells in disease, although the two terms are often used interchangeably. Sampled fluid and/or tissue from a patient is smeared onto a slide and stained (see techniques below). This is examined under the microscope by an anatomic pathologist (cytopathologist) to look at the number of cells on the slide, what types of cells they are, how they are grouped together, and what the cell details are (shape, size, nucleus, etc.). This information is useful in determining whether a disease is present and what is the likely diagnosis.

Cytology is most often used as a screening tool to look for disease and to decide whether or not more tests need to be performed. A common example of screening would be the investigation of a cervical (Pap) smear. The health practitioner inserts a speculum into the vagina to directly view the cervix, which is gently scraped or swabbed. The cells obtained are either smeared onto a glass slide or rinsed into a liquid-based fixative and submitted to the laboratory for examination.

The cells are reviewed by a cytotechnologist (see Who's Who in the Lab), who can issue a negative report if no abnormal cells are seen. If any suspicious cells are identified, a cytopathologist reviews the case and makes a final diagnosis. Follow-up may include a colposcopy (use of a magnifying device to examine the cervix more closely) to determine if the abnormality is a pre-cancerous or cancerous lesion caused by human papilloma virus (HPV) or some other lesion and if biopsies need to be taken.

Most of the immunohistochemical, cytogenetic, and molecular techniques described for histopathology can also be applied to cytopathology.

Techniques

Exfoliative cytology

This is the analysis of cells that are shed from body surfaces. The cervical (Pap) smear is the most common example, but samples from the urinary bladder, abdominal cavity, chest cavity, cerebrospinal fluid, and washings from the lung are also frequently examined.
Fine needle aspiration

Fluids that contain many individual cells (e.g., pleural fluid from around a lung) or very small pieces of tissue can be obtained via a fine needle aspiration (FNA). This is performed using a thinner needle than that used in a core biopsy but with a similar technique. This type of material is usually liquid or loosely packed cell mass rather than solid tissue. The FNA is particularly useful in evaluating the presence of normal or abnormal cell types.

The analysis of cells from a large mass or organ is a slightly more complicated process and may involve the use of imaging (e.g., ultrasound or CT scan) to ensure that the suspicious area is being sampled. While a simple FNA generally involves a visible or palpable lump or cyst, complex FNAs involve sampling of tissues located internally. Imaging assistance may be required to ensure the position of the needle is located correctly and an accurate sample is obtained.

Many different kinds of physicians perform FNAs, but a pathologist or cytotechnologist usually quickly examines the material obtained to determine if the sample is adequate for diagnosis and to triage the material for appropriate additional studies. Because a local anesthetic is used, the complications of surgery can be avoided and a diagnosis obtained quickly, typically at reduced cost.
Speciatl techniques

Cells obtained from fluid or tissue can be easily analyzed using flow cytometry, the technique commonly employed in the diagnosis of cancers of the blood cells (leukemias and lymphomas). Cells pass through a laser beam (single wave length light beam). One detector measures how the beam of light is scattered to determine size and internal structures of the cells, while another detects antibodies that have been added to the cells (labeled with special dyes) in order to determine which proteins are present on the surface of the cells.

Anatomic Pathology Results and Reports

Like clinical pathology reports (see Deciphering Your Lab Report), anatomic reports include essential elements such as patient name, identification number, sample type, and source. The report usually includes some very technical medical terms, so it is advisable to review and discuss the report with the health practitioner and ask questions about anything that is not understood.

An anatomic pathology report typically includes the following five main sections:

Specimen information—details the type of tissue or organ involved, where the sample was taken and how
Diagnosis—this may appear at the beginning or the end of the report; it includes the specific findings, the final diagnosis from the pathologist. It may be very short and to the point or may include several lines of language, depending on the complexity of the case.
Gross (obvious or macroscopic) description of the sample—describes the visible characteristics, those readily seen by the naked eye, including size, shape, weight, color and/or consistency
Microscopic findings—this is usually very detailed; it is what the pathologist sees under the microscope and includes size, shape, and type of cells seen. It may include several other specifics. For example, it may describe the characteristics of a tumor:
- Invasive or non-invasive—whether cancer has spread
- Grade—typically used only for cancers; describes how the abnormal cells compare to normal cells; low-grade cells look more like healthy cells while high-grade cells look more abnormal; this may be used to help determine the course of the disease (prognosis).
- Dividing cells—whether the cells seen are dividing
- Margins—this describes the appearance of the tissue at the edges of the sample; margins may be clear, indicating that no cancerous cells are seen at the edges. If the margins are not clear, additional surgery may be needed to fully excise the tumor.
- Nearby lymph nodes—included if lymph nodes are removed at the time of the biopsy; notes whether cancer has spread to nearby lymph nodes; may be described as "node negative" (no cancer spread) or "node positive" (cancer spread to nodes).
- Stage—this combines the information on the size, location, and whether a tumor has spread to lymph nodes or other organs/areas of the body. This information may be helpful in establishing the prognosis.
Comments —includes anything else the pathologist wishes to convey about the examination; it may include results of specialized tests that might impact treatment; however, these may also be provided in a separate report.

Depending on the reason for the biopsy and the type of tissue examined, the report may include additional details within these sections or additional sections. As mentioned in the Introduction, a frequent use of anatomic pathology is in the evaluation of cancers and there has been an attempt to standardize these types of anatomic pathology reports so that specific details can be more easily entered into databases that serve as a tumor registry. Each state has such a registry and the information is collected by the Centers for Disease Control and Prevention (CDC) so that researchers can evaluate trends in cancer prevalence as well as new approaches to treatment.

For an example of what an anatomic pathology report may look like, see The Doctor's Doctor: A Typical Pathology Report. The College of American Pathologists also has a resource: How to Read Your Pathology Report.