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Obtaining an Anorectal Cytology Specimen

The reason to do an anal Pap smear is to look for evidence of HPV-related cellular changes. When someone becomes infected with HPV and the infection persists, the cells that are infected with HPV may not appear normal. HPV infected cells also may divide and multiply more than normal uninfected cells. There are also other changes that interfere with normal cellular processes at a molecular level in such a way as to allow the development of lesions or growths. In some cases warts or condyloma grow, in other cases flat lesions occur, with thickening of the tissues and sometimes increased blood vessels, which are usually hallmarks of the precancerous kind of lesions. And in some cases with HPV, there are no visible changes seen at all.

By obtaining a sample of cells from the anus, we can get an idea of whether HPV has caused any changes and if so, how severe these changes might be. Most importantly, we can determine whether or not further examination is necessary. The anal Pap smear is a blind sampling of the anal canal and similar to a cervical Pap smear, is best looked at as a general indicator of whether the cells are normal or abnormal. If abnormal cells are present, then further examination is required to determine the exact nature of the abnormality.

As mentioned earlier, HPV infection is the principal cause of AIN, CIN, and anal and cervical cancer. So, why not do tests to look for HPV? One of the reasons not to do HPV screening is that in groups of patients known to be at increased risk for anal cancer, such as HIV-positive men who have sex with men (MSM) with a history of receptive anal intercourse, the likelihood of finding HPV is nearly 100%; even 60% of HIV-negative MSM with a history of receptive anal intercourse have been shown to have HPV. The point is that HPV infection is so widespread that knowing someone has HPV doesn't help discriminate who needs to be treated. Going back to cervical cancer screening, HPV testing is only recommended for women with ASC-US Pap smears and if high-risk types of HPV are found then women are referred for colposcopy or examination of the cervix using a microscope.

Remember not all people infected with HPV will have problems and we want to focus our attention on the ones in whom HPV has led to potentially precancerous changes. The best way to do that is by sampling the cells of the anus with a Pap smear. An anal Pap smear is performed to screen for anal HSIL (the correct terminology is to obtain a sample for anorectal cytology) in the following manner.

  1. Patients are asked not to douche or have an enema or insert anything into their anus for 24 hours prior to an anal cytology exam.
  2. Lubricants should not be used prior to obtaining a cytology sample because the lubricant may interfere with the processing and interpretation of the sample.
  3. We usually obtain the sample with the patient lying on their left side, but other positions are acceptable.
  4. The buttocks are retracted to visualize the anal opening and a Dacron or polyester tipped swab moistened in tap water is inserted for approximately 2 to 3 inches into the anus. The swab can be felt to pass through the internal sphincter so the sample is obtained from the junction of the anus and rectum, which is where most of the HPV-related lesions are found. This area is slightly above the region that corresponds anatomically to the dentate line.
  5. The swab is rotated 360 degrees with firm lateral pressure applied to the end of the swab, such that it is bowed slightly and then it is slowly withdrawn over a period of 15 to 30 seconds from the anus, continuing to rotate the swab in a circular fashion. The lateral pressure ensures that the mucosal surface, rather than rectal contents are sampled.
  6. The swab is either smeared directly onto a glass slide and fixed as a conventional Pap smear by placing it into alcohol, or the swab is placed in a preservative vial and vigorously agitated to disperse the cells for liquid based cytology.
  7. Regardless of method used, the sample must be fixed quickly within 15 seconds in order to avoid drying artifact, which occurs easily and makes interpretation difficult.
  8. The slides are stained using the Papanicolau stain, hence the term Pap smear, and then are examined by the pathologist.

What are the Possible Results from Anal Cytology and What Do They Mean?

As shown in this illustration, with increasing severity of SIL, of either the cervix or anus, the proportion of the epithelium replaced by immature cells with large nuclear-cytoplasmic ratios increases. Invasive cancer probably arises from one or more foci of high-grade squamous intraepithelial lesions (HSIL), which is depicted in the drawing by epithelial cells crossing the basement membrane. All epithelial cells sit on a basement membrane and below the basement membrane are connective tissue, blood vessels, nerves, and the muscles of the anus. Cancer arises when abnormal HSIL cells develop additional genetic changes, which then turns them into cancer cells that have the ability to invade across the basement membrane into normal tissues.
Adapted and used with permission of Joel Palefsky, MD

Examination of an anal cytology specimen will yield one of the following results:

  1. Insufficient: not enough cells were obtained for the pathologist to evaluate to make an interpretation: a new sample should be obtained. Some of the reasons why the specimen may have been inadequate include having an enema or sex within 24 hours prior to collecting the specimen or the swab may not have been inserted deep enough or rotated with enough pressure to dislodge cells from the lining of the anus.
  2. Negative for intraepithelial lesions: the sample had an adequate number of cells to be examined and they were normal in appearance with no evidence of HPV-related changes. It is still possible to have HSIL, because the swab may not have sampled an area of HSIL if it was very small or hidden deep in a fold and sometimes the lesions just don't shed cells.
  3. ASC-US (atypical squamous cell-undetermined significance): also known as atypia, which means that the cells were somewhat abnormal in appearance but not so much so as to be formally classified as a SIL. Based on our natural history studies, we currently recommend that patients with ASC-US on anal cytology be examined with a digital rectal examination (DRE) and using high-resolution anoscopy (HRA). HRA will be described below and is a more effective way of visually inspecting the anus for the presence of any lesions.
  4. ASC-H (atypical squamous cells-cannot exclude HSIL): this is another form of atypia in which the cells appear more abnormal and in some ways similar to HSIL, but the changes are not definitive enough to make a diagnosis of HSIL. Similar to the recommendations for ASC-US, patients with this type of atypia should be examined with DRE and HRA and are probably more likely to have either LSIL or HSIL found.
  5. LSIL (low-grade squamous intraepithelial lesion): also known as mild dysplasia or AIN 1, these changes include anal warts, which are also known as condyloma. As mentioned above, since cytology is not the best way to determine the exact level of abnormality, patients with LSIL on cytology should be examined with DRE and HRA to look for evidence of HSIL or anal cancer. More than half of patients with LSIL on cytology will have HSIL found during HRA.
  6. HSIL (high-grade squamous intraepithelial lesion): often subdivided into moderate or severe dysplasia, which are also known as AIN 2 or 3. Most patients with HSIL on anal cytology will have a lesion visible on HRA that when biopsied will also show HSIL. As with lower grades of abnormality, patients should undergo DRE and HRA to be sure that anal cancer is not present. Remember the swab is sampling only the cells on the surface, and as seen in the diagram, cancer develops when cells invade more deeply. This means that these cells may not be sampled with a Pap smear. Also since we believe that HSIL is a potentially precancerous lesion, then patients should be examined to determine how best to eliminate these areas as a means of preventing invasive anal cancer from developing.
  7. HSIL with features suggestive of invasion: the pathologist detects very abnormal cells that appear like cancer cells. As mentioned, patients should be examined as above with a DRE and HRA with biopsies. A biopsy is when samples of the most abnormal areas are cut out, so the pathologist can see the relationship of the cells to one another and evaluate the tissue underneath to see whether there are cells invading across the basement membrane as shown in the diagram above, which is how cancer is diagnosed.
  8. Squamous cell cancer (invasive anal cancer): this is the type of cancer that develops in the anus and is very different from colon or rectal cancer, although many people, including some health care providers, will incorrectly use the term rectal cancer for anal cancer. The anus is the opening to the rectum and is composed of skin type cells called squamous cells, which are susceptible to HPV, which in a very small percentage of all those infected develops into cancer. In contrast, cancers of the rectum and colon are called adenocarcinomas. They develop from the gland-like cells of the colon and rectum, which are different than the skin cells of the anus and do not become infected with HPV. Rectal or colon cancer is a type of cancer in which an increased tendency to develop it can be inherited from your parents; there is no inherited risk for anal cancer.

Obtaining an Anorectal Cytology Specimen
What are the possible results from anal cytology and what do they mean?
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