Anal Cytology and High-Resolution Anoscopy

Naomi Jay, RN, NP, PhD and Mary M. Rubin, RNC, PhD, CRNP

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Subject: Anal Cytology and High-Resolution Anoscopy

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The cervix is used as a model for anal human papilloma virus (HPV)-associated disease based on similar anatomy and pathophysiology. Both the cervix and anus consist of squamous epithelium, which abuts columnar epithelium inducing squamous metaplasia. These areas undergoing squamous metaplasia are most susceptible to abnormal changes caused by HPV. The same strains of HPV found in the female genital tract are found in the anal canal of women and men. They induce the same range of disease in the anus as in the cervix, vagina, and vulva. 
Anal disease is classified with similar cytology and histology taxonomies as the cervix, although in the anus, squamous intraepithelial lesions (SIL) is often called anal intraepithelial neoplasia (AIN) grades I, II, and III. High-grade AIN (HGAIN) is considered to be the precursor lesion to anal squamous cell cancer (SCC), and as such, screening procedures used for the cervix including cytology and colposcopy have been adapted for screening of anal HPV-associated disease. Sensitivity and specificity of anal cytology are similar to cervical cytology, and liquid-based cytology has been shown to improve quality of samples. As a screening test for anal cancer, anal cytology has been shown to be cost-effective. In the anal canal, colposcopy is called high-resolution anoscopy (HRA). Colposcopy techniques and terminology have been validated for anal canal disease. 
Figure 1
Figure 1
There are several principles of screening when using anal cytology and HRA. Anal cytology is used for identification of populations and individuals with HPV-associated diseases through cytology screening programs. HRA is used for detection of lesions, histologic diagnosis of disease, and treatment of disease, specifically HGAIN and prevention of cancer development. It is also used for early detection of nonsymptomatic cancer. 
Before an anal cytology exam or HRA, instruct the patient to avoid douching, enemas, or insertion of anything per rectum 24 hours prior to the procedure. Obtain relevant history, including current anal symptoms such as pruritus, bleeding, and pain. Determine prior history of anal or perianal condyloma and whether treatments were surgical or office based. Also determine prior history of any anal abnormalities such as fissures, fistula, abscesses, or hemorrhoids requiring intervention. Ask about any prior treatments that may have caused scarring or other alterations in the normal anal mucosa such as abscess lancing, fistula repairs, or hemorrhoidectomies. Obtain informed consent with explanation of the procedures to be performed. 


Much of the equipment is similar to that used for cervical examinations. Most gynecology or dysplasia practices have these supplies without significant additional cost for performing these procedures. A procedure tray for examination includes the following: 
Figure 2
Figure 2
Figure 3
Figure 3
  • Cytology liquid medium (or conventional slide with fixative solution)

  • Dacron swab

  • Anoscope (disposable or sterilized metal)

  • 3% acetic Acid

  • Nonsterile cotton swabs

  • Nonsterile Scopettes

  • Nonsterile 4 × 4 gauze pads

  • Lugol solution

  • K-Y Jelly mixed with 1% to 5% lidocaine gel

For intra-anal biopsies, the following additional equipment is needed: 
  • Monsel solution or silver nitrate sticks

  • Formalin

  • Baby-Tischler punch biopsy or endoscopy forceps

For perianal biopsies the following additional equipment is needed: 


The following specifications are recommended for colposcopes intended for HRA: 
  • Double objective lens with magnification up to 25 to 40×

  • Oculars that magnify 10 to 20×

  • Angled eye pieces, as the straight-on view is ergonomically difficult for HRA

  • Side-swing arm to brace clinician’s arm while holding the anoscope for long periods

  • Green filter for evaluation of vascular changes

Figure 4
Figure 4


Populations to screen include the following: 
  • HIV-seropositive individuals

  • Immune-compromised individuals (organ transplant recipients, autoimmune diseases)

  • HIV-seronegative women with a history of anal or perianal warts, genital high-grade SIL (HSIL), or cancer

  • HIV-seronegative men who have sex with men with a history of anal or perianal warts or prior receptive anal intercourse


  • There is no contraindication for cytology screening or HRA, although patients who have recently undergone anal procedures such as hemorrhoidectomy, fistula repair, or fulguration of anal warts should defer examination until healed.

  • Biopsy should be deferred in patients with platelets <65,000 or in patients who are neutropenic or who are on anticoagulant therapies.

The Procedure

Step 1

The anatomy of the anus is depicted. 

Step 2

The anus is composed of squamous epithelium. The rectum or colon is columnar epithelium. The anal canal is mucosa lined, and the anal margin is epidermal. The proximal end of the anal canal begins at the junction of the ani muscle and external anal sphincter and extends to the anal verge. It is 2 to 4 cm in length and is shorter in women compared with that found in men. The distal end of the anal canal is the dentate line, which is approximately equivalent to the original squamocolumnar junction (SCJ) in colposcopic terminology. The dentate line is considered to be a “fixed” anatomic zone, whereas the anal transformation zone (AnTZ) is dynamic and undergoing squamous metaplasia. The AnTZ is the current SCJ. The anal margin begins at the verge and represents the transition from mucosal to epidermal epithelium and extends to the perianal skin. 

Step 3

By consensus, perianal skin is considered to extend approximately 5 cm from the anal margin. Areas for screening include the SCJ, AnTZ, anal canal, verge, margin, and perianal skin. 

Performing Anal Cytology

Step 1
The anal cytology specimen should be performed first to provide the highest yield of cells. Gently separate the buttocks. The patient can hold his or her right cheek to facilitate the view. 
  • PITFALL: There must be no lubrication prior to obtaining a cytology sample, as the lubricant may interfere with the processing and interpretation of the sample.

Step 2
Insert a moistened Dacron swab approximately 3 to 4 cm into the anus to assure sampling of cells from the AnTZ. If initial resistance is encountered, change the position of the swab and reinsert. 
Step 3
Remove the swab in a circular motion in order to sample cells from all aspects of the anal canal. Apply pressure so that the swab bends while slowly removing it. Count slowly to ten as you remove it. Preserve quickly on slides or in liquid medium. Fewer cells exfoliate from the anal canal than the cervix, and it is easier to get air-dried artifacts. 

Performing High-Resolution Anoscopy

Step 1
Assist the patient into one of the following positions: left lateral, lithotomy if also performing cervical exam (but most women prefer to switch to left lateral for the HRA), or prone (if overhead colposcope is available). In the left lateral and prone positions, the patient should be as close to the bottom edge of the table as possible to facilitate focusing the colposcope. 
Step 2
Be clear and consistent in describing location of lesions and the position used. The “anal clock” is different from the “gynecologic clock.” In the prone position, posterior is 12:00, while in the lithotomy position, it is 6:00. When referring patients for follow-up to anal surgeons, it is helpful to use anatomic descriptors (posterior, anterior, left or right lateral) in place of or in addition to the “clock” positions. 
Step 3
Obtain a cytology specimen if needed (new patients or those referred with abnormal cytology specimens >3 months old). Lubricate the anal canal with K-Y Jelly mixed with 1% to 5% lidocaine. Perform a digital rectal exam, and palpate for warts, masses, ulcerations, fissures, and focal areas of discomfort or pain. The presence of hard and fixed lesions should increase your index of suspicion for cancer, since these are not the usual presentation of hemorrhoids and warts. 
Step 4
Insert the anoscope, and remove the obturator. 
Step 5
Insert a cotton swab wrapped in gauze that has been soaked in acetic acid. 
Step 6
Remove the anoscope, leaving the cotton swab–wrapped gauze pad inside. Soak for 1 to 2 minutes. 
Step 7
Remove the gauze, and reinsert the anoscope. Observe through the colposcope while slowly removing the anoscope until the AnTZ comes into focus. 
Step 8
Continue to apply acetic acid with Scopettes or cotton swabs during the exam. Using cotton swabs to manipulate the folds, hemorrhoids, or prolapsing mucosa as well as adjusting the anoscope will help to view all aspects of the AnTZ. In most cases, the entire AnTZ should be seen, and the exam will be considered satisfactory. Continue withdrawing the anoscope until the entire canal has been observed. 
Step 9
The AnTZ is seen here as a thin acetowhite line between the mature squamous and immature columnar epithelium. Early metaplasia can be seen as the columnar epithelium begins to coalesce adjacent to the SCJ. Acetic acid distinguishes anal squamous epithelium from colon columnar epithelium. Squamous epithelium will generally appear lighter and pinker in color, while columnar epithelium is darker and redder. 
Step 10
Lugol application may help determine areas of abnormality. Normal glycogenated squamous epithelium stains dark mahogany. Abnormal lesions lack glycogen and have a partial stain or no stain. Care must be taken to differentiate areas that do not pick up Lugol staining, such as columnar epithelium, scar tissue, and skin. In this case, a lesion can be seen, which is better delineated than with acetic acid alone. 
  • PITFALL: Review allergy to iodine during history taking. If patient has an allergic reaction to shellfish or has known allergy to iodine from prior procedures, do not use during the examination.

Step 10
Step 10
Step 11
Commonly recognized cervical lesion characteristics that help distinguish cervical low-grade SIL (LSIL) and HSIL are also seen in anal lesions and help guide the clinician in choosing areas for biopsy. A typical raised low-grade AIN (LGAIN) is shown in part A and a typical flat high-grade AIN (HGAIN) is shown in part B. 
Step 11
Step 11
Step 12
Biopsies are directed at areas thought to represent the highest grade of abnormality. Anal biopsies should be smaller than those typically taken of the cervix using forceps no larger than 2 to 3 mm. Internal biopsies do not require anesthesia. External biopsies require injecting a small amount of 1% lidocaine with epinephrine buffered with sodium bicarbonate (2 cc NaHC03: 10 cc lidocaine), similar to biopsies of the vulva. The injection can be preceded by numbing medication topically with lidocaine gel or spray. Monsel solution or silver nitrate is used for hemostasis, although the pressure of the anal walls will generally stop bleeding for internal biopsies. 
Step 13
Insert closed forceps through the anoscope while looking through the colposcope. 
  • Pearl: Closing the forceps will prevent unintentional injury.

Step 13
Step 13
Step 14
Once the forceps is adjacent to the lesion, open in the direction that allows for the forceps to grab the tissue. For some lesions, the forceps will need to be positioned upside down. 
  • PITFALL: Patients on warfarin (Coumadin) or daily aspirin may have increased bleeding with biopsy. If the platelet count is <65,000, approach the biopsy with caution or postpone until count improves. It is no longer considered necessary to provide antibiotic prophylaxis prior to biopsy in patients with history of endocarditis or otherwise at risk for heart valve disease.

Step 14
Step 14
Step 15
To obtain a small sample, the forceps should not be opened the entire width but rather should be partially closed before closing and grabbing the tissue. Monsel solution can be applied to the biopsy site for hemostasis, although most small biopsy samples will coagulate spontaneously once the anoscope is removed. 
Step 15
Step 15


  • Bleeding with bowel movements for several days post biopsy

  • Infection (rare)

  • Problematic bleeding (rare)

Pediatric Considerations

Since cytology screening is not usually done until a patient has been sexually active, this procedure is not routinely done in a pediatric population. 

Postprocedure Instructions

Patients should be told to expect slight bleeding with bowel movements for several days post biopsy. There may be mild postprocedure pain associated with biopsy of lesions in and around the anal canal. Rarely, a patient may require medication such as hydrocodone
Comfort measures include avoiding constipation by increasing fiber in the diet during a few days following biopsy. If the patient requires pain medications, stool softeners may be necessary, depending on their routine bowel habits. Avoid hot and spicy foods. Soaking in warm water will facilitate faster healing and relieve any pain associated with biopsy. Lidocaine 1% to 5% gel/cream can be applied to perianal tissue when biopsies have been performed. 
Follow-up will depend on the results of the cytology, histology, and the clinical indications for the referral. See 01412520

Triage for Anal Cytology and High-Resolution Anoscopy

Figure 5
Figure 5

Coding Information and Supply Sources

ICD-9 Codes

Supplies for HRA needed in addition to standard colposcopy supplies include the following: 


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