Principles of Vaginal Microscopy, pH, and KOH Testing

Complete discussion of all these topics is found in Annotation P.

VAGINAL pH

1. In healthy, asymptomatic women of reproductive age who are not immediately postpartum or breastfeeding, the vaginal pH is usually 4.5 or less. Recent studies show normal pH levels up to 5.0 in some ethnic groups. (See Annotation P for more on this.) Vaginal pH must be interpreted within the context of the patient’s history, physical exam, ethnic background, and the rest of the wet prep.
2. In healthy postmenopausal women (symptomatic or not), who are not using estrogen, the vaginal pH is greater than 4.5.
3. The pH of the vaginal secretions may be an indicator of pathology in the vagina and may direct the clinician to a diagnosis. In the presence of a normal pH, the only organism that can cause a problem is Candida. pH is always elevated with bacterial vaginosis or with trichomonas infection, as well with lichenoid vaginitis, desquamative inflammatory vaginitis, and with severe atrophy.
4. Women of reproductive age who are immediately postpartum or lactating will have elevated vaginal pH levels due to reduced systemic estrogen levels.

EPITHELIAL CELL TYPES (see examples in Tables A and B)

1. In healthy, asymptomatic women of reproductive age who are not immediately postpartum or breastfeeding, a preponderance of squamous epithelial cells is seen on wet prep. These cells are somewhat “squared off” and angular in appearance. Their nuclei are small and cytoplasm large. The presence of immature epithelial cells (parabasal cells), may be an indicator for a vaginal disorder. Parabasal cells are smaller and rounder than squamous epithelial cells, with smooth-edged walls, large nuclei and a small amount of cytoplasm. Healthy women who are immediately postpartum or lactating may have parabasal cells present on wet prep.
2. In healthy postmenopausal women (symptomatic or not), who are not using estrogen, parabasal cells predominate on wet prep.
3. Normal squamous epithelial cells may contain tiny dots on their surfaces, but their edges will be smooth. Squamous epithelial cells with edges dotted with bacteria look “moth eaten.” These are called “clue cells” and are one of the characteristic components of bacterial vaginosis.
4. The type of epithelial cell seen on wet prep will help in diagnosis of vaginal disorders.

WHITE BLOOD CELLS (see examples in Tables A and B)

1. In healthy, asymptomatic women of reproductive age who are not immediately postpartum or breastfeeding, the normal ratio of white blood cells (which look like white spheres on wet prep) to squamous epithelial cells is 1:1.
2. In healthy postmenopausal women who are not using estrogen, the WBC (white blood cell) population is not usually increased on wet prep. An increase in WBCs may indicate atrophy or another condition.
3. An increase in white blood cells on wet prep is an indicator of inflammation.
4. White blood cells are not increased with bacterial vaginosis. If bacterial vaginosis is identified in the setting of an increased number of WBCs, there is a second condition present.

LACTOBACILLI (see examples in Tables A and B)

1. Healthy, asymptomatic women of reproductive age who are not immediately postpartum or breastfeeding have many lactobacilli in their vaginal secretions. These hydrogen peroxide-producing bacteria help to maintain the normal acidity of the vagina. They produce anti-microbial compounds and compete with other organisms for adherence to cell walls. They depend on the presence of estrogen for survival. As estrogen wanes, lactobacilli wane.
2. Healthy postmenopausal women (symptomatic or not), who are not using estrogen, have a reduced number or complete absence of lactobacilli on wet prep. Immediately postpartum or lactating women may also have decreased or absent lactobacilli on wet prep.
3. The number of lactobacilli on wet prep may aid in diagnosis of vaginal disorders. The number of lactobacilli present on wet prep is usually correlated with pH levels. (More lactobacilli=lower pH).

KOH TESTING

1. A drop of KOH (potassium hydroxide 10%) added to a saline wet prep slide aids in diagnosis in two ways. A) A strong fishy (amine) odor to the solution may indicate bacterial vaginosis. B) KOH dissolves the cell walls of epithelial cells but does not dissolve the pseudohyphae or spores of Candida. It can be helpful in identifying yeast infections.
2. Non-albicans Candida usually presents with oval, shining,white spores on wet prep, but may not be visible at all.

YEAST CULTURES

1. Candida may be present even if it does not show up on KOH testing. In symptomatic women, a yeast culture will help to confirm the diagnosis and identify the species of yeast, if present. In most cases, a negative yeast culture will direct the clinician to look for other diagnoses. Pseudohyphae on wet prep confirm albicans species yeast. However, non-albicans yeast may also be present and not visible on wet prep.
2. Non-albicans yeast is treated differently than albicans yeast.
3. Yeast culture is an important adjuvant test in all symptomatic vulvovaginal cases.

OTHER LABORATORY TESTS

1. Routine vaginal cultures, other than yeast cultures, are not useful in vulvovaginal diagnosis.
2. Sexually transmitted infection testing may be appropriate, depending on history and/or cervical involvement.
3. Serum testing for herpes simplex may be useful to show whether or not the patient has ever had herpes, whether Type I or Type II.
4. Vulvar biopsy may be necessary in the case of skin involvement without a clear diagnosis.
5. DNA probe-based tests such as the BDA Affirm VPIII Microbial Identification Test, are used in many offices in lieu of microscopy. In some offices, results may be ready within one to two hours, but offices that require the laboratory to analyze results may not have an answer for the patient for one to two days.
   The Affirm test recognizes increased numbers of Gardnerella, a variety of species of Candida, and Trichomonas vaginalis. However, the presence of Gardnerella, without the rest of Amsel’s criteria, does not automatically mean that bacterial vaginosis can be diagnosed. Imbalances in vaginal flora may be present in cases of atrophy or desquamative inflammatory vaginitis and Gardnerella is present in normal vaginal secretions.
   In addition, Affirm does not speciate the type of Candida present. This may lead to incorrect treatment. The test is also proportionally more expensive than pH, KOH, and wet prep.
   Affirm may be useful in diagnosing Trichomonas when it is not clearly seen on wet prep; Trichomonas-specific cultures may also diagnose Trichomonas, with increased sensitivity.
   Affirm does not allow for the full evaluation of the vagina that pH, KOH, and wet prep allow.