Vaginal Wet Mount Interpretation
D. Microscopic Examination
1. Rapid examination performed to detect motile Trichomonas
2. Specimen swab is placed into a tube containing approximately 1.0ml of saline (0.9% NaCl)
3. Specimen is agitated or twirled for seconds to release the secretions.
4. Another slide is prepared with 10% KOH preparation added to the single drop of sample.
5. A gram stain may also be prepared.
Here copy and paste of the full pdf instructions
Analysis of Vaginal Secretions
I.
Introduction
The most common complaint encountered by the health professional working in
women’s health is the vaginal discharge or vaginal disorder. The three major
causes for these symptoms are bacterial infections, candidiasis, and
trichomoniasis. The causative agent for each of these conditions is different; the
clinical presentation can be very similar. The treatment for each of these is
unique so the diagnosis is critical. In addition, sexual partners are often treated to
avoid reinfection.
A. The Clinical Laboratory Improvement Ammendment
1.
The classification of direct examination (wet mount) and KOH
examination for vaginal secretions as provider-performed
microscopy tests were included.
2.
When non-laboratory personnel (NP, PA, and MD) perform these
tests the designated laboratory director is responsible for assuring
the accuracy and reliability of the testing performed.
3.
These tests enable health care providers to diagnose and treat the
common causes of vaginitis.
B. Specimen Collection and Handling
1.Optimal recovery from a pelvic examination of microorganisms and other elements for microscopic elucidation.
2. Pelvic sample using a non-lubricated speculum, moistened only with warm water.
a.Lubricants contain anti-microbial contaminates. b.Specimen collection device is a polyester (Dacron) tip.
1.Cotton is toxic to many organisms such as Neisseria. 2.Wooden shafts are toxic to Chlaymdia. c.Wire loop can also be used.
3. Vaginal secretions are collected from the posterior vaginal fornix
and the vaginal pool.
4. Sample transported with patient medical history such as menstrual status, exposure to sexually transmitted diseases, and use of lubricants, creams or douches.
5. Tests should be performed immediately; if unavoidably delayed then specimen must be held at room temperature. a. N. gonorrhoeae and Trichomonas are adversely affected by refrigeration. b. Refrigeration of Chlaymidia and Herpes is preferred because overgrowth of normal flora is prevented.C. pH 1. 2. The pH of vaginal secretion specimens should be determined using commercial pH paper before placing the swab into saline. Differential diagnosis using the pH of vaginal secretions a. pH 3.8-4.5 range for healthy vagina; this pH is maintained by lactobacilli in the normal flora that they produce hydrogen peroxide which enhances the acid environment. b. pH > 4.5 is associated with bacterial vaginosis, trichomoniasis, and atrophic vaginitis. c. pH < 4.5 limited diagnostic value but excludes Neisseria gonorrhoeae, Gardnerella vaginalis, Haemopholis influenzae, and Trichomonas vaginalis. D. Microscopic Examination 1. Rapid examination performed to detect motile Trichomonas. 2. Specimen swab is placed into a tube containing approximately 1.0ml of saline (0.9% NaCl). 3. Specimen is agitated or twirled for seconds to release the secretions. 4. Another slide is prepared with 10% KOH preparation added to the single drop of sample. 5. A gram stain may also be prepared. E. Wet Mount Examination – Direct Examination 1. The Dacron swab sample is put into a 1m tube of saline. 2. Roll the swab onto the sides of the test tube. 3. Remove the swab and a drop is placed on a glass slide. 4. A glass cover slip is placed on the drop of saline-suspended specimen. 5. Examine the sample using brightfield at low-power (10x) used to assess overall distribution and evaluate epithelial cells; amount, type, and groupings. 6. Examine using high-power (40x) magnification to quantify the elements present such as yeast and bacterial morphology. 7. Reportable elements are red and white cells, bacteria, yeast and hyphae/pseudohyphae, trichomonads, clue cells, parabasal and basal cells, and squamous epithelial cells. F. KOH Preparation and Amine Test 1. Prepare a KOH slide and the amine test by adding 1 drop of 10% KOH directly onto the drop of the specimen on the microscope slide. 2. Check the slide for a “fishy” odor; the name of the test is often called the “whiff” test. a. The distinct foul-smelling odor is trimethylamine caused by the volitilization product of polyamines, which results from3. 4. 5. 6. 7. II. the alkaline pH change when KOH is added. b. When microbial flora of the vagina is altered significantly by proliferation of microbes, there is an increase in polyamine production due to the development of the discharge and increased foliation of epithelial cells. Report the Amine test as either positive or negative. KOH preparation occurs at the same time as the direct examination but this slide is set aside to allow the KOH to dissolve the epithelial and blood cells in the specimen. Heat the KOH preparation to speed up the process of digestion. This enhances visualization of any fungal elements present. Low-power (10x) magnification is used to screen the preparation and high-power (40x) magnification is used identify and enumerate fungal elements or pseudohyphae. Limited to fungal identification and Amine testing. Cellular Components A. Blood Cells 1. White Blood Cells a. few seen in healthy women b. increase seen during ovulation and menses 2. Red Blood Cells a. usually contamination due to menstruation B. Bacterial Flora 1. Lactobacilli – 50% - 90% of bacteria in healthy women a. large, nonmotile, gram positive rods b. produce lactic acid as waste product c. produce hydrogen peroxide 2. Other Microflora; usually seen with imbalance of: a. G. vaginalis; small, nonmotile, gram variable coccobacilli b. Mobiluncus; thin, curved, gram variable, motile rods c. Peptostreptococcus; gram positive cocci d. Enterococcus; gram-negative cocci e. Prevotella; Bacteroides; Porphyromonas; gram-negative rods C. Yeast 1. often confused with red blood cells. 2. KOH prep lyses red blood cells. 3. Presence of hyphae or pseudohyphae is indication of candidiasis. D. Epithelial Cells 1. Predominant cell in direct examination/ wet mount specimens of healthy women. 2. Large, thin, flagstone-shaped appearance, central nucleus. E. Clue Cells 1. Epithelial cells with numerous bacteria adhering to the membrane. 2. Diagnostic indicator of bacterial vaginosis. 3. Appear soft and finely stippled with indistinct borders because of the adhesion of bacteria.4. 5. III. Nuclei may not be visible because of bacteria-laden shaggy edges. In order to be a clue cell a significant number of bacterial organisms must extend beyond and obscure visualization of cytoplasmic borders. F. Parabasal cells 1. Reside below the surface or luminal squamous epithelium of vaginal mucosa. 2. Not found in normal vaginal secretions. 3. Increased during menstruation or postmenapausal women 4. 14-40 um in diameter; oval to round with distinct cell borders. Look similar to transitional cells. 5. Most often seen in atrophic vaginitis; desquamative vaginitis. G. Basal cells 1. Basal layer of the vaginal stratified epithelium 2. Similar in size to a white blood cell; 10-16μm 3. If present, desquamative inflammatory vaginitis H. Trichomonads 1. Flagellated protozoans 2. Infect and cause inflammation of the vaginal epithelium. 3. Pear or turnip shaped with unicellular bodies averaging 15μm 4. Flitting or jerky motion; 4 anterior flagella; undulating membrane 5. Flagella provide propulsion and the wavelike motion of undulating membrane gives rotary motion 6. Posterior flagellum attaches to the mucosa causing tissue damage. 7. Trichomonads are not hardy and once removed from mucosa die. 8. Stains are toxic. 9. Hard to identify if dead because they may look like white blood cells. Clinical Correlations A. Bacterial vaginosis 1. Most common 2. Lack exogenous pathogen 3. Alteration in normal indigenous bacterial flora of vagina 4. Lactobacilli present in health are replaced a. Guardnerella vaginalis overgrowth b. Facultative anaerobic Mobiluncus species overgrowth 5. Acidic environment of vagina by the lactic acid production of lactobacilli limits proliferation. 6. Hydrogen Peroxide production by some Lactobacilli has bactericidal qualities preventing overgrowth. . Laboratory Findings 1. The presence of clue cells in the wet mount examination of vaginal secretions. 2. Lactobacilli are not present or rarely seen. 3. Small, gram-variable coccobacilli – G.vaginalis. 4. Thin, curved or comma-shaped, gram variable, motile rods, anaerobic-Mobiluncus.5. 6. 7. 8. B. Amine test is positive. KOH is negative. Absence of white blood cells in vaginal secretions. Vaginosis vs. vaginitis a. Lack of increase in white blood cells suggests no invasion of the subepithelial tissue. 9. Diagnosis of Vaginosis requires three out of four criteria a. presence of clue cells b. positive amine test c. vaginal pH >4.5 d. homogeneous vaginal discharge Treatment 1. Methronidazole is recommended treatment. 2. Reoccurrence in 30% of cases – failure to reestablish microbial balance. 3. New treatment lactobacillus-containing vaginal suppositories 4. No concurrent treatment for sexual partners. Candidiasis 1. Second most common vaginitis in women. 2. Most adult women have had vaginal candidiasis. 3. Occurs in both celibate and sexually active females. 4. Disease is less common in postmenopausal women. 5. Candida albicans is responsible for the majority of episodes of vulvovaginal candidiasis. a. Candida torulopsis (glabrata) is on the rise. 6. Overproliferation results when there is a disruption in the vaginal environment (pH) or a change in bacterial flora. 7. Predisposing factors a. treatment with broad spectrum antibiotics b. birth control pill usage c. pregnancy d. diabetes e. HIV f. Immunosuppression Laboratory Findings 1. Symptoms a. vulvovaginal itching b. external dysuria c. white, curd like discharge d. pain 2. pH is normal (3.8 to 4.5) 3. Wet mount examination shows increased wbcs and budding yeast. 4. Blastocondia and or pseudohype depending on species. 5. Squamous epithelials are often clumped or in sheets. 6. Lactobacilli is the predominant bacteria. 7. Amine Test is negative. 8. KOH shows budding yeast and/or pseudohyphae. 9. If negative wet mount, despite symptoms, perform DNA analysis.10. If negative wet mount, despite symptoms, perform culture. Treatment 1. Topical antimycotic agents from imidazole derivatives a. miconazole, clotrimazole 2. Oral agents a. fluconazole, ketoconazole 3. Treatment of the sexual partner not indicated or advised. C. Trichomoniasis 1. Most common gynecologic parasitic infection. 2. Trichomoniasis vaginalis is causative agent. 3. Classified as a sexually transmitted disease. 4. Human is only known host. 5. In women it resides in vaginal mucosa/ men it resides in urogenital tract. Laboratory Findings 1. Symptoms a. Infection in women can range from an asymptomatic carrier state to severe inflammatory condition. b. Asymptomatic in men or present with urethritis c. Recurrence is common if sexual partners are not treated i. 35% of all asymptomatic male partners are positive for T. vaginalis when tested. ii. non-sexual transmissions of T. vaginalis have occurred, particularly in older women, the mechanism is not clear. d. Trichomoniasis facilitates transmission of HIV. e. It can be a risk factor for preterm rupture of membranes for premature labor and delivery. f. 50% of women are asymptomatic. g. 50% of the women exhibit a discharge that is yellow, copious, malodrous, frothy with soreness of the vulvovaginal area, dysuria and dysparenunia. h. Pelvic exam reveals vaginal inflammation and exocervix that is strawberry due to the numerous punctuate hemorrhages. 2. 3. Testing a. Direct examination / wet mount i. motile trichomonads are observed in only 50-70% ii. usually related to microscopy skill iii. numerous white blood cells present with clumping iv. mixed bacterial flora with lactobacilli b. Negative wet mount i. If symptomatic, perform culture or DNA probe. c. pH of the secretion is elevated 5.0 – 6.0 d. positive amine test Treatment a. Metronidazoleb. E. Oral therapy preferred because it ensures that all potential sites that may harbor organism are treated. D. Atrophic Vaginitis 1. Occurs in perimenopausal and postmenopausal women 2. Vaginal epithelium changes because of reduction in estrogen. 3. Thinning of the vaginal epithelium due to the reduction in glycogen. 4. Lactobacilli presence is reduced due to lack of glycogen. 5. Fewer lactobacilli reduces the production of lactic acid. 6. These changes lead to atrophic vaginitis with mild to moderate symptoms. 7. Rare changes induce significant growth of nonacidophilic bacteria: a. vaginal dryness, vaginal soreness, and spotting b. vagina is thin, diffusely red mucosa and no vaginal folding 8. pH > 5.0 9. Wet mount reveals numerous white cells and few red blood cells. 10. Wet mount also includes squamous, parabasal and basal cells, and gram positive cocci and coliforms. 11. KOH and Amine test negative. 12. Treatment involves replacement of estrogen. Qualitative Semen Analysis 1. Post-Vasectomy specimens a. Presence or absence of sperm b. Direct microscopic examination 2. Negative results should be confirmed with a concentration method for rare sperm a. Qualitative semen analysis is approved for the PPM category. b. Quantitative semen analysis includes evaluating the color, appearance, time of liquefaction, volume, motility, count, pH, fructose, viability and the presence of abnormal forms. This analysis is classified as high complexity.
