CHAPTER 8 I



Vaginal Microscopy

I. Vaginal microscopy explained.

A. The value.

1. Vaginal microscopy is an important laboratory tool for the differential diagnosis of vaginitis. It is also used to assess normal vaginal flora (Box 8-1).

2. It observes living vaginal organisms in order to study the ecology of the lower genital tract in women.

3. It is a direct, rapid, inexpensive test with high sensitivity and specificity for most conditions.

4. It acts as an accessory tool to the patient history, inspection of the vulvar and vaginal mucosa, and pH determination in order to arrive at a presumptive etiologic diagnosis.

5. The two components involved are saline wet mount and potassium hydroxide (KOH) wet mount.

B. The wet mount.

1. Nomenclature. Microscopy involves use of the wet mount.

a. Other similar terms include wet smear, wet prep, vaginal smear, vaginalysis, or hanging drop.

2. Basic principles.

a. A 5-minute microscopic search is required before stating that a slide is negative.

b. Examination under oil immersion is rarely needed.

c. The sample should be taken from the vaginal side walls, the vaginal pool, or both; never from cervix.

d. The sensitivity rate depends on the expertise of the clinician and the adequacy of equipment.

e. Remember to consider cervical factors when working up studies of a patient complaining of vaginal discharge; if in the differential diagnosis phase, also perform a cervical wet mount.

C. Indications. Should be performed

1. On every patient presenting with vaginal symptoms or with clinical features suggestive of a cervical or vaginal condition.

2. Even if the diagnosis is clinically obvious (such as with a curdlike discharge associated with candidiasis), because many conditions can mimic other conditions.

3. In a patient with a urine sediment that contains white cells and many squamous epithelial cells to determine the exact source of infection (vagina or urinary tract).

The Five Conditions Causing the Majority of Vaginal Discharge or Infection (in Order)

Bacterial vaginoses

Candida vulvovaginitis

Cervicitis (usually caused by Chlamydia)

Excessive but normal secretions

Trichomonas vaginitis

4. To determine the reason a routine Pap smear shows an inflammatory response.

5. As a follow-up test in a woman after treatment for a vaginal or cervical infection.

6. During the routine health maintenance visit to assess for normal flora or asymptomatic vaginal infection.

7. A thorough and comprehensive health history is necessary to establish a differential diagnosis (Box 8-2). Table 8-1 presents the differential diagnoses.

II. Comments on the vaginal ecology.

A. The vagina has minimal nerve endings; therefore, the symptoms of vaginal disorders may become evident only when the vaginal discharge bathes and irritates the sensitive vulvar skin.

B. Normally, the vagina cleanses itself by the discharge of acidotic se cretions.

C. The pH is acidotic, at 3.8 to 4.2.

1. Organisms live symbiotically in an acid environment.

a. Factors that increase the glycogen content (high levels of estrogen in pregnancy or medication) increase the acidity of the vaginal secretions.

b. Glycogen present in the epithelial cells is used by the peroxide-producing lactobacilli to produce lactic acid, which maintains an acid environment.

c. Acidity allows for the overgrowth of the yeast organisms.

2. This level of acidotic secretions is antagonistic to harmful bacterial organisms.

D. Factors affecting normal vaginal flora.

1. Role of hormones.

a. Estrogen.

(1) Affects vaginal epithelium.

(2) Causes glycogen to be deposited in the vagina, mainly in the intermediate cells.

(3) Glycogen is metabolized to become lactic acid.

b. Progesterone causes shedding of these glycogen-rich cells into the vaginal pool. (This is the reason why symptoms of candidiasis increase premenstrually and are somewhat relieved after the menstrual flow.)

2. Effect of medications on the vaginal ecology.

a. Antibiotics may increase the incidence of Candida infection. There are many theories:

(1) Candida reproduce rapidly because they no longer have the competition from bacteria, which were destroyed by the antibiotic.

(2) Secretion of an antifungal substance by bacteria stops when the bacteria are killed (more recent theory).

(3) Possible direct stimulation of growth of Candida by the antibiotic.

(4) Other theories describe reduction of host defenses, as in human immunodeficiency virus (HIV).

b. Certain medications can affect the growth of lactobacilli and thus affect the vaginal milieu.

(1) Some drugs, such as oral and vaginal metronidazole and ampicillin (modest), increase the number of lactobacilli.

(2) Intravaginal clindamycin decreases the number of lactobacilli (very temporary, only lasts one week).

(3) Drugs such as doxycycline, azithromycin, clotrimazole, and fluconazole have little or no effect.

(4) Antifungals can reduce lactobacillus—possibly contributing to the problem of recurrence.

c. Corticosteroids.

(1) Reduce inflammatory response of host.

(2) Topical steroids do not aggravate candidiasis, as previously thought.

3. Douching. Decreases normal flora, and increases the risk of bacterial vaginoses.

4. Tampon use may also alter normal flora and increase the risk of vulvovaginal infection.

3. The position of the condenser.

a. For the low-power objective, need to drop the condenser for lower intensity setting.

b. For the high-power objective, need to increase for higher intensity setting.

C. Mechanics of observing the saline smear. (Always wear gloves.)

1. Position the slide on the stage of the microscope with saline preparation under the objective, and secure with stage clips.

2. Turn light on under stage.

3. Click low objective into place over the specimen—obtains a larger view of the slide area, although the images are small.

4. Turn the condenser to the lowest position; subdued light is best to accentuate fine details. (Try increasing the light by raising the condenser while viewing the specimen to see how the cells and bacteria disappear from view.)

5. Move objective and slide as close together as possible, until just barely touching.

6. Adjust the eyepiece until a single round field is seen.

7. While looking through eyepiece, turn the coarse adjustment knob in opposite direction until the microscopic field comes into focus. Use both eyes.

8. Turn fine adjust knob back and forth to adjust to the different planes, and bring image into sharper focus.

9. Adjust each eyepiece separately by closing one eye at a time.

10. Turn knob slowly to focus (some microscopes are very sensitive; turning too rapidly may result in missing the proper plane of visualization).

11. Move the saline specimen under the objective, and scan the slide at low magnification to locate representative sections.

a. Need to scan all fields because characteristic findings may be clumped in one section of the slide.

b. When the side of the coverslip is reached, move the slide over one field’s width, then start scanning in the opposite direction.

12. Switch to high-power objective (40X).

a. Facilitates identification of microbes by further magnifying the specimen.

b. It may be necessary to increase the light source slightly.

c. Note: Watch stage when switching to make sure that the objective does not break the slide. This should not happen if low power is properly adjusted.

13. Turn fine adjustment back and forth; coarse adjustment should not need to be readjusted.

14. Using the stage adjustment knobs, move the slide laterally up and down and back and forth to view all fields because pathogens may not be distributed evenly throughout the slide and may be found in a limited number of fields.

15. Scan the slide systematically to evaluate the specimen fully.

16. Move the slide until you have a general impression of the number of squamous cells and any other findings.

17. Evaluate at least 12 fields.

18. Hint: If you immediately see one of the organisms that cause vaginitis you still must continue to examine the specimen in order to prevent missing a concomitant infection.

D. Mechanics of observing the potassium hydroxide (KOH) preparation. Observe same principles of microscopy described earlier.

1. Move the KOH slide into position on stage.

2. Switch back to 10X objective to scan fields.

3. If yeast forms are noted, switch to high power to confirm their presence and type.

E. Concluding comments.

1. Thorough observation of both preparations should take at least 3 to 5 minutes.

2. Remember to turn off the light source and dispose of the slide in a special biohazard container.

3. Clean the microscope stage if it is soiled, and clean the lenses with special paper.

a. KOH can ruin the objective. Need to clean thoroughly.

4. Record findings, and review the findings with the patient.

IV. Preparation for the wet mount procedure.

A. Patient preparation. Any substance in the vagina can alter the accuracy of the microscopic findings. Instruct the woman in the following preparations:

3. Explain that the examination is similar to a Papanicolaou (Pap) smear and should not cause any discomfort.

4. Menses cannot be avoided if a woman happens to be symptomatic during that time of the month. However, it does make evaluation more difficult because of the presence of red blood cells in the smear.

1. Avoid coitus or douching for 24 hours before the examination.

2. Do not use over-the-counter preparations before the examination (avoid for as long as possible).

B. Perform clinical evaluation. (See Box 8-3 for the sequence of the examination.)

C. Observe characteristic of vaginal discharge (Table 8-2).

D. Equipment.

1. Gloves.

2. Speculum (metal or plastic).

3. Wooden handled cotton-tipped applicators or a wooden spatula, or both.

Sequence of Examination During an Infection Check

Perform a careful history

Review procedure and expected outcomes with patient

Insert speculum

Inspect genitalia, noting signs of infection

Determine vaginal pH

pH should be collected first because cervical specimens may cause cervical bleeding, which might raise the pH

Procure sample of discharge

Collect cervical culture or vaginal culture if indicated

Remove speculum

Label any specimens

Perform wet mount evaluation

Document results

Review results with patient

Treat any infection

V. Saline wet mount.

A. Obtaining and preparing the sample.

1. Obtain copious sample from the posterior and lateral vaginal walls using a wooden spatula or cotton-tipped applicator.

a. The collection technique may vary with the suspected diagnosis.

2. Place two separate samples of vaginal discharge on the same unfrosted glass slide. It takes practice to use only one slide, but it is both time and cost effective, so it is probably worthwhile to develop the skill. (Saline sample should be thin.)

a. Alternate procedure: Double slide method.

(1) May use two separate slides, placed in double cardboard container (Fig. S-2A).

(2) Place smear of sample on one slide, and a sample on the second slide (Fig. 8-2B). Add coverslip (Fig. 8-2C).

(3) This method had the advantage of eliminating the possibility of the two solutions contaminating each other.

3. Add one drop of normal saline to thinner sample, and one drop of KOH to thicker sample.

FIGURE 8-2 Preparation of wet mount of vaginal discharge for microscopic examination. (A) Using a cotton-tipped applicator or Pap stick, drops of the vaginal discharge are placed on two separate glass slides and spread thinly. (B) One drop of normal saline is added to one specimen for microscopic examination for Trichomonas vaginalis. One drop of 10%-20% potassium hydroxide (KOH) is added to the other specimen for microscopic examination for Candida albicans. (C) Separate coverslips are placed over each specimen. Slides are examined under high-and low-power lenses of microscope.

a. Be careful not to mix the solutions.

(1) If the solutions are mixed, the sample will have to be collected again because the KOH will dissolve the cellular material on the saline portion of the slide.

b. Mix each specimen, thoroughly stirring until smooth, to cre ate a turbid suspension. (Use separate utensils.)

(1) May use a wooden spatula or the opposite wooden end of a cotton-tipped applicator.

(2) The saline specimen should be fairly dilute in order to separate the epithelial cells from each other.

(3) If they are clumped on top of each other, the characteristic of the individual form is difficult to determine and sensitivity is reduced.

c. Alternate method. Saline immersion.

(1) Some believe that an undiluted smear is often far too thick to interpret accurately and dries too quickly.

(2) Place 7 to 10 drops (0.5 ml) of physiologic saline in a small test tube. (Saline must be room temperature or warmer.)

(3) Roll a cotton-tipped applicator along the posterolateral vaginal walls.

(4) Immediately immerse applicator into the saline-filled tube.

(5) Place a drop of the suspension on the slide using either method described earlier.

d. Some comments.

(1) It is controversial whether the dilutional effect of this method affects the sensitivity of this test.

(2) It may prevent drying of the specimen (therefore, the examiner may have more time—15 minutes—to read slide).

(3) This procedure may best be reserved for those examinations when trichomoniasis is suspected in order to ensure the motility of the organism, or when the examination does not allow the clinician to leave the room immediately.

(4) Because the KOH cannot be prepared in similar fashion, it is more time consuming to prepare the slides two different ways.

4. Immediately place separate coverslips over each specimen just before viewing to prevent drying.

a. Hold one edge of the coverslip against the side, and slowly drop (like a hinged door) over the liquid specimen in order to reduce the number of air bubbles.

5. Place a paper towel over entire slide, and lightly blot up any excess fluid.

a. Helps keep the microscope clean.

b. The pressure of the blotting stabilizes the mixture under the coverslip.

6. Interpret findings immediately, viewing saline slide first (allowing KOH time to lyse).

7. At least 12 fields should be analyzed for a total of 3 to 5 minutes.

B. Findings on saline wet mount.

1. Vaginal epithelial cells.

a. Slightly grainy cytoplasm-containing vacuoles.

b. Distinct cell walls.

c. Evaluate cells for the following features:

(1) Quantity of mature cells present.

(2) Presence of immature cells and their relative frequency (Fig. 8-3). May indicate

• Decreased estrogen.

• Significant inflammatory reaction of chronic inflammation.

FIGURE 8-3 Maturation of vaginal epithelical cells. Superficial and intermediate cells are considered mature. Parabasal and basal cells are considered immature.

• If many—indicates a severe inflammatory process of significant duration.

d. Epithelial cells change throughout the menstrual cycle (Table 8-3).

2. Presence of significant bacterial adherence to cell surfaces.

a. Often indicative of a virulent organism.

b. Dynamic process involving bacterial fimbriae and epithelial surface characteristic.

c. Attachment depends on pH, hydrophobic properties, and surface secretion.

|TABLE 8-3 Characteristics of Epithelial Cells in Relation to Menstrual Cycle |

|Early proliferative phase Few cells found in |Precornified |

|smear because desquamation is slight | |

| |Polygonal shape |

| |Little tendency toward folding of the edges |

| |Transparent cytoplasm |

| |Nucleus with granular chromatin |

| |PMNs present |

|Late proliferative phase |Under estrogenic stimulation |

| |Small deeply pigmented, homogeneous nuclei |

| |Polygonal shape |

| |May appear flat or folded |

| |Rare PMNs |

|Midsecretory phase |Progestational phase |

| |Increase in number of desquamated superficial |

| |cells |

| |Predominately precornified |

| |More angular with folded edges |

| |Nuclei are vesicular and elongated or oval |

| |Cytoplasm contains occasional granules |

| |Marked tendency towards folding and clumping |

| |Background clear |

| |Few PMNs |

|Late secretory (premenstrual) phase |Clusters of desquamated, precornified |

| |cells Fragments of cytoplasm, mucus, and |

| |PMNs Peak shedding |

|PMN, polymorphonuclear cells. |

FIGURE 8-4 Lactobacilli.

3. Lactobacilli (Fig. 8-4).

a. Easily visualized in saline preparation.

b. Pleomorphic, gram-positive, aerobic or facultative anaerobic, non-spore-forming organism.

c. Elongated rod-shaped bacilli that appear as straight rods, which may be slightly motile if smear is made properly and not excessively dried out.

d. Lactobacilli vary in length between 5 and 15 (m.

(1) Super-long bacilli may be a normal finding (previously termed Leptothrix); they may be longer than the diameter of an epithelial cell.

(2) May indicate lactobacillosis (see p. 187).

e. Lactobacilli usually dominate the flora of the normal estrogenized vagina (96%).

f. Predominance in vagina of acidophilic lactobacillus species. (1) Eighty species have been identified.

g. Maintains a low pH of vaginal discharge by making lactic acid, which inhibits adherence of bacteria to epithelial cells.

h. Known to inhibit growth of organisms that may normally be found in the vagina such as

(1) Gardnerella vaginalis.

(2) Mycoplasma hominis.

(3) Certain anaerobes.

i. Little effect on candidiasis (may even increase) or trichomoniasis.

j. Numbers increase following menarche, and markedly decrease following menopause.

4. White blood cells (leukocytes).

a. Present as dark and granular cells with clearly segmented nuclei.

(1). Termed polymorphonuclear (PMN).

(2). This lobulated nucleus is often fairly easy to distinguish.

d. May also appear as cytoplasmic granules with an indistinct nucleus (often with chronic infection).

c. Slightly larger than the nucleus of a mature epithelial cell.

d. Immobile trichomonal organisms are more like a teardrop in shape and slightly larger than mobile organisms, but they may be difficult to distinguish from PMNs.

f. Help diagnose extent of inflammation (Table 8-4).

g. Increased in many conditions (Box 8-4).

5. Motile trichomonads (see p. 182).

6. Clue cells (see p. 180).

7. False clue cells (see p. 186).

8. Immature squamous epithelial cells (see Ch. 13).

9. Eosinophils may indicate an allergic response.

10. Mobiluncus.

a. Easily visualized; be careful not to confuse with the rod-shaped lactobacilli.

b. Comma-shaped, highly motile bacteria.

c. Seen at one point as black dots which bounce off the coverslip and elongate in eyelash shape.

d. Gram stain is negative.

11. Red blood cells—visible as small concave spheres.

|TABLE 8-4 Significance of White Blood Cells |

| |Number in hpf |Ratio of WBC to epithelial cell |

|Normal |0-4 |1:1 to 5:1 |

|Moderate |10-20 |5:1 to 10:1 |

|Severe |>20 WBC/hpf |>10:1 to TNTC |

|hpf, high-power field; TNTC, too numerous to count. |

|Note: May be influenced by the concentration of the smear. |

|If inflammation is present, observe for the presence or absence of parabasal cells. |

BOX 8-4

Causes of the Presence of Leukocytes (White Blood Cells) on Wet Mount

Moderate increase of leukocytes found with

IUD use

Postpartum reparative process

Atrophic vaginitis

Allergic reaction to spermicides and douches (may also see eosinophils)

Depo-Provera users with low estrogen levels

Marked increase of leukocytes found with

Trichomoniasis

Candidiasis

Chlamydia or gonorrhea

Atrophic vaginitis with bacterial superinfection

Hint: If many leukocytes are seen but neither Candida nor Trichomonas are present, consider a cervical culture for infections such as chlamydia or gonorrhea. Must also consider dysplasia or metaplasia as a possible cause.

C. Normal findings include

1. Absence of or ................
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