Vaginal Secretions - AUBF LEC FINALS LESSONS PDF

VAGINAL SECRETIONS Leslee Anne Cortez, RMT, LPT, MPH Faculty, School of Medical Technology Emilio Aguinaldo College Cavite Learning Objectives At the end of this session, the students should be able to: 1. State the indications for collecting vaginal specimens. 2. Describe the specimen collection...

VAGINAL SECRETIONS Leslee Anne Cortez, RMT, LPT, MPH Faculty, School of Medical Technology Emilio Aguinaldo College Cavite Learning Objectives At the end of this session, the students should be able to: 1. State the indications for collecting vaginal specimens. 2. Describe the specimen collection and handling procedures for vaginal specimens and explain how deviations from the correct practice will affect test results. 3. Describe the appearance of normal and abnormal vaginal secretions. 4. Explain the significance of vaginal pH values. 5. List the diagnostic tests performed on vaginal secretions and explain the appropriate use for each. 6. Describe the microscopic constituents for the common syndromes associated with vaginitis. 7. Identify the most common causes of vaginitis, including the cause, clinical signs and symptoms, laboratory tests, and treatment. 8. Describe tests that can be performed on vaginal secretions to predict conditions of premature delivery and rupture of fetal membranes.  Introduction Vaginal secretions Diagnose infections Complications of pregnancy Forensic testing in sexual assault Vaginitis - one of the most common conditions in female patients Abnormal vaginal discharge Odor Pruritus Vaginal irritation Dysuria Dyspareunia Can also occur with noninfectious conditions Vaginitis Fresh secretions “Gold standard” Saline wet mount examination Potassium hydroxide (KOH) examination Gram stain Litmus pH levels DNA probe testing Culture Point of care test kits Specimen Collection and Handling Collection during a pelvic examination The specimen is collected by swabbing the vaginal walls and vaginal pool Sterile, polyester-tipped swabs on plastic shaft No cotton tip, wood shaft, or calcium alginate Swab in a tube containing 0.5 to 1.0 mL of sterile physiologic saline and agitated Properly labeled specimens should be placed in a biohazard bag Transport expediently for immediate analysis Specimen Collection and Handling Specimens must be kept at room temperature to preserve motility of Trichomonas vaginalis Neisseria gonorrhoeae Specimens must be refrigerated to prevent overgrowth of normal flora Chlamydia trachomatis Herpes Simplex Virus Trichomonas vaginalis specimens should be examined within 2 hours of collection for motility of organism Color and Appearance Normal: white with a flocculent discharge Microscopic Predominance of lactobacilli Squamous epithelial cells WBCs may be present Menstruation: RBCs Color and Appearance Abnormal Appear as an increased thin, homogeneous white- to-gray discharge White “cottage cheese”– like discharge Yellow-green, frothy, adherent discharge Yellow, opaque cervical discharge Diagnostic Tests Vaginal pH test (provider performed) Normal vaginal pH = 3.8 – 4.2 pH ~4.5 in women with vulvovaginal candidiasis pH > 4.5 in women with bacterial vaginosis, trichomoniasis, desquamative inflammatory vaginitis, atrophic vaginitis Microscopic Procedures Slide for each test (saline wet mounts, KOH mounts and the Gram stain) Wet mount/KOH: cells/organisms quantified at 40× HPO Gram stain: cells/organisms reported per 100× OIO  Wet Mount Examination Low power objective (10×) scan Even distribution of cellular components Types and numbers of epithelial cells Clumping of epithelial cells Presence of budding yeast or pseudohyphae High power (40×) for enumeration Squamous Epithelial Cells Originate from the linings of the vagina and female urethra 25 to 70 µm in diameter Polygonal “flagstone” appearance Prominent centrally located nucleus Clumps of epithelial cells are an indication of the presence of increased numbers of yeast.  Abnormal variation of the squamous epithelial cell Clue Cells Described as “shaggy” Diagnostic of bacterial vaginosis Gardnerella vaginalis  White Blood Cells 14 to 16 µm in diameter >3+ WBCs suggest Vaginal candidiasis Atrophic vaginitis Infections with Trichomonas, Chlamydia, Neisseria gonorrhoeae, or herpes simplex Red Blood Cells 7 to 8 µm in diameter Somewhat distorted in vaginal specimens Normally present during menstruation or desquamative inflammatory process RBCs can be confused with yeast cells Parabasal and Basal Cells Rare to find in vaginal secretions Round to oval shaped Marked basophilic granulation (“blue blobs”) 16 to 40 µm in diameter Found in desquamative inflammatory vaginitis Bacteria Lactobacillus spp Large, gram-positive, nonmotile rods, produce lactic acid pH 3.8-4.5 Anaerobic and alpha-hemolytic streptococci Diphtheroids Coagulase-negative staphylococci Vaginitis: occurs when alteration in the normal flora cause overgrowth of opportunistic flora Trichomonas vaginalis Atrial flagellated protozoan Oval shaped, measures 5 to 18 µm Four anterior flagella and an undulating membrane that extends half the length of the body “Jerky” motion of the flagella Undulating membrane Nonmotile trichomonads can be mistaken for WBCs and sperm cells Quickly lose their viability after collection Maximum of 2 hours Yeast Cells Occasional yeast in vaginal secretions is considered part of the normal flora Budding yeast cells (blastophores) Hyphae Pseudohyphae Differentiation can be made using the KOH test KOH Preparation and Amine Test Placing a drop of the saline vaginal specimen and one drop of 10% KOH solution on a clean slide Amine (“whiff”) test “Fishy” amine odor Positive (presence of fishy odor) - BV Negative (absence of fishy odor)  Gram Stain Gold standard in identifying the causative organisms for BV Weighted combination of the following morphotypes: Lactobacillus acidophilus (large, gram-positive rods) Gardnerella vaginalis and Bacteroides spp. (small, gram-variable or gram- negative rods) Mobiluncus spp. (curved, gram-variable rods) Gram Stain Nugent score 0 to 3 normal vaginal flora 4 to 6 is reported as intermediate >7 diagnostic of bacterial vaginosis Culture Gold standard test for detecting yeast and Trichomonas Diamond medium is required for T. vaginalis Commercial transport and culture pouch system for the detection of Trichomonas Specimen must be inoculated into the pouch within 30 minutes of collection Incubated for 5 days at 37°C in a CO2 atmosphere DNA Testing DNA hybridization probe is method to identify the causative pathogen for vaginitis Results are available in 1 hour with a sensitivity of 95% Trichomonas can also be detected by DNA probes amplified by polymerase chain reaction (PCR) Most accurate method Advantage of detecting nonviable organisms Point of Care Tests Rapid diagnostic tests to quickly screen for the causative agents of vaginitis Proline aminopeptidase activity in vaginal secretions for G. vaginalis OSOM trichomonas rapid test T. vaginalis antigen from vaginal swabs in 10 minutes Visible blue line and a red internal control indicate positive result OSOM BVBLUE test detects vaginal fluid sialidase Enzyme produced by Gardnerella, Bacteroides, Prevotella, and Mobiluncus 1 minute to perform Blue or green=positive result, Yellow=negative result Vaginal Disorders Bacterial Vaginosis (BV) is the most common cause of vaginitis Due to imbalance in the ratio of normal vaginal bacterial flora Lactobacilli is the predominant organism = pH 3.8 - 4.2 Malodor and increased abnormal vaginal discharge result from this mix of organisms and is more apparent after intercourse Vaginal Disorders Three out of four features must be present to diagnose BV: 1. Thin, white homogeneous discharge 2. Vaginal fluid pH greater than 4.5 3. Positive amine (whiff) test 4. Presence of clue cells on microscopic examination Trichomoniasis Causative agent: Trichomonas vaginalis Infection classified as a sexually transmitted infection (STI) Frequently occurs with gonorrhea or Chlamydia infections Vaginitis in women and sometimes urethritis in men S/S: green-to-yellow frothy vaginal discharge, malodor, irritation, dysuria and dyspareunia, vaginal mucosa erythema, “strawberry cervix” Trichomoniasis Diagnosed with a wet mount and microscopic examinations WBCs and lactobacilli are present pH is > 4.5 Amine test will be positive Treatment: metronidazole Vulvovaginal Candidiasis Common cause of vaginitis Causative agent: Candida albicans Change in the vaginal environment that permits the overgrowth of Candida Broad-spectrum antibiotics, oral contraceptives, or estrogen replacement therapy, hormonal changes that occur with pregnancy, ovulation, menopause, patients with diabetes mellitus, iron deficiency, and HIV infection Vulvovaginal Candidiasis S/S: genital itching or burning, dyspareunia, dysuria, abnormal thick, white, curd-like vaginal discharge pH remains normal (3.8 - 4.2) Saline and KOH wet prep and Gram stain Budding yeast and pseudohyphae forms, large numbers of WBCs, lactobacilli, and large clumps of epithelial cells Vulvovaginal Candidiasis Culture and DNA hybridization probe OTC Treatment: butoconazole, clotrimazole, tioconazole, and miconazole Other treatment: oral fluconazole or intravaginal butoconazole, nystatin, and terconazole Not transmitted through sexual intercourse Desquamative Inflammatory Vaginitis (DIV) Causative agent: Beta hemolytic Gram (+) streptococci S/S: profuse purulent vaginal discharge, vaginal erythema, and dyspareunia pH >4.5, amine test negative Can occur secondary to atrophic vaginitis in postmenopausal women High WBCs, RBCs, rare parabasal and basal cells, squamous epithelial cells, reduced or absent lactobacilli Treatment: 2% clindamycin, hormone replacement therapy Atrophic Vaginitis Syndrome found in postmenopausal women Thinning of the vaginal mucosa due to decreased estrogen and glycogen S/S: vaginal dryness and soreness, dyspareunia, inflamed vaginal mucosa, and purulent discharge Microscopic evaluation is similar to DIV Treatment: estrogen replacement Bronchoalveolar Lavage Fluid Leslee Anne Cortez, RMT, LPT, MPH Faculty, School of Medical Technology Emilio Aguinaldo College Cavite  Learning outcomes State the indications for performing a bronchoalveolar lavage (BAL). Describe the procedure for performing a BAL. Explain the procedures for collecting, handling, and transport of specimens for BAL. Describe the appearance of BAL fluid in normal and abnormal conditions. Discuss the normal and abnormal cellular composition of BAL fluid. Clinical Significance Bronchoalveolar lavage (BAL) is particularly useful in evaluating patients who are immunocompromised or patients with any number of possible diagnoses in the respiratory tract. BAL is used in conjunction with high-resolution computerized tomography (HRCT), medical history, and physical examination to determine the need for a biopsy. Specimen Collection Fiber-optic bronchoscope is used. Alveolar ground-glass opacity, prominent nodular profusion, or fine reticulation are optimal targets Aliquots of sterile saline are instilled into the alveolar spaces, mixed with bronchial contents, and aspirated for cellular examination and culture. Specimen Collection Instillation volume is between 100-300 mL of sterile saline in 20-50 mL aliquots. First aliquot is discarded; remaining aliquots are sent individually or pooled for analysis Desired volume 10-20 mL (minimum is 5 mL) Optimal sampling retrieves ≥ 30%, typical recovery range 50%-70% Low-volume recovery ≤ 25% Specimen Handling and Transport Specimens should be kept at room temperature when transporting to laboratory; process immediately If delivery is delayed longer than 30 minutes specimens must be transported on ice (4°C). Clarity of BAL Color of BAL Clear Cloudy Colorless Hazy Turbid Milky white Light-brown beige Gray-beige Red Color Clinical Significance Red (bloody) acute diffuse alveolar hemorrhage Orange-red older hemorrhagic syndrome Milky (light pulmonary alveolar brown-beige) proteinosis Cell Counts Counts for WBCs and RBCs are done using a hemocytometer. Cell viability determined by adding Trypan blue. WBC counts may be diluted using the BMP LeukoChek. Count all cells in the 18 squares on both sides and calculate the average of the two sides WBC/µL = Average number of cells x dilution factor x 10 9 squares Cell Counts RBC counts diluted with isotonic saline using an MLA pipette Count both sides of the hemocytometer Cells must be distributed evenly over the hemocytometer surface RBC/µL = Number of cells x dilution factor x 10 Number of squares counted REMEMBER! WBC count: There should be no more than a 15-cell difference between the highest and lowest total number of cells counted. RBC count: There should be no more than a 30-cell difference between the highest and lowest total number of cells counted. Cells counted on each side of the counting chamber should be within 10% of each other. Leukocytes Differential Count Differential slides are prepared by cytocentrifugation along with staining. At least 300 cells, but more often 500-1000 cells are counted and classified. Cells noted are macrophages, lymphocytes, CD4/CD8 ratio, neutrophils, eosinophils, ciliated columnar bronchial epithelial cells, and squamous epithelial cells. Normal BAL Differential Count Cells Percentage Alveolar 50 – 80% macrophages Lymphocytes 10 – 15% Neutrophils