Urinalysis and other Body Fluids Lab - 08 Tests for other Body Fluids

ANALYSIS OF URINE AND OTHER BODY FLUIDS TESTS FOR OTHER BODY FLUIDS OUTLINE • Sputum o Introduction o Macroscopic Examination o Microscopic Examination o Procedure: Gram Stain o Procedure: Acid Fast Stain • Bronchoalveolar Lavage o Bronchoalveolar Lavage o Tests for BAL • Vaginal Fluid/Secretions o Vaginal Secretions o Specimen Collection and Handling o Color and Appearance o Diagnostic Tests o Vaginal Disorders SPUTUM • Results of The Test/s: INTRODUCTION • The macroscopic or gross examination of sputum, it is important to determine first the acceptability of the specimen. Once acceptability criteria are satisfied, color of the sputum should be observed and noted. The presence of blood streak may be indicative of tuberculosis while a green-yellow appearance suggests bacterial infection. PROCEDURE: ACID FAST STAIN MACROSCOPIC EXAMINATION • • MATERIALS • • Sputum Specimen Applicator Stick PROCEDURE 1. 2. Obtain a small amount of the sputum specimen from different sources. Observe the color and consistency of sputum. MICROSCOPIC EXAMINATION MATERIALS • • • • • • • • • • • • • • • • • • Results of The Test/s: • • • Sputum Specimen Applicator stick Carbol fuchsin Acid alcohol Methylene blue Crystal/Gentian Violet Gram’s Iodine Safranin Microscope PROCEDURE: GRAM STAIN • • • • Flood the slide with acqueous carbol fuchsin solution 2 Steam it for 5 minutes over the low flame or burner (Make sure that the preparation does not dry up by adding more carbol fuchsin from time to time.) Wash the excess stain with water Decolorized the smear with acid alcohol. (Add the decolorizer in small amounts and pour it off after 30 seconds or more. Repeat this until no more stain comes off with the alcohol.) Counterstain with methylene blue for 30 seconds to 1 minute. Wash the stain with water Blot dry or air dry and examine under the microscope. Principle: presence of teichoic acid Cover the entire smear with some drops of crystal or gentian violet for 1 minute Wash the excess stain with water until no more stain comes off Cover the smear with Gram’s Iodine for 1 minute Wash the iodine with water Decolorized with 95% alcohol until no more stain comes off Wash with water Counterstain with safranin for 30 seconds Wash with water Blot dry or air dry and examine under the microscope BRONCHOALVEOLAR LAVAGE • BRONCHOALVEOLAR LAVAGE • • • • • Method for obtaining cellular, immunologic, and microbiologic information from the lower respiratory tract. It is often used in conjunction with high-resolution computerized tomography (HRCT), medical history, and physical examination to determine the need for a surgical biopsy. During bronchoscopy, a fiber-optic bronchoscope is guided into a selected bronchopulmonary segment, usually the right middle or lingular lobe Aliquots of sterile normal saline are instilled into the alveolar spaces through the bronchoscope to mix with the bronchial contents and are aspirated for cellular examination and culture. o The instillation volume is between 100 and 300 mL of sterile saline in 20- to 50-mL aliquots. o The first aliquot is discarded, the remaining aliquots are either sent individually for analysis or pooled for further analysis. o The desired fluid volume for analysis is 10 to 20 mL (minimal volume is 5 mL). Keep specimens at room temperature during transport to the laboratory and process them immediately. o When delivery to the laboratory is delayed for longer than 30 minutes, transport the specimens on ice (4°C). o Specimens that will not be analyzed immediately should be centrifuged, the cells resuspended in a nutrient-supplemented medium, and refrigerated at 4°C for up to 24 hours. o Specimens are unacceptable for testing after 24 hours. o Cell counts should be performed within 1 hour or are stable for up to 3 hours if the fluid is in a nutrientsupplemented medium. o Samples can be filtered through loose gauze (50 to 70 μm nylon filter) to remove mucus, phlegm, and dust. • • • TESTS FOR BAL • • Color / Clarity o Clear (colorless), milky white, light brown-beige, and red / Clear, hazy, cloudy, or turbid o Bloody BAL fluid with increasing intensities during the sequential aliquots indicates acute diffuse alveolar hemorrhage o Orange-red BAL fluid is the result of an older hemorrhagic syndrome and would be evaluated for intracellular iron content by cytochemistry o Milky or light brown-beige color BAL fluid indicates an accumulation of phospholipidprotein complexes derived from pulmonary surfactant in the lung alveoli and strongly suggests pulmonary alveolar proteinosis WBC and RBC Count o White blood cell (WBC) and red blood cell (RBC) counts are performed on BAL and may be diluted to facilitate counting using a hemocytometer. o Cell viability can be determined by adding Trypan blue. Erythrocytes o Presence of erythrocytes indicates an acute alveolar hemorrhage. o Phagocytosed erythrocytes suggest that an alveolar hemorrhage has occurred within the past 48 hours. o Hemosiderin-laden macrophages indicate an alveolar hemorrhage older than 48 hours. Epithelial Cells o Ciliated columnar bronchial epithelial cells are more numerous in bronchial wash specimens than in bronchial lavage specimens because of the more vigorous washing technique. o In a lavage specimen, these cells normally range from 4% to 17%. Fungi, Viruses, Bacteria o Organisms identified include Pneumocystis carinii, Toxoplasma gondii, Strongyloides stercoralis, Legionella pneumophila, Cryptococcus neoformans, Histoplasma capsulatum, Mycobacterium tuberculosis, Mycoplasma pneumoniae, influenza A and B viruses, and respiratory syncytial virus. o Bronchoalveolar lavage is becoming an important diagnostic test for P. carinii in immunocompromised patients. o C. neoformans has become a significant opportunistic pathogen in patients with AIDS Cytology o Oil Red O stain - observing sulfur granules (actinomycetes), hemosiderin-laden macrophages, Langerhans cells, cytomegalic cells, fat droplets seen in fat embolism o Sudan III stain - lipid-laden alveolar macrophages o Periodic acid Schiff staining or Oil Red O staining - useful in diagnosing pulmonary alveolar proteinosis or aspiration. o Dust particle inclusions indicate pneumoconiosis or asbestos exposure. o Specimens are evaluated by a pathologist in cytology whenever malignancy is suspected. VAGINAL FLUID/SECRETIONS VAGINAL SECRETIONS • • • Examined: to diagnose infections, complication of pregnancy, forensic testing in sexual assault patients. Vaginitis: most common conditions diagnosed in the laboratory. o Characterized by: vaginal discharge or odor, pruritus, vaginal irritation, dysuria, and dyspareunia o Secondary to bacterial vaginosis, vulvovaginal candidiasis or trichomoniasis o Can also occur with non-infectious condition such as: vaginal atrophy, allergies, and chemical irritation Microscopic Examination includes: o Saline wet mount examination o Potassium hydroxide (KOH) examination o Gram Stain - gold standard SPECIMEN COLLECTION AND HANDLING • • Speculum moistened with warm water is used to visualized vaginal fornices Collected by: swabbing the vaginal walls and vaginal pool to collect the epithelial cells along with vaginal secretions o • o Sterile, polyester-tipped swab on plastic shafts or swabs specifically designated by manufacturer should be used o Cotton swabs: toxic to Neisseria gonorrheae o Wood in wooden shafts: toxic to Chlamydia trachomatis o Calcium alginate: can inactivate herpes simplex virus (HSV) for viral cultures Specimen must be kept in: o Room Temperature ▪ Preserve motility of Trichomonas vaginalis ▪ Recovery of Neisseria gonorrheae o Refrigerated (for trachomatis and HSV) to prevent overgrowth of normal flora COLOR AND APPEARANCE • • • Normal vaginal fluid appears white with flocculent discharge Microscopically: o Normal vaginal flora: predominance of large, rodshaped, gram positive lactobacilli o Squamous Epithelial Cells o WBC and RBC may be present if the patient is menstruating Abnormal vaginal secretions: o Homogeneous white to gray discharge: often seen in bacterial vaginosis o White “cottage cheese” - like discharge: particular for Candida infections o Yellow-green frothy, adherent discharge: vaginalis o Yellow, opaque cervical discharge: trachomatis • DIAGNOSTIC TESTS • • pH o Normal pH: 3.8 - 4.2 o >4.5 : Bacterial vaginosis, Trichmoniasis, Desquamative Inflammatory vaginitis, Atrophic vaginitis o 3.8 - 4.5 : Candidiasis o Normal vaginal flora, bacteria lactobacilli: produces lactic acid (end product of glycogen metabolism), provides an acidic vaginal environment with pH between 3.8 to 4.5 o Some lactobacilli subgroups also produces hydrogen peroxide which keeps the vaginal pH acidic to provide protection from urogenital infections o Estrogen production is necessary to preserve an acidic vaginal environment Microscopic Procedures o Wet Mount Examination o Squamous Epithelial Cells - exhibit a polygonal “flagstone” appearance o Clue Cells - squamous epithelial cells with attached coccobacillus ▪ granular, irregular appearance; “shaggy” ▪ diagnostic for bacterial vaginosis (G. vaginalis) o White Blood Cells - are present in rare to scanty numbers in vaginal secretions ▪ greater then 3+ WBCs: vaginal candidiasis, atrophic vaginitis, or infections with Trichomonas, Chlamydia, N.gonorrheae or Herpes Simplex • • • • Red Blood Cells - present due to menstruation or desquamative inflammatory process structures (”blue blobs”) in surrounding cytoplasm. ▪ confused with yeast cells, can be distinguished by KOH ||> RBC will lyse and yeast cells will remain intact o 5. Parabasal Cells - round to oval shaped with marked basophilic granulation or amorphic basophilic ▪ Increased in desquamative inflammatory vaginitis accompanied by large numbers of WBCs o Basal Cells - round cells which can be distinguished from WBCs (multilobed nucleus) ▪ If present and accompanied by large number of WBCs: inidcative of desquamative inflammatory vaginitis o Bacteria - Lactobacillus spp., normal flora of vagina o Trichmonas vaginalis - an atrial flagellated protozoan that can cause vaginal inflammation and infection in women. ▪ exhibits “jerky” motion observed in wet mount ▪ dead trichomonad tends to appear oval and slightly larger than a WBC. o Yeast Cells - Candida albicans and non-Candida spp. Causes most fungal infections but an occasional yeast cells is considered as part of normal flora ▪ appears in wet mount as both budding yeast cells (blastophores) or as hyphae, which are long filaments and from a mycelium KOH Preparation and Amine (Whiff) Test o Performed by placing a drop of saline specimen prepared from the collection swab onto a properly labeled clean slide and adding one drop of 10% KOH o Checked for “fishy” amine odor o Positive (presence of fishy odor) o Negative (absence of fishy odor) Gram Stain o Considered as the GOLD STANDARD in identifying the causative organisms for bacterial vaginosis Culture o Using various types of media, is the Gold Standard for detecting yeast and Trichomonas o Diamond’s Medium - special media required for T. vaginalis DNA Testing Point of Care Tests VAGINAL DISORDERS • • Bacterial Vaginosis o most common cause of vaginitis, affecting 40% to 50% of women of childbearing age. o associated with new or multiple sex partners, frequent douching, use of intrauterine devices, pregnancy, and a lack of the protective lactobacilli Trichomoniasis o caused by the parasitic protozoon T. vaginalis o infection is transmitted by sexual intercourse and causes vaginitis in women and sometimes urethritis in men o classified as an STI, frequently occurs with gonorrhea and Chlamydia infections, and has been associated with enhanced transmission rates of HIV • • • Candidiasis o Vulvovaginal candidiasis is caused by an infection with the yeast Candida o common cause of vaginitis, and nearly 75% of adult women have at least one yeast infection in their lifetime Desquamative Inflammatory Vaginitis o β -hemolytic gram-positive streptococci can be cultured from most patient o Syndrome characterized by profuse purulent vaginal discharge, vaginal erythema, and dyspareunia Atrophic Vaginitis o caused by thinning of the vaginal mucosa because of reduced estrogen production and decreased glycogen production. Syndrome found in postmenopausal women.