SPUTUM EXAMINATION.pptx

Sputum examination in clinical laboratories.

1. SPUTUM EXAMINATION MITHUN VENUGOPAL

2. INTRODUCTION Sputum is a highly specialized watery, colorless and odorless product of the respiratory tract (bronchia, trachea & lungs). Sputum is mainly composed of mucus and also certain cellular and noncellular components of host origin. During expectoration, sputum gets contaminated with normal bacterial flora and cells from pharynx and mouth. It is the most frequently received specimen from the respiratory tract. Both its collection and examination are advantageous as samples are easily obtained and its cellular content is representative of the entire respiratory tract.

3. INDICATIONS Identification of causative agent or organism associated with a particular suspected infection of the lower respiratory tract like, Suspected tuberculosis. Pneumonia especially if severe or in an immuno - compromised host. Pneumocystis carinii pneumonia in HIV-positive patients. Suspected fungal infection. Chronic disease like bronchiectasis. Cytological examination for the investigation of viral infections (viral inclusions in cytomegalovirus and herpes simplex infections), fungal infection, asbestosis and malignant cells.

4. SPUTUM COLLECTION Sputum sample is ideally collected in the morning (since secretions accumulate overnight). Sputum sample is collected in a sterile, clean, dry and wide-mouthed plastic container with a securely fitting screw cap. The container should be of break resistant plastic and leak-proof to prevent desiccation and aerosol formation, and should have the capacity of about 30 ml. The patient is advised to take a deep breath 2-3 times filling his/her lungs, coughs deeply, and spit into the plastic container. About 2-5 ml of sputum is collected. Sample consisting only saliva (watery appearance, clear, and foamy) is not acceptable for laboratory investigations; in such case, another sample should be collected.

5. SPUTUM COLLECTION In those patients who cannot produce sputum spontaneously by deep coughing, a specimen of sputum may be induced. This is done by inhalation of 15% NaCl spray or propylene glycol for 20 minute which are aerosolized to stimulate sputum production. The container, containing sputum sample, is caped securely and labelled properly.

6. TRANSPORT For microbiological examination of sputum, sample should be sent to the laboratory immediately. If sputum is allowed to stand, rapid reproduction of contaminating bacterial flora from the throat and oral cavity will occur leading to incorrect results. Pathogenic organism especially Haemophilus influenza, do not survive for a long time in the collected sample. Sputum sample for bacterial culture should not be refrigerated. If the sample is to be transported to a remote laboratory for mycobacterial culture, sputum should be collected in 25 ml of the following solution: N-acetyl pyridinium chloride-5 gm Sodium chloride-10 gm Distilled water-1000 ml

7. SPUTUM CONCENTRATION METHOD (PETROFF’S METHOD) The sputum is transferred to a sterile test tube and equal amount of sterile 4% NaOH is added. The tube is incubated at 37’c for 30 minutes with vigorous shaking every 5 minutes. The mixture is centrifuged at 3000 rpm for 30 minutes and supernatant poured off. The deposit is neutralized by N/10 HCL using a drop of phenol red as indicator.

8. PHYSICAL EXAMINATION QUANTITY: L arge amount of purulent sputum is coughed out In bronchiectasis in bronchiectasis Large amount of watery sputum with pink tinge suggests pulmonary oedema. APPEARANCE/COLOR: White, viscid, mucoid: Asthma, tuberculosis. Serous, clear, watery: Pulmonary oedema. Frothy, pink, serous: Broncho-alveolar carcinoma. Clear or mucoid, grey, glassy, tenacious: Chronic bronchitis. Yellow due to pus/neutrophils: Acute lower respiratory tract (pulmonary) infections. Green: Long standing infection (bronchiectasis, lung abscess), pseudomonas.

9. PHYSICAL EXAMINATION Rusty due to lysis of red cells: Pneumonia (e.g. pneumococcal) and pulmonary infarction. Bright red due to fresh blood: Pulmonary tuberculosis, lung tumors, pulmonary infarction. Black due to coal dust: Coal workers, in heavy smokers. Anchovy sauce (chocolate brown): Rupture of amoebic liver abscess into lung. Blood tinged sputum: Mitral stenosis, pulmonary tuberculosis, carcinoma lung, pulmonary infarction.

10. MICROSCOPIC EXAMINATION Staining of sputum: Two to three smears are made on a clean dry glass slides and are stained with: Leishman's stain or Wright stain for differential count. Gram’s stain for microorganisms Ziehl-Neilsen stain for acid fast bacilli. Special stains for fungi. Papanicolaou stain for study of malignant cells. Other stains (depends on the clinical/pathological features).

11. Gram ’ s stain for microorganisms From the purulent portion of the sputum, a thin smear is made on the grease free sterile glass slide with a clean stick. The slide is air-dried, fixed and stained with Gram's stain. Entirely watery, mucoid, white, or frothy samples often show squamous epithelial cells covered with bunches of bacteria; this indicates that the sample consists mainly of secretions from the mouth and the throat. Such samples are not acceptable for bacteriological examination. Culture is not carried out if polymorphonuclear neutrophils are less than 10 per epithelial cell.

12. Gram ’ s stain for microorganisms Pat hogenic organisms found in sputum are; Gram-positive: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae. Gram-negative: Klebsiella pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Yersinia pestis, Pseudomonas aeruginosa

13. Ziehl-Neilsen stain for acid fast bacilli Ziehl - Neilsen -stained sputum smear is considered as positive if 5000-10000 tubercle bacilli/ml are present in the sputum. Possibilities of detection of tubercle bacilli are increased if multiple sputum samples are examined. Mycobacteria appear as bright red straight or slightly curved roads against a blue background. Minimum 100 fields are examined before reporting the smear as negative.

14. Parasites found in sputum Larvae of Strongyloides stercoralis and roundworm. Entamoeba histolytica: Cysts or the trophozoites may be found when an amoebic liver abscess ruptures into the lungs. Echinococcus granulosae : Scolices and hooklets of the larval form may be seen with the rupture of the hydatid cyst of the lungs into the bronchus.

15. Leishman stain or Wright stain for differential count Normal sputum consists of a few neutrophils, few lymphocytes, carbon laden macrophages, occasional eosinophils and red cells.

16. Papanicolaou stain for study of malignant cells Cytological examination of sputum is normally carried out for the diagnosis of bronchogenic carcinoma. For cytological examination, early morning sputum sample is preferred. A thin sputum smear is prepared on clean, sterile, grease-free glass slide from a yellowish, greyish, opaque, or blood-tinged portion, or from tissue fragments in sputum and stained with Papanicolaou technique.

17. SPUTUM CULTURE Culture media is inoculated with a floccule of the purulent portion of sputum for absolute identification of microorganism. Sputum sample is considered as unsuitable for the bacterial culture if it contains >25 squamous epithelial cells/low power field. An ideal sputum sample for bacterial culture contains bronchial epithelial cells, numerous neutrophils (>5/high power field), alveolar macrophages, and few squamous epithelial cells (<10/high power field). Saliva is washed away from sputum with sterile normal saline in order to reduce the amount of contaminating normal bacterial flora in the inoculum.

18. SPUTUM CULTURE Blood agar plate and chocolate agar are inoculated with the washed sputum. The chocolate agar plate is incubated in an atmosphere of extra carbon dioxide (CO2) and blood agar plate is incubated aerobically. After the incubation for 18 hours, inoculated agar plates are examined for growth; if growth is not sufficient, incubation for further 24 hours is indicated. Lowenstein-Jensen medium is used for culturing Mycobacterium Tuberculosis.