SPHYSICAL, AND MICROSCOPIC EXAMINATION OF SPUTUM

SPHYSICAL, AND MICROSCOPIC EXAMINATION OF SPUTUM

1. SPUTUM EXAMINATION MITHUN VENUGOPAL. A HOD, ASSISTANT PROFESSOR DEPT. OF PATHOLOGY

2. INTRODUCTION Sputum is a colorless, watery and odorless tracheobronchial secretion. Sputum is mainly composed of mucus and also certain cellular and noncellular components of host origin. Examination of sputum helps in the diagnosis of various lung diseases. 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. The consistency & contents of sputum gets altered in various conditions which affect the lungs and tracheobronchial tree.

3. Production & composition It is produced in the tracheobronchial tree. The principle cells for the production of sputum are goblet cells and submucous gland . The goblet cells of the surface epithelium produce thick Mucin which is diluted by the secretions of the submucous gland. As this secretion passes through the upper and lower respiratory tract, it gets contaminated with cellular exfoliations, nasal and salivary gland secretions and the normal bacterial flora of the oral cavity.

4. When the secretion spills over into the hypopharynx, it is swallowed into the oesophagus without the individual being aware of it. Only when the volume of these secretions increases, the person becomes aware of its presence. The excess of secretions is brought into the mouth and then expectorated by coughing. Normal sputum is a mixture of plasma, Mucin, electrolytes and water. 95% is water and 5% is solid (carbohydrates, proteins, lipids, DNA). Production & composition

5. 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.

6. collection Sputum sample is ideally collected in the morning (since secretions accumulate overnight). Instruct the patient to rinse his mouth with water before collecting the sample. 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.

7. COLLECTION The sputum should be collected from the lungs or the bronchi. Sample consisting only saliva (watery appearance, clear, and foamy) is not acceptable for laboratory investigations; in such case, another sample should be collected. Types of samples collected are; Early morning sample – for routine examination 24 hour sputum sample – for the demonstration of tubercle bacilli by concentration method. 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 capped securely and labelled properly.

8. 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

9. Concentration method for m. tuberculosis Collect 24 hour sputum sample in a sterile wide mouth container. Add equal volume of 6% sulphuric acid and allow it to stand for 20 minutes. Centrifuge at 3000 rpm for 30 minutes. Discard the supernatant and carefully wash the sediment with water. Repeat this for 3 times. Make a smear from the sediment, dry, fix and stain with Ziel-Neilsons staining.

10. Physical examination Examine the sputum for any colour difference, presence of materials. Select a portion where the consistency of the sputum showing abnormality. Quantity Normal – 2.5 ml morning sample and 100 ml for 240hour sample 24 hour sample >100 ml – pulmonary oedema, lung abscess, bronchiectasis pulmonary haemorrhage. 24 hour sample >500 ml – amoebic lung abscess, rupture of amoebic liver abscess into the lungs.

11. Physical examination Colour Normal – clear and colourless. Greenish – pseudomonas infection, rupture of liver abscess into the lungs. Rust colour – pneumonia, pulmonary infarction. Bright red – pulmonary tuberculosis, lung tumours, pulmonary infarctions. Black – inhalation of coal dust. Yellow – pulmonary infections. Odour Normal – odourless. Foul smelling – lung abscess, bronchiectasis, pulmonary infarction. Cheesy odour – malignant tumour with necrosis, perforating empyema.

12. Physical examination Consistency Normal – colourless, opalescent with slightly uneven consistency. Serous, frothy, colourless or yellow – pulmonary oedema. Mucoid, glossy, tenacious – acute bronchitis, asthma, lobar pneumonia. Purulent, foul smelling – ruptured empyema and bronchiectasis. Mucopurulent – lung cavitations, candidiasis.

13. Layer formation Place the sample in a test tube for 2-3 hours. Normally there is no layer formation. Formation of 2-3 layers of frothy mucous on the top watery material in the middle and the bottom sediment of pus cells, bacteria. This layer formation is seen in conditions like bronchiectasis, infarction and lung abscess. Cheesy masses (fragment of necrotic tissue) – seen in tuberculosis. Physical examination

14. Physical examination Bronchial casts – white branching tree like casts, made of fibrin, seen in bronchitis, bronchiolitis and pneumonia. Broncholiths (lung stones) – formed by calcification of the infected lung tissue or the contents of the lung cavities or dilated bronchi. Seen in tuberculosis and fungal infection of the lungs. Sulphur granules – present as yellow granular structure. These are made up of colonies of fungus. Seen in actinomycosis of the lungs. Foreign bodies – include pins, beads, ground nuts, coins, etc.

15. Microscopic examination WET SMEAR PREPARATION Place a drop of well mixed sputum on a glass slide and place a coverslip. Observe under microscope. Pus cells – normally few pus cells are seen. Numerous pus cells indicate inflammation of the respiratory tract. Heart failure cells ( hemosiderin laden macrophages) – seen in chronic venous congestion, mitral valve stenosis, pulmonary infarction and pulmonary haemorrhage . Anthracotic laden cells – normally few cells are seen. Increased numbers are seen in coal workers and those who live in smoky polluted atmosphere. Curschmanns spirals – appear as wiry, spiral structures with central thread. Seen in bronchial asthma.

16. Microscopic examination Elastic fibres – seen as wavy refractile fibres in bundles. Presence indicates breakdown of lung parenchyma. Myelin globules – seen normally. No clinical significance. Charcot laden crystals – fine needle shaped or hexagons, colourless crystals. Seen when sputum kept for long time. Seen in bronchial asthma due to the disintegration of eosinophils. Fatty acid crystals – seen in patients with chronic tuberculosis bronchitis and gangrene of the lungs. Cholesterol crystals – seen in empyema and chronic lung abscess. Haematoidin crystals – seen in case of haemorrhage in to the lungs. Parasites – cyts and trophozoites of entamoeba histolytica, larvae of echinococcus granulosa and larvae of strongyloides stercoralis .

17. Microscopic examination

18. Microscopic examination STAINED SMEAR PREPARATION Leishmans stain Wrights stain Ziel-Neilsons stain for AFB Special stains for fungi Papaniculaou stain for identifying malignant cells Normal sputum contains few neutrophils, lymphocytes and occasional eosinophils. Increased neutrophils indicate pyogenic infection. Increased eosinophils seen in asthma and parasitic infections. Increased lymphocytes are seen in early or mild cases of tuberculosis. RBCs are seen in the cases of haemorrhage into the lungs.

19. Grams stain 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. Pathogenic 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

20. Ziehl-Neilsen STAINING FOR MTB 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.

21. 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.

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

23. 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.

24. 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.