Sputum Analysis: Common Questions

Tracheobronchial secretions are often collectively referred to as sputum. Sputum is constituted by plasma, water, electrolytes and mucin.

Principal source of sputum/tracheobronchial secretions are the goblet cells and mucus glands. The surface lining of the respiratory tract is by ciliated columnar cells with goblet cells. The goblet cells produce a thick mucin type of sputum which is diluted by a more serous mixture of acid glycoproteins, sialoproteins and sulphoproteins secreted by submucus glands present in between surface epithelial cells and cartilaginous plate.

The stimulus for sputum production are
a. Vagal nerve stimulation
b. Cholinergic drugs
c. Immununologic and inflammatory stimulus, mast cells , Eosinophils and plasma cells may contribute to the secretions.

The sputum is viscoelastic, that is, some of the properties of a liquid and some that of solid.

The chemical composition is
a. 98% water
b. 5% solid comprising of carbohydrate, proteins, lipids and deoxyribonucleic acids.

The indications are
a. Smear and culture: for identification of causative organism in a suspected infection of lower respiratory tract.
b. Cytological examination to look for viral inclusions, asbestosis, fungal infection and malignant cells.

a. Before collecting or expectorating sputum, mouth should be pre rinsed to remove contaminants from oral cavity.
b. Early morning sample is preferred before any mouthwash or taking food
c. Patients should take deep breaths 2 -3 times for filling the lungs, coughs deeply and spits into the container.
d. About 2-5 ml of sputum is to be collected.

Early morning sample is best as it represents the pulmonary secretions accumulated overnight.

a. Nasopharyngeal swab may be taken which is quite representatiove of the bronchial pathogens
b. Cough plate is held before the child’s mouth and the child is urged to cough
c. Cough swab method gives the most representative, non contaminated sputum sample. Child’s mouth is held open by using tongue depressor. Epiglottis is visualized and touched with swab to induce cough. Material expelled from trachea is deposited on the swab, which can then be plated on the appropriate culture media.

The container should be
a. Clean and dry
b. Wide mouthed with 25 ml capacity
c. Securely fitted with screw cap
d. Leak proof to prevent aerosol formation

a. Sample consisting only of saliva ( foamy , clear and watery appearance)
b. Sample if only Squamous epithelial cells are present.

If the patient is not cooperative r cannot produce adequate sputum, induction should be tried. The commonly used inductants are
a. 15% NaCl aersol spray and 20% propylene glycol for 20 min.
b. Inhaling nebulized hypertonic saline or distilled water with chest physiotherapy
c. For the patient with history of bronchospasmodic disorders, bronchodilators should be given after inductants. Eg: Acetylcholine in aerosol form with a bronchodilator.

Samples should be immediately transported to laboratory if possible. If the sample is to be transported to a distant laboratory, transport in 25 ml of the following solution
N acetypyridinium chloride-5gm
Sodium chloride-10gm
Distilled water 1000ml.
Sputum should not be refrigerated in any case.

It will lead to
a. Rapid proliferation of contaminating bacterial flora from the oral cavity and throat
b. H Influenzae do not survive for long

Saccomano technique is the best & most widely used method of fixation & concentration of sputum. This method involves collection & fixation of sputum in a solution of 50% ethyl alcohol & 2% carbowax followed by blending & smearing. It was originally used in 1970’s to screen for lung cancer.

The advantages are
a. To preserve the specimen until preparation is possible
b) Transportation of the specimen
c) Higher diagnostic yield as this method enables concentration & use of representative sample of the entire specimen.

The color of the sputum signifies the following conditions.

Bloody (hemoptysis)	Pulmonary TB, Lung abscess, bronchiectasis, bronchogenic carcinoma, mitral stenosis, pulmonary infarction
Rusty	Pneumococcal lobar pneumonia
Bloody and gelatinous ( Red currant jelly)	Klebsiella pneumonia
Green	Pseudomonas infection
Purulent	Bronchiectasis, lung abscess
Pink, frothy	Pulmonary edema

Normal sputum is odourless
a. Putrid odour: seen in suppurative pulmonary disorders such as lung abscess, cavitary TB or gangrene
b. Fecal odour: in ruptures subphrenic or liver abscess.

The prerequisites are
a. There should not be Squamous cells covered with masses of bacteria- indicates sample is mostly from mouth or throat.
b. If Neutrophils are less than 10 per epithelial cells there is no need for culture
c. Knowledge of flora of mouth and pharynx is necessary before analysis

The normal flora are as follows

Gram positive	Gram Negative
Staphylococcus aureus and epidermidis	Enterobacteriaciae
Streptococcus viridians and pneumonia	Hemophilus influenza
Diptheroids	Eikenella corrodens
Enterococci	Actinobacillus
Micrococci	Actinomycetes
Lactobacilli
Yeasts ( candida species)

The morphological appearances are as follows

a) Streptococcus pneumonia	Gram positive diplococcic with surrounding clear space
b) Staphylococcus aureus	Gram positive cocci in grape like clusters
c) Candida	Gram positive yeast cells with budding & pseudohyphae
d) Moraxella catarrhalis	Gram negative diplococcic both in intra & extracellular
e) H. Influenzae	Gram negative coccobacilli
f) Actinomyces	Large granules with center gram negative & periphery gram positive

Ideal sample should contains:
a. Alveolar macrophages
b. numerous Neutrophils (>5/Hpf)
c. Bronchial epithelial cells (<10/Hpf)
d. Few Squamous cells (<10/Hpf)

These plugs are seen most commonly in chronic bronchitis, bronchiectasis & bronchial asthma
When expectorated, they are usually solitary of a variable size.
When crushed, they are found to be made of cellular debris, fatty acid crystals, fat globules & bacteria

a. AscarisLumbricoides
b. Echinococcusgranulosus
c. Toxocaracaris
d. Paragonimuswestermani

Curschmann’s Spirals are characteristic of bronchial asthma sputum but may be seen in other respiratory disorders.
Macroscopically, they can sometimes be recognized by naked eye & appear as yellow, white, mucoid, wavy threads frequently coiled into little balls. Their length may exceed 1-5cm.
Microscopically, they show a central thread around which mucus is wrapped, supported by a fibril network.

Charcot Leyden crystals are seen in sputum of bronchial asthma cases. The crystals are colorless,
pointed hexagons & variable in size (may look needle shaped). These are derived from
disintegration of eosinophils hence they stain strongly with eosin.

a. In acute Pulmonary oedema:Sputum is a abundant, frothy & pink (upto 1 liter a day) Microscopically, it shows numerous RBC’S & large hyaline masses
b. Mitral heart disease:Sputum is tenacious & blood is present, either in streaks or in dark masses mixed with mucus
c. In chronic congestive heart failure: Sputum is frothy & rust colored .Microscopically, presence of RBC’s & heart failure cells

Parainfluenza & amp;measles virus	Eosinophilic intracytoplasmic inclusion
Respiratory syncytial cytomegalovirus	Basophilic intracytoplasmic inclusion

a) AFB have more affinity for AR dye
b) The entire smear can be screened as the lower power (x10) objective is used
c) The black background in fluorescent microscopy males the bacilli stained out sharper to allow more rapid & accurate slide screening