Microscopic analysis of sputum

Microscopic examination of native and fixed stained sputum preparations allows for a detailed study of its cellular composition, and to a certain extent reflecting the nature of the pathological process in the lungs and bronchi, its activity, to identify various fibrous and crystalline formations, which also have important diagnostic value, and, finally, to roughly assess the state of the microbial flora of the respiratory tract (bacterioscopy).

Microscopy uses native and stained sputum preparations. To study the microbial flora (bacterioscopy), sputum smears are usually stained according to Romanovsky-Giemsa, according to Gram, and to identify Mycobacterium tuberculosis according to Ziehl-Neelsen.

Cellular elements and elastic fibers

Of the cellular elements that can be detected in the sputum of patients with pneumonia, epithelial cells, alveolar macrophages, leukocytes and erythrocytes have diagnostic value.

Epithelial cells. Squamous epithelium from the oral cavity, nasopharynx, vocal folds and epiglottis has no diagnostic value, although the detection of a large number of squamous epithelial cells, as a rule, indicates the low quality of the sputum sample delivered to the laboratory and containing a significant admixture of saliva.

In patients with pneumonia, sputum is considered suitable for examination if, under low-magnification microscopy, the number of epithelial cells does not exceed 10 in the field of view. A greater number of epithelial cells indicates an unacceptable predominance of oropharyngeal contents in the biological sample.

Alveolar macrophages, which can also be found in small quantities in any sputum, are large cells of reticulohistiocytic origin with an eccentrically located large nucleus and abundant inclusions in the cytoplasm. These inclusions may consist of tiny dust particles (dust cells) absorbed by macrophages, leukocytes, etc. The number of alveolar macrophages increases during inflammatory processes in the pulmonary parenchyma and respiratory tract, including pneumonia.

Columnar ciliated epithelium cells line the mucous membrane of the larynx, trachea and bronchi. They look like elongated cells, widened at one end, where the nucleus and cilia are located. Columnar ciliated epithelium cells are found in any sputum, but their increase indicates damage to the mucous membrane of the bronchi and trachea (acute and chronic bronchitis, bronchiectasis, tracheitis, laryngitis).

Leukocytes in small quantities (2-5 in the field of vision) are found in any sputum. In case of inflammation of the lung tissue or mucous membrane of the bronchi and trachea, especially in case of suppurative processes (gangrene, lung abscess, bronchiectasis) their quantity increases significantly.

When staining sputum preparations according to Romanovsky-Giemsa, it is possible to differentiate individual leukocytes, which sometimes has important diagnostic value. Thus, with pronounced inflammation of the lung tissue or bronchial mucosa, both the total number of neutrophilic leukocytes and the number of their degenerative forms with fragmentation of nuclei and destruction of the cytoplasm increase.

An increase in the number of degenerative forms of leukocytes is the most important sign of the activity of the inflammatory process and a more severe course of the disease.

Erythrocytes. Single erythrocytes can be found in almost any sputum. Their significant increase is observed in cases of impaired vascular permeability in patients with pneumonia, in cases of destruction of lung or bronchial tissue, congestion in the pulmonary circulation, pulmonary infarction, etc. Erythrocytes are found in large quantities in sputum in cases of hemoptysis of any genesis.

Elastic fibers. Another element of sputum should be mentioned - plastic fibers, which appear in sputum during destruction of lung tissue (lung abscess, tuberculosis, disintegrating lung cancer, etc.). Elastic fibers are presented in sputum in the form of thin, double-contour, twisted threads with a dichotomous division at the ends. The appearance of elastic fibers in sputum in patients with severe pneumonia indicates the occurrence of one of the complications of the disease - abscess formation of lung tissue. In some cases, when a lung abscess is formed, elastic fibers in sputum can be detected even somewhat earlier than the corresponding radiographic changes.

Often, in cases of lobar pneumonia, tuberculosis, actinomycosis, and fibrinous bronchitis, thin fibrin fibers can be found in sputum preparations.

Signs of an active inflammatory process in the lungs are:

1. the nature of sputum (mucopurulent or purulent);
2. an increase in the number of neutrophils in sputum, including their degenerative forms;
3. an increase in the number of alveolar macrophages (from single clusters of several cells in the field of view and more);

The appearance of elastic fibers in sputum indicates the destruction of lung tissue and the formation of a lung abscess.

Final conclusions about the presence and degree of activity of inflammation and destruction of lung tissue are formed only when they are compared with the clinical picture of the disease and the results of other laboratory and instrumental research methods.

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Microbial flora

Microscopy of sputum smears stained by Gram and the study of microbial flora (bacterioscopy) in some patients with pneumonia allows us to approximately determine the most likely causative agent of the lung infection. This simple method of express diagnostics of the causative agent is not accurate enough and should be used only in combination with other (microbiological, immunological) methods of sputum examination. Immersion microscopy of stained sputum smears is sometimes very useful for emergency selection and prescription of adequate antibacterial therapy. However, one should keep in mind the possibility of seeding the bronchial contents with microflora of the upper respiratory tract and oral cavity, especially if sputum is collected incorrectly.

Therefore, sputum is considered suitable for further examination (bacterioscopy and microbiological examination) only if it meets the following conditions:

• Gram staining reveals a large number of neutrophils in sputum (more than 25 in the field of view at low magnification of the microscope);
• the number of epithelial cells, more characteristic of the contents of the oropharynx, does not exceed 10;
• the preparation contains a predominance of microorganisms of one morphological type.

When staining a sputum smear according to Gram, it is sometimes possible to identify gram-positive pneumococci, streptococci, staphylococci and a group of gram-negative bacteria - Klebsiella, Pfeiffer's bacillus, Escherichia coli, etc. quite well. In this case, gram-positive bacteria acquire a blue color, and gram-negative bacteria - red.

Bacterial pathogens of pneumonia

Gram positive	Gram negative
Pneumococci Streptococcus pneumoniae. Streptococci Streptococcus pyogenes, Streptococcus viridans. Staphylococci: Staphylococcus aureus, Staphylococcus haemolyticus.	Klebsiella pneumoniae Haemophilus influenzae (Pfeiffer's bacillus) Haemophilius influenzae Pseudomonas aeruginosa Legionella Pneumophilia Escherichia coli

Preliminary sputum bacterioscopy is the simplest method of verifying the causative agent of pneumonia and is of certain importance for choosing the optimal antibiotic therapy. For example, if Gram-positive diplococci (pneumococci) or staphylococci are detected in Gram-stained smears, instead of broad-spectrum antibiotics that increase the risk of selection and spread of antibiotic-resistant microorganisms, targeted therapy active against pneumococci or staphylococci may be prescribed. In other cases, detection of Gram-negative flora prevailing in smears may indicate that the causative agent of pneumonia is Gram-negative enterobacteria (Klebsiella, Escherichia coli, etc.), which requires the appointment of appropriate targeted therapy.

True, a rough conclusion about the probable causative agent of a pulmonary infection can be made by microscopy only on the basis of a significant increase in bacteria in sputum, at a concentration of 10 ⁶ - 10 ⁷ m.c./ml and more (L.L. Vishnyakova). Low concentrations of microorganisms (< 10 ³ m.c./ml) are characteristic of accompanying microflora. If the concentration of microbial bodies fluctuates from 10 ⁴ to 10 ⁶ m.c./ml, this does not exclude the etiological role of this microorganism in the occurrence of a pulmonary infection, but does not prove it either.

It should also be remembered that "atypical" intracellular pathogens (mycoplasma, legionella, chlamydia, rickettsia) do not stain according to Gram. In these cases, suspicion of the presence of an "atypical" infection may arise if dissociation between a large number of neutrophils and an extremely small number of microbial cells is detected in sputum smears.

Unfortunately, the bacterioscopy method in general has a rather low sensitivity and specificity. The non-predictive value even for well-visualized pneumococci barely reaches 50%. This means that in half of the cases the method gives false-positive results. This is due to several reasons, one of which is that about 1/3 of patients have already received antibiotics before hospitalization, which significantly reduces the effectiveness of sputum bacterioscopy. In addition, even in the case of positive test results indicating a fairly high concentration of "typical" bacterial pathogens in the smear (e.g. pneumococci), the presence of co-infection with "atypical" intracellular pathogens (mycoplasma, chlamydia, legionella) cannot be completely ruled out.

The method of bacterioscopy of sputum smears stained by Gram, in some cases helps to verify the causative agent of pneumonia, although in general it has a very low predictive value. "Atypical" intracellular pathogens (mycoplasma, legionella, chlamydia, rickettsia) are not verified by bacterioscopy at all, since they are not stained by Gram.

It is worth mentioning the possibility of microscopic diagnostics in patients with pneumonia of fungal lung infection. The most relevant for patients receiving long-term treatment with broad-spectrum antibiotics is the detection of Candida albicans in the form of yeast-like cells and branched mycelium during microscopy of native or stained sputum preparations. They indicate a change in the microflora of the tracheobronchial contents, which occurs under the influence of antibiotic treatment, which requires significant correction of therapy.

In some cases, patients with pneumonia need to differentiate existing lung damage from tuberculosis. For this purpose, sputum smear staining according to Ziehl-Neelsen is used, which in some cases allows identifying tuberculosis mycobacteria, although a negative result of such a study does not mean that the patient does not have tuberculosis. When staining sputum according to Ziehl-Neelsen, tuberculosis mycobacteria are stained red, and all other elements of sputum are stained blue. Tuberculosis mycobacteria have the appearance of thin, straight or slightly curved rods of varying length with individual thickenings. They are located in the preparation in groups or singly. The detection of even single tuberculosis mycobacteria in the preparation is of diagnostic value.

To increase the efficiency of microscopic detection of tuberculosis mycobacteria, a number of additional methods are used. The most common of these is the so-called flotation method, in which homogenized sputum is shaken with toluene, xylene or gasoline, the drops of which, floating up, capture the mycobacteria. After the sputum has settled, the upper layer is applied to a glass slide with a pipette. The preparation is then fixed and stained according to Ziehl-Neelsen. There are also other methods of accumulation (electrophoresis) and microscopy of tuberculosis bacteria (luminescent microscopy).

Microscopic examination (analysis) of sputum allows detection of mucus, cellular elements, fibrous and crystalline formations, fungi, bacteria and parasites.

Cells

• Alveolar macrophages are cells of reticulohistiocytic origin. A large number of macrophages in sputum are detected in chronic processes and at the stage of resolution of acute processes in the bronchopulmonary system. Alveolar macrophages containing hemosiderin ("heart defect cells") are detected in pulmonary infarction, hemorrhage, and congestion in the pulmonary circulation. Macrophages with lipid droplets are a sign of an obstructive process in the bronchi and bronchioles.
• Xanthomatous cells (fat macrophages) are found in abscesses, actinomycosis, and echinococcosis of the lungs.
• Columnar ciliated epithelial cells are the cells of the mucous membrane of the larynx, trachea and bronchi; they are found in bronchitis, tracheitis, bronchial asthma, and malignant neoplasms of the lungs.
• Flat epithelium is detected when saliva enters sputum and has no diagnostic value.
• Leukocytes are present in any sputum in varying quantities. A large number of neutrophils are found in mucopurulent and purulent sputum. Sputum is rich in eosinophils in cases of bronchial asthma, eosinophilic pneumonia, helminthic lung lesions, and pulmonary infarction. Eosinophils may appear in sputum in cases of tuberculosis and lung cancer. Lymphocytes are found in large quantities in cases of whooping cough and, less frequently, tuberculosis.
• Red blood cells. The detection of single red blood cells in sputum has no diagnostic value. If fresh blood is present in the sputum, unchanged red blood cells are detected, but if blood that has been in the respiratory tract for a long time is released with the sputum, leached red blood cells are detected.
• Malignant tumor cells are found in malignant neoplasms.

Fibers

• Elastic fibers appear during the disintegration of lung tissue, which is accompanied by the destruction of the epithelial layer and the release of elastic fibers; they are found in tuberculosis, abscess, echinococcosis, and neoplasms in the lungs.
• Coral-shaped fibers are found in chronic lung diseases such as cavernous tuberculosis.
• Calcified elastic fibers are elastic fibers impregnated with calcium salts. Their detection in sputum is characteristic of the disintegration of tuberculous petrification.

Spirals, crystals

• Kurshman's spirals are formed in the case of a spastic condition of the bronchi and the presence of mucus in them. During a cough push, viscous mucus is thrown into the lumen of a larger bronchus, twisting into a spiral. Kurshman's spirals appear in bronchial asthma, bronchitis, lung tumors that compress the bronchi.
• Charcot-Leyden crystals are breakdown products of eosinophils. They usually appear in sputum containing eosinophils; they are characteristic of bronchial asthma, allergic conditions, eosinophilic infiltrates in the lungs, and pulmonary fluke.
• Cholesterol crystals appear in abscesses, pulmonary echinococcosis, and neoplasms in the lungs.
• Hematoidin crystals are characteristic of lung abscess and gangrene.
• Actinomycete drusen are found in pulmonary actinomycosis.
• Echinococcus elements appear in pulmonary echinococcosis.
• Dietrich plugs are yellowish-gray lumps with an unpleasant odor. They consist of detritus, bacteria, fatty acids, and fat droplets. They are characteristic of lung abscess and bronchiectasis.
• Ehrlich's tetrad consists of four elements: calcified detritus, calcified elastic fibers, cholesterol crystals, and tuberculosis mycobacteria. It appears during the decay of a calcified primary tuberculosis lesion.

Mycelium and budding fungal cells appear during fungal infections of the bronchopulmonary system.

Pneumocystis bacteria appear in Pneumocystis pneumonia.

Fungal spherules are detected in coccidioidomycosis of the lungs.

Ascaris larvae are detected in ascariasis.

Eelworm larvae are found in strongyloidiasis.

Lung fluke eggs are detected during paragonimiasis.

Elements found in sputum in bronchial asthma. In bronchial asthma, a small amount of mucous, viscous sputum is usually secreted. Macroscopically, Curschmann spirals can be seen. Microscopic examination typically reveals eosinophils, cylindrical epithelium, and Charcot-Leyden crystals.