Microscopic analysis of sputum

Microscopic examination of native and fixed stained sputum specimens allows detailed study of its cellular composition, and to a certain extent reflects the nature of the pathological process in the lungs and bronchi, its activity, reveal various fibrous and crystalline formations, which also have important diagnostic significance, and, finally, microbial flora of the respiratory tract (bacterioscopy).

At a microscopy use native and painted preparations of a sputum. To study the microbial flora (bacterioscopy), sputum smears are usually stained by Romanovsky-Giemsa, according to Gram, and for the detection of Mycobacterium tuberculosis, but Cilu-Nielsen.

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 are of diagnostic importance.

Epithelial cells. Flat epithelium from the oral cavity, nasopharynx, vocal folds and epiglottis is not of diagnostic significance, although the detection of a large number of flat epithelial cells usually indicates a low quality of a sputum specimen delivered to the laboratory and containing a significant admixture of saliva.

In patients with pneumonia, sputum is considered suitable for investigation if, with a microscopy with a small increase, the number of epithelial cells does not exceed 10 in the field of vision. A larger number of epithelial cells indicates an unacceptable predominance in the biological sample of the contents of the oropharynx.

Alveolar macrophages, which in small amounts can also be found in any sputum, are large cells of reticulohystocytic origin with an eccentrically located large nucleus and abundant inclusions in the cytoplasm. These inclusions can consist of macrophages absorbed by the smallest dust particles (dust cells), leukocytes, and the like. The number of alveolar macrophages increases with inflammatory processes in the pulmonary parenchyma and the airways, including pneumonia.

Cells of cylindrical ciliated epithelium lining 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. Cells of cylindrical ciliated epithelium are found in any sputum, but their increase indicates damage to bronchial mucosa and trachea (acute and chronic bronchitis, bronchiectasis, tracheitis, laryngitis).

Leukocytes in small amounts (2-5 in the field of vision) are found in any sputum. When inflammation of the lung tissue or bronchial mucosa and trachea, especially when suppuration (gangrene, lung abscess, bronchiectasis), their number is significantly increased.

When staining sputum preparations according to Romanovsky-Giemsa, it is possible to differentiate individual leukocytes, which sometimes has an 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 the 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 the more severe course of the disease.

Erythrocytes. Single erythrocytes can be detected practically and any sputum. A significant increase is observed when vascular permeability is impaired in patients with pneumonia, with destruction of lung or bronchial tissue, stagnation in a small circle of circulation, lung infarction, etc. In a large number of red blood cells in sputum are found during hemoptysis of any genesis.

Elastic fibers. One more element of sputum plastic fibers that appear in sputum when destruction of lung tissue (lung abscess, tuberculosis, disintegrating lung cancer, etc.) should also be mentioned. Elastic fibers are presented in sputum in the form of thin two-contour, crimped filaments with 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 - abscessing of lung tissue. In some cases, in the formation of lung abscess, elastic fibers in sputum can be detected even slightly earlier than the corresponding radiographic changes.

Often, with croupous pneumonia, tuberculosis, actinomycosis, fibrinous bronchitis in sputum preparations, thin fibrin fibers can be detected.

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

The 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 methods of investigation.

[1], [2], [3], [4], [5]

Microbial flora

Smear microscopy, stained according to Gram, and the study of microbial flora (bacterioscopy) in a part of patients with pneumonia allows tentatively to establish the most likely causative agent of pulmonary infection. This simple method of express diagnostics of the pathogen is not accurate enough and should be used only in combination with other (microbiological, immunological) methods of sputum examination. Immersion microscopy of stained smears is sometimes very useful for emergency selection and administration of adequate antibiotic therapy. However, one should keep in mind the possibility of contamination of the bronchial contents of the microflora of the upper respiratory tract and oral cavity, especially when sputum collection is incorrect.

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

• Gram staining in sputum reveals a large number of neutrophils (more than 25 in the field of view with a small magnification of the microscope);
• The number of epithelial cells, more characteristic of the contents of the oropharynx, does not exceed 10;
• in the preparation there is a predominance of microorganisms of the same morphological type.

When Gram stains in smear sputum, it is sometimes possible to identify gram-positive pneumococci, streptococci, staphylococci and a group of gram-negative bacteria - Klebsiella, Pfeiffer's stick, E. Coli, etc. - at the same time. Gram-positive bacteria acquire a blue color, and Gram-negative bacteria - red.

Bacterial pathogens of pneumonia

Gram-positive	Gram-negative
Pneumococcus Streptococcus pneumoniae. Streptococcus Streptococcus pyogenes, Streptococcus viridans. Staphylococci: Staphylococcus aureus, Staphylococcus haemolyticus.	Klebsiella  (Klebsiella pneumoniae) Hemophilus influenzae (Pfeiffer) Haemophilius influenzae Pseudomonas aeruginosa  (Pseudomonas aeruginosa) Legionnella (Legionella Pneumophilia) E. Coli (Escherichia coli)

Preliminary sputum smear is the simplest way to verify the causative agent of pneumonia and has definite implications for the selection of optimal antibiotic therapy. For example, if smear-positive diplococci (pneumococci) or staphylococci are found in smears stained with Grammar instead of broad-spectrum antibiotics that increase the risk of selection and spread of antibiotic-resistant microorganisms, it may be possible to administer targeted therapy active against pneumococci or staphylococci. In other cases, the detection of the predominant gram-negative flora 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.

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

It should also be remembered that "atypical" intracellular pathogens (mycoplasma,  legionella, chlamydia, rickettsia) do not stain Gramm. In these cases, suspicion of having an "atypical" infection can occur if smears show a dissociation between a large number of neutrophils and an extremely small number of microbial cells.

Unfortunately, the method of bacterioscopy is generally quite low in sensitivity and specificity. Not predictive value, even for well-visualized pneumococci, barely reaches 50%. This means that in half the cases the method gives false positive results. This is due to several reasons, one of which is that about 1/3 of the patients before the hospitalization have already received antibiotics, which significantly reduces the effectiveness of sputum smear-microscopy. In addition, even in the case of positive results of the study, indicating a sufficiently high concentration in the smear of "typical" bacterial pathogens (eg pneumococci), the presence of co-infection by "atypical" intracellular pathogens (mycoplasma, chlamydia, legionella) can not 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 it generally has very low predictive value. Atypical intracellular pathogens (mycoplasma, legionella, chlamydia, rickettsia) are not verified at all by the method of bacterioscopy, since they do not stain Gramm.

It should be mentioned the possibility of microscopic diagnosis 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 sputum in the form of yeast-like cells and branched mycelium by 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 a substantial correction of therapy.

In some cases in patients with pneumonia, there is a need to differentiate the existing lung disease with tuberculosis. For this purpose, the color of the sputum smear according to Tsiol-Nielsen is used, which in some cases makes it possible to identify the mycobacterium tuberculosis, although the negative result of such a study does not mean that the patient does not have tuberculosis. When staining sputum according to Tsil-Nielsen, mycobacterium tuberculosis is colored red, and all other sputum elements are blue. Tuberculous mycobacteria have the appearance of feces, straight or slightly curved sticks of different lengths with separate thickenings. They are located in the preparation in groups or singly. Diagnostic value is the detection in the preparation of even single mycobacteria tuberculosis.

To increase the effectiveness of microscopic detection of mycobacteria tuberculosis use a number of additional methods. The most common of these is the so-called flotation method, in which homogenized sputum is shaken with toluene, xylene or gasoline, the droplets of which pop up and capture mycobacteria. After settling the sputum, the top layer is pipetted onto the slide. Then the drug is fixed and stained by Tsilyu-Nielsen. There are other methods of accumulation (electrophoresis) and microscopy of tuberculosis bacteria (luminescence microscopy).

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

Cells

• Alveolar macrophages are cells of reticulogistocyte origin. A large number of macrophages in sputum is detected in chronic processes and at the stage of resolving acute processes in the bronchopulmonary system. Alveolar macrophages containing hemosiderin ("cells of cardiac defects") are detected with a mild infarction, hemorrhage, stagnation in a small circle of blood circulation. Macrophages with lipid droplets are a sign of obstructive process in the bronchi and bronchioles.
• Xantom cells (fatty macrophages) are found in abscess, actinomycosis, echinococcosis of the lungs.
• Cells of the cylindrical ciliated epithelium are cells of the mucous membrane of the larynx, trachea and bronchi; they are found in bronchitis, tracheitis, bronchial asthma, malignant neoplasms of the lungs.
• The flat epithelium is detected when spittle enters the sputum, it has no diagnostic significance.
• Leukocytes in one or another quantity are present in any sputum. A large number of neutrophils are detected in mucopurulent and purulent sputum. Eosinophils are rich in sputum in bronchial asthma, eosinophilic pneumonia, glottis lung lesions, and lung infarction. Eosinophils may appear in sputum for tuberculosis and lung cancer. Lymphocytes in large numbers are found in whooping cough and, more rarely, with tuberculosis.
• Erythrocytes. Detection of single red blood cells in sputum is not of diagnostic significance. If fresh blood is present in the sputum, unchanged erythrocytes are determined, but if the blood that has been in the respiratory tract for a long time leaves with sputum, then leached red blood cells are found.
• Cells of malignant tumors are found in malignant neoplasms.

Fibers

• Elastic fibers appear in the decay 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, neoplasms in the lungs.
• Coronal fibers are detected in chronic lung diseases, such as cavernous tuberculosis.
• Calcined elastic fibers are elastic fibers impregnated with calcium salts. Detection of them in sputum is characteristic for the breakdown of tubercular petrichitis.

Spirals, crystals

• Kurshman spirals are formed in the spastic state of the bronchi and the presence of mucus in them. During a cough thrust, viscous mucus is released into the lumen of a larger bronchus, twisting in a spiral. Kurshman spirals appear with bronchial asthma, bronchitis, lung tumors, compressing bronchi.
• The Charcot-Leiden crystals are the products of the decay of eosinophils. Usually appear in a sputum containing eosinophils; are characteristic for bronchial asthma, allergic conditions, eosinophilic infiltrates in the lungs, pulmonary flukes.
• Cholesterol crystals appear with abscess, lung echinococcosis, neoplasms in the lungs.
• Crystals of hematoidin are characteristic for abscess and gangrene of the lung.
• Druses of actinomycete are detected in the actinomycosis of the lungs.
• Elements of echinococcus appear with echinococcosis of the lungs.
• Corks Dietrich - lumps of a yellowish-gray color, having an unpleasant smell. They consist of detritus, bacteria, fatty acids, droplets of fat. They are typical for an abscess of lung and bronchiectasis.
• Ehrlich's tetrad consists of four elements: calcified detritus, calcified elastic fibers, cholesterol crystals and mycobacterium tuberculosis. Appears in the decay of the calcified primary tubercular focus.

Mycelium and budding fungal cells appear in fungal lesions of the bronchopulmonary system.

Pneumocystis occurs with pneumocystis pneumonia.

Spherules of fungi are detected in coccidioidomycosis of the lungs.

The ascarid larvae are detected with ascariasis.

Larvae of the intestinal ugristic are identified with strongyloidiasis.

Eggs of the pulmonary fluke are identified with paragonimosis.

Elements found in sputum in bronchial asthma. When bronchial asthma is usually separated by a small amount of mucous, viscous sputum. Macroscopically you can see the Kurshman spiral. When microscopic research is characteristic of the presence of eosinophils, cylindrical epithelium, there are crystals of Charcot-Leiden.