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Learning Objectives

  • The student will be able to describe the best method for sputum collection and the diagnostic information to be gained from its analysis.

  • The student will be able to recognize the special stains used on sputum and pleural fluids to aid in making a diagnosis.

  • The student will be able to list the characteristics of a normal pleural fluid and mechanisms for the formation of a pleural effusion.

  • The student will be able to distinguish between an exudate and a transudate and list laboratory testing used to make such a determination.

  • The student will be able to identify the major cell types found in pleural effusions and explain how these correlate with various disease processes.

Pneumonia is an infectious process affecting the parenchyma of the lungs. Infectious agents that are potentially responsible for such pneumonia include bacteria, fungi, viruses, and rarely parasites. The goal when evaluating sputum in the laboratory is to confirm the diagnosis of pneumonia, identify the etiologic agent(s) responsible for producing the present disease, and guide selection of appropriate antibiotic therapy.

Sputum Collection and Gram Staining

Expectorated sputum is the most common lower respiratory tract specimen received in the clinical microbiology laboratory. Sputum can be collected either by spontaneous expectoration or after sputum induction, which is collected following instillation or inhalation of an irritating aerosol such as hypertonic saline. Sputum induction is performed when the patient cannot produce sputum. Generally, the first morning sputum samples are the best specimens; a volume of 5-10 mL is usually adequate. Because many received specimens consist mostly of pharyngeal secretions and upper airway cells rather than sputum, the first step in evaluating a specimen's suitability uses a Gram stain (Fig. 19.1). Use of such a Gram stain assesses specimen acceptability for further processing, and aids interpretation by identifying the morphology of any likely etiologic agents of pneumonia in a specimen that is acceptably purulent.


A typical sputum sample that has been Gram-stained to show neutrophils, epithelial cells, and gram-positive bacteria, in this case diplococci. Courtesy of C. Sinave, MD.

Ideally, a sputum sample examined microscopically should contain <10 epithelial cells per low power field (LPF), or from 10-25 epithelial cells and >25 leukocytes/LPF. Sputum specimens containing >25 squamous epithelial cells are rejected because these represent oropharyngeal secretions that will be heavily contaminated with normal throat flora. Likewise, a sputum specimen containing no epithelial cells, leukocytes, or bacteria is rejected because the specimen is an inadequate representation of conditions in the intermediate airways. Most hospital laboratories have well-established guidelines of this type, including stipulations as to the handling of such potentially dangerous biological specimens. These guidelines can be adjusted when a particular patient's needs are communicated to the laboratory personnel.


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