Sputum Analysis

Specimen Requirements and Procedure

Sputum specimen collection is typically a noninvasive and straightforward procedure. However, in some clinical settings, a more intensive approach may be necessary when patients have difficulty expelling fluid from the upper respiratory tract. In such cases, physiotherapy maneuvers may be considered as an adjuvant to obtain material for analysis. A deep cough sample, often collected early in the morning before eating or drinking, helps reduce potential biases in test results. First, the patient should rinse their mouth with clear water for 10 to 15 seconds to eliminate contaminants in the oral cavity. After expelling saliva, the patient should take 3 deep breaths and then cough at 2-minute intervals until bringing up some sputum. The sputum is then collected in a sterile, sealed container provided by medical professionals.

Medical professionals assess the volume and quality of the sputum, which should be thick to facilitate proper laboratory investigation. In several institutions, clear and runny samples are unacceptable for further microscopic or microbiological studies. In some cases, collection may be repeated until 10 to 20 mL of sputum is obtained. Changing the lid of the collection container after the initial sample helps prevent contamination. If the patient has trouble producing enough sputum, medical professionals may use physiotherapeutic maneuvers to facilitate the progressive release of sputum. For routine sputum cultures, a single sample is collected and sent to the laboratory on the same day of collection. If the patient leaves the specimen in the refrigerator after collection, there is often a tolerance range that may extend well over 24 hours. In tuberculosis, 3 sputum samples must be collected on 3 consecutive days and returned to the clinical laboratory daily.

Sputum Induction

Sputum induction is a procedure used to collect adequate lower respiratory secretions from patients who have difficulty producing sputum, aiding in the diagnosis of tuberculosis. This procedure is essential for patients suspected of having miliary tuberculosis or tuberculous pleural effusion. The patient inhales a nebulized hypertonic saline solution to liquefy airway secretions, stimulating coughing and promoting the expectoration of mucus. Medical professionals prepare a 20 mL 3% hypertonic saline solution and inject it into the water-filled nebulizer cup. Similar to the non-adjuvant procedure, patients must thoroughly rinse their mouths before beginning sputum induction to minimize contamination.

Moreover, after sitting upright, the patient wears the nebulizer cup to cover the face and nose. The patient inhales and exhales through the mouthpiece. Expectorated saliva is placed into an emesis bowl, and expectorated sputum coughed up is collected into a sterile, sealed container. The medical staff activates the nebulizer device, allowing the patient to inhale the hypertonic mist for approximately 5 minutes. The patient then takes several deep breaths before attempting to cough. If the patient has difficulty producing sputum, the medical staff may use gentle chest physiotherapy to aid the patient in producing sputum. During the procedure, close observation by medical staff is essential to monitor for potential complications, such as the potential rupture of pleural bullae, which can lead to a life-threatening pneumothorax. The procedure should be stopped when a 1- to 2-mL sputum specimen is collected, after 15 minutes of nebulization, or if the patient complains of chest tightness, dyspnea, or wheezing. Imaging is advised if these symptoms persist at the end of the sputum collection.

Bronchoscopy

Bronchoscopy is a procedure used to visualize the throat and airways with a thin, lighting camera. This technique can also be used to collect sputum samples in specific situations, such as persistent infections, coughs, or unusual findings on clinical laboratory tests or chest x-rays. The collected sputum specimen is examined under a microscope to detect the presence of abnormal cells. Flexible bronchoscopy is more commonly used compared to rigid bronchoscopy to collect sputum samples. Before a flexible bronchoscopy, the clinician may administer an anesthetic to relax the throat muscles and numb the mouth, nasal passages, or throat. The procedure uses a thin, lighted bronchoscope inserted through the mouth or nose, down the throat, into the windpipe (trachea), and then to the major bronchi leading to the lungs. Sputum samples can be collected using instruments passed through the bronchoscope by the clinician.

Diagnostic Tests

Clinical diagnostic sputum tests are designed to identify the causes of lower respiratory tract infections and other diseases, making them a valuable tool for assessing the effectiveness of clinical treatments. Sputum culture is the most commonly performed test when a patient is diagnosed with pneumonia, as it helps identify the bacteria or fungi causing the airway or lung infection.

Sputum smear microscopy is the initial step in laboratory sputum analysis. This fast and cost-effective technique is beneficial in resource-limited settings. The Gram stain is the initial staining technique used in preliminary bacterial identification, which helps determine whether there is an adequate number of pathogens in the culture, making it a definitive diagnosis. This technique is also crucial because it can address antibiotic therapy more specifically. The Gram stain categorizes bacterial species into two groups, gram-positive and gram-negative, based on the differences in the physical and chemical properties of their cell walls.

Gram-positive bacteria have a thick peptidoglycan layer in their cell walls, stained with crystal violet. In contrast, gram-negative bacteria have a thinner peptidoglycan layer, stained red or pink with a counterstain.[2] When tuberculosis is suspected, acid-fast bacilli (AFB) stain testing must be performed. Tuberculosis is a lung infection caused by Mycobacterium tuberculosis. Mycobacteria are a group of rod-shaped AFB. These bacteria can be distinguished under the microscope after an AFB staining procedure, where the bacilli retain the stain color after an acid-fast wash. The Grocott-Gomori's methenamine silver stain (GMS) is a standard staining method used to detect fungal microorganisms, especially Pneumocystis jirovecii, which was first identified in patients with HIV infection in the 1980s. This organism, initially classified as a protozoan and known as Pneumocystis carinii, is now recognized as a fungus. Colony morphology is a method that describes the characteristics of an individual bacterial colony growing on agar in a petri dish. This method can help lab technologists identify specific bacterial species.

However, relying solely on microscopic observation and colony morphology may not be sufficient to obtain relevant information about the species and genus of etiologic microorganisms. Biochemical tests of bacterial growth are the next step in recognizing bacteria. The standard biochemical tests used to identify bacterial growth include fluid thioglycollate medium, catalase, and oxidase tests. 

Respiratory viruses can be detected in sputum specimens from patients with cystic fibrosis, asthma, and chronic obstructive pulmonary disease.[3][4][5] Viral testing is typically performed on upper airway samples such as nasopharyngeal swabs or nasal washes. However, some viral pathogens, such as SARS-CoV, H1N1 influenza, Middle Eastern respiratory syndrome coronavirus, and SARS-CoV-2, the causative agent of COVID-19, may be absent in upper airway secretions.[6][7][8][9] Consequently, sputum samples are frequently used for viral diagnosis testing using real-time polymerase chain reaction (RT-PCR) or the newly developed next-generation sequencing method. Potentially, face masks, which reduce the aerosol-related risk of transmission in the current era of the COVID-19 pandemic, may also represent a useful source for next-generation sequencing investigations.[10]

Sputum cytology involves examining sputum samples under a microscope to detect the presence of abnormal cells. A thin layer of sputum is placed on a slide, stained with specific dyes, and analyzed directly under the microscope to identify cellular abnormalities. Sputum cytology is useful for detecting lung cancer cells and non-cancerous cellular and acellular materials. These findings assist in diagnosing conditions such as pneumonia, tuberculosis, interstitial lung diseases, and pneumoconiosis, including asbestosis. Hematoxylin and eosin staining is the most commonly used method in medical laboratory diagnosis and is often considered the gold standard.[11][12] This stain is primarily used for samples suspected of containing lung cancer cells. The periodic acid–Schiff stain is used to detect polysaccharides and mucosubstances in tissue specimens. This stain is primarily used to identify living fungi in sputum specimens.

Wright, Giemsa, and Wright-Giemsa mixture stains are commonly used for sputum smears, aiding in the differentiation of blood cell types through specific staining solutions. These methods are instrumental in detecting abnormal white blood cells in sputum, which are critical indicators of lung infections.

Sputum molecular analysis is a novel and advanced technique used to detect lung cancer–related biomarkers, aiding in the early diagnosis of lung cancer. Many DNA mutations, including p53, KRAS, EML4-ALK, and GFR, have been investigated in sputum specimens.[13][14][15] DNA hypermethylation has also been reported in sputum samples from patients with lung cancer. Loss of heterozygosity and microsatellite instability have been found in sputum specimens of patients with lung cancer using DNA markers.[16] MicroRNAs, such as miR-21 and miR-155, and proteins, such as a proliferation-inducing ligand (APRIL) and complement factor H, were significantly overexpressed. Some messenger RNAs, such as APRIL, MAGE, Telomerase, and CEA, have been found to rapidly degrade in the sputum specimens of patients with lung cancer, as detected by RT-PCR and immunocytochemistry.[17] Other molecular biomarkers, such as free and mitochondrial DNA variants, also appear promising.[17]

Lastly, sputum antimicrobial susceptibility testing is performed on bacteria or fungi identified in sputum cultures to determine effective treatment options. The most common approaches include the disk diffusion and minimum inhibitory concentration methods. These tests are used to assess the effectiveness of specific antibiotics against bacteria or to detect whether the bacteria have already developed resistance to certain antibiotics. The results of antimicrobial susceptibility testing assist in selecting the most appropriate antibiotics in treating lung infections.

Testing Procedures

The following procedures outline the diagnostic techniques used to analyze sputum samples for identifying pathogens, assessing cellular abnormalities, and determining antimicrobial susceptibility. These tests are crucial for diagnosing a variety of respiratory conditions, including infections and lung diseases.

Sputum Culture Procedure

The sputum sample is inoculated onto a culture plate containing a medium that promotes bacterial or fungal growth. The plate is covered and incubated at 37 °C for bacterial growth and 30 °C for fungal growth. The laboratory specialist should monitor bacterial or fungal growth in the sputum plate daily. Once the sputum culture is positive, further tests, such as microscopy, colony morphology assessment, and biochemical testing, are performed to identify the specific bacterium or fungus.

Sputum Staining Test Procedure

A smear of the sputum specimen is prepared on a microscope slide. Various staining dyes are applied to the cells, bacteria, or fungi on the slide, followed by rinsing with water, alcohol, or acid solutions. The slide is then examined under a microscope, and results are considered positive if bacteria, fungi, or specific cell types are identified.

Sputum Biochemical Test Procedure

Bacteria are first inoculated into a series of differential media to identify the suspected organism. Various indicators are then used to observe the specific end products of metabolism inside the medium. 

Sputum Cytology Examination Procedure

The sputum smear slide is stained with different dyes according to the instructions. A pathology specialist examines the stained slide under a microscope to detect the abnormal cells in the sputum specimen.

Sputum Nucleic Acid Amplification Test Procedure

RNA or DNA is extracted from the sputum specimen following the instructions provided by different commercial kits. The DNA or RNA is added to a PCR reaction tube containing designed primers, Taq polymerase, deoxynucleoside triphosphates, and a fluorescent-labeled probe. The tube with the RT-PCR reaction mixture is then placed in an RT-PCR device to amplify the molecules at specific temperatures. 

Sputum Antimicrobial Susceptibility Test Procedure

For the minimum inhibitory concentration method, the bacteria or fungi isolated from sputum specimens were diluted in saline and swabbed onto the minimum inhibitory concentration panels. Different antibiotic concentrations are placed directly onto the bacteria-swabbed agar plates for the dish diffusion method. Panels or plates are incubated at 35 °C for about 16 to 18 hours or longer. The minimum concentration of the antibiotic that inhibits the growth of organisms, known as the minimum inhibitory concentration, is determined according to the guidelines of various manufacturers. The results are then documented and reported.

Interfering Factors

Various factors can interfere with results at each stage of sputum diagnosis. Any deviation from the standard procedure for sample collection, culture, staining, biochemical, molecular, and antimicrobial susceptibility tests can significantly impact the diagnostic result, directly affecting the patient's clinical management. Therefore, strict adherence to laboratory workflow procedures and the involvement of well-trained laboratory technologists are essential for sputum analysis.

Obtaining a high-quality sputum sample is crucial for accurate diagnosis. If the specimen is transparent, laboratory technologists may not be able to detect the pathogens or cells associated with the disease. Moreover, pathogens identified from sputum culture do not always originate from lower respiratory tract infections because they may be part of contaminant sites or preexist in the oral flora. Thus, standard microbiological procedures for isolating and identifying organisms are critical for assessing sputum quality.

Quality assurance is crucial for differentiating actual respiratory pathogens from potential colonizing flora. The quality of commercial laboratory products—such as culture plates, staining kits, and molecular biology kits—must meet the approval standards set by regulatory bodies, including the United States Food and Drug Administration, the Public Health Agency of Canada, and similar agencies in Europe and Australia. Substandard products or expired materials can compromise test performance. Several agencies determine the quality of laboratory work, and the College of American Pathologists plays a significant role in establishing laboratory standards and quality control procedures, which are essential for avoiding various interfering factors.

Results, Reporting, and Critical Findings

Sputum Culture

A sputum culture is considered positive if pathogenic organisms grow after 24 hours of incubation in the culture dish. Some sample dishes may require longer incubation times depending on the microbial flora, the need to identify semiquantitative isolates, and the requirement for antimicrobial susceptibility testing. Conversely, a negative result is determined if no bacteria or fungi grow in 6 to 8 weeks for solid culture media or 6 weeks for liquid culture media. 

Sputum Staining Tests

Gram stain test: This test detects whether the bacteria are gram-positive or gram-negative.

• Common gram-positive bacteria include Staphylococcus, Streptococcus, Bacillus, Listeria, Enterococcus, and Clostridium.
• Common gram-negative bacteria include Escherichia coli, Klebsiella species, Proteus species, and Pseudomonas aeruginosa.

Acid-fast bacilli stain test: This test determines whether the bacteria are AFB-positive or AFB-negative.

• AFB-positive result: AFB, such as Mycobacterium tuberculosis, retains a red or pink color.
• AFB-negative result: No red or pink bacteria are observed on the stained slide.

Grocott-Gomori's methenamine silver stain test: This test determines whether fungal organisms are present, yielding a positive or negative result.

• GMS-positive result: Black or brown walls are observed from fungal organisms or worms such as Pneumocystis jirovecii.
• GMS-negative result: No black or brown stained fungal organisms or worms are observed.

Sputum Biochemical Tests

Several biochemical tests are used to characterize bacteria isolated from sputum samples, with results typically reported as positive or negative.

Motility test: The test is used to determine whether gram-negative enteric bacilli are motile or nonmotile.

• Motile (positive): Organisms spread out from the stab line, producing cloudiness or turbidity throughout the medium.
• Nonmotile (negative): Organisms remain along the stab line of inoculation.

McFarland standard test: This test is used to standardize the concentration of bacteria in liquid suspensions by comparing their turbidity to that in the standard McFarland solution vial or tube. The results of the test are obtained by measuring the turbidity of a bacterial suspension against various concentrations of McFarland standard solutions.

Fluid thioglycollate medium test: The test is used to detect the aerotolerance of bacteria. 

• Obligate aerobes, such as Pseudomonas species, require oxygen for growth and only grow toward the oxygen-rich surface layer.
• Obligate anaerobes cannot grow in the presence of oxygen and only grow at the bottom of the tube.
• Microaerophiles frequently grow below the oxygen-rich layer.
• Gram-negative, facultative, or aerotolerant anaerobes can generally grow throughout the broth, but mostly grow between the oxygen-rich and oxygen-free areas.

Catalase test: This test is used to differentiate staphylococci from streptococci by detecting the presence of catalase.

• Catalase-positive: The organisms can produce catalase, which generates oxygen bubbles after adding 3% hydrogen peroxide.
• Catalase-negative: The organisms cannot produce catalase, and there is no reaction after adding 3% hydrogen peroxide.

Oxidase test: This test is used to detect the presence of cytochrome c oxidases.

• Oxidase-positive: There is a deep purple-blue or blue color change within 10 to 30 seconds.
• Oxidase-negative: No color change is observed.

Sputum Nucleic Acid Amplification Test

The result is positive if the RT-PCR amplification is successful. However, if it is not successful, the result is negative.

Sputum Cytology Examination

If a sputum sample shows a few white blood cells and no abnormal cells, the sputum cytology examination is considered normal, and other factors may be responsible for the patient's symptoms.

Sputum Antimicrobial Susceptibility Test

If antibiotics inhibit the growth of the organism, they effectively treat the infection, indicating that the organism is susceptible. Conversely, if the antibiotic does not inhibit the growth of the organism, it is not adequate for the patient's treatment, indicating resistance.

Clinical Significance

Sputum analysis plays a critical role in identifying the causes of various airway and lung diseases. An accurate diagnosis is directly related to the clinician's treatment strategy. In cases of infectious diseases, a lab technician first attempts to identify the pathogens and then tests various drugs that can be used for treatment. For suspected lung cancers and certain non-cancerous pulmonary conditions, sputum specimens are examined for the presence of abnormal cells.

Quality Control and Lab Safety

Quality control in sputum analysis is essential to ensure accurate diagnostic results. Every step—from specimen collection, transport, and storage to test procedure environments and result reporting—should strictly follow standardized guidelines. In 1988, the United States Congress passed the Clinical Laboratory Improvement Amendments. The Clinical and Laboratory Standards Institute was established to minimize analysis errors and maximize the control of test variables. Similar regulations are present in other countries. A standard positive, sensitivity, and negative control should always be included during diagnostic tests when a laboratory technician performs sputum sample tests. No diagnostic results should be reported when quality control measurements are not authorized. 

Laboratory staff must adhere to strict safety protocols. Depending on specific diagnostic tests, the procedural risk assessment results may differ. Sputum samples should always be considered potentially infectious, and essential biosafety measures should be set to limit or reduce the risks of laboratory infection when specific procedures are performed. The Clinical and Laboratory Standards Institute has issued updated practice guidelines for applying risk management to quality control plans and statistical quality control.[18][19]

Enhancing Healthcare Team Outcomes

Sputum analysis plays a crucial role in diagnosing various conditions, including respiratory infections, lung cancers, and non-cancerous lung diseases. Pneumonia is the most common lung infection and can spread easily. Untreated pneumonia may lead to severe complications. Lung cancer is ranked the fourth most commonly diagnosed malignancy in developed countries and the first in cancer-caused death worldwide.[20] Despite the high incidence and mortality of pneumonia and lung cancer, the treatments are limited.[21][22][23] Therefore, an interprofessional approach is essential to ensure patients receive the most appropriate evidence-based diagnosis, treatment, and healthcare support. This team includes a laboratory diagnostic specialist, a radiologist, a pulmonologist, an infectious disease expert, a pharmacist, and a public health nurse.

The pulmonologist or infectious disease specialist orders laboratory tests based on the patient's symptoms. A nurse or a healthcare professional collects the sputum sample and sends it to the clinical lab. The laboratory specialist conducts the diagnostic tests for the sputum specimen and reports the results. The radiologist may also need to carry out a chest x-ray for the patient. When the clinician receives the sputum diagnostic report, they may prescribe the drugs for the patient's treatment. Then, the nurse performs the clinical therapy.

Once the patient is discharged from the hospital, the nurse should educate the patient and family members on maintaining good health. The pharmacist should monitor the outpatient therapy and ensure the patient follows the treatment. When a patient has an allergy to a particular drug or difficulty adhering to the treatment, the pharmacist should report the situation to the interprofessional team. A social worker may also provide financial and emotional support for the patient. Regular follow-up testing in the clinical lab and imaging studies are essential to monitor progress and adjust treatment as needed.

Outcomes

The multidisciplinary team approach facilitates the delivery of high-quality healthcare services to patients. Previous evidence suggests that patients are more satisfied with the traditional care model and experience improved quality of life if they receive multidisciplinary care.[24][25]