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, some physiotherapy maneuvers may be considered adjuvant to get some material for the 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 the medical professionals.

The 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, the medical professionals may use some physiotherapeutic maneuvers, which allow the progressive release of the sputum. For routine sputum cultures, a single sample is collected and sent to the lab on the same day of collection. If the patient leaves the specimen in the refrigerator after collection, there is often a tolerance range, which may run well over 24 hours. In tuberculosis, 3 sputum samples must be collected on 3 consecutive days and returned to the clinical lab 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. The 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, the patients must always wash their mouths thoroughly.

Moreover, after sitting upright, the patients wear the nebulizer cup to cover the face and nose. The patients inhale and exhale through the mouthpiece. An expectorate saliva is placed into an emesis bowl, and expectorate sputum coughed up is collected into a sterile, sealed container. The medical staff turns on the nebulizer device to allow the patient to inhale the hypertonic mist for approximately 5 minutes. Then, the patients take several deep breaths before attempting to cough. If patients have difficulty producing sputum, the medical staff may use gentle chest physiotherapy to aid the patients 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 used to visualize the throat and airway through a thin lighting camera. This technique can also be used to collect sputum samples in special situations, such as a persistent infection, cough, or unusual findings in clinical laboratory tests or chest X-rays. The sputum specimen is examined under a microscope to detect the presence of abnormal cells. Flexible bronchoscopy is used more often compared to rigid bronchoscopy to collect the sputum samples. Before undergoing 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 and lighted bronchoscope inserted through the mouth or nose, down to 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 aim to detect the causes of lower respiratory tract infections and other diseases, serving as a valuable tool for monitoring the effectiveness of clinical treatment. Sputum culture is the most common test performed when the patient has 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 performed in preliminary bacterial identification, which helps determine whether there is an adequate amount 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 distinguishes the bacterial species into gram-positive and gram-negative groups based on the differences in their cell walls' physical and chemical properties.

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 human HIV infection in the 1980s, and it was used to be classified as a protozoan. This microorganism, now classified as a fungus, was initially called Pneumocystis carinii. Colony morphology is a method that describes the characteristics of an individual colony of bacteria growing on agar in a Petri dish. This method can help the lab technologist to identify some specific bacteria. 

However, relying only on microscopic observation and colony morphology may not be enough to get the 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 severe acute respiratory syndrome (SARS) coronavirus, H1N1 influenza, Middle Eastern respiratory syndrome coronavirus (MERS-CoV), and SARS coronavirus 2 (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 (NGS) 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 NGS investigations.[10]

Sputum cytology examination uses a microscope to determine the presence of abnormal cells in sputum samples. The thin layer of sputum placed on a slide before specific staining and diagnosed directly under the microscope helps find some abnormal cells. Sputum cytology helps detect lung cancer cells and non-cancer cellular and acellular materials useful for diagnosing conditions such as pneumonia, tuberculosis, interstitial lung diseases, or pneumoconiosis, for example, asbestosis. The hematoxylin and eosin stain is the most commonly performed tissue stain in medical laboratory diagnosis and is often considered the gold standard.[11][12] This stain is primarily used for samples suspected of lung cancer. The periodic acid–Schiff stain is used to detect polysaccharides and mucosubstances in tissue specimens. This stain is mainly helpful for detecting living fungi in sputum specimens.

Wright, Giemsa, and Wright-Giemsa mixture stains are commonly used for sputum smears, aiding in differentiating 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 new insight and advanced technique used to detect lung cancer–related biomarkers, assisting in early-stage lung cancer diagnosis. Many DNA mutations, such as p53, KRAS, EML4-ALK, and GFR, have been investigated on sputum specimens.[13][14][15] DNA hypermethylation has also been reported in lung cancer sputum samples. Loss of heterozygosity and microsatellite instability have been found in sputum specimens of lung cancer patients using DNA markers.[16] MicroRNAs, such as miR-21 and miR-155, and proteins, such as 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 lung cancer patients, as detected by RT-PCR and immunocytochemistry.[17] Still, other molecular biomarkers, such as free and mitochondrial DNA variants, appear promising.[17]

Finally, sputum antimicrobial susceptibility testing is performed on the bacteria or fungi identified in sputum cultures to determine effective treatment options. The most common approaches include the disk diffusion and minimum inhibitory concentration (MIC) methods. These tests are used to detect the effectiveness of the specific antibiotics on the bacteria or to detect whether the bacteria have already developed resistance to certain antibiotics. Antimicrobial susceptibility testing results help select the most likely effective antibiotics in treating lung infections.

Testing Procedures

Sputum Culture Procedure

The sputum sample is added to 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 lab specialist should check 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 in a series of differential media to identify a suspected organism. Then, different indicators are used to observe the specific end products of metabolism inside the medium. 

Sputum Cytology Examination Procedure

The smear sputum 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 from 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 with designed primers, Taq polymerase, deoxynucleoside triphosphates, and a fluorescent-labeled probe. The tube with the RT-PCR reaction mixture is then placed in a real-time PCR device to amplify the molecules at specific temperatures. 

Sputum Antimicrobial Susceptibility Test Procedure

For the MIC method, the bacteria or fungi isolated from sputum specimens were diluted in saline and swabbed onto the MIC 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 minimal concentration of the antibiotic that inhibits the growth of organisms, known as the MIC, is read according to the guidelines of various manufacturers. The results are then documented and reported.

Interfering Factors

Various factors can interfere with the results at each stage of sputum diagnosis. Any deviation from the standard procedure of 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 laboratory workflow procedures and well-trained laboratory technologists are required to perform the sputum analysis.

Obtaining a high-quality sputum sample is crucial for accurate diagnosis. If the specimen is transparent, laboratory technologists may not find the pathogens or cells related to 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 organism isolation and identification are critical for the sputum quality assessment.

Quality assurance is essential for distinguishing the actual respiratory pathogens from the possible colonizing flora. The quality of commercial laboratory products—such as culture plates, staining kits, and molecular biology kits—must meet approval standards from regulatory bodies such as the United States Food and Drug Administration (FDA), 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 dictating laboratory standards and quality control procedures, which are essential to avoid different interfering factors.

Results, Reporting, and Critical Findings

Sputum Culture

The result is considered positive if the pathogenicity organisms grow after 24 hours of incubation in the culture dish. Some sample dishes may require longer incubation times based on microbial flora, the need to identify semiquantitative isolates, and to perform antimicrobial susceptibility tests. 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, retain 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 test positive result: Black or brown walls are observed from fungal organisms or worms such as Pneumocystis jirovecii.
• GMS test negative result: No black or brown stained fungal organisms or worms are observed.

Sputum Biochemical Tests: The motility, McFarland standard, catalase, and oxidase tests are positive or negative.

Motility test: The test is used for Gram-negative enteric bacilli to determine whether organisms 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 bacterial concentration in liquid suspensions by comparing the turbidity of bacteria in the McFarland standard vial or tube. The test results are obtained by comparing the turbidity of a bacterial suspension with different 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: The 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: The 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 few white blood cells and no abnormal cells have been found in the sputum sample, the sputum cytology examination is regular, and other reasons may cause the patient's symptoms.

Sputum antimicrobial susceptibility test: If antibiotics inhibit the organism's growth, they effectively treat the infection, indicating susceptibility. Conversely, if the antibiotic does not inhibit the organism's growth, the antibiotics are not adequate for the patient's treatment and are resistant.

Clinical Significance

Sputum analysis is essential for discovering the causes of different airway and lung diseases. An accurate diagnosis is directly related to the clinician's treatment strategy. For infectious diseases, a lab technician first tries to find the pathogens and then tests different drugs that can be used for treatment. The lab inspects whether abnormal cells exist in the sputum specimens for suspected lung cancers and certain non-cancerous lung conditions.

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 the 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 performed during the diagnostic tests when the laboratory technician carries out the 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 provided new 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 few.[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 expert orders the laboratory tests according to the patient's symptoms. A nurse or a healthcare supporter 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 reportings, they may prescript 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 the patient has an allergy to some drugs or difficulties obeying 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 delivering high-quality healthcare services to the patient. Previous evidence suggested that patients are more satisfied with the traditional care model and have improved life quality if they receive multidisciplinary care.[24][25]