Introduction

Mucus is the fluid secreted by the airways (also known as bronchial and windpipes) and lungs. In the setting of an infection or a longstanding health condition, the term phlegm is also used. The mixture of saliva and mucus specifically coughed up from the respiratory tract, often either following an infection or an irritation of the mucosa, is precisely labeled "sputum." The term phlegm arises from the Greek word "φλ?γμ?" which in the ancient books of medicine was used to label humor caused by heat.

The sputum is examined grossly and microscopically to aid medical diagnosis. The sputum contains various cells and molecular compounds such as soluble lipids and proteins. Its analysis is crucial in medicine. The sputum analysis involves an analytical approach to investigate the cellular and acellular components expelled from the patient's upper respiratory tract. This procedure is essential in the evaluation and management of lower respiratory infections or other longstanding health conditions.[1] Clinically, sputum molecular biomarkers or gene sequencing of the microorganisms have increased medicine accuracy and represent a milestone in the current evaluations of the algorithms running for precision medicine.

Specimen Requirements and Procedure

The procedure of sputum specimen collection is usually non-invasive. In medicine, it is comparatively simple. However, in some clinical settings, the approach may be more vigorous due to the inability of the patient to expel such fluid from the upper respiratory tract. Thus, some maneuvers of physiotherapy may be considered adjuvant for getting some material for the analysis. Commonly, the "deep cough" sample of the early morning is collected before eating or drinking anything to avoid bias in interpreting the results. At first, the patients need to rinse out the mouth with clear water for 10-15 seconds to eliminate any contaminants in the oral cavity. After expelling saliva, the patients then breathe in deeply three times to cough at 2-minutes intervals until bringing up some sputum. The sputum is then released in a sterile well-closed container provided by the medical professionals to the patient.

The medical professionals will check the amount and gross qualities of the sputum, which should be thick to allow a proper investigation by the laboratory medical staff. In several institutions, clear and runny samples are not acceptable for further microscopic or microbiological studies. In some settings, the procedure can be repeated until 10 to 20 mL of sputum sample has been collected. It is paramount that the lid of the container where the first fluid is collected is changed to avoid contaminations. If the patient has difficulties coughing up enough sputum, the medical professionals may apply some physiotherapeutic maneuvers, which allow the progressive release of the sputum. Routine sputum culture requires that one 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 (TB), three sputum samples must be collected on three consecutive days and be returned to the clinical lab each day.

Sputum induction is a procedure used to collect adequate lower respiratory secretions from patients who have trouble producing sputum to aid the diagnosis of TB. In particular, patients with suspicion of miliary tuberculosis and/or tuberculous pleural effusion are often targeted using this adjuvant procedure. In such settings, the patient inhales nebulized hypertonic saline solution to liquefy airway secretions. This solution stimulates the patient's coughing and promotes the expectoration of airway secretions. The medical professionals prepare a 20 ml 3% hypertonic saline solution and inject it into the nebulizer cup filled with water. Similar to the non-adjuvant procedure, the patients are always required to wash their mouths thoroughly.

Moreover, the patients wear the nebulizer cup to cover the face and nose after sitting in an upright position. The patients inhale and exhale through the mouthpiece. An expectorate saliva into an emesis bowl and expectorate sputum coughed up are collected into a sterile well-closed container. The medical staff turns on the nebulizer device to allow the patient to inhale the hypertonic mist for approximately five minutes. Then the patients take several deep breaths before attempting to cough. If there are difficulties for the patients to cough up the sputum, the medical staff may use gentle chest physiotherapy to aid the patients to produce sputum. During the procedure, the patients should be observed closely by the medical staff to identify any potential rupture of pleural bullae, triggering a life-threatening pneumothorax. The patients should stop when 1 to 2 mL sputum specimen is collected for each sample or reach 15 minutes of nebulization, or the patient complains of chest tightness, dyspnoea, or wheeze. Imaging is advised if there is the persistence of these symptoms at the end of the sputum collection.

Bronchoscopy is a procedure used to investigate the throat and airway through a thin viewing camera. It is also used to collect the sputum samples in some special situations such as a persistent infection, cough, or something unusual seen on clinical laboratory tests or chest X-rays. The sputum specimen will be examined under a microscope to detect whether abnormal cells are present. Flexible bronchoscopy is used more often than rigid bronchoscopy to collect the sputum samples. Before having a flexible bronchoscopy, the doctor may give the patient anesthetic to relax the throat muscles and numb the mouth, nasal passages, or throat. The procedure is performed using 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 may be taken using the devices passed through the bronchoscope by the doctor.

Diagnostic Tests

Clinical diagnostic sputum tests aim to detect the causes of lower respiratory tract infections and some other diseases. It also provides an efficacious tool for monitoring the effectiveness of clinical treatment. Sputum culture is the most common test needed to be performed when the patient has pneumonia. It is used to identify the bacteria or fungi causing the airways or lung infection.

Sputum smear microscopy is the initial step taken in laboratory sputum analysis. It is a fast and inexpensive technique, precisely, in resource-limited settings. Gram stain is used to differentiate bacteria into two broad groups (gram-positive and gram-negative microorganisms). The Gram stain is the first staining technique performed in preliminary bacterial identification, which helps determine if there is an adequate amount of pathogens in the culture and make a definitive diagnosis. It is also crucial because it can address antibiotic therapy more specifically. With the Gram stain, the bacterial species are distinguished into gram-positive and gram-negative groups by the differences in cell walls' physical and chemical properties. Some bacteria have a thick peptidoglycan layer cell wall stained with crystal violet (gram-positive).

In contrast, some other bacteria have a thinner peptidoglycan layer stained red or pink by counterstain (gram-negative).[2] When the physician suspects that the patient may have TB, acid-fast bacilli (AFB), stain testing must be performed. TB is a lung infection disease caused by Mycobacterium tuberculosis. Mycobacteria are a group of rod-shaped acid-fast bacilli. They 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. GMS staining is critical in identifying Pneumocystis jirovecii. This microorganism first appeared in patients with human immunodeficiency virus (HIV) infection in the 1980s, and it was used to be classified as a protozoan. This microorganism, which is 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. It can help the lab technologist to identify some specific bacteria. 

However, only relying on microscopic observation and colony morphology maybe not be enough to get the relevant information of the species and genus of etiologic microorganisms. Biochemical tests of bacterial growth are the next step to perform to recognize the bacteria. The common biochemical tests used to identify bacterial growth include motility, McFarland standard, fluid thioglycollate medium (FTM), catalase, and oxidase tests. 

Respiratory viruses have been tested in sputum specimens from patients with cystic fibrosis, asthma, and chronic obstructive pulmonary disease (COPD).[3][4][5] Typically, viral testing is also 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 Coronavirus Virus disease 2019 (COVID-19), may be absent in upper airway secretions.[6][7][8][9] So the sputum samples are also frequently used for viral diagnosis testing by using the clinical real-time polymerase chain reaction (RT-PCR) method 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 is using a microscope to determine whether abnormal cells are present in sputum samples. The thin layer of sputum placed on a slide before specific staining and diagnosed directly under the microscope helps find out some abnormal cells. Sputum cytology helps detect both lung cancer cells and non-cancer cellular and acellular material useful for the diagnosis of conditions such as pneumonia, tuberculosis, interstitial lung diseases, or pneumoconiosis (e.g., asbestosis). Hematoxylin and eosin stain is the worldwide most performed tissue stain in medical laboratory diagnosis. It is often considered the gold standard.[11][12]. It is mostly used for suspected lung cancer samples. Periodic acid–Schiff (PAS) stain is used to detect polysaccharides and mucosubstances in tissue specimens. It is mainly helpful for the detection of living fungi in sputum specimens.

Further, Wright stain, Giemsa stain, and Wright-Giemsa mixture stain are used for staining the sputum smears. These stain methods facilitate the differentiation of blood cell types by using specific solutions. These staining methods help detect abnormal white blood cells of sputum, which are vital signs of lung infection.

Sputum molecular analysis is a new insight and advanced technique used to detect lung cancer-related biomarkers to assist in the early stage of a lung cancer diagnosis. Many DNA mutations, such as p53, KRAS, EML4-ALK, and GFR mutations, have been investigated on sputum specimens.[13][14][15] DNA hypermethylation has also been reported in lung cancer sputum samples. Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been found in lung cancer patients' sputum specimens by using DNA markers.[16] MicroRNAs (miRNAs) such as  miR-21 and miR-155, proteins such as proliferation-inducing ligand (APRIL) and complement factor H were significantly overexpressed, and some messenger RNAs (mRNAs) such as APRIL, MAGE, Telomerase, CEA, et al., have been found rapidly degraded in sputum specimens of lung cancer patients by RT-PCR and immunocytochemistry.[17] Still, other molecular biomarkers such as free DNA and mitochondrial DNA (mtDNA) variants seem to exhibit some promise.[17]

Finally, sputum antimicrobial susceptibility testing is performed on the bacteria or fungi, leading to lung infection after being identified in a sputum culture sample. The most common approaches consist of 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 or not. The results of antimicrobial susceptibility testing help to select the most likely effective antibiotics in treating lung infection.

Testing Procedures

Sputum Culture Procedure

The sputum sample is added to a culture plate with a specific substance that promotes the growth of bacteria or fungi. Then cover the lid of the dish and place it in a 37 degree C incubator for bacteria and 30 degrees C for fungus. The lab specialist should check the bacteria or fungi growth in the sputum plate every day. Once the sputum culture is positive, microscopy, colony morphology, or biochemical tests of bacterial growth will be performed to identify the specific type of bacterium or fungus.

Sputum Staining Tests Procedure

The sputum specimen is a smear on a microscope slide. Different staining dyes are added to the cells, bacteria, or fungi of the sample on the slides and then washed with water, alcohol, or acid solutions. The slides are then diagnosed under a microscope. If the bacteria, fungi, or specific cells are identified in the specimens, the results are positive.

Sputum Biochemical Tests Procedure

To identify a suspected organism, at first, the bacteria will be inoculated in a series of differential media. Then use different indicators to observe the specific end products of metabolism inside of the medium. 

Sputum Cytology Examination Procedure

The smear sputum slide is stained with different dyes according to the instructions. Then the pathology specialist examines the stained slide under the microscope to find the abnormal cells from the sputum specimen. 

Sputum Nucleic Acid Amplification Test Procedure

The RNA or DNA is extracted from the sputum specimen according to the instruction of different commercial kits. The DNA or RNA is added to a PCR reaction tube with designed primers, Taq polymerase, deoxynucleoside triphosphates [dNTPs], and a fluorescent-labeled probe. Then the tube with RT-PCR reaction mixture is placed in a real-time PCR device for amplifying the molecules at specific temperatures. 

Sputum Antimicrobial Susceptibility Tests Procedure

For the MIC method, the bacteria or fungi isolated from sputum specimens were diluted in saline and swabbed onto the MIC panels. For the dish diffusion method, selected different concentrations of antibiotics are placed directly onto the bacteria swabbed agar plates. Panels or plates are incubated at 35 degrees C for about 16 to 18 hours or longer. The minimal concentration of the antibiotic that inhibits the growth of organisms or MIC panel is read according to the guidelines of different manufacturers. Then the result is reported. 

Interfering Factors

Many interfering factors affect the results of every step of the 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.

Collecting a good quality sputum sample is the first step for getting an accurate diagnosis. If the specimen is transparent, the laboratory technologists may not find the pathogens or cells related to the disease. Moreover, the pathogens identified from sputum culture do not always originate from lower respiratory tract infections because they may be part of contaminant sites or preexisting in the oral flora. Thus, standard microbiological procedures for organisms' isolation and identification are critical for the sputum quality assessment (QA).

QA remains an essential tool in the lab for distinguishing the real respiratory pathogens from the possible colonizing flora. Finally, it is vital to check the quality of commercial products that need to be approved by the United States Food and Drug Administration, the Public Health Agency of Canada, and European and Australian similar agencies. Inferior quality culture plates, expired staining kits, or ineffective molecular biology kits are directly related to poor performance. Several agencies determine the quality performed in a laboratory, and the College of the American Pathologists plays a major in dictating laboratory standards and quality control procedures, which are essential to avoid different interfering factors.

Results, Reporting, and Critical Findings

Sputum Culture

If the pathogenicity organisms grow after 24 hours of incubation in the culture dish, the result is positive. Some sample dishes will keep incubating longer, depending on microbial flora present and the need to identify and semiquantitative isolates and perform antimicrobial susceptibility tests. Conversely, if no bacteria or fungi grow in 6 to 8 weeks for solid culture media or six weeks for liquid culture media, the result is negative. 

Sputum Staining Tests

•  Gram stain test: The bacteria detected by the test will be Gram-positive or Gram-negative.
  • Common Gram-positive bacteria include Staphylococcus, Streptococcus, Bacillus, Listeria, Enterococcus, and Clostridium.
  • Common Gram-negative bacteria include E. coli, Klebsiella species, Proteus species, and Pseudomonas aeruginosa.
•  AFB stain test: The bacteria detected by the test will be positive or negative.
  • AFB stain test positive result: the acid-fast bacilli, such as Mycobacterium tuberculosis, retain the red or pink color.
  • AFB stain test negative result: no red or pink bacteria are found in the stained slide.
•  GMS stain test: The fungal organisms detected by the test will be positive or negative.
  • GMS test positive result: black or brown wall from fungal organisms or worms such as Pneumocystis jirovecii are found.
  • GMS test negative result: no black or brown stained fungal organisms or worms are found.

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

•  Organisms motility test: the test is performed in Gram-negative enteric bacilli, and the result will be either motile or nonmotile.
  • Motile (positive): organisms will spread out from the stab line and produce cloudiness or turbidity throughout the medium.
  • Nonmotile (negative):  organisms will remain along the stab line of inoculation.
•  McFarland standard test: The test is used to standardize the number of bacteria in liquid suspensions by the turbidity of bacteria in the McFarland standard vial or tube.        The test result arises by comparing the turbidity of a bacterial suspension to different concentrations of McFarland standard solutions.
•  FTM test: the test is used to detect the aerotolerance of bacteria. 
  • Obligate aerobes, such as Pseudomonas spp., requiring oxygen for growth, will only grow toward the oxygen-rich surface layer.
  • Obligate anaerobes, which cannot grow with oxygen, will only grow on the bottom of the tube. 
  • Microaerophiles frequently grow below the oxygen-rich layer.
  • Gram-negative, facultative or aerotolerant anaerobes generally can grow throughout the broth but will mostly grow between the oxygen-rich and oxygen-free area.
• 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 will generate 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 purple-blue color or no color change.

Sputum Nucleic Acid Amplification Test 

If the RT-PCR amplification is successful, the result is positive. 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, that means the sputum cytology examination is regular, and other reasons may cause the patient's symptoms.

Sputum Antimicrobial Susceptibility Test

If antibiotics inhibit the growth of an organism, that means the antibiotics are working to treat the patient infected by the organism, and the antibiotics are susceptible. Conversely, if the antibiotic does not inhibit the organism's growth, it means the antibiotics are not adequate for the patient's treatment, and the antibiotics are resistant.

Clinical Significance

The analysis of sputum is essential for discovering the reasons leading to different airway and lung diseases. The accurate diagnosis is directly related to the strategy that the physician uses to treat the patients. For infectious diseases, a lab technician first tries to find out the pathogens, and then they test different drugs that can be used for the treatment. For the suspected lung cancers and certain non-cancerous lung conditions, the lab will inspect whether abnormal cells exist in the sputum specimens or not.

Quality Control and Lab Safety

Sputum analysis quality control is essential and directly affects the diagnostic results. From sputum specimens collection, samples transport, samples storage, diagnostic test procedures environment, to tests results reporting, every step should be strictly performed under the standard guidelines. In 1988, the Clinical Laboratory Improvement Amendments (CLIA) was passed by the Congress of the United States of America. The Clinical and Laboratory Standards Institute (CLSI) was established to minimize the analysis errors and maximize the control of test variables. Similar bills are present for other countries. During the diagnostic tests, a standard positive, sensitivity, and negative control should always be performed when the laboratory technician carries out the sputum samples tests. No diagnostic results should be reported when measurements of the quality control are not authorized. 

Strict lab safety rules and measures should be conducted by the staff working in the lab. Depending on specific diagnostic tests, the results of the procedural risk assessment may be different. It is always good to consider the sputum samples as potentially infectious, and essential biosafety measures should be set to limit or reduce the risks of laboratory infection when specific procedures are performed. The CLSI has provided new practice guidelines for applying risk management to both QC plans and statistical QC.[18][19]

Enhancing Healthcare Team Outcomes

Sputum analysis's diagnostic results involve many different diseases, such as respiratory infections, lung cancers, and non-cancerous lung conditions. Pneumonia is the most common lung infection disease and may relate to potential spreading. 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]. Thus, requiring an interprofessional approach is essential for the patient to receive the most appropriate evidence-based laboratory diagnosis decisions, clinical treatment, and healthcare support. This interdisciplinary team approach includes a laboratory diagnostic specialist, a radiologist, a pulmonologist, an infectious disease expert, a pharmacist, and a public health nurse.

According to the symptoms of the patient, the pulmonologist or infectious disease expert orders the laboratory tests. A nurse or a healthcare supporter will then collect the sputum sample and send it to the clinical lab. The laboratory specialist will conduct the diagnostic tests for the sputum specimen and report the results. The radiologist may also need to carry out a chest X-ray for the patient. When the physician receives the sputum diagnostic reportings, they may prescript the drugs for the patient's treatment. Then the nurse will perform the clinical therapy.

Once the patient is discharged from the hospital, the nurse should educate the patient and the family members on how to take care of good health. After that, a pharmacist will observe the outpatient therapy and make sure the patient follows the treatment. When the patient has an allergy to some drugs or has difficulties in obeying the treatment, the pharmacist should report the situation to the interprofessional team. Besides, a social worker is needed to support the outpatient spiritually and financially. To ensure the treatment at home is working for the patient, regular following up serial monitoring in the clinical lab and imaging studies by the radiologist are essential. 

Outcomes

The multidisciplinary team approach facilitates the delivery of 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]