This work was supported by the University Hospital of Liege (CHU Liege). We acknowledge "Instants productions" for the video production. We also acknowledge Cedric Fran&#231;ois, the patient who appeared in the film.

All methods described in this section have been approved by the Ethics Committee of the University Hospital of Liege and all healthy subjects gave written informed consent for participation.
1.Sputum Induction
<ol>
	<li>Pre- and post-bronchodilator spirometry
	<ol>
		<li>Perform a spirometry (forced expiratory volume in 1 second [FEV1] and forced vital capacity [FVC] maneuver) according to the American Thoracic Society (ATS)/European Respiratory Society (ERS) standard criteria12.</li>
		<li>Administer 400 &#181;g of inhaled salbutamol from a metered dose inhaler (MDI) through a spacer device. 
		NOTE: In adults, adverse events of inhaled salbutamol include tremor and tachycardia13.</li>
		<li>Repeat spirometry (FEV1 and FVC maneuver) 15 min later, according to the ATS/ERS standard criteria12.</li>
	</ol>
	</li>
	<li>Preparation of the ultrasonic nebulizer 
	NOTE: The nebulizer must be thoroughly cleaned and disinfected before each use, in accordance with the manufacturer&#39;s instructions.
	<ol>
		<li>Fill the nebulizer chamber with distilled water up to the recommended level.</li>
		<li>Place a clean cup in the nebulizer chamber.</li>
		<li>Cover the cup with the appropriate lid.</li>
		<li>Fill the cup with either 50 mL of hypertonic saline (5%) for a patient with post-bronchodilator FEV1 &#62;65% predicted or 50 mL of isotonic saline (0.9%) for a patient with post-bronchodilator FEV1&#8804;65% predicted.</li>
		<li>Add 1.75 mL of salbutamol sulfate solution (5 mg/mL) to the cup.</li>
		<li>Connect the tubes and valves in accordance with the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Nebulization and sputum collection 
	NOTE: Perform the technique under medical supervision.
	<ol>
		<li>Explain the procedure to the patient.</li>
		<li>Ask the patient to use a nose clip.</li>
		<li>Turn the nebulizer on and select the lowest level of aerosol and fan settings.</li>
		<li>Ask the patient to inhale the aerosol through the mouth piece with tidal breathing for 5 min. 
		NOTE: Aerosol and fan settings may be increased depending on the patient&#39;s tolerance.
		<ol>
			<li>In case of unbearable cough or nausea, discontinue the procedure and go to step 1.3.5.</li>
			<li>In the event of chest tightness or respiratory discomfort, go to step 1.3.8.</li>
		</ol>
		</li>
		<li>Turn the nebulizer off.</li>
		<li>Ask the patient to remove the nose clip, rinse his mouth with water, and gargle twice before discarding it in the sink. 
		NOTE: Saliva cells may contaminate the sputum sample and lead to an unusable sample.</li>
		<li>Ask the patient to cough up sputum into a plastic container.</li>
		<li>Perform a spirometry (forced expiratory maneuver) to measure FEV1.</li>
		<li>Evaluate the FEV1 fall as follows: (FEV1 measured at step 3.8 [mL]) - (post-bronchodilator FEV1 measured at step 1.3 [mL]) / (post-bronchodilator FEV1 measured at step 1.3 [mL])*100.
		<ol>
			<li>If the FEV1 falls by 20% or more from the post-bronchodilator value, discontinue the procedure. Measure FEV1 again 10 min later. If the FEV1 falls 20% or more, ask the physician to administer nebulized ipratropium bromide (0.25 mg/2 mL) and keep the patient under medical observation. Go to step 1.3.10.</li>
			<li>If the FEV1 does not fall by 20% or more from the post-bronchodilator value, repeat steps 3.2 to 3.9 one to three times to reach a total nebulization time of 10 to 20 min. 
			NOTE: The total nebulization time will depend on quality (presence of plugs or viscous parts) and quantity of the sputum sample. If the sample quality and/or quantity is poor after 10 min of nebulization, the procedure should be extended to reach a total nebulization time of 15 to 20 min.</li>
		</ol>
		</li>
		<li>Keep the sputum sample refrigerated until processing.</li>
	</ol>
	</li>
</ol>
2. Sputum Processing
NOTE: Process the sputum within 3 hours of sampling for optimum cell viability.
Caution: Wear a lab coat, protective gloves, and goggles.
<ol>
	<li>Preliminary Preparations
	<ol>
		<li>Make a 10-fold dilution of the concentrated dithiothreitol solution (mucolytic agent) with sterile distilled water to obtain 10 mL of a 6.5 mM solution, in accordance with the manufacturer&#39;s instructions. 
		NOTE: In order to prevent concentrated dithiothreitol solution mixing with ambient air, withdraw the solution from the vial with a sterile syringe and needle. Once opened, the concentrated solution vial must be kept at room temperature and used within 5 days. The diluted solution is prepared daily. 
		CAUTION: Because of eye and skin irritation hazard, wear protective equipment (clothing, gloves, and goggles).</li>
		<li>Make a 5-fold dilution of the trypan blue solution (0.4%) with Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) to obtain a 0.08% solution. Keep this diluted solution at room temperature and use within 2 weeks. 
		CAUTION: Because of carcinogenic hazards, wear protective equipment (clothing, gloves and goggles).</li>
	</ol>
	</li>
	<li>Collection of Sputum Supernatant
	<ol>
		<li>Transfer the whole sputum in a 50 mL conical bottom plastic tube and weigh the sample.</li>
		<li>Add a 3-fold weight of DPBS solution.</li>
		<li>Slowly vortex the sample for 30 s.</li>
		<li>Centrifuge at 800 x g and 4 &#176;C for 10 min.</li>
		<li>Filter the sample through 2 single layers of sterile gauze and collect the supernatant in a 50 mL conical bottom plastic tube.</li>
		<li>Aliquot the supernatant in 2 mL plastic tubes and store them at -80 &#176;C. 
		NOTE: Supernatant samples are useful to assay sputum fluid phase components.</li>
	</ol>
	</li>
	<li>Sputum Mucolysis
	<ol>
		<li>If necessary, add DPBS to the cell pellet to reach a total volume of cell suspension of 5 mL.</li>
		<li>Dilute the cell suspension with 1 volume of 6.5 mM dithiothreitol. Use a bench rocker to rock the cell suspension for 20 min at room temperature.
		<ol>
			<li>Repeat at least 3 times with DPBS.</li>
		</ol>
		</li>
		<li>Centrifuge the diluted cell suspension at 550 x g and 4 &#176;C for 10 min. Discard the supernatant.</li>
		<li>Resuspend the cell pellet in around 1 mL of DPBS.</li>
	</ol>
	</li>
	<li>Assessment of qualitative properties of the sputum sample (cell concentration, squamous cell contamination, and viability by Trypan Blue exclusion method) by hemocytometer count
	<ol>
		<li>Assess and record the exact volume of the cell suspension.</li>
		<li>Add 50 &#181;L of cell suspension to 50 &#181;L of the 0.08% trypan blue solution and homogenize.</li>
		<li>Put the sample under the coverslip of a hemocytometer (Thoma chamber) and place it under the optical microscope (400X).</li>
		<li>Count the squamous cells, the living non-squamous cells (uncolored cells), and the dead non-squamous cells (blue colored cells). 
		NOTE: In case of too low or too high cell density, concentrate or dilute the cell suspension and repeat steps 2.4.1-2.4.3.</li>
		<li>Assess the cell concentration of the suspension, the percentage of squamous cells, and the percentage of viability of non-squamous cells. 
		NOTE: Calculate the total non-squamous cell count/g of sputum: cell concentration of the suspension (step 5.4) * volume of the cell suspension (step 4.8) *% of non-squamous cells / weight of the sputum sample (step 2.2.1).</li>
	</ol>
	</li>
	<li>Preparation of a stained cytospin slide with the cell suspension and storage of residual cells, if needed 
	NOTE: If the cell suspension contains more than 80% squamous cells, the sample is considered poor quality and unsuitable for a cytospin slide14. In that case, discontinue the processing and consider this sample unsuccessful.
	<ol>
		<li>Dilute an aliquot of the cell suspension with DPBS to obtain at least 350 &#181;L of a cell suspension at 500,000 cells/mL.</li>
		<li>Assemble the cell concentrator in accordance with the manufacturer&#39;s instructions.</li>
		<li>Fill each of the 3 compartments of the cell concentrator with 100 &#181;L of this cell suspension.</li>
		<li>In a cytocentrifuge, spin for 2 min at 150 x g and room temperature. Discard the supernatants.</li>
		<li>Disassemble the cell concentrator in accordance with the manufacturer&#39;s instructions.</li>
		<li>Allow the slide to air dry. 
		NOTE: At this stage, residual cells can be stored in an adequate buffer if needed for further experiments.</li>
		<li>Stain the slide with a commercial staining kit (see the Table of Materials), in accordance with the manufacturer&#39;s instructions.</li>
		<li>Mount the slide with a suitable mounting medium and a cover slip.</li>
		<li>Place the slide under the optical microscope (600X) with oil immersion.</li>
		<li>Count at least 500 non-squamous cells: neutrophils, eosinophils, macrophages, lymphocytes, and epithelial cells. 
		NOTE: Differential cell count is usually performed by only one or two dedicated and trained observers to limit interobserver discrepancies.</li>
		<li>Record absolute values and percentages of each cell type.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

The induced sputum method is a useful tool to study the airway compartment. There are several possible applications of this technique. First, it may improve knowledge about immune cells and mechanisms involved in various respiratory diseases. For example, this technique has allowed the investigation of airway inflammation in large cohorts of patients, and it has been shown that about half of asthmatic patients are characterized by an abnormal airway eosinophilic inflammation15,16,17, while COPD patients usually exhibit a raised sputum neutrophil count18. This technique has also contributed to better characterization of airway inflammation in patients with idiopathic pulmonary fibrosis and to evidence mediators that may contribute to this disease19. Second, the technique of induced sputum may be useful to predict response to treatment. For example, the presence of an abnormal percentage of sputum eosinophils has been shown to be a predictive marker of corticosteroid responsiveness11,18. In asthma and COPD, tailoring the dose of corticosteroids to normalize the percentage of sputum eosinophils was shown to be more effective in decreasing the number of exacerbations than adjusting treatment according to the current clinical guidelines11,20,21. Third, sputum analysis may help to develop targeted therapies. For example, the presence of an abnormal number of sputum neutrophils in COPD and some asthmatic patients has led to the development of antineutrophilic treatments11. Fourth, the technique may play a role in diagnosis. For example, the presence of sputum eosinophilia is necessary to make a diagnosis of non-asthmatic eosinophilic bronchitis11.
Besides obtaining a differential cell count, the technique of inducing sputum also allows the performance of many additional analyses, by studying either sputum supernatant or sputum cells. Examples with sputum supernatant include the analysis of mediators8,19,22 and the assessment of the chemotactic activity of the sample for eosinophils22. From sputum cells, RNA can be extracted and used for microRNA or gene expression analyses19,23 . The sputum cells can also be analyzed by flow cytometry9,23 which allows, among others, immunophenotyping and sorting cells. Furthermore, sputum cells can be cultured10, and their mediator production can be measured in vitro24. It is worth noting that in this case, the mediator content is different from what can be found in sputum supernatant. Indeed, mediators in sputum supernatant may be influenced by secretions from airway resident cells and by plasma exudation, contrary to the sputum cell culture model24. Finally, immunocytochemistry and in situ hybridization can also be performed using sputum cells7.
The induced sputum technique has some limitations. The induction must be performed under medical supervision. It is also essential for the operator to give thorough instructions to patients. Other limitations include the cooperation and medical condition of patients, as respiratory efforts are needed. Regarding the feasibility of this technique in children, several studies have reported that it was successful and safe in children older than 6 years old25. Data on children of &#60;6 years of age are lacking25, but it is likely that sputum induction is difficult to perform in those children14. The technique is currently restricted to research services and specialized centers because it is technically demanding, time-consuming, and it requires trained staff1. Another limitation is that the technique of sputum induction and analysis is not always successful, meaning that a readable cytospin is not always obtained26. However, the success rate is usually around 80% in different cohorts of patients4,26,27,28,29. Finally, caution must be taken when comparing different cohorts of patients regarding the matching of age as it was shown to impact the sputum cell count30,31. Likewise, other parameters such as gender and tobacco habits have to be matched as they may also interfere with the sputum cell count29,32. When patient matching is not possible, another way to take age, gender or smoking status into account is to adjust the statistical analysis for these characteristics.
Compared to other techniques that allow collecting cells from airways, like biopsies and bronchoalveolar lavages, the induced sputum method has the advantages of being simple, well-tolerated, safe, reproducible, cost-effective, and non-invasive. These advantages allow the induced sputum technique to be performed at a large scale and repeatedly over time, and make it an alternative of choice for airway sampling. Bronchosorption is another technique allowing the collection of the mucosal lining fluid in order to assess airway mediators33. While this technique (requiring bronchoscopy) is more invasive than induced sputum, it has the advantages of avoiding any contamination with saliva and obtaining higher concentrations of mediators than in bronchoalveolar lavage33.
Critical steps need attention in the protocol. First, caution must be taken concerning the saline concentration and the time of induction, because these two parameters can influence the results and therefore have to be standardized34,35. Second, the mucolysis with DTT is an important step of the processing. As compared with PBS, DTT was shown to better disperse sputum cells7, which improves the cytopsin slide quality and is essential to have a reproducible cell count2. However, the mucolytic agent may interfere with the measurement of biochemical components. Spiking experiments should then be performed when measuring a new biochemical compound36. Finally, another critical step of the procedure is the cell counting step, which must be performed by a trained technician to ensure reliable and interpretable results.
An alternative method using sputum plugs (viscid or denser parts), instead of the whole sputum, is also described in the literature7. Both techniques have advantages and disadvantages. The whole sputum technique allows more rapid processing7, but is frequently contaminated with saliva, which dilutes the sample and can reduce the quality of cytospins2,7. With the plug selection technique, the saliva contamination is reduced, which means that samples are often of better quality for the dosage of fluid phase mediators and for cytospin slides7. The processing is however longer7 and selected plugs may not be representative of the whole sample37. It is also worth noting that not all samples contain plugs, which can be limiting. Both methods are validated and reproducible, and there is currently no evidence that the differential cell counts obtained from both these methods are different14,38.
In the future, induced sputum could be used as a clinical tool to provide biomarkers for the investigation, diagnosis, and management of all kinds of inflammatory respiratory diseases.

Several methods are used to investigate airway inflammation: direct measurements (like bronchial biopsies or bronchoalveolar lavages) and indirect methods (like symptom assessment, blood sample analysis and lung function tests)1. The direct techniques have the advantage of reliably assessing the airway inflammation, but they are invasive and not feasible at large scale because of patient discomfort and the risk incurred1. As for the indirect methods, they poorly correlate with the direct assessment of airway inflammation1.
Sputum collection is another way to sample cells from the airways and allows the direct assessment of airway inflammation. Nevertheless, producing sputum spontaneously may lead to samples of poor quality and is not possible for all patients2. This issue has been overcome by using ultrasonically nebulized hypertonic saline to induce the sputum production2. This method was initially used in patients infected with human immunodeficiency virus (HIV) for the diagnosis of Pneumocystis carinii pneumonia3 and was adapted for healthy subjects and asthmatics in 19924. Sputum induction is also feasible in more severe patients by using isotonic saline5. Although generally well-tolerated, the inhalation of saline may cause bronchospasm in patients with hyperresponsive airways5. Therefore, it is recommended to administer a short-acting beta agonist before the procedure1. Moreover, we have previously shown that adding salbutamol to the saline solution in the ultrasonic nebulizer further decreased this risk6. The advantages of sputum induction is that it is non-invasive2 and safe when appropriate precautions are taken5. 
For the processing of sputum samples, two methods are currently used in the literature: the whole sputum technique and the plug selection7. In our laboratory, the whole sputum technique is performed. The primary aim of sputum processing is to obtain a differential cell count to study the type of inflammation present in the airway lumen. However, many additional analyses are also possible to further investigate inflammatory processes and immune mechanisms, either by studying mediators in the sputum supernatant8 or by performing detailed investigation on sputum cells (e.g., flow cytometry9, cell cultures10, genomics10, proteomics10, immunocytochemistry7, in situ hybridization7, etc.)
The technique of sputum induction and analysis is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff1. Airway inflammation may be investigated with this method for various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis11.

<table><tbody><tr><td>Spirometer - Spirobank</td><td>MIR France</td><td></td><td></td></tr><tr><td>Salbutamol MDI - Ventolin</td><td>GLAXOSMITHKLINE</td><td>CNK: 0135-913</td><td>See drug available in the country</td></tr><tr><td>Nebulizer UltraNeb</td><td>DeVilbiss Healthcare USA</td><td>UltraNeb U3000</td><td></td></tr><tr><td>Devilbiss UltraNeb Disposable Cups &amp;amp; Lids</td><td>DeVilbiss Healthcare USA</td><td>100HD-647</td><td></td></tr><tr><td>DeVilbiss Bacterial Filter For UltraNeb Nebulisers</td><td>DeVilbiss Healthcare USA</td><td>1001005879</td><td></td></tr><tr><td>One-way Valves</td><td>Hudson RCI USA</td><td>41664</td><td></td></tr><tr><td>One-way Valves</td><td>Hudson RCI USA</td><td>41665</td><td></td></tr><tr><td>Aerosol T-connector&amp;nbsp;</td><td>Hudson RCI USA</td><td>41077</td><td></td></tr><tr><td>Ventilation circuit monobranch corrugated</td><td>Int&#039;Air Medical France</td><td>TA30A</td><td></td></tr><tr><td>NaCl 5 %&amp;nbsp;</td><td>/</td><td>/</td><td>Produced by our hospital pharmacy</td></tr><tr><td>Mini-Plasco NaCl 0.9%</td><td>B.Braun Medical&amp;nbsp; Diegem Belgium</td><td></td><td></td></tr><tr><td>Salbutamol sulfate 5 mg/mL - Ventolin</td><td>GLAXOSMITHKLINE</td><td>CNK: 0094-987</td><td>See drug available in the country</td></tr><tr><td>Ipratropium bromide 0.25 mg/2 mL - Atrovent</td><td>Boehringer Ingelheim Germany</td><td>CNK: 1543-305</td><td>See drug available in the country</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>LONZA Belgium</td><td>BE17512F</td><td></td></tr><tr><td>Sputolysin reagent</td><td>Calbiochem</td><td>56000</td><td></td></tr><tr><td>Trypan blue 0.4% solution 100 mL</td><td>LONZA Bio Whittaker Belgium</td><td>17-942E</td><td></td></tr><tr><td>Hemocytometer - Thoma chamber</td><td>Labor Optik Lancing UK</td><td>1500000</td><td></td></tr><tr><td>Hemacolor Stainig set</td><td>Merck Belgium</td><td>1116610001</td><td>Alternative product&amp;nbsp; : Diff Quik Staining Set&lt;br/&gt; &amp;nbsp;Medion Diagnostic&amp;nbsp;</td></tr><tr><td>Mounting medium - Entellan</td><td>Merck Belgium</td><td>107960</td><td></td></tr><tr><td>Statspin Cytofuge 12&amp;nbsp;</td><td>Beckman Coulter Belgium</td><td>X00-003066-001</td><td></td></tr></tbody></table>

methodology for sputum induction and laboratory processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Hemocytometer 
A typical image is shown in Figure 1 that is visualized with the microscope using the hemocytometer. Squamous cells are easily identified, as they are much larger than the non-squamous cells. These squamous cells are epithelial cells coming from the mouth. Both types of cells are stained by Trypan Blue when dead. Caution must be taken to avoid counting yeast, bacteria, and scrap.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56612/56612fig1.jpg" /> 
Figure 1: Hemocytometer picture showing (A) a living non-squamous cell, (B) a dead non-squamous cell, and (C) a squamous cell. Scale bar = 50 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56612/56612fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cytospin slide: Figure 2 shows a representative image of a cytospin slide obtained after sputum processing. The different cell types (neutrophils, eosinophils, macrophages, lymphocytes, and epithelial cells) can be differentiated by means of their morphology and coloration. In some cases, contamination by squamous cells may be important and, if the percentage of squamous cells is greater than 80%, the sample is considered unsuccessful (Figure 3).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56612/56612fig2.jpg" /> 
Figure 2: Cytospin slide picture showing (A) a neutrophil, (B) a macrophage, (C) an eosinophil, (D) a lymphocyte, and (E) an epithelial cell. Scale bar = 20 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56612/56612fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56612/56612fig3.jpg" /> 
Figure 3: Example of a poor quality cytospin slide with &#62;80% of squamous cells. Scale bar = 20 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56612/56612fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Success rate 
In our department, the success rate of the procedure (combining a successful induction and a readable cytospin), based on a sample of 1,129 patients (healthy subjects, asthmatics, or COPD patients), is 82% (924/1,129). In a sub-analysis according to the type of patients, the success rate is 75% (57/76) in healthy subjects, 82% (827/1,004) in asthmatics, and 82% (40/49) in COPD patients.
Results in healthy subjects 
In a retrospective analysis of a series of 289 healthy subjects from our department, the median (interquartile range) sputum weight was 3.72 g (interquartile range of 2.46 g - 5.54 g) and the median total non-squamous cell count/g of sputum was 0.59 x 106 (interquartile range of 0.37 x 106 - 1.29 x 106).
In those healthy subjects, the proportion of squamous cells is low at 19% (10%-34%) and the viability is high at 66% (54-78%). Regarding the percentage of the different cell types, results are summarized in Figure 4A. We can observe that the percentage of macrophages (49% [31-68%]) is higher than the percentage of neutrophils (34% [14%-60%]), while the percentages of lymphocytes (2% [1-3%]), eosinophils (0% [0%-0%]) and epithelial cells (4% [2-11%]) are low. These results are similar when data are expressed in absolute values (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56612/56612fig4.jpg" /> 
Figure 4: Representative results of the differential cell count observed in healthy subjects expressed as (A) percentages or (B) absolute values. Results are shown as median (interquartile range). <a href="https://cloudflare.jove.com/files/ftp_upload/56612/56612fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It is also important to take the age of patients into consideration. Indeed, a strong correlation is present between a patient&#39;s age and the proportion of neutrophils in sputum samples (Figure 5). Likewise, when classifying patients according to 10 year age groups (Figure 6), we observed a significant increase in the neutrophil percentage with increasing age group. Therefore, this variable must be considered when comparing results from different cohorts, and caution must be taken in the matching of subjects.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56612/56612fig5.jpg" /> 
Figure 5: Correlation between age and sputum neutrophil percentage. The correlation was calculated with the Spearman test. <a href="https://cloudflare.jove.com/files/ftp_upload/56612/56612fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56612/56612fig6.jpg" /> 
Figure 6: Evolution of the neutrophil percentage according to the age category. The p-value of ANOVA was &#60;0.0001 for the comparison of neutrophil percentage between age classes. Multiple comparisons were made with Dunn&#39;s multiple comparisons test. The p-values are represented as follows: * p &#60;0.05, ** p &#60;0.01, and *** p &#60;0.001. Results are shown as median (interquartile range). <a href="https://cloudflare.jove.com/files/ftp_upload/56612/56612fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Results in patients suffering from respiratory diseases 
The technique of induced sputum is commonly used to assess the inflammatory cell profile in asthmatic patients. This technique may also be applied to patients suffering from COPD, another inflammatory respiratory disease. When comparing healthy subjects, asthmatics, and COPD patients (the 3 groups being matched for age, gender, and tobacco habits), we observed that the inflammatory cell profile is quite different between these cohorts (Figure 7). Indeed, asthmatic patients are usually characterized by raised sputum eosinophils, while the proportion of sputum neutrophils is usually higher in COPD patients compared to healthy controls, which is linked to the disease severity.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56612/56612fig7.jpg" /> 
Figure 7: Sputum inflammatory cell profile of healthy subjects (n = 45), asthmatic patients (n = 108), and COPD patients (n = 54). The three groups were matched for gender, age, and tobacco habits. The p-values of ANOVA were &#60;0.05, &#60;0.0001, and &#60;0.0001 for the comparison of neutrophils, eosinophils, and macrophages between groups, respectively. Multiple comparisons were made with Dunn&#39;s multiple comparisons test. The p-values are represented as follows: * p &#60;0.05 and *** p &#60;0.001. Results are shown as median (interquartile range). <a href="https://cloudflare.jove.com/files/ftp_upload/56612/56612fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Methodology for Sputum Induction and Laboratory Processing at JoVE.com

Methodology for Sputum Induction and Laboratory Processing

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

methodology-for-sputum-induction-and-laboratory-processing

Research

JoVE Journal

Medicine

27.5K Views.  University of Liege. Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis. 

Video: Methodology for Sputum Induction and Laboratory Processing

Quantifying Carbon in Pulmonary Macrophages via Induced Sputum Analysis

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with ...

Environment

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

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A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

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The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

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Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

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The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

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Biology

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Sputum Studies I: Gram Stain, cytology, and Acid-fast smear and culture

Sputum studies are a critical part of diagnosing and treating numerous respiratory conditions. These studies involve obtaining sputum samples for analysis to identify pathogenic organisms and assess the presence of abnormal cells indicative of malignant conditions. This lesson will delve into three fundamental sputum studies: Gram Stain, Cytology, and Acid-fast Smear and Culture.
Gram Stain
The Gram Stain is an integral part of sputum studies. It involves the staining of sputum, which permits the classification of bacteria into gram-negative and gram-positive types. This process guides therapy until culture and sensitivity results are obtained.
To carry out this study, patients should be instructed to expectorate sputum into the container after coughing deeply. The goal is to obtain sputum (mucoid-like), not saliva. Specimens should ideally be collected early after mouth care because secretions tend to accumulate overnight. If the patient fails to produce a sample, increasing oral fluid intake may be beneficial unless fluids are restricted. Once the sputum is collected, it should be sent promptly to the laboratory for analysis.
Cytology
Cytology is another essential sputum study that aims to determine the presence of abnormal cells that may indicate a malignant condition. A single sputum specimen is typically collected in a special container with a fixative solution.
Patients need clear instructions on how to produce a good specimen, similar to the Gram Stain procedure. If the patient cannot produce a specimen, bronchoscopy may be employed as an alternative collection method. As with the Gram Stain, the Cytology specimen should promptly be sent to the laboratory for analysis.
Acid-fast Smear and Culture
The Acid-Fast Smear and Culture study assesses sputum for acid-fast bacilli (e.g., Mycobacterium tuberculosis). This study usually requires a series of three early-morning specimens.
As with the previous methods, patients must cover their specimens and send them to the laboratory for analysis. Detailed instructions on producing a quality sample are essential for achieving accurate results.
In conclusion, sputum studies play a valuable role in diagnosing and treating respiratory conditions. Whether it is identifying the type of bacteria through a Gram Stain, detecting abnormal cells via Cytology, or finding Acid-Fast bacilli through an Acid-Fast Smear and Culture, these studies provide clinicians with the necessary information to guide their treatment plans. To ensure the accuracy of these studies, proper collection and prompt delivery of the sputum samples are essential.

Sputum studies are key in diagnosing respiratory issues, identifying pathogens, and detecting cancer cells. The three crucial sputum studies include Gram ...

Methodology for Sputum Induction and Laboratory Processing

Summary

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Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum Studies I: Gram Stain, cytology, and Acid-fast smear and culture