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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V., Liu, J., Wong, H., Boushey, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+Biochemical+Analysis+of+Induced+Sputum+from+Asthmatic+and+from+Healthy+Subjects.">Cellular and Biochemical Analysis of Induced Sputum from Asthmatic and from Healthy Subjects.</a> American Review of Respiratory Disease. 147 (5), 1126-1131 (1993).</li><li>Pizzichini, E., Pizzichini, M. M., Efthimiadis, A., Hargreave, F. E., Dolovich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+inflammatory+indices+in+induced+sputum:+effects+of+selection+of+sputum+to+minimize+salivary+contamination.">Measurement of inflammatory indices in induced sputum: effects of selection of sputum to minimize salivary contamination.</a> The European Respiratory Journal. 9 (6), 1174-1180 (1996).</li></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.

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			<td height="21" style="height:21px;width:513px;">Ventilation circuit monobranch corrugated</td>
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			<td height="20" style="height:20px;">NaCl 5 %&#160;</td>
			<td style="width:265px;">/</td>
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			<td height="26" style="height:26px;">Mini-Plasco NaCl 0.9%</td>
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			<td height="21" style="height:21px;">Salbutamol sulfate 5 mg/mL - Ventolin</td>
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			<td>See drug available in the country</td>
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			<td height="21" style="height:21px;">Ipratropium bromide 0.25 mg/2 mL - Atrovent</td>
			<td style="width:265px;">Boehringer Ingelheim Germany</td>
			<td style="width:140px;">CNK: 1543-305</td>
			<td>See drug available in the country</td>
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			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:265px;">LONZA Belgium</td>
			<td style="width:140px;">BE17512F</td>
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			<td height="21" style="height:21px;width:513px;">Sputolysin reagent</td>
			<td style="width:265px;">Calbiochem</td>
			<td style="width:140px;">56000</td>
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			<td height="21" style="height:21px;width:513px;">Trypan blue 0.4% solution 100 mL</td>
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			<td style="width:140px;">17-942E</td>
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			<td height="21" style="height:21px;">Hemocytometer - Thoma chamber</td>
			<td style="width:265px;">Labor Optik Lancing UK</td>
			<td style="width:140px;">1500000</td>
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			<td height="42" style="height:42px;">Hemacolor Stainig set</td>
			<td style="width:265px;">Merck Belgium</td>
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			<td height="21" style="height:21px;">Mounting medium - Entellan</td>
			<td style="width:265px;">Merck Belgium</td>
			<td style="width:140px;">107960</td>
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			<td height="21" style="height:21px;width:513px;">Statspin Cytofuge 12&#160;</td>
			<td style="width:265px;">Beckman Coulter Belgium</td>
			<td>X00-003066-001</td>
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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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

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27.3K 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

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...