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.

Transcript

The technique of induced sputum has been developed in the early 90's. The technique was proposed to investigate new information in obstructive airway disease like asthma, COPD, and more recently there has been an interest also to look at new information in pulmonary fibrosis. The main advantage of this technique is that it's relatively non-invasive as opposed to bronchoscopy.

The processing requires lab work, and it takes time, so it's quite demanding, time consuming, and it needs to have some expertise in the laboratory, so it's clearly a technique which is difficult to obtain in every centers. The technique of induced sputum is composed of two parts, the first part is the induction, and the second part is the processing. Before inducing sputum, we need to perform a spirometry, which is an assessment of the volume and the flow rate that the patient is able to produce.

And we need to do that because the sputum induction by itself may cause bronchiospasm, so we absolutely need to know the baseline value of the spirometry of the patients. So we first perform a spirometry, then we give the patients bronchodilators, we usually give 400 microgram salbutamol, to open maximally the airways of the patients, and also to prevent a bronchiospasm that may occur during the induction phase. And after 15 minutes, we measure again the spirometry, to have what we call the baseline value of spirometry, and in particular we look at the FEV1 which is the volume that the patient is able to exhale within one second.

Pour distilled water into the nebulizer chamber until the recommended level. Place a clean cup in the nebulizer chamber. Cover the filled cap with the appropriate lid.

Fill the cup with 50mL hypertonic saline 5%if post-bronchodilator FEV1 is greater than 65%predicted, or 50mL isotonic saline 9%if post-bronchodilator FEV1 is less than 65%predicted. Add 1.75 salbutamol sulfate solution at the concentration of five milligrams per ml to the cup. Connect the tubes and valves in accordance with the manufacturer instructions.

The technique has to be performed under medical supervision. Explain the principle of the test to the patient. Ask the patient to use a nose clip.

Start the nebulizer and select the lowest level of aerosol and fan setting. Ask the patient to inhale the aerosol through the mouthpiece with tidal breathing. The level of aerosol and fan setting may be increased depending on the patient's tolerance.

In case of chest tightness of respiratory discomfort, stop the procedure and perform a spirometry to measure the patient FEV1 value. After the five minute of inhalation, or in case of cough or nausea, stop the nebulizer. Ask the patient to remove the nose clip, to rinse the mouth, and gargle twice with tap water, and to discard this water in the sink.

Then, as the patient to cough up sputum and spit what is coming out from the throat in a plastic container. Perform the first respiratory maneuver to measure FEV1. After each tentative of sputum production, we measure the FEV1 value.

If the FEV1 value drops by more than 20%we stop the procedure and we give the patients nebulization of ipratropium bromide, which is an anticholinergic that will act in combination with beta-two agonists that we have already administered during the procedure. If the FEV1 value does not drop by more than 20%we carry on the procedure for total time of 10 to 20 minutes according to the quality and the volume of sputum produced by the patient. Once you get the sample of sputum, keep it in the fridge until processing.

So the second part consists of the sputum processing, and for this, we have first to prepare the two solutions that we are going to use for this processing. So we have to prepare the Dithiothreitol also known as DTT. The solution must be prepared by dilution of the DTT with sterile water in order to obtain a 6.5 millimolar solution.

So you have to avoid to expose the DTT to ambient air, and for this we use a syringe and needle. Secondly, you have to prepare the trypan blue solution, and for this you have to do a dilution of DPBS with trypan blue, to obtain a solution of 0.08%So concerning the sputum, you have to transfer the entire sputum in a 50mL conical bottom plastic tube, and weigh the sample. Add a three-fold weight of DPBS solution.

Slowly vortex the sample for 30 seconds, centrifuge at 800 g for 10 minutes at four degrees Celsius. Collect the supernatant in a 50mL conical bottom plastic tube, after filtration through two single layers of sterile gauze. Allocate the supernatant in 2mL plastic tubes and store at minus 80 degrees Celsius.

Note that the supernatant samples are useful to us as three phase component of sputum. If the cell pallet volume is less than 5mL, add DPBS to reach a 5mL volume of cell suspension. Dilute the cell suspension with one volume of DTT at 6.5 millimolar.

Rock the cell suspension for 20 minutes at room temperature with a bench worker. Dilute the cell suspension at least three times with DPBS. Centrifuge the diluted cell suspension at 550 g for 10 minutes at four degrees Celsius.

Discard the supernatant. Suspend the cell palette in around one ml DPBS. Assess and record the exact volume of the cell suspension.

Add 50 microliter of cell suspension to 50 microliter of the diluted trypan blue solution and homogenize. Put the sample under the cover slip of a hemocil cytometer, and place it under an optical microscope. Assess the cell concentration of the cell suspension, the percentage of squamous cells, and the percentage of viability of non-squamous cells.

Dilute an allocate of the suspension with DPBS to obtain at least 350 microliter of cell suspension at 500, 000 cell per mL. Assemble the cell concentrator in accordance with the manufacturer instructions. Add 100 microliter of the cell suspension in each of the three compartments of the cell concentrator.

Spin two minutes at 150 g at room temperature in a cyto centrifuge. Discard the supernatant. Allow the slide to air-dry.

Stain the slide with div quick or a nickel valence stain kit in accordance with the manufacturer instructions. Mount with a mounting medium and cover slip. Place the slide under the optical microscope with oil immersions.

Count at least 500 non-squamous cells, the neutrophils, eosinophils, macrophages, lymphocytes, and epithelial cells, and record the absolute values and percentage and cell type. Concerning the results, using the hemocytometer, we have to count the living non-squamous cells that are uncolored. Also, the dead non-squamous cells that are colored in blue.

And the squamous cells. These cells are epithelial cells that are coming from the mouth. They are large, so they are easy to recognize compared to the small human cells.

So you have to avoid also to count bacteria, yeast, or scrap. If the cell suspension contains more than 80%of squamous cells, this sample is considered of poor quality and unsuitable for a cytospin slide. This sample is therefore considered as unsuccessful.

The count on the stained cytospin is performed according to the color of the cells and their morphology. The neutrophils, the main characteristics is the multilobulated nucleus that made the cells easy to identify. These cells are round, small, and colored in neutral pink.

The eosinophils, these cells are composed of red granules, easy to identify. The size of these cells is close to the neutrophils. The macrophages, they are two to five times bigger than the neutrophils, and are colored in blue.

The cytoplasm can contain debris and vacuoles. The lymphocytes, these cells are round and small, colored in blue, and possess a big and dense nucleus. The epithelia cells have a columnar shape, are colored in pink, and often present cilia at the top.

Here are some examples of an unreadable cytospin samples with greater than 80%of squamous cells. I think it's a technique which is very useful, however we must be very cautious because the sputum induction must be done under medical supervision, but the technique of sputum induction gives you a lot of information about the inflammatory status of the patients in the airways. It can allows you to measure different biochemical markers in supernatant, and you can also measure different things on the cellular parts, so induced sputum allows you to perform flow cytometry, proteomics, transcriptomics, and even genomics.

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Methodology for Sputum Induction and Laboratory Processing

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