Name: quality-controlled sputum analysis by flow cytometry
Uploaded: 2021-08-09T13:00:00
Description: Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

We want to thank David Rodriguez for his assistance with the figure preparation. Sputum samples were run on the BD LSR II at the UT Health San Antonio Flow Cytometry Shared Resource Facility, supported by UT Health, NIH-NCI P30 CA054174-20 (CTRC at UT Health) and UL1 TR001120 (CTSA grant).

All steps of the sputum processing are performed in a biological safety cabinet with appropriate personal protective equipment.
1. Reagent preparation before starting sputum dissociation
<ol>
	<li>Thaw 1% Paraformaldehyde (PFA), 25 mL per sample on ice, and keep cold until use. 
	CAUTION: PFA is toxic by inhalation and skin contact. Prepare the fixative according to the manufacturer&#39;s instructions and freeze at -20 &#176;C in 25 mL aliquots until use.</li>
	<li>Approximate the weigh...

The cellular content of sputum includes a large variety of wide-ranging cells, often accompanied by a lot of debris37. In addition, sputum analysis requires a quality control that confirms the sample is collected from the lung instead of the oral cavity38. Therefore, it is not as simple to analyze sputum by flow cytometry as it is for blood, for example, which releases a much cleaner and homogeneous cell suspension. This protocol has addressed all these issues: providing in...

Technical advancements in the hardware and software of flow cytometers have made it possible to identify many distinct cell populations simultaneously1,2,3,4. The utilization of the flow cytometer in hematopoietic cell research, for example, has led to a much better understanding of the immune system2 and the cellular hierarchy of the hematopoietic system5, as well as the diagnostic distinction of a multitude of different blood cancers6,7,8. Although most sputum cells are of hematopoietic origin9,10,11, flow cytometry has not been widely applied to sputum analysis for diagnostic purposes. However, several studies suggest that the evaluation of immune cell populations in sputum (the most significant subset of cells) may be of great help in diagnosing and/or monitoring diseases such as asthma and chronic obstructive pulmonary disease (COPD)12,13,14,15. Moreover, the existence of epithelial-specific markers that can be used in flow cytometry allows the interrogation of the following most significant subset of cells in sputum, lung epithelial cells.
In addition to the ability to analyze many distinct cell populations of different tissue origins, a flow cytometer can evaluate large numbers of cells in a relatively short period. In comparison, slide-based, cytological types of analyses often require highly specialized personnel and/or equipment. These analyses can be labor-intensive, which leads to only a proportion of the sputum sample being analyzed16.
Three critical issues limit the widespread use of sputum in flow cytometry. The first issue relates to the collection of sputum. Sputum is collected through a huff cough that expels mucus from the lungs into the oral cavity, subsequently spitting into a collection cup. Since the mucus travels through the oral cavity, there is a high chance of SEC contamination. This contamination complicates the specimen analysis, but the problem is easily rectified on a flow cytometric platform, as shown in this study.
Not everyone can produce sputum spontaneously; therefore, several devices have been developed to assist with the sputum collection in a non-invasive manner17. The nebulizer is one such device and has been shown to produce reliable sputum samples18,19,20. Although the nebulizer is a very effective way of non-invasively collecting sputum, its use still requires a setting at a medical facility with specialized personnel21. In contrast, handheld devices such as the lung flute22,23,24 and the acapella16,25 can be used at home since they are very user-friendly. These assist devices are both safe and cost-effective.
For us, the acapella gave consistently better results than the lung flute16, and therefore, the acapella device has been chosen for sputum collections. A 3-day collection sample was decided because the primary purpose for using sputum is to develop a lung cancer detection test16. It has been shown that a 3-day sample increases the likelihood of lung cancer detection compared to a 1- or 2-day sample26,27,28. However, other methods of sputum collection may be preferable for different purposes. If a different sputum collection method is used than the one described here, it is recommended to carefully titrate each antibody or dye used for flow cytometric analysis; very little data is available on how different sputum collection methods affect the targeted proteins for flow cytometry.
The second issue dampening the enthusiasm for using sputum for diagnostics, primarily related to flow cytometry, is cell number. The problem is the collection of sufficient viable cells for a reliable analysis. Two studies demonstrated that sputum samples collected by non-invasive methods, with the help of an assist device, contain enough viable cells that can be utilized in clinical diagnosis or research studies16,24. However, neither of these studies addressed the issue of cell numbers concerning flow cytometry.
For the studies that form the basis for this protocol, sputum samples were collected from participants at high risk for developing lung cancer following approved institutional guidelines for each study site. High-risk participants were defined as between 55-75 years, having smoked 30 pack-years and having not quit smoking within the past 15 years. Patients were shown how to use the acapella device according to the manufacturer&#39;s instructions29 and collected sputum for three consecutive days at home. The sample was kept in the refrigerator until the last collection. On the final collection day, the sample was shipped to the laboratory overnight with a frozen cold pack. The samples were processed into a single cell suspension on the day they were received. With this method of sputum collection, more than enough viable cells are obtained for a reliable flow cytometric analysis.
Lastly, and related to the previous cell number issue, is the question of how to release the sputum cells from its mucinous environment. How can the cells be kept viable and create a single cell suspension that does not clog the flow cytometer? Based on initial work by Pizzichini et al.30 and Miller et al.31, this protocol describes an easy and reliable method for sputum processing into a single cell suspension that is suitable for flow cytometric analysis. This method has used well-established guidelines in flow cytometry32,33,34 to develop an efficient antibody labeling strategy to identify hematopoietic and epithelial cells in sputum and provide instrument settings, quality control measures, and analysis guidelines standardizing sputum analysis on a flow cytometric platform.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% Paraformaldehyde Flow-Fix</td>
			<td>Polysciences</td>
			<td>25037</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;M nylon cell strainers, Falcon #352360</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M NaOH</td>
			<td>EMD</td>
			<td>SX0593-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical falcon tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 anti-human CD19</td>
			<td>BioLegend</td>
			<td>302219</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 anti-human CD3</td>
			<td>BioLegend</td>
			<td>300415</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 anti-human cytokeratin</td>
			<td>BioLegend</td>
			<td>628608</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 PanCK, CD3, and CD19 Isotype</td>
			<td>BioLegend</td>
			<td>400129</td>
			<td></td>
		</tr>
		<tr>
			<td>BV510 anti-human CD45</td>
			<td>BioLegend</td>
			<td>304036</td>
			<td></td>
		</tr>
		<tr>
			<td>CD66b FITC isotype</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td></td>
		</tr>
		<tr>
			<td>CompBead Plus Compensation Beads</td>
			<td>BD Biosciences</td>
			<td>560497</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Polystyrene dispoable sterile bottle 250 mL</td>
			<td>Fisher Scientific</td>
			<td>09-761-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Polystyrene dispoable sterile bottle 500 mL</td>
			<td>Fisher Scientific</td>
			<td>09-761-10</td>
			<td></td>
		</tr>
		<tr>
			<td>CS&#38;T beads</td>
			<td>BD Biosciences</td>
			<td>655051</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD66b</td>
			<td>GeneTex</td>
			<td>GTX75907</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Stain</td>
			<td>BD Biosciences</td>
			<td>564406</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowCheck</td>
			<td>Beckman Coulter</td>
			<td>A69183</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowSet</td>
			<td>Beckman Coulter</td>
			<td>A69184</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Fisher Scientific</td>
			<td>14-175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>NAC</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>NIST Beads, 05 &#956;M</td>
			<td>Polysciences</td>
			<td>64080</td>
			<td></td>
		</tr>
		<tr>
			<td>NIST Beads, 20 &#956;M</td>
			<td>Polysciences</td>
			<td>64160</td>
			<td></td>
		</tr>
		<tr>
			<td>NIST Beads, 30 &#956;M</td>
			<td>Polysciences</td>
			<td>64170</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD45</td>
			<td>BioLegend</td>
			<td>304039</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 anti-human EpCAM</td>
			<td>BD Biosciences</td>
			<td>565399</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 CD206/EpCAM Isotype</td>
			<td>BD Biosciences</td>
			<td>562292</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CR594 anti-human CD206</td>
			<td>BD Biosciences</td>
			<td>564063</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>EMD</td>
			<td>SX0445-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution, 0.4%</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 was developed with a clinical laboratory setting in mind. The focus during the development of the protocol was on simplicity, efficiency, and reproducibility. It was found that the most time-consuming step in the processing of sputum was counting the cells. Therefore, the protocol is set up in such a way that sputum processing and cell labeling can be performed independently from cell counting without loss of time. An accurate cell count, which is still necessary to dilute the sample appropriately for an un...

Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

Quality-Controlled Sputum Analysis by Flow Cytometry

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 ...

This protocol resolves common challenges experienced with sputum processing that have made it difficult in the past to use with flow cytometry for high-throughput clinical diagnostic purposes. This protocol is designed to save time by using sputum weight to determine antibody and dye staining volumes. During the staining incubation, cell count can be performed for the downstream steps.It is important to achieve an accurate cell count to determine the correct re-suspension volume to prevent any problems when running on the flow cytometer. This protocol can provide insight into areas of research that investigate lung health. For example, illnesses such as COPD, asthma, lung cancer, or the effects of COVID could be studied.Demonstrating the procedure will be Dr.Michael Lai, a research and development scientist from my laboratory. To begin with, weigh the sputum sample to determine the volumes of dissociation reagents. Transfer the sample to the appropriately sized vessel based on the sputum weight, then add one milliliter per gram of 0.5%NAC and four milliliters per gram of 0.1%DTT.Vortex the samples at maximum speed for 15 seconds and rock at maximum speed for 15 minutes at room temperature. Dilute this sample with 4X Hanks balanced salt solution or HBSS to neutralize the NAC and DTT. And after quick vortexing, rock the sample at room temperature for five minutes.Filter cell suspension through a 100 micrometer nylon mesh cell strainer into one or more 50 milliliter conical centrifuge tubes to create a single-cell suspension. Pellet down the cells. After aspirating the supernatant, combine pellets in one 15 milliliter conical tube and wash with HBSS under the same conditions.Resuspend the cell pellet in a volume of buffer determined by the initial weight of the sputum sample. Take an aliquot from the cell suspension and mix 10 microliters of the sputum dilution with 30 microliters of 0.4%trypan blue, then load into the counting chambers of a hemocytometer for a live/dead cell count. Label sputum in compensation tubes.Add HBSS antibody and dye to each sputum cell tube and compensation tubes as indicated in the text. Add the sputum cells to the assay tubes and add compensation beads to the compensation tubes as mentioned in the text. Incubate all the tubes on ice protected from light for 35 minutes.Then after filling the tubes with ice cold HBSS, centrifuge at four degrees Celsius for 10 minutes at 800 times G.For compensation tubes, aspirate the supernatant as close to the pellets as possible and flick the pellets to loosen. Next, add 0.5 milliliters of cold HBSS to the compensation tubes and store the tubes on the ice in a light protected area until needed for flow cytometry analysis. Then aspirate the supernatant from the sputum unstained, isotype, blood, and epithelial tubes.Loosen the pellets by flicking the tubes. Add two milliliters of 1%PFA to the unstained and isotype tubes and 10 milliliters to the blood and epithelial tubes. Incubate the tubes on ice in a light protected manner for 30 minutes, then quickly vortex at maximum speed and again incubate for another 30 minutes.Then fill the tubes with ice cold HBSS. Next, centrifuge the tubes at four degrees Celsius for 10 minutes at 1, 600 times G.Aspirate as much supernatant as possible without disturbing the cell pellet and flick the tube with the fingers to loosen the cells, then add 200 microliters of cold HBSS to the unstained and isotype tubes. Calculate and add the required volume of HBSS for resuspension of the blood and epithelial tube according to the total cell count.Store all the samples in compensation tubes protected from light on the ice at four degrees Celsius until flow cytometry analysis is performed. Apply appropriate startup procedures for the flow cytometer being used. Use the mixture of National Institute of Standards and Technology or NIST beads to ensure that forward scatter and side scatter voltages are set to place the NIST beads to span the plot without placing the beads too close to the axes.Set the flow rate to medium for LSRII or high for Navios EX.Adjust the voltages for each parameter used for the scatter and fluorescent parameters to place the cell populations on scale. Use the figures with the gating strategy as guidance to adjust the voltages accordingly. Acquire data for the unstained sputum sample first, followed by the isotype stained sample, and then the blood tube and the epithelial tube.Sputum weight was used to determine antibody or dye volumes for staining. The correlation between sputum weight and cell yield was not strong. Sputum samples were divided into four weight categories, which clustered in the distribution of cell yield.Each antibody used was titrated in a concentration on the plateau phase with the highest staining index was chosen as the working concentration. In all weight categories, the average SEC contamination was less than 20%with the percentage of viable cells shown. Presented is the gating strategy used for analysis.NIST beads were used to set a gate which was applied to the sputum sample to eliminate debris. Small debris was further eliminated by the width gate. Viability dye was used to eliminate dead cells, while the singlet gate removed doublets.The anti-CD45 antibody labeling allowed the separation of live single sputum cells into a blood cell and a non-blood cell compartment. The profiles obtained from Navios EX, LSRII, and Lyric were compared. The gating strategy for eliminating the debris, dead cells, doublets, and comparing blood and non-blood profiles is shown.This technique allowed us to apply these methods to detect lung cancer risk for use as a non-invasive clinical test.

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