Quality-Controlled Sputum Analysis by Flow Cytometry

Summary

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.

Abstract

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.

Introduction

Technical advancements in the hardware and software of flow cytometers have made it possible to identify many distinct cell populations simultaneously^1,2,3,4. 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 cancers^6,7,8. Although most sputum cells are of hematopoietic origin^9,10,11, 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 samples^18,19,20. 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 flute^22,23,24 and the acapella^16,25 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 sample^26,27,28. 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 studies^16,24. 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'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 cytometry^32,33,34 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.

Protocol

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

1. 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's instructions and freeze at -20 °C in 25 mL aliquots until use.
2. Approximate the weight of the sample and thaw enough 0.1% Dithiothreitol (DTT) for step 2.2 and bring it to 37 °C. (Aliquots of DTT should be stored at -20 °C before use.)
3. Bring enough 0.5% N-acetyl- L-cysteine (NAC) up to 37 °C for step 2.2. (NAC should be made fresh weekly and stored at 4 °C before use.)

2. Sputum dissociation

1. Weigh the sputum sample to determine the volumes of dissociation reagents.
   NOTE: A sample is considered to be small if the initial weight is ≤3 g, medium if >3 g but ≤8 g, large if >8 but ≤16 g, and extra-large if a sample weighs >16 g. The indications small, medium, large, and extra-large will be used throughout the protocol. The quantity of reagents required for dissociation and labeling differ according to the size of the sputum sample.
2. Transfer a small sample to a clean 50 mL conical tube, a medium sample to a clean 250 mL plastic disposable bottle, or a large and extra-large sample to a clean 500 mL plastic disposable bottle. Add 1 mL/g sample weight of 0.5% NAC and 4 mL/g sample weight of 0.1% DTT.
3. Vortex at maximum speed (for 15 s), and then rock at room temperature (at maximum speed) for 15 min.
4. Dilute the sample with four volumes of Hank's Balanced Salt Solution (HBSS) (based on the total volume of sample + reagents) to neutralize the NAC and DTT; vortex quickly at maximum speed and rock at room temperature for 5 min at maximum speed.
5. Filter the cell suspension through 100 µm nylon mesh cell strainer(s) into one or more 50 mL conical centrifuge tube(s) to create a single-cell suspension.
6. Centrifuge the cells at 800 x g for 10 min at 4 °C. Aspirate the supernatant, combine all pellets in one 15 mL conical tube, and then wash the pellets with HBSS using the same conditions.
7. Resuspend the cell pellet in a volume of buffer determined by the initial weight of the sputum sample.
   NOTE: A small sample is resuspended in 250 µL of HBSS. A medium sample is resuspended in 760 µL of HBSS. Large and extra-large samples are resuspended in 1460 µL of HBSS.
8. Take an aliquot of the cell suspension for a live/dead cell count using Trypan Blue.
   NOTE: For a small sample, use 5 µL. For a medium, large or extra-large sample, use 10 µL. Dilute 1:10 with HBSS.
   1. Mix 10 µL of the sputum dilution with 30 µL of 0.4% Trypan Blue to achieve a final sample dilution of 1:40. Load into the counting chambers of a hemocytometer for a cell count.
      NOTE: It may be necessary to adjust the final dilution if the cell numbers are too low or too high to achieve an accurate count. Consulting Guiot et al.²⁰ for correctly distinguishing sputum cells from SECs and debris is strongly recommended here. This is essential for an accurate cell count.
9. From the extra-large sample, remove 50 x 10⁶ cells from the total and add to a new tube with enough added HBSS to create a total volume of 1700 µL.
   ​NOTE: Consider this a large sample for the remainder of the protocol. Leftover samples can be discarded or used for other purposes.

3. Antibody and viability dye staining

1. Choice of antibody and staining dye
   NOTE: Table 1 shows the antibodies and viability dye that are used in this protocol and the cell populations they identify.
   1. Label the tubes containing the sputum cells (see Table 2 for labels).
   2. Use 5 mL flow cytometry tubes (compatible with the flow cytometer used) for the sample tube with the unstained cells and the tube with the isotype control. Use 15 mL conical tubes for the blood and epithelial tube samples.
      NOTE: These samples will be transferred to flow cytometry tubes following antibody staining and fixation.
2. Label the compensation tubes (Table 3).
   NOTE: Use 5 mL flow cytometry tubes compatible with the flow cytometer being used.
3. Add the amount of HBSS, antibody and/or dye to each of the sputum cell tubes and compensation tubes as indicated in Table 2 and Table 3, respectively.
   NOTE: Add buffer (HBBS), antibodies, and dye to all the tubes before adding cells or compensation beads to ensure that all tubes' staining time is consistent.
4. Add the amounts of sputum cell volume listed in Table 2 to the assay tubes.
5. Add compensation beads to the compensation tubes as listed in Table 3.
6. Incubate all the tubes (assay and compensation tubes) on ice, protected from light, for 35 min. Then, fill the tubes with ice-cold HBSS and centrifuge at 4 °C for 10 min at 800 x g.
7. For the compensation tubes, aspirate the supernatant as close to the pellets as possible and flick the pellets to loosen.
8. Add 0.5 mL of cold HBSS to the compensation tubes, store them on the ice at 4 °C and protect them from light until needed for flow cytometry analysis.
9. Aspirate the supernatant from the unstained isotype, blood, and epithelial tubes after centrifugation (step 3.6 from the antibody staining section) and loosen the pellets by flicking the tubes.

4. Fixation with 1% Paraformaldehyde (PFA)

1. Add cold 1% PFA (which should be thawed by now) to the unstained, isotype, blood, and epithelial tubes; 2 mL to the unstained and isotype tubes, and 10 mL to the blood and epithelial tubes.
2. Incubate the tubes on ice, protected from light for 1 h. Vortex quickly at maximum speed after 30 min.
3. Fill the tubes with ice-cold HBSS. Then, centrifuge tubes at 4 °C for 10 min at 1600 x g.
4. Aspirate as much supernatant as possible without disturbing the cell pellet and flick the tube with the fingers to loosen the cells.
5. Add 200 µL of cold HBSS to the unstained and isotype tubes.
6. Calculate the volume of HBSS for resuspension of the blood and epithelial tube according to the total cell count.
   NOTE: Resuspension volume = 0.15 x [total cell count (step 8 of sputum dissociation) / 10⁶]. Use 50 x 10⁶ as the cell count for an extra-large sample.
7. Store all sample and compensation tubes, protected from light on the ice at 4 °C until flow cytometry analysis is performed.
   ​NOTE: This protocol has not been tested for storage for more than 24 h.

5. Data acquisition on the flow cytometer

1. Apply appropriate startup procedures for the flow cytometer being used.
   NOTE: This section of the protocol assumes that the person operating the flow cytometer is trained in the use of the instrument available to them, especially concerning daily procedures including checking the stability of the optics and fluidics systems, techniques for standardizing light scatter and fluorescence intensity, as well as calculating and applying the correct compensation matrix.
2. Use the mixture of National Institute of Standards and Technology (NIST) beads to ensure that forward scatter and side scatter voltages are set to place the NIST beads to span the entire plot without placing the beads too close to the axes.
   NOTE: This step is crucial to ensure that debris smaller than 5 µm can be eliminated by gating post-acquisition analysis. Depending on the flow cytometer used, be mindful that the smallest beads are not being excluded with the threshold (when using the LSR II or the Lyric) or with the high discriminator (using the Navios EX flow cytometer). For the Navios EX, a gain of 2 was used for the forward and side scatter, a voltage of 236 for forward scatter, and 250 for side scatter. For the LSR II, a forward scatter voltage of 165 and a side scatter voltage of 190 was used.
3. Set the flow rate to medium (LSR II) or high (Navios EX).
   NOTE: The medium or high flow rate for most instruments can be used to acquire the sputum tubes. It is important to note that using too slow of a flow rate or too dilute of the sample may result in the cells settling, resulting in increased vortexing which is not desired. Therefore, adhering to the calculated resuspension volume in step 4.6 should result in cellular density that can be acquired quickly but not clog the machine.
4. Adjust the voltages for each parameter used for the scatter and fluorescent parameters to place the cell populations on the scale. Use the figures with the gating strategy as guidance on how to adjust the voltages accordingly.
   NOTE: Ensure that all the parameters needed are selected in the parameter selection window before the acquisition or that data will not be acquired.
5. Acquire data for the unstained sputum sample first, followed by the isotype-stained sample, and then the blood tube and the epithelial tube.
   NOTE: If the cell suspension is too concentrated to allow the flow cytometer to run the samples without clogging, the samples may be further diluted with HBSS.

Results

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...

Discussion

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...

Materials

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