Patent Publication Number: US-2021177384-A1

Title: Airway sampling device and associated methods

Description:
FIELD OF THE INVENTION 
     The present invention relates to an airway sampling device and associated methods. In particular, embodiments of the present invention seek to provide a non-invasive lower airway mucosal lining fluid sampling device and associated methods. 
     BACKGROUND OF THE INVENTION 
     Current methods to monitor inflammation in the airways utilise blood samples, exhaled breath samples, sputum samples, nasal samples and samples obtained during invasive bronchoscopy. 
     However, various problems are associated with these existing respiratory sampling techniques, and overall there is failure of the prior art when measuring inflammation with non-invasive sampling methods (blood, breath, sputum and nasal methods) when studying lung diseases. The following takes the example of measuring inflammation in asthma in order to illustrate the range of problems with blood, breath, sputum and nasal samples; but these samples are deficient in a range of lung diseases, and not merely asthma. 
     Blood analysis: blood sampling is from a site too distant from the airways; blood is influenced by many organs through the circulation around the body, and there is considerable dilution in a volume of approximately 5 L. In modern clinical practice in asthma there is a tendency to use the blood eosinophil count to assess the level of airway inflammation. This is reflected in a minimum level of blood eosinophils being required before selection of asthmatic patients for a monoclonal antibody therapy (anti-interleukin-5 or anti-IL-5). However, blood eosinophil counts vary greatly during the day with exercise and due to circadian steroid rhythms. 
     The eosinophil is regarded as an important target for patients with asthma, since it is a pro-inflammatory cell that migrates from the bloodstream into inflamed respiratory and gut sites (1, 2). Historically, the humble blood eosinophil count has been extensively used in the management of asthma (3-5). Recently, there has been renewed interest in using blood eosinophil counts to select asthmatic patients for monoclonal antibody therapy (6-10). A mathematical algorithm has been used to predict elevated sputum eosinophils: the eosinophil/lymphocyte and eosinophil/neutrophil index (ELEN) index (9). Moreover, the blood eosinophil count is favoured by recent American Thoracic Society/European Respiratory Society international guidelines on severe asthma, that suggest that the utility of other biomarkers in identifying asthma phenotypes needs further validation (11). However, blood eosinophil counts are notoriously variable, with levels increasing during the day (12) and exercise having the capacity to increase the eosinophil count (13). A recent study of 24-hour blood eosinophil counts noted increased variability in the blood eosinophil count of patients with moderate asthma (14). 
     Breath NO: levels of exhaled nitric oxide (NO, or FENO) are a crude measure of airway inflammation in asthma. However, levels are variable and very non-specific and can be changed by therapy, dietary factors, and the menstrual cycle in women. They do not provide a specific marker for asthma, where we need to study a range of protein, lipid and prostanoid mediators. 
     Exhaled breath condensate (EBC) analysis is confounded by the influence of condensed water vapour and the oropharynx; A major problem with current non-invasive sampling methods from the respiratory tract, including breath and sputum analysis, is contamination from the mouth (or oropharynx). Exhaled breath has been extensively studied as a non-invasive means to assess airway inflammation, including by measurement of mediators in exhaled breath condensate (EBC) (15). Richard Effros and colleagues have elegantly highlighted the issues of salivary contamination and dilution in condensed water vapour that occurs during collection of EBC (16-18); and this is likely to be a serious obstacle to measuring EBC pH (19) (20) and levels of inflammatory mediators that are in breath droplets. 
     Breath volatile organic compound (VOC) analysis and metabolomics looks to be more promising (21-24). However, VOCs do not include proteins such as cytokines, chemokines and antibodies. 
     Sputum contains dead and dying cells and mediator levels are influenced by bacteria, saliva, proteases, and sticky mucus proteins. Sputum was used to measure eosinophilia by the late Morrow Brown in his original studies from the 1950s showing the efficacy of oral prednisolone in asthma (25), although sputum has been of interest to clinicians since before the time of Hippocrates (26). The clinical application of quantitation of levels of eosinophils in induced sputum was pioneered by the late Freddy Hargreave (27). As an extension of this work, normalisation of sputum eosinophil counts has been shown by Ian Pavord and colleagues (Leicester and Oxford) to be effective in the reduction of asthma exacerbations (28). In addition, adult asthma phenotypes have been defined by sputum eosinophil and neutrophil percentages (29) (30). There are reports that blood eosinophil counts are a poor surrogate for sputum eosinophil counts (31, 32), while another group found that blood eosinophil counts can be used to predict sputum eosinophil counts (33, 34). The analysis of fluid-phase mediators derived from sputum samples has a large number of technical problems (35): these range from degradation by proteases and bacteria, loss of protein secondary structure due to reduction by dithiothreitol (DTT), binding to mucus, contamination with saliva and oropharyngeal contents, and variable leakage of mediators from dead and dying cells. Elegant attempts have been made to validate measurement of fluid phase levels of IL-5 in sputum (36), and this has highlighted the effects of proteases (37). 
     Nasal sampling is from the airways or respiratory tract, but the mucociliary escalator (MCE) takes nasal molecules from the anterior to posterior, from the nares to the pharynx. Hence the nasal MCE is non-continuous with the MCE up from the lower airways through bronchi and trachea. However, nasosorption is looking preferable to nasal lavage to measure inflammatory mediators, and does inform about airway inflammation from the upper respiratory tract. 
     Bronchoscopy sampling includes bronchial biopsy, bronchoalveolar lavage (BAL), bronchial brushes, and bronchosorption. Carrying out bronchoscopy to obtain bronchial mucosal biopsies and bronchial brush samples requires a team of specialist staff in an endoscopy suite, and the patient requires sedation and local anaesthesia. Biopsies, BAL, bronchial brushing samples and bronchosorption from the airways are useful samples for analysis: but the procedure is too erroneous for most asthmatics. Bronchoscopy is generally performed in selected patients with lung cancer, tuberculosis (TB) and interstitial lung diseases at specialised centres. 
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     STATEMENTS OF INVENTION 
     Aspects of the present invention seek to provide improved airway sampling devices and methods which seek to overcome or ameliorate one or more of the problems associated with the prior art. In particular, embodiments of the present invention aim to provide a non-invasive airway sampling device and sampling method for sampling airway mucosal lining fluid (MLF), and especially to obtain lower respiratory tract samples (originating from beyond the vocal cords) free from (or with only minimal) salivary and oropharyngeal contamination. 
     An aspect of the current invention is based on sampling droplets from the vocal cords and lower respiratory tract (the peripheral airways beyond the vocal cords). The aspect samples mucosal lining fluid (MLF) that is expelled from the lower respiratory tract by forced expiration or coughing. A key feature of an aspect of the invention is to minimise salivary contamination of the obtained sample. A further aspect of the invention is to non-invasively obtain a lower respiratory tract specimen without employing bronchoscopy. An important feature of lower airway MLF is that it passes continuously up the respiratory tract through the mucociliary escalator (MCE), and then passages through the vocal cords before being swallowed. Hence MLF from the vocal cords reflect airway events in the peripheral lower respiratory tract. The MLF in the small airways contains molecules and biomarkers that reflect disease in the underlying tissue. The small airway MLF is transmitted by the MCE to larger airways and up to the vocal cords. The inventors of the present invention have appreciated that it is of great benefit to assess respiratory diseases to capture the fluids from the vocal cords and lower airways in a non-invasive and precise manner, obtaining a sample from the lower respiratory tract (the trachea, bronchi and bronchioles) that is free from (or with only minimal) saliva and oropharyngeal contamination. 
     According to a first aspect of the present invention, there is provided an airway sampling device for taking a sample from a subject&#39;s airway, the device comprising a handle to be gripped by a user when taking the sample and a sampling head carried by the handle, the sampling head comprising a cavity with an opening for entry by the sample and a sample collection membrane located within the cavity for receiving the sample. 
     Preferably, the sample collection membrane comprises absorbent and/or adsorbent material. 
     Preferably, the sample collection membrane is detachable from the sampling head. 
     Preferably, the sample collection membrane comprises a perforation to facilitate its removal from the sampling head. 
     Preferably, the sample collection membrane comprises a notch to facilitate grasping of the sample collection membrane when detaching the sample collection membrane from the sampling head. 
     Preferably, the cavity has a gutter provided at least partly around its opening. 
     Preferably, the cavity is defined within a peripheral wall provided at least partly around the sampling head, and wherein outer surfaces of the peripheral wall are configured to be perpendicular to the tonsils of the subject when the sampling head is inserted into and/or removed from the subject&#39;s pharynx. 
     Preferably, the cavity is defined within a peripheral wall provided at least partly around the sampling head, and wherein an outer surface of the peripheral wall is configured to be perpendicular to the uvula and/or posterior wall of the oropharynx of the subject during capture of the sample. 
     Preferably, an outer surface of the peripheral wall is configured to deflect the uvula of the subject, allowing the sampling head to enter the pharynx from the oral cavity. 
     Preferably, the sampling head is connected to the handle via a stem. 
     Preferably, the sampling head, stem and handle are integrally formed. 
     Preferably, the handle is provided with a protrusion for engagement by a finger of the user, to facilitate grip of the handle. 
     Preferably, the handle is provided with a chamber, and the sampling head is movable relative to the handle between a first condition in which the sampling head is distal from the handle and a second condition in which the cavity is located over the chamber to define an enclosure which encloses the sample collection membrane between the interior of the cavity and the interior of the chamber. 
     Preferably, the enclosure is fluid-tight. 
     Preferably, a weakened area is provided in one of the chamber or the cavity. 
     Preferably, the weakened area is configured to rupture when pressure is applied to the enclosure. 
     Preferably, the weakened area is provided in the chamber, and the chamber is formed from a deformable material to allow a user to apply pressure to the enclosure. 
     Preferably, the weakened area is configured to permit a syringe needle to be inserted into the enclosure. 
     Preferably, the weakened area is configured to rupture when the sampling device is spun by a centrifuge. 
     Preferably, the interior of the cavity is provided with one or more protrusions on which the sample collection membrane is located. 
     Preferably, the interior of the cavity is provided with a plurality of protrusions in a chevron pattern on which the sample collection membrane is located. 
     Preferably, the interior of the chamber is provided with one or more protrusions which contact the sample collection membrane when the sampling device is placed into its second condition. 
     Preferably, the one or more protrusions are configured to push against the sample collection membrane when the user applies pressure to the enclosure. 
     Preferably, the sampling device comprises retaining means to retain the sampling device in its first condition and in its second condition. 
     Preferably, an edge of the handle comprises a scalloped area to facilitate movement of the sampling device into the first condition from the second condition. 
     Preferably, the sampling device further comprises an illumination module, and the sampling head is configured as a light guide to guide and emit light emitted from the illumination module. 
     Preferably, the illumination module is removably mounted on the sampling device. 
     Preferably the illumination module comprises a switch and the handle comprises a projection for actuating the switch to an on position when the sampling device is mounted to the sampling device. 
     Preferably the illumination module comprises a switch and the handle comprises a projection for actuating the switch to an on position when the sampling device is placed into its first condition. 
     Preferably the illumination module comprises an LED light source or a laser light source. 
     Preferably, the sampling head is provided at a first end of the sampling device distal from a second end of the sampling device at which the handle is provided, and the sampling device further comprises a shield mounted between the first and second ends of the sampling device, for shielding the user from sample from the subject. 
     Preferably, the airway sampling device is shaped and dimensioned so as to locate the opening of the cavity over the vocal cords and within the oropharynx posterior to the uvula of a subject when the sampling head is located at a sampling position in the patient&#39;s airway for taking the sample. 
     Preferably, the sampling head is angled relative to the handle, so as to present the plane of the opening of the cavity at an angle of between 25° to 45° downwardly from horizontal when the sampling head is located at the sampling position in the patient&#39;s airway. 
     Most preferably, the sampling head is angled relative to the handle, so as to present the plane of the opening of the cavity at an angle of 39° downwardly from horizontal when the sampling head is located at the sampling position in the patient&#39;s airway. 
     Preferably, the depth of the sampling device, from an uppermost surface of the handle to a lowermost tip of the sampling head is from 17 mm to 23 mm. 
     Preferably, the length of the opening is between 15 mm to 30 mm. 
     Most preferably, the length of the opening is 26 mm. 
     Preferably, the maximum width of the sampling head is between 10 mm to 16 mm. 
     Most preferably, the maximum width of the sampling head is 16 mm. 
     Preferably, an outer surface of the sampling head is designed so as to be perpendicular to at least one of the tonsils, uvula, and back of a subject&#39;s throat during placement, sample capture, and removal of the sampling device from the subject&#39;s airway. 
     According to a second aspect of the present invention, there is provided an airway sampling device for taking a sample from a sampling position within a subject&#39;s airway, the device comprising a handle to be gripped by a user when taking the sample and a sampling head for insertion into the subject&#39;s airway and being carried by the handle, the sampling head comprising a cavity with an opening for entry by the sample, and wherein the airway sampling device is shaped and/or dimensioned so as to locate the opening over the vocal cords and within the oropharynx, posterior to the uvula, of a subject when the sampling head is located at the sampling position in the patient&#39;s airway. 
     Preferably, the sampling head is angled relative to the handle, so as to present the plane of the opening of the cavity at an angle of between 25° to 45° downwardly from horizontal when the sampling head is located at the sampling position in the patient&#39;s airway. 
     Most preferably, the sampling head is angled relative to the handle, so as to present the plane of the opening of the cavity at an angle of 39° downwardly from horizontal when the sampling head is located at the sampling position in the patient&#39;s airway. 
     Preferably, the depth of the sampling device, from an uppermost surface of the handle to a lowermost tip of the sampling head is from 17 mm to 23 mm. 
     Preferably, the length of the opening is between 15 mm to 30 mm. 
     Most preferably, the length of the opening is 26 mm. 
     Preferably, the maximum width of the sampling head is between 10 mm to 16 mm. 
     Most preferably, the maximum width of the sampling head is 16 mm. 
     Preferably, an outer surface of the sampling head is designed so as to be perpendicular to the tonsils, uvula, and back of a subject&#39;s throat during placement, sample capture, and removal of the sampling device from the subject&#39;s airway. 
     According to a third aspect of the present invention, there is provided an airway sampling device for taking a sample from a subject&#39;s airway, the device comprising a handle to be gripped by a user when taking the sample and a sampling head carried by the handle, wherein the handle is provided with a chamber, and the sampling head is movable relative to the handle between a first condition of the sampling device in which the sampling head is distal from the handle and a second condition of the sampling device in which the sampling head is located adjacent the chamber. 
     Preferably, an enclosure is defined between the sampling head and the chamber when the sampling device is in the second condition. 
     Preferably, the enclosure is fluid-tight. 
     Preferably, a weakened area is provided in one of the chamber or the sampling head. 
     Preferably, the weakened area is configured to rupture when pressure is applied to the enclosure. 
     Preferably, the weakened area is provided in the chamber, and the chamber is formed from a deformable material to allow a user to apply pressure to the enclosure. 
     Preferably, the weakened area is configured to permit a syringe needle to be inserted into the enclosure. 
     Preferably, the weakened area is configured to rupture when the sampling device is spun by a centrifuge. 
     Preferably, a sample collection membrane is located within the sampling head. 
     Preferably, the interior of the sampling head is provided with one or more protrusions on which the sample collection membrane is located. 
     Preferably, the interior of the sampling head is provided with a plurality of protrusions in a chevron pattern on which the sample collection membrane is located. 
     Preferably, the interior of the chamber is provided with one or more protrusions which contact the sample collection membrane when the sampling device is placed into its second condition. 
     Preferably, the sampling device comprises retaining means to retain the sampling device in its first condition and in its second condition. 
     Preferably, an edge of the handle comprises a scalloped area to facilitate movement of the sampling device into the first condition from the second condition. 
     According to a fourth aspect of the present invention, there is provided a method of taking a sample from a subject&#39;s airway, the method comprising collecting a sample from a sampling position located above the vocal cords and within the oropharynx and posterior to the uvula of a subject. 
     Preferably, the step of collecting the sample comprises:
         positioning a sample collector within the subject&#39;s airway at the sampling position; and   prompting the subject to cough or give a forced exhalation, so as to produce the sample.       

     Preferably, the sample comprises mucosal lining fluid projected from the subject&#39;s vocal cords by the subject&#39;s cough or forced exhalation. 
     According to a fifth aspect of the present invention, there is provided a method of preparing a sample from a subject taken using the sampling device of the first aspect, the method comprising exposing the sample collection membrane to an elution buffer to elute the sample into the elution buffer. 
     Preferably, the method comprises removing the sample collection membrane from the cavity and placing it into the elution buffer. 
     Preferably, the handle of the sampling device is provided with a chamber, and the sampling head is movable relative to the handle between a first condition in which the sampling head is distal from the handle and a second condition in which the cavity is located over the chamber to define an enclosure which encloses the sample collection membrane between the interior of the cavity and the interior of the chamber, and wherein the method comprises introducing the elution buffer into the chamber and placing the sampling device into its second condition, to expose the sample collection membrane to the elution buffer. 
     Preferably, the method further comprises agitating the sampling device after the sampling device has been placed into its second condition. 
     Preferably, a weakened area is provided in one of the chamber or the cavity, and the method further comprises applying pressure to the enclosure to rupture the weakened area, to remove the elution buffer, containing the eluted sample, from the enclosure. 
     Preferably, the method further comprises inserting the needle of a syringe into the enclosure, and extracting the elution buffer, containing the eluted sample, from the enclosure using the syringe. 
     Preferably, a weakened area is provided in one of the chamber or the cavity, and the method further comprises placing the sampling device, still in its second condition, into a vessel and spinning the vessel using a centrifuge, rupturing the weakened area and introducing the elution buffer, containing the eluted sample, into the vessel. 
     Preferably, the method further comprises freezing the sampling device, still in its second condition, with the elution buffer, containing the eluted sample, still located within the enclosure. 
    
    
     
       LIST OF FIGURES 
       In order that the present invention may be more readily understood, embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which: 
         FIG. 1  is a schematic view showing a detail of part of the muco-ciliary escalator of a human subject; 
         FIGS. 2A TO 2E  schematically illustrate the cough function of a human subject; 
         FIG. 3  shows a first embodiment of a sampling device according to the present invention; 
         FIGS. 4A to 4C  show details of a sampling head of the first embodiment; 
         FIGS. 5A and 5B  show the sampling device of the first embodiment in a sampling position within a subject; 
         FIGS. 6 to 10  describe various preferable dimension and angling features of the first embodiment; 
         FIG. 11  shows a protective hood of the sampling device of the first embodiment; 
         FIG. 12  is a flow chart of a first sampling method according to an embodiment of the present invention; 
         FIGS. 13 to 15  show a second embodiment of a sampling device according to the present invention and various details thereof; 
         FIG. 16  shows assembly of the second embodiment; 
         FIGS. 17 and 18  show various preferable dimension and angling features of the second embodiment; 
         FIGS. 19A to 19E  show the second embodiment in various free standing conditions; 
         FIG. 20  shows the second embodiment being held by a user; 
         FIG. 21  is a flow chart illustrating a second embodiment of a sampling method according to the present invention, with  FIGS. 22 to 27  illustrating various steps of that method; 
         FIG. 28  shows a third embodiment of a sampling device according to the present invention; 
         FIGS. 29 to 31  shows a fourth embodiment of a sampling device according to the present invention; 
         FIG. 32  shows a fifth embodiment of a sampling device according to the present invention; 
         FIGS. 33 and 34  illustrate alternative sampling methods according to further embodiments of the present invention; and 
         FIGS. 25 and 36  show details of the second embodiment. 
     
    
    
     DESCRIPTION OF EXEMPLARY EMBODIMENTS 
       FIG. 1  is a schematic diagram showing a detail of a small section of the Muco-Ciliary Escalator (MCE) (shown generally at  1 ) in a human subject  3 . The MCE  1  transports Mucosal Lining Fluid (MLF) from the small airways up to the larynx and the vocal cords. In particular, ciliary beating carries MLF upwards from small bronchioles to larger bronchi and onwards to the trachea and to the larynx through the vocal cords. The MLF is then normally swallowed (at a rate of approximately 30 ml/day). 
     The vocal cords (in the larynx) are “the gateway to the lower respiratory tract” and airways. The MLF provides the body with a barrier against infection clearing out the airways carrying with it foreign particles and microorganisms. Due to the MCE, vocal cord MLF (from part of the larynx) reflects large and small airway molecular events. The surface MLF reflects information in the underlying airway wall and peripheral airway. This is relevant to biomarkers for example for vaccination, lung cancer, infection (whether viral, bacterial or fungal), inflammation, asthma/chronic obstructive pulmonary disease (COPD)/lung fibrosis/cystic fibrosis. 
     Embodiments of the present invention aim to collect pure vocal cord MLF, free (or with only minimal contamination) from saliva. To do so, embodiments of the present invention take advantage of the fact that the cough function of the human body expels MLF from the vocal chords to the oropharynx. By sampling this expelled MLF from a position within the oropharynx, pure vocal cord MLF, uncontaminated (or with only minimal contamination) by saliva, may be obtained, e.g. to allow analysis of biomarkers contained in the MLF. 
     The cough function is schematically illustrated with reference to  FIGS. 2A to 2E . Coughing forces air through the vocal cords at high speed (typically, air is expelled in a cough at velocities ranging from around 75 to 100 miles/hour). Tracheal and vocal cord MLF is expelled from the mouth by coughing, along with saliva from the uvula, tongue and oropharynx. 
     In more detail,  FIG. 2A  illustrates an inhalation phase of the cough function (typically triggered by airway irritation), which fills the lungs (generally at  5 ) with air. In the next stage of the cough function, shown in  FIG. 2B , the glottis is closed, and the abdominal muscles are compressed, to create pressure. In the following stage of the cough function, shown in  FIGS. 2C and 2E  (the latter being a cross-sectional view through the oropharynx, at the position indicated by the arrowhead in  FIG. 2D ), the glottis is opened and a cough-cloud  7  is emitted. As part of this process, MLF  9  is transmitted from the vocal cords to the oropharynx (see  FIG. 2E ). 
     A first embodiment of an airway sampling device  11  is shown in  FIG. 3 . The device  11  comprises a handle  13  to be gripped by a user (facilitated by a locator  14  provided on the upper surface of the handle  13  for contact with the user&#39;s forefinger), a stem  15  extending from the handle  13  and a sampling head  17  provided at the end of the stem distal from the handle  13 , and angled relative to the longitudinal axis of the handle  13 . 
     In the present embodiment, the handle  13 , stem  15  and sampling head  17  are provided as an integrally formed, unitary body e.g. by moulding. An integrally formed stem  15 , handle  13  and sampling head  17  is preferred to minimise the chances of any one of those components coming loose and being swallowed. However, in other embodiments, one or more of these parts of the sampling device  11  may be formed as separate parts which may then be attached, releasably or non-releasably, to the other parts to assemble the device. Also in the present embodiment, the sampling device  11  may be formed for example from plastics materials such as acrylonitrile butadiene styrene (ABS) or polypropylene (PP); however, different materials (either plastics or otherwise) may be used, as appropriate. 
     As shown in  FIG. 4A  (and in cross section in  FIG. 4B ), the sampling head  17  is provided with a perimeter wall  19  which extends generally perpendicularly to the axis of the stem  15  to create a protective hood  21  having a cavity or recess  22  to accommodate a sample collection membrane  23  in the form of a patch or small piece of (preferably absorbent and/or adsorbent) sampling material  23 , to collect the sample from the subject  3 . The perimeter wall  19  includes a gutter  25  around its upper edge, the purpose of which is explained later. 
     The sample collection membrane  23  of the present embodiment preferably comprises absorbent and/or adsorbent material, and may for example be Synthetic Absorptive Matrix (SAMT™) material. More generally, the sample collection membrane  23  materials could for example include, without limitation, a variety of synthetic and functionalised polymers in foam, fibrous or solid format. For example, and without limitation: polyurethane, fibrous hydroxylatred polyester (FHPE), polycaprolactone (PCL), nylon, cellulose acetate, cellulose, nitrocellulose, polyethersulfone, polysulfone, polypropylene, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), acrylic copolymer, white blood cell isolation media; also assay membranes for Point-of-Care (POC) diagnostics, lateral flow and flow through assays, blotting; also materials with antibodies and/or aptamers for diagnostic assays; and the like. 
     The sample collection membrane  23  is retained within the hood  21  by, but not limited to, adhesive bond, chemical weld, ultrasonic weld, or an overmoulding. 
     The sample collection membrane  23  is provided with an integral perforation  26  for its removal, post sample collection, with forceps or tweezers T, e.g. for analysis or retention by a clinician or other user (see  FIG. 4C ). To this end, the sample collection membrane  23  further includes a notch  27  at one end, to allow ready insertion of e.g. tweezers, to facilitate removal. 
       FIGS. 5A and 5B  show the sampling device  11  in situ according to an airway sampling method aspect of the present invention. In this condition, the sampling head  17  is located at a sampling position according to the present embodiment, which sampling position is above (for example, a few centimetres above) the vocal cords of a subject  3 , within the oropharynx and posterior to the uvula of the subject  3 . This allows for MLF, uncontaminated (or with only minimal contamination) by saliva, to be adsorbed or absorbed onto the sample collection membrane  23  carried by the sampling head  17  when the subject is prompted to give a small cough or forced expiration (i.e. a short, sharp breath out). 
     The sampling device  11  of the present embodiment is specifically designed to facilitate the placement of the sampling head  17  into the sampling position shown in  FIGS. 5A and 5B . 
     Firstly, various features of the sampling device  11  are dimensioned, angled and/or shaped to facilitate placement of the sampling head  17  into the sampling position shown in  FIGS. 5A and 5B . In a preferred embodiment, different variants of the sampling device  11  are provided, each version being dimensioned, angled and/or shaped for usage with a subject, allowing ready placement by a user of the sampling head at the sampling position, based upon an age grading of the subject. Ultimately, the decision of which sized sampling device  11  to use will be determined by a clinician e.g. to account for a subject who is significantly larger or smaller than average for their age. However, the present embodiment seeks to provide, for example, three different sizes, intended for use by subjects coarsely graded according to three different age groups. 
       FIGS. 6A and 6B  show dimensions (all in millimetres) and angles according to a currently most preferred embodiment for usage with an adult human subject (aged 16 or over);  FIGS. 7A and 7B  show preferred ranges for these dimensions (all in millimetres) and angles;  FIGS. 7C and 7D  show currently preferred dimensions (all in millimetres) and angles for a large-sized sampling device  11  intended for an adult subject (aged 16 or over);  FIGS. 7E and 7F  show currently preferred dimensions and angles for a medium-sized sampling device  11  intended for a subject aged between 12 to 15; and  FIGS. 7G and 7H  show currently preferred dimensions and angles for a small-sized sampling device  11  intended for a child subject (aged 8 to 11). 
     According to the present embodiment, the width of the sampling head  17  (this width being the dimension labelled in  FIGS. 7C, 7E and 7G ) is selected to maximise sampling material size (and hence maximising sample capture), without causing undue discomfort for a patient or subject  3 . In particular, the width X of the sampling head  17  is designed so as to comfortably clear the corresponding distance X′ between the tonsils of the subject  3  (see  FIG. 8A ) and especially to avoid and/or minimise interreference with the tonsils of a subject during the sample collection process, especially for a subject suffering from a viral or bacterial infection causing the tonsils to swell. In the present embodiment, the width ranges from 10 mm to 16 mm and, as an illustrative example only, may preferably be 16 mm for an adult/large sized device, 14 mm for an intermediate aged/medium sized device and 12 mm for a child/small sized device. A head width of 10 mm or more is advantageous, as it maximises sample capture and allows for a good-sized sample collection membrane  23  to be located within the protective hood  21  of the sampling head  17 . A head width of 16 mm or less is also desirable, to avoid discomfort for the subject, and especially to avoid and/or minimise interreference with the tonsils of a subject during the sample collection process, especially for a subject suffering from a viral or bacterial infection causing the tonsils to swell. 
     Next, and referring to  FIGS. 8C and 8B , respectively, the open angle θ of the sampling head  17 , along with the overall vertical depth Z of the sampling device  11  (measured from the uppermost point of the sampling device handle  11  to the lower-most point (the tip) of the downwardly-angled sampling head  17 ), are designed to maximise the sampling material sampling area (i.e. to maximise the exposure to expelled MLF), without significantly restricting airflow. Here, the “open angle” of the head  17  means the angle θ of the sampling head  17 , and more specifically the plane of the opening of the recess  22  within the hood  21  (which plane also preferably corresponds to the plane of the sample collection membrane  23 ) relative to horizontal, when the sampling device  11  is positioned in situ in the sampling position shown in  FIG. 8C , with the sampling head  17  located at the desired sampling position (i.e. above the vocal cords within the oropharynx, posterior to the uvula). In the present embodiment, the preferred range of open angle θ° of the sampling head  17  is 25° to 45°, with a most preferred angle of 39°, regardless of the age of the subject. 
     Here, and as explained with reference to  FIG. 9 , an open angle of at least 25° relative to horizontal is preferred, to avoid significantly restricting the airflow of the subject (and hence to avoid reducing the volume of the airborne sample). On the other hand, an open angle of 45° or less relative to horizontal is preferred, to avoid reducing the amount of airborne sample landing on the sample collection membrane, either as a result of sample escaping around the back of the sampling head  17 , without coming into contact with the sample collection membrane  23 , or simply impinging upon the protective hood  21  of the sampling device  11 . As shown in  FIG. 10 , the preferred angling and length of the sampling head  17  maximise airflow and the amount of sample impacting on the sample collection membrane  23 . 
     The overall depth Z of the sampling device  11  is preferably varied according to the age of the subject; purely as an illustration, for a sampling device  11  intended for use with an adult (aged 16 or over), the depth Z may for example be 23 mm; for a sampling device  11  intended for use with an intermediate-aged subject adult (aged 12 to 15), the depth Z may for example be 20 mm or 21 mm; for a sampling device  11  intended for use with a child (aged 8 to 11), the depth Z may for example be 17 mm. 
     The following table  1  recites currently preferred optimal values for the head width X, depth Z and sampling head angle θ. It is however to be appreciated that the following preferred optimal values, as well as all of the foregoing described angles and dimensions, are strictly non-limiting and illustrative only, and that other angles and dimensions may be used as appropriate. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Optimal Sizes 
                 Age 
                 X 
                 Z 
                 θ 
               
               
                   
                   
               
             
            
               
                   
                 Small 
                  8-11 
                 12 
                 17 
                 39° 
               
               
                   
                 Medium 
                 12-15 
                 14 
                 20 
                 39° 
               
               
                   
                 Large 
                 16+ 
                 16 
                 23 
                 39° 
               
               
                   
                   
               
            
           
         
       
     
     Next, and also with reference to  FIG. 10 , the outside surface  29  of the sampling head  17  is smooth and radiused so as to readily deflect the uvula  31  of the subject  3  towards the rear of the oropharynx, allowing the sampling head  17  to adopt the optimal sampling position shown in  FIG. 10 , centrally above the airway of the subject. For example, and simply as an illustration, a mid-point of the outside surface of the sampling head  17  may present the angles such as shown in  FIGS. 7D and 7F  (38° and 36° relative to horizontal when in the sampling position, respectively) to facilitate deflection of the uvula. However these angles are merely illustrative and other angles nay be used, as appropriate. 
     The sampling head  17  is further configured to minimise and/or eliminate sample collection membrane contamination e.g. from saliva or from lymph fluid from the tonsils. Firstly, and as explained above, the sampling head  17  is provided with a wrap-around hood  21  which encloses the sample collection membrane  23  on all sides (other than at the opening to the recess within the hood  21 ), and hence enables the sampling head  17  to push past the tonsils, to upwardly deflect the uvula, and potentially to also contact the back of a subject&#39;s throat, without any (or with only minimal) fluid contamination of the sample collection membrane  23 . To prevent direct surface contact contamination from these areas the outer surface of the hood  21  is designed to be perpendicular to these landmarks, as shown in  FIG. 11 , during placement, sample capture, and removal of the sampling device  11  from the subject&#39;s airway. 
     As a further measure, and as noted above, the hood  21  is provided with an integral gutter  25 . When the sampling device  11  is inverted for sample processing, there is a risk that fluids such as saliva or lymph fluid could flow over the peripheral edge of the sampling head  17 , potentially contaminating the sample collection membrane  23 . The integral gutter  25  avoids or ameliorates this risk by capturing these fluids, and allowing them to safely drain away as indicated by the pointed arrows in  FIG. 4A . Flow is gravity fed and dependent on fluid viscosity, and allows fluid to drain off safely outside of the sampling area of the sampling device  11 . 
     In addition to the design of the sampling head  17 , the stem  15  is designed to be thin to minimise contact with the tongue and mouth of a subject  3 , thus minimising the gag reflex. For example, and as illustrated in  FIG. 6A , the stem width may preferably be 8 mm, for a sampling device  17  intended for use with an adult. Also, the sampling device  11  is preferably flexible, to minimise accidental trauma to the subject under testing. 
     In summary, the sampling device  11  of the present embodiment is designed to position the sample collection membrane in the oropharynx (behind the uvula), protected from saliva and other fluids from the mouth, tongue and uvula. On coughing, the sample collection membrane  23  catches (by impingement) tiny droplets of MLF from the vocal cords and originating from the lower airways. 
     An airway sampling method according to an embodiment of the present invention, using the sampling device  11  described above, will now be described with reference to  FIG. 12 . 
     As a preliminary step  1201 , the back of the subject&#39;s throat is sprayed with lignocaine or other local anaesthetic, to minimise discomfort and to reduce the risk of a gag-reflex. 
     Next, at step  1202 , and with the subject&#39;s mouth wide open, the sampling head  17  of the sampling device  11  is inserted into the patient&#39;s mouth, taking care to avoid saliva contamination to the sampling material from the tongue. Although not necessary, a tongue depressor may optionally be used during this step, to depress the tongue of the subject for greater visibility of the mouth and throat. 
     At step  1203 , the rear surface of the sampling head  17  is used to upwardly lift the uvula, as necessary, so that the sampling head  17  is positioned centrally over the subject&#39;s airway, and in particular over the subject&#39;s vocal chords, within the oropharynx and posterior to the uvula. 
     Next, at step  1204 , the subject is prompted to cough or give a forcible expiration (i.e. a sharp exhalation). As explained above, this results in MLF expelled from the vocal cords to be collected, uncontaminated (or with only minimal contamination) from saliva and other fluids. 
     Finally, at step  1205 , the sampling device is removed from the patient&#39;s airway, allowing the sample collection membrane  23  to be removed from the sampling head  17  e.g. for analysis or storage. 
     A second embodiment of a sampling device  11  according to the present invention is shown in  FIG. 13 , in which the same or similar features are given the same reference numerals, and only the differences from the first embodiment will be described. As explained in the following, the primary distinction is that the second embodiment allows for an integrated sample washing and elution function. 
     To this end, and unlike the first embodiment, the second embodiment is firstly provided with a washing and elution chamber  33  at the far end of the handle. As best shown in  FIGS. 14A to 14C , the washing and elution chamber  33  has a perimeter wall  35  upstanding from and surrounding the entire circumference of a bottom wall  37 , to define a cavity  39  within. Within the perimeter wall  35 , a plurality of upstanding columns  41  are provided, spaced at regular intervals. In use, and as explained below, the chamber  33  is designed to be compressed (i.e. squeezed) by a user, as part of the sample washing and elution function. The 41 columns push in a spreading motion against the saturated sample collection membrane (e.g. SAM) to maximise the recovery of the eluted MLF, when the user compresses the chamber  33  e.g. with their thumb T, as shown in  FIGS. 35 and 36 . Accordingly, the chamber  33  is preferably formed from a deformable, resilient material (such as thermoplastic vulcanizate, TPV, although other suitable materials may equally be employed). In the present embodiment, the chamber  33  is further provided with a peripheral undercut feature  43  which locates over an annular flange  45  within the handle, holding the chamber  33  in place, with the bottom wall  37  protruding from the handle  13  to define a button, to facilitate squeezing of the chamber  33  by a user. The outer surface of the bottom wall  37  is provided with a roughened or textured area  41  to facilitate a user&#39;s steady grip on the bottom wall  37  during squeezing of the chamber  33 . Preferably, the opening of the chamber  33  is closed by a removable protective cover  46  (see  FIG. 14D ), to prevent/reduce contamination risks prior to and during the sample collection process. The bottom wall  37  of the chamber  33  is further provided with a generally circular weakened area  47  of reduced thickness (see  FIG. 13 ), the purpose of which is explained later. 
     Secondly, the device  11  of the present embodiment is provided with a hinge  49  connecting the handle  13  to the stem  15  (see  FIG. 15A ), and thus permitting the stem  15  and sampling head  17  to be rotated relative to the handle  13  between the unfolded condition shown in  FIG. 13  or  FIG. 15A  (for use in sample collection) and a folded condition as shown in  FIG. 15B  (for sample washing and elution, as well as sample storage and, optionally, initial shipping). 
     As in the first embodiment, the sampling head  17  of the present embodiment is configured to carry a sample collection membrane  23  such as a piece of absorbent and/or adsorbent sampling material (e.g. SAM™), to collect a sample from a subject&#39;s airway. In this embodiment, however, the interior of the sampling head  17  is further provided with a series of protrusions  50 , arranged in a chevron pattern (see  FIGS. 15C and 15D ), on which the sample collection membrane  23  is placed, with its edges located on a peripheral ledge  51  of the interior head surrounding the chevron-patterned protrusions (see  FIG. 15E ). The sample collection membrane  23  may be attached to the peripheral edge of the sampling head  17  by, but not limited to, adhesive bond, chemical weld, ultrasonic weld, an overmoulding. Again, the sample collection membrane  23  is provided with an integral perforation  26  for optional removal with forceps or tweezers T (see  FIG. 15F );  FIG. 15G  shows the interior of the sampling head  17  with the sample collection membrane  23  removed. 
     As shown in  FIGS. 16A to 16G , the hinge  49  is preferably a snap-fit hinge, in which the stem  15  and sampling head  17  assemble to the handle  11  via a snap-fit. To assemble, the hinge centre  51  of the stem  15  is pushed between hinge studs  53  provided in the handle  13 , which causes the handle sides to flex rotate, allowing the stem  15  to pass the hinge studs. When the hole  55  at the hinge centre of the stem  15  and the hinge studs  53  in the handle  15  are in-line, the pre-loaded force within the flexible sides of the handle  15  force the hinge studs to snap-in, captivating the hinge assembly. 
     Preferably, tapered hinged studs  53  are used. Tapered studs offer two advantages—firstly, they significantly improves assembly; secondly, the increased surface area contact gives the hinge greater transverse stability. 
     In the present embodiment, the hinge centre of the stem  15  is designed with a deliberate interference, therefore, once assembled, there is a frictional contact between both components (stem  15  and handle  17 ). 
     Once assembled, and as shown in  FIG. 16G , there are two positional snap-fits, 165° apart, one in the extended “sampling” position and the other in the housed “washing and elution” position. Movement between these positions is facilitated by the scalloped finger locater curves  57  provided in the handle  17  (see  FIG. 16B ). 
     As with the first embodiment, the present embodiment may be provided in different sizes, shapes and dimensions for usage with different sized-subjects, preferably based as a guideline on the age of the subject. As with the first embodiment, for example, an adult/large size sampling device  11  may be produced for preferred use with a subject aged 16 or over—see  FIGS. 17A and 17B, 18C and 18D  for (merely illustrative) preferred dimensions and angles, with a preferable range of dimensions and angles as shown by  FIGS. 18A and 18B . Further, a medium sized device  11  may preferably be dimensioned and angled as shown in  FIGS. 18E and 18F , for preferable usage with a subject of intermediate age (age 12 to 15) and a small sized device  11  may preferably be dimensioned and angled as shown in  FIGS. 18G and 18H , for preferable usage with a subject of child age (age 8 to 11). Here, the benefits of using the angles and dimensions shown is the same as for the first embodiment described above, again all dimensions shown are in millimetres, and again, although preferable, all dimensions and angles shown are illustrative and non-limiting and other sizes and dimensions and angles may be used e.g. for different sized subjects. 
       FIGS. 19A to 19E  show various free standing positions of the sampling device  11  of the present embodiment. As shown in  FIG. 19A , the sides of the handle  13 , stem  15  and sampling head  17  are designed to allow the sampling head  17  to stay motionless when placed on its side, without rolling.  FIGS. 19B and 19C  show the device  11  in its unfolded/sampling condition, and placed on a level surface so as to rest on the sampling head  17  and sides of handle  13 .  FIGS. 19D and 19E  shows the sampling device  11  placed on a level surface in an inverted condition, resting on a finger-grip portion of the handle  13  and the button provided by the bottom wall  37  of the washing and elution chamber  33 , e.g. for insertion of a washing and elution buffer as explained below in connection with  FIG. 21 . 
       FIG. 20  shows the sampling device  11  of the present embodiment in a hand grip position for a user to conduct the sampling process described below in connection with  FIG. 21 . 
     Operation of the second embodiment of the sampling device, according to a second embodiment of a sampling method of the present invention, will now be described with reference to the flow chart of  FIG. 21 . 
     Firstly, in step  2101 , and aided by the scalloped finger locators  57 , a user pinches/pulls the stem  15  to open device  11  (see  FIG. 22 ), and rotates the stem  15  and sampling head  17  relative to the handle  11 , to click it into the fully unfolded sampling position. 
     In Step  2102 , to reduce the risk of a gag-reflex, the back of the subject&#39;s throat is sprayed with lignocaine or other local anaesthetic. As will be appreciated, the order of steps  2101  and  2102  may be reversed, or these steps may be performed simultaneously e.g. by two clinicians working in tandem. 
     In step  2103 , with the subject&#39;s mouth wide open, the sampling head  17  of the device  11  is inserted into the subject&#39;s mouth (see  FIG. 23 ), taking care to avoid saliva contamination from the tongue. Although the device is useable on its own, for greater visibility of the mouth and throat, the device  11  may optionally be inserted whilst the subject&#39;s tongue is depressed using a suitable tongue depressor. 
     In step  2104 , the sampling head  17  is used to deflect the subject&#39;s uvula, as necessary, until the device  11  is position centrally over the subject&#39;s airway (see  FIG. 24 , with the stem shown in cross-section and the handle omitted, for clarity) and the sampling head located at the desired sampling position namely over the vocal cords, within the oropharynx and posterior to the uvula of the subject. 
     In step  2105 , the subject  3  is asked to cough or give a forced expiration (a sharp exhalation), thus allowing a sample of MLF to be collected by the sample collection membrane  23  located within the sampling head  17  of the device  11 , uncontaminated (or with only minimal contamination) by saliva or other fluids. 
     In step  2106 , the sampling device  11  is entirely removed from the subject&#39;s airway. 
     If the sample is to be stored for future sample preparation, the process proceeds to step  2107 , in which the protective cover  46  is removed from the chamber  33 , and the stem  15  and sampling head  17  are rotated towards the handle  13  until the closed condition is adopted, protecting the sample from extraneous contamination; the closed sampling device  11 , including its collected sample, may then be frozen. 
     On the other hand, if a user wishes to directly wash and elute the sample, the process proceeds to step  2108 . In this step, the protective cover  46  is again removed from the chamber  33 , and elution buffer is introduced into the chamber  33  e.g. via a pipette P as shown in  FIG. 25A . In the present embodiment, the chamber  33  has, merely as an example, a maximum capacity of 504.1, although other sized chambers may of course be employed, as appropriate. 
     Next, in step  2109 , the stem  15  and sampling head  17  are rotated towards the handle  13  to bring the device  11  into its fully folded condition (see  FIG. 25B ). As will be appreciated, the sample collection membrane  23  will now be located between the chevron-patterned protrusions of the sampling head  17  on one side, and the tops of the protruding columns  41  provided within the washing and elution chamber  33  on the other side. 
     Next, in step  2110 , the user shakes the folded device  11 , causing the elution buffer to wash the sampling material  23  now located within the chamber (see  FIG. 26 ). Here, the washing of the sample collection membrane  23  is facilitated by the fact that the elution buffer is able to travel freely around and between the columns  41  within the chamber and the chevron-patterned protrusions within the sampling head  17 , thus readily exposing both sides of the sampling material of the sample collection membrane  23  to the elution buffer and hence maximising MLF capture from the sample collection membrane  23 . 
     Next, in step  2111 , the user orientates the device  11  with the circular weakened area  47  located over a suitable collection vessel V (see  FIG. 27A ). 
     Finally, in step  2112 , the user squeezes the button defined by the bottom wall  37  of the chamber  33 . The resultant pressure increase within the chamber  33  causes the weakened area  47  to rupture, ejecting the liquid contents (i.e. the elution buffer containing MLF washed from the sampling material) (see  FIG. 27B ) into the collection vessel V, e.g. for analysis or storage. 
     Hence, the process described above provides a user with a ready and convenient means of sample extraction. However, the sample extraction process of  FIG. 21  is only one example, and other sample extraction processes are possible. Some exemplary alternative sample extraction process are described later. First, some further sampling device embodiments are described, in which like features are given the same reference numerals, and the discussion will focus only on the distinctions from the first and/or second embodiments of the sampling device described above. 
     A third embodiment of a sampling device  11  is shown in  FIGS. 28A to 28E . The sampling device  11  of the present embodiment is very similar to the second embodiment described above, but is additionally provided with a transverse slot or groove  57 , located forward (i.e. towards the sampling head end) of the finger locator  14  of the handle, into which a generally circular cough shield  59  is located (as shown in  FIGS. 28A and 28B ) to form the completed device  11  shown in  FIGS. 28C and 28D . The third embodiment is otherwise the same as the second embodiment. 
     The cough shield  59  is preferably made from a thin sheet of plastics material (e.g. Polyethylene Terephthalate Glycol (PETG) or Polycarbonate (PC)) although other suitable materials (e.g. metals) may be used, as appropriate. In the present embodiment, the cough shield  59  offers a user ≃315° protective coverage from the cough cloud generated by the subject during airway sampling, with the remaining ≃45° of the cough cloud passing underneath the winged sides of the handle. A slot  61  is provided in the cough shield  59 , offering sufficient clearance for the sampling head  17  to be freely rotated between the folded and unfolded conditions of the sampling device  11  (see  FIG. 28E ). 
     As will be appreciated, the first embodiment of a sampling device  11  described above may likewise be modified to similarly include a cough shield  59 , locating into a slot  57  to be provided, according to this modification, in the handle of the device  11 . 
     A fourth embodiment of a sampling device  11  is shown in  FIGS. 29 and 30 . This embodiment modifies the second embodiment described above, to include an illumination module  65  within the handle  13 , and to configure the stem  15  and sampling head  17  as a light guide device, beneficially allowing for the interior of a subject&#39;s mouth to be illuminated to facilitate the correct positioning of the sampling device  11  during the sampling process. 
     In more detail, and as shown in the various parts of  FIG. 29 , the stem  15  and sampling head  17  of the present embodiment are formed from a suitable light-transmissive material or materials so as to act as a light guide. For example, the stem  15  and sampling head  17  of the present embodiment may be formed from optically clear thermoplastic styrene-butadiene copolymers (SBC) or optically clear polycarbonates (PC) which are designed to glow with light from an external light source. 
     Next, the handle  13  of the present device is adapted to include a location groove  63  (see  FIG. 29A ) for accommodating an illumination module  65 . In the present embodiment, the illumination module  65  includes a snap hook  67  (see  FIG. 29 b   ), and the handle  13  further comprises a snaphook hole (not shown) to receive the same, to securely retain the illumination module  65  in the handle  13  (see FIGS.  29 C and  29 D). In the present embodiment, the snap hook  67  may be disengaged from the snaphook hole, allowing the illumination module  65  to be removed for insertion into one or more other sampling devices  11 ; that is, one illumination module  65  may be re-used (after appropriate cleansing) and shared amongst a plurality of different illuminated sampling devices  11 . The illumination module  65  is sealed against liquid and dirt ingress and for example may be constructed with an ABS moulded housing, with the necessary electronics potted in place using e.g. a TPE overmoulding process. 
     Preferably, the illumination module  65  may include a switch  71 , which may be actuated by a light activating spigot optionally provided within the handle  13 . This arrangement may for example allow for the light to be automatically switched on when the illumination module  65  is inserted into the handle  13  and switched off when the illumination module  65  is removed from the handle  13 . Alternatively, the switch  71  may allow for the light to be automatically switched on when the sampling device  11  is brought into its unfolded (sampling) condition, and switched off when the sampling device  11  is in the folded condition. Alternatively, a manual on/off switch may be provided for manual activation by a user. 
     As for the illumination module  65 , any suitable illumination device may be employed, but for example these may include e.g.: 
     1) A laser light source, for example a laser with a wavelength between 450-500 nm (blue-cyan). 
     2) An LED light source, for example an Ultrabright White directional LED. 
     An example of a laser light source is shown in  FIG. 30A , with the light emitting element shown in detail in  FIG. 30B . The latter may include, for example, a suitable power supply  69  such as two 3 Volt batteries wired in parallel, for example 5.5 mAh Lithium Manganese Silicon Batteries having Dimensions 06.8 mm, 2.1 mm thick (Part number: MS621). Also shown in  FIG. 30B  is a low profile, tactile, surface mount switch  71  e.g. for automatic activation by a light activating spigot optionally provided within the handle  11  as described above. The light emitting element further comprises a laser diode  73 , for example a 3.3 mm laser diode with driver module. 
     An example of an LED light source is shown in  FIG. 31A , with the light emitting element shown in detail in  FIG. 31B . The latter may include, for example, a suitable power supply  69  such as two 1.55 Volt batteries wired in series, for example 16 mAh Silver Oxide Batteries having dimensions 06.8 mm, 1.65 mm thick (Part number: SR65). Also shown in  FIG. 31B  is a low profile, tactile, surface mount switch  71  e.g. for automatic activation by a light activating spigot optionally provided within the handle, as described above. The light emitting element further comprises an LED light source  75 , for example a 3 mm ultra bright directional LED. 
     In the same way that the second embodiment may be modified to include an illumination feature, according to a fifth embodiment of the present invention, the first embodiment of the sampling device  11  described above may also be modified as shown in  FIG. 32  to include a light module  65  within the handle  13 , beneficially allowing for the interior of a subject&#39;s mouth to be illuminated to facilitate the correct positioning of the sampling device  11  during the sampling process. In this embodiment, as it is provided integrally with the sampling head  17  and stem  15  of the device  11 , the handle  13  is also configured as a light guide device. In particular, the integral handle  13 , stem  15  and sampling head  17  of the present embodiment are preferably formed from a suitable light-transmissive material or materials so as to act as a light guide. For example, the handle  13 , stem  15  and sampling device  17  of the present embodiment may be integrally formed from optically clear thermoplastic styrene-butadiene copolymers (SBC) or optically clear polycarbonates (PC) which are designed to glow with light from the illumination module  65 . 
     As the present fifth embodiment (like the first embodiment) does not have a folding function, a light activating spigot is not provided in the handle  11 . However, a manual light switch is provided for activation by a user, so as to switch on the illumination device during the sampling process. In other respects, such as the nature of the illumination module, the fifth embodiment may generally be the same as for the fourth embodiment described above, and hence is not re-described here. 
     It will be appreciated that, according to further embodiments of the present invention, the cough shield feature of the third embodiment may also be combined with the fourth and fifth embodiments having the light guide feature. 
     The following describes some alternative sample extraction methods, suitable for usage with embodiments of the sampling device having a washing and elution chamber (e.g. the second, third and fourth embodiments described above). 
     According to a further embodiment of a sample extraction method, as shown in  FIG. 33 , rather than a user squeezing the chamber to cause rupture of the weakened area, a user may instead extract the liquid content (elution buffer containing MLF) by inserting a needle N of a syringe S into the weakened area, and pulling back on the plunger of the syringe to extract the sample. The extracted sample may then be processed as desired e.g. ejected from the syringe into a suitable vessel for direct analysis or transferred into a cryogenic storage container and frozen. 
     According to a still further embodiment of a sample extraction method, as shown in  FIG. 34 , a centrifuge method may for example be employed. According to this embodiment, after conducting the sample gathering process, introducing elution buffer introduced into the chamber and placing the sampling device into its folded condition, a user places the sampling device into a suitable centrifuge tube T (e.g. a cryogenic 50 ml centrifuge tube), with the tube then being closed by a cap C. The centrifuge tube is then located into a suitable centrifuge, which is then operated to spin the centrifuge tube e.g. to spin-down for 30 seconds @ 4000 rpm. This causes the weakened area to rupture, so that the liquid contents (elution buffer containing sampled MLF) collect at the bottom of the centrifuge tube T. A user then removes the cap from the tube, removes the device, and re-caps the tube e.g. for freezing or analysis. 
     The embodiments described above relate to airway sampling from a human subject. However, this is merely exemplary, and according to further embodiments the present invention may instead be applied to sampling devices and associated sampling methods for airway sampling performed on non-human subjects e.g. livestock such as cattle or pets such as cats and dogs. 
     The embodiments above assume that a user e.g. a nurse, doctor or other clinician would take a sample from a subject. However, potentially, a subject may take a sample from themselves, in which case the “user” and the “subject” are the same person. 
     The foregoing description has been given by way of example only and it will be appreciated by a person skilled in the art that modifications can be made without departing from the scope of the present invention as defined by the claims.