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WO2025099417A1 - Appareil d'échantillonnage - Google Patents

Appareil d'échantillonnage Download PDF

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Publication number
WO2025099417A1
WO2025099417A1 PCT/GB2024/052810 GB2024052810W WO2025099417A1 WO 2025099417 A1 WO2025099417 A1 WO 2025099417A1 GB 2024052810 W GB2024052810 W GB 2024052810W WO 2025099417 A1 WO2025099417 A1 WO 2025099417A1
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WO
WIPO (PCT)
Prior art keywords
lumen
fluid
distal end
probe
sampling
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Pending
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PCT/GB2024/052810
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English (en)
Inventor
Craig MCDOUGALL
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University of Edinburgh
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University of Edinburgh
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Publication of WO2025099417A1 publication Critical patent/WO2025099417A1/fr
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Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • A61B10/04—Endoscopic instruments, e.g. catheter-type instruments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/012—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor characterised by internal passages or accessories therefor
    • A61B1/015—Control of fluid supply or evacuation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/267—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor for the respiratory tract, e.g. laryngoscopes, bronchoscopes
    • A61B1/2676—Bronchoscopes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
    • A61B10/0045—Devices for taking samples of body liquids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2217/00—General characteristics of surgical instruments
    • A61B2217/002—Auxiliary appliance
    • A61B2217/005—Auxiliary appliance with suction drainage system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2217/00—General characteristics of surgical instruments
    • A61B2217/002—Auxiliary appliance
    • A61B2217/007—Auxiliary appliance with irrigation system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/71—Suction drainage systems
    • A61M1/77—Suction-irrigation systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/84—Drainage tubes; Aspiration tips
    • A61M1/85—Drainage tubes; Aspiration tips with gas or fluid supply means, e.g. for supplying rinsing fluids or anticoagulants

Definitions

  • the present invention relates to an apparatus and method for sampling a biological substance.
  • the invention relates to an apparatus and method for endoscopic sampling.
  • Pulmonary Epithelial Lining Fluid is an aqueous layer coating surfaces in the lung comprised of phospholipids, proteins and surfactant that promotes healthy lung function and optimal gas exchange.
  • the ELF acts as a barrier to irritants and pathogens. Furthermore, it enhances mucociliary clearance and plays a role in immune response.
  • the ELF is distributed across the conducting airways and alveolar regions of the lungs.
  • the ELF contains a variety of cellular and molecular components that can provide information on lung disease and infection.
  • the ELF reveals information on inhaled components and penetration of anti-infective agents which is crucial in monitoring inhaled drugs and modelling lung deposition.
  • Cytological analysis of ELF cellular components in the airway secretions may be used for differential diagnosis of disease, however much work remains in developing an understanding of the distributions of different cell populations in lung regions throughout the bronchial tree. Such work relies on the capabilities of available sampling tools.
  • Proteomic analysis of the ELF allows evaluation of protein content and provides information on protein function and expression in the lungs, informing understanding of fundamental lung biology and disease. It may also provide information on the efficacy of gene therapy treatments, by demonstrating evidence of expression of a delivered transgene in protein secretions.
  • a fundamental challenge is detection of low abundance protein targets in the ELF.
  • the ELF When the ELF is sampled using any of the methods discussed below, it becomes diluted in either the fluid used for sampling or the fluid used for elution. This necessitates an accurate estimation of ELF concentration in the sample fluid for providing useful conclusions in downstream analysis.
  • urea-based methods Endogenous markers are preferable and use of the urea-based method commonly employed.
  • urea-based method commonly employed to allow significant diffusion of urea from regions outside the sampled region has been observed from blood and surrounding locations due to the high mobility of urea.
  • urea diffusion into the sample region is greatly impacted by the state of pulmonary surfactants in the ELF confounding difficulties. It is widely understood that urea levels may be artificially inflated resulting in an overestimation of the ELF concentration in the sample.
  • Methods for sampling pulmonary fluids generally fall into blind and guided methods.
  • Blind lung fluid sampling covers a variety of non-bronchoscopic methods including mini-BAL (Broncho-Alveolar Lavage), non-bronchoscopic NB BAL, s-Cath/s- BAL or protected BAL that were first reported in the 1980s.
  • Non-bronchoscopic sampling involves blindly advancing a suction catheter into the distal airways where it becomes wedged enabling either sampling of oedema fluid (s-cath) or a lavage wash (mini- BAL/NB-BAL/protected BAL).
  • Protected BAL minimises contamination from the upper respiratory tract by employing an endotracheal tube to wedge in the airway and introducing the sampling catheter through this tube, however the principal sampling mechanism of performing a low volume BAL through a single lumen sampling catheter is consistent between methods.
  • the reported sensitivity and specificity of these methods is similar to bronchoscopic BAL. Chest radiography suggests the catheter becomes wedged in the right bronchial tree. However, there is concern that blindly sampling in this way may lead to false negatives if sampling from an area of the lung that is unaffected by infection.
  • Blind methods are not standardised and vary on the catheter type, position of catheter, volume of fluid instilled.
  • BAL by flexible bronchoscope was first reported introduced in the 1970s and is the current standard diagnostic tool for pulmonary infection and disease.
  • BAL is the current clinical gold standard for sampling of pulmonary fluids.
  • BAL introduces a large volume of liquid into the lungs before subsequent removal by suction. Samples are captured within the fluid for downstream analysis. Reviews exist for diagnostic and therapeutic applications of BAL in the literature.
  • BAL is performed following bronchoscopic inspection to guide the sampling.
  • the procedure is not standardised and varies based on application/location, however the general principal involves advancing the bronchoscope until the distal tip becomes wedged, blocking the airway leading to the targeted region.
  • saline washes are typically performed and pooled (up to ⁇ 200 mL) by wedging the bronchoscope in the airway and dispensing/aspirating through the biopsy port of the scope.
  • BAL is well tolerated in healthy patients however, several complications are reported including lung and airway irritation, transient hypoxemia, fever (-30% of patients), bronchospasm, respiratory distress syndrome, and on rare occasions pneumothorax. Results are vulnerable to false positives and there are several methodological problems for consideration when evaluating the clinical relevance of obtained samples, suggesting that BAL should always be used in conjunction with comprehensive clinical information in any diagnosis.
  • BAL recovers low volumes of ELF relative to the instilled volume, reported as low as 2 mL ELF recovered in from 200mL pooled BAL fluid (four 50 mL lavages). In this case, dwell times were around 2 minutes, which suggests possible inflation of the ELF levels due to dilution estimation errors. This low recovery of ELF in the BAL fluid and inability to perform localised sampling has led to the development of other more targeted ELF sampling methods.
  • BAL is the only method currently capable of sampling alveolar regions.
  • Bronchoscopic microsampling is a method in which a device consisting of a wire with an absorbent probe at the tip is used to collect bronchial epithelial lining fluid with bronchoscopy.
  • microsampling probes could enable fundamental research in areas where BAL sensitivity is inadequate, such as Covid-19.
  • absorbent microsampling approaches are limited in sampling capability, introduce elution sample dilution/contamination, and are prone to causing tissue damage. This method makes it very difficult to estimate sample dilution since the true volume of ELF collected is estimated and the elution step and sampling step are separate. The probe is also unable to obtain cytological information.
  • Microdialysis was first described in the 1950s, however use of microdialysis to study tissue biochemistry in began in animals during 1960s and humans in the 1990s. Microdialysis allows for continuous sampling providing excellent information in pharmacokinetic studies and many other applications under constant perfusion, including those for calibration methods and those specific to pulmonary applications.
  • Microdialysis probes mimic blood capillaries, exchanging substances via extracellular fluid.
  • the dual lumen probe is slowly perfused with a physiological liquid known as the perfusate.
  • Substances in the fluid surrounding the probe perfuse into the perfusate through the semi-permeable membrane of the probe tip and are collected as dialysate at the exit of the system for downstream analysis.
  • BMD bronchoscopic microdialysis
  • the semi-permeable membrane only allows analytes of ⁇ 100kDA to pass into the dialysate and therefore excludes collection of cytological data.
  • the limited sample capacity of the tip ( ⁇ 1.4 pL) and the underpinning diffusion mechanism demanding slow sampling rates (1-2 pL/min) and hour perfusion before sampling may yield limited utility outside a continuous monitoring context.
  • a number of devices such as endoscopes, exist which combine a conduit for injecting a fluid and a conduit for removing a fluid, for example as disclosed in US5823940 (Newman), US4750902 (Wuchinich), US2006/1073244 (Boulais) or US2023/0190078 (Clayman).
  • these devices are not configured to allow sampling of a biological sample in a subject for analysis, for example, of Pulmonary Epithelial Lining Fluid (ELF).
  • ELF Pulmonary Epithelial Lining Fluid
  • ELF Pulmonary Epithelial Lining Fluid
  • the present invention is based on the finding that continuous or semi-continuous endoscopic sampling may be carried out using a multi-lumen device, by simultaneous dispensing a fluid via a dispensing lumen and aspirating via a different lumen.
  • a continuous and/or self-sustaining fluid interface may be created between the distal tip of the device and a target sample, and a continuous flow of fluid may be generated between the dispensing lumen and the aspirating lumen, which may allow continuous sampling under soft (fluid) contact with the target sample, thereby minimising potential damage to a subject’s tissue.
  • the present apparatus has an outer diameter (O.D.) small enough to be compatible with the biopsy port of commercially available endoscopes, and can thus be deployed at a target site via a standard lumen in a conventional endoscope. This ensures that the distal end of the apparatus is not contaminated through contact with other parts of the subject’s tissue before reaching the target sample site.
  • O.D. outer diameter
  • a sampling apparatus comprising a probe, wherein the probe comprises: a first lumen configured to dispense a first fluid at a distal end thereof; a second lumen configured to aspire the first fluid at a distal end thereof, wherein a distal end of the first lumen is adjacent a distal end of the second lumen, wherein the sampling apparatus is configured to dispense the first fluid via the first lumen and to aspirate the first fluid via the second lumen simultaneously.
  • this allows continuous sampling, for example within an endoscope, e.g. a bronchoscope.
  • an endoscope e.g. a bronchoscope.
  • a self-sustaining volume of fluid may be created at the distal tip of the sampling apparatus, which may allow simultaneous and/or continuous sampling under soft (fluid) contact with the target, thereby minimising potential damage to a subject’s tissue.
  • a self-sustaining flow of fluid may be created at the distal tip of the sampling apparatus between the first (dispensing) lumen and the second (aspirating) lumen, which may allow simultaneous and/or continuous sampling under soft (fluid) contact with the target, thereby minimising potential damage to a subject’s tissue.
  • the present apparatus may also allow translation of the apparatus during sampling, allowing normal function of the target (tissue), for example respiration.
  • normal function of the target for example respiration.
  • BAL conventional sampling methods, such as BAL, which involves wedging the scope in the airway and introducing a large volume of liquid into the lungs before subsequent removal by suction, thus impeding respiration during the sampling procedure.
  • the present apparatus may also allow sampling of both fluid and dry analytes.
  • distal will be herein understood to refer to a location or end nearest a sampling location and/or farthest from a user or operator.
  • proximal will be herein understood to refer to a location or end opposite a sampling location and/or nearest a user or operator.
  • the apparatus may be configured to generate a self-sustaining flow of fluid between the first (dispensing) lumen and the second (aspirating) lumen at a distal end thereof.
  • the apparatus may be configured to generate a volume of the first fluid at or near the distal end of the probe, e.g. of the first lumen and/or of the second lumen, sufficient to create a continuous fluid interface between the probe and a target, e.g. a subject’s tissue, in use.
  • the second lumen may be capable of aspiring the first fluid.
  • the fluid aspired by the second lumen may be the first fluid which is dispensed via the first lumen, thereby creating a self-sustaining and/or continuous flow of fluid between the first (dispensing) lumen and the second (aspirating) lumen at a distal end thereof.
  • the first fluid “wets” and/or interacts with the target so as to generate a second fluid.
  • the second lumen is configured to aspire the second fluid at a distal end thereof.
  • the fluid aspired by the second lumen may be a second fluid generated via interaction between the first fluid and the target sample.
  • the second fluid may be a mixture of the first fluid and the target sample.
  • the probe may comprise one first lumen configured to dispense a first fluid at a distal end thereof.
  • the probe may comprise a plurality of first lumens configured to dispense a first fluid at a distal end thereof.
  • the probe may comprise one second lumen configured to aspire the first fluid or the second fluid at a distal end thereof.
  • the probe may comprise a plurality of second lumens configured to aspire the first fluid or the second fluid at a distal end thereof.
  • the probe may comprise one first lumen configured to dispense a first fluid at a distal end thereof, and one second lumen configured to aspire the first fluid or the second fluid at a distal end thereof.
  • the apparatus may be configured to dispense the first fluid via the first lumen at a distal end thereof and to aspire the first fluid or the second fluid via the second lumen at a distal end thereof simultaneously at least temporarily, e.g. for at least 1 ms, e.g. at least 10ms, e.g. at least 0.1s, e.g. at least 1s.
  • the apparatus may be configured to generate a continuous and/or self-sustained, e.g. a substantially constant or stable, flow of the first fluid between the first (dispensing) lumen and the second (aspirating) lumen at a distal end thereof.
  • the apparatus may be configured to generate a continuous and/or self-sustained, e.g. a substantially constant or stable, volume of the second fluid at or near the distal end of the first lumen and/or the distal end of the second lumen, in use, and/or the apparatus may be configured to generate a continuous and/or self-sustained, e.g. a substantially constant flow of fluid between the first (dispensing) lumen and the second (aspirating) lumen at a distal end thereof, in use.
  • a continuous and/or self-sustained e.g. a substantially constant or stable
  • the apparatus may be capable of creating a self-sustaining fluid volume at a distal end thereof, it will be understood that, upon contact of the first fluid with a/the target, e.g. tissue, surface tension will cause the first fluid to “wet” the target (tissue) so as to create a fluid interface, preferably a continuous and/or self-sustained fluid interface.
  • a fluid interface preferably a continuous and/or self-sustained fluid interface.
  • the flow of fluid between the first (dispensing) lumen and the second (aspirating) lumen may be such that a continuous fluid interface may be generated between the probe and a target, e.g. a subject’s tissue, in use, thereby continuously sampling the target at or near the fluid interface.
  • the sampling apparatus may be configured to continuously dispense a first fluid via the first lumen at a distal end thereof, and may be configured to continuously aspire the first fluid or the second fluid via the second lumen at a distal end thereof.
  • the term “continuous” will be herein understood to mean that there is no interruption in the dispensing and/or aspiration of the fluid.
  • the term may, however include one or more types of flow, for example steady state (substantially constant rate) flow, ramping of flow rates between values over a time period, square wave flow, oscillating flow, pulsatile flow, or the like.
  • the sampling apparatus may be configured to dispense a first fluid via the first lumen at a distal end thereof, and/or to aspire the first fluid or the second fluid via the second lumen at a distal end thereof, semi-continuously.
  • intermittent flow itself may be steady, or non-steady, e.g. ramped, square wave flow, oscillating flow, pulsatile flow, or the like.
  • the sampling apparatus may be configured to aspire the first fluid continuously via the second lumen at a distal end thereof, and to dispense the first fluid via the first lumen at a distal end thereof intermittently. It was found that dispensing the first fluid in an intermittent fashion may not adversely affect the fluid interface between the probe and the target (tissue), and may promote mixing at the interface. However, it will be understood that, as explained above, the intermittent nature of the flow should still allow or maintain a continuous or self-sustained fluid interface between the apparatus, e.g. the tip of the probe, and the target. This may be achieved by ceasing dispensing and/or aspiration for short periods of time, typically in the order of a few ps. However, it will be appreciated that the duration of the intermittent cycles will depend in the specific system being used and the associated conditions, including for example probe dimensions, target wettability, presence of surfactants, nature of the first fluid, nature of the target, flow parameters, temperature, etc.
  • the second lumen may be provided adjacent the first lumen.
  • the first lumen may have a proximal end and a distal end.
  • the distal end may be provided at a sampling end of the first lumen.
  • the proximal end may be provided at an end opposite the distal end or sampling end.
  • the distal end of the first lumen may define a dispensing end and/or may be configured to dispense the first fluid.
  • the second lumen may have a proximal end and a distal end.
  • the distal end may be provided at a sampling end of the second lumen.
  • the proximal end may be provided at an end opposite the distal end or sampling end.
  • the distal end of the second lumen may define an inlet end and/or may be configured to aspire the first fluid or the second fluid.
  • the first lumen may be defined by a first conduit, e.g. first tube.
  • the second lumen may be defined by a second conduit, e.g. second tube.
  • first conduit and the second conduit may be in contact with each other, at least at a distal end thereof.
  • first conduit and the second conduit may be provided side-by- side, at least at a distal end thereof.
  • the apparatus e.g. the probe, may comprise a sheath.
  • first lumen and the second lumen may be provided within the sheath.
  • first conduit and the second conduit be provided within the sheath.
  • the first conduit and the second conduit may be in contact with each other within the sheath.
  • the first conduit and the second conduit may be provided side-by-side within the sheath.
  • the sheath may have a distal end at or near the sampling end of the apparatus, and a proximal end opposite the distal end.
  • the sheath may have a width, e.g. an inner width or inner diameter, of about 0.5- 5mm, e.g. about 1-3mm, typically about 1.-1 ,7mm.
  • the sheath may have a width, e.g. an outer width or outer diameter, of about 0.5-5mm, e.g. about 1-3mm, typically about 1- 1 ,9mm or 1-1 ,8mm, for example about 1 ,8mm.
  • the outer width or outer diameter of the sheath may be substantially equal to or less than the inner diameter of a port or internal conduit of a standard endoscope, e.g. bronchoscope, (typically about 2mm in inner diameter), which may allow the present apparatus, e.g.
  • probe to be provided within or to be compatible with conventional endoscopes, e.g. bronchoscopes.
  • conventional endoscopes e.g. bronchoscopes.
  • the apparatus e.g. probe
  • contamination of a distal end of the apparatus e.g. through contact with tissue surfaces of the subject, may be avoided.
  • This may ensure that the distal end of the apparatus, e.g. probe, may remain sterile and/or free of contamination, until it is deployed at the sample site through a distal end of the endoscope.
  • the sheath may have a width, e.g. an outer width or outer diameter, of about 1- 1.8mm or 1-1.9mm, e.g. about 1.8mm, so as to provide a degree of clearance between an outer surface thereof and an inner surface of a/the port or internal conduit of a/the endoscope.
  • the sheath may have a wall thickness of less than or equal to about 300pm, typically less than or equal to about 250pm.
  • the sampling apparatus e.g. probe
  • the sampling apparatus may be sufficiently flexible to be easily deployed to a sampling site.
  • the sampling apparatus when deployed via an endoscope, the sampling apparatus may be sufficiently flexible to be deployed via a port and/or internal conduit of an endoscope.
  • the distal end of the first lumen may be adjacent and/or may be substantially flush or level with the distal end of the second lumen.
  • the distal ends of the first lumen and second lumen may be substantially flush or level, and/or the first lumen and second lumen may have a substantially equal z-height relative to a proximal end of the probe. This may promote the formation of a constant or stable volume of the first fluid at or near the end of the probe, in use.
  • the apparatus may be configured to generate a continuous and/or self-sustained, e.g. a substantially constant or stable, flow of the first fluid between the first (dispensing) lumen and the second (aspirating) lumen at a distal end thereof. This may cause the formation of a substantially constant or stable volume of the first fluid at a distal end or tip of the probe may, which in some embodiments may define or may form a droplet.
  • the volume of the first fluid and/or droplet may be about 0-200 pl, e.g. about 1-200pl, e.g. about IQ- 200 pl.
  • the volume of the first fluid and/or droplet may be less than about 200 pl, typically less than about 183 pl. It will be appreciated that the precise volume of the first fluid and/or droplet may depend on a number of factors such as lumen/conduit diameters, relative flow rates, first fluid viscosity, and the like.
  • a fluid interface between a distal end of the probe and a target (tissue) may be established upon temporary contact between the fluid at a distal end of the probe and the target (tissue), and may be maintained when the distal end of the probe is no longer in physical contact with, but is in close proximity with, the target (tissue), for example due to surface tension.
  • the distal end of the sheath may be adjacent and/or may be substantially flush with the distal end of the first lumen or first conduit and/or of the second lumen or second conduit.
  • the distal end of the first lumen or first conduit and/or of the second lumen or second conduit may extend beyond (i.e. in a more distal direction) the distal end of the sheath. This may promote the formation of a constant or stable volume of the first fluid and/or second fluid, in use, by minimising possible interaction and/or capillary action with the distal end of the sheath.
  • a space between the first lumen and second lumen, and the sheath may be filled or sealed with a filling material at least at or near a distal end of the sheath.
  • the filling material may comprise or may be an adhesive, e.g. an epoxy resin.
  • the first lumen and/or first conduit may have a first width or diameter, e.g. a first inner width or diameter.
  • the second lumen and/or second conduit may have a second width or diameter, e.g. a second inner width or diameter.
  • the second width or diameter may be equal to or greater than the first width or diameter, e.g. first inner width or diameter.
  • the second width or diameter, e.g. second inner width or diameter may be greater than the first width or diameter, e.g. first inner width or diameter.
  • the first fluid may preferentially migrate or flow towards or into the second or aspiration lumen or second conduit.
  • this may prioritise flow of the first fluid into the aspiration lumen in the event of failure of an associated pumping mechanism, e.g. pump(s).
  • this may cause the fluid in the first or dispensing lumen or first conduit to be provided at a higher pressure than the first fluid in the second or aspiration lumen or second conduit. This may help provide a liquid barrier to physical blockage of the aspiration lumen, and/or may help prevent blockages.
  • the first lumen or first conduit may have a first width or diameter, e.g. a first inner width or diameter, of about 50-1000pm, e.g. about 100-500pm, e.g. about 100-200pm, e.g. about 150 pm.
  • the second lumen or second conduit may have a second width or diameter, e.g. a second inner width or diameter, of about 50-1000
  • the small dimensions of the first conduit and second conduit allow, in use, the completion of a sampling event using a very small total volume of sampling fluid, typically in the range of about 300pl - 1.5ml.
  • the minimum volume of sampling fluid required to perform a sampling event is greater than the “dead volume”, i.e. the volume of first conduit of the probe, which may typically be about 300pl.
  • the volume of first fluid required to perform a sampling event may be greater than about 300pl, e.g. greater than about 350pl, and/or may be about 300pl - 1.5ml, e.g. about 350pl - 1 ,5ml.
  • the exact minimum volume of sampling fluid will depend on the exact dimensions of the probe, including first lumen diameter and/or probe length. These small sample volumes may help minimise dilution of the sample collected.
  • these dimensions also allow for fluid flows that generate shear rates similar to those experienced under normal physiological conditions to protect the tissues and samples, similarly to the range of shear rates associated with commercially available syringe drivers thus protecting the tissues being samples from and/or the collected samples.
  • the ratio of the second width or diameter, e.g. a second inner width or diameter, to the first width or diameter, e.g. a first inner width or diameter, may be about 5:1 to 0.8:1 , e.g. about 5:1 to 1 :1 , e.g. about 4:1 to 2:1.
  • the ratio of the second width or diameter, e.g. a second inner width or diameter, to the first width or diameter may be greater than 1 :1.
  • first lumen and/or second lumen may depend on a number of factors, including the type of target, e.g. tissue or cells, being sampled, and that the above dimensions are provided for context only, particularly in relation to the sampling of ELF from a subject’s lungs.
  • the sampling apparatus may further comprise circulation or pumping means, e.g. one or more pumps.
  • the sampling apparatus may comprise a first pump configured to dispense the first fluid via the first lumen.
  • the first pump may be configured to operate at a first flow rate.
  • the sampling apparatus may comprise a second pump configured to aspirate the fluid, e.g. first fluid, via the second lumen.
  • the second pump may be configured to operate at a second flow rate.
  • the first pump and the second pump may be the same.
  • the first flow rate and the second flow rate may be the same.
  • using a single pump may reduce costs, size and/or complexity of the apparatus.
  • the first pump and the second pump may be different or distinct.
  • a first pump configured to dispense the first fluid via the first lumen and a second pump configured to aspirate the second fluid via the second lumen.
  • the first flow rate and the second flow rate may be the same or may be different.
  • using two separate pumps may allow adjustment of the relative values between the first flow rate and the second flow rate. This may help adjust the performance of the apparatus, in use.
  • the circulation or pumping means may form or may define a sealed circuit or system.
  • the circulation or pumping means e.g. one or more pumps, may comprise gas tight syringes.
  • the first flow rate and/or the second flow rate may be in the range of about 50-7000 pl/min, e.g. about 100-2000 pl/min, e.g. about 500 pl/min.
  • the first flow rate and the second flow rate may be relatively low.
  • shear rates may be minimised, thereby avoiding or reducing damage to cellular material in the fluid, e.g. first fluid.
  • the first flow rate and the second flow rate may be less than about 5000 pl/min, e.g. less than about 1000 pl/min.
  • the shear rate in the first lumen may be kept under about 1700 Dyn/cm 2 , e.g. under about 1000 Dyn/cm 2 .
  • the shear rate in the second lumen may be kept under about 20 Dyn/cm 2 , e.g. under about 10 Dyn/cm 2 .
  • flow rate parameters used may depend on a number of factors, including the type of target, e.g. tissue or cells, being sampled, and that the above parameters are provided for context only, particularly in relation to the sampling of ELF from a subject’s lungs.
  • the sampling apparatus may be used in combination with an endoscope, e.g. a bronchoscope.
  • the endoscope or bronchoscope may further comprise a camera.
  • the endoscope or bronchoscope may comprise or may use a fibre optics camera.
  • the apparatus may be used without an endoscope.
  • the sampling apparatus e.g. probe thereof, may be deployed directly, e.g. in a “blind” manner to a sampling location.
  • the first fluid may comprise or may be a liquid, e.g. a biologically compatible liquid, such as a saline solution, preferably a sterile saline solution, which may help minimise diffusion, to allow for accurate localised sampling.
  • a biologically compatible liquid such as a saline solution, preferably a sterile saline solution, which may help minimise diffusion, to allow for accurate localised sampling.
  • the sampling apparatus may be configured to sample a target area, e.g. a target tissue, of a subject.
  • the sampling apparatus may be configured to sample a lining mucosal tissue, e.g. an epithelial lining fluid (ELF).
  • ELF epithelial lining fluid
  • the second fluid may comprise or may be a mixture of the first fluid and ELF.
  • the endoscope or bronchoscope may further comprise illumination means, e.g. one or more lights.
  • the endoscope or bronchoscope may comprise a housing, typically a tubular housing.
  • the endoscope or bronchoscope may comprise of may define a port (e.g. a biopsy port) and/or internal conduit, though which the sampling apparatus, e.g. probe, may be inserted and/or deployed.
  • the port and/or internal conduit may have a size, e.g. inner width or diameter, of around 2mm.
  • One or more components of the endoscope or bronchoscope e.g. the components of the endoscope or bronchoscope, may be provided within the housing.
  • a sheath of the apparatus may have a width, e.g. an outer width or outer diameter, of about 1-1.9mm, e.g. 1-1.8mm, e.g. 1-1.7mm, so as to allow the probe to be deployed via a/the endoscope cavity or lumen.
  • This may allow the apparatus, e.g. probe, to be inserted within a conventional endoscope in order to deploy a distal end of the apparatus to a sampling site.
  • contamination of a distal end of the apparatus e.g. through contact with tissue surfaces of the subject, may be avoided. This may ensure that the distal end of the apparatus, e.g. probe, may remain sterile and/or free of contamination, until it is deployed at the sample site through a distal end of the endoscope.
  • the endoscope or bronchoscope may comprise orientation/steering means and/or an orientation/steering mechanism configured to adjust the orientation of the endoscope or bronchoscope, e.g. at a distal end thereof.
  • the endoscope or bronchoscope may comprise Bowden cables, or the like.
  • a system comprising: an endoscope; and a sampling apparatus, wherein the sampling apparatus comprises a probe, wherein the probe comprises: a first lumen configured to dispense a first fluid at a distal end thereof; and a second lumen configured to aspire the first fluid at a distal end thereof, wherein the distal end of the first lumen is adjacent the distal end of the second lumen, wherein the sampling apparatus is configured to dispense the first fluid via the first lumen and to aspirate the first fluid via the second lumen simultaneously.
  • the sampling apparatus may be a sampling apparatus according to the first aspect.
  • the probe may be configured to be inserted or deployed via a port or internal conduit of the endoscope.
  • the probe may have an external width or diameter less than an internal width or diameter of the port or internal conduit of the endoscope.
  • the endoscope may comprise or may be a bronchoscope.
  • the first fluid may comprise or may be a liquid, e.g. a biologically compatible liquid, such as a saline solution, preferably a sterile saline solution.
  • a biologically compatible liquid such as a saline solution, preferably a sterile saline solution.
  • the sampling apparatus may be configured to sample a target area, e.g. a target tissue, of a subject.
  • the sampling apparatus may be configured to sample an epithelial lining fluid (ELF).
  • ELF epithelial lining fluid
  • the second fluid may comprise or may be a mixture of the first fluid and ELF.
  • the endoscope e.g. bronchoscope
  • the bronchoscope may comprise or may use a fibre optics camera.
  • the endoscope e.g. bronchoscope may further comprise illumination means, e.g. on or more lights.
  • the endoscope e.g. bronchoscope may comprise a housing, typically a tubular housing.
  • One or more components of the endoscope may be provided within the housing.
  • the probe and/or the camera preferably the probe and the camera may be provided within the housing.
  • the endoscope, e.g. bronchoscope may be a flexible endoscope, e.g. bronchoscope.
  • the endoscope e.g. bronchoscope
  • the endoscope may comprise orientation/steering means and/or an orientation/steering mechanism configure to adjust the orientation of the endoscope, e.g. bronchoscope, e.g. at a distal end thereof.
  • the endoscope, e.g. bronchoscope may comprise Bowden cables, or the like.
  • the sampling apparatus may further comprise pumping means, e.g. one or more pumps.
  • the sampling apparatus may comprise a first pump configured to dispense the first fluid via the first lumen.
  • the first pump may be configured to operate at a first flow rate.
  • the sampling apparatus may comprise a second pump configured to aspirate the second fluid via the second lumen.
  • the second pump may be configured to operate at a second flow rate.
  • a method of sampling a target sample comprising: providing a sampling apparatus comprising a probe, wherein the probe comprises a first lumen configured to dispense a first fluid at a distal end thereof and a second lumen configured to aspire the first fluid at a distal end thereof, wherein a distal end of the first lumen is adjacent a distal end of the second lumen; dispensing the first fluid via the first lumen; and simultaneously aspirating the first fluid via the second lumen.
  • the method may comprise establishing a self-sustaining and/or continuous flow of fluid between the first (dispensing) lumen and the second (aspirating) lumen at a distal end thereof.
  • the flow of the first fluid from the first lumen may help prevent the second lumen from becoming blocked during aspiration.
  • the present method allows continuous sampling, for example by deploying the sampling apparatus via an endoscope, e.g. a bronchoscope.
  • the method may allow continuous sampling by directed using the sampling apparatus, e.g. probe thereof, e.g. by “blind” deployment of the sampling apparatus.
  • the method may comprise deploying the apparatus, e.g. placing the apparatus, e.g. a distal end of the probe, at or near the target sample, before circulating the first fluid.
  • the method may comprise “blind” deployment of the sampling apparatus to a sampling location.
  • the method may comprise inserting the apparatus, e.g. probe, within a lumen or internal conduit of an endoscope, so as to deploy the probe to a target site and/or near the target sample in a guided manner.
  • the probe may be protected within the lumen or internal conduit of the endoscope until the target site is reached, thus maintaining the sterility of samples and avoiding contamination and confusion with other tissues/pathologies providing truly localised sampling.
  • the method may comprise placing a distal end of the apparatus, e.g. probe, in contact with or in proximity with the target sample, e.g. biological tissue.
  • the term “in proximity with” will be herein understood to mean that a fluid interface between a distal end of the probe and the target may be maintained, in use, e.g. during dispensing of the first fluid and/or aspiration of the first fluid or second fluid.
  • the method may comprise contacting the first fluid with the target.
  • the method may comprise establishing a fluid interface between the first fluid and the target sample, and/or between the fluid flowing between the first (dispensing) lumen and the second (aspirating) lumen, and the target sample.
  • the method may comprise placing the distal end of the apparatus, e.g. probe, at less than 2mm, e.g. less than 1 mm, e.g. less than 0.5mm, from the target.
  • the method may comprise contacting the distal end of the apparatus, e.g. probe, with the target.
  • the present method may allow temporary and/or gentle contact between the probe and the target in order to position the probe effectively, whilst minimising or avoiding damage to the target, e.g. biological tissue.
  • the method may comprise placing the distal end of the apparatus, e.g. probe, in proximity with or in contact with the target (tissue).
  • the method may comprise placing the distal end of the apparatus, e.g. probe, at an angle, e.g. less than 90°, e.g. less than 80°, relative to the target (tissue).
  • this may generate at least a partial gap or space between the distal end of the probe and the target, which may allow the fluid interface to be maintained, in use, e.g. during dispensing of the first fluid and/or aspiration of the second fluid, whilst avoiding obstruction of the probe, e.g. of the first lumen or the second lumen upon contact with the target (tissue).
  • the method may comprise dispensing the first fluid via the first lumen at a distal end thereof, and/or to aspiring the first fluid or the second fluid via the second lumen at a distal end thereof, continuously, or semi-continuously.
  • the method may comprise dispensing a total volume of fluid, e.g. first fluid, in the range of about 300pL - 1.5mL.
  • the method may comprise dispensing a minimum volume of sampling fluid required to perform a sampling event which is greater than the “dead volume”, i.e. the volume of first conduit of the probe, which may typically be about 300pl.
  • the method may comprise dispending a volume of first fluid greater than about 300pl, e.g. greater than about 350pl, and/or about 300pl - 1 ,5ml, e.g. about 350pl - 1 ,5ml.
  • the exact minimum volume of sampling fluid will depend on the exact dimensions of the probe, including first lumen diameter and/or probe length. These small sample volumes may help minimise dilution of the sample collected.
  • these dimensions also allow for fluid flows that generate shear rates similar to those experienced under normal physiological conditions to protect the tissues and samples, similarly to the range of shear rates associated with commercially available syringe drivers thus protecting the tissues being samples from and/or the collected samples.
  • the method may comprise actuating one or more pumps.
  • the one or more pumps are provided within a sealed fluid circuit.
  • the method may comprise actuating a first pump configured to dispense the first fluid via the first lumen.
  • the first pump may be configured to operate at a first flow rate.
  • the method may comprise actuating a second pump configured to aspirate the first fluid or second fluid via the second lumen.
  • the second pump may be configured to operate at a second flow rate.
  • the first pump and the second pump may be the same.
  • a single pump configured to dispense the first fluid via the first lumen and to aspirate the second fluid via the second lumen.
  • the first flow rate and the second flow rate may be the same.
  • using a single pump may reduce costs, size and/or complexity of the apparatus.
  • the first pump and the second pump may be different or distinct.
  • the method may comprise actuating a first pump configured to dispense the first fluid via the first lumen and actuating a second pump configured to aspirate the second fluid via the second lumen.
  • the first flow rate and the second flow rate may be the same or may be different.
  • using two separate pumps may allow adjustment of the relative values between the first flow rate and the second flow rate. This may help adjust the performance of the apparatus, in use.
  • the method may comprise creating a fluid interface, e.g. a permanent and/or stable fluid interface, between a distal end of the probe and the target sample.
  • a fluid interface e.g. a permanent and/or stable fluid interface
  • the method may comprise moving, e.g. translating, the apparatus, e.g. probe.
  • this may allow sampling a larger area of the target sample whilst maintaining the fluid interface between the distal end of the probe and the target sample.
  • the first fluid may comprise or may be a liquid, e.g. a biologically compatible liquid, such as a saline solution, preferably a sterile saline solution.
  • a biologically compatible liquid such as a saline solution, preferably a sterile saline solution.
  • the sampling apparatus may be configured to sample a target area, e.g. a target tissue, of a subject.
  • the sampling apparatus may be configured to sample a lining mucosal tissue, e.g. an epithelial lining fluid (ELF).
  • ELF epithelial lining fluid
  • the second fluid may comprise or may be a mixture of the first fluid and ELF.
  • the method may comprise collecting the fluid, e.g. first fluid or second fluid, aspirated via the second lumen.
  • the method may comprise analysing the fluid, e.g. first fluid or second fluid, aspirated via the second lumen.
  • Figure 1 shows a system according to a first embodiment
  • Figure 2 shows an embodiment of the distal end of a probe of a sampling apparatus according to another embodiment
  • Figures 3a to 3c illustrate a distal end of a probe of a sampling apparatus according to another embodiment
  • Figure 4 shows analyte measurement in a fluid sampled in vivo using a conventional BAL method
  • Figure 5 shows analyte measurement in a fluid sampled in vivo in the same animal as Figure 4 but using the sampling apparatus of Figure 1 ;
  • Figure 6 shows a graph illustrating the urea levels in a fluid sampled using BAL vs the sampling apparatus of Figure 1 ;
  • Figure 7 shows a graph illustrating the GM-CSF levels in a fluid sampled using BAL vs the sampling apparatus of Figure 1 ;
  • Figure 8 shows a graph illustrating the GM-CSF concentration in a fluid sampled using the sampling apparatus of Figure 1 , based on the total sample volume;
  • Figure 9 shows a microscopy image of Epithelial Lining fluid sampled using the apparatus of Figure 1 ;
  • Figure 10 shows the experimental timescale relating to the investigation of potential inflammatory response
  • Figure 11 shows the standard curve plot relating to the investigation of potential inflammatory response of Figure 10.
  • Figures 1 shows a system 100 according to a first embodiment.
  • the system 100 comprises a sampling apparatus 115 and, in this embodiment, an endoscope 110.
  • the sampling apparatus 115 comprises a probe 120 and pumps 151 ,161.
  • the probe 120 is described in more detail below with reference to Figures 2- 3, like parts being denoted by like numerals, but incremented by ‘100’.
  • the endoscope 110 has a housing 111 , and includes an internal conduit (not shown) through which the probe 120 can be inserted and deployed.
  • the sampling apparatus 115 is configured for sampling an epithelial lining fluid (ELF) from a patient’s lung(s) 140 at the distal end 130 of the probe 120.
  • ELF epithelial lining fluid
  • Figures 2 and 3 show the distal end 130,230 of a probe 220,320 which may be used in conjunction with a scope as depicted in Figure 1.
  • the probe 220 comprises a first lumen 222 configured to dispense a first fluid at a distal end 230 of the probe, and a second lumen 223 configured to aspire the first fluid at a distal end 230 of the probe.
  • the distal end of the first lumen 222 is adjacent the distal end of the second lumen 223.
  • a space 224 between the first lumen 222 and second lumen 223, and the sheath 221 is filled or sealed with a filling material at least at or near a distal end of the sheath, which prevents a sampling fluid from being drawn into the sheath 221 , e.g. by surface tension or capillary action, which may help promote steady flow of the sampling fluid between the first or dispensing lumen 222 and the second or aspiration lumen 223.
  • the sampling apparatus 100 is configured to dispense the first fluid via the first lumen 222,322 and to aspirate the first fluid via the second lumen 223,323 simultaneously.
  • this allows continuous sampling, for example when used in combination with endoscope 110 in the system 100 of Figure 1.
  • FIGS 3(a)-3(c) This is illustrated in Figures 3(a)-3(c).
  • a first fluid 325 is dispensed via first lumen 322 at a distal end 330 of the probe 320.
  • aspiration via the adjacent second lumen 323 causes the first fluid 325 to be aspired into the second lumen 323 at a distal end 330 of the probe 320.
  • a self-sustaining flow of fluid 325 may be created at the distal end 330 of the probe 320 between the first (dispensing) lumen 322 and the second (aspirating) lumen 323, which may allow simultaneous and/or continuous sampling under soft (fluid) contact with the target, thereby minimising potential damage to a subject’s tissue.
  • this may allow translation of the probe 120 during sampling, allowing normal function of the target (tissue), for example respiration.
  • the apparatus may be configured to generate a substantially constant or stable volume of the first fluid 325 at or near the distal end 330 of the probe 320 in use.
  • the constant or stable volume of the first fluid 325 may define or may form, in this embodiment and represented schematically for easy of understanding, a droplet 325.
  • the volume of the first fluid and/or droplet 325 may be about 0-200 pl, e.g. about 1 -200pl, e.g. about 10-200 pL in the case of a sheath 321 having an outer diameter of about 1.8mm.
  • the apparatus 100 may be capable of creating a self-sustaining fluid volume 325 at a distal end of the probe 320, it will be understood that, upon contact of the fluid droplet with a/the target, e.g. tissue, surface tension will cause the fluid to “wet” the target (tissue) so as to create a fluid interface, preferably a continuous fluid interface.
  • a target e.g. tissue
  • Figures 3 (a)-3(c) illustrates the probe 320 on its own, and therefore the fluid aspirated into second lumen 323 is the same fluid as the fluid dispensed via first lumen 322.
  • the first fluid dispensed from first lumen 322 “wets” and/or interacts with the target so as to generate a second fluid 326.
  • the second lumen 323 is configured to aspire the second fluid 326 at a distal end 330.
  • the fluid 326 aspired by the second lumen 323 is a second fluid 326 generated via interaction between the first fluid 325 and the target sample 341.
  • the probe comprises a sheath 221 ,321 .
  • the first lumen 222,322 and the second lumen 223,323 are provided within the sheath 221 ,231.
  • the sheath has a distal end 235 at or near the sampling end 130 of the apparatus 100.
  • the sheath typically has a width, e.g. an inner diameter, of about 0.5-5mm, e.g. about 1-3mm, e.g. about 1.8mm. Conveniently, this may be similar to or less than the inner diameter (typically about 2mm) of a biopsy port of a standard bronchoscope, which may allow the present 120 probe, to be provided within or compatible with conventional bronchoscopes 110.
  • the distal end 235 of the sheath 221 is adjacent and substantially flush with the distal end of the first lumen 222 and of the second lumen 223.
  • the space between the first lumen 222 and second lumen 223, and the sheath 221 may preferably be filled with a filling material (not shown) at least at or near a distal end 235 of the sheath 221 .
  • the first fluid 325 may be prevented from being drawn into the sheath 221 , e.g. by surface tension, which may help promote steady flow of the first fluid between the first or dispensing lumen 222 and the second or aspiration lumen 223.
  • the distal end of the first lumen 322 and of the second lumen 323 extends beyond (i.e. in a more distal direction) the distal end 335 of the sheath 321. This may promote the formation of a constant or stable volume of the first fluid 325, in use, by minimising possible interaction and/or surface tension with the distal end 335 of the sheath 321 .
  • the sampling apparatus 115 includes circulation or pumping means, here in the form of pumps 151 ,161.
  • a first pump 151 is in fluid communication with the first lumen 222 via first conduit 153, and is configured to dispense the first fluid from first fluid reservoir 152.
  • the first pump is configured to operate at a first flow rate, in this embodiment at about 500 pl/min.
  • a second pump 161 is in fluid communication with the second lumen 223 via second conduit 163, and is configured to aspirate the first fluid into second fluid reservoir 162.
  • the second pump is configured to operate at a second flow rate, in this embodiment at about 500 pl/min, so as to generate rates shear forces no greater than physiologically experienced by system under study.
  • using two separate pumps 151 ,161 may allow adjustment of the relative values between the first flow rate and the second flow rate. This may help adjust the performance of the apparatus 115, in use.
  • the first flow rate and the second flow rate are substantially the same.
  • using a single pump may reduce costs, size and/or complexity of the apparatus.
  • the first flow rate and/or the second flow rate may be in the region of about50-7000 pl/min, e.g. about 100-2000 pl/min, e.g. about 500 pl/min.
  • the first flow rate and the second flow rate may be relatively low.
  • shear rates may be minimised, thereby avoiding or reducing damage to cellular material in the fluid, e.g. first fluid.
  • the first flow rate and the second flow rate may be less than about 5000 pl/min, e.g. less than about 1000 pl/min.
  • the shear rate in the first lumen may be kept under about 1700 Dyn/cm 2 , e.g. under about 1000 Dyn/cm 2 .
  • the shear rate in the second lumen may be kept under about 20 Dyn/cm 2 , e.g. under about 10 Dyn/cm 2 .
  • the endoscope 110 may comprise or may be associated with any device that would typically be useful in an endoscope but are not shown here for clarity, such as a camera, e.g. a fibre optics camera, illumination means, e.g. one or more lights, and/or orientation means and/or an orientation mechanism configured to adjust the orientation of the probe 220 at a distal end 230 thereof, e.g. Bowden cables, or the like.
  • a camera e.g. a fibre optics camera
  • illumination means e.g. one or more lights
  • orientation means and/or an orientation mechanism configured to adjust the orientation of the probe 220 at a distal end 230 thereof, e.g. Bowden cables, or the like.
  • GM-CSF granulocyte/macrophage colony stimulation factor
  • Propofol was administered systemically followed by general anaesthesia under isoflurane maintained by the LARIF anaesthetist.
  • the right lobe was designated the control side and left lobe the treated side.
  • BAL was performed on RA for confirming lung health by lack of inflammatory status (differential cell counts).
  • Treatment involved delivery of a murine granulocyte/macrophage colony stimulation factor (mGM-CSF) packaged in a Lentiviral vector (vGM173) introduced to the left lung.
  • mGM-CSF murine granulocyte/macrophage colony stimulation factor
  • vGM173 Lentiviral vector
  • a custom 2.2-meter long 1 ,8mm outer diameter dual lumen sampling probe was cleaned by 3x6 ml Virkon followed by 3x6 ml H20 flushes prior to use.
  • the dispensing lumen 222 was connected to the Luer lock of a 1 m gastight glass syringe (Hamilton), pre-filled with PBST minimise the possibility of air inclusion in the system, the delivery lumen 222 was then backfilled with PBS to minimise the risk of airlocks in the small diameter lumen.
  • the collection lumen 223 was connected to the Luer lock of an empty 1 ml airtight glass syringe (Hamilton) and both syringes loaded to syringe pumps (Harvard instruments). For sampling, both pumps operated at identical flow rates, in this embodiment 0.5mL/min.
  • the experiment completion point was determined when no GM-CSF could be detected in the probe sample or BAL at week 16 time point.
  • the animal was culled, tissue was collected for HCR RNA-FISH imaging and DNA analysis to ascertain whether GM-CSF function has been silenced by the immune system or if transduced cells have been actively removed by an immune t-cell response.
  • Sample GM-CSF levels were obtained by ELISA using a commercially available kit to the manufacturer’s instructions (Quantikine ELISA mouse GM-CSF, R&D systems). Colorimetric assessment was performed by plate reader (Synergy HT, Biotek) using manufacturer’s instructions and software (Gen5, Biotek). Sample urea levels were measured by a commercially available kit (MAK006 Sigma-Aldrich, Merck) as per manufacturer’s instructions. Colorimetric assessment by plate reader (Synergy, Biotek) followed manufacturer instructions and software (Gen5, Biotek). ELF dilution was estimated by comparison of ELF urea levels and blood serum levels obtained from Easter Bush pathology analysis of the blood samples obtained by the LARIF team immediately prior to probe sampling.
  • the ELISA inter-plate controls remained comparable between weeks allowing comparison between runs.
  • Figure 4 shows measurements of GM-CSF in fluid sampled using BAL.
  • Figure 5 shows measurements of GM-CSF in fluid sampled using an embodiment of the present methodology as described above.
  • GM-CSF was detectable in ELF sampled by the probe up to 77 days after instillation, using both BAL and new probe methods. At day 112 (16 weeks) no GM-CSF was detectable and the animal culled.
  • the present methodology allows accurate measurement of a target analyte, matching the performance obtained using the current “gold standard”, BAL.
  • Figure 6 shows a graph illustrating the urea levels in a fluid sampled using BAL vs the sampling apparatus of Figure 1. This illustrates that the urea concentrations measured using the present apparatus are comparable to urea levels measured using BAL, confirming the suitability of the present apparatus and method as a sampling apparatus, in this case in the context of a bronchoscope.
  • Figure 7 shows a graph illustrating the GM-CSF levels in a fluid sampled using BAL vs the sampling apparatus of Figure 1. This leads to the same conclusions as highlighted above in relation to Figure 6.
  • Figure 8 shows a graph illustrating the GM-CSF concentration in a fluid sampled using the sampling apparatus of Figure 1 , based on the total sample volume.
  • the experiment was conducted in a first sample of overall low volume obtained over a sampling period of 40 seconds, and in a second sample of greater volume obtained over a sampling period of 120 seconds. It can be seen that the sample volume does not affect analyte concentration in the sample. A smaller volume sample does not concentrate the analyte in the fluid sample. Running the sampling longer just averages over a larger sampling region. In other words, longer samplings leads to a larger sample volume, not a more diluted sample.
  • Figure 9 shows a microscopy image of Epithelial Lining Fluid sampled using the apparatus of Figure 1.
  • the ELF sample was spun at 450g for 5 minutes. 1 ml of supernatant was removed for downstream ELISA analysis. The remaining supernatant was discarded and the cell pellet re-suspended in 1 ml PBS.
  • a cell count was performed on a haemocytometer and concentration adjusted to 5x10 5 / ml. 100ul at 5x105/ml was loaded for cytospins conducted at 600rpm for 5 minutes. The slides were then Giemsa stained following manufacturer’s instructions and the slide was then imaged at 20x to produce the image shown in Figure 1 .
  • the present invention provides a significant advantage over the methods of the prior art in that provision of a self-sustaining volume of fluid at the distal tip of the sampling apparatus may allow simultaneous and/or continuous sampling under soft (fluid) contact with the target, thereby minimising potential damage to a subject’s tissue compared to absorptive methods.
  • LVD1 left ventral diaphragmatic 1
  • LVD2 left ventral diaphragmatic 2
  • BAL was performed by wedging the scope, instilling 10ml pbs through bronchoscope biopsy channel and then aspirating.
  • the plate plan was as shown below in Table 1.
  • “semi-continuously” using an intermittent flow which may itself may be steady, or nonsteady, e.g. ramped, square wave flow, oscillating flow, pulsatile flow, or the like.
  • the present study investigates the type of the type of flow on the performance of the probe. We hypothesized that keeping the collection flow rate as constant whilst changing the dispensing flow rate modality, that effective sampling would still be possible, i.e. that we did not need to operate under exclusively continuous flow regimes. Additionally, it is believed that non-continuous mode may be advantageous in that changes in flow rate may generate greater mixing at the sampling site, allowing for more analyte to be mixed into the aspirate.
  • sampled RC and LCD were sampled using a different flow rate configuration.
  • sampling events were:

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Abstract

Un appareil d'échantillonnage (100) comprend une sonde (220), la sonde comprenant une première lumière (222) conçue pour distribuer un premier fluide au niveau d'une extrémité distale (230) de celle-ci ; une seconde lumière (223) conçue pour aspirer le premier fluide au niveau d'une extrémité distale (230) de celle-ci, une extrémité distale de la première lumière étant adjacente à une extrémité distale de la seconde lumière, l'appareil d'échantillonnage (100) étant conçu pour distribuer le premier fluide par l'intermédiaire de la première lumière (322) et pour aspirer le premier fluide par l'intermédiaire de la seconde lumière (323), simultanément.
PCT/GB2024/052810 2023-11-07 2024-11-06 Appareil d'échantillonnage Pending WO2025099417A1 (fr)

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GBGB2317067.3A GB202317067D0 (en) 2023-11-07 2023-11-07 Sampling apparatus
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