Methods and systems for determining respiratory health

Abstract

The present application provides methods for determining a respiratory health of a test subject based on mass spectrometry data of a sample of exhaled breath of the test subject. Also provided are methods for assessing the efficacy of a therapeutic treatment of a respiratory disease in a test subject based on mass spectrometry data of one or more samples of exhaled breath of the test subject. Further provided are systems or devices for use in said methods and the use of said systems or devices.

Description

[0001] METHODS AND SYSTEMS FOR DETERMINING RESPIRATORY HEALTH

[0002] Field of the invention

[0003] A method for determining a respiratory health of a test subject using mass spectrometry data of a sample of exhaled breath of the test subject, and identifying, from molecular ion mass signals of the mass spectrometry data, at least one molecular mass corresponding to a volatile organic compound (VOC).

[0004] Background to the invention

[0005] Respiratory complications such as bronchopulmonary dysplasia (BPD) and ventilator-induced lung injury (VI LI) are common after preterm birth. They cause significant morbidity during the neonatal period and beyond, such as more frequent severe respiratory infections during infancy, obstructive lung disease, exercise intolerance and an increased risk of early development of chronic obstructive pulmonary disease (COPD). The origins of BPD are complex and multifactorial and involve, among others, incomplete lung development after preterm birth, pre- and postnatal exposure to inflammation and oxidative stress, and therapeutic interventions such as oxygen administration and mechanical ventilation. Nowadays, BPD is diagnosed based on oxygen and / or ventilation requirement at day 28 of life and at 36 weeks gestational age (GA). Even though respiratory support indirectly reflects clinical disease severity, current diagnostics lack pathophysiological disease indicators that directly reveal local tissue processes in the lungs and airways, thus allowing early identification and potential interventions for newborns at high risk of BPD.

[0006] A limited number of studies investigated diseases of prematurity using applied sensor technology on faecal samples for diagnosing BPD (among other neonatal diseases). Although fecal sample collection is easily feasible in the neonatal intensive care unit (NICU), faecal studies might not be an accurate reflection of the lung microenvironment - even whilst considering the gut-lung axis. This may hamper the accuracy of faecal analytes as a biomarker for lung diseases and particularly BPD with accuracies ranging from 50 to 70%. Additionally, fecal sensor technology does not allow (tentative) analyte identification and it is thus difficult to link the presence of certain analyte to underlying pathophysiological changes.

[0007] There is a need for a method for early detection of respiratory health in a subject, in particular in neonatal subjects. Summary of the invention

[0008] In a first aspect, a method for determining a respiratory health of a test subject is provided. Said method comprises generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject, identifying, from the mass spectrometry data, at least one molecular ion mass (m / z), wherein the molecular ion mass is a molecular ion mass (m / z) of Table 1 selected from the group consisting of #1 to #18 of Table 1 , and determining from the at least one molecular ion mass (m / z), the respiratory health of the test subject.

[0009] In another aspect, a method for assessing the efficacy of a therapeutic treatment of a respiratory disease in a test subject is provided. Said method comprises generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject at a time point before the start of the treatment and at one or more subsequent time points, such as at regular intervals, after the start of the treatment; identifying, from the mass spectrometry data at least one molecular ion mass (m / z) in the sample at start of the treatment and in one or more of the subsequent samples, wherein the molecular ion mass is a molecular ion mass (m / z) of Table 1 selected from the group consisting of #1 to #18 of Table 1 ; comparing said at least one molecular ion mass (m / z) in the first sample with said at least one molecular ion mass (m / z) in one or more of the subsequent samples, wherein a deviation or no deviation is indicative for the efficacy of the therapeutic treatment in the subject.

[0010] A further aspect provides a device for use in any of the methods disclosed herein, said device comprising detection means for detection of one or more molecular ion masses (m / z) of Table 1 , in a sample of exhaled breath from said subject.

[0011] Provided herein is a method for determining a respiratory health of a test subject, said method comprising:

[0012] - generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject;

[0013] - identifying, from the mass spectrometry data at least one molecular ion mass (m / z), wherein the molecular ion mass is a molecular ion mass (m / z) of Table 1 selected from the group consisting of #1 to #18; and

[0014] - determining from the at least one molecular ion mass (m / z), the respiratory health of the test subject.

[0015] According to one aspect, the method further comprises:

[0016] - comparing a presence and / or amount of the least one identified molecular ion mass (m / z) of Table 1 with a reference; - finding a deviation or no deviation of the presence and / or amount of said at least identified molecular ion mass (m / z) from said reference; and

[0017] - attributing said finding of deviation or no deviation to an indication of respiratory health of the subject.

[0018] Provided herein is a method for assessing the efficacy of a therapeutic treatment of a respiratory disease in a test subject, said method comprising:

[0019] - generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject at a time point before the start of the treatment and at one or more subsequent time points, such as at regular intervals, after the start of the treatment;

[0020] - identifying, from the mass spectrometry data at least one molecular ion mass (m / z) in the sample at start of the treatment and in one or more of the subsequent samples, wherein the molecular ion mass is a molecular ion mass (m / z) of T able 1 selected from the group consisting of #1 to #18; and

[0021] - comparing said at least one molecular ion mass in the first sample with said at least one molecular ion mass (m / z) in one or more of the subsequent samples, wherein a deviation or no deviation is indicative for the efficacy of the therapeutic treatment in the subject.

[0022] According to one aspect:

[0023] - at least one molecular ion mass (m / z) is identified from the mass spectrometry data selected from the group consisting of #13 to #18 of Table 1 ; and / or

[0024] - at least four molecular ion masses (m / z) are identified from the mass spectrometry data selected from the group consisting of #1 to #10 of Table 1 ; and / or

[0025] - at least three molecular ion masses (m / z) are identified from the mass spectrometry data that are #3, #6, and #11 of T able 1.

[0026] According to a further aspect: at least one molecular ion mass (m / z) is identified from the mass spectrometry data selected from the group consisting of #13 to #18 of Table 1 and the determining the respiratory health is diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating lung tissue inflammation and / or oxidative stress in the lungs over time.

[0027] According to a further aspect at least two, preferably at least three, molecular ion masses (m / z) are identified from the mass spectrometry data selected from the group consisting of #13 to #18 of Table 1 and the determining the respiratory health is diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating lung tissue inflammation and / or oxidative stress in the lungs over time.

[0028] According to a further aspect:

[0029] - at least one molecular ion mass (m / z) is #13, #16, #17, or #18 of T able 1 , and wherein an increase in an amount of said at least one molecular ion mass (m / z) compared to a reference is indicative of an increase in lung tissue inflammation and / or oxidative stress in the lungs; and / or

[0030] - at least one molecular ion mass (m / z) is #14, or #15 of Table 1 , and wherein a decrease in an amount of said at least one molecular ion mass (m / z) compared to a reference is indicative of an increase in lung tissue inflammation and / or oxidative stress in the lungs.

[0031] According to a further aspect at least four molecular ion masses (m / z) are a molecular ion mass of Table 1 selected from the group consisting of #1 to #10, and the determining the respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is BDP and / or lung tissue inflammation; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time.

[0032] According to a further aspect at least five, preferably at least six, ion masses (m / z) are identified from the mass spectrometry data selected from the group consisting of #1 to #10 of Table 1 , and the determining the respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is BPD and / or lung tissue inflammation; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time.

[0033] According to a further aspect the at least four or five molecular ion masses are a molecular ion mass (m / z) of Table 1 selected from the group consisting of #1 to #5 and #7 to #10, and the determining the respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is bronchopulmonary dysplasia (BPD) and / or lung tissue inflammation; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time. According to a further aspect:

[0034] - at least four molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #4 of Table 1 , and / or

[0035] - at least five molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #5 of Table 1 , and / or

[0036] - at least six molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #6 of Table 1 , and / or

[0037] - at least seven molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #7 of Table 1 , and / or

[0038] - at least eight molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #8 of Table 1 , and / or

[0039] - at least nine molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #9 of Table 1 , and / or

[0040] - at least ten molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #10 of T able 1 , and / or

[0041] - at least three molecular ion masses (m / z) are identified from the mass spectrometry data that are #3, #6, and #11 of Table 1 , and, the determining respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is BPD and / or lung tissue inflammation or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time; optionally, wherein

[0042] - a decrease in an amount of said at least four, five, six, seven, eight, nine, ten molecular ion masses of the test subject compared to a reference is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject compared with the reference, and / or over time is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject over time, and / or

[0043] - an increase in an amount of said at least four, five, six, seven, eight, nine, ten molecular ion masses of the test subject compared to a reference is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject compared with the reference, and / or over time is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject over time. According to a further aspect the method is performed using a computer.

[0044] Further provided is a device for use in a method for determining a respiratory health of a test subject according to a method as described herein, said device comprising detection means for one or more molecular ion masses (m / z) of Table 1 , in a sample of exhaled breath from said subject; optionally, further comprising a processing unit, said processing unit receiving and processing signals from said detection means; optionally, further comprising a breath collector.

[0045] Further provided is a use of the device as described herein for determining a respiratory health in a test subject; preferably by performing the method as described herein for diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs; or preferably by performing the method as described herein for evaluating the efficacy of a therapeutic treatment of a respiratory disease; or preferably by performing the method as described herein for diagnosing, prognosing and / or monitoring respiratory dysfunction that is BPD and / or lung tissue inflammation.

[0046] Further provided is a computing device or system configured for performing the as described herein.

[0047] Another aspect provides a method of treatment of a respiratory disease, in particular BPD or a respiratory disease associated with lung inflammation and / or oxidative stress in a test subject, said method comprising assessing whether a test subject suffers from a respiratory disease, in particular from BPD ora disease associated with lung inflammation and / or oxidative stress in the lungs, according to any of the methods disclosed herein, and based on the outcome, treating the subject with a therapeutic treatment for said respiratory disease.

[0048] Figure Legends

[0049] FIG. 1. 2D (A) and 3D (B) s-PLS-DA showing the discriminatory potential of VOCs in distinguishing BPD patients from preterm controls. The main components and loadings on each component are also shown. (C). Components correlated with BPD diagnosis are indicated in dark grey, components correlated with preterm controls in light grey. FIG. 2. Summary of logistic regression models predicting allocation to the post-CPAP (model 1) or post-intubation (models 2 and 3) group after lasso regression with LOOCV. All three models contained only the VOCs detected in the breath samples. Sensitivity and specificity are shown per model.

[0050] FIG. 3. PCA with 2D and 3D representations of the respective models. (A) Model 1 : pre-CPAP versus post-CPAP; (B) Model 2: pre-intubation versus post-intubation ; (C) Model 3 : post- CPAP versus post-intubation

[0051] FIG. 4. sPLS-DA for the respective models. Corresponding component loadings are also shown. For model one, an sPLS-DA model with 2 components, consisting of 20 and 7 variables was chosen. Similarly, we chose a model with 2 components consisting of 4 and 4 variables for model two, distinguishing pre- versus post-intubation breath profiles. For model three, we again chose an optimal model with 2 components, consisting of 150 and 60 variables.

[0052] Detailed description of invention

[0053] Before the present system and method of the invention are described, it is to be understood that this invention is not limited to particular systems and methods or combinations described, since such systems and methods and combinations may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0054] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0055] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms "comprising", "comprises" and "comprised of" as used herein comprise the terms "consisting of", "consists" and "consists of".

[0056] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0057] The term "about" or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1 % or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or “approximately” refers is itself also specifically, and preferably, disclosed.

[0058] Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0059] “Directly proportional” in respect of two observations means that as one increases (or decreases) the other also increases (or decreases).

[0060] “Inversely proportional” in respect of two observations means that as one increases (or decreases), the other decreases (or increases), or vice versa.

[0061] All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.

[0062] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0063] In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0064] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0065] In the present description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. Parenthesized or emboldened reference numerals affixed to respective elements merely exemplify the elements by way of example, with which it is not intended to limit the respective elements. Unless otherwise indicated, all figures and drawings in this document are not to scale and are chosen for the purpose of illustrating different embodiments of the invention. In particular the dimensions of the various components are depicted in illustrative terms only, and no relationship between the dimensions of the various components should be inferred from the drawings, unless so indicated.

[0066] It is to be understood that other embodiments may be utilised and structural or logical changes may be made without departing from the scope of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

[0067] The methods and systems described herein are based on the finding that a respiratory health of a subject can be indicated by presence and / or amounts of single or multiple molecular ion mass signals in a mass spectrum of exhaled in the breath of a test subject. The molecular ion masses correspond to volatile organic compounds produced in biochemical pathways as intermediates, and some can be unique to a given pathway. The inventors have found that certain molecular ion masses are produced in larger or smaller amounts when the subject is suffering from bronchopulmonary dysplasia (BPD), lung tissue inflammation, or lung tissue haemorrhage. In addition, the inventors have found that amounts of certain molecular ion masses are produced in proportion (direct or inverse) to biochemical markers for inflammation and / or oxidative stress in the lung tissue (IL-1 p, IL-1 , IL-6, IL-8, IL-10, SOD2 mRNA, NOS3, A4-HNE, 4-HNE, VEGF-a, catalase, actin 4-HNE and / or oxyblot, and / or Aoxyblot). The inventors have used mass spectrometry to identify and measure the presence and / or amount of VOCs in the exhaled breath. The present invention relates to a method for determining a respiratory health of a test subject, said method comprising:

[0068] - generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject,

[0069] - identifying, from the mass spectrometry data at least one molecular ion mass (m / z),

[0070] - determining from the at least one molecular ion mass (m / z), the respiratory health of the test subject.

[0071] The at least one molecular ion mass is preferably selected from the group consisting of #1 to #18 in Table 1 herein.

[0072] The present invention also relates to a method for assessing the efficacy of a therapeutic treatment of a respiratory disease in test subject, said method comprising:

[0073] - generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject at a time point before the start of the treatment and at one or more subsequent time points, such as at regular intervals, after the start of the treatment,

[0074] - identifying, from the mass spectrometry data, at least one molecular ion mass (m / z) in the sample at start of the treatment and in one or more of the subsequent samples, wherein the molecular ion mass is a molecular ion mass (m / z) of Table 1 , selected from the group consisting of #1 to #18; and

[0075] - comparing said at least one molecular ion mass in the first sample with said at least one molecular ion mass (m / z) in one or more of the subsequent samples, wherein a deviation or no deviation is indicative for the efficacy of the therapeutic treatment in the subject.

[0076] In some embodiments, the test subject is a subject whose (respiratory) health is not known at the time of carrying out the method and is to be determined. In some embodiments, the test subject is a subject that has been diagnosed with a respiratory disease but wherein the severity of said respiratory disease is not known. In some embodiments, the test subject is a subject that has been diagnosed with a respiratory disease and wherein the efficacy of a therapeutic treatment for said disease is to be assessed.

[0077] The test subject is a mammalian subject, preferably human (more preferably neonatal human) or lamb. The subject is preferably an infant subject; preferably the subject is a neonate; more preferably a preterm neonate. A preterm neonate is defined as a baby that is born alive before 37 weeks of pregnancy are completed.

[0078] The respiratory health may be diagnosis, prognosis and / or monitoring, and / or determining the risk of developing of a lung dysfunction or a respiratory disease. The lung dysfunction or respiratory disease may be bronchopulmonary dysplasia (BPD). The lung dysfunction or respiratory disease may be lung inflammation. The lung dysfunction or respiratory disease may be oxidative stress present in the lung tissue.

[0079] In some embodiments, the lung dysfunction or respiratory disease is BPD, the presence of lung inflammation or both. In preferred embodiments, the lung dysfunction or respiratory disease is BPD.

[0080] In some embodiments, the lung dysfunction or respiratory disease is the presence of lung inflammation, the presence of oxidative stress in the lungs, or both the presence of lung inflammation and oxidative stress in the lungs.

[0081] The presence of lung inflammation and / or oxidative stress may be evaluated by determining or measuring the status of one or more biochemical markers present in respiratory tissue. The one or more biochemical markers present in respiratory tissue that can be used to determine the presence of lung inflammation and / or oxidative stress may be selected from I L-1 p, IL-1 , IL- 6, IL-8, IL-10, SOD2 mRNA, NOS3, A4-HNE, 4-HNE, VEGF-a, catalase, actin 4-HNE and / or oxyblot, and / or Aoxyblot.

[0082] In the context of the present application, “diagnosis” and diagnosing” generally include a determination of a subject’s susceptibility to a disease or disorder, a determination as to whether a subject is presently affected by a disease or disorder, a prognosis of a subject affected by a disease or disorder, and therametrics (e.g., monitoring a subject’s condition to provide information as to the effect or efficacy of therapy). For example, as used herein, the terms diagnosing or diagnosis refer to diagnosing a disease or condition based on the presence, and / or amount of one or more molecular ion masses detected in a sample of exhaled breath.

[0083] The terms “prognosis” or “prognosing” refer to the fact or art of foretelling the course of a disease. Additionally, the terms refer to the prospect of survival recovery from a disease as anticipated from the usual course of that disease or indicated by special features of the individual case. Further, the terms refer to the art or act of identifying a disease from its signs and symptoms.

[0084] The terms “treatment”, “treating”, “treat” and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptoms thereof and / or may be therapeutic in terms of partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. “Treatment” covers any treatment of a disease in a mammal, particular a human, and includes: (a) preventing the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptoms, i.e. arresting its development: or (c) relieving the disease symptoms, i.e. causing regression of the disease or symptom. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Non-limiting examples of therapeutic treatment of lung dysfunction or respiratory disease, such as BPD, include but are not limited to non- invasive or invasive ventilation, oxygen supplementation, diuretics, inhaled and / or systemic corticosteroids, diuretics, antibiotics for the treatment of respiratory infections, prophylactic antibiotics, and adequate nutritional support.

[0085] In some embodiments, the methods disclosed herein comprise generating mass spectrometry data of a sample of exhaled breath of the test subject. In some embodiments, the methods disclosed herein comprise receiving mass spectrometry data of sample of exhaled beath of the test subject.

[0086] The mass spectrometry data is thus generated or recorded using mass-spectrometry. The mass-spectrometer may be any type suitable for analysis of volatile organic compounds. The mass spectrometry may be performed with or without chromatography. For example, the mass spectrometry may be any of: proton-transfer-reaction mass spectrometry (PTR-MS), gas chromatography mass spectrometry (GC-MS), selected ion flow tube mass spectrometry (SIFT-MS), liquid chromatography-tandem mass spectrometry (LC-MS), ion mobility spectrometry-mass spectrometry (IMS-MS), tandem gas chromatography mass spectrometry (GCxGC-MS), secondary electron-spray ionization MS (SESI-MS).

[0087] Mass spectrometry methods which may be used with the present invention include, but are not limited to, electron ionization, electrospray ionization, glow discharge, field desorption (FD), fast atom bombardment (FAB), thermospray, desorption / ionization on silicon (DIOS), Direct Analysis in Real Time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), spark ionization and thermal ionization (TIMS). Preferably, the mass spectrometry is PTR-MS.

[0088] The mass spectrometry produces a mass spectrum for the exhaled breath sample. The “molecular ion mass signals” refers to a signal(s) in the mass spectrum that allow identification of an ion form of a molecule (molecular ion mass). The molecular ion mass refers to a mass of that ion form of a molecule, in particular. It is preceded with a “m / z” herein to distinguish from molecular mass. The molecular mass refers to a mass of a non-ionised molecule determined from the molecular ion mass signals or molecular ion mass. It is typically expressed in g / mol. It has no prefix herein. The molecular ion mass (and molecular mass where identifiable) is identified from the molecular ion mass signals by standard techniques known the in art. The molecular ion mass (and molecular mass where identifiable) is identified automatically from the molecular ion mass signals by techniques known the in art. Typically software applications built-in or supplied with the mass spectrometer automatically identify the molecular ion mass and optionally molecular mass. The processing steps for identifying molecular ion mass (and molecular mass where identifiable) depends on the type of mass-spectrometry (e.g. PTR- HRMS, GC-MS, SIFT-MS). Databases and / or protocols are available in the art, for instance the National Institute of Standards and Technology (NIST) library (available at http: / / www.nist.gov / srd / nist1a.cfm). For example, when using PTR-HRMS, as a general rule a value of 1 is subtracted from the molecular ion mass signal to arrive at a molecular mass.

[0089] An advantage of mass spectrometry is the accuracy and precision of the measurement of molecular ion mass (m / z), and the molecular ion mass (m / z) measurement produced for a VOC is the same for different mass spectrometry techniques.

[0090] The sample of exhaled breath is collected during non-invasive ventilation or minimally invasive ventilation of the test subject. The sample of exhaled breath is collected by passing the exhaled breath into a gas sampling system. The gas sampling system may pass the exhaled breath directly to the mass spectrometer. Alternatively the gas sampling system may comprise a solid support (e.g. beads or fibres) through / over which the exhaled breath passes and to which the VOCs bind. The solid support may be contained in a (thermal) desorption tube such as a Tenax column, SPME-column or canister. The exhaled breath may be drawn through / over the solid support assisted by a vacuum. VOCs are subsequently desorbed from the solid support (e.g. using thermal desorption), and the desorbed VOCs are analysed using mass-spectrometry.

[0091] The mass spectrometry data comprises at least a part of a mass spectrum recorded for a plurality of VOCs that have been captured over a period of time (e.g. 1 to 5 hours) in a recording session.

[0092] The present method is performed in vitro. In some embodiments, the present method is performed in silico (is computer implemented).

[0093] A presence of at least one molecular ion mass may be identified from the mass spectrometry data. The presence may be based on a standard threshold. An “amount”, also referred to as “quantity” or “level”, of at least one molecular ion mass may be further determined from the mass spectrometry data. The terms as used herein may particularly refer to an absolute quantification of one or more molecular ion masses in a sample, or to a relative quantification of one or more molecular ion masses in a sample, i.e. , relative to another value such as relative to a reference value as taught herein, or to a range of values indicating a baseline expression of the one or more molecular ion mases. These values or ranges can be obtained from a single subject or from a group of subjects. As used herein, the amount is based on a signal (e.g. peak height(s) or peak area(s)) of the molecular ion mass signal(s). The amount is an indication of the amount of the VOC having the identified the molecular ion mass.

[0094] An internal reference (a substance present in a known amount which provides a mass spectrometry signal) may be used to calibrate the amount of the at least one molecular ion mass in the sample.

[0095] The reference as used herein represents a known respiratory health of a reference group of one or more reference subjects. The reference subject of the reference group is healthy or has normal levels of respiratory health, and preferably no reported history of respiratory unhealth. In particular, by healthy it is meant the reference subject is not affected by bronchopulmonary dysplasia (BPD) or lung tissue inflammation or oxidative stress in the lungs or lung tissue haemorrhage and preferably no reported history of BPD or lung tissue inflammation or oxidative stress in the lungs or lung tissue haemorrhage. In particular, by healthy it is meant that the reference subject has reference (normal) levels of I L-1 p, IL-1 , IL-6, IL-8, IL-10, SOD2 mRNA, NOS3, A4-HNE, 4-HNE, VEGF-a, catalase, actin 4-HNE and / or oxyblot, and / or Aoxyblot in the lung tissue. The reference is a comparable reference, meaning that the presence or amounts are being compared for the same at least one molecular ion mass in the test subject and reference. More in particular, the comparable reference is a presence or amount of the least one molecular ion mass in the reference group, wherein the least one molecular ion mass (e.g. one or multiple ion masses from Table 1) in the reference group is the same as in the test subject. The reference or comparable reference is a value.

[0096] In some embodiments, the reference refers to the first sample of the test subject that is obtained, such as the sample that is obtained from the test subject before the start of a therapeutic treatment.

[0097] Where a presence and / or amount of the least two identified molecular ion masses is compared, the presence and / or amount of the least two identified molecular ion masses in the test subject may be statistically combined to provide a scalar value. For instance, the presence and / or amount of the least two identified molecular ion masses in the test subject may be averaged, preferably weighted averaged, and compared with the reference that comprises the presence and / or amount of the same least two identified molecular ion masses in the reference group that have been averaged, preferably weighted averaged.

[0098] The (comparable) reference is determined for the reference subject(s) of the reference group and the test subject using the same or similar protocols for collection of a sample of exhaled breath and receiving mass spectrometry data, and identification of the molecular ion masses.

[0099] A deviation of the amount in the test subject, preferably an increased or decreased amount, of said one or more molecular ion masses, in particular one or more VOCs represented by said one or more molecular ion masses, from said reference is indicative (diagnostic or prognostic) of the respiratory health or respiratory dysfunction in the subject or of the therapeutic response to a particular treatment.

[0100] The term “no deviation of the amount” refers to similar or unchanged amounts of one or more molecular ion masses or VOCs in a sample of exhaled breath from a subject compared to a reference value. By “similar or unchanged level” is meant that the difference of the amount of said one or more molecular ion masses or VOCs in a sample of exhaled breath from the subject compared to the reference value is not statistically significant. Preferably, the reference value is obtained in samples of exhaled breath obtained from one or more subjects of the same species and the same sex and age group as the subject in which the respiratory health is to be determined, prognosed or monitored. Alternatively, the reference value may be a previous value for the amount of one or more molecular ion masses or VOCs obtained in a sample of exhaled breath from a test subject. This kind of reference value may be used if the method is to be used for monitoring respiratory health in a subject, e.g., over time, or to monitor the response of a subject to a particular treatment.

[0101] Preferably, the reference value is the average amount of the same one or more molecular ion masses or VOCs found in samples of exhaled breath from a population of subjects. Preferably, said average expression level is determined once and then stored in a database for reference.

[0102] As explained, the present methods, uses, or products may involve finding a deviation or no deviation between at least one molecular ion mass (m / z) as taught herein measured in a sample from a subject and a given reference value or threshold value.

[0103] A ’’deviation” of a first value from a second value or a “difference” between a first value and a second value may generally encompass any direction (e.g., increase: first value > second value; or decrease: first value < second value) and any extent of alteration. For example, a deviation or a difference may encompass a decrease in a first value by, without limitation, at least about 10% (about 0.9-fold or less), or by at least about 20% (about 0.8-fold or less), or by at least about 30% (about 0.7-fold or less), or by at least about 40% (about 0.6- fold or less), or by at least about 50% (about 0.5-fold or less), or by at least about 60% (about 0.4-fold or less), or by at least about 70% (about 0.3-fold or less), or by at least about 80% (about 0.2-fold or less), or by at least about 90% (about 0.1 -fold or less), relative to a second value with which a comparison is being made.

[0104] For example, a deviation or a difference may encompass an increase of a first value by, without limitation, at least about 10% (about 1.1-fold or more), or by at least about 20% (about 1 .2-fold or more), or by at least about 30% (about 1 .3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second value with which a comparison is being made.

[0105] Preferably, a deviation or a difference may refer to a statistically significant observed alteration. For example, a deviation or a difference may refer to an observed alteration which falls outside of error margins of reference values in a given population (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., ±1xSD or ±2xSD or ±3xSD, or ±1xSE or ±2xSE or ±3xSE). Deviation or a difference may also refer to a value falling outside of a reference range defined by values in a given population (for example, outside of a range which comprises >40%, > 50%, >60%, >70%, >75% or >80% or >85% or >90% or >95% or even >100% of values in said population).

[0106] In a further embodiment, a deviation or a difference may be concluded if an observed alteration is beyond a given threshold or cut-off. Such threshold or cut-off may be selected as generally known in the art to provide for a chosen accuracy, sensitivity and / or specificity of the prediction methods, e.g., accuracy, sensitivity and / or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.

[0107] For example, receiver-operating characteristic (ROC) curve analysis can be used to select an optimal threshold or cut-off value of the quantity of a given biomarker for clinical use of the present diagnostic tests, based on acceptable global accuracy, sensitivity and / or specificity, or related performance measures which are well-known perse, such as positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), Youden index, or similar.

[0108] For example, an optimal threshold or cut-off value may be selected for each individual biomarker as a local extremum of the receiver operating characteristic (ROC) curve, i.e. a point of local maximum distance to the diagonal line, as described in Robin X., PanelomiX: a threshold-based algorithm to create panels of biomarkers, 2013, Translational Proteomics, 1 (1):57-64.

[0109] The person skilled in the art will understand that it is not relevant to give an exact threshold or cut-off value. A relevant threshold or cut-off value can be obtained by correlating the sensitivity and specificity and the sensitivity / specificity for any threshold or cut-off value.

[0110] It is to the diagnostic engineers to determine which level of positive predictive value / negative predictive value / sensitivity / specificity is desirable and how much loss in positive or negative predictive value is tolerable. The chosen threshold or cut-off level could be dependent on other diagnostic parameters used in combination with the present method by the diagnostic engineers.

[0111] The respiratory health of the test subject may be monitored over time. Over time means that respiratory health of the test subject is determined at different time points that are preferably at least 1 day apart, for instance, at least one week, at least one month, or at least one year apart. A trend in respiratory health (e.g. improvement, decline, no trend) over time may be derived from test subject. From the trend, a response to a treatment can be monitored, for instance, or a worsening or improvement in the condition without treatment..

[0112] In some embodiments, mass spectrometry data are generated or received from a sample of exhaled breath of the test subject at a time point before the start of a treatment of a respiratory disease and at one or more subsequent time points, such as at regular intervals, after the start of the treatment. Preferably, the sample that is obtained before the start of the treatment is the reference sample from which the presence and / or amount of one or more molecular ion masses or VOCs are determined. Said presence and / or amount of one or more molecular ion masses or VOCs is then a reference value. In some embodiments, the presence and / or amount of one or more molecular ion masses or VOCs is determined in one or more samples that are collected at one or more subsequent time points after the start of the treatment. The amount and / or presence of the one or more molecular ion masses or VOCs in said one more samples is then compared to the reference value of the sample that was collected before the start of the treatment.

[0113] The molecular ion mass corresponds to an ion (charged) species of a volatile organic compound (VOC). The term “volatile organic compounds” (abbreviated VOC, VOCs, or VOCS) refers to organic chemicals, or derivatives thereof, present in exhaled breath of a subject. The VOCs that are identified from the molecular ion masses of interest in the present application (see Table 1) are represented in Table 5. The skilled person is well aware that VOCs may be referred to by different names or synonyms.

[0114] At least one molecular ion mass is identified from the molecular ion mass signals of the mass spectrometry data, wherein the identified molecular ion mass corresponds to a volatile organic compound. Preferably, multiple molecular ion masses are identified from the molecular ion mass signals of the mass spectrometry data, wherein each identified molecular ion mass corresponds to a different volatile organic compound.

[0115] Preferably, the molecular ion mass is a molecular ion mass corresponding to one of #1 to #18 in Table 1. In other words, the identified molecular ion mass is a molecular ion mass (m / z) in the tolerance range mentioned in Column II that corresponds to one of #1 to #18 in Table 1. Table 1 also shows the molecular mass (Column III) that may be identified from the molecular ion mass signals of the mass spectrometry data, and corresponding compound name (Column IV) that may be identified from the molecular ion mass signals of the mass spectrometry data.

[0116] Table A: Code (column I), molecular ion mass (m / z) and tolerance range (Column II), assigned molecular mass (Column III), assigned (tentative) compound name (Column IV). ND - not determined.

[0117] Preferably, at least one molecular ion mass is identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 in Table 1. In some embodiments, at least two, at least three, or preferably at least four molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 in Table 1. Preferably, the determining the respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least one molecular ion mass selected from the group consisting of #1 to #10 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least one molecular ion mass selected from the group consisting of #1 to #10 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least one molecular ion mass selected from the group consisting of #1 to #10 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in amount of the at least one molecular ion mass selected from the group consisting of #1 to #10 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time. [Likely not novel]

[0118] Preferably, at least one molecular ion marker is identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 in Table 1. In some embodiments, at least two, at least three, or preferably at least four molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 in Table 1.

[0119] Preferably, the determining the respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. [Likely not novel] According to one aspect, an amount of the at least one molecular ion mass selected from the group consisting of #1 to #5 and #7 to #10 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least one molecular ion mass selected from the group consisting of #1 to #5 and #7 to #10 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least one molecular ion mass selected from the group consisting of #1 to #5 and #7 to #10 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least one molecular ion mass selected from the group consisting of #1 to #5 and #7 to #10 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0120] Preferably, at least four molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #4 of Table 1 . Preferably the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least four molecular ion masses that are #1 to #4 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least four molecular ion masses that are #1 to #4 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least four molecular ion masses that are #1 to #4 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least four molecular ion masses that are #1 to #4 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0121] Preferably, at least five molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #5 of Table 1 . Preferably, the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least five molecular ion masses that are #1 to #5 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least five molecular ion masses that are #1 to #5 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least five molecular ion masses that are #1 to #5 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least five molecular ion masses that are #1 to #5 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0122] Preferably, at least six molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #6 of Table 1 . Preferably, the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least six molecular ion masses that are #1 to #6 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least six molecular ion mass that are #1 to #6 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least six molecular ion masses that are #1 to #6 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least six molecular ion masses that are #1 to #6 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0123] Preferably, at least seven molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #7 of Table 1. Preferably, the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least seven molecular ion masses that are #1 to #7 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least seven molecular ion mass that are #1 to #7 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least seven molecular ion masses that are #1 to #7 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least seven molecular ion masses that are #1 to #7 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0124] Preferably, at least eight molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #8 of Table 1 . Preferably, the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least eight molecular ion masses that are #1 to #8 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least eight molecular ion mass that are #1 to #8 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least eight molecular ion masses that are #1 to #8 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least eight molecular ion masses that are #1 to #8 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0125] Preferably, at least nine molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #9 of Table 1 . Preferably, the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least nine molecular ion masses that are #1 to #9 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least nine molecular ion mass that are #1 to #9 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least nine molecular ion masses that are #1 to #9 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least nine molecular ion masses that are #1 to #9 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0126] Preferably, at least ten molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #10 of Table 1. Preferably, the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least ten molecular ion masses that are #1 to #10 is inversely proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A lower amount of the at least ten molecular ion mass that are #1 to #10 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. A decrease in an amount of the at least ten molecular ion masses that are #1 to #10 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. A decrease in an amount of the at least ten molecular ion masses that are #1 to #10 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0127] Preferably, at least three molecular ion masses (m / z) are identified from the mass spectrometry data that are #3, #6, #11 of Table 1. Preferably the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is BDP or lung tissue inflammation. According to one aspect, an amount of the at least four molecular ion masses that are #3, #6, #11 is directly proportional to a severity of the BDP or to a level of lung tissue inflammation of the test subject. A higher amount of the at least four molecular ion masses that are #3, #6, #11 indicates an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject. An increase in an amount of the at least four molecular ion masses that are #3, #6, #11 of the test subject, compared to a reference is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject compared with the reference. An increase in an amount of the at least four molecular ion masses that are #3, #6, #11 of the test subject over time is indicative of an increase in severity of the BDP or an increase in the level of lung tissue inflammation of the test subject over time.

[0128] According to one aspect, at least one molecular ion mass (m / z) is identified from the mass spectrometry data that are #13 to #18 of T able 1 . The at least one molecular ion mass #13 to #18 of Table 1 is indicative of respiratory health, in particular for diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs of the subject. The at least one molecular ion mass #13 to #18 of T able 1 may also be indicative for the response to a particular therapeutic treatment for a respiratory disease in the test subject.

[0129] In some embodiments, the methods disclosed herein include the identification of at least two, preferably at least three, more preferably at least four or at least five, molecular ion masses (m / z) from the mass spectrometry data that are #13 to #18 of Table 1 , and the determining the respiratory health in a test subject, in particular diagnosing, prognosing, and / / or monitoring lung tissue inflammation and / or oxidative stress in the lungs of the subject.

[0130] In some embodiments, the presence and / or amount of at least one molecular ion mass (m / z) of #13, #16, #17, or #18 of Table 1 is determined and an increase in the presence and / or amount of said at least one molecular ion mass (m / z) compared to a reference is indicative of an increase in lung tissue inflammation and / or oxidative stress in the lungs of the subject.

[0131] In some embodiments, the presence and / or amount of at least one molecular ion mass (m / z) of #14 or #15 of T able 1 is determined and a decrease in the presence and / or amount of said at least one molecular ion mass (m / z) compared to a reference is indicative of an increase in lung tissue inflammation and / or oxidative stress in the lungs of the subject. According to one aspect, at least one molecular ion mass is identified from the mass spectrometry data that is #13 in Table 1. The molecular ion mass #13 in Table 1 is indicative of respiratory health, in particular, of lung-tissue inflammation and oxidative stress in the lungs. In some embodiments, the molecular ion mass #13 in Table 1 is indicative of Nitric Oxide Synthase 3 (NOS3) levels, and / or A4-HNE level, which are both markers for oxidative stress.

[0132] According to one aspect, an amount of the molecular ion masses that is #13 is inversely proportional to the respiratory health of the test subject. A lower amount of the molecular ion masses that is #13 indicates an increase in the respiratory health of the test subject. A decrease in an amount of the molecular ion masses that is #13 of the test subject, compared to a reference is indicative of an increase in the respiratory health of the test subject compared with the reference. A decrease in an amount of the molecular ion masses that is #13 is indicative of an increase of the respiratory health of the test subject over time.

[0133] Preferably, the determining respiratory health is diagnosing, prognosing and / or monitoring a respiratory dysfunction that is lung tissue inflammation. According to one aspect, an amount of the molecular ion masses that is #13 is directly proportional to a level of lung tissue inflammation of the test subject. A higher amount of the molecular ion masses that is #13 indicates an increase in the level of lung tissue inflammation of the test subject. An increase in an amount of the molecular ion masses that is #13 of the test subject, compared to a reference is indicative of an increase in the level of lung tissue inflammation of the test subject, compared with the reference. An increase in an amount of the molecular ion masses that is #13 of the test subject over time is indicative of an increase in the level of lung tissue inflammation of the test subject, over time.

[0134] Preferably, the determining respiratory health is determining a level of oxidative stress in the test subject; in particular determining a level of Nitric Oxide Synthase 3 (NOS3) in the test subject, more in particular a level of NOS3 in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #13 is directly proportional to oxidative stress, in particular a level of NOS3 in the test subject, more in particular of NOS3 in the lung tissue of the test subject. A higher amount of the molecular ion mass that is #13 indicates an increase in oxidative stress, reflected by an increase in the level of NOS3 in the test subject, in particular of NOS3 in the lung tissue of the test subject. An increase in an amount of the molecular ion mass that is #13 of the test subject, compared to a reference is indicative of an increase in oxidative stress in the lungs of the test subject, in particular indicative of an increase in the level of NOS3 in the test subject, more in particular of NOS3 in the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion mass that is #13 of the test subject over time is indicative of an increase of oxidative stress, reflected in the level of NOS3 in the test subject, in particular of NOS3 in the lung tissue of the test subject, over time.

[0135] Preferably, the determining respiratory health by determining oxidative stress is determining a level of A4-HNE in the test subject, in particular a level of A4-HNE in the lung tissue of the test subject. According to one aspect, an amount of the molecular ion mass that is #13 is directly proportional to a level of A4-HNE in the test subject, in particular of A4-HNE in the lung tissue of the test subject. A higher amount of the molecular ion mass that is #13 indicates an increase in the level of A4-HNE in the test subject, in particular of A4-HNE in the lung tissue of the test subject. An increase in an amount of the molecular ion mass that is #13 of the test subject, compared to a reference is indicative of an increase in the level of A4-HNE in the test subject, in particular of A4-HNE in the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion mass that is #13 of the test subject over time is indicative of an increase in the level of A4-HNE in the test subject, in particular of A4-HNE in the lung tissue of the test subject, over time.

[0136] According to one aspect, at least one molecular ion mass identified from the mass spectrometry data that is #14 in Table A. The molecular ion mass #14 in Table A is indicative of respiratory health, in particular, a level of I L-1 p, and / or a level of IL-6, and / or a level of IL-8, and / or a level of IL-10, and / or a level of SOD2, and / or a level of A 4-HNE, and / or a level of 4- HNE in the test subject.

[0137] According to one aspect, an amount of the molecular ion masses that is #14 is directly proportional to the respiratory health of the test subject. A higher amount of the molecular ion masses that is #14 indicates an increase in the respiratory health of the test subject. An increase in an amount of the molecular ion masses that is #14 of the test subject, compared to a reference is indicative of an increase in the respiratory health of the test subject compared with the reference. An increase in an amount of the molecular ion masses that is #14 is indicative of an increase of the respiratory health of the test subject over time.

[0138] Preferably, the determining respiratory health is determining a level of interleukin-1 p (I L-1 p) in the test subject, in particular a level of I L-1 p in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #14 is inversely proportional to a level of I L-1 p in the test subject, in particular of IL-1 p in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #14 indicates an increase in the level of I L-1 p in the test subject, in particular of I L-1 p in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #14 of the test subject, compared to a reference is indicative of an increase in the level of I L-1 in the test subject, in particular of IL-1 p in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #14 of the test subject over time is indicative of an increase in the level of I L-1 p in the test subject, in particular of I L-1 p in the lung tissue of the test subject, over time.

[0139] Preferably, the determining respiratory health is determining a level of interleukin-6 (IL-6) in the test subject, in particular a level of IL-6 in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #14 is inversely proportional to a level of IL-6 in the test subject, in particular of IL-6 in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #14 indicates an increase in the level of IL-6 in the test subject, in particular of IL-6 in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #14 of the test subject, compared to a reference is indicative of an increase in the level of IL-6 in the test subject, in particular of IL-6 in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #14 of the test subject over time is indicative of an increase in the level of IL-6 in the test subject, in particular of IL-6 in the lung tissue of the test subject, over time.

[0140] Preferably, the determining respiratory health is determining a level of interleukin-8 (IL-8) in the test subject, in particular a level of IL-8 in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #14 is inversely proportional to a level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #14 indicates an increase in the level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #14 of the test subject, compared to a reference is indicative of an increase in the level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #14 of the test subject over time is indicative of an increase in the level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject, over time.

[0141] Preferably, the determining respiratory health is determining a level of interleukin-10 (IL-10) in the test subject, in particular a level of IL-10 in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #14 is inversely proportional to a level of IL-10 in the test subject, in particular of IL-10 in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #14 indicates an increase in the level of IL-10 in the test subject, in particular of IL-10 in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #14 of the test subject, compared to a reference is indicative of an increase in the level of IL-10 in the test subject, in particular of IL-10 in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #14 of the test subject over time is indicative of an increase in the level of IL-10 in the test subject, in particular of IL-10 in the lung tissue of the test subject, over time.

[0142] Preferably, the determining respiratory health is determining a level of superoxide dismutase 2 (SOD2) in the test subject, in particular a level of SOD2 in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #14 is inversely proportional to a level of SOD2 in the test subject, in particular of SOD2 in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #14 indicates an increase in the level of SOD2 in the test subject, in particular of SOD2 in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #14 of the test subject, compared to a reference is indicative of an increase in the level of SOD2 in the test subject, in particular of SOD2 in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #14 of the test subject over time is indicative of an increase in the level of SOD2 in the test subject, in particular of SOD2 in the lung tissue of the test subject, over time.

[0143] Preferably, the determining respiratory health is determining a level of A 4-HNE in the test subject, in particular a level of A4-HNE in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #14 is inversely proportional to a level of A 4-HNE in the test subject, in particular of A 4-HNE in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #14 indicates an increase in the level of A 4- HNE in the test subject, in particular of A 4-HNE in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #14 of the test subject, compared to a reference is indicative of an increase in the level of A 4-HNE in the test subject, in particular of A 4-HNE in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #14 of the test subject over time is indicative of an increase in the level of A 4-HNE in the test subject, in particular of A 4-HNE in the lung tissue of the test subject, over time.

[0144] Preferably, the determining respiratory health is determining a level of 4-HNE in the test subject, in particular a level of 4-HNE in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #14 is inversely proportional to a level of 4-HNE in the test subject, in particular of 4-HNE in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #14 indicates an increase in the level of 4-HNE in the test subject, in particular of 4-HNE in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #14 of the test subject, compared to a reference is indicative of an increase in the level of 4-HNE in the test subject, in particular of 4-HNE in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #14 of the test subject over time is indicative of an increase in the level of 4-HNE in the test subject, in particular of 4-HNE in the lung tissue of the test subject, over time.

[0145] According to one aspect, at least one molecular ion mass identified from the mass spectrometry data that is #15 in Table A. The molecular ion mass #15 in Table A is indicative of respiratory health, in particular, a level of IL-1 p, and / or a level of IL-8, and / or a level of VEGF- a in the test subject.

[0146] According to one aspect, an amount of the molecular ion masses that is #15 is directly proportional to the respiratory health of the test subject. A increase amount of the molecular ion masses that is #15 indicates an increase in the respiratory health of the test subject. An increase in an amount of the molecular ion masses that is #15 of the test subject, compared to a reference is indicative of an increase in the respiratory health of the test subject compared with the reference. A increase in an amount of the molecular ion masses that is #15 is indicative of an increase of the respiratory health of the test subject over time.

[0147] Preferably, the determining respiratory health is determining a level of interleukin-1 p (I L-1 p) in the test subject, in particular a level of I L-1 p in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #15 is inversely proportional to a level of I L-1 p in the test subject, in particular of I L-1 p in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #15 indicates an increase in the level of I L-1 p in the test subject, in particular of I L-1 p in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #15 of the test subject, compared to a reference is indicative of an increase in the level of I L-1 p in the test subject, in particular of IL-1 p in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #15 of the test subject over time is indicative of an increase in the level of I L-1 p in the test subject, in particular of I L-1 p in the lung tissue of the test subject, over time. Preferably, the determining respiratory health is determining a level of interleukin-8 (IL-8) in the test subject, in particular a level of IL-8 in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #15 is inversely proportional to a level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #15 indicates an increase in the level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #15 of the test subject, compared to a reference is indicative of an increase in the level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #15 of the test subject over time is indicative of an increase in the level of IL-8 in the test subject, in particular of IL-8 in the lung tissue of the test subject, over time.

[0148] Preferably, the determining respiratory health is determining a level of vascular endothelial growth factor-a (VEGF-a) in the test subject, in particular a level of VEGF-a in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #15 is directly proportional to a level of VEGF-a in the test subject, in particular of VEGF-a in the lung tissue of the test subject. A higher amount of the molecular ion mass that is #15 indicates an increase in the level of VEGF-a in the test subject, in particular of VEGF-a in the lung tissue of the test subject. An increase in an amount of the molecular ion mass that is #15 of the test subject, compared to a reference is indicative of an increase in the level of VEGF-a in the test subject, in particular of VEGF-a in the lung tissue of the test subject compared with the reference. An increase in an amount of the molecular ion mass that is #15 of the test subject over time is indicative of an increase in the level of VEGF-a in the test subject, in particular of VEGF-a in the lung tissue of the test subject over time.

[0149] According to one aspect, at least one molecular ion mass identified from the mass spectrometry data that is #16 in Table A. The molecular ion mass #16 in Table A is indicative of respiratory health, in particular, a level of oxyblot, and / or a level of Aoxyblot in the test subject. In some embodiments, the test subject is a sheep.

[0150] According to one aspect, an amount of the molecular ion masses that is #16 is directly proportional to the respiratory health of the test subject. A higher amount of the molecular ion masses that is #16 indicates an increase in the respiratory health of the test subject. An increase in an amount of the molecular ion masses that is #16 of the test subject, compared to a reference is indicative of an increase in the respiratory health of the test subject compared with the reference. An increase in an amount of the molecular ion masses that is #16 is indicative of an increase of the respiratory health of the test subject over time.

[0151] Preferably, the determining respiratory health is determining a level of oxyblot in the test subject, in particular a level of oxyblot in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #16 is directly proportional to a level of oxyblot in the test subject, in particular of oxyblot in the lung tissue of the test subject. A higher amount of the molecular ion mass that is #16 indicates an increase in the level of oxyblot in the test subject, in particular of oxyblot in the lung tissue of the test subject. An increase in an amount of the molecular ion mass that is #16 of the test subject, compared to a reference is indicative of an increase in the level of oxyblot in the test subject, in particular of oxyblot in the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion mass that is #16 of the test subject over time is indicative of an increase in the level of oxyblot in the test subject, in particular of oxyblot in the lung tissue of the test subject, over time.

[0152] Preferably, the determining respiratory health is determining a level of A oxyblot in the test subject, in particular a level of A oxyblot in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #16 is directly proportional to a level of A oxyblot in the test subject, in particular of A oxyblot in the lung tissue of the test subject. A higher amount of the molecular ion mass that is #16 indicates an increase in the level of A oxyblot in the test subject, in particular of A oxyblot in the lung tissue of the test subject. An increase in an amount of the molecular ion mass that is #16 of the test subject, compared to a reference is indicative of an increase in the level of A oxyblot in the test subject, in particular of A oxyblot in the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion mass that is #16 of the test subject over time is indicative of an increase in the level of A oxyblot in the test subject, in particular of A oxyblot in the lung tissue of the test subject, over time.

[0153] According to one aspect, at least one molecular ion mass identified from the mass spectrometry data that is #17 in Table A. The molecular ion mass #17 in Table A is indicative of respiratory health, in particular, of haemorrhage of the lung tissue, and / or a level of catalase, and / or level of actin 4-HNE in the test subject.

[0154] According to one aspect, an amount of the molecular ion masses that is #17 is proportional to the respiratory health of the test subject. A lower amount of the molecular ion masses that is #17 indicates an increase in the respiratory health of the test subject. A decrease in an amount of the molecular ion masses that is #17 of the test subject, compared to a reference is indicative of an increase in the respiratory health of the test subject compared with the reference. A decrease in an amount of the molecular ion masses that is #17 is indicative of an increase of the respiratory health of the test subject over time.

[0155] According to one aspect, an amount of the molecular ion masses that is #17 is directly proportional to the respiratory health that is haemorrhage of the lung tissue of the test subject. An increase amount of the molecular ion masses that is #17 indicates an increase in the level of haemorrhage of the lung tissue of the test subject. An increase in an amount of the molecular ion masses that is #17 of the test subject, compared to a reference is indicative of an increase in the in the level of haemorrhage of the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion masses that is #17 is indicative of an increase of the in the level of haemorrhage of the lung tissue of the test subject, over time.

[0156] Preferably, the determining respiratory health is determining a level of catalase in the test subject, in particular a level of catalase in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #17 is directly proportional to a level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject. A higher amount of the molecular ion mass that is #17 indicates an increase in the level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject. An increase in an amount of the molecular ion mass that is #17 of the test subject, compared to a reference is indicative of an increase in the level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion mass that is #17 of the test subject over time is indicative of an increase in the level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject, over time.

[0157] Preferably, the determining respiratory health is determining a level of actin 4-HNE in the test subject, in particular a level of actin 4-HNE in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #17 is inversely proportional to a level of actin 4-HNE in the test subject, in particular of actin 4-HNE in the lung tissue of the test subject. A lower amount of the molecular ion mass that is #17 indicates an increase in the level of actin 4-HNE in the test subject, in particular of actin 4-HNE in the lung tissue of the test subject. A decrease in an amount of the molecular ion mass that is #17 of the test subject, compared to a reference is indicative of an increase in the level of actin 4-HNE in the test subject, in particular of actin 4-HNE in the lung tissue of the test subject, compared with the reference. A decrease in an amount of the molecular ion mass that is #17 of the test subject over time is indicative of an increase in the level of actin 4-HNE in the test subject, in particular of actin 4-HNE in the lung tissue of the test subject, over time.

[0158] According to one aspect, at least one molecular ion mass identified from the mass spectrometry data that is #18 in Table A. The molecular ion mass #18 in Table A is indicative of respiratory health, in particular, of haemorrhage of the lung tissue, and / or a level of catalase in the test subject.

[0159] According to one aspect, an amount of the molecular ion masses that is #18 is proportional to the respiratory health of the test subject. A lower amount of the molecular ion masses that is #18 indicates an increase in the respiratory health of the test subject. A decrease in an amount of the molecular ion masses that is #18 of the test subject, compared to a reference is indicative of an increase in the respiratory health of the test subject compared with the reference. A decrease in an amount of the molecular ion masses that is #18 is indicative of an increase of the respiratory health of the test subject over time.

[0160] According to one aspect, an amount of the molecular ion masses that is #18 is directly proportional to the respiratory health that is haemorrhage of the lung tissue of the test subject. An increase amount of the molecular ion masses that is #18 indicates an increase in the level of haemorrhage of the lung tissue of the test subject. An increase in an amount of the molecular ion masses that is #18 of the test subject, compared to a reference is indicative of an increase in the in the level of haemorrhage of the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion masses that is #18 is indicative of an increase of the in the level of haemorrhage of the lung tissue of the test subject, over time.

[0161] Preferably, the determining respiratory health is determining a level of catalase in the test subject, in particular a level of catalase in the lung tissue of in the test subject. According to one aspect, an amount of the molecular ion mass that is #18 is directly proportional to a level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject. A higher amount of the molecular ion mass that is #18 indicates an increase in the level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject. An increase in an amount of the molecular ion mass that is #18 of the test subject, compared to a reference is indicative of an increase in the level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject, compared with the reference. An increase in an amount of the molecular ion mass that is #18 of the test subject over time is indicative of an increase in the level of catalase in the test subject, in particular of catalase in the lung tissue of the test subject, over time. The methods mentioned herein, wherein: at least one molecular ion mass is identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least two molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least three molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least four molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least five molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least six molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least seven molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least eight molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least nine molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least ten molecular ion masses are identified from the mass spectrometry data that is selected from the group consisting of #1 to #10 of Table 1 , or at least one molecular ion marker is identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of Table 1 , or at least two molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of Table 1 , or at least three molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of T able 1 , or at least four molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of Table 1 , or at least five molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of Table 1 , or at least six molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of Table 1 , or at least seven molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of T able 1 , or at least eight molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of Table 1 , or at least nine molecular ion markers are identified from the mass spectrometry data that is selected from the group consisting of #1 to #5 and #7 to #10 of Table 1 , or at least four molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #4 of Table 1 , or at least five molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #5 of Table 1 , or at least six molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #6 of Table 1 , or at least seven molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #7 of Table 1 , or at least eight molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #8 of Table 1 , or at least nine molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #9 of Table 1 , or at least ten molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #10 of Table 1 , or at least three molecular ion masses (m / z) are identified from the mass spectrometry data that are #3, #6 and #11 of Table 1 , or at least one molecular ion mass is identified from the mass spectrometry data that are #13 to #18 of Table 1 ; or at least two molecular ion masses are identified from the mass spectrometry data that are #13 to #18 of T able 1 ; or at least three molecular ion masses are identified from the mass spectrometry data that are #13 to #18 of T able 1 ; or at least four molecular ion masses are identified from the mass spectrometry data that are #13 to #18 of T able 1 ; or at least five molecular ion masses are identified from the mass spectrometry data that are #13 to #18 of T able 1 ; or at least six molecular ion masses are identified from the mass spectrometry data that are #13 to #18 of T able 1 ; or at least one molecular ion mass is identified from the mass spectrometry data that is #13 in Table 1 , or at least one molecular ion mass is identified from the mass spectrometry data that is #14 in Table 1 , or at least one molecular ion mass is identified from the mass spectrometry data that is

[0162] #15 in Table 1 , or at least one molecular ion mass is identified from the mass spectrometry data that is

[0163] #16 in Table 1 , or at least one molecular ion mass is identified from the mass spectrometry data that is

[0164] #17 in Table 1 , at least one molecular ion mass is identified from the mass spectrometry data that is

[0165] #18 in Table 1 , may be applied to one or more methods as described in the aspects below.

[0166] According to one aspect, a method of determining respiratory health in a test subject is provided as described elsewhere herein, which method further comprises:

[0167] - comparing a presence and / or amount of the least one identified molecular ion mass with a reference,

[0168] - finding a deviation or no deviation of the presence and / or amount of said at least identified molecular ion mass from said reference; and

[0169] - attributing said finding of deviation or no deviation to an indication of respiratory health of the subject.

[0170] According to one aspect, a method of determining respiratory health over time in a test subject is provided using a method described herein, wherein

[0171] - multiple instances of mass spectrometry data of a sample of exhaled breath of the test subject are received, wherein each mass spectrometry data instance is of a sample of exhaled breath obtained at a different time from the test subject,

[0172] - respiratory health is determined at each different time, thereby determining respiratory health over time.

[0173] In particular changes in presence and / or amounts each molecular ion mass or VOC over time allow determination of respiratory health over time.

[0174] According to one aspect, a method of determining respiratory health over time in a test subject is provided using a method described herein, wherein:

[0175] - multiple instances of mass spectrometry data of a sample of exhaled breath of the test subject are received, wherein each mass spectrometry data instance is of a sample of exhaled breath obtained at a different time from the test subject,

[0176] - for each instance:

[0177] - a presence or amount of at least one identified molecular ion mass is compared with a reference, - a deviation or no deviation of the amount of said identified # from said reference is determined; and

[0178] - a finding of deviation or no deviation to an indication of respiratory health of the subject is determined,

[0179] - respiratory health over time from the deviation over time.

[0180] The different time points are preferably at least 1 day apart, for instance, at least one week, at least one month, or at least one year apart.

[0181] According to one aspect, a method of diagnosing, prognosing and / or monitoring respiratory dysfunction in a test subject is provided, which method further comprises:

[0182] - comparing a presence and / or amount of an identified molecular ion mass with a reference,

[0183] - finding a deviation or no deviation of the presence and / or amount of said at least one identified molecular ion mass from said reference; and

[0184] - attributing said finding of deviation or no deviation to a particular diagnosis, prognosis and / or monitoring status of respiratory dysfunction in the test subject.

[0185] According to one aspect, a method of determining the risk of developing a respiratory dysfunction in a test subject is provided, which method further comprises:

[0186] - comparing a presence and / or amount an identified molecular ion mass with a reference,

[0187] - finding a deviation or no deviation of the presence and / or amount of said identified molecular ion mass from said reference; and

[0188] - attributing said finding of deviation or no deviation to a particular risk of developing a respiratory dysfunction in the test subject.

[0189] According to one aspect, a method of in the selection of a prophylactic or therapeutic treatment of a lung dysfunction in a test subject is provided, which method further comprises:

[0190] - exposing the subject to the prophylactic or therapeutic treatment,

[0191] - comparing a presence and / or amount of an identified molecular ion mass in the test subject prior to and after exposing to the subject to the prophylactic or therapeutic treatment.

[0192] The selection of a prophylactic or therapeutic treatment is determined based on an increase or decrease in an amount of one or more one molecular masses prior to and after the exposing.

[0193] According to one aspect, a method of assessing an efficacy of a therapeutic treatment in a test subject is provided, which method further comprises:

[0194] - exposing the subject to the therapeutic treatment, - comparing a presence and / or amount of an identified molecular ion mass in the test subject prior to and after exposing to the subject to the therapeutic treatment.

[0195] The efficacy of the therapeutic treatment is determined based on an increase or decrease in an amount of one or more one molecular masses prior to and after the exposing.

[0196] According to one aspect, a method of treatment of a respiratory disease, in particular BPD or a respiratory disease associated with lung inflammation and / or oxidative stress in a test subject is provided, which method comprises assessing whether a test subject suffers from a respiratory disease, in particular from BPD or a disease associated with lung inflammation and / or oxidative stress in the lungs, according to any of the methods disclosed herein, and based on the outcome, treating the subject with a therapeutic treatment for said respiratory disease. Therapeutic options include but are not limited to non-invasive or invasive ventilation, oxygen supplementation, diuretics, inhaled and / or systemic corticosteroids, diuretics, antibiotics for the treatment of respiratory infections, prophylactic antibiotics, and adequate nutritional support.

[0197] According to one aspect, a system or device for use in a method described herein is provided, wherein said system or device comprises detection means for detecting one or more molecular ion masses (m / z) of Table 1 , in a sample of exhaled breath from said subject. In some embodiments, the system or device further comprises a processing unit, said processing unit receiving and processing signals from the detection means. In some embodiments, the device comprises a breath collector.

[0198] According to an embodiment, the system or device comprises a mass spectrometer.

[0199] According to one aspect, a use of a system or device is provided for use in a method described herein, wherein said device comprises a mass spectrometer.

[0200] In some embodiments, said use is by performing a method described herein for diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs of the subject.

[0201] In some embodiments, said use is by performing a method described herein for diagnosing, prognosing and / or monitoring respiratory dysfunction that is BPD and / or lung tissue inflammation in the test subject.

[0202] The system or device for use is a method may further comprise one or more of:

[0203] - a computer system for executing the method described herein;

[0204] - a gas sampling system for collection of the sample of exhaled breath;

[0205] - a ventilator for obtaining exhaled breath from the subject while the subject is being ventilated; The present method is performed using a computer. The present method is a computer- implemented method.

[0206] Further provided is a computing device or system configured for performing the method described herein.

[0207] Further provided is a computer program or computer program product having instructions which when executed by a computing device or system cause the computing device or system to perform (each of the steps of) a method as described herein.

[0208] Further provided is a computer readable medium having stored thereon a computer program (product) having instructions which when executed by a computing device or system cause the computing device or system to perform (each of the steps of) a method as described herein.

[0209] Further provided is a data stream which is representative of a computer program or computer program product having instructions which when executed by a computing device or system cause the computing device or system to perform (each of the steps of) the method as described herein.

[0210] The method may be performed using a computer system such as an Intel Architecture IA-32 based computer system 2 or using a supercomputing system, and implemented as programming instructions of one or more software modules stored on non-volatile (e.g. hard disk or solid-state drive) storage associated with the corresponding computer system.

[0211] However, it will be apparent that at least some of the steps of any of the described processes could alternatively be implemented, either in part or in its entirety, as one or more dedicated hardware components, such as gate configuration data for one or more field programmable gate arrays (FPGAs), or as application-specific integrated circuits (ASICs), for example.

[0212] The method or system may produce an output that is: displayed on a screen, or saved to a file.

[0213] The present method of system may be regarded as a method for measuring or detecting the at least one molecular mass corresponding to a volatile organic compound (VOC). Non-limiting examples of a method for determining respiratory health, in accordance with the description, are now disclosed below. These examples are merely for the purpose of illustration and are not to be regarded as limiting the scope of the invention or the manner in which it can be practiced. Other examples will be appreciated by a person having ordinary skill in the art.

[0214] EXAMPLES

[0215] MATERIALS AND METHODS

[0216] Study population

[0217] Preterm infants - neoVOC study Patients born preterm at a gestational age < 30 weeks admitted to the NICU of the Antwerp University Hospital were eligible for inclusion. We included patients receiving respiratory support (endotracheal ventilation, nasal CPAP, high flow nasal cannula (HFNC)) or low flow oxygen (BPD cases) and patients not receiving any form of respiratory support at the time of inclusion (controls). Patients were excluded if they had a major congenital defect or disorder, if they suffered from late-onset sepsis or fulminant inflammation (e.g. necrotising enterocolitis) at the time of inclusion in the study, and / or if they were not deemed stable enough for enrolment by the attending neonatologist. We included 8 patients for sampling and analysis optimisation, and 10 patients for preliminary data analysis. This study was approved by the Ethical Committee of the Antwerp University Hospital (B300201838156). Relevant maternal and neonatal data were retrieved from the infant’s medical files. BPD was diagnosed if there was a need for oxygen therapy or respiratory support at day 28 of life and its severity was assessed at 36 weeks GA.

[0218] Preterm lambs - oviVOC study Experiments were conducted on 9 mixed-bred Dorset- Romanov preterm lambs obtained from a local breeder. The lambs were vaginally delivered 14 days prematurely (at day 133; normal gestation 147 days) — corresponding to about 34 weeks of gestation in humans. Preterm labour was induced at 90% gestation by mifepristone (8 mg / kg), after intramuscular administration of betamethasone (2 doses of 12 mg) 48 and 24 h before birth to promote lung maturation. All lambs surviving the first 48 postnatal hours were included in this study. Vital signs, including heart and respiratory rates, oxygen saturation level, rectal temperature, blood glucose levels and weight were regularly monitored throughout the experiment. During the first 48h of life, a researcher was continuously present to monitor vital signs and correct any hypothermia, hypoglycaemia or desaturation. From birth, the preterm lambs stayed with and fed on their ewe. The ewes were returned to the sheep farm at the end of the experiment and were eligible for subsequent gestations in the current research program on preterm lambs. As part of a concurrent study investigating the impact of non-invasive respiratory support on sucking-swallowing-breathing coordination during bottle feeding, animals underwent surgery on postnatal day 6 to allow chronic instrumentation during the remainder of the experiment. Surgical instrumentation was performed under conscious sedation (intramuscular ketamine 2.5 mg / kg) and local anesthesia (xylocaine 2%) and included the insertion of (i) bipolar electrodes in the thyroarytenoid muscles (laryngeal constrictors) for recording swallowing activity, (ii) a catheter in the left carotid artery for arterial blood gas measurements (RapidLab 348, Siemens, Saint-Laurent, Canada) and (iii) a transcutaneous catheter with the tip between the fifth and sixth tracheal rings for monitoring the effect of each respiratory support condition on tracheal pressure. From this point on, all animals received daily prophylactic ampicillin (50 mg BID) and tobramycin (5 mg / kg SID). VOCs sampling was planned at least 24h before or after the experiments with induced tachypnoea.

[0219] Breath sampling was performed at day 8 and day 15 of life in 9 preterm lambs. After the completion of the final breath sampling on day 15, animals were euthanised by an intravenous injection of pentobarbital (90 mg / kg). Snap-frozen samples from the right lung were obtained for subsequent mRNA and protein analyses. Samples from the anterior lower right lobe were obtained for histological assessment. Experiments were carried out in accordance with the recommendations of the Canadian Council on Animal Care. This study was approved by the Ethics Committee for Animal Care and Experimentation of the University of Sherbrooke, Canada (protocol number 2018-2051)

[0220] Exhaled breath sampling

[0221] We developed an exhaled breath sampling method that allows offline measurements in infants receiving respiratory support or low flow oxygen, as well as in infants no longer receiving any form of respiratory support. In human neonates, samples were obtained at day 28 of life. For infants under non-invasive respiratory support or not ventilated, exhaled breath was obtained via a face mask using a custom-built VOCs adaptor (filed in iDepot with reference number 122217 by the University of Antwerp in February 2020). In intubated patients, exhaled breath was sampled locally at the tracheal end of the endotracheal tube via the surfactant application side port. Prior to every patient sample, a background sample from the set-up was obtained under similar conditions (sampling in the room of the infant, tubing installed, and respiratory support devices switched on, after hand disinfection, but with the face mask held against an inert, sterilised glass plate) to account for VOCs specific to the ventilatory circuit. Medical grade synthetic air provided via the central hospital supply was administered to all patients during sampling. Five hundred ml of exhaled breath was collected at a continuous flow rate of 30 ml / min and stored onto a Tenax®GR tube (Markes, Llantrisant, UK). In the first two patients, 2 Tenax®GR tubes were connected in series to confirm that the beforementioned sampling protocol was adequate and did not cause breakthrough of breath analytes onto the second Tenax®GR tube; so that overflow of potentially important VOCs with a single Tenax® tube setup was excluded.

[0222] Prior to use in humans, the Tenax® tubes were conditioned for one hour at 300°C while being flushed with helium (50 ml / min). After conditioning but before sampling, the tubes were loaded with 10.7 ng toluene-d8 internal standard, by making a two-phase system and using a homemade injector system. After sampling, Tenax® tubes were capped, wrapped in aluminum foil and stored in air-tight glass containers until analysis at Ghent University to prevent contamination. Human samples were registered at the Biobank of the University Hospital of Antwerp (ID: BE71030031000), in accordance with the Royal Decree of January 9th, 2018 (C- 2018 / 30209) and the Royal Decree of December 30th, 2008.

[0223] VOCs analysis via PTR-HRMS

[0224] PTR-HRMS Breath samples were analysed by a newly developed method in the research group Environmental Organic Chemistry and Technology (EnVOC) of Ghent University. Breath samples were first dry purged to remove excess water arising from condensation (only needed in ovine samples). The breath volume sampled on the Tenax® sorbent tube (about 500 ml) was registered to ascertain standardisation with regards to the dilution of the breath sample (since this can differ on an individual basis). The sampling tubes were closed with brass storage caps and PTFE ferrules, packed in a glass jar. Sorbent tubes were consequently analysed using PTR-HRMS. After sampling, sorbent tubes were analysed at Ghent University by thermal desorption using a Unity Thermal Desorption system (Markes, Llantrisant, UK) by heating the tube (1 min at 50°C; 7 min at 250°C at 20 ml per minute). Next, analytes were refocussed on a microtrap filled with Tenax®TA 35 / 60 and Carbograph 1TD 40 / 60, cooled at - 10°C. After heating the microtrap at 280°C (24°C / s), analytes were carried by a N2 stream and were injected in the PTR-HRMS. In the proton transfer reaction mass spectrometer, VOCs present in the breath sample were ionised using HaO+precursor ions. A proton transfer from HaO+to the VOC took place if the VOC’s affinity for the proton was greater than the affinity of H2O. Typical air constituents such as nitrogen (N2) and oxygen gas (O2), have lower affinity for the protons in comparison to H2O and were therefore not ionised. The proton transfers took place under stabilised conditions regarding temperature, pressure and electric fields. The electric fields and a difference in pressure drove the ionised VOCs to the mass analyser. Here, the ionised VOCs were identified based on their mass / charge ratio of their product ion and the measured signal intensity is commensurate with the sampled mass of the VOC. Finally, protonized VOCs could be identified based on their mass / charge ratio of their product ion and the quantity of the compounds was determined based on the measured signal intensity. While molecular mass can be inferred, identification is not unambiguous and relies on compound attribution based on the literature.

[0225] Raw data processing Raw data were further processed before statistical analysis. All concentrations were first normalized to the internal standard toluene-d8 (PTR mass 101 .12088). Relevant peaks were selected by means of the ptairMS package for R. After the calibration of the mass axis, peaks were detected with an untargeted peak picking algorithm and the peak area (AUCpeak) for each ion was calculated. The different samples were aligned, and missing values were imputed. To allow sparse partial least squares discriminant analysis (sPLS-DA), VOCs with an AUCpeak equal to zero in at least one of the two compared groups were omitted from the analysis (for both the human and ovine data). For the human data, the raw patient VOCs concentrations were used for further statistical analysis, since background samples were not analysable for all included patients. For the ovine data analysis, we computed so-called ‘alveolar gradients’, i.e. measured concentration in the ovine sample minus the respective concentration in a background sample to account for environmental confounders. This was not possible for human samples (background samples were not analyzable for all included patients), so that raw patient VOCs concentrations were used for further statistical analysis.

[0226] Tissue analyses in preterm lambs ddPCR Droplet digital PCR (ddPCR) was performed to assess mRNA expression levels of IL- 1 P, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)- a, mucin 1 (MUC1), catalase, superoxide dismutase 2 (SOD2), nitric oxide synthase 3 (NOS3) and vasculo-endothelial growth factor alpha (VEGF-a). First, RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA). Reverse transcription was performed on 1.7 pg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs (Roche Diagnostics, Basel, Switzerland), and 10 units of RNAseOUT (Invitrogen, Massachusetts, USA) following the manufacturer’s protocol in a total volume of 10 pl. All forward and reverse primers were individually resuspended to 20-100 pM in Tris-EDTA buffer (IDT, Iowa, USA) and diluted as a primer pair to 1 pM in RNase DNase-free water (IDT, Iowa, USA). Primer validation was performed in 10 pl in 96 well plates on a CFX-96 thermocycler (BioRad, California, USA) with 5 pL of 2X PerfeCTa® SYBR® Green Supermix (Quantabio, Massachusetts, USA), 10 ng (3 pl) cDNA (universal RNA), and 200 nM final (2 pl) primer pair solutions. The following cycling conditions were used: 3 min at 95°C; 50 cycles: 15 sec at 95°C, 30 sec at 60°C, 30 sec at 72°C. The amplified products were analyzed by automated chip-based microcapillary electrophoresis on Labchip GX Touch HT instruments (Perkin Elmer). As part of the ddPCR set-up, quantitative PCR (qPCR) reactions were performed in 10 pl in 96 well plates on a CFX-96 thermocycler (BioRad, California, USA) with 5 pL of 2X PerfeCTa SYBR Green Supermix (Quantabio, Massachusetts, USA), 10 ng (3 pl) cDNA, and 200 nM final (2 pl) primer pair solutions. The following cycling conditions were used: 3 min at 95°C; 50 cycles: 15 sec at 95°C, 30 sec at 60°C, 30 sec at 72°C. In every qPCR run, a no-template control was performed for each primer pair and these were consistently negative. All primer sequences are available in Table 2. ddPCR reactions were composed of 10 pl of 2X QX200 ddPCR EvaGreen Supermix (BioRad), 60 ng (6 pl) cDNA, 200 nM final (4 pl) primer pair solutions for a 20 pl total reaction. Each reaction mix (20 pl) was converted to droplets with the QX200 droplet generator (BioRad). Droplet-partitioned samples were then transferred to a 96-well plate, sealed and cycled in a C1000 deep well Thermocycler (BioRad, California, USA) under the following cycling protocol: 95 °C for 5 min (DNA polymerase activation), followed by 50 cycles of 95 °C for 30 s (denaturation), 59 °C for 1 min (annealing) and 72°C for 30 s (extension) followed by postcycling steps of 4°C for 5 min and 90 °C for 5 min (signal stabilization) and an infinite 12°C hold. The cycled plate was then transferred and read using the QX200 reader (Bio-Rad) either the same or the following day post-cycling. The concentration reported is copies / pl of the final 1x ddPCR reaction (obtained using QuantaSoft software from Bio-Rad).

[0227] Table 2. Sequences of the primers used forddPCR.

[0228] Oxidative stress Carbonylated proteins (Oxyblot Protein Detection Kit, Millipore, Darmstadt, Germany) and 4-hydroxynonenal (4-HNE; R&D Systems, Minneapolis, USA) were quantified as markers for oxidative stress. Protein was extracted from frozen lung tissue and antibody dilutions were selected according to the manufacturer’s instructions.

[0229] Histological assessment Samples were obtained from the anterior lower right lobe. The samples were conserved in 10% formaldehyde, consequently paraffin embedded and cut into 5 pm sections for haematoxylin and eosin staining. The samples were examined using a previously developed histological score of lung inflammation in the newborn lamb: septation thickness, inflammatory cell infiltration, epithelial sloughing and hemorrhage were each assessed on a scale of 0 to 2, adding up to a maximal inflammation score of 8.

[0230] Statistical analysis

[0231] Descriptive statistics on the clinical (human and ovine) data were computed in SPSS version 28 (IBM Corporation, USA). To assess differences across groups, non-parametric testing was used by means of a Mann-Whitney U test or Kruskal-Wallis test (with post-hoc Bonferroni tests). All other analyses were performed in R v4.1.1. For the human data, Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was used to investigate the differentiating potential of VOCs to distinguish preterm controls from BPD patients. sPLS-DA reduces the contribution of noise variables by minimising their coefficients. This technique is suitable for data with high multicollinearity and / or when there are more variables than the number of observations. sPLS- DA allows a differentiation between the two groups based on different components, that each consist of several loadings ( / .e. VOCs). The optimal number of components was assessed so that a minimal classification error was obtained, and finally the 10 most important contributors for assignment to the BPD group and the preterm control group respectively, were identified in each sPLS-DA component. These VOCs were then correlated to the clinical data (Spearman correlation). Tentative component identification for these VOCs was attempted via a previously composed literature review by our group.

[0232] For ovine data, we used penalised logistic (lasso) regression with leave-one-out cross- validation (LOOCV) to further analyse the pre-processed VOCs data. Three models were computed: (i) pre-CPAP versus post-CPAP breath profiles, (ii) pre-endotracheal ventilation versus post-endotracheal ventilation breath profiles and (iii) post-CPAP versus post- endotracheal ventilation breath profiles. By using ‘alveolar gradient’ data, we accounted for potential confounders originating from the ventilatory circuit and local environment in the ovine care facility. Only matched samples (both samples used for the respective comparison in each model obtained from the same animal) were included in the analysis. The glmnet R package (v4.1-1) was used to perform these analyses. Corresponding receiver operating characteristic (ROC) curves with sensitivity, specificity, positive (PPV) and negative predictive value (NPV), diagnostic accuracy of the model and 95% confidence intervals for all estimates were computed. We identified VOCs with important contribution to the respective models as having a variable frequency of > 50% in the cross-validation models. These VOCs were then correlated (Spearman correlation) to the abovementioned tissue markers. Thereafter, sPLS- DA was used to visualize discrimination in the three models. Finally, model attributes of the sPLS-DA were computed, and the main contributing parameters were identified.

[0233] RESULTS

[0234] Population characteristics

[0235] Preterm neonates

[0236] Eighteen preterm infants were included in this part of the study. Samples from the first eight patients were used for optimisation of the sampling and analysis method. Of these eight patients, one patient was intubated at the time of sampling. The final statistical analysis contained data from 10 patients, of whom 5 had BPD and 5 were included as preterm controls. All BPD patients received respiratory support via HFNC on the day of VOCs sampling. Patient characteristics are summarised in Table 3.

[0237] Table 3. Patient characteristics. Expressed as mean ± standard deviation for normally distributed continuous variables and as median, minimum and maximum for not normally distributed continuous variables. Categorical variables are expressed as total number per group in which the symptom was present and % affected per group. Corresponding P-values are shown in the last column. Significant P-values are indicated with an asterisk.

[0238] Preterm lambs

[0239] Nine animals (7 males and 2 females) were included in this part of the study. Mean birth weight was 2673.33 ± 296.65g (mean ± standard deviation) and there was a mean daily weight gain of 124.89 ± 30.19g. All animals thrived throughout the experiment.

[0240] Feasibility and sampling protocol optimisation

[0241] Breath sampling was safe and well-tolerated in all infants and lambs, during both invasive and non-invasive respiratory support. While minor discomfort due to positioning of the face mask was noted in some subjects, all samples could be obtained in standardised conditions and no adverse events (desaturations, bradycardia...) were recorded.

[0242] Exhaled breath sampling allowed detection of volatiles via PTR-HRMS, despite very low VOCs concentrations in our highly diluted breath samples due to high flow volumes necessary for neonatal non-invasive respiratory support. Sampling protocol assessment showed no volatiles on the second Tenax® tube (placed in series after the first tube), indicating that there was no saturation, nor breakthrough.

[0243] VOCs as markers for preterm lung disease

[0244] Untargeted analysis of the raw PTR-HRMS data based on algorithms identified 143 relevant VOCs in the human samples and 169 in the ovine samples.

[0245] VOCs relevant to BPD diagnosis at day 28 of life

[0246] Based on the VOCs data alone, sPLS-DA distinguished BPD patients from preterm controls with good characteristics (FIGs. 1A-B). Minimal classification error was obtained when including 3 sPLS-DA components. The loadings on each component (the contribution of the VOCs to that specific sPLS-DA component) are represented in Table 4 and FIG. 1C. Tentative component identification yielded identification of 13 out of 24 VOCs (Table 5). Correlations between the main VOCs based on the sPLS-DA and the clinical variables are shown in Table 6.

[0247] VOCs patterns characteristic of specific ventilation strategies

[0248] Lasso regression was performed to identify relevant VOCs specific to ventilation by means of CPAP or intubation in preterm lambs. Breath samples pre-CPAP were differentiated from post- CPAP with 100% sensitivity and specificity; m / z104.0489 was an important discriminator (Table 7 and FIG. 2). Based upon VOCs m / z149.0418 and m / z149.024, pre- and post-intubation exhaled breath samples were differentiated with a sensitivity and specificity of 100% and 85.7% respectively. Differentiation of post-CPAP and post-intubation again showed a sensitivity and specificity of 100% for both based upon m / z118.9031 and m / z47.0487. m / z104.0489 was tentatively identified as benzonitrile; m / z149.0418 and m / z149.024 as phthalic anhydride, 2-methoxymethylethoxy / propanol or 3-(1-methylethyl)-benzene, m / z118.9031 as indole and m / z47.0487 as dimethyl ether (Table 5).

[0249] Finally, PCA and sPLS-DA were performed, which confirmed the results of the lasso regression, as both techniques showed moderate to good discrimination of the respective groups in each of the three differentiating models (FIGs. 3 and 4). For pre- versus post CPAP breath samples, the model with 2 components consisting of 20 and 7 variables ( / .e. VOCs) was chosen in order to obtain minimal classification error. Similarly, we chose a model with 2 components consisting of 4 and 4 variables for distinguishing pre- versus post-intubation breath profiles. When comparing post-CPAP to post-intubation breath patterns, we again chose an optimal model with 2 components, consisting of 150 and 60 variables.

[0250] VOCs in relation to lung tissue markers

[0251] Spearman correlations between lung tissue markers and alveolar gradients of exhaled VOCs are shown in Table 8. The alveolar gradients of m / z74.06 (tentatively identified as as N,N- dimethylformamide) and m / z104.0489 showed significant negative correlations with inflammatory cytokines, while correlations with oxidative stress parameters were more heterogeneous. There was also a significant negative correlation between the total histological score and the alveolar gradient of m / z104.0489 (r=-0.66; p<0.001). Background uncorrected correlations are shown in Table 9, indicating that exhaled concentrations of m / z47.0487 and m / z104.0489 significantly correlated with inflammatory cytokines. We also observed a positive correlation between exhaled concentrations of m / z149.0418 and the total histological score.

[0252] CONCLUSION

[0253] The present examples show the feasibility of exhaled breath sampling in preterm neonates with and without BPD. Distinctive VOCs patterns linked to BPD diagnosis and breathprints specific to invasive and non-invasive ventilation (m / z47.0487, m / z104.0489, m / z149.0418, m / z118.9031 and m / z149.024) were identified.

[0254] Three VOCs (m / z47.09, m / z74.06 and m / z118.90) were relevant discriminators in both the human and ovine setting. PTR-HMS analysis allowed tentative component identification, which is unique in the field. Furthermore, correlations between exhaled VOCs and local tissue markers for inflammation, oxidative stress and histological markers for inflammation are shown. Table 4. Main contributing ions on the sPLS-DA component loadings relevant to BPD diagnosis (see also FIG. 1); ranked from most to least contributing per group on each component; with (+) indicating a positive correlation and (-) indicating a negative correlation to the designated group. If applicable, only the 10 most contributing ions per group are listed.

[0255] Table 5. Tentative component identification of VOCs in the neoVOC and oviVOC study.

[0256] The left column shows the protonated exact mass of the ions detected via PTR-HRMS, whereas the far-right column indicates the known (non-protonated) molar mass of the proposed tentatively identified VOCs in column 2. If compounds remained unidentified, this is indicated by means

[0257] Table 6. Correlations between clinical parameters and the abundance of the main

[0258] VOCs in the patient samples upon sPLS-DA. Spearman correlations coefficients are reported. No significant correlations were found for single markers.

[0259] Table 7. Summary of logistic regression models predicting allocation to the post-CPAP (model 1) or post-intubation (models 2 and 3) group after lasso regression with LOOCV. All three models contained only the VOCs detected in the breath samples. (A) Sensitivity, specificity, PPV, NPV, accuracy and AUG with corresponding 95% confidence intervals are shown per model. In addition, variable frequencies (B) yielded by the lasso regression with LOOCV are shown to indicate the importance of variables included in the three models respectively. Only variables that had a variable frequency > 50% of the number of observations in each group ( / .e. a cut off of variable frequency > 2 for model 1 , > 4 for model 2 and > 3.3 for model 3; indicated in italic) were considered important and included in further analyses.

[0260] A B

[0261] Table 8. Correlations between clinical parameters, inflammatory and oxidative stress tissue markers and the abundance of the main VOCs in the post-intubation VOCs samples (alveolar gradients). Spearman correlation coefficients are reported. Significant correlations are indicated with * for significance at the 0.05 to 0.1 level, ** at the 0.05 level and *** at the 0.001 level. Table 9. Correlations between clinical parameters, tissue markers and the abundance of the main VOCs in the post-endotracheal ventilation VOCs samples (sheep samples uncorrected for background). Spearman correlation coefficients are reported. Significant correlations are indicated with * for significance at the 0.05 level, ** at the 0.01 level.

Claims

Claims1 . A method for determining a respiratory health of a test subject, said method comprising:- generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject;- identifying, from the mass spectrometry data at least one molecular ion mass (m / z), wherein the molecular ion mass is a molecular ion mass (m / z) of Table 1 selected from the group consisting of #1 to #18; and- determining from the at least one molecular ion mass (m / z), the respiratory health of the test subject.

2. The method according to claim 1 , further comprising:- comparing a presence and / or amount of the least one identified molecular ion mass (m / z) of Table 1 with a reference;- finding a deviation or no deviation of the presence and / or amount of said at least identified molecular ion mass (m / z) from said reference; and- attributing said finding of deviation or no deviation to an indication of respiratory health of the subject.

3. A method for assessing the efficacy of a therapeutic treatment of a respiratory disease in a test subject, said method comprising:- generating or receiving mass spectrometry data of a sample of exhaled breath of the test subject at a time point before the start of the treatment and at one or more subsequent time points, such as at regular intervals, after the start of the treatment;- identifying, from the mass spectrometry data at least one molecular ion mass (m / z) in the sample at start of the treatment and in one or more of the subsequent samples, wherein the molecular ion mass is a molecular ion mass (m / z) of Table 1 selected from the group consisting of #1 to #18; and- comparing said at least one molecular ion mass in the first sample with said at least one molecular ion mass (m / z) in one or more of the subsequent samples, wherein a deviation or no deviation is indicative for the efficacy of the therapeutic treatment in the subject.

4. The method according to any one of claim 1 to 3, wherein:- at least one molecular ion mass (m / z) is identified from the mass spectrometry data selected from the group consisting of #13 to #18 of Table 1 ; and / or- at least four molecular ion masses (m / z) are identified from the mass spectrometry data selected from the group consisting of #1 to #10 of Table 1 ; and / or- at least three molecular ion masses (m / z) are identified from the mass spectrometry data that are #3, #6, and #11 of T able 1.

5. The method according to any one of claims 1 to 4, wherein at least one molecular ion mass (m / z) is identified from the mass spectrometry data selected from the group consisting of #13 to #18 of Table 1 and the determining the respiratory health is diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating lung tissue inflammation and / or oxidative stress in the lungs over time.

6. The method according to claim 5, wherein at least two, preferably at least three, molecular ion masses (m / z) are identified from the mass spectrometry data selected from the group consisting of #13 to #18 of Table 1 and the determining the respiratory health is diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating lung tissue inflammation and / or oxidative stress in the lungs over time.

7. The method according to claim 5 or 6,- wherein at least one molecular ion mass (m / z) is #13, #16, #17, or #18 of Table 1 , and wherein an increase in an amount of said at least one molecular ion mass (m / z) compared to a reference is indicative of an increase in lung tissue inflammation and / or oxidative stress in the lungs; and / or- wherein at least one molecular ion mass (m / z) is #14, or #15 of Table 1 , and wherein a decrease in an amount of said at least one molecular ion mass (m / z) compared to a reference is indicative of an increase in lung tissue inflammation and / or oxidative stress in the lungs.

8. The method according to claim 4, wherein the at least four molecular ion masses (m / z) are a molecular ion mass of Table 1 selected from the group consisting of #1 to #10, and the determining the respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is BDP and / or lung tissue inflammation; orthe efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time.

9. The method according to claim 8, wherein at least five, preferably at least six, ion masses (m / z) are identified from the mass spectrometry data selected from the group consisting of #1 to #10 of T able 1 , and the determining the respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is BPD and / or lung tissue inflammation; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time.

10. The method according to claim 8 or 9, wherein the at least four or five molecular ion masses are a molecular ion mass (m / z) of Table 1 selected from the group consisting of #1 to #5 and #7 to #10, and the determining the respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is bronchopulmonary dysplasia (BPD) and / or lung tissue inflammation; or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time.

11. The method according to any one of claims 1 to 3 or claims 8 to 10, wherein:- at least four molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #4 of Table 1 , and / or- at least five molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #5 of Table 1 , and / or- at least six molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #6 of Table 1 , and / or- at least seven molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #7 of Table 1 , and / or- at least eight molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #8 of Table 1 , and / or- at least nine molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #9 of Table 1 , and / or- at least ten molecular ion masses (m / z) are identified from the mass spectrometry data that are #1 to #10 of T able 1 , and / or- at least three molecular ion masses (m / z) are identified from the mass spectrometry data that are #3, #6, and #11 of Table 1 , and, the determining respiratory health is diagnosing, prognosing and / or monitoring respiratory dysfunction that is BPD and / or lung tissue inflammation or the efficacy of the therapeutic treatment of the respiratory disease is assessed by evaluating symptoms of BPD and / or lung tissue inflammation in the lungs over time; optionally, wherein- a decrease in an amount of said at least four, five, six, seven, eight, nine, ten molecular ion masses of the test subject compared to a reference is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject compared with the reference, and / or over time is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject over time, and / or- an increase in an amount of said at least four, five, six, seven, eight, nine, ten molecular ion masses of the test subject compared to a reference is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject compared with the reference, and / or over time is indicative of an increase in severity of the BPD or an increase in the level of lung tissue inflammation of the test subject over time.

12. The method according to any one of claims 1 to 11 performed using a computer.

13. A device for use in a method for determining a respiratory health of a test subject according to any one of the claims 1 to 11 , said device comprising detection means for one or more molecular ion masses (m / z) of Table 1 , in a sample of exhaled breath from said subject; optionally, further comprising a processing unit, said processing unit receiving and processing signals from said detection means; optionally, further comprising a breath collector.

14. Use of the device according to claim 13 for determining a respiratory health in a test subject;preferably by performing the method according to any one of claims 1 to 2 or 4 to 7 for diagnosing, prognosing and / or monitoring lung tissue inflammation and / or oxidative stress in the lungs; or preferably by performing the method according to any one of claims 3 to 11 for evaluating the efficacy of a therapeutic treatment of a respiratory disease; or preferably by performing the method according to any one of claims 1 to 2 or 8 to 11 for diagnosing, prognosing and / or monitoring respiratory dysfunction that is BPD and / or lung tissue inflammation.

15. A computing device or system configured for performing the method according to any one of claims 1 to 11.

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