Biofluid sample collection and stabilisation for subsequent analyte quantification
Patent Information
- Application Number
- PCT/EP2026/057288
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-17
- Filing Date
- 2026-03-16
- Publication Date
- 2026-09-24
Smart Images
Figure EP2026057288_24092026_PF_FP_ABST
Abstract
Description
[0001] P2671PC00
[0002] 1
[0003] BIOFLUID SAMPLE COLLECTION AND STABILISATION FOR SUBSEQUENT ANALYTE QUANTIFICATION
[0004] TECHNICAL FIELD
[0005] The present disclosure relates to a method of collecting a biofluid sample and stabilising an analyte of interest of the biofluid sample for subsequent analysis as well as biofluid sampling kit for performing the method.
[0006] BACKGROUND
[0007] The present invention is situated in the technical field of biofluid sampling for analyte quantification. Biofluid sampling is a critical procedure in medical diagnostics, and several established methods are employed for collecting different types of biofluids, such as blood, saliva, urine, and more.
[0008] Blood sampling is one of the most commonly performed procedures in this field, with venipuncture being the standard technique for obtaining larger volumes of blood. It is primarily used for routine blood tests and analyses requiring significant sample volumes. Despite its reliability, venipuncture requires trained healthcare professionals and can be inconvenient for patients, especially in settings lacking adequate medical personnel.
[0009] Fingerstick or pin-prick micro-sampling offers a minimally invasive alternative for collecting small blood samples, often used for glucose monitoring and point-of-care testing. While convenient and not requiring specialized personnel, this method is unsuitable for tests needing larger volumes or precise quantification of analytes, due to issues like coagulation.
[0010] Arterial sampling, used for assessing arterial blood gases, is more invasive and carries risks such as bleeding complications, limiting its application to specific critical care scenarios.
[0011] Dried blood spot (DBS) testing, a finger-prick variant, is valued for its simplicity and costeffectiveness, particularly in remote or resource-limited settings. However, DBS faces challenges such as haematocrit bias, which affects analyte quantification due to variability in blood spread, and issues with protein analysis because of coagulation and drying processes.
[0012] Beyond blood, sampling of other biofluids like saliva, urine, and nasal secretions is also integral to diagnostic procedures. These fluids are easier to obtain but present their own challenges in terms of stability, transport, and analysis.P2671PC00
[0013] 2
[0014] The current methods of biofluid sampling highlight a need for solutions that provide the convenience and accessibility of minimally invasive techniques while ensuring analytical precision and reliability across various biofluid types. The growing demand for home-based sampling methods, driven by socio-economic benefits and technological advancements, underscores the market need for improved approaches that address the limitations of existing solutions to also enable e.g. proteomics analysis and other diagnostic tests.
[0015] SUMMARY
[0016] On this background, it may be seen as an object of the present disclosure to provide a method of collecting a biofluid sample that provides the convenience and accessibility of minimally invasive sample collection techniques while ensuring analytical precision and reliability across various biofluid types. Another object is to provide a biofluid sampling kit for performing this method. One or more of these objects may be met by aspects of the present disclosure as described in the following.
[0017] A first aspect of the present disclosure relates to a method of collecting a biofluid sample and stabilising an analyte of interest of the biofluid sample on a structural support for subsequent analysis, e.g. mass spectrometry (MS) quantification, of an analyte of interest. The method comprises the steps of providing a biofluid sample comprising one or more analytes of interest, such as proteins, for subsequent MS quantification, diluting the biofluid sample in a first aqueous solution, and transferring at least an aliquot of the diluted biofluid sample to a second anti-solvent to precipitate and adsorb the analyte of interest, such as proteins, onto the structural support. The structural support may also be known as a sorbent.
[0018] Biofluids typically have inherent high viscosity and often start to coagulate immediately. High viscosity can lead to poor penetration onto or into a structural support and difficult mixing of the biofluid with reagents. Coagulation of the biofluid sample leads to the formation of clots that can interfere with the uniform distribution of the sample within the structural support. This can result in uneven spreading and inconsistent sample volumes, affecting the reproducibility of the sample preparation process. Additionally, coagulation can also hinder the effective mixing of the sample with reagents.
[0019] The inventors found that reducing the inherently high viscosity of the biofluid sample by diluting it to a viscosity of approximately that of the first aqueous solution provides several benefits. First, the lowering of the viscosity in the biofluid sample facilitates more effective mixing with reagents, such an anticoagulant. Second, the adjustment in viscosity ensures that the biofluid can penetrate into a structural support in a consistent and reproducible manner unlike undiluted DBS samples. Furthermore, any variation in the initial sample volume, such as between 1 pL and 3 pL obtained inP2671PC00
[0020] 3
[0021] DBS samples, has a diminished impact when the biofluid sample is diluted. This results in a more predictable behaviour of the biofluid sample allowing for a more accurate analysis. Furthermore, quantification by mass spectrometry allows for the analysis of necessary analytes of interest from a tiny sample volume, such as 20 nL, whereas biofluid samples are often obtained in volumes that are orders of magnitude larger. For example, in pinprick blood sampling, patients cannot provide much less than 1 pL. Thus, the inventors found that the above advantages of diluting can be obtained while still allowing for an accurate analysis. This contrasts with conventional biofluid sampling where larger (undiluted) sample volumes are often preferred. This method also allows the collection of biofluid samples to be performed in one location, and the subsequent quantification of the analyte of interest may be performed in another, external location. The adsorbed analyte of interest may be sent safely by a carrier, either in wetted or dried format for remote analysis, following sample preparation according to the present method. An analyte of interest, thus precipitated and adsorbed on purpose on the structural support, is more amenable to proteomic analysis than proteins coagulated and dried as in the case of DBS samples.
[0022] The first aqueous solution is preferably an aqueous buffer solution, such as phosphate buffers, Tris buffers, HEPES buffers, citrate buffers, acetate buffers, bicarbonate buffers and the like. While the first aqueous buffer is preferably entirely without anti-solvent, e.g. an organic solvent, it may also contain up to 49% anti-solvent, e.g. an organic solvent. The anti-solvent of the first aqueous solution may be the same or different from the second anti-solvent. The first aqueous solution may comprise an anticoagulant.
[0023] Additionally, the method may comprise a step of providing the structural support wetted, e.g. prewetted, with the first aqueous solution. The step of diluting the biofluid sample may involve subjecting the wetted structural support to the provided biofluid sample so as to dilute the biofluid sample in the first aqueous solution.
[0024] Alternatively, the structural support may be provided in a dry condition. The dry structural support may be subjected to the first aqueous solution comprising at least the aliquot of the diluted biofluid sample so that the aliquot of the diluted biofluid sample is received on the structural support. The aliquot of diluted biofluid sample may then be transferred via the structural support to the second anti-solvent.
[0025] Additionally or alternatively, the biofluid sample may be vol u metrically diluted by the first aqueous solution by a ratio in the range of 1:2 to 1:100, preferably 1:5 to 1:100, more preferably 1:10 to 1:100. For example, the biofluid sample, e.g. a blood sample, may have a volume in the range of 1 pL to 10 pL, and about lOOpL of the first aqueous solution may be provided.P2671PC00
[0026] 4
[0027] Additionally or alternatively, the method may comprise a step, performed prior to the step of providing the biofluid sample, of pre-loading at least one internal standard without the analyte of interest on the structural support, and / or pre-loading at least one internal standard without the analyte of interest in the first aqueous solution. The European patent application EP25164085.0 describes how at least one internal standard can be pre-loaded onto a stationary phase of a solid phase extraction cartridge. The at least one internal standard can be pre-loaded onto the structural support without the analyte of interest in the same manner. As described in the referenced patent application, the inventors have found that such adsorbed internal standard(s) remained highly stable after prolonged storage. In the present disclosure, the inventors found such stable pre-loaded internal standard(s) can be advantageously used in the context of biofluid sample collection and stabilisation for use in subsequent LCMS quantification of the analyte of interest.
[0028] The at least one internal standard pre-loaded on the structural support or present in the first aqueous solution may be any internal standard commonly used in MS quantification. Typically, such internal standards may be e.g. isotopically labelled versions of the analyte such that they are isobaric to the analyte but differ in the isotope pattern. The internal standard may also be e.g. a protein or peptide labelled with Tandem Mass Tags (TMT), or a protein or peptide labelled with isobaric tags for relative and absolute quantification (iTRAQ). The internal standard may also be a surrogate internal standard having a different nominal mass to charge ratio (m / z). Thus, in a preferred embodiment, the at least one adsorbed internal standard is an isotopically labelled version of the analyte, a protein or peptide labelled with Tandem Mass Tags (TMT), a protein or peptide labelled with isobaric tags for relative and absolute quantification (iTRAQ), or a surrogate internal standard. In some embodiments, the internal standard may also be an isotopically labelled biological sample comprising a plurality of labelled internal standards. The biological sample may e.g. be derived from cells grown in a mixture of isotopically labelled nutrients. In yet some embodiments, the internal standard may be an ortholog peptide or -protein to the analyte of interest.
[0029] The internal standard(s) may be present in a known concentration in the first aqueous solution and used to compute the volume of the first aqueous solution transferred to the structural support and thus help quantify the analyte of interest. That is, the amount of the internal standard(s) transferred, precipitated and adsorbed onto the structural support from the first aqueous solution depends solely on the concentration of the internal standard(s) in the first aqueous solution and the volume transferred to the structural support. Standard curves can be made by transferring a known volume of a solution comprising the internal standard(s) in a known concentration to the structural support. These standard curves can subsequently be used to compute the absolute amount of the internal standard(s) and the absolute amount of the analyte(s) of interest in the sample based on the relativeP2671PC00
[0030] 5
[0031] MS response between the internal standard(s) and the analyte of interest(s). Once the absolute amount of internal standard(s) in a sample is known, the volume of the first aqueous solution transferred to the structural support from the diluted biofluid can also be computed and hence the concentration of the analyte of interest in the first aqueous solution (i.e. in the diluted biofluid) calculated.
[0032] To correct for any variation in initial sampling volume of the undiluted biofluid the concentration of the analyte of interest may be normalized by comparing the signal from the internal standard to one or more of most abundant plasma proteins, e.g. albumin, as the concentration of many high-abundance proteins remain fairly constant between samples and between individuals.
[0033] Additionally or alternatively, the at least one internal standard may be pre-loaded on the structural support by subjecting the structural support to a third aqueous solution comprising at least one internal standard without the analyte of interest, subjecting the third aqueous solution to a fourth anti-solvent to precipitate and adsorb the internal standard without the analyte of interest onto the structural support, and removing excess liquid from the third aqueous solution and the fourth antisolvent to adsorb the at least one internal standard on the structural support.
[0034] Additionally or alternatively, the second anti-solvent (and / or the anti-solvent of the first aqueous solution, if any) may comprise one or more organic solvent(s) or preferably essentially consist of one or more organic solvent(s). The organic solvent may be selected from acetonitrile, isopropanol, n-propanol, methanol, ethanol, acetone, dioxane, tetra hydrofuran or mixtures thereof, most preferably acetonitrile, methanol, ethanol, isopropanol or mixtures thereof. Alternatively, the second anti-solvent (and / or the anti-solvent of the first aqueous solution, if any) may comprise or, preferably, essentially consist of an aqueous solution of suitable ionic strength (i.e. a sufficiently high content of electrolytes to cause precipitation). Such solutions are well-known in the art and are sometimes referred to as salting-out solutions, high-salt buffers or salt precipitation solutions. Thus, in an embodiment, the second anti-solvent (and / or the anti-solvent of the first aqueous solution, if any) is a water miscible organic solvent, an aqueous electrolyte solution or a mixture thereof. In a preferred embodiment, the aqueous electrolyte solution is an acidic aqueous electrolyte solution with a pH below 4. Preferably, the concentration of electrolytes is at least 100 mM.
[0035] Additionally, after transferring at least the aliquot of the diluted biofluid sample to the second antisolvent so as to form a mixture. The cumulative anti-solvent (e.g. the second anti-solvent and the anti-solvent of the first aqueous solution, if any) constitutes at least 50% by volume, preferably at least 80% by volume, most preferably at least 90% of the mixture.P2671PC00
[0036] 6
[0037] Additionally or alternatively, the undiluted biofluid sample and the cumulative anti-solvent (e.g. the second anti-solvent and the anti-solvent of the first aqueous solution, if any) may have a volume ratio in the range of 1:5 to 1:500, preferably in the range of 1:10 to 1:100. For example, the biofluid sample, e.g. a blood sample, may be provided with a volume in the range of 1 pL to 10 pL, and 500pL of the second anti-solvent may be provided.
[0038] Additionally or alternatively, the biofluid sample may be blood, lymph, interstitial fluid, urine, feces, semen, vaginal fluid, saliva, bile, gastric juice, mucus, sweat, tears, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, cerebrospinal fluid, amniotic fluid, and breast milk.
[0039] Additionally or alternatively, the analyte of interest may comprise one or more selected from protein(s) (e.g. glycoprotein(s)), peptide(s), carbohydrate(s), glycopeptides, lipids, nucleic acids, metabolites, or drug(s). Preferably, the biofluid sample is a blood sample and the analyte of interest is a protein.
[0040] Blood samples often present challenges due to their high viscosity and immediate tendency to coagulate, leading to clot formation. These factors can interfere with the uniform distribution of the blood sample on or within a structural support, resulting in uneven spreading and inconsistent sample volumes, thus affecting the reproducibility of sample preparation. Coagulation also complicates the mixing of blood with reagents, e.g. anticoagulants. By diluting the blood sample, its viscosity can be reduced to a level similar to that of the first aqueous solution, enhancing the mixing efficiency with reagents, such as anticoagulants. This viscosity adjustment ensures consistent and reproducible penetration of the blood into the structural support. This leads to more predictable sample behaviour and accurate analysis. Additionally, mass spectrometry can quantify analytes from very small volumes, like 20 nL, while typical blood samples collected via pinprick are much larger, often above 1 pL. Thus, the inventors identified that dilution not only facilitates these advantages but also maintains accurate analytical outcomes, contrasting with traditional blood sampling methods that favour larger sample volumes.
[0041] Additionally or alternatively, the structural support may be a surface, a gauze, a mesh, a web, a weave, or a filter. The structural support may be made of glass, cotton, knitted polyester, microfiber, paper, or foam, or a sorbent such as those used for solid phase extraction, or microspheres, such as magnetic beads with a suitable surface chemistry for capturing analytes that are being precipitated from solution.
[0042] Additionally or alternatively, the structural support may form part of a vial and may be made of glass. However, it is preferred that the structural support forms part of a swap (e.g. a sampleP2671PC00
[0043] 7
[0044] collection device of the second aspect of the present disclosure). The swap may comprise a stick of e.g. plastic with the structural support at one end.
[0045] In a preferred embodiment, the biofluid sample may be a blood sample, and the first aqueous solution may comprise an anticoagulant.
[0046] Additionally, the anticoagulant may be a calcium binder, e.g. ethylenediaminetetraacetic acid or citric acid. The concentration of the calcium binder is chosen low, such as to minimize any toxic or environmentally adverse effects, yet high enough to halt the coagulation cascade of the blood when presented with a blood sample of up to around 10 pL.
[0047] Additionally or alternatively, the blood sample may be provided as a droplet on a skin surface. The step of diluting the blood sample may involve arranging, e.g. temporarily sealing, an opening of a first container comprising the first aqueous solution around the blood sample on the skin surface and washing the blood sample off the skin surface and into the first container with the first aqueous solution. For example, the first container and the skin surface may be shaken together to wash off the blood sample.
[0048] Alternatively, the blood sample may be provided as a droplet on a skin surface. The step of diluting the blood sample may involve removing, e.g. by wiping, the blood sample from the skin surface using the structural support, preferably forming part of a swab, wetted with the first aqueous solution so as to dilute the blood sample in the first aqueous solution. Alternatively, the blood sample may be wiped from the skin surface using a dry structural support, preferably forming part of a swab, but a-priori impregnated with an anticoagulant, and immediately after wiping off the blood sample, transferring said structural support to the first aqueous solution, e.g. to a first container comprising the first aqueous solution, or directly to the second anti-solvent.
[0049] Alternatively, the blood sample may be provided as a droplet on a skin surface. The step of diluting the blood sample may involve removing, e.g. by wiping, the blood sample from the skin surface using a dry structural support (e.g. not wetted with the first aqueous solution), preferably forming part of a swab, but a-priori impregnated with an anticoagulant, and immediately after wiping off the blood sample, transferring said swab to a vial with the first aqueous solution or directly to the second anti-solvent.
[0050] Additionally or alternatively, the method may further comprise a step, performed during or after the step of diluting the blood sample, of at least partially separating the diluted blood sample constituents on the structural support.P2671PC00
[0051] 8
[0052] Additionally or alternatively, the structural support may comprise affinity agents, such as antibodies or nanobodies, for an analyte of interest wherein the affinity agents are pre-loaded or chemically linked to the structural support or onto a portion adjacent to the structural support of a sample collection device. The affinity agents may be beneficial to capture analytes that are present in low concentrations, to increase the sensitivity.
[0053] Additionally or alternatively, the structural support may reside inside a vial with the second antisolvent to which an aliquot of the diluted body fluid sample is added. The structural support may in this case preferably be magnetic beads with at least one pre-loaded internal standard, onto which the analyte(s) of interest are going to precipitate on mixing the first solution with the analyte(s) of interest with the second anti-solvent.
[0054] Additionally or alternatively, the method may further comprise the steps of removing the sample collection device including the structural support with the adsorbed analyte of interest from the second anti-solvent, optionally drying the structural support with the adsorbed analyte of interest, and preparing, e.g. packaging, the sample collection device for sending to an external location for subsequent quantification of the analyte of interest, preferably proteomics analysis.
[0055] Additionally or alternatively, the method may comprise the steps of sealing the second container comprising the structural support with the adsorbed analyte of interest and preparing, e.g. packaging, the sealed second container comprising the structural support with the adsorbed analyte of interest for sending to an external location for subsequent quantification of the analyte of interest, preferably using liquid chromatography-mass spectrometry. The subsequent quantification of the analyte of interest may, for example, advantageously be performed by liquid-chromatography, e.g. high-performance liquid chromatography-mass spectrometry analysis.
[0056] A second aspect of the present disclosure relates to a biofluid sampling kit for performing the method according to the first aspect. The biofluid sampling kit comprises a sample collection device including a structural support, a first aqueous solution for diluting the biofluid sample, and a second container comprising a second anti-solvent to precipitate and adsorb the analyte of interest of the diluted biofluid sample onto the structural support.
[0057] Additionally or alternatively, the structural support may comprise at least one pre-loaded adsorbed internal standard. The structural support may contain a set of pre-loaded and previously adsorbed internal standards that may be used as a reference to quantify how much analyte of interest was sampled by the sample collection device, and how well the further sample processing was performed,P2671PC00
[0058] 9
[0059] e.g. the digestion and peptide extraction steps, and for calibrating the measured mass spectrometric signal response from the detected sample proteins.
[0060] Additionally or alternatively, the structural support may be pre-wetted with the first aqueous solution. Alternatively, the structural support may be provided in a dry condition and may be impregnated by an anticoagulant which is especially advantageous when the biofluid sample is a blood sample. The biofluid sampling kit may further comprise a first container comprising the first aqueous solution. The structural support may be submersible in the first aqueous solution in the first container so as to allow wetting of the structural support with the first aqueous solution.
[0061] Alternatively, the structural support may be provided within the second container comprising the second anti-solvent. The second anti-solvent is for precipitating and adsorbing the analyte of interest of the diluted biofluid sample onto the structural support once the first aqueous solution comprising at least an aliquot of the diluted biofluid sample is transferred to the second container while the structural support is present within the second container.
[0062] Additionally or alternatively, the first aqueous solution may comprise at least one internal standard. Additionally or alternatively, the biofluid sampling kit may further comprise a skin puncturing device, such as a lancet, for puncturing skin to provide a blood sample in the form of a droplet on the skin surface. Additionally or alternatively, the lancet may be a spring-loaded lancet with a 23-gauge needle and about 1.8 mm penetration. The blood droplet sample may be in the range of 1 pL to 10 pL.
[0063] Additionally or alternatively, the sample collection device may comprise a cover that is entirely or partly impervious to the biofluid sample. The structural support may form a core within the cover so that the structural support has an exposed portion and a covered portion. Accordingly, the sample collection device may be configured for, when the exposed portion of the structural support is submerged in the first aqueous solution comprising the diluted biofluid sample, receiving and at least partly separating the constituents in the aliquot of the diluted biofluid sample within the structural support. This arrangement of the sample collection device allows the different components of the biofluid sample to penetrate into the structural support to varying degrees, thereby causing at least a partial separation (or fractionation) of the biofluid sample components as the biofluid sample traverses the structural support. Hence, small molecules, e.g. peptides or short polypeptides, may penetrate further into the structural support than the larger molecules, e.g. proteins, and proteins may penetrate further into the structural support than cells, such as erythrocytes. As an alternative, the core may be cylindrically shaped, and the cover may be a sleeve so that the exposed portion is an end of the structural support. In another implementation, the core may be disc shaped and theP2671PC00
[0064] 10
[0065] core may be sandwiched between covers so that the exposed portion of the covered structural support is a circumferential portion.
[0066] Additionally or alternatively, the sample collection device may comprise a filter arranged so that structural support receives a filtered diluted biofluid sample, preferably the filter being configured to prevent entry of cells. By filtering the biofluid sample, unwanted matter can be excluded from the analyte of interest. For example, it is frequently desired to analyse peptides and proteins present in blood plasma but not have any cellular matter included in the analysis. Advantageously, the filter is configured to permit entry of proteins but not entry of cells.
[0067] Additionally or alternatively, the biofluid sampling kit may be a biofluid sampling kit configured for self-collection. Accordingly, the biofluid sampling kit may further comprise a packaging for sending (for example via a carrier, such as via mail) the sample collection device to an external location for subsequent quantification of the analyte of interest, preferably using mass spectrometry. Selfcollection sampling may also be known as home-sampling.
[0068] Whereas the above aspects of this disclosure and embodiments thereof are described for sampling body fluids, they may equally be used to sample other biomolecular samples, such as growth media in fermenters, microbial samples, and food samples that all may need to be conveniently collected and stabilized for subsequent analysis.
[0069] Whereas the above aspects of this disclosure and embodiments thereof are described for sampling of analytes that are predominantly hydrophilic, such as proteins, they may equally be used for sampling analytes that are predominantly hydrophobic, such as steroids. In such case the biofluid sample can be diluted in an organic solution, instead of an aqueous solution as in the first aspect, and at least an aliquot of the diluted biofluid sample transferred to an anti-solvent in the form of an aqueous solution, instead of solution comprising e.g. an organic solvent as in the first aspect, to precipitate and adsorb the hydrophobic analyte of interest, such as a steroid, onto the structural support.
[0070] Hence, a third aspect of the present disclosure relates to a method of collecting a biofluid sample and stabilising a hydrophobic analyte of interest of the biofluid sample on a structural support for mass spectrometry, MS, quantification of an analyte of interest, comprising the steps of:
[0071] providing a biofluid sample comprising a hydrophobic analyte of interest, such as a steroid, for subsequent MS quantification;
[0072] diluting the biofluid sample in a third organic solution; andP2671PC00
[0073] 11
[0074] transferring at least an aliquot of the diluted biofluid sample to a fourth anti-solvent to precipitate and adsorb the analyte of interest, such as steroids, onto the structural support.
[0075] Additionally or alternatively, the third organic solution may comprise one or more organic solvent(s) or preferably essentially consist of one or more organic solvent(s). The organic solvent may be selected from acetonitrile, isopropanol, n-propanol, methanol, ethanol, acetone, dioxane, tetrahydrofuran or mixtures thereof, most preferably acetonitrile, methanol, ethanol, isopropanol or mixtures thereof. The third organic solution may comprise an anticoagulant.
[0076] Additionally or alternatively, the fourth anti-solvent is an aqueous solution, e.g. an aqueous buffer solution, such as phosphate buffers, Tris buffers, HEPES buffers, citrate buffers, acetate buffers, bicarbonate buffers and the like. While the fourth anti-solvent is preferably entirely without an organic solvent, it may also contain up to 49% of an organic solvent. The organic solvent of the fourth anti-solvent, if any, may be the same or different from the third organic solution.
[0077] Additionally or alternatively, the hydrophobic analyte of interest, such as a steroid, has a LogP (the partition coefficient between octanol and water) above 1, preferably above a LogP 1.5, most preferably a LogP above 2.
[0078] It should be understood that all the embodiments described in the first and second aspect with regard to e.g. the structural support, dilution of the body fluid, the internal standard(s), the anticoagulants) apply equally to third aspect.
[0079] A person skilled in the art will appreciate that anyone or more of the above aspects of this disclosure and embodiments thereof may be combined with any one or more of the other aspects of this disclosure and embodiments thereof.
[0080] BRIEF DESCRIPTION OF THE DRAWINGS
[0081] Embodiments of this disclosure will be described in more detail in the following with regard to the accompanying figures. The figures show one way of implementing the present invention and are not to be construed as being limiting to other possible embodiments falling within the scope of the attached claim set.
[0082] Fig. 1 illustrates a process for dried blood testing according to the prior art.
[0083] Fig. 2 illustrates an exemplary method according to the present disclosure for collecting and preparing a biofluid sample, in this case a blood sample, for subsequent analysis.P2671PC00
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[0085] Fig. 3A-3C illustrate different implementations of a sample collection device.
[0086] DETAILED DESCRIPTION
[0087] In the following figure description, the same reference numbers refer to the same elements and may thus not be described in relation to all figures.
[0088] Fig. 1 is an example of the conventional dried blood spot method of collecting and preparing a blood sample for subsequent testing. This method is widely used in scenarios requiring minimal blood volume, such as paediatric and home-based testing. The process begins with the skin surface 1, typically the fingertip, which is punctured with a lancet (not shown) to obtain a small sample droplet 2 of blood. The droplet, typically ranging from 1 pL to 10 pL, is transferred onto a filter paper 4 made from cellulose. As the blood spreads across this filter paper 4 and dries under ambient conditions, a stable dried blood spot 3 (DBS) is formed. The filter paper 4 with the dried blood spot 3 is then packaged in a packing 40 conducive to transport and storage, allowing for the sample to be conveniently mailed to an external laboratory for subsequent analysis. The DBS method is advantageous for its simplicity, cost-effectiveness, and ability to facilitate remote sampling without requiring healthcare personnel or specialized facilities. However, there are drawbacks associated with this method. A significant limitation is haematocrit bias, which affects the quality and reliability of the analysis due to its impact on blood spreading and analyte distribution on the filter paper. This bias, along with inaccuracies in analyte quantification, particularly in hormone and drug concentration measurements, can lead to variability in results. Furthermore, the packaging of the DBS is important to maintain sample integrity during transit, protecting it from environmental factors like moisture and temperature fluctuations.
[0089] Moreover, several researchers have attempted to use DBS for proteomics analysis but encountered significant challenges. The blood coagulation process and the specimen's drying out tend to render proteins far less available for analysis compared to plasma obtained from venipuncture. As a result, the protein identification depth from DBS specimens has been low, and the quantitative precision and accuracy have typically been poor compared to proteomics data obtained from venipuncture plasma samples. Consequently, DBS samples, and finger-prick specimens in general, have not shown wide adoption for proteome analysis, highlighting the need for a replacement technology that can overcome these limitations.
[0090] Fig. 2 shows an exemplary method of collecting and preparing a biofluid sample comprising an analyte of interest according to the present disclosure. In this example, the biofluid sample is provided 100 in the form of a blood droplet 2 of about 1 mm in diameter or about 1 pL to 10 pL byP2671PC00
[0091] 13
[0092] puncturing, e.g. using a lancet (not shown), the skin surface 1, at e.g. a fingertip, in the same manner as for the DBS method described above. The present method differs, however, in the following steps.
[0093] The blood droplet 2 is then diluted 200A, 200B by a first aqueous solution comprising an anticoagulant according to a first option or second option of the present method.
[0094] In the first option of diluting 200A, a first container 20, e.g. a vial, comprising a first aqueous solution 24 and an opening 22, is provided. The opening 22 is placed around the blood droplet 2 and forms a temporary seal against the skin surface 1. The first container 20 and the skin surface 1, e.g. the fingertip, is shaken together, e.g. two to ten times, to wash the blood droplet 2 from the skin surface 1 and into the first aqueous solution 24 in the first container 20. Thereby, the blood droplet 2 is diluted 200 by the first aqueous solution 24. A structural support 12 of a sampling collection device 10, e.g. a swab, is provided in dry condition and then submerged in the first aqueous solution now containing the diluted blood sample. The structural support 12 is held submerged, preferably without shaking, to absorb the diluted blood sample for a first period of time, e.g. a few seconds, such as, at least 2 seconds and preferably up to 60 seconds.
[0095] In the second option of diluting 200B, the sample collection device 10 comprises a structural support 14 of a known volume, e.g. a known volume of gauze or filter, wetted with the first aqueous solution. The structural support 14 may be delivered pre-wetted in a suitable container or simply impregnated by the first aqueous solution. The wetted structural support 14 is brought into contact with the blood droplet 2 so as to wipe off the blood droplet 2 from the skin surface 1. The structural support 16 now comprising the diluted blood sample is allowed to rest for a few seconds to let the blood distribute within the structural support 16.
[0096] The sample collection device 10 with the structural support 16 now comprising an aliquot of the diluted blood sample is then removed from the first container 20 and transferred 300, preferably without delay, to a second container 30, e.g. a vial, comprising a second anti-solvent 34. The structural support 16 with an aliquot of the diluted blood sample is then submerged in the second anti-solvent and held submerged, preferably without shaking, for a second period of time longer than the first period of time, for example at least one minute and preferably more than ten minutes. During this second period of time, the analyte of interest, such as proteins, precipitates and adsorbs onto the structural support 18.P2671PC00
[0097] 14
[0098] The sample collection device 10 including the structural support 18 with the adsorbed analyte of interest is then packaged 400A, 400B according to a first option or second option of the present method.
[0099] In the first option of packaging 400A, the sample collection device 10 is removed from the second container 30, the structural support 18 is dried out, and the sample collection device 10 including the dried structural support with the adsorbed analyte of interest is then packed within a packaging 40 for sending to an external location for subsequent analysis, preferably LC-MS, of the adsorbed analyte of interest.
[0100] In the second option of packaging 400B, the structural support 18 with the adsorbed analyte of interest is sealed, e.g. by closing a lid of the second container 30, within the second container 30 and the sealed second container 32 is packaged in a packaging 40.
[0101] Fig. 3A shows a first sample collection device 10 as also shown in Fig. 2. The sample collection device 10 comprises a swap 11 with a structural support 12 arranged at one end. The other end of the swap 11 forms ofa handle and / or lid as shown in Fig. 2. Fig. 3B shows a second sample collection device 10' similar to the first sample collection device 10 but differing in that the structural support 12 is sandwiched between two covers 13 so that the structural support 12 forms a core with a circumferential exposed portion and an inner covered portion. The covers 13 are entirely or partly impervious to the biofluid sample. Fig. 3C shows a third sample collection device 10" differing from the first sample device 10 in that the structural support 12 is sleeved by a cylindrical cover 13 so that the exposed portion of the structural device 12 is circular. The structural support 12 of the third sample collection device 10" may also extend slightly beyond the cover 13. The second and third sample collection devices 10', 10" are configured for, when the exposed portion of the structural support 12 is submerged in the first aqueous solution comprising the diluted biofluid sample as described for Fig. 2, receiving and at least partly separating the constituents in the aliquot of the diluted biofluid sample within the structural support 12. Thus, the different constituents of the biofluid sample penetrate into the structural support 12 to varying degrees, thereby causing at least a partial separation (or fractionation) of the biofluid sample constituents as the biofluid sample traverses the structural support 12. Hence, small molecules, e.g. metabolites, peptides, or short polypeptides, may penetrate further into the structural support 12 than the larger molecules, e.g. proteins, and proteins may penetrate further into the structural support 12 than cells, such as erythrocytes. The structural support 12 can thus be divided into sections before analysing to at least partially separate the constituents of the biofluid sample.P2671PC00
[0102] 15
[0103] LIST OF ITEMS
[0104] The following is a list of items relating to various aspects and embodiments of the present disclosure.
[0105] 1. A method of collecting a biofluid sample and stabilising an analyte of interest of the biofluid sample on a structural support for mass spectrometry, MS, quantification of an analyte of interest, comprising the steps of:
[0106] providing a biofluid sample comprising an analyte of interest, such as proteins, for subsequent MS quantification;
[0107] diluting the biofluid sample in a first aqueous solution; and
[0108] transferring at least an aliquot of the diluted biofluid sample to a second anti-solvent to precipitate and adsorb the analyte of interest, such as proteins, onto the structural support.
[0109] 2. A method according to item 1, comprising a step of:
[0110] - wetting the structural support with the first aqueous solution, or providing the structural support pre-wetted with the first aqueous solution; and wherein the step of diluting the biofluid sample comprises subjecting the provided biofluid sample to the wetted structural support so as to dilute the biofluid sample in the first aqueous solution.
[0111] 3. A method according to item 1, comprising the steps of:
[0112] providing the structural support in a dry condition; and
[0113] subjecting the dry structural support to the first aqueous solution comprising at least the aliquot of the diluted biofluid sample so as to receive the aliquot of the diluted biofluid sample on the structural support;
[0114] wherein the aliquot of diluted biofluid sample is transferred via the structural support to the second anti-solvent.
[0115] 4. A method according to any one of the previous items, wherein the biofluid sample is volumetrically diluted by the first aqueous solution by a ratio in the range of 1:2 to 1:100, preferably 1:5 to 1:100, more preferably 1:10 to 1:100.
[0116] 5. A method according to any one of the previous items, comprising a step, performed prior to the step of providing the biofluid sample, of:
[0117] pre-loading at least one internal standard without the analyte of interest on the structural support; and / orP2671PC00
[0118] 16
[0119] pre-loading at least one internal standard without the analyte of interest in the first aqueous solution.
[0120] A method according to item 5, wherein the at least one internal standard is pre-loaded on the structural support by:
[0121] - subjecting the structural support to a third aqueous solution comprising at least one internal standard without the analyte of interest;
[0122] subjecting the third aqueous solution to a fourth anti-solvent to precipitate and adsorb the internal standard without the analyte of interest onto the structural support; and
[0123] removing excess liquid from the third aqueous solution and the fourth anti-solvent to adsorb the first internal standard on the structural support.
[0124] A method according to any one of the previous items, wherein the second anti-solvent consists essentially of an organic solvent, such as acetonitrile, ACN; isopropanol, i-PrOH; n-propanol; methanol, MeOH; ethanol, EtOH; acetone; dioxane; or tetra hydrofuran THF, or the second anti-solvent consists essentially of an aqueous solution with high ionic strength.
[0125] A method according to any one of the previous items, wherein, after the step of transferring at least the aliquot of the diluted biofluid sample to the second anti-solvent so as to form a mixture, wherein the cumulative anti-solvent constitutes at least 50% by volume, preferably at least 80% by volume, most preferably at least 90% by volume of the mixture.
[0126] A method according to any one of the previous items, wherein the undiluted biofluid sample and the cumulative anti-solvent have a volume ratio in the range of 1:5 to 1:500, preferably in the range of 1:10 to 1:100.
[0127] A method according to any one of the previous items, wherein the biofluid sample is blood, lymph, interstitial fluid, urine, feces, semen, vaginal fluid, saliva, bile, gastric juice, mucus, sweat, tears, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, cerebrospinal fluid, amniotic fluid, and breast milk.
[0128] A method according to any one of the previous items, wherein the analyte of interest comprises one or more selected from proteins, peptides, carbohydrates, glycopeptides, lipids, nucleic acids, metabolites, small biomolecules.P2671PC00
[0129] 17
[0130] A method according to any one of the previous items, wherein the structural support is a surface, a mesh, a web, or a weave, a filter, or microparticles such as magnetic beads.
[0131] A method according to any one of the previous items, wherein the structural support is made of glass, cotton, knitted polyester, microfiber, paper, a sorbent such as that of an SPE cartridge, or foam.
[0132] A method according to any one of the previous items, wherein the structural support forms part of a vial, or, preferably, a swab.
[0133] A method according to any one of the previous items, wherein the biofluid sample is a blood sample, and wherein the first aqueous solution comprises an anticoagulant.
[0134] A method according to item 15, wherein the anticoagulant is a calcium binder, e.g. ethylenediaminetetraacetic acid, EDTA; or citric acid.
[0135] A method according to any one of items 15-16, wherein the blood sample is provided as a droplet on a skin surface, and wherein the step of diluting the blood sample comprises: arranging, e.g. temporarily sealing, an opening of a first container comprising the first aqueous solution around the blood sample on the skin surface and washing the blood sample off the skin surface and into the first container with the first aqueous solution.
[0136] A method according to any one of items 15-16, wherein the blood sample is provided as droplet on a skin surface, and wherein the step of diluting the blood sample comprises: removing, e.g. by wiping, the blood sample from the skin surface using the structural support, preferably forming part of a swab, wetted with the first aqueous solution so as to dilute the blood sample in the first aqueous solution.
[0137] A method according to any one of the previous items, further comprising a step, performed during or after the step of diluting the biofluid sample, of at least partially separating the diluted biofluid sample constituents on the structural support.
[0138] A method according to any one of items 15-19, wherein the structural support comprises affinity agents, such as antibodies, for an analyte of interest wherein the affinity agents are pre-loaded or chemically linked to the structural support or onto a portion adjacent to the structural support of a sample collection device.P2671PC00
[0139] 18
[0140] 21. A method according to any one of the previous items, comprising the steps of:
[0141] removing the sample collection device including the structural support with the adsorbed analyte of interest from the second anti-solvent;
[0142] - optionally drying the structural support with the adsorbed analyte of interest; and preparing, e.g. packaging, the sample collection device for sending to an external location for subsequent quantification of the analyte of interest, preferably proteomics analysis.
[0143] 22. A method according to any one of the items 1-20, comprising the steps of:
[0144] - sealing the second container comprising the structural support with the adsorbed analyte of interest; and
[0145] preparing, e.g. packaging, the sealed second container comprising the structural support with the adsorbed analyte of interest for sending to an external location for subsequent quantification of the analyte of interest, preferably using liquid chromatography-mass spectrometry, LC-MS.
[0146] 23. A biofluid sampling kit for performing the method according to any one of the previous items, the biofluid sampling kit comprising:
[0147] - a sample collection device comprising a structural support;
[0148] - a first aqueous solution for diluting the biofluid sample; and
[0149] - a second container comprising a second anti-solvent to precipitate and adsorb the analyte of interest of the diluted biofluid sample onto the structural support.
[0150] 24. A biofluid sampling kit according to item 23, wherein the structural support comprises at least one pre-loaded adsorbed internal standard.
[0151] 25. A biofluid sampling kit according to any one of item 23-24, wherein the first aqueous solution comprises at least one internal standard.
[0152] 26. A biofluid sampling kit according to any one of items 23-25, wherein the structural support is pre-wetted with the first aqueous solution.
[0153] 27. A biofluid sampling kit according to any one of items 23-25, wherein the structural support is provided in a dry condition, and wherein the biofluid sampling kit further comprises a first container comprising the first aqueous solution, and the structural support is submersible inP2671PC00
[0154] 19
[0155] the first aqueous solution in the first container so as to allow wetting of the structural support with the first aqueous solution.
[0156] 28. A biofluid sampling kit according to any one of items 23-25, wherein the structural support is within the second container comprising a second anti-solvent to precipitate and adsorb the analyte of interest of the diluted biofluid sample onto the structural support once the first aqueous solution comprising at least an aliquot of the diluted biofluid sample is transferred to the second container while the structural support is present within the second container.
[0157] 29. A biofluid sampling kit according to any one of items 23-28, further comprising:
[0158] a skin puncturing device, such as a lancet, for puncturing skin to provide a blood sample in the form of a droplet on the skin surface.
[0159] 30. A biofluid sampling kit according to any one of items 23-29, wherein the sample collection device comprises a cover partly or, preferably, entirely impervious to the biofluid sample and wherein the structural support forms a core within the cover so that the structural support has an exposed portion and a covered portion, wherein the sample collection device is configured for, when the exposed portion of the structural support is submerged in the first aqueous solution comprising the diluted biofluid sample, receiving and at least partly separating the constituents in the aliquot of the diluted biofluid sample within the structural support.
[0160] 31. A biofluid sampling kit according to any one of items 23-30, wherein the sample collection device comprises a filter arranged so that structural support receives a filtered diluted biofluid sample, the filter preferably being configured to prevent entry of cells.
[0161] 32. A biofluid sampling kit of according to any one of items 23-31, wherein the biofluid sampling kit is a self-collection biofluid sampling kit, preferably further comprising a packaging for sending the sample collection device to an external location for subsequent quantification of the analyte of interest, preferably using mass spectrometry.
[0162] 33. A method of collecting a biofluid sample and stabilising a hydrophobic analyte of interest of the biofluid sample on a structural support for mass spectrometry, MS, quantification of an analyte of interest, comprising the steps of:P2671PC00
[0163] 20
[0164] providing a biofluid sample comprising an analyte of interest, such as a steroid, for subsequent MS quantification;
[0165] diluting the biofluid sample in a third organic solution; and
[0166] transferring at least an aliquot of the diluted biofluid sample to a fourth anti-solvent to precipitate and adsorb the analyte of interest, such as steroids, onto the structural support.
[0167] A method according to item 33, wherein the third organic solution comprise one or more organic solvent(s) or preferably essentially consist of one or more organic solvent(s) selected from acetonitrile, isopropanol, n-propanol, methanol, ethanol, acetone, dioxane, tetra hydrofuran or mixtures thereof, most preferably acetonitrile, methanol, ethanol, isopropanol or mixtures thereof.
[0168] A method according to any one of items 33-34, wherein the fourth anti-solvent is an aqueous solution, e.g. an aqueous buffer solution, such as phosphate buffers, Tris buffers, HEPES buffers, citrate buffers, acetate buffers, bicarbonate buffers.
[0169] A biofluid sampling kit for performing the method according to any one of the items 33-35, the biofluid sampling kit comprising:
[0170] - a sample collection device comprising a structural support;
[0171] a third organic solution for diluting the biofluid sample; and
[0172] - a second container comprising a fourth anti-solvent to precipitate and adsorb the analyte of interest of the diluted biofluid sample onto the structural support.P2671PC00
[0173] 21
[0174] LIST OF REFERENCES
[0175] 1 skin surface
[0176] 2 sample droplet
[0177] 3 dried blood spot
[0178] 4 filter paper
[0179] 10 sample collection device
[0180] 11 swap
[0181] 12 structural support
[0182] 13 cover
[0183] 14 structural support wetted, e.g. pre-wetted, with first solution 16 structural support with aliquot of sample droplet
[0184] 18 structural support with adsorbed analyte of interest
[0185] 20 first container
[0186] 22 opening
[0187] 24 first solution
[0188] 30 second container
[0189] 32 sealed second container
[0190] 34 second anti-solvent
[0191] 36 lid
[0192] 40 packaging
[0193] 100 providing step
[0194] 200 diluting step
[0195] 300 precipitation step
[0196] 400 preparing step
Claims
P2671PC0022CLAIMS1. A method of collecting a biofluid sample and stabilising an analyte of interest of the biofluid sample on a structural support for mass spectrometry, MS, quantification of an analyte of interest, comprising the steps of:providing a biofluid sample comprising an analyte of interest, such as proteins, for subsequent MS quantification;diluting the biofluid sample in a first aqueous solution; andtransferring at least an aliquot of the diluted biofluid sample to a second anti-solvent to precipitate and adsorb the analyte of interest, such as proteins, onto the structural support.
2. A method according to claim 1, comprising a step of:- wetting the structural support with the first aqueous solution, or providing the structural support pre-wetted with the first aqueous solution;wherein the step of diluting the biofluid sample comprises subjecting the provided biofluid sample to the wetted structural support so as to dilute the biofluid sample in the first aqueous solution.
3. A method according to claim 1, comprising the steps of:providing the structural support in a dry condition; andsubjecting the dry structural support to the first aqueous solution comprising at least the aliquot of the diluted biofluid sample so as to receive the aliquot of the diluted biofluid sample on the structural support;wherein the aliquot of diluted biofluid sample is transferred via the structural support to the second anti-solvent.
4. A method according to any one of the previous claims, wherein the biofluid sample is volumetrically diluted by the first aqueous solution by a ratio in the range of 1:2 to 1:100, preferably 1:10 to 1:100.
5. A method according to any one of the previous claims, comprising a step, performed prior to the step of providing the biofluid sample, of:pre-loading at least one internal standard without the analyte of interest on the structural support; and / orP2671PC0023pre-loading at least one internal standard without the analyte of interest in the first aqueous solution.
6. A method according to claim 5, wherein the at least one internal standard is pre-loaded on the structural support by:- subjecting the structural support to a third aqueous solution comprising at least one internal standard without the analyte of interest;subjecting the third aqueous solution to a fourth anti-solvent to precipitate and adsorb the internal standard without the analyte of interest onto the structural support; andremoving excess liquid from the third aqueous solution and the fourth anti-solvent to adsorb the first internal standard on the structural support.
7. A method according to any one of the previous claims, wherein the biofluid sample is a blood sample, and wherein the first aqueous solution comprises an anticoagulant.
8. A method according to claim 7, wherein the blood sample is provided as a droplet on a skin surface, andwherein the step of diluting the blood sample comprises:arranging, e.g. temporarily sealing, an opening of a first container comprising the first aqueous solution around the blood sample on the skin surface, and washing the blood sample off the skin surface and into the first container with the first aqueous solution; orwherein the step of diluting the blood sample comprises:removing, e.g. by wiping, the blood sample from the skin surface using the structural support, preferably forming part of a swab, wetted with the first aqueous solution so as to dilute the blood sample in the first aqueous solution; orwherein the step of diluting the blood sample comprises:removing, e.g. by wiping, the blood sample from the skin surface using a dry structural support that has been impregnated with an anti-coagulant, preferably forming part of a swab, and immediately thereafter immersing the structural support in the first aqueous solution so as to dilute the blood sample in the first aqueous solution.
9. A method according to any one of the previous claims, further comprising a step, performed during or after the step of diluting the biofluid sample, of at least partially separating the diluted biofluid sample constituents on the structural support.P2671PC002410. A method according to any one of the previous claims, comprising the steps of:removing the sample collection device including the structural support with the adsorbed analyte of interest from the second anti-solvent;- optionally drying the structural support with the adsorbed analyte of interest; and preparing, e.g. packaging, the sample collection device for sending to an external location for subsequent quantification of the analyte of interest, preferably proteomics analysis; orwherein the method comprises the steps of:- sealing the second container comprising the structural support with the adsorbed analyte of interest; andpreparing, e.g. packaging, the sealed second container comprising the structural support with the adsorbed analyte of interest for sending to an external location for subsequent quantification of the analyte of interest, preferably using liquid chromatography-mass spectrometry, LC-MS.
11. A biofluid sampling kit for performing the method according to any one of the previous claims, the biofluid sampling kit comprising:- a sample collection device comprising a structural support;- a first aqueous solution for diluting the biofluid sample; and- a second container comprising a second anti-solvent to precipitate and adsorb the analyte of interest of the diluted biofluid sample onto the structural support.
12. A biofluid sampling kit according to claim 11, wherein the structural support comprises at least one pre-loaded adsorbed internal standard and / or wherein the first aqueous solution comprises at least one internal standard.
13. A biofluid sampling kit according to any one of claims 11-12, wherein:- the structural support is pre-wetted with the first aqueous solution; or wherein - the structural support is provided in a dry condition, and wherein the biofluid sampling kit further comprises a first container comprising the first aqueous solution, and the structural support is submersible in the first aqueous solution in the first container so as to allow wetting of the structural support with the first aqueous solution; or wherein- the structural support is within the second container comprising a second antisolvent to precipitate and adsorb the analyte of interest of the diluted biofluid sample onto the structural support once the first aqueous solution comprising atP2671PC0025least an aliquot of the diluted biofluid sample is transferred to the second container while the structural support is present within the second container.
14. A biofluid sampling kit according to any one of claims 11-13, wherein the sample collection device comprises a cover partly or entirely impervious to the biofluid sample and the structural support forms a core within the cover so that the structural support has an exposed portion and a covered portion, wherein the sample collection device is configured for, when the exposed portion of the structural support is submerged in the first aqueous solution comprising the diluted biofluid sample, receiving and at least partly separating the constituents in the aliquot of the diluted biofluid sample within the structural support.
15. A biofluid sampling kit according to any one of claims 11-14, wherein the sample collection device comprises a filter arranged so that structural support receives a filtered diluted biofluid sample, preferably the filter is configured to prevent entry of cells.