Automated detection of particulate compounds in a sample

Abstract

The present invention relates to an automated method for determining a particulate compound in a liquid sample, said method comprising (a) dispensing a thin blot aliquot of said sample on a first working area of a substrate; and / or (b) dispensing an overprint aliquot of said sample on a subarea of said first working area; and / or (c) distributing a thick blot aliquot of said sample on a subarea of said first working area and / or on a second working area of the substrate; and (d) determining the particulate compound in said first working area, in said subarea of said first working area, and / or in said second working area, wherein said dispensing comprises translating an applicator tip in relation to the substrate while dispensing the liquid sample through the applicator tip onto the substrate. The present invention further relates to systems and to diagnostic methods related thereto.

Description

[0001] Automated detection of particulate compounds in a sample

[0002] The present invention relates to an automated method for determining a particulate compound in a liquid sample, said method comprising (a) dispensing a thin blot aliquot of said sample on a first working area of a substrate; and / or (b) dispensing an overprint aliquot of said sample on a subarea of said first working area; and / or (c) distributing a thick blot aliquot of said sample on a subarea of said first working area and / or on a second working area of the substrate; and (d) determining the particulate compound in said first working area, in said subarea of said first working area, and / or in said second working area, wherein said dispensing comprises translating an applicator tip in relation to the substrate while dispensing the liquid sample through the applicator tip onto the substrate. The present invention further relates to systems and to diagnostic methods related thereto.

[0003] Detection of blood-borne parasites can be challenging, in particular due to the frequently very low numbers of detectable parasites in sample materials. Thus, while automated analysis methods have been proposed, the gold standard still is preparation of blood smears on glass microscopic slides, either as a thin smear spreading a drop of blood over most of the area of a microscopic slide, or as a thick smear spreading a blood drop over a relatively small area of a microscopic slide (cf. e.g. www.cdc.gov / dpdx / diagnosticprocedures / blood / specimenproc.html).

[0004] Nonetheless, since the frequency of parasites in the sample typically cannot be predicted, the resulting density of sample material may still be suboptimal for determining the number of parasites and at the same time identifying the parasites. Moreover, the methods of preparing blood smears typically do not allow to determine the volume of sample applied to a given area of a microscopic slide, so the number of parasites per volume of sample typically cannot be determined, and e.g. the number of parasites per red blood cell or similar replacement parameters have to be determined. There is, thus, a need for improved methods for determining particulate compounds such as blood stages of parasites in samples, avoiding the drawbacks of the prior art. This problem is solved by the means and methods disclosed herein.

[0005] Thus, the present invention relates to an automated method for determining a particulate compound in a liquid sample, said method comprising

[0006] (a) dispensing a thin blot aliquot of said sample on a first working area of a substrate; and / or

[0007] (b) dispensing an overprint aliquot of said sample on a subarea of said first working area; and / or

[0008] (c) distributing a thick blot aliquot of said sample on a subarea of said first working area and / or on a second working area of the substrate; and

[0009] (d) determining the particulate compound in said first working area, in said subarea of said first working area, and / or in said second working area, wherein said dispensing comprises translating an applicator tip in relation to the substrate while dispensing the liquid sample through the applicator tip onto the substrate.

[0010] In general, terms used herein are to be given their ordinary and customary meaning to a person of ordinary skill in the art and, unless indicated otherwise, are not to be limited to a special or customized meaning. As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" in an embodiment refer to "comprising one or more", i.e. are equivalent to "comprising at least one". In accordance, expressions relating to one item of a plurality, unless otherwise indicated, in an embodiment relate to at least one such item, in a further embodiment a plurality thereof; thus, e.g. identifying "a cell" relates to identifying at least one cell, in an embodiment to identifying a multitude of cells.

[0011] Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

[0012] The methods specified herein below are in vitro methods. The methods may comprise steps in addition to those explicitly mentioned above, in particular those described herein below. The methods are at least partially automated methods, i.e. are at least partially assisted or performed by automated equipment, in an embodiment a system as described herein below. In particular, the method may be a method for automated generation of a substrate with dispensed sample for determining a particulate compound; in a further embodiment, the method is a fully automated method for determining a particulate compound, as discussed in more detail herein below. In an embodiment, the method is performed on a sample known or suspected to comprise a particulate compound, in a further embodiment on a sample suspected to comprise a particulate compound.

[0013] The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but in an embodiment are performed in one of the indicated sequences. As discussed in more detail herein below, without restriction, the following sequences may be envisaged in particular: steps (a), (b), (d), and (d); steps (d), (a), optionally (b), and (d), steps (a) and optionally (b), (c), and (d), and steps (d), optionally (a) and (b), and (d). Also, e.g. one or more of steps (a) to (c) may be performed contingent on the determination in step (d) for one of the other steps; thus the order may also be steps (c), (d), and if a particulate compound is detected in step (d), steps (a), optionally (b), and (d). As the skilled person understands from the above, method steps may be repeated; in particular step (b) may be repeated to create an increasing number of overprints; and / or step (d) in an embodiment is performed each time after at least one of steps (a) to (c) was performed.

[0014] As used herein, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, in an embodiment relates to the indicated value ± 20%, in a further embodiment ± 10%, in a further embodiment ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, in a further embodiment ± 10%, in a further embodiment ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. In an embodiment, a composition consisting essentially of a set of components will comprise less than 5% by weight, in a further embodiment less than 3% by weight, in a further embodiment less than 1% by weight, in a further embodiment less than 0.1% by weight, of non-specified component(s).

[0015] The term “determining” as used herein refers to qualitative, semi quantitative, or quantitative determination of a particulate compound. Determining a particulate compound may be carried out by any technique which allows for establishing the presence or absence of the particulate compound in the sample (qualitative determination), in an embodiment above background levels, or to establishing a measure of quantity of the particulate compound in a semi quantitative or quantitative manner. Suitable techniques, e.g. staining techniques and optical determination methods are, in principle, known in the art, and include in particular staining of the particulate compounds and counting the number of detectable particulate compounds within a pre-determined area of a working area. Determining includes all methods of determining at least one detectable feature of a particulate compound. The detectable feature may be any feature deemed appropriate by the skilled person. In an embodiment, the detectable feature is selected from the list consisting of a physical feature, a chemical feature, a structural feature, and an electrochemical feature. Physical features are all features detectable by physical means, e.g. optical features such as absorption, transmission, color, reflection, fluorescence, radiation, and the like, or features detectable by physical interaction with a composition of matter, e.g. in atomic force microscopy. Chemical features are all features detectable by at least one chemical reaction, e.g. the activity of producing a chemical compound, in an embodiment when provided with a co-substrate and / or a catalyst. Structural features are all detectable features conferred by the chemical structure of particulate compound, in particular immunological features, i.e. the property of being recognized or recognizable by a binding compound such as an antibody. Electrochemical features include all features based on a chemical reaction and detectable by electric means, e.g. by measuring current, voltage, and / or chemiluminescence, in an embodiment after applying an external voltage; electrochemical features include in particular electrochemiluminescence (ECL) features. As the skilled person understands, determining a detectable feature of an analyte such as a particulate compound in an embodiment also includes detecting the absence of said feature. In an embodiment, determining comprises determining a multitude of features, e.g. stainability and physical form and / or size, immunogenicity and physical form and / or size, or the like. If a multitude of features is determined, it may in particular be envisaged to determine features which can be determined by a single detection device; e.g. stainability and physical form can be determined with an image processing unit, optionally coupled to a microscopy device.

[0016] In an embodiment, determining a particulate compound is establishing whether a particulate compound is present or absent in the sample at a concentration above the detection limit of the method, i.e., in an embodiment, the determining is qualitative. Methods of determining a detection limit are known to the skilled person. In a further embodiment, determining is determining semi-quantitatively or quantitatively the amount or concentration of the particulate compound in a sample. For semi-quantitative determining, the amount may be assigned e.g. to two or more pre-defined categories, e.g. above or not above a reference value, or low, medium, or high. For quantitative determination, either the absolute or precise amount of the particulate compound will be determined or the relative amount of the particulate compound will be determined. The relative amount may be determined in a case were the precise amount of particulate compound can or shall not be determined. In said case, it can be determined whether the amount in which the particulate compound is present is increased or diminished with respect to a reference sample or a plurality of reference samples comprising the particulate compound in a pre-determined amount or in pre-determined amounts. For quantitative determination, any parameter correlating with the amount or concentration of the particulate compound in the sample or any value derived therefrom by standard mathematical and / or evaluation operations, including in particular multiplication, division, reciprocal formation, scaling, normalization, standardization, error correction, background correction, or mean or median calculation, may be determined and / or output. Further options of parameters which may be determined are described herein below. In an embodiment, in case the sample is a blood sample, the quantitative measure is a number of particulate compounds per red blood cell, a number of particulate compounds per area of the working area, a number of particulate compounds per sample volume, or an equivalent parameter.

[0017] In an embodiment, the particulate compound is determined by a physical method, in a further embodiment by any optical method deemed appropriate by the skilled person. Thus, the particulate compound may be determined by a method comprising absorption measurement, transmission measurement, reflection measurement, fluorescence measurement, or the like. In such case, the substrate in an embodiment is transparent and the measurement is performed by passing light through the substrate and at least a part of the sample distributed thereon. In a further embodiment, the particulate compound is determined by a method comprising illuminating at least a part of the sample distributed on the substrate and taking at least one digital image of said part of the sample. Methods including such taking at least one digital image are known in the art and are described herein elsewhere in more detail. The determining may further comprise providing an optical enlargement of a working area of interest or a section thereof; such enlargement may be accomplished e.g. by one or more optical lenses, such as in a microscope unit.

[0018] According to the method described herein, the particulate compound may be determined, semi- quantitatively or quantitatively, in an embodiment as described herein above. Alternatively or in addition, determining the particulate compound may comprise identifying the particulate compound. Means and methods for identifying a particulate compound are known in principle to the skilled person and are selected, in particular in dependence on the particulate compound of interest and on sample type, without further ado. E.g., a particulate compound for which a specific antiserum is available may be identified based on its immunologic properties alone. A particulate compound which is stainable by a pre-determined staining method may be identified by said staining, in particular in case the staining is specific; or may be identified by a combination of staining and determination of physical form. In particular blood parasites may have characteristic intracellular and / or extracellular forms in a sample.

[0019] Also according to the method described herein, quantification and identification may be performed on the same or on different working areas or subareas thereof. E.g. identification may be performed on a (sub)area onto which sample was only dispensed once, e.g. the first working area obtained in step (a); while (semi)quantitative determination may be performed on a (sub)area with higher sample density, e.g. the subarea of the first working area, or the second working area, obtained in step (b).

[0020] The term "particulate compound" is in principle known to the skilled person. In an embodiment, the term relates to any solid composition of matter known or suspected to be dispersed in a liquid sample. The form of the particulate compound may be regular or essentially regular, such as is the case with e.g. coccoid bacteria. The form may, however, also be irregular, i.e. showing a distribution of size, volume, and / or general form over a predetermined range. The form of the particulate compound may also be characteristic of the particulate compound, such as is the case with e.g. red blood cells, Spirillum bacteria, and the like. In an embodiment, the particulate compound has a diameter or an average diameter of from 0.2 pm to 2 mm, in an embodiment of from 0.5 pm to 500 pm, in a further embodiment of from 1 pm to 100 pm. The particulate compound may be a rare particulate compound, wherein the term "rare" relates to the frequency of the particulate compound in the specific sample; thus, the rare particulate compound in an embodiment is a particulate compound present in the sample at a frequency of at most 1 particle per pl, in an embodiment at most per 10 pl, in a further embodiment per 100 pl, of sample. In case the liquid sample is a blood sample, the rare particulate compound in an embodiment is a particulate compound present in the sample at a frequency of at most 1 per 103red blood cells, in an embodiment at most 1 per 104red blood cells, in a further embodiment of at most 1 per 105red blood cells, in a further embodiment of at most 1 per 106red blood cells. The particulate compound may be any composition of matter, i.e. a pure chemical compound, or a, homogenous or heterogeneous, mixture of chemical compounds. In an embodiment, the particulate compound is a biological particulate compound. In a further embodiment, the particulate compound is a cell or a substructure or fragment thereof, in a further embodiment is a cell. The particulate compound, thus, may be a pathogenic microorganism, in particular in case the liquid sample is a biological liquid sample. In a further embodiment, the particulate compound is a parasite, in an embodiment a human parasite, in a further embodiment from the group Haemospororida, Fila, Trypanosomatida, Piroplasmorida, or Schistosomatidae. Thus, the particulate compound may in particular be a blood form of a member of the genus Plasmodium, Wuchereria or Brugia, Trypanosoma, Babesia, Leishmania, or Schistosoma. Thus, the particulate compound may e.g. be a trophozoite, a schizont, a sporozoite, a merozoite, and / or a microfilarion. In an embodiment, the particulate compound is a parasite from the genus Plasmodium. As used herein, the term “sample”, also referred to as "test sample", relates to any type of composition of matter known or suspected to comprise a particulate compound; thus, the term may refer, without limitation, to any arbitrary sample such as a biological sample. As referred to herein, the sample is a liquid sample, in a further embodiment an aqueous sample at the time of performing the method described herein. Thus, the sample may, in principle, be a solid sample at the time of sample taking; e.g. a tissue sample may be taken and cells in said tissue sample may be dissociated in a liquid medium to produce a liquid sample. In an embodiment, the test sample is selected from the group consisting of a physiological fluid, including whole blood, serum, plasma, saliva, ocular lens fluid, lacrimal fluid, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, synovial fluid, peritoneal fluid, and amniotic fluid; and lavage fluid. The sample is known or suspected to comprise at least one particulate compound of interest, i.e. a particulate compound which shall be determined, which may also be referred to as "analyte". The sample may comprise one or more further chemical compounds, which are not to be determined and which are commonly referred to as "matrix". The sample may be used directly as obtained from the respective source or may be subjected to one or more pretreatment and / or a sample preparation step(s). Thus, the sample may be pretreated by physical and / or chemical methods, in an embodiment by centrifugation, filtration, mixing, dilution, concentration, contacting with a binding and / or detection reagent, and / or any other method deemed appropriate by the skilled person. In an embodiment, the liquid sample is a biological sample, in a further embodiment is a blood sample or a blood-derived sample, in a further embodiment is a whole blood sample. In case the sample is a blood sample or a blood-derived sample, red blood cells in said liquid sample may be or may have been hemolyzed at least partially.

[0021] The term "aliquot" for which also the term "sample aliquot" may be used, as used herein, relates to any subportion of a sample which may have any volume deemed appropriate by the skilled person. In concurrence with the general use of the term, the composition of aliquots and of the sample is essentially identical. In contrast, subportions of a sample which differ in composition from the sample are generally referred to as "fractions". Thus, the skilled person understands that in an embodiment the thin blot aliquot, the overprint aliquot, and the thick blot aliquot do not differ in composition, but in a further embodiment only differ in their volume and the area or subarea they are distributed onto. The aliquot may have any volume deemed appropriate by the skilled person, and will typically be pre-defined to be sufficient to cover the intended area or subarea of a working area. As the skilled person understands in view of the description herein, the pre-defined volume of the thin blot aliquot used in step (a) may be selected independently of a volume of an overprint aliquot used in a step (b) and of a thick blot aliquot used in step (c). E.g. the pre-defined volume of the thin-blot aliquot used in step (a) may be larger than the volume of an overprint aliquot used in step (b). I.e., in case a working area is overprinted, the overprint generated in step (b) may have a smaller area, and thus may require a smaller sample volume, than the dispensing in step (a). Similarly, any repetition of step (b) may be performed on a smaller subarea of the first working area than any preceding step (b), again requiring a smaller overprinting aliquot volume. Thus, in step (a) the thin blot aliquot may have a first pre-defined volume, e.g. Vi, and in optional step (bl) the overprint aliquot may have a second pre-defined volume, e.g. V2, with, in an embodiment, Vi > V2. This way, an array of subareas, e.g. stripes, may be generated, with each subarea having a distinct number of overprints.

[0022] Also, in case step (c) is performed, the pre-defined volume of the thick-blot aliquot may be selected independently of a volume of any aliquot used in a step (a) and / or (b), Thus, the thick blot aliquot in step (c) may have a third pre-defined volume, e.g. V3. In an embodiment, the third volume is at least five times, in an embodiment at least six times, in a further embodiment at least seven times, in a further embodiment at least eight times, in a further embodiment at least nine times, in a further embodiment at least ten times, the first volume, i.e. the volume of the thin blot aliquot used in step (a). Also, the thick blot aliquot in step (c) may be distributed over a smaller area than e.g. the thin blot aliquot of step (a); e.g. the subarea of the first working area and / or the area of the second working area onto which the thick blot aliquot of the sample is distributed in step (c) may be most one fifth, in an embodiment at most a sixth, in a further embodiment at most a seventh, in a further embodiment at most an eighth, in a further embodiment at most a ninth, in a further embodiment at most one tenth, of the area of the first working area. Thus, the density of sample constituents in the area or subarea covered by the thick blot aliquot in step (c) may be considerably higher than the one generated in step (a) and optionally in step(s) (b). Also, while the distribution of sample constituents in the working area generated in step (a) and optional subareas generated in step (b) in an embodiment is essentially homogenous, the distribution of sample constituents in any area or subarea generated in step (c) may be inhomogeneous. Nonetheless, as indicated above, the thick blot aliquot in an embodiment has a pre-defined volume.

[0023] The "substrate", as the term is used herein, can be any substrate that the person skilled in the art would consider suitable for use in the context of the description herein. The substrate can have a shape at least in two dimensions, in an embodiment selected from the group consisting of round, oval, angular or a mixture of at least two thereof. The substrate can furthermore have a shape selected from the group consisting of a plate, a strip, a tape, or a mixture of at least two thereof. In an embodiment, the substrate is of a material providing for sufficient rigidity to allow handling and dispensing of a liquid sample. Also in an embodiment, the substrate has binding capacity for biological material such as cells and / or the particulate compound of interest. In a further embodiment, the substrate is transparent. The skilled person selects appropriate materials in dependence on the properties required. Thus, in an embodiment, the substrate is made of glass or polycarbonate, in particular is a glass slide or a polycarbonate slide.

[0024] A "working area", as the term is used herein, is a subportion of a surface of a substrate, which may be virtual, i.e. assigned without being delimited by structural features of the substrate, or may be structurally delimited, e.g. by an engraving or by a physical boundary, which structural delimitation may be structured to prevent a fluid applied to a first working area from entering a second working area. A first and a second working area may, in principle be located on two different substrates; in an embodiment, however, the first and the second working area are located on the same substrate. Thus, step (c) may be dispensing a thick blot aliquot of the liquid sample on a second working area of the substrate. In such case, the first working area and the second working area may be spatially separated on said substrate, in an embodiment wherein said spatial separation enables independently contacting the first working area and / or the second working area with a detection solution.

[0025] In concurrence with the above, a "subarea", as the term is used herein, may be any subportion of a working area, which may be virtual, i.e. assigned without being delimited by structural features of the substrate, or may be structurally delimited, e.g. by an engraving or by a physical boundary, which structural delimitation may be structured to prevent a fluid applied to a subarea from entering a second subarea and / or a further working area. A subarea may, in principle, have the same area as the working area, in particular the first working area, e.g. in cases where it is known that the concentration of particulate compounds is too low for determining after only one dispensation. In an embodiment, however, the subarea is smaller than the working area. A subarea may in particular be created by dispensing an overprinting aliquot of a liquid sample onto a working area, e.g. in step (b), onto which an aliquot of said sample has already been dispensed, e.g. in step (a), i.e. by overprinting (step (b)). Thus, subareas of a working area may in particular differ in the amount of sample dispensed onto the respective subarea. As a consequence, subareas of a working area may in particular differ in the amount of particulate compound which may be expected to be present on the respective subarea. A working area may comprise an essentially unlimited number of subareas; in view of the above, however, a working area may in particular comprise a number of subareas correlating with the maximal number of overprints; i.e. if e.g. step (b) is performed three times, the first working area may comprise four subareas, i.e. one corresponding to the dispensation of step (a), one corresponding to the first overprint, one corresponding to cumulated two overprints, and one corresponding to all cumulated prints.

[0026] The term "distributing", as referred to herein, includes each and every proceeding causing an aliquot of a sample to cover an area or subarea of a working area. Thus, distributing may be actively causing the aliquot of a sample to cover a pre-defined working area or subarea thereof, in particular may be dispensing as the term is specified herein below. Distributing may, however, also be applying a thick blot aliquot of a sample to a point on the surface of a substrate and leaving the thick blot aliquot to spread over the support, in an embodiment by forces of cohesion, adhesion, and / or surface tension. In an embodiment, a pre-defined volume of a sample is distributed; in a further embodiment of step (c), all sample remaining in the applicator tip after step (a) and optionally step (b) and optional repetitions thereof, is distributed ("needle drop"). As specified herein above, the density of sample constituents produced by the distributing in step (c) may be considerably higher than the one produced in steps (a) and (b), and thus may also be referred to as a "thick blot". As used herein, distributing an aliquot of a sample on a working area of a substrate may also be referred to as "printing" said aliquot onto said working area. In accordance, the process of distributing an aliquot of a sample onto a working area are or part thereof onto which sample has already been distributed at least once is also referred to as "overprinting".

[0027] The term "dispensing" relates to actively causing an aliquot of a sample to become distributed over at least one working area of a substrate. As the term is referred to herein, dispensing comprises translating an applicator tip in relation to the substrate while dispensing the liquid sample through the applicator tip onto the substrate as part of an automated method. Said dispensing, in particular the dispensing in step (a), in an embodiment comprises causing a monolayer of blood cells to form on the substrate. However, dispensing may also comprise causing a multilayer of blood cells to form on the substrate. In an embodiment, dispensing comprises translating the applicator tip in essentially linear motions, e.g. zig-zag or striped, or in a circular motion, in relation to the substrate. However, also any other type of motion deemed appropriate by the skilled person may be used, e.g. an x / y movement generating a checkered array of overprints. Also, during the dispensing, the substrate, the applicator tip, or both may be moved, i.e. the substrate may be moved under a fixed applicator tip, the applicator tip may be moved over a fixed substrate, or the substrate and the applicator tip may both move relative to each other.

[0028] Adjacent tracks of dispensing may be selected to overlap, causing an essentially homogenous distribution of cells and optionally particulate compounds over a working area or subarea thereof. Adjacent tracks may, however, also be selected to be spaced such that they do not overlap, which may cause a striped appearance of the working area. Also, adjacent track of dispensing may be arranged such that each track can spread over the substrate; such spreading may be more pronounced for the liquid phase of the sample than for its particulate constituents, such that particulate sample constituents may be concentrated towards the middle of the dispensing track.

[0029] The term "applicator tip" is used herein to relate to any means configured to allow a liquid to be dispensed on a substrate. Thus, the applicator tip may in particular comprise an orifice fluidly connected to at least one other liquid handling means of a liquid handling device, e.g. via a tubing. Typically such an orifice will be contained in a tip that is two to five millimeters in outside diameter with an inner diameter of about 0.5 millimeters. The applicator tip may be disposable or washable. The applicator tip may be rounded to facilitate insertion and cleaning of the tip. Fluid flow through the applicator tip in an embodiment is controlled to allow a thin layer of sample to be deposited onto the substrate. By optimizing flow rate through the applicator tip and the relative speed and height of the tip over the substrate, an appropriate sample volume per area can be deposited onto the slide, e.g. to deposit essentially a monolayer of cells comprised in the sample. Each of the aforesaid factors may influence the other, so the proper combination of height, flow rate through the applicator tip, and speed over the substrate may be determined and / or optimized. In an embodiment and solely on an exemplary basis, the applicator tip is positioned less than about 110 microns above the substrate. In an embodiment, the applicator tip is positioned of from 5 pm to 100 pm, in a further embodiment of from 8 pm to 25 pm, in a further embodiment about 12 pm above the substrate. Also in an embodiment, the system maintains, e.g. directed by software instructions, relative movement between the tip and the substrate at a speed of about 10 to 100 mm per second while the ejection of the fluid onto the substrate is occurring. Thus, software instructions may cause the system to dispense a known volume of sample out of the applicator tip. Also in an embodiment, the flow rate through the applicator tip is of from 0.01 pl / s to 5 pl / s, in an embodiment of from 0.02 pl / s to 2 pl / s, in a further embodiment of from 0.025 pl / s to 1 pl / s, in a further embodiment of about 0.055 pl / s. Also in an embodiment, the aforesaid flow rates are applied while the applicator tip is moving at a speed of from 5 mm / s to 250 mm / s, in an embodiment of from 10 mm / s to 200 mm / s, in a further embodiment of from 50 mm / s to 100 mm / s, in a further embodiment of about 65 mm / s. In an embodiment, and on a strictly exemplary basis, the flow rate through the applicator tip may be 0.055 pl / s while the applicator tip is moving at a speed of 65 mm / s over the substrate surface at a height of about 12 pm. In a further embodiment, for example when the sample comprises undiluted blood, the flow rate through the applicator tip may be approximately 0.04 pl / s while the tip is moving at a speed relative to a point on the substrate of 65 mm / s at a height of about 12 pm above the substrate surface. The viscosity and consistency of the liquid sample may influence the flow rate through the applicator tip and the relative speed and height of the tip over the substrate required to ensure that an appropriate volume per area is deposited on the substrate for examination.

[0030] The method described herein comprises at least one of steps (a) to (c), in an embodiment comprises steps (a) and (c), (c) and (a), or (a) and (b); in a further embodiment comprises steps (a), (b), and (c) or steps (c), (a), and (b). The method further comprises step (d). In an embodiment, the method does not comprise generating a blood smear as is known in the art, in an embodiment comprises neither generating a thin blood smear nor a thick blood smear known in the art.

[0031] The method may comprise step (a) dispensing a thin blot aliquot of said sample on a first working area of a substrate. As described herein elsewhere, said dispensing comprises translating an applicator tip in relation to the substrate while dispensing the liquid sample through the applicator tip onto the substrate. The volume of sample dispensed on the first working area depends in particular on the desired area of the first working area and on the desired density of sample constituents. In an embodiment, 0.2 to 10 pl, in a further embodiment about 1 pl, of sample are dispensed on the first working area in case the sample is an essentially undiluted whole blood sample. For diluted samples, appropriately higher volumes per area may be used. In an embodiment, a pre-defined volume of sample is dispensed over a pre-defined area in step (a), such that sample volume per area of working area can be calculated, such that it may be possible to calculate a concentration of a particulate compound in the sample in step (d).

[0032] Step (a) may comprise a further step of drying the first working area after said dispensing. Appropriate methods are known in the art, and include in particular air drying, optionally enhanced by a gas movement device. As the skilled person will understand, drying conditions may in particular be selected so as to not negatively affect the structural integrity of any particulate compound possibly present in the sample.

[0033] The method may further comprise step (b) dispensing an overprint aliquot of said sample on a subarea of said first working area. To said dispensing in step (b), the description for step (a) applies mutatis mutandis. In an embodiment, compared to step (a), a smaller sample aliquot is dispensed on a subarea of the first working area, i.e. on an area smaller than the first working area. In an embodiment, sample volume and area are reduced correspondingly, i.e. in an embodiment such that sample volume dispensed per area is the same in all step(s) (b) and optionally in step (a). Thus, the sample volume per area in an embodiment increases linearly with each overprint. E.g., the first dispensing in step (b) (the first overprint) may generate twice the sample volume per area compared to step (a) on a first subarea, the second dispensing in step (b) (the second overprint) may generate three times the sample volume per area compared to step (a) on a second subarea, and so forth. It may, however, also be envisaged to use the same pre-determined sample volume in steps (a) and (b), while reducing the area onto which dispensing is performed. Also after step (b), the method may comprise a further step of drying the respective subarea after said dispensing, in an embodiment as described herein for step (a). In an embodiment, step (b) is performed at least once, in a further embodiment at least twice, in a further embodiment at least three times. Also in an embodiment, step (b) is performed at most ten times. Thus, in an embodiment, step (b) may be performed of from one to ten times, in a further embodiment of from two to eight times, in a further embodiment three or four times, in a further embodiment four times.

[0034] The method also may further comprise step (c) distributing a thick blot aliquot of said sample on a subarea of said first working area and / or on a second working area of the substrate. As described herein above, said distributing may, but not necessarily has to be, dispensing a thick blot aliquot. Thus, said distributing may also comprise applying an aliquot of the sample on the substrate without movement of the substrate relative to the applicator tip. The sample may then distribute on the substrate by physical forces. Thus, step (c) may be or comprise a thick blot, in particular a needle dump, as described herein above, in an embodiment creating an area or subarea of high sample volume per area. Such a needle dump may in particular be suitable in the determination of particulate compounds which are known or expected to be very rare in the sample, such as blood forms of malaria. Also after step (c), the method may comprise a further step of drying the respective area or subarea after said distributing, in an embodiment as described herein for step (a).

[0035] The method also comprises step (d) determining the particulate compound in said first working area, in said subarea of said first working area, and / or in said second working area. Means and methods of determining a particulate compound are, in principle, known in the art and are described herein elsewhere. The result of determining may be qualitative, e.g. determining that the particulate compound is present or is not present in the sample, in an embodiment above background and / or above the detection limit; or may be semi quantitative, e.g. determining that the particulate compound of interest is present at an undetectable, low, medium, or high number or concentration; or may be quantitative, e.g. determining a number of particulate compounds per volume of sample. The result of determining may be an absolute value or a relative value, e.g. compared to a reference.

[0036] Step (d) may, in particular in case the working area was not dried after at least one of steps (a), (b), and (c), comprise step (dl) drying said working area or working areas, in an embodiment as described herein above for step (a). Thus, in an embodiment, all working areas are dried before determining the particulate compound. Step (d) may also comprise a further step of fixing sample constituents, e.g. preceding a staining step. Appropriate methods are known in the art.

[0037] Step (d) may further comprise step (d2) contacting at least fraction of the working area or working areas with a detection agent, in an embodiment wherein contacting with a detection agent is staining. For said staining, in principle, all staining methods deemed appropriate by the skilled person for a particulate compound of interest may be used; appropriate staining methods are known in the art and comprise in particular methods comprising contacting at least a part of the first working area and / or of the second working area with a detection agent for said particulate compound, wherein said detection agent in an embodiment is a dye or a dye-coupled binding agent. In an embodiment, the detection agent comprises eosin Y (Cas No.17372-87-1) or eosin B (das No. 548-24-3) and methylene blue (das No. 61-73-4). In a further embodiment, the determining comprises a staining selected from the list consisting of a Romanowsky-type stain, a Giemsa-type stain, a Wright-Giemsa-type stain, a Jenner-type stain, a Wright-type stain, a Field-type stain, a May-Grunwald-type stain, and a Leishman-type stain, all of which are known in the art. In case the particulate compound is a causative agent of malaria, the staining may in particular be a Romanowsky stain or Giemsa stain, in an embodiment is a Romanowsky stain, in a further embodiment is a Giemsa stain. In the dye-coupled binding agent, the binding agent may e.g. be an antibody specifically recognizing a particulate compound of interest, in an embodiment coupled to a suitable dye.

[0038] Step (d) may further comprise step (d3) removing unbound detection agent from said at least part of the first working area and / or of the second working area. Said step may comprise removing any surplus of a staining solution comprising the staining agent(s), partially or completely, and optionally one or more wash steps. Again, appropriate means and methods are known in the art.

[0039] Step (d) may further comprise step (d4) capturing at least one digital image of the first working area and / or of the second working area, or parts thereof, wherein in an embodiment the at least one digital image corresponds to light transmitted or reflected by sample constituents in the first and / or second working area. As the skilled person is aware of, said digital image may be captured at a pre-determined wavelength or wavelength range, e.g. visible light and / or UV- light. The at least one digital image may be evaluated by a qualified person, e.g. a medical practitioner, to determine the particulate compound; in such case the method is an automated method of providing a substrate for determining a particulate compound, i.e. the method is at least partially automated. In a further embodiment, also evaluation of the aforesaid at least one digital image is automated. In such case, the method in an embodiment is a fully automated method for determining a particulate compound in a liquid sample. In such case, the method may comprise further step (d5) automatically analyzing the at least one digital image with an automated imaging system to automatically provide a determination of the particulate compound.

[0040] Step (d) may further comprise step (d6) refining said at least one image by sharpening or compensating for spatial shifts or other distortions, and / or step (d7) determining spatial, densitometric, colorimetric, and / or texture features within said at least part of the first working area and / or of the second working area.

[0041] As the skilled person understands, the distributed thick blot aliquot generated in step (c) may in particular be used to detect that particulate compound, i.e. to establish its presence or absence in the sample, and optionally to quantify the particulate compound, i.e. to establish the number of particulate compounds per sample volume and / or relative to other compounds, e.g. red blood cells. The dispensation produced in step (a) and optional step (b) may in particular be used to identify the particulate compound, e.g. its species and optionally its subspecies.

[0042] In step (d), the various working areas and subareas thereof may be examined in any order, or may not be examined, as deemed appropriate by the skilled person. On an exemplary basis, the following evaluation protocols may envisaged:

[0043] - The first working area produced in step (a) and the subarea or second working produced in step (c) may be evaluated to identify and / or quantify particulate compounds, if present; in case determining is by microscopy, different magnifications may be used in different working areas or subareas.

[0044] - The subarea or second working area produced in step (c) may be evaluated; only in case a particulate compound is detected, the particulate compound may be identified; moreover, also only in such case, the working area produced in step (a) and / or at least one subarea produced in step (b) may be evaluated to determine the amount of particulate compound in the sample.

[0045] - Only the second working area of step (c) may be produced and evaluated initially in step (d); only in case a particulate compound is determined, step (a) and optionally step (b) may be performed, and the working area produced may be evaluated by performing step (d) on these. As the skilled person will understand, in the first performance of step (d), an amount of particulate compound may be determined, which may be used to adjust the amount of sample constituents per area when performing step (a) and optionally step (b).

[0046] Advantageously, it was found in the work underlying the present invention that the means and methods described herein enable automated analysis of samples to determine particulate compounds, such as agents causing malaria. In contrast to known methods, no manual slide preparation is necessary. Also, a very homogenous distribution of sample, with different degrees of overprinting, can be achieved, making automated and very exact sample analysis possible. Moreover, since it is not necessary to determine the number of particulate compounds relative to the number of red blood cells, hemolyzed blood samples can be used, thus reducing background material (matrix) in the working areas.

[0047] The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

[0048] The present invention also relates to a system comprising

[0049] (i) a liquid handling device comprising an applicator tip for dispensing a fluid sample onto a working area of a substrate;

[0050] (ii) a microprocessor; and

[0051] (iii) a storage device comprising stored software instructions for controlling the system, wherein the software instructions, when executed on the microprocessor, cause the system to perform a method as specified herein.

[0052] The term “system”, as used herein, relates to a system of means, such as units and devices, comprising at least the aforementioned means operatively linked to each other as to allow the determination. Typical means for determining particulate compounds, and means for carrying out the determination are disclosed above in connection with the methods of the invention. How to link the means in an operating manner will depend on the type of means included into the device. In an embodiment, the means are comprised by a single device. Said device may accordingly include (i) a liquid handling device; (ii) a microprocessor; and (iii) a storage device. In an embodiment, the system comprises means as described in WO 2010 / 126903 Al, US 8,815,537 B2, or US 9,017,610 B2.

[0053] Typical means for detection are disclosed in connection with embodiments relating to the method of the invention above. The means may be operatively linked in that the user of the system brings together the result of a determination performed by the detection device due to the instructions and interpretations given in a manual, or said instructions and interpretations are comprised in an executable program code comprised in the device, such that, as a result of determination, an identity, indication of presence or absence, and / or quantification of a particulate compound in the sample applied is output to the user. The person skilled in the art will realize how to link the means without further ado. Typical systems are those which can be applied without the particular knowledge of a specialized technician, e.g., a system only requiring loading of a sample, e.g. in a tube or a multiwell plate, and handling all determination steps automatically. The results may be given as output of raw data which need interpretation by a technician. In an embodiment, the output of the device is, however, processed, i.e. evaluated, raw data, the interpretation of which does not require a technician. Further typical devices which may optionally be included in the system comprise further analysis devices, which may perform auxiliary or additional measurements, or evaluation units / devices, e.g. as referred to above in accordance with the methods of the invention.

[0054] The term "liquid handling device" is used in its common meaning understood by the skilled person. Thus, the term includes in principle each and every device configured to handle a liquid sample and, optionally, a detection reagent, such as a staining solution. Thus, the liquid handling device may comprise units for transporting and / or storing liquid, such as a syringe, a manual or motor driven pipettor, or a motor controlled pump, in an embodiment fluidly connected via appropriate tubing. The pump may be a peristaltic pump, a syringe pump, or other similar device that allows small volumes of liquid samples to be aspirated and dispensed through an applicator tip as specified herein elsewhere. The liquid handling device further comprises a substrate mount configured to hold the substrate in place. The substrate mount may be fixed in a position relative to the liquid handling device; in such case, in an embodiment the sample applicator tip may be movable relative to the substrate mount. In a further embodiment, the substrate mount is movable relative to the sample applicator tip, at least in the two directions of the horizontal plane. Thus, to place a sample on the substrate, the sample mount may be moved horizontally under the applicator tip so that it is tracing the eventual location to which the sample shall be applied. In an embodiment, the applicator tip moves in a wavy, arcuate, or circular path in relation to the substrate. Thus, in an embodiment, the substrate is positioned under the applicator tip and the substrate in the substrate mount is moved relative to the applicator tip while the applicator tip applies a flow of sample onto the substrate.

[0055] The liquid handling device may comprise, e.g. in the applicator tip, a volume of sample, such as of from 2 pl to 250 pl, in an embodiment of from 5 pl to 100 pl, in a further embodiment about 30 pl. Some samples may need to be pre-processed, e.g. to disperse cells that may be clumped together or to minimize mucus or other protein material that may cause cells to stick together. The liquid handling unit may mix the sample or an aliquot thereof with a fixing agent, a detection agent, a diluent, and / or the like. The liquid handling device may further comprise or be configured to function as a staining station, i.e. as a unit configured to handle one or more staining reactions on optionally related washing steps on one or both working areas. Appropriate staining stations are in principle known in the art, e.g. from WO 2012 / 064873 Al. In an embodiment, the staining station comprises means to restrict a treatment solution and / or a detection solution to one of the working areas, in an embodiment the first working area or the second working area, or a subarea of any of the aforesaid. Thus, the staining station may comprise a staining chamber restricting staining to one or more subsections of the substrate, in particular of a working area or a subarea thereof.

[0056] The liquid handling device, in particular the staining station, may further contain at least one reagent applicator. The reagent applicator may be used to direct a detection reagent and / or a wash reagent, onto the substrate. In such case, the liquid handling device may contain one or more fluid chambers to eject at least one treatment solution. Some reagent applicators may be able to store both fixative and detection reagent, and direct them sequentially onto the first working area of the support, or in alternate embodiments two reagent applicators may be used, one for the fixative and one for the detection solution. In an embodiment, the reagent applicator is the applicator tip as described herein above. Excess treatment solution may be removed from the slide, e.g. by tilting the substrate mount relative to the liquid handling device.

[0057] The term "applicator tip" has been specified herein above. In an embodiment, the system comprises software instructions causing the system to fill the applicator tip with a thin blot aliquot of the sample in step (a), with an overprinting aliquot in step (b), and / or with a thick blot aliquot in step (c), and dispensing said aliquots in steps step (a), (b), and / or (c), respectively. Said aliquots and optional further liquids may be present in the applicator tip simultaneously, optionally separated one or more air gaps.

[0058] The term "detection device", as used herein, generically relates to any device configured to allow the measurements underlying the determination in step (d) to be made, i.e. in an embodiment allows measurement of at least one value of a detectable feature, wherein said detectable feature in an embodiment is selected from the list consisting of a physical feature, a chemical feature, a structural feature, and an electrochemical feature. As the skilled person will understand in view of the description herein, the detectable feature determined in an embodiment is the same in all working areas and subareas of a substrate; i.e. in an embodiment, is the same for a given particulate compound of interest. Nonetheless, also more than one detectable feature may be determined during determination. E.g. the amount of particulate compound may be determined after staining in a distribution produced according to step (c), while the particulate compound may be identified after antibody staining, e.g. in a dispensation obtained according to step (a). Appropriate devices for performing the aforesaid measurements are in principle known in the art.

[0059] In an embodiment, the detectable feature is a physical feature, in particular an optical feature. Thus, the detection device may in particular comprise an optical device comprising at least one light source and at least one light detector. Thus, the detection device may comprise a photometric unit, in an embodiment comprising at least one photocell and at least one light source. Appropriate devices, in particular for use with substrates on a solid support such as a slide dish, are known in the art. The detection device may also comprise, in an embodiment, in addition to the photometric unit, an optical unit comprising an imaging unit configured to capture at least one image of at least a section of the first working area and / or the second working area. The imaging unit and the photometric may, however, also be comprised of essentially the same components; e.g. one or more LEDs as light source and a CCD camera, an LED detector, a CMOS sensor or a combination of at least two thereof as an optical detector. The detection device may further comprise a unit configured for providing an optical enlargement of an area of interest; such enlargement may be accomplished e.g. by one or more optical lenses, such as in a microscope unit. Thus, the system may comprise (iv) a detection device, e.g. an optical device comprising a light source and a light detector, which may, in an embodiment, further comprise a microscope device intervening the light source and the light detector.

[0060] The term "microprocessor" is understood by the skilled person. In an embodiment, the term includes each and every device or unit thereof comprising at least one integrated circuit, in an embodiment comprising the arithmetic, logic, and / or control circuitry required to perform the function(s) as indicated. Thus, the microprocessor may in particular be a microprocessor of a computer.

[0061] The system comprises or has access to a storage device comprising stored software instructions for controlling the system, such as a memory. A "storage device", as referred to herein, comprises a computer readable information storage medium, wherein appropriate media are known to the skilled person. The system may comprise or have access to a single storage device or multiple storage devices, located either locally with the computing device or accessible to the computing device across a network, for example. Computer-readable media may be any available media that can be accessed by the computing device and includes both volatile and non-volatile media. Further, computer readable-media may be one or both of removable and non-removable media. By way of example, and not limitation, computer-readable media may comprise computer storage media. Exemplary computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, Digital Versatile Disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used for storing a plurality of instructions capable of being accessed by the computing device and executed by the processor of the computing device.

[0062] The term "stored software instructions" is understood by the skilled person. The stored software instructions in an embodiment include any sequence of instructions configured to cause the system to perform at least the indicated steps. In an embodiment, the software instructions cause an optical device comprised in the system to capture an image of at least a section of the first working area and / or the second working area. In an embodiment, the system further comprises an analysis device and, in an embodiment the software instructions further cause the analysis device to analyze the image to determine the particulate compound; and / or to determine the particulate compound from a measurement of the optical device and / or to analyze the image to determine the particulate compound.

[0063] Thus, further disclosed and proposed herein is a computer program including computerexecutable instructions for performing the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the computer program may be stored on a computer-readable data carrier. Thus, specifically, one, more than one or even all of the method steps as indicated above may be performed by using a computer or a computer network, in an embodiment by using a computer program.

[0064] Also disclosed and proposed is a computer program product having program code means, in order to perform the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the program code means may be stored on a computer-readable data carrier.

[0065] Further disclosed and proposed is a data carrier having a data structure stored thereon, which, after loading into a computer or computer network, such as into a working memory or main memory of the computer or computer network, may execute the method according to one or more of the embodiments disclosed herein.

[0066] Moreover disclosed and proposed is a computer program product with program code means stored on a machine-readable carrier, in order to perform the method according to one or more of the embodiments disclosed herein, when the program is executed on a computer or computer network. As used herein, a computer program product refers to the program as a tradable product. The product may generally exist in an arbitrary format, such as in a paper format, or on a computer-readable data carrier. Specifically, the computer program product may be distributed over a data network.

[0067] In addition disclosed and proposed is a modulated data signal which contains instructions readable by a computer system or computer network, for performing the method according to one or more of the embodiments disclosed herein.

[0068] In an embodiment, referring to the computer-implemented aspects of the invention, one or more of the method steps or even all of the method steps of the method according to one or more of the embodiments disclosed herein may be performed by using a computer or computer network. Thus, generally, any of the method steps including provision and / or manipulation of data may be performed by using a computer or computer network. Generally, these method steps may include any of the method steps, typically except for method steps requiring manual work, such as providing the samples and / or certain aspects of performing the actual measurements.

[0069] Specifically, the present invention further discloses:

[0070] A computer or computer network comprising at least one processor, wherein the processor is adapted to perform the method according to one of the embodiments described in this description, a computer loadable data structure that is adapted to perform the method according to one of the embodiments described in this description while the data structure is being executed on a computer, a computer program, wherein the computer program is adapted to perform the method according to one of the embodiments described in this description while the program is being executed on a computer, a computer program comprising program means for performing the method according to one of the embodiments described in this description while the computer program is being executed on a computer or on a computer network, a computer program comprising program means according to the preceding embodiment, wherein the program means are stored on a storage medium readable to a computer, a storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to perform the method according to one of the embodiments described in this description after having been loaded into a main and / or working storage of a computer or of a computer network, and a computer program product having program code means, wherein the program code means can be stored or are stored on a storage medium, for performing the method according to one of the embodiments described in this description, if the program code means are executed on a computer or on a computer network.

[0071] In an embodiment, the system comprises an automated imaging device. Said imaging device may comprise the detection device and the analysis device, both as specified herein above. The imaging device in an embodiment automatically provides a determination of a particulate compound. In such case, step (d4) may comprise obtaining a first digital image of the first working area and a second image of the second working area, wherein in an embodiment the first image corresponds to light transmitted or reflected by particulate compounds in the first working area; and the second image corresponds to light transmitted or reflected by particulate compound in the second working area. In a further embodiment, step (d4) is followed by automatically analyzing the first and / or the second digital image with an automated imaging system to automatically provide a determination of the particulate compound.

[0072] The system may optionally comprise (v) a substrate mover, i.e. a device or unit moving the substrate and the substrate mount relative to the applicator tip. In such case, step (d) may comprise using the substrate mover to move the substrate to the optical device for detection; and / or step (d) may comprise using the substrate mover to position the substrate relative to a light source in the optical device, activating the light source to generate illumination light and illuminating the first and / or the second working area.

[0073] The system may also optionally comprise further components, such as a fixing device configured to fix particulate compounds, e.g. by application of methanol; and / or a staining device configured to stain a first working area and, optionally a second working area; and / or a washing device configured to wash the first working area, e.g. with a wash solution. Corresponding devices are known in the art, e.g. from WO 2012 / 064873 Al.

[0074] The present invention also relates to a method for diagnosing an infection with a parasite comprising at least one blood stage, comprising

[0075] (A) contacting at least a part of a working area of a substrate produced or producible according to the method described herein with a detection agent for said parasite;

[0076] (B) determining at least one particulate compound being a parasite in the working area; and, thereby

[0077] (C) diagnosing an infection with a parasite comprising at least one blood stage.

[0078] The diagnosis method of the present invention is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to, e.g., providing or having provided a substrate produced or producible according to the method described herein for step (A), or performing additional diagnostic steps before or after step (C). Moreover, one or more of said steps may be assisted or performed by automated equipment, in an embodiment as described herein above.

[0079] The term “diagnosing”, as used herein, refers to assessing the probability according to which a subject is suffering from a disease or condition or is at risk of developing a disease or condition. Accordingly, the method may provide an aid for diagnosis, since it may be necessary to further strengthen or confirm said diagnosis by, e.g., a medical practitioner. In particular, as will be understood by those skilled in the art, such an assessment, although preferred to be, may be not correct for 100% of the subjects to be diagnosed. The term, in an embodiment, requires that a statistically significant portion of subjects can be identified as suffering from the disease. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values are, in an embodiment, 0.1, 0.05, 0.01, or 0.005. It will be understood, moreover, that the methods of the present invention essentially provide an aid for diagnosis and may be included into or supplemented by other diagnostic measures. Diagnosing according to the present invention includes monitoring, confirmation, and classification of the relevant disease or its symptoms. Monitoring relates to keeping track of an already diagnosed disease, or a complication, e.g. to analyze the progression or regression of the disease, the influence of a particular treatment on the progression of disease or complications arising during the disease period or after successful treatment of the disease. Confirmation relates to the strengthening or substantiating a diagnosis already established using other indicators or markers. Classification relates to allocating the diagnosis according to the strength or kind of symptoms into different classes, e.g. the severity of malaria.

[0080] The term "infection with a parasite comprising at least one blood stage" is understood by the skilled person; corresponding diseases are known in the art. In an embodiment, included are any and all diseases in which a parasite is detectable or may be detectable in the blood of a subject, wherein the term "parasite" is used in a broad sense to relate to any organism that lives and feeds on or in an organism of a different species and causes harm to its host. Thus, the parasite may be a bacterial or a eukaryotic organism, which may be unicellular or multicellular. Thus, the parasite may in particular be a unicellular parasite, such as a Plasmodium, a Trypanosoma, a Babesia, a Leishmania or a multicellular parasite, such as a nematode, e.g. from the genus Wuchereria or Brugia, or a Trematoda, e.g. from the genus Schistosoma. However, also other parasites comprising at least one blood stage are known to the skilled person, e.g. from medical textbooks. Thus, the disease to be diagnosed may in particular be malaria, African sleeping disease, Chagas disease, Babesiosis, Leishmaniosis, a Filariosis, e.g. Elephantiasis tropica, and Schistosomiasis. As the skilled person understands, there is no range of normal value for any of the aforesaid parasites, i.e. detecting one of said parasites in a sample typically is diagnostic of an infection with said parasite. Whether symptoms of disease occur and their severity may depend on a variety of factors, such a as constitution, gender and age of the infected subject, time of being infected, therapy applied in the past, and other factors known to the skilled person. Summarizing the findings of the present invention, the following embodiments are particularly envisaged:

[0081] Embodiment 1 : An automated method for determining a particulate compound in a liquid sample, said method comprising

[0082] (a) dispensing a thin blot aliquot of said sample on a first working area of a substrate; and / or

[0083] (b) dispensing an overprint aliquot of said sample on a subarea of said first working area; and / or

[0084] (c) distributing a thick blot aliquot of said sample on a subarea of said first working area and / or on a second working area of the substrate; and

[0085] (d) determining the particulate compound in said first working area, in said subarea of said first working area, and / or in said second working area, wherein said dispensing comprises translating an applicator tip in relation to the substrate while dispensing the liquid sample through the applicator tip onto the substrate.

[0086] Embodiment 2: The method of embodiment 1, wherein said liquid sample is a biological sample.

[0087] Embodiment 3 : The method of embodiment 1 or 2, wherein said sample is a blood sample or a blood-derived sample.

[0088] Embodiment 4: The method of any one of embodiments 1 to 3, wherein said sample is a whole blood sample.

[0089] Embodiment 5: The method of any one of embodiments 1 to 4, wherein red blood cells in said sample are hemolyzed at least partially.

[0090] Embodiment 6: The method of any one of embodiments 1 to 5, wherein said particulate compound is a rare particulate compound.

[0091] Embodiment ?: The method of any one of embodiments 3 to 6, wherein said rare particulate compound is a particulate compound present in the sample at a frequency of at most 1 per 103blood cells, in an embodiment at most 1 per 104blood cells, in a further embodiment of at most 1 per 105blood cells, in a further embodiment of at most 1 per 106blood cells.

[0092] Embodiment 8: The method of any one of embodiments 1 to 7, wherein said particulate compound is a human parasite.

[0093] Embodiment 9: The method of any one of embodiments 1 to 8, wherein said particulate compound is a parasite comprising at least one blood stage. Embodiment 10: The method of any one of embodiments 1 to 9, wherein said particulate compound is a parasite from the genus Plasmodium, Wuchereria or Brugia, Trypanosoma, Babesia, Leishmania, or Schistosoma, in an embodiment of the genus Plasmodium.

[0094] Embodiment 11 : The method of any one of embodiments 1 to 10, wherein in step (a) the thin blot aliquot has a first pre-defined volume.

[0095] Embodiment 12: The method of any one of embodiments 1 to 11, comprising performing step (a) and optionally comprising performing step (b) at least once, in an embodiment at least twice.

[0096] Embodiment 13: The method of any one of embodiments 1 to 12, wherein step (b) is performed at least three times.

[0097] Embodiment 14: The method of any one of embodiments 1 to 13, wherein step (b) is performed at most ten times.

[0098] Embodiment 15: The method of any one of embodiments 1 to 14, wherein the sample on the first working area is dried after each performing of step (b).

[0099] Embodiment 16: The method of any one of embodiments 1 to 15, wherein in step (b) the overprint aliquot has a pre-defined second volume.

[0100] Embodiment 17: The method of embodiment 16, wherein said first volume and said second volume are identical.

[0101] Embodiment 18: The method of any one of embodiments 1 to 17, wherein in step (c) the thick blot aliquot has a pre-defined third volume.

[0102] Embodiment 19: The method of embodiment 18, wherein the third volume is larger than the first volume and the second volume.

[0103] Embodiment 20: The method of any one of embodiments 1 to 19, comprising performing at least steps (a) and (c), wherein the volume of the thick blot aliquot is at least five times, in an embodiment at least six times, in a further embodiment at least seven times, in a further embodiment at least eight times, in a further embodiment at least nine times, in a further embodiment at least ten times, the volume of the thin blot aliquot.

[0104] Embodiment 21 : The method of embodiment 20, wherein the subarea of the first working area and / or the area of the second working area onto which the thick blot aliquot is distributed in step (c) is at most one fifth, in an embodiment at most a sixth, in a further embodiment at most a seventh, in a further embodiment at most an eighth, in a further embodiment at most a ninth, in a further embodiment at most one tenth, of the area of the first working area.

[0105] Embodiment 22: The method of any one of embodiments 1 to 21, wherein said step (d) comprises step (dl) drying said working area or working areas. Embodiment 23 : The method of any one of embodiments 1 to 22, wherein said step (d) comprises step (d2) contacting at least fraction of the working area or working areas with a detection agent, in an embodiment wherein contacting with a detection agent is staining.

[0106] Embodiment 24: The method of embodiment 23, wherein said staining comprises contacting at least a part of the first working area and / or of the second working area with a detection agent for said particulate compound.

[0107] Embodiment 25: The method of any one of embodiments 1 to 24, wherein said detection agent is a dye or a dye-coupled binding agent.

[0108] Embodiment 26: The method of any one of embodiments 1 to 25, wherein the determining in step (d) comprises a staining selected from the list consisting of a Romanowsky-type stain, a Giemsa-type stain, a Wright-Giemsa-type stain, a Jenner-type stain, a Wright-type stain, a Field-type stain, a May-Grunwald-type stain, and a Leishman-type stain.

[0109] Embodiment 27: The method of embodiment 25 or 26, wherein said binding agent is an antibody specifically recognizing said particulate agent.

[0110] Embodiment 28: The method of any one of embodiments 23 to 27, wherein said step (d) comprises step (d3) removing unbound detection agent from said at least part of the first working area and / or of the second working area.

[0111] Embodiment 29: The method of any one of embodiments 1 to 28, wherein said step (d) comprises step (d4) capturing at least one digital image of the first working area and / or of the second working area, or parts thereof.

[0112] Embodiment 30: The method of embodiment 29, wherein step (d) further comprises step

[0113] (d5) automatically analyzing the at least one digital image with an automated imaging system to automatically provide a determination of the particulate compound.

[0114] Embodiment 31 : The method of embodiment 29 or 30, wherein said step (d) comprises step (d6) refining said at least one image by sharpening or compensating for spatial shifts or other distortions.

[0115] Embodiment 32: The method of any one of embodiments 1 to 31, wherein said method comprises step (d7) determining spatial, densitometric, colorimetric, and / or texture features within said at least part of the first working area and / or of the second working area.

[0116] Embodiment 33: The method of any one of embodiments 1 to 32, wherein step (c) comprises determining said particulate compound via microscopy.

[0117] Embodiment 34: The method of any one of embodiments 1 to 33, wherein said determining the particulate compound comprises determining the number of particulate compounds per volume of sample or, optionally in case the sample is a blood or blood-derived sample, the number of particulate compounds per number of red blood cells.

[0118] Embodiment 35: The method of any one of embodiments 1 to 34, wherein said method is a fully automated method.

[0119] Embodiment 36: A system comprising

[0120] (i) a liquid handling device comprising an applicator tip for dispensing a fluid sample onto a working area of a substrate;

[0121] (ii) a microprocessor; and

[0122] (iii) a storage device comprising stored software instructions for controlling the system, wherein the software instructions, when executed on the microprocessor, cause the system to perform the method according to any one of embodiments 1 to 35.

[0123] Embodiment 37: The system of embodiment 36, wherein the software instructions cause the system to fill the applicator tip with an aliquot of the sample.

[0124] Embodiment 38: The system of embodiment 36 or 37, wherein the software instructions cause the system to position the applicator tip less than about 110 microns above the substrate. Embodiment 39: The system of any one of embodiments 36 to 38, wherein the software instructions cause the system to dispense a known volume of sample out of the applicator tip.

[0125] Embodiment 40: The system of any one of embodiments 36 to 39, wherein the software instructions cause the system to maintain relative movement between the applicator tip and the substrate at a speed of about 10 to 100 mm per second while the dispensation of the fluid onto the substrate is occurring.

[0126] Embodiment 41 : The system of any one of embodiments 36 to 40, wherein in step (c) the entire volume of sample present in the applicator tip is distributed.

[0127] Embodiment 42: The system of any one of embodiments 36 to 41, wherein the system further comprises an optical device comprising a light source and a light detector.

[0128] Embodiment 43 : The system of any one of embodiments 36 to 42, wherein the optical system further comprises a microscope unit intervening the light source and the light detector. Embodiment 44: The system of embodiment 42 or 43, wherein the software instructions cause the system to capture an image of at least a section of the first working area and / or the second working area.

[0129] Embodiment 45: The system of any one of embodiments 36 to 44, wherein the system further comprises an analysis device. Embodiment 46: The system of any one of embodiments 36 to 45, wherein the software instructions cause the analyzing device to analyze the image to determine the particulate compound.

[0130] Embodiment 47: A method for diagnosing an infection with a parasite comprising at least one blood stage, comprising

[0131] (A) contacting at least a part of a working area of a substrate produced or producible according to the method according to embodiments 1 to 35 with a detection agent for said parasite;

[0132] (B) determining at least one particulate compound being a parasite in the working area; and, thereby

[0133] (C) diagnosing an infection with a parasite comprising at least one blood stage.

[0134] Embodiment 48: The method of embodiment 54, wherein said method is a method of treating an infection with a parasite comprising at least one blood stage, and further comprises step (D) treating said infection.

[0135] Embodiment 49: Use of the substrate according to embodiment 47 or 48 for the manufacture of a diagnostic, in an embodiment for diagnosing an infection with a parasite comprising at least one blood stage.

[0136] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0137] Figure Legends

[0138] Fig. 1 : Exemplary analytic slides 100 prepared according to the method of the invention; (A) rectangular first working area 120, of which a subarea may be overprinted (not shown); additional subarea with a needle dump 130 is shown in the upper left comer of the first working area 120; (B) as in (A), but circular dispensation of the first working area 120; dispensation was started at the periphery of the first working area and was performed in a spiraling manner with decreasing diameter, so the final subarea with a needle dump 130 is at the center; (C) as in (B), but dispensation in a spiraling manner starting at the center and with increasing diameter, so the final subarea with a needle dump 130 is at the periphery. Fig. 2: Comparison of the thick drop reference method with a thick blot according to step (c) of the method described herein. Slides were stained with Romanowsky stain and photographed at lOOx magnification (oil immersion); (A) reference thick drop created manually, (B) thick blot according to step (c).

[0139] Fig. 3: Results of overprinting; slides were stained with Romanowsky stain and photographed at lOOx magnification (oil immersion); (A) lx print, (B) overprint 2x, (C) overprint 4x, (D) overprint 8x.

[0140] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0141] Example 1 : Samples

[0142] In all Examples, whole blood samples known to comprise malaria parasites were used.

[0143] Example 2: Automated slide preparation

[0144] As shown in Fig. 1, analytic slides 100 comprising blood samples can be prepared according to the automated method of the present invention, e.g. by applying a first working area 120 to a support 110 according to step (a) of the method described herein. The first working area 120 may be overprinted completely or partially, e.g. according to step (b) of the method without negative effect on the results (cf. below). An area of high sample density may e.g. be created as a subarea comprising thick blot, e.g. a needle drop 130, e.g. according to step (c) of the method described herein. The method of printing the sample provides for full flexibility as to the form of the first working area 120; also, the amount of sample per area can be exactly defined.

[0145] Example 3: Comparison of the thick drop reference method with a thick blot according to step (c)

[0146] As shown in Fig. 2, the thick drop reference method and the needle dump according to step (c) of the method described herein provide for similar results with regards to stainability and detectability of parasites, in particular malaria ringforms. Example 4: Overprinting

[0147] As shown in Fig. 3, overprinting according to step (b) of the method described herein is well possible. Increasing overprinting (Fig. 3(A)-(D)) leads to an increase of detectable parasites without negative influence on the staining results or sample stability. Thus, a variety of defined amounts of sample can be applied in a multitude of subareas of the first working area 120, increasing the probability that a subarea with an optimal density is comprised on the analytic slide 100.

[0148] Reference signs:

[0149] 100 analytic slide

[0150] 110 support

[0151] 120 first working area

[0152] 130 sub area with needl e dump

Claims

Claims1. An automated method for determining a particulate compound in a liquid sample, said method comprising(a) dispensing a thin blot aliquot of said sample on a first working area of a substrate; and / or(b) dispensing an overprint aliquot of said sample on a subarea of said first working area; and / or(c) distributing a thick blot aliquot of said sample on a subarea of said first working area and / or on a second working area of the substrate; and(d) determining the particulate compound in said first working area, in said subarea of said first working area, and / or in said second working area, wherein said dispensing comprises translating an applicator tip in relation to the substrate while dispensing the liquid sample through the applicator tip onto the substrate.

2. The method of claim 1, wherein said sample is a blood sample or a blood-derived sample.

3. The method of claim 1 or 2, wherein said particulate compound is a parasite comprising at least one blood stage.

4. The method of any one of claims 1 to 3, wherein said particulate compound is a parasite from the genus Plasmodium, Wuchereria or Brugia, Trypanosoma, Babesia, Leishmania, or Schistosoma, in an embodiment of the genus Plasmodium.

5. The method of any one of claims 1 to 4, comprising performing step (a) and comprising performing step (b) at least once.

6. The method of any one of claims 1 to 5, wherein in step (b) the overprint aliquot has a pre-defined second volume.

7. The method of any one of claims 1 to 6, wherein the volume of the overprint aliquot is smaller than the volume of the thin blot aliquot and wherein the subarea of said firstworking area is smaller than the working area.

8. The method of any one of claims 1 to 7, wherein step (b) is performed at least three times to create an array of subareas with each subarea having a distinct number of overprints.

9. The method of any one of claims 1 to 4, comprising performing at least steps (a) and (c), wherein the volume of the thick blot aliquot is at least five times, in an embodiment at least six times, in a further embodiment at least seven times, in a further embodiment at least eight times, in a further embodiment at least nine times, in a further embodiment at least ten times, the volume of the thin blot aliquot.

10. The method of claim 9, wherein the subarea of the first working area and / or the area of the second working area onto which the thick blot aliquot is distributed in step (c) is at most one fifth, in an embodiment at most a sixth, in a further embodiment at most a seventh, in a further embodiment at most an eighth, in a further embodiment at most a ninth, in a further embodiment at most one tenth, of the first working area.

11. The method of any one of claims 1 to 10, comprising performing steps (a) to (c), wherein step (b) is performed at least once.

12. The method of any one of claims 1 to 11, wherein said step (d) comprises step(dl) drying said working area or working areas;(d2) contacting at least fraction of the working area or working areas with a detection agent, in an embodiment wherein contacting with a detection agent is staining selected from the list consisting of a Romanowsky-type stain, a Giemsa-type stain, a Wright- Giemsa-type stain, a Jenner-type stain, a Wright-type stain, a Field-type stain, a May- Griinwald-type stain, and a Leishman-type stain;(d3) removing unbound detection agent from said at least part of the first working area and / or of the second working area;(d4) capturing at least one digital image of the first working area and / or of the second working area, or parts thereof;(d5) automatically analyzing the at least one digital image with an automated imaging system to automatically provide a determination of the particulate compound;(d6) refining said at least one image by sharpening or compensating for spatial shifts or other distortions;(d7) determining spatial, densitometric, colorimetric, and / or texture features within said at least part of the first working area and / or of the second working area; or (d8) any combination of (dl) to (d7).

13. The method of any one of claims 1 to 12, wherein said determining the particulate compound comprises determining the number of particulate compounds per volume of sample or, optionally in case the sample is a blood or blood-derived sample, the number of particulate compounds per number of red blood cells.

14. The method of any one of claims 3 to 9, wherein the method further comprises(A) contacting at least a part of said first and / or second working area with a detection agent for said parasite;(B) determining at least one particulate compound being a parasite in the working area; and, thereby(C) diagnosing an infection with a parasite comprising at least one blood stage.

15. A system compri sing(i) a liquid handling device comprising an applicator tip for dispensing a fluid sample onto a working area of a substrate;(ii) a microprocessor; and(iii) a storage device comprising stored software instructions for controlling the system, wherein the software instructions, when executed on the microprocessor, cause the system to perform the method according to any one of claims 1 to 14.

16. The system of claim 15, wherein the software instructions cause the system to maintain relative movement between the applicator tip and the substrate at a speed of about 10 to 100 mm per second while the dispensation of the fluid onto the substrate is occurring.

17. The system of claim 15 or 16, wherein the system further comprises an optical device comprising a light source and a light detector, wherein the optical system optionally further comprises a microscope unit intervening the light source and the light detector.

18. The system of claim 17, wherein the software instructions cause the system to capture an image of at least a section of the first working area and / or the second working area.

19. The system of any one of claims 15 to 18, wherein the system further comprises an analysis device, wherein the software instructions cause the analyzing device to analyze the image to determine the particulate compound.

20. A method for diagnosing an infection with a parasite comprising at least one blood stage, comprising (A) contacting at least a part of a working area of a substrate produced or producible according to the method according to claims 1 to 14 with a detection agent for said parasite;(B) determining at least one particulate compound being a parasite in the working area; and, thereby (C) diagnosing an infection with a parasite comprising at least one blood stage.

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