Hemoglobin determination methods and systems

Abstract

The present invention relates to a method for determining hemoglobin in a sample comprising red blood cells, said method comprising (a) dispensing a first aliquot of said sample on a first working area of a substrate; (b) providing a hemolyzed second aliquot of said sample and dispensing said second aliquot on a second working area; and (c) determining the hemoglobin in said first and second working area, wherein said method comprises a further step of contacting said first working area, but not the second working area, with a treatment solution. The present invention also relates to systems and diagnostic methods related to said method.

Description

[0001] Hemoglobin determination methods and systems

[0002] The present invention relates to a method for determining hemoglobin in a sample comprising red blood cells, said method comprising (a) dispensing a first aliquot of said sample on a first working area of a substrate; (b) providing a hemolyzed second aliquot of said sample and dispensing said second aliquot on a second working area; and (c) determining the hemoglobin in said first and second working area, wherein said method comprises a further step of contacting said first working area, but not the second working area, with a treatment solution. The present invention also relates to systems and diagnostic methods related to said method.

[0003] Hemoglobin (HB) is the iron-containing oxygen transport protein in red blood cells of almost all vertebrate animals. Thus, HB in healthy subjects is mostly cell bound in red blood cells. However, in particular in physiological states causing increased lysis of red blood cells, HB may also be found free in blood and blood-derived samples. Under physiological conditions, however, about 80-90% of erythrocytes are destroyed without releasing hemoglobin into plasma.

[0004] In accordance, HB, mostly measured as cell-bound HB, free HB, and / or as total HB, has been used as a diagnostic marker for a long time. Typically, total HB in a blood sample is measured after lysing red blood cells; free HB may be measured after removing red blood cells, e.g. by centrifugation. Cellular HB can be determined as the difference between total and free HB, or from red blood cells obtained from a sample. Thus, determining a HB parameter other than total HB in any case requires separation of red blood cells from plasma. Manual methods for the aforesaid analyses have been known, e.g. as the CyanmetHB method, Drabkin's or Sahli's method, or as the alkaline hematin method, reviewed e.g. in Karakochuk et al. (2019) Ann NY Acad Sci 1450: 126. A set of other parameters has been introduced over the years as surrogate markers of HB, as have been automated and point of care methods and devices for HB determination (cf. e.g. Karakochuk et al. (2019), ibd.).

[0005] In parallel, methods and devices for analyzing cells in samples, such as blood cells in blood samples, have been described, such as flow cytometry or solid surface based systems, as described e.g. in WO 2010 / 126903 Al, US 9,017,610 B2, US 8,815,537 B2, and WO 2013 / 016037 Al. Such systems make possible, among others, to determined cell-bound HB, however, free HB or total HB cannot be determined, since the measurement in these systems is cell-based.

[0006] There is, thus, a need for improved methods for determining hemoglobin in samples, avoiding the drawbacks of the prior art. This problem is solved by the means and methods disclosed herein.

[0007] In accordance, the present invention relates to a method for determining hemoglobin in a sample comprising red blood cells, said method comprising

[0008] (a) dispensing a first aliquot of said sample on a first working area of a substrate;

[0009] (b) providing a hemolyzed second aliquot of said sample and dispensing said second aliquot on a second working area; and

[0010] (c) determining the hemoglobin in said first and second working area, wherein said method comprises a further step of contacting said first working area, but not the second working area, with a treatment solution.

[0011] 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. 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] 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).

[0013] The methods specified herein below are in vitro methods. 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 the indicated sequence; also, one or more, in an embodiment all, of said steps may be assisted or performed by automated equipment. Thus, the methods may be automated methods, in an embodiment fully automated methods. Moreover, the methods described comprise the steps as specified; the methods may, however, comprise steps in addition to those explicitly specified. Exemplary further steps are specified herein elsewhere.

[0014] The term “determining” as used herein refers to semi quantitative or quantitative determination of hemoglobin. Determining the amount of hemoglobin may be carried out by any technique which allows for establishing a measure of quantity of hemoglobin in a semi quantitative or quantitative manner. Suitable techniques, e.g. staining techniques and optical determination methods are, in principle, known in the art. Thus, determining includes all methods of determining at least one detectable feature of hemoglobin. The detectable feature may be any feature deemed detectable 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. 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 cosubstrate and / or a catalyst. Structural features are all detectable features conferred by the chemical structure of hemoglobin, 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 a hemoglobin in an embodiment also includes detecting the absence of said feature.

[0015] In an embodiment, determining hemoglobin is establishing whether hemoglobin 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 hemoglobin 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 hemoglobin will be determined or the relative amount of hemoglobin will be determined. The relative amount may be determined in a case were the precise amount of hemoglobin can or shall not be determined. In said case, it can be determined whether the amount in which hemoglobin is present is increased or diminished with respect to a reference sample or a plurality of reference samples comprising hemoglobin in a pre-determined amount or in pre-determined amounts. For quantitative determination, any parameter correlating with the amount or concentration of hemoglobin 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.

[0016] In an embodiment, hemoglobin is determined by a physical method, in a further embodiment by any optical method deemed appropriate by the skilled person. Thus, hemoglobin 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 dispensed thereon. In a further embodiment, the hemoglobin is determined by a method comprising illuminating at least a part of the sample dispensed 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 described herein elsewhere in more detail.

[0017] In an embodiment, determining hemoglobin is determining free hemoglobin, total hemoglobin and / or cellular hemoglobin, terms well-known to the skilled person. In an embodiment, total hemoglobin is the entirety of hemoglobin in a sample, irrespective of its localization within the sample; cellular hemoglobin is the fraction of hemoglobin which is bound to cells, in particular red blood cells (erythrocytes), and which can in principle be removed from a sample by removing cells; and / or free hemoglobin is the fraction of hemoglobin in a sample which is not cellular hemoglobin. Free hemoglobin typically is determined indirectly as the difference between total hemoglobin and cellular hemoglobin in a sample. Thus, determining hemoglobin may comprise determining free hemoglobin, wherein the value of said free hemoglobin in an embodiment is determined as the difference between a value of total hemoglobin determined in the second working area and a value of cellular hemoglobin determined in the first working area. Values determined for hemoglobin in a sample are typically expressed as concentrations, e.g. with a mass per volume unit, such as mg / dL, but also amount of substance per volume, %of a pre-defined reference, and the like may be used. Values for cellular hemoglobin may also be expressed as hemoglobin per red blood cell, e.g. in pg; said value may also be referred to as "mean corpuscular hemoglobin" or "mean cell hemoglobin", both abbreviated as "MCH".

[0018] The term "hemoglobin" is known to the skilled person to relate to the iron-containing oxygentransport protein present in red blood cells (erythrocytes) of almost all vertebrates. Similarly, the terms "erythrocytes" and "red blood cells", which may also be abbreviated as "RBCs", are used essentially interchangeably herein to refer to the type of blood cells carrying hemoglobin. As the skilled person understands, the hemoglobin subtype in an embodiment corresponds to sample type; thus, in case the sample is a sample from a human, the hemoglobin in an embodiment is human hemoglobin.

[0019] The term "hemolysis" is known to the skilled person as well. In an embodiment, the term relates to lysis of red blood cells in a sample, in an embodiment to lysis of at least 80% of RBCs, in a further embodiment at least 90% of RBCs, in a further embodiment at least 95% of RBCs, in a further embodiment at least 98% of RBCs, in a further embodiment at least 99% of RBCs, in a sample. Methods and conditions of inducing hemolysis are known in the art and include in particular incubation of RBCs in a hypotonic medium, ammonium chloride treatment, application of shearing forces, or the like. In view of the description herein above, the skilled person knows how to provide a hemolyzed aliquot of a sample, such as a hemolyzed second aliquot. As is known to the skilled person, induction of hemolysis and a detection reaction may be elicited by the same reagent; e.g. in the sodium lauryl sulfate (SLS) method, sodium lauryl sulfate causes lysis of blood cells, as well as an oxidation of heme to create a stable, colored SLS-hemoglobin complex, which can be detected e.g. photometrically.

[0020] The term “sample”, as used herein, relates to a sample known or suspected to comprise hemoglobin and optionally erythrocytes. In an embodiment, the sample is or comprises a sample of a body fluid, a sample from a tissue or an organ, or a sample of wash / rinse fluid or a swab or smear obtained from an outer or inner body surface. Body fluid samples include samples of blood, plasma, serum, urine, saliva, and lacrimal fluid. Samples can be obtained by use of brushes, (cotton) swabs, spatula, rinse / wash fluids, punch biopsy devices, puncture of cavities with needles or lancets, or by surgical instrumentation. However, samples obtained by well-known techniques including, in an embodiment, scrapes, swabs or biopsies from the urogenital tract, perianal regions, anal canal, the oral cavity, the upper aerodigestive tract and the epidermis are also included as samples. Cell-free fluids may be obtained from the body fluids or the tissues or organs by lysing techniques such as homogenization and / or by separating techniques such as filtration or centrifugation. It is to be understood that the sample may be further processed in order to carry out the method. Particularly, cells may be removed from the sample by methods and means known in the art, calcium ions may be complexed to prevent coagulation, coagulation may be induced, hemolysis may be induced in an aliquot of the sample, and the like. Moreover, at least one analyte such as hemoglobin may be enriched, extracted, and / or purified from the sample by methods and means known in the art. Thus, the term sample also may relate to preparations comprising or suspected to comprise hemoglobin which are diluted, enriched, purified and / or extracted from a sample. In an embodiment, the sample is a blood sample or a blood-derived sample, in a further embodiment the sample is a whole blood sample.

[0021] The term "aliquot" is used herein in its common meaning relating to a subpart or subportion of an entity; thus, an aliquot of a sample in an embodiment is a subportion of a sample. As the skilled person is aware of, the composition of all aliquots of a sample, at the time of aliquoting, is essentially the same, while subportions of a sample differing in composition generally are referred to as "fractions". In an embodiment, at least a first and a second aliquot of a sample are used in the methods described herein. Said first and second aliquots may have the same, but may also have different volumes, so, in an embodiment, the first aliquot has a first pre-defined volume, and / or the second aliquot as a second pre-determined volume. In a further embodiment, said first volume and said second volume are pre-determined such that the sample volume / area ratios in the first and the second working area are essentially identical.

[0022] The term "dispensing", in an embodiment, includes each and every proceeding causing a sample or an aliquot thereof to become distributed over at least one working area of a substrate. Said dispensing in an embodiment comprises causing a monolayer of red blood cells to form on the substrate. As will be described in more detail herein below, dispensing may in particular comprise translating an applicator in relation to the substrate while dispensing the sample through the applicator onto the substrate.

[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, 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 sample. Also in an embodiment, the substrate has binding capacity for biological material such as cells, in particular erythrocytes. 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 (b) may be providing a hemolyzed second aliquot of said sample and dispensing said second aliquot on a second working area of said support. In such case, the first working area and the second working area are spatially separated on said substrate, in an embodiment wherein said spatial separation enables contacting the first working area with a treatment solution while not contacting said second working area with said treatment solution. The product of step (b) of the method, i.e. a solid support comprising a first and a second working area as specified herein, may also be referred to as "analytic slide".

[0025] The term “contacting”, as used in the context of the methods descried herein, is understood by the skilled person. In an embodiment, the term relates to bringing a compound, such as a staining agent or a wash solution, in physical contact with at least one second compound, such as an aliquot of a sample, thereby allowing the compounds contacted to interact.

[0026] The term "treatment solution", as used herein, includes any and all solutions contacted with at least one aliquot of a sample on a first working area. The treatment solution may thus be a fixing solution, a wash solution, and / or a detection solution, in an embodiment is a wash solution and / or a detection solution. In a further embodiment, the treatment solution is a solution contacted with a first working area and removed therefrom before step (c). The treatment solution may have any composition deemed appropriate by the skilled person. In an embodiment, the treatment solution is an aqueous solution, and may comprise additional components selected by the skilled person depending on the required properties of the treatment solution; thus, the treatment solution may comprise one or more of a buffer, salts, one or more fixing agent(s), such as methanol, and the like. In an embodiment, the treatment solution is a wash solution. Appropriate wash solutions are known in the art, and include in particular solutions comprising one or more buffer compound(s) and / or one or more salts, such as phosphate buffered saline, physiological saline solution, ringer solution, and the like. The wash solution may comprise further compounds, such as detergents, organic solvents, and the like. In an embodiment, the treatment solution is a detection solution. Appropriate detection solutions are prepared by the skilled person depending on the staining intended; thus, the detection solution in an embodiment comprises at least one detection reagent as specified herein elsewhere. The treatment solution, as referred to herein, is contacted with a sample on the first working area, and is removed after said contacting, at least before step (c). As the skilled person understands in view of the description herein, contacting the hemolyzed sample in the second working area with a treatment solution and removing said treatment solution before step (c) could wash away at least parts of the free hemoglobin in the second working area. Thus, the second working area is not contacted with a treatment solution. The second working area may, however, be contacted with a detection solution which is not removed after contacting; thus, a detection solution may be a treatment solution as referred to herein, i.e. in case it is removed from the working area before step (c), but a detection solution may also be not a treatment solution as referred to herein, i.e. in case it is not removed from the working area before step (c). As is understood by the skilled person, the expression "removing a solution" from a working area relates to removing removable fractions of the solution, i.e. the solution, e.g. the detection solution, in an embodiment is not removed completely, e.g. in case detection reagent comprised in the detection solution remains bound to the working area.

[0027] The term "detection reagent", as is understood by the skilled person, relates to any chemical compound or mixture of compounds enabling or improving detection of an analyte. On an exemplary basis, the detection reagent may be e.g. Romanowsky stain, Giemsa stain, Wright’s stain, Leishman stain, Jenner’s stain, May-Grunwald stain, or Field stain, all of which are known to the skilled person, including their constituents. In the context of hemoglobin determination as referred to herein, the detection reagent is a chemical compound or mixture thereof causing hemoglobin to become detectable or improving its detectability. Appropriate detection reagent are known to the skilled person, e.g. from the hemoglobin determination methods from the prior art. In an embodiment, the detection reagent comprises methylene blue (CAS No. 61-73-4) and Eosine Y (CAS No. 17372-87-1). However, other detection reagents may be used, such as azure A (CAS No. 531-53-3), Azure B (CAS No. 531-55-5), or Azure C (CAS No. 531-57-7), and / or Eosine B (CAS 548-24-3).

[0028] The method for determining hemoglobin comprises steps (a) dispensing a first aliquot of said sample on a first working area of a substrate; and (b) providing a hemolyzed second aliquot of said sample and dispensing said second aliquot on a second working area. Methods for dispensing an aliquot of a sample have been described herein above, as have methods of providing a hemolyzed aliquot of a sample. As detailed herein above, in step (a) the first aliquot in an embodiment has a first pre-defined volume; and / or in step (b) the second aliquot in an embodiment as a second pre-determined volume.

[0029] The method for determining hemoglobin further comprises step (c) determining the hemoglobin in said first and second working area. Methods for determining hemoglobin have been described herein above. In an embodiment, the method comprises step (cl) contacting at least a part of the first working area and, optionally of the second working area, with a detection solution, in an embodiment, with a detection solution comprising at least one detection reagent; said step (cl), in an embodiment, precedes step (c). In an embodiment, the method further comprises step (c2) spectrophotometrically determining absorption in at least a part of the first working area and / or in at least a part of the second working area, wherein said step may in particular comprise spectrophotometrically determining absorption of the reaction product from step (cl). In an embodiment, step (c2) comprises obtaining at least one first digital image of the first working area and at least one second image of the second working area, wherein in an embodiment the first image corresponds to light transmitted or reflected by the cells in the first working area in a first wavelength interval; and the second image corresponds to light transmitted or reflected by the hemoglobin in the second working area in the first wavelength interval. In a further embodiment, step (c2) is followed by automatically analyzing the first and / or the second digital image. Also in an embodiment, the aforesaid first wavelength interval is from about 500 nm to 600 nm, in an embodiment from 530 nm to 550 nm.

[0030] The method, in an embodiment, further comprises step (c3) determining total hemoglobin in the second working area and / or cellular hemoglobin in the first working area. Advantageously, it was found in the work underlying the present invention that by using two working areas, e.g. on the same solid support, as described herein, it is possible to determine free hemoglobin, total hemoglobin and / or cellular hemoglobin in a sample in easily, automatable steps.

[0031] 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.

[0032] The present invention also relates to a system comprising

[0033] (i) a liquid handling device comprising an applicator tip for dispensing a fluid sample onto a working area of a substrate;

[0034] (ii) a detection device configured for determining hemoglobin;

[0035] (iii) a microprocessor; and

[0036] (iv) 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 of the present invention.

[0037] 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 hemoglobin, 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 detection device; (iii) a microprocessor; and (iv) 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.

[0038] 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 amount or concentration of hemoglobin 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, such as hemolysis, icterus, and / or lipemia (HIL) detection, or evaluation units / devices, e.g. as referred to above in accordance with the methods of the invention.

[0039] 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 treatment solution and / or a detection 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 fluid samples containing cells 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 comprising the first and the second working area 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.

[0040] 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. In a preferred embodiment, the liquid handling device comprises, e.g. in the applicator tip, a volume of sample, such as of from 0.1 pl to 250 pl, in an embodiment of from 0.1 pl to 100 pl, in a further embodiment about 0.5 pl to 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.

[0041] 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. Thus, the staining station may comprise a staining chamber restricting staining to one of the working areas, in an embodiment the first working area.

[0042] 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 treatment solution, e.g. 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.

[0043] 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 of the other liquid handling means of the 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 0.5 millimeters. In a preferred embodiment, the orifice is contained in a tip that is 0.4 to five millimeters in outside diameter and / or the inner diameter is of from 0.3 to 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 body fluid 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 particular 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. In an embodiment, the system comprises software instructions causing the system to fill the applicator tip with an aliquot of the sample in step (a) and with an aliquot of hemolyzed sample in step (b); or causing the system to fill the applicator tip with an aliquot of the sample and with an aliquot of the hemolyzed sample, in an embodiment separated by an air gap, and dispensing said aliquots in steps step (a) and (b), respectively. The term "detection device", as used herein, generically relates to any device configured to allow the measurements underlying the determination in step (c) 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 the first working area may be different from the detectable feature determined in the second working area; also, more than one detectable feature may be determined during determination. Thus total hemoglobin may be determined in the second working area via absorption measurement, while cellular hemoglobin may be determined in the first working area by a combination of a physical and a structural feature, e.g. absorption within a cell. Appropriate devices for performing the aforesaid measurements are in principle known in the art.

[0044] 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 sample 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 as a detection device, (v) 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.

[0045] In an embodiment, the detection device provides at least a measurement of a first parameter of cellular hemoglobin in first working area, and a measurement of a second parameter correlating with total hemoglobin content in the second working area. The aforesaid first and second parameters may be the same parameter, e.g. a spectrophotometric measurement of an absorption of hemoglobin in an area of interest within the first and / or second working area; such may be the proceeding e.g. in cases where free hemoglobin was removed from the first working area, e.g. by one or more wash steps. The aforesaid first and second parameters may, however, also be the different parameters; e.g. in an embodiment, the first parameter is measured via digital image analysis within the first working area, while the second parameter is measured spectrophotometrically as an absorption of hemoglobin within the second working area. Digital imaging analysis may in particular comprise multispectral imaging, in which a plurality of features in a digital image of at least a subsection of the first working area are determined and an MCH as specified herein above is determined as a weighted sum of the aforesaid plurality of features. In such case, the plurality of features in an embodiment comprises RBC area, absorption of blue light by said RBC, and optionally further parameters such as RBC volume, absorption of red, yellow, and / or green light by said RBC. The optical principles of detecting hemoglobin, be it free hemoglobin, cellular hemoglobin, or total hemoglobin, are known in the art, as are means and methods for putting these principles into practice. From the MCH, in combination with the RBC count, the cellular hemoglobin may be determined. In case free hemoglobin was removed from the first working area, e.g. by washing, also spectrophotometric determination of hemoglobin in both working areas may be performed. In a further embodiment, digital image analysis is performed to determine both total and cellular hemoglobin in the respective working areas.

[0046] 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.

[0047] 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.

[0048] 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 hemoglobin; and / or to determine the hemoglobin from a measurement of the optical device and / or to analyze the image to determine the hemoglobin.

[0049] 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.

[0050] 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. 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Specifically, the present invention further discloses:

[0055] 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.

[0056] 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 value of a free hemoglobin, of a total hemoglobin and / or of a cellular hemoglobin. In such case, step (c2) 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 the cells in the first working area in the first wavelength interval; and the second image corresponds to light transmitted or reflected by the hemoglobin in the second working area in the first wavelength interval. In a further embodiment, step (c2) is followed by automatically analyzing the first and / or the second digital image with an automated imaging system to automatically provide a value of a free hemoglobin, of a total hemoglobin and / or of a cellular hemoglobin.

[0057] The system may optionally comprise (vi) 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 (cl) may comprise using the substrate mover to move the substrate to the optical device after contacting at least part of the first and the second working area with a detection reagent; and / or step (c2) 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 in a first wavelength interval and illuminating the first and the second working area.

[0058] The system may also optionally comprise further components, such as a fixing device configured to fix cells in the first working area, 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.

[0059] The present invention further relates to a method for diagnosing a disease associated with a change in free hemoglobin, total hemoglobin and / or cellular hemoglobin in a subject, said method comprising

[0060] (A) determining free hemoglobin, total hemoglobin and / or cellular hemoglobin in a sample of said subject by the method for determining hemoglobin described herein,

[0061] (B) comparing said free hemoglobin, total hemoglobin and / or cellular hemoglobin determined in step (b) to a reference, and

[0062] (C) thereby diagnosing said disease in said subject.

[0063] 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 or as having a predisposition therefor. 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-te st, 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, 0.2, 0.1, 0.05. 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 anemia. Being at risk as used herein means that a subject has not yet developed the disease or condition but, nevertheless, will develop it in the future with a certain likelihood. Diagnosis of a predisposition may sometimes be referred to as prediction of the likelihood that a subject will develop the disease.

[0064] The term "disease associated with a change in free hemoglobin, total hemoglobin and / or cellular hemoglobin" is understood by the skilled person; corresponding diseases are known in the art. In an embodiment, included are diseases in which free hemoglobin is increased, in particular intravascular hemolysis; hemolytic anemia, e.g. immunohematologic hemolytic anemia, drug- induced hemolytic anemia, anemia in hemolytic-uremic syndrome, or anemia induced by toxic chemicals; blackwater fever; and paroxysmal cold haemoglobinuria. Also included are diseases in which total hemoglobin is increased, in particular exsiccosis, polycythaemia, polyglobulia, lung diseases, heart disease, kidney disease, and dehydration; total hemoglobin may, however, also be increased by adaptation to oxygen deficiency, e.g. in adaptation to high altitudes. Also included are diseases in which total hemoglobin is decreased, in particular acute or chronic anemia, blood loss, cancer, nephropathy, iron deficiency, vitamin B12 deficiency, and hemolytic anemia. Also included are diseases in which cellular hemoglobin is changed, in particular hypochromic anemia, hyperchromic anemia, and thalessemia.

[0065] The terms "increased", "decreased", and "changed" hemoglobin are understood by the skilled person to refer to a comparison of a measured value to a reference, wherein the reference typically is a healthy reference. The term “reference”, as used herein, relates to a value, e.g. an amount or any value derived therefrom, e.g. a score, or to a range of values of hemoglobin used as a basis for comparison to a measured value. Thus, a reference may in particular be an amount or a value representing an amount, i.e. data of characteristic features of an analyte such as hemoglobin, which can be correlated to the presence or absence of a disease or condition. Appropriate reference values or ranges of healthy subjects are known in the art. Such a reference may depend on parameters of the subject to evaluate, e.g. on gender, age, and medical history. Thus, the healthy reference range for adult male subjects is 14 to 18 g / dL, while for adult female subjects it is 12 to 16 g / dL.

[0066] The term “comparing”, as used herein, encompasses comparing the determined amount of hemoglobin as referred to herein to a reference. It is to be understood that comparing as used herein refers to any kind of comparison made between the value for the amount with the reference. However, it is to be understood that, in an embodiment, identical types of values are compared with each other, e.g., if a total hemoglobin is determined, the reference shall also be a total hemoglobin value or range, if a cellular hemoglobin is determined, the reference shall also be a cellular hemoglobin, etc. The term comparing also encompasses comparing a calculated score with a suitable reference score. The comparison may be carried out manually or computer assisted. The value of the amount and the reference can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison. The computer program carrying out the said evaluation will provide the desired assessment in a suitable output format.

[0067] The present invention also relates to a method for treating a disease associated with a change in free hemoglobin, total hemoglobin and / or cellular hemoglobin in a subject, said method comprising

[0068] (I) diagnosing said disease according to the method described herein, and

[0069] (II) treating said disease in case said disease is diagnosed in said subject.

[0070] The term "treating", as used herein, refers to any improvement, cure or amelioration of a disease or condition as referred to herein. It will be understood that treatment may not be successful in 100% of the subjects to which the cancer therapy has been administered. The term, however, requires that the treatment in an embodiment is successful in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, preferably as specified herein above.

[0071] In view of the above, the following embodiments are particularly envisaged: Embodiment 1 : A method for determining hemoglobin in a sample comprising red blood cells, said method comprising

[0072] (a) dispensing a first aliquot of said sample on a first working area of a substrate;

[0073] (b) providing a hemolyzed second aliquot of said sample and dispensing said second aliquot on a second working area; and

[0074] (c) determining the hemoglobin in said first and second working area, wherein said method comprises a further step of contacting said first working area, but not the second working area, with a treatment solution.

[0075] Embodiment 2: The method of embodiment 1, wherein said sample is a blood sample or a blood-derived sample.

[0076] Embodiment 3 : The method of embodiment 1 or 2, wherein said sample is a whole blood sample.

[0077] Embodiment 4: The method of any one of embodiments 1 to 3, wherein said determining hemoglobin is determining free hemoglobin, total hemoglobin and / or cellular hemoglobin.

[0078] Embodiment 5: The method of any one of embodiments 1 to 4, wherein said determining hemoglobin is determining free hemoglobin, wherein the value of said free hemoglobin in an embodiment is determined as the difference between a value of total hemoglobin determined in the second working area and a value of cellular hemoglobin determined in the first working area.

[0079] Embodiment 6: The method of any one of embodiments 1 to 5, wherein said further step of contacting the first working area, but not the second working area, with a treatment solution, precedes step (c).

[0080] Embodiment 7: The method of any one of embodiments 1 to 6, wherein said treatment solution is a solution contacted with a working area and removed therefrom before step (c). Embodiment 8: The method of any one of embodiments 1 to 7, wherein said first working area and said second working area are located on the same substrate.

[0081] Embodiment 9: The method of embodiment 8, wherein the first working area and the second working area are spatially separated on said substrate.

[0082] Embodiment 10: The method of embodiment 9, wherein said spatial separation enables contacting the first working area with a treatment solution while not contacting said second working area with said treatment solution.

[0083] Embodiment 11 : The method of any one of embodiments 1 to 10, wherein said treatment solution is a wash solution and / or a detection solution. Embodiment 12: The method of any one of embodiments 1 to 11, wherein dispensing comprises translating an applicator in relation to the substrate while dispensing the sample through the applicator onto the substrate.

[0084] Embodiment 13: The method of any one of embodiments 1 to 12, wherein dispensing comprises causing a monolayer of red blood cells to form on the substrate.

[0085] Embodiment 14: The method of any one of embodiments 1 to 13, wherein said first working area and said second working area are on two non-identical substrates.

[0086] Embodiment 15: The method of any one of embodiments 1 to 14, wherein in step (a) the first aliquot has a first pre-defined volume.

[0087] Embodiment 16: The method of any one of embodiments 1 to 15, wherein in step (b) the second aliquot as a second pre-determined volume.

[0088] Embodiment 17: The method of any one of embodiments 1 to 16, wherein the first volume and the second volume are pre-determined such that the sample volume / area ratios in the first and the second working area are essentially identical.

[0089] Embodiment 18: The method of any one of embodiments 1 to 17, wherein in step (b) the hemolyzed second aliquot is provided by hemolyzing red blood cells in an aliquot of said sample.

[0090] Embodiment 19: The method of any one of embodiments 1 to 18, wherein step (c) comprises step (cl) contacting at least a part of the first working area and optionally of the second working area with a detection reagent.

[0091] Embodiment 20: The method of embodiment 19, wherein the second working area is not contacted with a detection reagent.

[0092] Embodiment 21 : The method of any one of embodiments 1 to 19, wherein said detection reagent comprises methylene blue (CAS No. 61-73-4) and Eosine Y (CAS No. 17372-87-1).

[0093] Embodiment 22: The method of any one of embodiments 1 to 20, wherein step (c) comprises step (c2) spectrophotometrically determining absorption in at least a part of the first working area and / or in at least a part of the second working area.

[0094] Embodiment 23 : The method of embodiment 22, wherein said step (c2) comprises spectrophotometrically determining absorption of the reaction product from step (cl).

[0095] Embodiment 24: The method of embodiment 23, wherein step (c2) comprises 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 digital image corresponds to light transmitted or reflected by the cells in the first working area, in an embodiment in a first wavelength interval; and the second image corresponds to light transmitted or reflected by the hemoglobin in the second working area, in an embodiment in said first wavelength interval.

[0096] Embodiment 25: The method of any one of embodiments 1 to 24, wherein step (c) comprises step (c3) determining total hemoglobin in the second working area and / or cellular hemoglobin in the first working area.

[0097] Embodiment 26: The method of embodiment 25, further comprising determining free hemoglobin as the difference between total hemoglobin and cellular hemoglobin.

[0098] Embodiment 27: The method of embodiment 25 or 26, wherein said cellular hemoglobin is determined as hemoglobin per red blood cell.

[0099] Embodiment 28: The method of any one of embodiments 1 to 27, wherein said method is an automated method, in an embodiment a fully automated method.

[0100] Embodiment 29: A system comprising

[0101] (i) a liquid handling device comprising an applicator tip for dispensing a fluid sample onto a working area of a substrate;

[0102] (ii) a detection device configured for determining hemoglobin;

[0103] (iii) a microprocessor; and

[0104] (iv) 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 28.

[0105] Embodiment 30: The system of embodiment 29, wherein the software instructions cause the system to fill the applicator tip with an aliquot of the sample for step (a) and with an aliquot of hemolyzed sample for step (b).

[0106] Embodiment 31 : The system of embodiment 29 or 30, wherein the software instructions cause the system to position the applicator tip less than about 110 microns above the substrate. Embodiment 32: The system of any one of embodiments 29 to 31, wherein the software instructions cause the system to dispense a known volume of sample out of the applicator tip.

[0107] Embodiment 33: The system of any one of embodiments 29 to 32, wherein the software instructions cause the system to maintain 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.

[0108] Embodiment 34: The system of any one of embodiments 29 to 33, further comprising (v) an optical device comprising a light source and a light detector.

[0109] Embodiment 35: The system of embodiment 34, wherein the optical device further comprises a microscope device intervening the light source and the light detector. Embodiment 36: The system of embodiment 34 or 35, wherein the software instructions cause the optical device to capture an image of at least a section of the first working area and / or the second working area.

[0110] Embodiment 37: The system of any one of embodiments 29 to 36, further comprising (vi) an analysis device.

[0111] Embodiment 38: The system of embodiment 37, wherein the software instructions cause the analysis device to analyze the image to determine the hemoglobin.

[0112] Embodiment 39: The system of embodiment 37 or 38, wherein the software instructions cause the analysis device to determine the hemoglobin from a measurement of the optical device and / or to analyze the image to determine the hemoglobin.

[0113] Embodiment 40: The system of any one of embodiments 28 to 39, wherein step (c2) is followed by automatically analyzing the first and / or the second digital image.

[0114] Embodiment 41 : The system of any one of embodiments 28 to 40, comprising an automated imaging device to automatically provide a value of a free hemoglobin, of a total hemoglobin and / or of a cellular hemoglobin.

[0115] Embodiment 42: The system of any one of embodiments 28 to 41, wherein the first wavelength interval is from about 500 nm to 600 nm, in an embodiment from 530 nm to 550 nm.

[0116] Embodiment 43 : The system of any one of embodiments 28 to 42, further comprising (vi) a substrate mover.

[0117] Embodiment 44: The system of embodiment 43, wherein step (cl) comprises using the substrate mover to move the substrate to the optical device after contacting at least part of the first and the second working area with a detection reagent.

[0118] Embodiment 45: The system of embodiment 43 or 44, wherein step (c2) comprises 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 in a first wavelength interval and illuminating the first and the second working area.

[0119] Embodiment 46: The system of any one of embodiments 28 to 45, wherein step (c2) is followed by automatically analyzing the first and / or the second digital image with an automated imaging system to automatically provide a value of a free hemoglobin, of a total hemoglobin and / or of a cellular hemoglobin.

[0120] Embodiment 47: The system of any one of embodiments 28 to 46, wherein said liquid handling device comprises a reagent applicator for applying a detection reagent and / or a treatment solution. Embodiment 48: A method for diagnosing a disease associated with a change in free hemoglobin, total hemoglobin and / or cellular hemoglobin in a subject, said method comprising

[0121] (A) determining free hemoglobin, total hemoglobin and / or cellular hemoglobin in a sample of said subject by the method of any one of embodiments 1 to 27 and / or using a system according to any one of embodiments 28 to 47,

[0122] (B) comparing said free hemoglobin, total hemoglobin and / or cellular hemoglobin determined in step (b) to a reference, and

[0123] (C) thereby diagnosing said disease in said subject.

[0124] 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.

[0125] Figure Legends

[0126] Fig. 1 : Schematic representation of an analytic slide 100.

[0127] Fig. 2: Schematic representation of an exemplary allocation of sample aliquots to working areas.

[0128] Fig. 3: Schematic representation of a cross-section of the analytic slide of Fig. 2 along axis A - with additional optional staining chamber 190 covering the first working area 120 or parts thereof.

[0129] Fig. 4: As in Fig. 2, but with applicator 140 and applicator 140' as described in the text.

[0130] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0131] Taking into account Figure 1, the analytic slide 100 may comprise a support 110, a first working area 120 and a second working area 130. Lines outside the exemplary working areas show the course of dispensation of cells. In view of Figure 2, applicator 140 may be e.g. a hollow needle. The applicator may comprise a system fluid 180, such as a buffer; the applicator may aspirate a hemolyzed aliquot of a sample 170, an air gap 150, an aliquot of a sample 160, and optionally a further air gap. Thus, the applicator may comprise an air gap 150, an aliquot of a whole blood sample 160, an air gap 150, a hemolyzed aliquot of the whole blood sample 170, and system fluid 180. The whole blood sample aliquot 160 may be dispensed on the first working area 120, and the hemolyzed blood sample aliquot 170 may be dispended over the second working area 130.

[0132] Taking into account Figure 3, the first working area 120 of an analytic slide 100 may be contacted with a treatment solution. To avoid contacting the second working area with the same treatment solution, which might cause at least a portion of the free hemoglobin dispensed on the second working area to be washed away, said contacting may be performed in a staining chamber 190, fluidly separating the first working area 120 and the second working area 130.

[0133] In view of Figure 4 referring to a preferred embodiment, application may be accomplished by applicator 140 and applicator 140', wherein applicator 140' may be the same applicator as applicator 140 but at a different point in time, or applicator 140' may be an applicator nonidentical to applicator 140. Otherwise, the description relating to Fig. 2 may apply. Thus, the applicator 140 may first apply a whole blood sample 160, and then, e.g. after an optional step comprising washing the lumen of the applicator 140, may apply a hemolyzed aliquot of the whole blood sample 170. As the skilled person will understand, the application sequence may also be inverted, i.e. a hemolyzed aliquot of the whole blood sample 170 may be applied first, followed by an optional washing step, and then the whole blood sample 160 may be applied. Also in a preferred embodiment, non-identical applicators 140 and 140' may be provided, such that applicator 140 may apply a whole blood sample 160 and applicator 140' may apply a hemolyzed aliquot of the whole blood sample 170. As the skilled person understands, use of two non-identical applicators 140 and 140' permits sequential sample application, but also concomitant application of the whole blood sample 160 and the hemolyzed aliquot of the whole blood sample 170. References

[0134] Karakochuk et al. (2019) Ann NY Acad Sci 1450: 126

[0135] US 8,815,537 B2

[0136] US 9,017,610 B2

[0137] WO 2010 / 126903 Al

[0138] WO 2012 / 064873 Al

[0139] WO 2013 / 016037 Al

[0140] Reference signs

[0141] 100 analytic slide

[0142] 110 support

[0143] 120 first working area

[0144] 130 second working area

[0145] 140 applicator

[0146] 150 air gap

[0147] 160 whole blood

[0148] 170 hemolyzed blood

[0149] 180 system fluid

[0150] 190 staining chamber

Claims

Claims1. A method for determining hemoglobin in a sample comprising red blood cells, said method comprising(a) dispensing a first aliquot of said sample on a first working area of a substrate;(b) providing a hemolyzed second aliquot of said sample and dispensing said second aliquot on a second working area; and(c) determining the hemoglobin in said first and second working area, wherein said method comprises a further step of contacting said first working area, but not the second working area, with a treatment solution.

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

3. The method of claim 1 or 2, wherein said determining hemoglobin is determining free hemoglobin, total hemoglobin and / or cellular hemoglobin.

4. The method of any one of claims 1 to 3, wherein said treatment solution is a solution contacted with said first working area and removed therefrom before step (c), in an embodiment wherein said treatment solution is a wash solution and / or a detection solution.

5. The method of any one of claims 1 to 4, wherein said first working area and said second working area are located on the same substrate, in an embodiment wherein the first working area and the second working area are spatially separated on said substrate.

6. The method of any one of claims 1 to 5, wherein dispensing comprises translating an applicator in relation to the substrate while dispensing the sample through the applicator onto the substrate.

7. The method of any one of claims 1 to 6, wherein dispensing comprises causing a monolayer of red blood cells to form on the substrate.

8. The method of any one of claims 4 to 7, wherein the detection reagent comprisesmethylene blue (CAS No. 61-73-4) and Eosine Y (CAS No. 17372-87-1).

9. The method of any one of claims 1 to 8, wherein step (c) comprises step (c2) spectrophotometrically determining absorption in at least a part of the first working area and / or in at least a part of the second working area.

10. The method of claim 9, wherein step (c2) comprises 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 digital image corresponds to light transmitted or reflected by the cells in the first working area; and the second image corresponds to light transmitted or reflected by the hemoglobin in the second working area.

11. The method of any one of claims 1 to 10, wherein step (c) comprises step (c3) determining total hemoglobin in the second working area and / or cellular hemoglobin in the first working area, in an embodiment further comprising determining free hemoglobin as the difference between total hemoglobin and cellular hemoglobin.

12. A system comprising(i) a liquid handling device comprising an applicator tip for dispensing a fluid sample onto a working area of a substrate;(ii) a detection device configured for determining hemoglobin;(iii) a microprocessor; and(iv) 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 11.

13. The system of claim 12, wherein the software instructions cause the system to dispense a known volume of sample out of the applicator tip.

14. The system of claim 12 or 13, further comprising (v) an optical device comprising a light source and a light detector, in an embodiment wherein the software instructions cause the optical device to capture an image of at least a section of the first working area and / or the second working area.

15. A method for diagnosing a disease associated with a change in free hemoglobin, total hemoglobin and / or cellular hemoglobin in a subject, said method comprising(A) determining free hemoglobin, total hemoglobin and / or cellular hemoglobin in a sample of said subject by the method of any one of claims 1 to 11 and / or using a system according to any one of claims 12 to 14,(B) comparing said free hemoglobin, total hemoglobin and / or cellular hemoglobin determined in step (b) to a reference, and(C) thereby diagnosing said disease in said subject.

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