Detection of Hemolysis Using a Chromatographic Chemical Analyzer

Abstract

A chromatographic chemical analyzer for detecting the presence or absence of free hemoglobin in a liquid biological sample is disclosed. The apparatus includes a sample application pad and a chromatographic detection pad, and the sample application pad is formed of two material layers. In addition to the medical diagnostic apparatus used together with this chromatographic chemical analyzer, a kit and a method for using this chromatographic chemical analyzer and / or the medical diagnostic apparatus are also disclosed.

Description

Technical Field

[0001] Cross - reference to related applications / incorporation by reference description This application claims the benefit of U.S. Provisional Patent Application No. 63 / 366,558, filed on June 17, 2022, under 35 U.S.C. § 119(e). The entire content of the above - mentioned patent application is hereby expressly incorporated by reference into this specification.

[0002] Description of research and development funded by the federal government Not applicable.

Background Art

[0003] Hemolysis represents the destruction or lysis of red blood cells (RBCs), as a result of which hemoglobin (“free hemoglobin”) is released into the surrounding liquid. In the case of whole - blood samples, free hemoglobin is released into the surrounding plasma. In the case of urine, free hemoglobin is released into the surrounding moisture. The occurrence of RBC hemolysis is thought to be due to the result of a patient's medical condition or mishandling of the sample itself.

[0004] Hemolysis is a pre - analytical error that is a concern when testing patient samples. When hemolysis is severe, laboratory test results are thought to be inaccurate. For example, in blood gas and electrolyte tests, hemolysis is known to cause an increase in the potassium level of the sample. Also, cardiac troponin T (cTnT) levels are known to decrease in samples with hemolysis, and cardiac troponin I (cTnI) levels are known to increase in samples with hemolysis.

[0005] The detection of hemolysis in whole - blood samples has conventionally been difficult and time - consuming. In major laboratory settings, whole - blood samples are centrifuged to produce plasma, which is then optically examined in the near - infrared (NIR) or visible wavelength regions. Although this technique is very effective, it is complex and time - consuming, so its effectiveness is diminished for point - of - care (POC) applications.

[0006] In some systems, hemolysis is electrochemically detected in the point-of-care area. However, the electrochemical detection of hemoglobin and hematocrit is known to be inaccurate; also, since hemolysis is only detected after the electrochemical reagent has been consumed, samples, reagents, and time may be wasted.

Summary of the Invention

Problems to be Solved by the Invention

[0007] After collecting a whole blood sample, it is important to perform the analysis as quickly as possible; delays are thought to affect the results of the sample. Also, the sample requires aliquoting and centrifugation before hemolysis measurement. However, since centrifugation is required, the settings where hemolysis measurement is determined are limited, and the time between sample collection and testing will necessarily be long.

[0008] Therefore, in this technical field, there is a need for a novel and improved device and method for hemolysis detection that overcomes the disadvantages and deficiencies of the prior art.

Brief Description of the Drawings

[0009]

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DETAILED DESCRIPTION OF THE INVENTION

[0010] Before describing at least one embodiment of the present disclosure in detail, it is understood that the present disclosure is not limited in its application to the details of the components, processes, or method configurations and arrangements described in the following description or drawings. The present disclosure is capable of other embodiments and of various implementations or executions. It is also understood that the expressions and terminology employed herein are for the purpose of description and should not be regarded as limiting in any way.

[0011] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. The above techniques and procedures are generally well known in the art and are performed according to conventional methods as described in various general and specific references cited and discussed throughout this specification. The terminology systems utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as these laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0012] All patents, published patent applications, and non-patent publications cited in this specification are indicative of the level of skill of those of ordinary skill in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications cited anywhere in this application are hereby expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication were specifically and individually indicated to be incorporated by reference.

[0013] All of the articles, compositions, kits, and / or methods disclosed herein can be constructed and executed without undue experimentation in light of the present disclosure. Although the articles, compositions, kits, and / or methods have been described in terms of specific embodiments, it will be apparent to those of ordinary skill in the art that variations can be applied to the articles, compositions, kits, and / or methods, and to the steps or series of steps of the methods, described herein without departing from the concepts, ideas, and scope of the present disclosure. All such apparent similar substitutions and modifications are considered to be within the scope of the spirit, scope, and concepts of the present disclosure as defined by the appended claims.

[0014] For the purposes of the present disclosure, the following terms are to be understood to have the following meanings unless otherwise indicated:

[0015] In the claims and / or the specification, the use of the term "a" or "an" when used in conjunction with the term "comprising" can mean "one", while also being consistent with the meanings of "one or more", "at least one", and "one or more than one". For this reason, the terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" can represent one or more compounds, two or more compounds, three or more compounds, four or more compounds, or more compounds. The term "plurality" represents "two or more".

[0016] The use of the term "at least one" is understood to include, in addition to one, any quantity greater than or equal to two, such as two, three, four, five, ten, fifteen, twenty, thirty, forty, fifty, one hundred, etc., but is not limited thereto. The term "at least one" can be extended to one hundred or more than one thousand depending on the terms associated with it; also, the quantity of one hundred / one thousand is not considered a limitation because satisfactory results can be obtained with higher upper limits. Also, the use of the term "at least one of X, Y, and Z" is understood to include only X, only Y, and only Z, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is only for the purpose of distinguishing between two or more items and does not imply, for example, a sequence, order, or importance in which one item is preferred over another, nor does it imply an additional order.

[0017] The use of the term "or" in the claims is used in the inclusive sense of "and / or" unless there is an explicit indication that it is meant to represent alternatives only and the alternatives are not mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: namely, A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0018] As used herein, any reference to "one embodiment" or "an embodiment", "some embodiments", "one example", "for example", or "an example" means that a particular element, structure, configuration, or characteristic described in connection with that embodiment is included in at least one embodiment. For example, the appearance of the phrases "in some embodiments" or "one example" in various places in this specification does not necessarily refer to all the same embodiments. Further, all references to one or more embodiments or examples should be construed as not being limited to the claims.

[0019] Throughout this application, the terms "about" and "approximately" are used to indicate that the inherent variability of error in the composition / apparatus / device, method employed to determine a value, or the variability that exists between the items being considered is included in the value. That is, the terms "about" and "approximately" and their variations are intended to include not only the exact values limited by the terms, but also some deviation from these (e.g., measurement error, manufacturing tolerances, wear of components or structures, sedimentation or precipitation of cells or particles from a suspension or solution, chemical or biological degradation of a solution over time, stress exerted on a structure, and deviations due to combinations of these, etc.). In particular, for example, when the term "about" is utilized, the specified value can vary by ±20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value, as understood by a person skilled in the art, since it is suitable for the execution of the disclosed method, but is not limited thereto.

[0020] As used in this specification and the claims, the word "comprising" (and any form of "comprising" ("comprise" and "comprises", etc.)), "having" (and any form of "having" ("have" and "has", etc.)), "including" (and any form of "including" ("includes" and "include", etc.)), or "containing" (and any form of "containing" ("contains" and "contain", etc.)) is inclusive or open-ended and does not exclude additional elements, method steps not listed. For example, a composition, process, method, article, or apparatus that includes a list of elements is not necessarily limited to these elements and can include other elements not explicitly listed or other elements that were not present.

[0021] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations that include repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are explicitly included. One of ordinary skill in the art will understand that, unless otherwise explicitly stated in the context, there is usually no limit to the number of items or terms in any combination.

[0022] As used herein, the term "substantially" means that the event or situation described thereafter occurs completely, or that most of the event or situation described thereafter occurs. For example, when associated with a particular event or situation, the term "substantially" means that the event or situation described thereafter occurs with a probability of at least 80%, at least 85%, at least 90%, or at least 95%. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, that two items are close to each other but not 100% adjacent to each other, or that a part of one of the two items is close to but not 100% adjacent to the other item.

[0023] As used herein, the expressions "associated with" and "coupled to" include, in addition to the direct association / coupling of two parts with each other, the indirect association / coupling of two parts with each other. Examples of association / coupling include, but are not limited to, for example, a covalent bond of one part to another part by a direct bond or a spacer group, a non-covalent bond of one part to another part by a direct bond or a specific binding pair member that is bound, incorporation of one part into another part by dissolution or synthesis of one part into another part, and coating of one part on another part.

[0024] As used herein, it is understood that the term "sample" includes any kind of biological sample utilized in accordance with the present disclosure. Examples of fluid biological samples utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural effusion, nasopharyngeal fluid, combinations thereof, and the like.

[0025] As used herein, the terms "liquid sample" and variations thereof are intended to include, but are not limited to, for example, biological fluids (such as urine and whole blood), chemical fluids, chemical substances, suspensions, solutions, slurries, mixtures, aggregates, tinctures, slides or other biological fluid preparations, synthetic analogs of biological fluids, and combinations thereof.

[0026] Turning now to various non - limiting embodiments of the inventive concept, described herein is a simple disposable chromatographic chemical analyzer for alerting a medical professional when a sample is thought to be compromised and produce inaccurate test results by rapidly and efficiently detecting hemolysis in a liquid sample. This chromatographic chemical analyzer can rapidly detect hemolysis in a liquid sample while keeping the sample size small and the cost per test low. This chromatographic chemical analyzer provides rapid and centrifugation - free hemolysis detection. Centrifugation lengthens the period from collection to testing and is a limitation in the environment where the detection test is performed. Unlike prior - art methods that require centrifugation, the chromatographic chemical analyzer of the present disclosure can be used for testing in all types of settings, including (but not limited to) primary testing laboratories, point - of - care locations, and in - home settings. For example, the chromatographic chemical analyzer of the present disclosure can provide results in less than one minute, inexpensively and ubiquitously, for hemolysis detection, and can be used with or without an analyzer (but not limited to this). Also, visual or optical detection methods are used that enable more reliable detection.

[0027] Certain non-limiting embodiments of the present disclosure are directed to a chromatographic chemical analysis device for detecting the presence or absence of free hemoglobin in a liquid biological sample (including, but not limited to, whole blood samples, urine, or other erythrocyte-containing liquid samples). The device includes a sample application pad in fluid contact with a chromatographic detection pad. The sample application pad is configured to apply a portion of the liquid biological sample to the chromatographic chemical analysis device. The sample application pad comprises at least two different material layers. For example, the sample application pad can comprise a first material laminated or attached to a second different material (however, it is not limited thereto). At least two different material layers of the sample application pad can differ, for example, with respect to pore size, pore size uniformity or non-uniformity, material type, and / or material thickness. In an exemplary embodiment, the sample application pad is formed of a first upper layer of pre-filter material and a second lower layer of filter material, and the pre-filter material and the filter material differ with respect to material type and / or porosity (e.g., pore size and / or pore size uniformity / non-uniformity). In certain (however, non-limiting) embodiments, the sample application pad is formed of a first upper layer of pre-filter material and a second lower layer of asymmetric filter material. As used herein, the term "asymmetric filter material" refers to a filter material or membrane having an asymmetric pore structure in which voids (pores) are non-uniform or size-varying throughout the thickness of the material (e.g., the voids can be made small near one surface (e.g., the bottom surface) and large near the opposite surface (e.g., the upper surface adjacent to the bottom surface of the pre-filter layer)). Exemplary asymmetric filter materials used as the second lower layer can include, for example, polysulfone, polyethersulfone, or mixed cellulose esters. Other exemplary types of materials used as the filter material of the second lower layer can include, for example, potato lectin, or a treated glass fiber membrane treated with, for example, potato lectin. In another exemplary embodiment, the second lower layer of the sample application pad is formed of a symmetric material such as a material containing potato lectin (e.g., glass fiber treated with potato lectin).On one hand, the first upper layer of the pre-filter material is formed of different materials such as a material that does not contain potato lectin (for example, glass fiber not treated with potato lectin).

[0028] The sample application pad is porous to plasma and free hemoglobin but not porous to red blood cells. Thus, the red blood cells present in the liquid biological sample are retained within the two material layers of the sample application pad and do not flow through the interior. In particular, the first and / or second material layer has a pore size that varies and decreases in size from the upper surface to the lower surface of the sample application pad (i.e., in the direction towards the chromatographic detection pad), having at least one pore size smaller than the size of red blood cells, thereby preventing red blood cells from flowing through the sample application pad, but not limited thereto. In an exemplary embodiment, the first pre-filter layer has less restricted (e.g., larger) pore size compared to the pore size of the second filter layer. In another exemplary embodiment, the first pre-filter material layer has a more uniform or symmetric pore size structure across the entire thickness of the material compared to the second filter layer having a more non-uniform or asymmetric pore size structure.

[0029] Also, since there are different pore sizes in the two layers of the sample application pad, larger cell components (i.e., red blood cells) are retained and do not lyse, while free fluids such as plasma can pass through the pores. Also, due to the combination of the two materials, the first layer can hold more than the second layer, enabling all red blood cells to be retained without overloading the second layer.

[0030] In the present disclosure, by a chromatographic chemical analyzer including a sample application pad with a multi-layer design (composed of at least two different materials, such as filter materials like a pre-filter material and an asymmetric filter material), while removing or improving the filtration of red blood cells (RBCs) present in a liquid biological sample such that the RBCs are captured within multiple layers of the sample application pad, it has been unexpectedly found to be advantageous that plasma and free hemoglobin present in the liquid biological sample can flow (e.g., by gravity) through the pores of the sample application pad to the chromatographic detection pad. Thereby, for example, due to the improvement in the filtration of red blood cells by multiple layers of the sample application pad, the sample application pad does not require treatment with a red blood cell-binding or aggregating agent. Further, since the multi-layer or two-layer sample application pad is directly connected to the chromatographic detection pad, in this chromatographic chemical analyzer, the removal of RBCs and the detection of hemolysis (by detecting the presence or absence of free hemoglobin) in a single or integrated device are improved. Therefore, in this chromatographic chemical analyzer, it is advantageous that there is no need to transfer the plasma and free hemoglobin filtered by the sample application pad to a separate device for detecting the presence or amount of free hemoglobin.

[0031] The first layer of the sample application pad can be formed of any material known in the art or any material contemplated herein that can hold at least a portion of the red blood cells and other larger cell components in a blood sample (without cell lysis), thereby reducing the amount of red blood cells and other larger cell components flowing into the second layer of the sample application pad so as not to overload the second filtration layer. A non-limiting example of a material utilized for forming the pre-filtration layer is a glass fiber material or the like; however, it is understood that other materials that can function in this manner are known in the art and are included within the scope of the term "prefiltration material" for use herein.

[0032] The chromatographic detection pad defines a path for capillary fluid flow. The chromatographic detection pad has a first end and a second end. The first end is in fluid contact with the sample application pad and forms a sample application site on the chromatographic detection pad. Also, the chromatographic detection pad has a detection site spaced from (and in some non-limiting embodiments, downstream of) the first end / sample application site. The detection site is disposed between the first and second ends or is substantially adjacent to the second end.

[0033] When a liquid biological sample (including, but not limited to, whole blood samples, urine, or other red blood cell-containing liquid samples) is applied to the chromatographic chemical analyzer, free hemoglobin flows through the sample application pad into the chromatographic detection pad and then from the sample application site of the chromatographic detection pad to its detection site. Thus, free hemoglobin (indicating hemolysis) can be detected by a color change at the detection site.

[0034] The chromatographic detection pad is formed of a white (to enable visual reading) any material having a pore size capable of defining a path for capillary flow for the detection of free hemoglobin in the sample by the free flow of free hemoglobin and plasma. In a particular (non-limiting) one embodiment, the chromatographic detection pad is formed of a nitrocellulose membrane.

[0035] The sample application pad and the chromatography detection pad are each provided with any pore size that enables the sample application pad and the chromatography chemical analyzer to function in accordance with the present disclosure. Non-limiting examples of pore sizes utilized in accordance with the present disclosure include about 0.1 micron, about 0.22 micron, about 0.3 micron, about 0.45 micron, about 0.5 micron, about 1 micron, about 2 micron, about 3 micron, about 4 micron, about 5 micron, about 6 micron, about 7 micron, about 8 micron, about 9 micron, about 10 micron, about 11 micron, about 12 micron, about 13 micron, about 14 micron, about 15 micron, about 16 micron, about 17 micron, about 18 micron, about 19 micron, about 20 micron, about 21 micron, about 22 micron, about 23 micron, about 24 micron, about 25 micron, about 26 micron, about 27 micron, about 28 micron, about 29 micron, about 30 micron, about 31 micron, about 32 micron, about 33 micron, about 34 micron, about 35 micron, about 36 micron, about 37 micron, about 38 micron, about 39 micron, about 40 micron, about 41 micron, about 42 micron, about 43 micron, about 44 micron, about 45 micron, about 46 micron, about 47 micron, about 48 micron, about 49 micron, and about 50 micron, and in addition, ranges formed by any of the above values (i.e., the range of about 0.1 micron to about 6 microns, the range of 8 microns to about 40 microns, the range of about 8 microns to about 13 microns, etc.) are included.

[0036] In certain (non-limiting) embodiments, the chromatography chemical analyzer can further include a backing material to which the chromatography detection pad is attached or associated.

[0037] In certain (non-limiting) embodiments, the chromatographic detection pad is a lateral flow strip. In a particular (non-limiting) embodiment, the lateral flow strip is configured to be disposed or attached to a medical diagnostic device that detects color changes at the detection site. In another particular (non-limiting) embodiment, the lateral flow strip is designed for personal manual use. Thus, the color change at the detection site is detected by the individual by visual comparison with a reference device that includes a plurality of reference colors corresponding to different hemolysis levels.

[0038] In certain non-limiting embodiments, the sample application pad is provided with one or more additional agents that further function to prevent red blood cells from passing through the sample application pad and entering the chromatographic detection pad. For example, the sample application pad is treated with, but not limited to, at least one type of red blood cell (RBC) binding or aggregating agent such that the RBC binding or aggregating agent is present in a free soluble form. The RBC binding or aggregating agent aggregates with any RBCs in the whole blood sample to produce aggregated RBCs, but these aggregated RBCs are too large to pass through the pore size of the sample application pad and thus cannot flow through the sample application pad. On the other hand, any free RBC binding or aggregating agent can enter the chromatographic detection pad without interfering with the detection of free hemoglobin. Non-limiting examples of RBC binding or aggregating agents utilized in accordance with the present disclosure include lectins, anti-human red blood cell (anti-hRBC) binding or aggregating protein antibodies, and non-antibody human red blood cell (hRBC) binding or aggregating proteins.

[0039] The devices and methods of the present disclosure rely on visual or optical detection of hemolysis by a color change (such as, but not limited to, a change from the pad's default transparent or white color to a cream, yellow, pink, or red color tone) of the chromatographic detection pad due to the red color of free hemoglobin. Thus, in certain non-limiting embodiments, the chromatographic detection pad does not contain a compound that reacts with free hemoglobin in the liquid biological sample downstream of the sample application site.

[0040] The chromatographic detection pad can contain one or more reagents that, instead of containing reactive compounds, react with free hemoglobin present in the liquid sample and flowing through the chromatographic detection pad. Exemplary reagents present in the chromatographic detection pad can enhance the color change resulting from free hemoglobin in the detection zone. Non-limiting exemplary reagents utilize the peroxidase-like activity of hemoglobin that catalyzes the reaction of diisopropylbenzene dihydroperoxide and 3,3’,5,5’-tetramethylbenzidine. The resulting color range is from orange to green and, in some cases, to blue. Exemplary reagents in the detection pad can be arranged (but not limited to) in a strip disposed perpendicular to the direction of flow, for example.

[0041] Certain non-limiting embodiments of the present disclosure relate to a kit comprising at least one of the above-described chromatographic chemical analyzers or any chromatographic chemical analyzer contemplated herein, in combination with a reference device for personal use in the visual detection of hemoglobin. The reference device includes a plurality of reference colors, each corresponding to a different hemolysis level, degree, or amount. The reference device can assume any form or format known in the art or any form or format contemplated herein. For example, the kit can include a container in which the chromatographic chemical analyzer is housed prior to use, and the reference device can be attached or formed on a portion of the outer surface of the container (but not limited to this). Alternatively, an insertion card present within the container can be considered as the reference device.

[0042] In addition to the chromatographic chemical analysis apparatus and the reference device, the kit can further include other components or reagents that are used when performing either the specific chemical analysis described herein or a specific chemical analysis contemplated herein. The nature of these additional reagents will depend on the specific chemical analysis format, but their identification is within the skill of those in the art; thus, further description is considered unnecessary. Also, the compositions / reagents present in the kit can each be present in separate containers / compartments. Alternatively, the various compositions / reagents can be combined in one or more containers / compartments depending on the reactivity and stability of the compositions / reagents. For example, the kit can further include (but is not limited to) positive and / or negative control reagents. The kit can also further include a set of instructions that explain how to use the kit. A kit of this nature is used in either the method described herein or a method contemplated herein.

[0043] One non-limiting embodiment of the present disclosure is directed to a method for testing a liquid biological sample for hemolysis. In this method, a liquid biological sample is applied to the sample application pad of either the chromatographic chemical analysis apparatus disclosed above or a chromatographic chemical analysis apparatus contemplated herein, allowing the plasma and any free hemoglobin present in the liquid biological sample to flow through the sample application pad to the chromatographic detection pad and from the sample application site to the detection site. Thereafter, the color change at the detection site is visually compared to a reference device that includes a plurality of reference colors corresponding to different hemolysis levels. The visual comparison step can be performed manually by an individual or by a diagnostic device.

[0044] Certain non-limiting embodiments of the present disclosure are directed to a medical diagnostic device assembly. This assembly includes, in combination with a medical diagnostic device, either the chromatographic chemical analyzer of the above disclosure or any chromatographic chemical analyzer contemplated herein. The medical diagnostic device includes a light source, an optical sensor, and a processor. The optical sensor detects the amount of red light reflected by the detection site of the chromatographic chemical analyzer and outputs a detection signal; the amount of red light reflected by the detection site is due to the amount of free hemoglobin present in the liquid biological sample. The processor receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample.

[0045] Certain non-limiting embodiments of the present disclosure are directed to a method for testing a liquid biological sample (including, but not limited to, whole blood samples, urine, or other red blood cell-containing liquid samples, etc.) for hemolysis. In this method, a liquid biological sample is applied to the sample application pad of either the chromatographic chemical analyzer disclosed herein or any chromatographic chemical analyzer contemplated herein, allowing the plasma and any free hemoglobin present in the liquid biological sample to flow through the sample application pad to the chromatographic detection pad and from the sample application site to the detection site. The chromatographic chemical analyzer is disposed within a medical diagnostic device that includes a light source, an optical sensor, and a processor. The optical sensor detects the amount of red light reflected by the detection site of the chromatographic chemical analyzer and outputs a detection signal, and the processor receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample. Then, the amount of red light reflected by the detection site of the chromatographic chemical analyzer is measured. The amount of red light reflected by the detection site is due to the amount of free hemoglobin present in the liquid biological sample. Thereafter, based on the measured amount of reflected red light, the amount of free hemoglobin present in the liquid biological sample is determined.

[0046] In certain (non-limiting) embodiments, the method can further include a step of displaying a notification that the liquid biological sample is hemolyzed when the measured amount of reflected red light exceeds a predetermined reference value. If this occurs, in certain (non-limiting) embodiments, the method can further include a step of preventing subsequent tests using the liquid biological sample from being performed when a notification that the liquid biological sample is hemolyzed is displayed.

[0047] In certain (non-limiting) other embodiments, the method can further include a step of enabling subsequent tests using the liquid biological sample to be performed when the measured amount of reflected red light does not exceed a predetermined reference value; and a step of reporting the results of the subsequent tests to a healthcare provider.

[0048] Referring now to FIGS. 1 and 2, these illustrate a chromatographic chemical analyzer 10 for detecting the presence or absence of free hemoglobin in a liquid biological sample 12 (including, but not limited to, whole blood, urine, or other red blood cell-containing samples). The chromatographic chemical analyzer 10 includes a sample application pad 14 in fluid contact with a chromatographic detection pad 16. The sample application pad 14 is configured to apply a portion of the liquid biological sample 12 to the chromatographic chemical analyzer 10. After the sample application pad 14 receives and absorbs the liquid biological sample 12, the liquid biological sample 12 is absorbed from the sample application pad 14 to the chromatographic detection pad 16.

[0049] The sample application pad 14 has a first end 20, a second end 22, an upper surface 24, and a lower surface 26. The sample application pad 14 is formed of a first upper layer 30 formed of a pre-filter material (such as, but not limited to, a glass fiber material or other suitable pre-filter material), and a second lower layer 32 formed of an asymmetric filter material (such as, but not limited to, a polysulfone material or other suitable asymmetric filter material). However, it is understood that the second lower layer 32 can be formed of a symmetric material, and in this case, the second lower layer 32 is formed of a material different from the first upper layer 30 as described above. The first layer 30 of the pre-filter material forms the upper surface 24 of the sample application pad 14. On the other hand, the second layer 32 of the asymmetric filter material forms the lower surface 26 of the sample application pad 14. The first layer 30 has a first surface and a second surface. The upper surface of the first layer 30 is the upper surface 24 of the sample application pad 14 configured to receive the liquid biological sample 12, and directly receives (but is not limited to) the liquid biological sample 12 from, for example, a syringe, pipette, dropper, valve, capillary, or other dispensing device (but is not limited to these). The second surface of the first layer 30 has a direct formation and / or direct attachment to the first surface of the second layer 32. On the other hand, the second surface of the second layer 32 is the lower surface 26 of the sample application pad 14 in direct fluid contact with the chromatographic detection pad 16. Therefore, in the embodiment shown in FIG. 2, the sample application pad 14 is formed of the first layer 30 having a direct formation and / or direct attachment to the second layer 32, and is convenient because it does not involve an additional intervening layer (does not include means for forming and / or attaching the first layer 30 to the second layer 32).

[0050] The sample application pad 14 is porous to plasma and free hemoglobin present in the liquid biological sample 12, but not porous to red blood cells. Therefore, the red blood cells present in the liquid biological sample 12 are retained within the two material layers 30 and 32 of the sample application pad 14 and do not flow through the interior to the chromatographic detection pad 16.

[0051] The chromatography detection pad 16 defines a path for capillary fluid flow. Components of the liquid biological sample 12 that can flow through the sample application pad 14 then flow through the chromatography detection pad 16 by capillary action (also referred to as capillary flow). The chromatography detection pad 16 has a first end 40 and a second end 42. The chromatography detection pad 16 is made of any suitable material through which plasma and free hemoglobin of the liquid biological sample 12 can freely flow by capillary action. As a non-limiting example, the chromatography detection pad 16 is a nitrocellulose membrane. The chromatography detection pad 16 can have pores through which certain components of the liquid sample 12 move by capillary action. Most of the pores of the chromatography detection pad 16 can all have substantially the same size or can be included within a certain range of values.

[0052] The first end 40 of the chromatography chemical analysis device 10 is in fluid contact with the lower surface 26 of the sample application pad 14 and forms a sample application site 48 on the chromatography detection pad 16. Also, the chromatography detection pad 16 has a detection site 50 that is spaced from the first end 40 / sample application site 48 (and, in certain non-limiting embodiments, downstream of the first end 40 / sample application site 48). The detection site 50 is disposed between the first and second ends 40 and 42, is substantially adjacent to the second end 42, or is closer to the second end 42 than the first end 40.

[0053] Also, the sample application pad 14 covers only the portion of the chromatography detection pad 16 adjacent to the first end 40 and its sample application site 48 and does not cover its detection site 50; thereby, the flow of the sample through the chromatography detection pad 16 and into its detection site 50 is visible. For example, since the second end 22 of the sample application pad 14 is located adjacent to or extends only slightly beyond the downstream end of the sample application site 48, the flow of the sample from the sample application site 48 toward the detection site 50 is visible (but not limited thereto).

[0054] The chromatography chemical analyzer 10 can further include a backing material 18 to which the lower surface 46 of the chromatography detection pad 16 is attached or associated (examples include, but are not limited to, methods via a double-sided adhesive).

[0055] When a liquid biological sample 12 (examples include, but are not limited to, whole blood samples, urine, or other red blood cell-containing liquid samples) is applied to the chromatography chemical analyzer 10, free hemoglobin flows through the sample application pad 14 into the chromatography detection pad 16, and then flows from the sample application portion 48 of the chromatography detection pad 16 to its detection site 50. In this way, free hemoglobin (indicating hemolysis) can be detected by a color change at the detection site due to the red color of free hemoglobin.

[0056] A specific non-limiting embodiment of the chromatography chemical analyzer 10 is shown in FIGS. 1 and 2, but it is understood that the illustrated design and configuration of the chromatography chemical analyzer 10 are for illustrative purposes only. The scope of the present disclosure includes adapting the design and configuration of the chromatography chemical analyzer of the present disclosure as long as the chromatography chemical analyzer can function in accordance with the present disclosure.

[0057] For example, it is understood (but not limited to) that the first layer (of the pre-filter material) and the second layer (of the asymmetric filter material) of the sample application pad need not be symmetric with respect to each other (i.e., their sizes, lengths, widths, and / or thicknesses can be different from each other). Also, since the first and second layers of the sample application pad need not match each other, each layer can have an area that does not overlap with the other layer. After the sample flows through the first layer and into the second layer, at least a portion of the first layer only needs to cover a sufficient portion of the second layer so that the sample can flow from the second layer into the sample application site of the chromatographic detection pad. FIG. 3A shows a chromatographic chemical analyzer 10a similar to the chromatographic chemical analyzer 10 of FIGS. 1 and 2, except that it has a sample application pad 14a formed of two separate layers that differ in size and dimensions and do not completely overlap each other. The sample application pad 14a includes a first layer 30a formed of a pre-filter material and a second layer 32a formed of an asymmetric filter material. The first layer 30a has a first end 140, a second end 142, an upper surface 144, and a lower surface 146. The second layer 32a has a first end 148, a second end 150, an upper surface 152, and a lower surface 154. At least a portion adjacent to the second end 142 of the first layer 30a overlaps a portion between the first and second ends 148 and 150 of the second layer 32a. The overlapping portions of the first and second layers 30a and 32a are attached to each other. Alternatively, since the overlapping portion of the first layer 30a is simply laminated on the second layer 32a, a portion of the lower surface 146 of the first layer 30a is in contact with a portion of the upper surface 152 of the second layer 32a.

[0058] FIG. 3B shows the workflow of the chromatographic chemical analyzer 10a of FIG. 3A, but it should be noted that the workflow of the chromatographic chemical analyzer 10 of FIG. 2 is also substantially the same. A blood sample 12a is applied to the first layer 30a of the sample application pad 14a and enters the first layer 30a. The second panel of FIG. 3B shows the blood sample 12a as being applied to the upper surface 144 of the first layer 30a, but if the first and second layers 30a and 32a do not completely overlap each other, it is understood that the blood sample 12a may be applied to the lower surface 146 of the first layer 30a in the non-overlapping portion. Thereafter, the blood sample 12a saturates the first layer 30a and flows into the second layer 32a through the overlapping portion (third panel). Then, the plasma of the blood sample 12a passes through the second layer 32a and enters the chromatographic detection pad 16a, flows from the sample application site 48a to the detection site 50a, and any hemolysis present is detected (fourth panel). In one non-limiting embodiment, the plasma reaching the detection site 50a changes the control line from yellow to blue, indicating that the device 10a is ready for reading.

[0059] FIG. 4 shows a non-limiting embodiment of a reference device 120 that can visually determine the hemolysis level in a liquid sample 12 by use in conjunction with the chromatographic chemical analyzer 10. The reference device 120 includes a plurality of reference colors (including, but not limited to, reference colors 122, 124, 126, 128, and 130, etc., where color 122 has the white / default color of the chromatographic detection pad 16 and functions as a negative control, and colors 124, 126, 128, and 130 are pink / red of various color tones with increasing intensity / hue, and the darker the intensity / hue, the greater the amount / degree of hemolysis); five reference colors are shown, but this is for illustrative purposes only). The reference device 120 also includes a key 132 that correlates each of the reference colors 122, 124, 126, 128, and 130 with a specific free hemoglobin concentration. That is (merely for illustrative purposes), while color 122 of the key 132 is the negative control, color 124 indicates the presence of 0 mg / dL of free hemoglobin, color 126 indicates the presence of 100 mg / dL of free hemoglobin, color 128 indicates the presence of 250 mg / dL of free hemoglobin, and color 130 indicates the presence of 500 mg / dL of free hemoglobin. Thus, by comparing the color of the detection site 50 with the reference colors 124 - 130 of the reference device 120, an individual can determine the hemolysis level in a liquid biological sample at any setting (including, but not limited to, point-of-care or in-home settings).

[0060] The design and configuration of the reference device 120 in FIG. 4 are merely shown for illustrative purposes; the reference device 120 may be provided with less than 5 reference colors or more than 5 reference colors (examples include, but are not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more reference colors). Also, the shape and arrangement of the reference colors can be made different. Further, the key 132 can be given a shape / arrangement different from that shown in FIG. 4. That is, the design and configuration of each of the various components of the reference device 120 (including, but not limited to, the reference colors and the key 132) can be easily adapted by those skilled in the art to have any settings and configurations that enable the reference device 120 to function in accordance with the present disclosure.

[0061] For example, FIG. 5 shows another non-limiting embodiment (but not limited thereto) of a reference device (herein designated by reference numeral 120a) that can visually determine the hemolysis level in a liquid sample 12 by use in conjunction with the chromatographic chemical analyzer 10. While the reference device 120 of FIG. 4 includes individual reference colors, the reference device 120a of FIG. 5 includes a plurality of reference color ranges (including, but not limited to, reference color ranges 122a, 124a, 126a, and 128a, etc.; color 122a has the white / default color of the chromatographic detection pad 16 and functions as a negative control; color ranges 124a, 126a, and 128a each include pink / red of various color tone ranges with increasing intensity / hue, and the darker the intensity / hue, the greater the amount / degree of hemolysis); four reference color ranges are shown, but for illustrative purposes only). The reference device 120a also includes a key 132a that correlates specific free hemoglobin concentration ranges with the reference color ranges 122a, 124a, 126a, and 128a respectively. That is (but for illustrative purposes only), while color 122a of key 132a is the negative control, color range 124a indicates the presence of free hemoglobin less than 0 - 100 mg / dL, color range 126a indicates the presence of free hemoglobin less than 100 - 500 mg / dL, and color range 128a indicates the presence of free hemoglobin less than 500 - 1000 mg / dL. Thus, by comparing the color of the detection site 50 with the reference color ranges 124a - 128a of the reference device 120a, an individual can determine the hemolysis level in a liquid biological sample in any setting (including, but not limited to, point-of-care or in-home settings).

[0062] Alternatively, by using a medical diagnostic device, the hemolysis level in a liquid biological sample can be optically detected. Referring to FIG. 6 here, this shows a medical diagnostic device 100. The medical diagnostic device 100 includes an optical sensor 102, a processor 104, and a light source 106, and is directed toward the detection site of a chromatographic chemical analyzer (such as the detection site 50 of the chromatographic chemical analyzer 10 in FIGS. 1 and 2, but not limited thereto). The optical sensor 102 acquires one or more images of the detection site 50 and transmits them to the processor 104 in the detection signal 108. Then, the processor 104 analyzes the characteristics of the light reflected by the detection site 50 of the chromatographic chemical analyzer 10 based on the received image. The observable colors of the characteristics of the light reflected by the detection site 50 (such as red, orange, green, and blue, etc.) are due to the presence or absence of free hemoglobin in the liquid sample 12. Therefore, the processor can use the characteristics of the reflected light to quantify the amount of free hemoglobin present in the liquid sample 12. For example, when the detection site / zone 50 does not contain a compound downstream of the sample application site 48 of the sample application pad 14 or the chromatographic detection pad 16, the amount of free hemoglobin present in the liquid sample 12 can be quantified by using the amount / intensity of the red light reflected by the detection site 50. When the chromatographic detection pad 16 contains a reagent that reacts with free hemoglobin and is arranged downstream of the sample application site 48, the amount of free hemoglobin present in the liquid sample 12 can be quantified by using the amount / intensity of one or more of the red light, orange light, green light, and blue light reflected by the detection site 50. In this way, the processor 104 can determine the amount of free hemoglobin in the liquid sample 12, for example, by comparing the measured amount of the observable color of the light reflected by the detection site 50 with a known reference value. Also, it is understood that the processor 104 does not need to be arranged inside the device 100 and can be arranged at an external location.

[0063] In a non-limiting embodiment, the light source 106 can be a broadband light source, and the optical sensor 102 can employ a two-dimensional pixel array that captures a two-dimensional image of the detection site 50. The processor 104 selects a specific region of interest within the image of the chromatographic chemical analysis substrate, analyzes the spectral components and surface topography of the region of interest on the substrate, determines the porosity and depth variations of the region of interest, and algorithmically improves the selectivity, dynamic range, and signal-to-noise ratio of the primary signal of interest, which may be degraded by variations in the detection region, turbidity of the residual sample, and chemical interference.

[0064] A method for testing a liquid sample for hemolysis can include applying a portion of the liquid sample 12 to the sample application pad 14, allowing free hemoglobin to flow through the sample application pad 14 into the chromatographic detection pad 16, and after flowing from the sample application site 48 to the detection site 50, measuring the characteristics of the light reflected by the detection site 50 of the chromatographic chemical analysis device 10 as described above. Then, the level of free hemoglobin can be determined, for example, by using the measured amounts of red light, orange light, green light, and / or blue light and comparing the measured amounts to one or more reference values. In an exemplary embodiment, this method is performed by the device 100 or a healthcare provider. The healthcare provider can visually determine the hemolysis of the liquid sample 12 by comparing the completed chromatographic chemical analysis device 10 to a reference device (such as, but not limited to, the reference devices shown in FIGS. 4 and 5) that includes a plurality of reference colors corresponding to different hemolysis levels.

[0065] This method can be used to detect hemoglobin levels exceeding a predetermined interference value (e.g., the manufacturer's interference level). If the sample exceeds the interference value, a flag is set to inform the end user (i.e., the relevant healthcare provider) that the sample is hemolyzed and damaged. After the medical diagnostic device 100 determines that the liquid sample 12 is hemolyzed, if additional tests can be performed on the liquid sample 12, it can prevent subsequent tests using the liquid sample 12 from being executed, or it can also enable subsequent tests using the liquid sample 12 to be executed. However, when interpreting the results of the subsequent tests, the end user is informed to consider that the liquid sample 12 is hemolyzed.

[0066] The processor 104 can have any suitable architecture such as a general-purpose processor, a central processing unit, a digital signal processor, an application-specific integrated circuit, a field programmable gate array, a digital circuit, an analog circuit, combinations thereof, or any other device currently known or later developed for processing data. Similarly, the processing method can include multiprocessing, multitasking, parallel processing, etc. Also, a program can be uploaded and executed by the processor. The processor can execute the program alone or include multiple processors for parallel or sequential processing in a network or system.

[0067] The processor outputs the status and / or related information to a display, memory, network, printer, or another medium. The display is a display of text, graphics, etc.

[0068] The display is a CRT, LCD, plasma, projector, monitor, printer, or other output device for presenting data. The display is operable to output to the user a state associated with a patient. This state indicates whether a medical concept is documented in the medical record. Also, this state is whether a disease, condition, symptom, or test result is indicated. In one embodiment, this state is limited to true and false, or true, false, and unknown. In other embodiments, this state is one level of a range of levels or other non-boolean state.

[0069] Processor 104 operates according to instructions. The instructions are embodied in a program. As the program, when executed by at least one processor 104, there is a non-transitory computer-readable medium that stores instructions for causing the processor 104 to quantify the amount of free hemoglobin present in the liquid sample 12 based on the image of the detection site 50 according to any one of the techniques described herein. The program can be arranged in a non-transitory computer-readable memory such as external storage, ROM, and / or RAM. Instructions for implementing the processes, methods, and / or techniques discussed herein are provided on a computer-readable storage medium or memory such as a cache, buffer, RAM, removable medium, hard drive, or other computer-readable storage medium. The computer-readable storage medium includes various types of volatile and non-volatile storage media. The functions, operations, or tasks described in the drawings or this specification are executed in response to one or more sets of instructions stored in or on the computer-readable storage medium. These functions, operations, or tasks are independent of a particular type of instruction set, storage medium, processor, or processing method, and are executed by software, hardware, integrated circuit, firmware, microcode, etc. that operate alone or in combination. In one embodiment, these instructions are stored on a removable media device and read by a local or remote system. In other embodiments, these instructions are stored in a remote location and transmitted through a computer network or telephone line, wireless GSM, or WiFi. In yet other embodiments, these instructions are stored in a given computer, CPU, GPU, or system. Since some of the components of the configured system and the method operations shown in the accompanying drawings are realized by software, the actual connection between the components of the system (or the process steps) can be varied according to the programming mode.

[0070] Non-limiting exemplary embodiments The following is a list of non-limiting exemplary embodiments disclosed herein:

[0071] Exemplary Embodiment 1. A chromatographic chemical analyzer for detecting the presence or absence of free hemoglobin in a liquid biological sample, comprising: a sample application pad for applying a liquid biological sample, wherein the sample application pad is formed of a first layer of a pre-filtering material and a second layer of a filtering material different from the pre-filtering material, and wherein the sample application pad is porous to plasma and free hemoglobin but not porous to red blood cells, so that red blood cells present in the liquid biological sample are retained within the first and second layers of the sample application pad and do not flow through the interior; the sample application pad; a chromatographic detection pad defining a path of capillary fluid flow, the chromatographic detection pad being in fluid contact with the sample application pad and having a first end forming a sample application site on the chromatographic detection pad, the chromatographic detection pad further having a detection site spaced from the sample application site; the chromatographic detection pad; and free hemoglobin flows through the sample application pad into the chromatographic detection pad, flows from the sample application site to the detection site, and is detectable by a color change at the detection site. The chromatographic chemical analyzer.

[0072] Exemplary Embodiment 2. The chromatographic chemical analyzer according to Exemplary Embodiment 1, wherein the pre-filtering material of the sample application pad comprises a glass fiber material and the filtering material is an asymmetric material containing polysulfone.

[0073] Exemplary Embodiment 3. The chromatographic chemical analyzer according to Exemplary Embodiment 1 or 2, wherein the chromatographic detection pad is formed of a nitrocellulose membrane.

[0074] Exemplary Embodiment 4. The chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 3, wherein the chromatographic detection pad has a pore diameter in the range of about 8 microns to about 40 microns.

[0075] Exemplary Embodiment 4A. The pore diameter of the chromatographic detection pad ranges from about 8 microns to about 13 microns, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 4.

[0076] Exemplary Embodiment 5. The chromatographic detection pad is a lateral flow strip, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 4A.

[0077] Exemplary Embodiment 6. The lateral flow strip is configured to be disposed in a medical diagnostic device that detects a color change at a detection site, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 5.

[0078] Exemplary Embodiment 7. The chromatographic detection pad is configured to provide a visual comparison of the color change of the detection site with respect to a reference device including a plurality of reference colors corresponding to different hemolysis levels, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 6.

[0079] Exemplary Embodiment 8. The sample application pad is treated with at least one type of red blood cell (RBC) binding or aggregating agent, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 7.

[0080] Exemplary Embodiment 9. The RBC binding or aggregating agent includes lectin, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 8.

[0081] Exemplary Embodiment 10. The RBC binding or aggregating agent includes an anti-human red blood cell (anti-hRBC) binding or aggregating protein antibody, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 9.

[0082] Exemplary Embodiment 11. The RBC binding or aggregating agent is a human red blood cell (hRBC) binding or aggregating protein that is not an antibody, and the chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 10.

[0083] Exemplary Embodiment 12. The chromatographic detection pad does not contain a compound that reacts with free hemoglobin in a liquid biological sample downstream of the sample application site, and the free hemoglobin is detected by a red color change at the detection site. The chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 11.

[0084] Exemplary Embodiment 13. A kit comprising: at least one chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 12; and a reference device including a plurality of reference colors corresponding to different hemolysis levels.

[0085] Exemplary Embodiment 14. A method for testing a liquid biological sample for hemolysis, comprising: applying the liquid biological sample to a sample application pad of a chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 13, enabling plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to a chromatographic detection pad and from the sample application site to the detection site; and visually comparing the color change at the detection site with a reference device including a plurality of reference colors corresponding to different hemolysis levels.

[0086] Exemplary Embodiment 15. A medical diagnostic device assembly comprising: a chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 14; and a medical diagnostic device including: a light source; an optical sensor that detects the amount of red light reflected by the detection site of the chromatographic chemical analyzer and outputs a detection signal, wherein the amount of red light reflected by the detection site is due to the amount of free hemoglobin present in the liquid biological sample; and a processor that receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample.

[0087] Exemplary Embodiment 16. A method for testing a liquid biological sample for hemolysis, comprising: applying the liquid biological sample to a sample application pad of a chromatographic chemical analyzer according to any one of Exemplary Embodiments 1 to 15, enabling plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to a chromatographic detection pad and from the sample application site to the detection site; arranging the chromatographic chemical analyzer in a medical diagnostic device including a light source, an optical sensor that detects the amount of red light reflected by a detection site of the chromatographic chemical analyzer and outputs a detection signal, and a processor that receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample; measuring the amount of red light reflected by the detection site of the chromatographic chemical analyzer, wherein the amount of red light reflected by the detection site is due to the amount of free hemoglobin present in the liquid biological sample; and determining the amount of free hemoglobin present in the liquid biological sample based on the measured amount of reflected red light.

[0088] Exemplary Embodiment 17. The method according to any one of Exemplary Embodiments 1 to 16 further includes a step of displaying a notification indicating that the liquid biological sample is hemolyzed when the measured amount of reflected red light exceeds a predetermined reference value.

[0089] Exemplary Embodiment 18. The method according to any one of Exemplary Embodiments 1 to 17 further includes a step of preventing a subsequent test using the liquid biological sample from being performed when a notification indicating that the liquid biological sample is hemolyzed is displayed.

[0090] Exemplary Embodiment 19. The method according to any one of Exemplary Embodiments 1 to 18 further includes a step of enabling a subsequent test using the liquid biological sample to be performed when the measured amount of reflected red light does not exceed a predetermined reference value, and a step of reporting the result of the subsequent test to a healthcare provider.

[0091] Exemplary Embodiment 20. The liquid biological sample is one of a whole blood sample and urine, according to the method of any one of Exemplary Embodiments 1 to 19.

[0092] As described above, according to the present disclosure, in addition to apparatuses and kits that fully meet the above-described objects and advantages, methods for manufacturing and using these are provided. Although the present disclosure has been described in conjunction with the specific drawings, experiments, results, and representations described above, it will of course be apparent to those skilled in the art that many changes, improvements, and modifications will be possible. Accordingly, it is intended to embrace all such changes, improvements, and modifications that fall within the spirit and broad scope of the present disclosure.

Claims

Claim 1 A chromatographic chemical analyzer for detecting the presence or absence of free hemoglobin in a liquid biological sample, comprising: A sample application pad for applying a liquid biological sample, wherein the sample application pad is formed of a first layer of a pre-filtering material and a second layer of a filtering material different from the pre-filtering material, and wherein the sample application pad is porous to plasma and free hemoglobin but not to red blood cells, so that red blood cells present in the liquid biological sample are retained within the first and second layers of the sample application pad and do not flow through the interior, said sample application pad; A chromatographic detection pad defining a path for capillary fluid flow, said chromatographic detection pad being in fluid contact with the sample application pad and having a first end forming a sample application site on the chromatographic detection pad, said chromatographic detection pad further having a detection site spaced from the sample application site, said chromatographic detection pad; comprising Free hemoglobin flows through the sample application pad into the chromatographic detection pad, flows from the sample application site to the detection site, and is detectable by a color change at the detection site, said chromatographic chemical analyzer. Claim 2 The chromatographic chemical analyzer according to claim 1, wherein the pre-filtering material of the sample application pad comprises a glass fiber material and the filtering material is an asymmetric material comprising polysulfone. Claim 3 The chromatographic chemical analyzer according to claim 1, wherein the chromatographic detection pad is formed of a nitrocellulose membrane. Claim 4 The chromatographic chemical analyzer according to claim 1, wherein the chromatographic detection pad has a pore size in the range of about 8 microns to about 40 microns. Claim 5 The chromatographic chemical analyzer according to claim 1, wherein the chromatographic detection pad is a lateral flow strip. Claim 6 The chromatographic chemical analyzer according to claim 5, wherein the lateral flow strip is configured to be disposed in a medical diagnostic device for detecting a color change at the detection site. Claim 7 The chromatographic chemical analyzer according to claim 1, wherein the chromatographic detection pad is configured to provide a visual comparison of the color change at the detection site with a reference device comprising a plurality of reference colors corresponding to different hemolysis levels. Claim 8 The sample application pad is treated with at least one type of red blood cell (RBC) binding or aggregating agent, and the chromatographic chemical analyzer according to claim 1.

9. The RBC binding or aggregating agent contains lectin, and the chromatographic chemical analyzer according to claim 8.

10. The RBC binding or aggregating agent contains an anti-human red blood cell (anti-hRBC) binding or aggregating protein antibody, and the chromatographic chemical analyzer according to claim 8.

11. The RBC binding or aggregating agent is a human red blood cell (hRBC) binding or aggregating protein that is not an antibody, and the chromatographic chemical analyzer according to claim 8.

12. The chromatographic detection pad does not contain a compound that reacts with free hemoglobin in the liquid biological sample downstream of the sample application site, and the free hemoglobin is detected by a red color change at the detection site. The chromatographic chemical analyzer according to claim 1.

13. A kit comprising: At least one chromatographic chemical analyzer according to claim 1; A reference device including a plurality of reference colors corresponding to different hemolysis levels The kit comprising.

14. A method for testing a liquid biological sample for hemolysis, comprising: Applying a liquid biological sample to the sample application pad of the chromatographic chemical analyzer according to claim 1, and allowing the plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to the chromatographic detection pad and from the sample application site to the detection site; Visually comparing the color change at the detection site with a reference device including a plurality of reference colors corresponding to different hemolysis levels The method comprising.

15. A medical diagnostic device assembly comprising: The chromatographic chemical analyzer according to claim 1; A medical diagnostic device: A light source; An optical sensor that detects the amount of red light reflected by the detection site of the chromatographic chemical analyzer and outputs a detection signal, wherein the amount of red light reflected by the detection site is due to the amount of free hemoglobin present in the liquid biological sample, the optical sensor; A processor that receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample The medical diagnostic device comprising The medical diagnostic device assembly comprising.

16. A method for testing a liquid biological sample for hemolysis, comprising: Applying a liquid biological sample to a sample application pad of the chromatographic chemical analyzer according to claim 1, enabling plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to a chromatographic detection pad and from the sample application site to the detection site; Placing the chromatographic chemical analyzer in a medical diagnostic device including a light source, an optical sensor that detects the amount of red light reflected by a detection site of the chromatographic chemical analyzer and outputs a detection signal, and a processor that receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample; Measuring the amount of red light reflected by a detection site of the chromatographic chemical analyzer, wherein the amount of red light reflected by the detection site is due to the amount of free hemoglobin present in the liquid biological sample; Determining the amount of free hemoglobin present in the liquid biological sample based on the measured amount of reflected red light The method comprising the above.

17. The method further comprises: Displaying a notification indicating that the liquid biological sample is hemolyzed when the measured amount of reflected red light exceeds a predetermined reference value The method according to claim 16, further comprising the above.

18. The method further comprises: Preventing subsequent tests using the liquid biological sample from being performed when a notification indicating that the liquid biological sample is hemolyzed is displayed The method according to claim 17, further comprising the above.

19. The method further comprises: Enabling subsequent tests using the liquid biological sample to be performed when the measured amount of reflected red light does not exceed a predetermined reference value; and Reporting the results of the subsequent tests to a healthcare provider The method according to claim 16, further comprising the above.

20. The method according to claim 16, wherein the liquid biological sample is one of a whole blood sample and urine.

Images