Hemolysis detection using chromatographic assay device

Abstract

Chromatographic assay devices for detecting the presence of free hemoglobin in a liquid biological sample are disclosed. The apparatus includes a sample application pad and a chromatographic detection pad, where the sample application pad is formed of two layers of material. Also disclosed are medical diagnostic devices for use with the chromatographic assay devices, as well as kits and methods of using the chromatographic assay devices and / or medical diagnostic devices.

Description

[0001] CROSS-REFERENCE / INCORPORATION STATEMENT TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 366,558, filed on June 17, 2022. The entire contents of the above patent application are hereby expressly incorporated herein by reference.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] not applicable. Background Art

[0005] Hemolysis refers to the destruction or dissolution of red blood cells (RBCs), which results in the release of hemoglobin ("free hemoglobin") into the surrounding fluid. In the case of a whole blood sample, free hemoglobin is released into the surrounding plasma. In the case of urine, free hemoglobin is released into the surrounding water. Hemolyzed RBCs may occur due to a patient's medical condition or due to improper handling of the sample itself.

[0006] Hemolysis is a pre-analytical error of concern when testing a patient's sample. When severe enough, hemolysis may lead to inaccurate laboratory test results. For example, in blood gas and electrolyte tests, hemolysis is known to cause an increase in the potassium level of the sample. In addition, cardiac troponin T (cTnT) levels are known to be reduced in samples with hemolysis, and cardiac troponin I (cTnl) levels have been shown to be increased in samples with hemolysis.

[0007] Traditionally, hemolysis detection in whole blood samples is difficult and time-consuming. In a central laboratory setting, whole blood samples are subjected to centrifugation, which produces plasma that is optically detected in the near-infrared (NIR) or visible wavelength region. While this technique is very effective, it is complex and time-consuming, making it ineffective for point-of-care (POC) applications.

[0008] In point-of-care diagnostic scenarios, some systems detect hemolysis electrochemically. However, electrochemical detection of hemoglobin and hematocrit is known to be inaccurate; in addition, hemolysis will only be detected after the electrochemical reagents are consumed, which may waste samples, reagents, and time.

[0009] It is important to perform the whole blood sample test as soon as possible after it is drawn; any delay may affect the sample results. In addition, the sample must be aliquoted and centrifuged before hemolysis measurements can be made. However, the requirement for centrifugation limits the circumstances in which hemolysis measurements can be determined, and this requirement necessarily increases the time between sample collection and testing.

[0010] Therefore, there is a need in the art for new and improved devices and methods for hemolysis detection that overcome the shortcomings and deficiencies of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a top view of one non-limiting embodiment of a chromatographic assay device constructed in accordance with the present disclosure.

[0012] Figure 2 yes Figure 1 A cross-sectional view of a chromatographic assay device.

[0013] Figure 3A is a perspective view of another non-limiting embodiment of a chromatographic assay device constructed in accordance with the present disclosure.

[0014] Figure 3B Contains diagrams for use Figure 3A A perspective view of the workflow of the chromatographic assay apparatus.

[0015] Figure 4 One non-limiting embodiment of a reference device for use with a chromatographic assay device constructed in accordance with the present disclosure is schematically depicted.

[0016] Figure 5 Another non-limiting embodiment of a reference device for use with a chromatographic assay device constructed in accordance with the present disclosure is schematically depicted.

[0017] Figure 6 One non-limiting embodiment of a medical diagnostic device constructed in accordance with the present disclosure is schematically depicted. DETAILED DESCRIPTION

[0018] Before explaining at least one embodiment of the present disclosure in detail, it is to be understood that the present disclosure is not limited in its application to the details of the construction and arrangement of the components or steps or methods set forth in the following description or illustrated in the accompanying drawings. The present disclosure is capable of other embodiments or can be practiced or performed in various ways. In addition, it is also to be understood that the wording and terminology used herein are for descriptive purposes and should not be considered to be limiting in any way.

[0019] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural terms and plural terms should include singular terms. The aforementioned techniques and procedures are usually performed according to conventional methods known in the art and as described throughout this specification quoting and discussing various general and more specific references. The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein and its laboratory chamber procedures and techniques are those known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0020] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the technical levels of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0021] In view of the present disclosure, all articles, compositions, kits and / or methods disclosed herein can be made and performed without undue experimentation. Although the articles, compositions, kits and / or methods have been described in accordance with specific embodiments, it will be apparent to those skilled in the art that changes may be applied to the articles, compositions, kits and / or methods described herein and in the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the present disclosure. All such similar substitutions and modifications obvious to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0022] As utilized in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0023] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the terms "a," "an," or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" means "two or more."

[0024] The use of the term "at least one" will be understood to include one and any amount more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can be extended to up to 100 or 1000 or more, depending on the term it is associated with; in addition, the amount of 100 / 1000 should not be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is used solely for the purpose of distinguishing two or more items and is not intended to imply any order or sequence or importance of one item relative to another, or any order of addition, for example.

[0025] The term "or" used in the claims is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives, or unless the alternatives are mutually exclusive. For example, any of the following satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0026] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "an example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. For example, the phrases "in some embodiments" or "an example" appearing in various places in the specification do not necessarily refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be interpreted as non-limiting to the claims.

[0027] Throughout this application, the terms "about" and "approximately" are used to indicate that a value includes the inherent error variation of the composition / device / apparatus, the method used to determine the value, or the variation between the subjects of study. That is, the terms "about" and "approximately" and variations thereof are intended to include not only the exact value defined by the term, but also some slight deviations therefrom, such as, for example, deviations caused by measurement errors, manufacturing tolerances, wear of parts or structures, sedimentation or precipitation of cells or particles in suspensions or solutions, chemical or biological degradation of solutions over time, stresses applied to structures, and combinations thereof. In particular, for example and not as a limitation, when the term "about" is used, the specified value may vary from the specified value by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, as long as such variation is suitable for performing the disclosed methods and is understood by a person of ordinary skill in the art.

[0028] As used in this specification and claims, the terms "comprising" (and any forms of comprising, such as "comprise" and "comprises"), "having" (and any forms of "having", such as "have" and "has"), "including" (and any forms of containing, such as "includes" and "include"), or "containing" (and any forms of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent therein.

[0029] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is important in the particular context. Continuing with this example, expressly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. The skilled artisan will understand that there is generally no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

[0030] As used herein, the term "substantially" means that the subsequent event or situation described completely occurs, or the subsequent event or situation described occurs to a great extent or degree. For example, when associated with a particular event or situation, the term "substantially" means that the subsequent event or situation described occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to each other, or that two items are closely adjacent to each other but not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item but is closely adjacent to the other item.

[0031] As used herein, the terms "associated with" and "coupled to" include both direct association / binding of two moieties to each other and indirect association / binding of two moieties to each other. For example, non-limiting examples of association / coupling include: covalent binding of one moiety to another moiety through a direct bond or through a spacer; non-covalent binding of one moiety to another moiety directly or by means of a specific binding pair member bound to the moiety; incorporation of one moiety into another moiety, such as by dissolving one moiety in the other moiety or by synthesis; and coating one moiety on another moiety.

[0032] The term "sample" as used herein will be understood to include any type of biological sample that can be utilized according to the present disclosure. Examples of fluid biological samples that can be 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, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, stool, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0033] As used herein, the term "liquid sample" and variations thereof are intended to include, but are not limited to, for example, biological fluids (e.g., urine and whole blood), chemical fluids, chemicals, suspensions, solutions, slurries, mixtures, agglomerates, tinctures, slides or other preparations of biological fluids, synthetic analogs of biological fluids, and combinations thereof.

[0034] Turning now to various non-limiting embodiments of the concepts of the present invention, a simple disposable chromatographic assay device is generally disclosed herein for rapid and efficient detection of hemolysis in a liquid sample, thereby notifying a medical professional when a sample is damaged and may produce inaccurate test results. The chromatographic assay device is capable of rapidly detecting hemolysis in a liquid sample using a small sample size and at a low cost per test. The chromatographic assay device rapidly provides hemolysis detection and does not require centrifugation, which prolongs the time period from collection to testing and also limits the environment in which the detection test can be performed. Unlike the prior art methods that require centrifugation, the chromatographic assay device of the present disclosure can be used for testing in all types of environments, including central laboratories and point-of-care locations and home environments. For example (but not as a limitation), the chromatographic assay device of the present disclosure can provide results in a cheap and versatile manner in less than a minute, and the chromatographic assay device can be used in hemolysis detection with or without an analyzer. Visual or optical detection methods can be used, which provides more reliable detection.

[0035] Certain non-limiting embodiments of the present disclosure relate to a chromatographic assay device for detecting the presence of free hemoglobin in a liquid biological sample (e.g., but not limited to, a whole blood sample, urine, or other liquid sample containing red blood cells). 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 assay device. The sample application pad includes at least two different material layers. For example (but not by way of limitation), the sample application pad may include a first material that is laminated on or otherwise attached to a different second material. The at least two different material layers of the sample application pad may differ in, for example, pore size, uniformity or non-uniformity of pore size, material type, and / or material thickness. In an exemplary embodiment, the sample application pad is formed by an upper first layer of a pre-filter material and a lower second layer of a filter material, wherein the pre-filter material and the filter material differ in material type and / or porosity (e.g., pore size and / or uniformity / non-uniformity of pore size). In certain specific (but non-limiting) embodiments, the sample application pad is formed by an upper first layer of pre-filter material and a lower second 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 the voids (pores) are not uniform or their size varies throughout the thickness of the material, for example, the voids may be smaller near one surface (e.g., the bottom surface) and larger near the opposite surface (e.g., the top surface adjacent to the bottom surface of the pre-filter layer). For example, exemplary asymmetric filter materials for use as the lower second layer may include polysulfone, polyethersulfone, or mixed cellulose esters. For example, other exemplary material types for use as filter materials for the lower second layer include potato lectin or treated glass fiber membranes treated with, for example, potato lectin. In another illustrative embodiment, the lower second layer of the sample application pad is formed from a non-asymmetric material, such as a material that includes potato lectin (e.g., glass fibers treated with potato lectin), while the upper first layer of pre-filter material is formed from a different material, such as a material that does not include potato lectin (e.g., glass fibers that are not treated with potato lectin).

[0036] The sample application pad is permeable to plasma and free hemoglobin, but impermeable to red blood cells, such that red blood cells present in the liquid biological sample are retained within the two material layers of the sample application pad and are thereby prevented from flowing through the sample application pad. In particular, but not by way of limitation, the first and / or second material layers have different pore sizes that decrease in size when moving from the upper surface of the sample application pad to its lower surface (i.e., moving in a direction toward the chromatographic detection pad), wherein the first and / or second material layers have at least one pore size therein that is smaller than the size of a red blood cell, thereby preventing red blood cells from flowing through the sample application pad. In an exemplary embodiment, the first pre-filter layer has a less restrictive (e.g., larger) pore size than the pore size of the second filter layer. In another exemplary embodiment, the first pre-filter material layer has a more uniform or symmetrical pore size structure throughout the thickness of the material compared to the second filter layer having a more non-uniform or asymmetric pore size structure.

[0037] Additionally, the various pore sizes present in the two layers of the sample application pad allow larger cellular components (i.e., red blood cells) to be retained but not lysed, while plasma and other free fluids are able to pass through the pores. The combination of the two materials allows the first layer to retain more than the second layer, such that the combination of the two layers allows all of the red blood cells to be retained without overburdening the second layer.

[0038] The present disclosure unexpectedly and advantageously discovered that a chromatographic assay device including a sample application pad having a multi-layer design (e.g., composed of at least two different materials, including a pre-filter material and a filter material, such as an asymmetric filter material) improves the removal or filtration of red blood cells (RBCs) present in a liquid biological sample, such that the RBCs are captured within the multiple layers of the sample application pad, while the plasma and free hemoglobin present in the liquid biological sample are allowed to flow (e.g., by gravity) through the pores of the sample application pad to the chromatographic detection pad. Thus, for example, due to the improved filtration of red blood cells through the multiple layers of the sample application pad, the sample application pad does not need to be treated with a red blood cell binding or agglutinating material. In addition, because the multi-layer or dual-layer sample application pad is directly coupled to the chromatographic detection pad, the chromatographic assay device advantageously provides improved RBC removal and hemolysis detection (by detecting the presence of free hemoglobin) in a single or integrated device. Therefore, the chromatographic assay device advantageously avoids the need to transfer the plasma and free hemoglobin filtered through the sample application pad to a separate device for detecting the presence or amount of free hemoglobin.

[0039] The first layer of the sample application pad may be formed of any material known in the art or otherwise contemplated herein that is capable of retaining at least a portion of the red blood cells and other larger cellular components in the blood sample (without lysing the cells) and thereby reducing the amount of red blood cells and other larger cellular components that flow into the second layer of the sample application pad so as not to overburden the second filter layer. One non-limiting example of a material that can be used to form the pre-filter layer is a fiberglass material or equivalent; however, it is to be understood that other materials that can function in this manner are known in the art and therefore fall within the scope of the term "pre-filter material" as used herein.

[0040] The chromatographic detection pad defines a path for the capillary fluid to flow. The chromatographic detection pad has a first end portion and a second end portion. The first end portion is in fluid contact with the sample application pad and forms a sample application site on the chromatographic detection pad. The chromatographic detection pad also has a detection site, which is separated from the first end portion / sample application site (and in some non-limiting embodiments, is downstream thereof), wherein the detection site is arranged between the first and second end portions or substantially adjacent to the second end portion.

[0041] When a liquid biological sample (such as, but not limited to, a whole blood sample, urine, or other liquid sample containing red blood cells) is applied to the chromatographic assay device, free hemoglobin flows through the sample application pad into the chromatographic detection pad, and then flows from the sample application site of the chromatographic detection pad to its detection site. In this way, free hemoglobin (indicating hemolysis) can be detected at the detection site by a color change.

[0042] The chromatographic detection pad can be formed of any material that is white in color (thereby allowing visual reading) and has a pore size that allows free hemoglobin and plasma to flow freely therethrough, whereby the chromatographic detection pad is able to define a capillary flow path for detecting free hemoglobin in the sample. In a specific (but non-limiting) embodiment, the chromatographic detection pad is formed of a nitrocellulose membrane.

[0043] Each of the sample application pad and the chromatographic detection pad can be provided with any pore size that will allow the sample application pad and the chromatographic assay device to function according to the present disclosure. Non-limiting examples of pore sizes that can be utilized according to the present disclosure include about 0.1 microns, about 0.22 microns, about 0.3 microns, about 0.45 microns, about 0.5 microns, about 1 micron, about 2 microns, about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, about 10 microns, about 11 microns, about 12 microns, about 13 microns, about 14 microns, about 15 microns, about 16 microns, about 17 microns, about 18 microns, about 19 microns, about 20 microns, about 21 microns, about 22 microns, about 23 microns, about 24 microns, about 25 microns, about 26 microns, about 27 microns, about 28 microns, about 29 microns, about 30 microns, about 31 microns, about 32 microns, about 33 microns, about 34 microns, about 35 microns, about 36 microns, about 37 microns, about 38 microns, about 39 microns, about 40 microns, about 41 microns, about 42 microns, about 43 microns, about 44 microns, about 45 microns, about 46 microns, about 47 microns, about 48 microns, about 49 microns, about 50 microns, about 51 microns, about 52 microns, about 53 microns, about 54 microns, about 55 microns, about 56 microns, about 57 microns, about 58 microns, about 59 microns, about About 27 microns, about 28 microns, about 29 microns, about 30 microns, about 31 microns, about 32 microns, about 33 microns, about 34 microns, about 35 microns, about 36 microns, about 37 microns, about 38 microns, about 39 microns, about 40 microns, about 41 microns, about 42 microns, about 43 microns, about 44 microns, about 45 microns, about 46 microns, about 47 microns, about 48 microns, about 49 microns and about 50 microns, and ranges formed by any of the above values ​​(i.e., a range from about 0.1 micron to about 6 microns, a range from 8 microns to about 40 microns, a range from about 8 microns to about 13 microns, etc.).

[0044] In certain specific (but non-limiting) embodiments, the chromatographic assay device may further include a backing material to which the chromatographic test pad is attached or otherwise associated.

[0045] In some specific (but non-limiting) embodiments, the chromatographic detection pad is a lateral flow strip. In a specific (but non-limiting) embodiment, the lateral flow strip is configured to be arranged in a medical diagnostic device for detecting the color change at the detection site or is attached to the medical diagnostic device. In another specific (but non-limiting) embodiment, the lateral flow strip is designed to be used manually by an individual. In this way, the color change at the detection site is detected by an individual by visual comparison with a reference device, and the reference device comprises a plurality of reference colors corresponding to different hemolysis levels.

[0046] In certain non-limiting embodiments, the sample application pad may be provided with one or more additives that act to further ensure that no red blood cells pass through the sample application pad and enter the chromatographic detection pad. For example, but not by way of limitation, the sample application pad may be treated with at least one type of red blood cell (RBC) binding or agglutination material such that the RBC binding or agglutination material is present therein in a free, soluble form. The RBC binding or agglutination material agglutinates with any RBC in the whole blood sample to produce agglutinated RBCs, wherein the agglutinated RBCs have a size greater than the pore size of the sample application pad and are unable to flow through the sample application pad, while any free RBC binding or agglutination material can enter the chromatographic detection pad without interfering with the detection of free hemoglobin. Non-limiting examples of RBC binding or agglutination materials that can be utilized according to the present disclosure include agglutinins, anti-human red blood cell (anti-hRBC) binding or agglutination protein antibodies, and non-antibody human red blood cell (hRBC) binding or agglutination proteins.

[0047] The devices and methods of the present disclosure rely on visual or optical detection of hemolysis by a color change in the chromatographic detection pad (e.g., but not limited to, a change from the default clear or white color of the pad to a shade of milky white, yellow, pink, or red) due to the red color of free hemoglobin. Thus, in certain non-limiting embodiments, the chromatographic detection pad lacks compounds that react with free hemoglobin in the liquid biological sample downstream of the sample application site.

[0048] Instead of a chromatographic detection pad lacking a reactive compound, the chromatographic detection pad may include one or more reagents that react with free hemoglobin present in a liquid sample flowing through the chromatographic detection pad. Exemplary reagents present in the chromatographic detection pad may exacerbate color changes in the detection zone that are attributable to free hemoglobin. Non-limiting exemplary reagents utilize the peroxidase-like activity of hemoglobin, which catalyzes the reaction of diisopropylbenzenedihydroperoxide and 3,3',5,5'-tetramethylbenzidine. The resulting color ranges from orange to green, and possibly to blue. Exemplary reagents located in the detection pad may be arranged (for example, but not by way of limitation) in strips arranged perpendicular to the direction of flow.

[0049] Certain non-limiting embodiments of the present disclosure relate to a kit comprising at least one of any chromatographic assay devices described above or otherwise contemplated herein, in combination with a reference device for use in visual detection of hemoglobin by an individual. The reference device comprises a plurality of reference colors, wherein each reference color corresponds to a different level, degree or amount of hemolysis. The reference device may take any form or style known in the art or otherwise contemplated herein. For example (but not as a limitation), the kit may include a container in which the chromatographic assay device is stored before use, and a portion of the outer surface of the container may have a reference device attached thereto or formed thereon. Alternatively, the reference device may be an insert card present in the container.

[0050] In addition to the chromatographic assay device and the reference device, the kit may also include other components or reagents for use when performing any specific assay described herein or otherwise contemplated. The properties of these additional reagents will depend on the specific assay format, and their identification is within the skill of those of ordinary skill in the art; therefore, it is believed that there is no need to further describe them. In addition, the compositions / reagents present in the kit may each be in a separate container / compartment, or various compositions / reagents may be combined in one or more containers / compartments, depending on the reactivity and stability of the compositions / reagents. For example (but not limited to), the kit may also include positive and / or negative control reagents. In addition, the kit may also include a set of written instructions explaining how to use the kit. A kit with this property may be used in any method described herein or otherwise contemplated.

[0051] Certain non-limiting embodiments of the present disclosure relate to methods for testing a liquid biological sample for hemolysis. In the method, the liquid biological sample is applied to a sample application pad of any chromatographic assay device disclosed above or otherwise contemplated herein, and the plasma and any free hemoglobin present in the liquid biological sample is allowed to flow through the sample application pad to the chromatographic detection pad, and from its sample application site to the detection site. The color change at the detection site is then visually compared to a reference device containing a plurality of reference colors, wherein each reference color corresponds to a different level of hemolysis. The visual comparison step may be performed manually by an individual, or may be performed by a diagnostic device.

[0052] Certain non-limiting embodiments of the present disclosure relate to a medical diagnostic device assembly. The assembly includes any chromatographic assay device disclosed above or otherwise contemplated herein in combination with a medical diagnostic device. 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 a detection site of the chromatographic assay device and outputs a detection signal; the amount of red light reflected by the detection site is attributable to the amount of free hemoglobin present in a liquid biological sample. The processor receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample.

[0053] Certain non-limiting embodiments of the present disclosure relate to methods for testing a liquid biological sample (e.g., but not limited to, a whole blood sample, urine, or other liquid sample containing red blood cells) for hemolysis. In the method, the liquid biological sample is applied to a sample application pad of any chromatographic assay device disclosed herein or otherwise contemplated, and the plasma and any free hemoglobin present in the liquid biological sample are allowed to flow through the sample application pad to the chromatographic detection pad and from its sample application site to the detection site. The chromatographic assay device is placed in a medical diagnostic device including a light source, an optical sensor, and a processor, wherein the optical sensor detects the amount of red light reflected by the detection site of the chromatographic assay device and outputs a detection signal, and wherein the processor receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample. The amount of red light reflected by the detection site of the chromatographic assay device is measured, and the amount of red light reflected by the detection site can be attributed to the amount of free hemoglobin present in the liquid biological sample. Then, the amount of free hemoglobin present in the liquid biological sample is determined based on the measured amount of reflected red light.

[0054] In certain specific (but non-limiting) embodiments, the method may further include the 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. When this occurs, in certain (but non-limiting) embodiments, the method may further include the step of: preventing subsequent tests from being performed using the liquid biological sample when the notification that the liquid biological sample is hemolyzed is displayed.

[0055] In another specific (but non-limiting) embodiment, the method may further include the steps of: allowing subsequent testing to be performed using the liquid biological sample when the measured amount of reflected red light does not exceed a predetermined reference value; and reporting the results of the subsequent testing to a healthcare provider.

[0056] Reference now Figure 1 and Figure 2 , a chromatographic assay device 10 for detecting the presence of free hemoglobin in a liquid biological sample 12 (such as, but not limited to, whole blood, urine, or other samples containing red blood cells) is shown. The chromatographic assay device 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 assay device 10. The sample application pad 14 can receive and absorb the liquid biological sample 12, and the liquid biological sample 12 can then be absorbed from the sample application pad 14 into the chromatographic detection pad 16.

[0057] 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 two layers of material: an upper first layer 30 formed of a pre-filter material (such as, but not limited to, a fiberglass material or other suitable pre-filter material); and a lower second 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 should be understood that the lower second layer 32 can be formed of a non-asymmetric material, wherein the lower second layer 32 is formed of a different material than the upper first layer 30, as described above. The first layer 30 of pre-filter material forms the upper surface 24 of the sample application pad 14, while the second layer 32 of 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 first surface of the first layer 30 is the upper surface 24 of the sample application pad 14, which is configured to receive the liquid biological sample 12, such as (but not limited to) directly receiving the liquid biological sample 12 from, for example (but not limited to), a syringe, a pipette, a dropper, a balloon, a capillary or other dispensing device. The second surface of the first layer 30 is directly formed on and / or directly attached to the first surface of the second layer 32, and 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 Figure 2 In the embodiment shown in , the sample application pad 14 is advantageously formed by a first layer 30 that is directly formed on and / or attached to a second layer 32 without an additional intervening layer (excluding means for forming and / or attaching the first layer 30 to the second layer 32).

[0058] The sample application pad 14 is permeable to plasma and free hemoglobin present in the liquid biological sample 12, but is impermeable to red blood cells, so that the red blood cells present in the liquid biological sample 12 are retained in the two material layers 30 and 32 of the sample application pad 14 and are thereby prevented from flowing therethrough to the chromatographic detection pad 16.

[0059] The chromatographic detection pad 16 defines a path for capillary fluid flow. The components of the liquid biological sample 12 that can flow through the sample application pad 14 then flow through the chromatographic detection pad 16 by capillary action (which may also be referred to as capillary flow). The chromatographic detection pad 16 has a first end portion 40 and a second end portion 42. The chromatographic detection pad 16 can be made of any suitable material that allows plasma and free hemoglobin from the liquid biological sample 12 to flow freely through the material by capillary action. As a non-limiting example, the chromatographic detection pad 16 can be a nitrocellulose membrane. The chromatographic detection pad 16 can have holes, and certain components of the liquid sample 12 move through these holes by capillary action. Most of the holes in the chromatographic detection pad 16 can all be substantially the same size or fall within a value range.

[0060] The first end portion 40 of the chromatographic assay 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 chromatographic test pad 16. The chromatographic test pad 16 also has a detection site 50 spaced apart from (and in certain non-limiting embodiments, downstream of) the first end portion 40 / sample application site 48, wherein the detection site 50 is disposed between the first and second end portions 40 and 42, or substantially adjacent to the second end portion 42 or closer to the second end portion 42 than the first end portion 40.

[0061] In addition, the sample application pad 14 covers only the portion of the chromatographic detection pad 16 adjacent to its first end portion 40 and the sample application site 48, but not its detection site 50; in this manner, the flow of the sample through the chromatographic detection pad 16 into its detection site 50 is visible. For example (but not by way of limitation), the second end 22 of the sample application pad 14 is positioned adjacent to the downstream end of the sample application site 48 or extends only slightly beyond the downstream end, so that the flow of the sample beyond the sample application site 48 toward the detection site 50 is visible.

[0062] The chromatographic assay device 10 may also include a backing material 18 to which the lower surface 46 of the chromatographic test pad 16 is attached or otherwise associated (eg, but not limited to, via double-sided tape).

[0063] When a liquid biological sample 12 (such as, but not limited to, a whole blood sample, urine, or other liquid sample containing red blood cells) is applied to the chromatographic assay device 10, free hemoglobin flows through the sample application pad 14 into the chromatographic detection pad 16, and then flows from the sample application site 48 of the chromatographic detection pad 16 to its detection site 50. In this way, free hemoglobin (indicative of hemolysis) can be detected at the detection site by a color change due to the red color of free hemoglobin.

[0064] Although Figure 1-2 A specific non-limiting embodiment of the chromatographic assay device 10 is shown in FIG. 1 , but it will be understood that the design and construction of the chromatographic assay device 10 shown is for illustrative purposes only. The scope of the present disclosure includes adjustments to the design and construction of the chromatographic assay device of the present disclosure, as long as the chromatographic assay device is still able to function according to the present disclosure.

[0065] For example (but not by way of limitation), it will be appreciated that the first layer (of pre-filter material) and the second layer (of asymmetric filter material) of the sample application pad need not be symmetrical to one another (i.e., they may differ from one another in size, length, width, and / or thickness). Additionally, the first and second layers of the sample application pad need not be identical to one another, and thus each layer may have areas that do not overlap with the other layer. The only requirement is that at least a portion of the first layer must overlap a sufficient portion of the second layer so that the sample can flow through the first layer into the second layer, and then from the second layer into the sample application site of the chromatographic detection pad. Figure 3A A chromatographic assay device 10a is depicted, which is Figure 1-2 The chromatographic assay device 10 of the present invention is similar to that of the chromatographic assay device 10 of the present invention, except that the chromatographic assay device 10a has a sample application pad 14a formed of two separate layers, which have different sizes 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 of the first layer 30a adjacent to its second end 142 overlaps a portion of the second layer 32a between its first end 148 and the second end 150. The overlapping portions of the first layer 30a and the second layer 32a may be attached to each other, or the overlapping portions of the first layer 30a may simply be stacked on the second layer 32a so that a portion of the lower surface 146 of the first layer 30a contacts a portion of the upper surface 152 of the second layer 32a.

[0066] Figure 3B Picture shows Figure 3A The workflow of the chromatographic assay device 10a, however, should be noted that Figure 2 The workflow of the chromatographic assay device 10 is substantially the same. The blood sample 12a is applied to the first layer 30a of the sample application pad 14a and enters the first layer 30a. Figure 3BThe second figure illustrates a blood sample 12a applied to the upper surface 144 of the first layer 30a, but it will be understood that when the first layer 30a and the second layer 32a do not completely overlap each other, the blood sample 12a can optionally be applied to the lower surface 146 of the first layer 30a in its non-overlapping portion. The blood sample 12a then penetrates the first layer 30a and flows into the second layer 32a through the overlapping portion (third figure). The plasma from the blood sample 12a then passes through the second layer 32a and enters the chromatographic detection pad 16a and flows from the sample application site 48a to its detection site 50a to detect any hemolysis present (fourth figure). In a non-limiting embodiment, the plasma reaching the detection site 50a changes the control line from yellow to blue, thereby indicating that the device 10a is ready to be read.

[0067] Figure 4 One non-limiting embodiment of a reference device 120 is illustrated that can be used in conjunction with the chromatographic assay device 10 to visually determine the level of hemolysis in the liquid sample 12. The reference device 120 includes a plurality of reference colors (e.g., but not limited to, reference colors 122, 124, 126, 128, and 130, where color 122 has the white / default color of the chromatographic detection pad 16 and serves as a negative control, and colors 124, 126, 128, and 130 are various shades of pink / red that increase in intensity / hue, where darker intensities / hues are associated with higher amounts / degrees of hemolysis); the five reference colors shown are for illustrative purposes only). In addition, the reference device 120 also includes a legend 132 that associates each of the reference colors 122, 124, 126, 128, and 130 with a specific concentration of free hemoglobin. That is, (and for purposes of illustration only), color 122 of legend 132 is the negative control, while 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. In this manner, an individual can determine the level of hemolysis in a liquid biological sample in any setting, including but not limited to a point of care or home setting, by comparing the color at the detection site 50 to the reference colors 124-130 of the reference device 120.

[0068] Figure 4 The design and construction of reference device 120 is shown for illustrative purposes only; it will be understood that reference device 120 may have less than five reference colors or more than five reference colors disposed thereon (e.g., but not limited to, two, three, four, five, six, seven, eight, nine, ten, or more reference colors disposed thereon). Additionally, the shapes and arrangements of the reference colors may vary. In addition, legend 132 may also be provided with reference colors that are similar to those of FIG. Figure 4That is, the design and construction of each of the various components of the reference device 120 (such as, but not limited to, the reference colors and legend 132) can be easily adjusted by one of ordinary skill in the art to have any design and construction that will allow the reference device 120 to function in accordance with the present disclosure.

[0069] For example (but not by way of limitation), Figure 5 Another non-limiting embodiment of a reference device is illustrated, indicated herein by reference numeral 120a, which may be used in conjunction with the chromatographic assay device 10 to visually determine the level of hemolysis in the fluid sample 12. Figure 4 The reference device 120 contains discrete reference colors, but Figure 5 The reference device 120a includes a plurality of reference color ranges (such as, but not limited to, reference color ranges 122a, 124a, 126a, and 128a, wherein color 122a has the white / default color of the chromatographic detection pad 16 and serves as a negative control, and color ranges 124a, 126a, and 128a each include a range of various shades of pink / red of increasing intensity / hue, wherein darker intensities / hues are associated with higher amounts / degrees of hemolysis); four reference color ranges are shown for illustrative purposes only). The reference device 120a also includes a legend 132a that associates a specific concentration range of free hemoglobin with each of the reference color ranges 122a, 124a, 126a, and 128a. That is, (and for purposes of illustration only), color 122a of legend 132a is the negative control, while color range 124a indicates the presence of 0-<100 mg / dL of free hemoglobin, color range 126a indicates the presence of 100-<500 mg / dL of free hemoglobin, and color range 128a indicates the presence of 500-1000 mg / dL of free hemoglobin. In this manner, an individual can determine the level of hemolysis in a liquid biological sample in any setting, including but not limited to a point of care or home setting, by comparing the color at the detection site 50 to the reference color range 124a-128a of the reference device 120a.

[0070] Alternatively, a medical diagnostic device may be used to optically detect the level of hemolysis in a liquid biological sample. Figure 6 , depicting a medical diagnostic device 100. The medical diagnostic device 100 includes an optical sensor 102, a processor 104, and a detection site directed to a chromatographic assay device (such as but not limited to Figure 1-2The optical sensor 102 captures one or more images of the detection site 50 and transmits the images to the processor 104 as a detection signal 108. The processor 104 then analyzes the characteristics of the light reflected by the detection site 50 of the chromatographic assay device 10 based on the received images. The characteristics of the light reflected by the detection site 50, such as observable colors (e.g., red, orange, green, and blue), can be attributed to the presence of free hemoglobin in the liquid sample 12. Therefore, the characteristics of the reflected light can be used by the processor to quantify the amount of free hemoglobin present in the sample liquid 12. For example, when the detection site / area 50 lacks compounds located downstream of the sample application site 48 of the sample application pad 14 or the chromatographic detection pad 16, the amount / intensity of red light reflected by the detection site 50 can be used to quantify the amount of free hemoglobin present in the liquid sample 12. When the chromatographic detection pad 16 contains a reagent that reacts with free hemoglobin and is located downstream of the sample application site 48, the amount / intensity of one or more of the red, orange, green, or blue light reflected by the detection site 50 can be used to quantify the amount of free hemoglobin present in the liquid sample 12. Thus, for example, the processor 104 can determine the amount of free hemoglobin in the liquid sample 12 by comparing the measured amount of observable color of the light reflected by the detection site 50 to a known reference value. It should also be understood that the processor 104 need not be located within the device 100 and can be located at an external location.

[0071] In one non-limiting embodiment, the light source 106 may be a broadband light source and the optical sensor 102 may employ a two-dimensional pixel array that captures a two-dimensional image of the detection site 50. The processor 104 may be configured to select specific regions of interest within the image of the chromatographic assay substrate, analyze the spectral content and surface topography of the region of interest on the substrate, determine porosity and depth variations in the region of interest, and algorithmically improve the selectivity, dynamic range, and signal-to-noise ratio of primary signals of interest that would otherwise be degraded by variations in the detection region, residual sample turbidity, and chemical interferents.

[0072] A method for testing a liquid sample for hemolysis may include measuring a characteristic of light reflected by a detection site 50 of a chromatographic assay device 10 after a portion of a liquid sample 12 has been applied to a sample application pad 14 and free hemoglobin has flowed from a sample application site 48 through the sample application pad 14 and into a chromatographic detection pad 16 to a detection site 50, as described above. The measured amount of, for example, red, orange, green, and / or blue light may then be used to determine the level of free hemoglobin by, for example, comparing the measured amount to one or more reference values. In an exemplary embodiment, the method may be performed by the device 100 or by a medical provider. For example, a medical provider may compare a completed chromatographic assay device 10 to a reference device (such as, but not limited to, Figure 4 and Figure 5 ), wherein the reference device comprises a plurality of reference colors, each of which corresponds to a different hemolysis level, so as to visually determine the hemolysis of the liquid sample 12.

[0073] The method can be used to detect hemoglobin levels that exceed a predetermined interference value (e.g., a manufacturer's interference level). If the sample is above the interference value, the sample will be marked to inform the end user (i.e., the associated healthcare provider) that the sample is hemolyzed and therefore damaged. When the medical diagnostic device 100 is capable of performing additional tests on the liquid sample 12 after determining that the liquid sample 12 is hemolyzed, the device 100 can prevent the subsequent tests from being performed using the liquid sample 12, or allow the subsequent tests to be performed using the liquid sample 12, but inform the end user to take into account the hemolysis of the liquid sample 12 when interpreting the results of the subsequent tests.

[0074] Processor 104 may 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, a combination thereof, or any other now known or later developed device for processing data. Likewise, processing strategies may include multi-processing, multi-tasking, parallel processing, etc. Programs may be uploaded to and executed by the processor. The processor implements the program individually, or multiple processors may be included in a network or system for parallel or sequential processing.

[0075] The processor outputs the status and / or associated information on a display, into a memory, over a network, to a printer, or in another medium. The display is a text, graphic, or other display.

[0076] The display is a CRT, LCD, plasma, projector, monitor, printer or other output device for displaying data. The display is operable to output a state associated with the patient to the user. The state provides an indication of whether the medical concept is noted in the medical record. The state may be whether to indicate a disease, condition, symptom or test result. In one embodiment, the state is limited to true and false, or true, false and unknown. In other embodiments, the state can be a level or other non-Boolean state in a level range.

[0077] The processor 104 operates according to the instructions. These instructions can be implemented in a program. The program can be a non-temporary computer-readable medium storing instructions, which, when executed by at least one processor 104, causes 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 of the techniques described herein. The program may be located in a non-temporary computer-readable memory, such as an external memory, 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 media, hard disk, or other computer-readable storage medium. Computer-readable storage media include various types of volatile and non-volatile storage media. The functions, actions, or tasks shown in the drawings or described herein are performed in response to one or more groups of instructions stored in or on a computer-readable storage medium. These functions, actions, or tasks are independent of a particular type of instruction set, storage medium, processor, or processing strategy, and can be performed by software, hardware, integrated circuits, firmware, microcode, etc., operating alone or in combination. In one embodiment, the instructions are stored on a removable media device for reading by a local or remote system. In other embodiments, the instructions are stored in a remote location to be transmitted over a computer network or over a telephone line, wireless GSM or WiFi. In yet other embodiments, the instructions are stored in a given computer, CPU, GPU or system. Since some of the component system components and method actions depicted in the accompanying drawings can be implemented with software, the actual connection between the system components (or process steps) may vary depending on the programming method.

[0078] Non-limiting illustrative embodiments

[0079] The following is a list of non-limiting illustrative embodiments disclosed herein:

[0080] Illustrative embodiments 1. A chromatographic assay device for detecting the presence of free hemoglobin in a liquid biological sample, the device comprising: a sample application pad for applying the liquid biological sample, wherein the sample application pad is formed by a first layer of a pre-filter material and a second layer of a filter material different from the pre-filter material, wherein the sample application pad is permeable to plasma and the free hemoglobin, but impermeable to red blood cells, so that the red blood cells present in the liquid biological sample are retained within the first layer and the second layer of the sample application pad and are thereby prevented from flowing through the sample application pad; and a chromatographic detection pad defining a path for capillary fluid flow, the chromatographic detection pad having a first end portion that is in fluid contact with the sample application pad and forms a sample application site on the chromatographic detection pad, the chromatographic detection pad also having a detection site separated from the sample application site; wherein the free hemoglobin flows into the chromatographic detection pad through the sample application pad and flows from the sample application site to the detection site and can be detected by a color change at the detection site.

[0081] Illustrative Embodiment 2. The chromatographic assay device of Illustrative Embodiment 1, wherein the pre-filter material of the sample application pad comprises a glass fiber material, and wherein the filter material is an asymmetric material and comprises polysulfone.

[0082] Illustrative embodiment 3. The chromatographic assay device of Illustrative embodiment 1 or 2, wherein the chromatographic detection pad is formed of a nitrocellulose membrane.

[0083] Illustrative Embodiment 4. The chromatographic assay device of any of Illustrative Embodiments 1-3, wherein the chromatographic detection pad has a pore size in a range from about 8 microns to about 40 microns.

[0084] Illustrative Embodiment 4A. The chromatographic assay device of any of the preceding illustrative embodiments, wherein the pore size of the chromatographic test pad is in a range from about 8 microns to about 13 microns.

[0085] Illustrative Embodiment 5. The chromatographic assay device of any one of the preceding illustrative embodiments, wherein the chromatographic test pad is a lateral flow strip.

[0086] Illustrative Embodiment 6. The chromatographic assay device of any of the preceding illustrative embodiments, wherein the lateral flow strip is configured for placement in a medical diagnostic device that detects the color change at the detection site.

[0087] Illustrative Embodiment 7. The chromatographic assay device of any of Illustrative Embodiments 1-6, wherein the chromatographic test 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 levels of hemolysis.

[0088] Illustrative Embodiment 8. The chromatographic assay device of any of Illustrative Embodiments 1-7, wherein the sample application pad is treated with at least one type of red blood cell (RBC) binding or agglutinating material.

[0089] Illustrative Embodiment 9. The chromatographic assay device of any of the preceding illustrative embodiments, wherein the RBC binding or agglutinating material comprises a lectin.

[0090] Illustrative Embodiment 10. The chromatographic assay device of any of the preceding illustrative embodiments, wherein the RBC binding or agglutinating material comprises an anti-human red blood cell (anti-hRBC) binding or agglutinating protein antibody.

[0091] Illustrative Embodiment 11. The chromatographic assay device of any of the preceding illustrative embodiments, wherein the RBC binding or agglutinating material is a non-antibody human red blood cell (hRBC) binding or agglutinating protein.

[0092] Illustrative Embodiment 12. The chromatographic assay device of any one of Illustrative Embodiments 1-11, wherein the chromatographic detection pad lacks a compound that reacts with the free hemoglobin in the liquid biological sample downstream of the sample application site, and wherein the free hemoglobin is detected by a red color change at the detection site.

[0093] Illustrative embodiment 13. A kit comprising: at least one chromatographic assay device as described in any one of illustrative embodiments 1-12; and a reference device comprising a plurality of reference colors, wherein each reference color corresponds to a different level of hemolysis.

[0094] Illustrative embodiment 14. A method for testing a liquid biological sample for hemolysis, the method comprising the steps of: applying the liquid biological sample to a sample application pad of a chromatographic assay device as described in any of illustrative embodiments 1-13, and allowing plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to a chromatographic detection pad and from its sample application site to a detection site; and visually comparing the color change at the detection site with a reference device containing multiple reference colors, wherein each reference color corresponds to a different level of hemolysis.

[0095] Illustrative embodiment 15. A medical diagnostic device component, the component comprising: a chromatographic assay device as described in any one of illustrative embodiments 1-14; and a medical diagnostic device, the medical diagnostic device comprising: a light source; an optical sensor, which detects the amount of red light reflected by a detection site of the chromatographic assay device and outputs a detection signal, the amount of red light reflected by the detection site being attributable to the amount of free hemoglobin present in the liquid biological sample; and a processor, which receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample.

[0096] Illustrative embodiment 16. A method for testing a liquid biological sample for hemolysis, the method comprising the following steps: applying the liquid biological sample to a sample application pad of a chromatographic assay device as described in any of illustrative embodiments 1-15, and allowing plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to a chromatographic detection pad and from its sample application site to a detection site; placing the chromatographic assay device in a medical diagnostic device including a light source, an optical sensor, and a processor, wherein the optical sensor detects an amount of red light reflected by a detection site of the chromatographic assay device and outputs a detection signal, and wherein the processor 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 assay device, wherein the amount of red light reflected by the detection site can be attributed 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.

[0097] Illustrative Embodiment 17. The method as recited in any one of the preceding illustrative embodiments, further comprising the 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.

[0098] Illustrative Embodiment 18. The method as recited in any of the preceding illustrative embodiments, further comprising the step of preventing subsequent tests from being performed using the liquid biological sample when the notification that the liquid biological sample is hemolyzed is displayed.

[0099] Illustrative embodiment 19. The method as described in any of the preceding illustrative embodiments further includes the following steps: when the measured amount of reflected red light does not exceed a predetermined reference value, allowing subsequent testing to be performed using the liquid biological sample; and reporting the results of the subsequent testing to a healthcare provider.

[0100] Illustrative Embodiment 20. The method as recited in any one of the preceding illustrative embodiments, wherein the liquid biological sample is one of a whole blood sample and urine.

[0101] Therefore, according to the present disclosure, devices and kits, as well as methods of producing and using them, are provided, which fully meet the purposes and advantages set forth above. Although the present disclosure has been described in conjunction with the specific figures, experiments, results and language set forth above, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. A chromatographic assay device for detecting the presence of free hemoglobin in a liquid biological sample, the device comprising: a sample application pad for applying the liquid biological sample, wherein the sample application pad is formed by a first layer of a pre-filter material and a second layer of a filter material different from the pre-filter material, wherein the sample application pad is permeable to plasma and the free hemoglobin but impermeable to red blood cells, such that red blood cells present in the liquid biological sample are retained within the first and second layers of the sample application pad and thereby prevented from flowing through the sample application pad; and a chromatographic detection pad defining a path for capillary fluid flow, the chromatographic detection pad having a first end portion in fluid contact with the sample application pad and forming a sample application site on the chromatographic detection pad, the chromatographic detection pad also having a detection site spaced apart from the sample application site; The free hemoglobin flows into the chromatographic detection pad through the sample application pad and flows from the sample application site to the detection site, and can be detected by a color change at the detection site.

2. The chromatographic measuring device according to claim 1, wherein: The pre-filter material of the sample application pad comprises a glass fiber material, and wherein the filter material is an asymmetric material and comprises polysulfone.

3. The chromatographic measuring device according to claim 1, wherein: The chromatographic detection pad is formed of nitrocellulose membrane.

4. The chromatographic measuring device according to claim 1, wherein: The chromatographic test pad has a pore size in a range from about 8 microns to about 40 microns.

5. The chromatographic measuring device according to claim 1, wherein: The chromatographic detection pad is a lateral flow strip.

6. The chromatographic measuring device according to claim 5, wherein: The lateral flow strip is configured for placement in a medical diagnostic device that detects the color change at the detection site.

7. The chromatographic assay device according to claim 1, wherein: The chromatographic test pad is configured to provide a visual comparison of the color change at the test site with a reference device comprising a plurality of reference colors corresponding to different levels of hemolysis.

8. The chromatographic assay device according to claim 1, wherein: The sample application pad is treated with at least one type of red blood cell (RBC) binding or agglutinating material.

9. The chromatographic assay device according to claim 8, wherein: The RBC binding or agglutinating material comprises a lectin.

10. The chromatographic assay device according to claim 8, wherein: The RBC binding or agglutinating material includes anti-human red blood cell (anti-hRBC) binding or agglutinating protein antibodies.

11. The chromatographic assay device according to claim 8, wherein: The RBC binding or agglutinating material is a non-antibody human red blood cell (hRBC) binding or agglutinating protein.

12. The chromatographic assay device according to claim 1, wherein: The chromatographic detection pad lacks a compound that reacts with the free hemoglobin in the liquid biological sample downstream of the sample application site, and wherein the free hemoglobin is detected by a red color change at the detection site.

13. A kit comprising: At least one chromatographic assay device according to claim 1; as well as A reference set comprising a plurality of reference colors, wherein each reference color corresponds to a different level of hemolysis.

14. A method for testing a liquid biological sample for hemolysis, the method comprising the steps of: Applying the liquid biological sample to A sample application pad of the chromatographic assay device according to claim 1, and allowing plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to the chromatographic detection pad and from its sample application site to the detection site; as well as The color change at the detection site is visually compared to a reference device containing a plurality of reference colors, wherein each reference color corresponds to a different level of hemolysis.

15. A medical diagnostic device assembly, the assembly comprising: The chromatographic assay device according to claim 1; as well as A medical diagnostic device, comprising: light source; an optical sensor that detects an amount of red light reflected by a detection site of the chromatographic assay device and outputs a detection signal, the amount of red light reflected by the detection site being attributable to an amount of free hemoglobin present in the liquid biological sample; and A processor receives the detection signal and determines the amount of free hemoglobin in the liquid biological sample.

16. A method for testing a liquid biological sample for hemolysis, the method comprising the steps of: Applying the liquid biological sample to A sample application pad of the chromatographic assay device according to claim 1, and allowing plasma and free hemoglobin present in the liquid biological sample to flow through the sample application pad to the chromatographic detection pad and from its sample application site to the detection site; placing the chromatographic assay device in a medical diagnostic device including a light source, an optical sensor, and a processor, wherein the optical sensor detects an amount of red light reflected by a detection site of the chromatographic assay device and outputs a detection signal, and wherein the processor receives the detection signal and determines an amount of the free hemoglobin in the liquid biological sample; measuring an amount of red light reflected by the detection site of the chromatographic assay device, wherein the amount of red light reflected by the detection site is attributable to an amount of the free hemoglobin present in the liquid biological sample; and Based on the measured amount of reflected red light, the amount of free hemoglobin present in the liquid biological sample is determined.

17. The method according to claim 16, further comprising the steps of: When the measured amount of reflected red light exceeds a predetermined reference value, a notification that the liquid biological sample is hemolyzed is displayed.

18. The method according to claim 17, further comprising the steps of: When the notification that the liquid biological sample is hemolyzed is displayed, subsequent tests are prevented from being performed using the liquid biological sample.

19. The method according to claim 16, further comprising the steps of: allowing subsequent testing to be performed using the liquid biological sample when the measured amount of reflected red light does not exceed a predetermined reference value; as well as The results of such follow-up tests are reported to a healthcare provider.

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

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