A rapid test card for ABO, RhD blood typing and sickle cell anemia

Abstract

This invention discloses a rapid test card for ABO, RhD blood typing and sickle cell anemia, comprising a card holder, a card cover, an ABD test strip, and an SCD test strip. The ABD and SCD test strips are installed in the card holder, and the card cover is affixed to the card holder. This invention can simultaneously detect ABO, RhD blood typing, and SCD. By improving sample processing, test point layout, and test strip design, it overcomes the shortcomings of existing technologies, significantly improving the accuracy, simplicity, and efficiency of the test, making it particularly suitable for point-of-care testing.

Description

Technical Field

[0001] This utility model specifically relates to a rapid detection card for ABO, RhD blood types and sickle cell anemia. Background Technology

[0002] The ABO and RhD blood group systems are crucial in clinical medicine, transfusion safety, maternal and infant health, and organ transplantation. Transfusions of different blood types can trigger severe immune reactions and even be life-threatening. Therefore, rigorous ABO blood typing before transfusion is a necessary prerequisite for safe transfusion. Sickle cell anemia (SCD) is an autosomal recessive genetic disorder. Heterozygotes (AS) are carriers of the sickle gene, while homozygotes (SS) are affected. When sickle cell hemoglobin (HbS) is deoxygenated, it polymerizes, causing red blood cells to sickle. Sickened red blood cells have poor deformability, short lifespan, and weak oxygen-carrying capacity. Generally, AS symptoms are milder than SS, but the SC phenotype is more severe. Therefore, timely and accurate detection of HbS and HbC is of great significance for the prevention of SCD. Current technologies for detecting ABO, RhD blood types, and SCD have several limitations, especially in resource-constrained environments, limiting their widespread application.

[0003] For blood typing, CN210626494U discloses a blood group antigen detection element based on immunochromatography. This method is simple and cost-effective, but for samples with high hematocrit or high viscosity, the migration speed of red blood cells (RBCs) on the reaction membrane is slow, resulting in a reddish background and control line tailing, which interferes with the accurate interpretation of RhD detection results.

[0004] Regarding SCD detection, US20160116489A1 discloses a lateral flow immunoassay method that can simultaneously detect hemoglobin S (Hb S), hemoglobin C (Hb C), and hemoglobin A (Hb A). However, this method requires pre-dissolving the blood sample to release hemoglobin, increasing operational complexity. Glass fiber sampling leads to competition between bound and unbound hemoglobin, affecting color intensity and potentially causing misdiagnosis. Furthermore, the parallel arrangement of the test lines increases the risk of cross-reactivity, reducing result accuracy.

[0005] Other SCD detection methods include CN106796215A, which discloses a technique using a porous membrane under hypoxic conditions to differentiate between normal and sickle cells based on erythrocyte deformability. While this method avoids the need for expensive equipment, it relies on unstable reagents (such as sodium sulfite), limiting the shelf life of the kit, and it cannot specifically identify Hb C, potentially leading to misdiagnosis between Hb AS and Hb SC genotypes.

[0006] WO / 2017 / 106334 discloses a method for screening sickle cells (SCD) based on capillary flow rate under deoxygenated conditions. This technique utilizes the poor deformability of sickle cells, but relies on unstable deoxygenating agents, and the results are subjective due to small differences in flow rate. Variations in sample viscosity further complicate the interpretation of results, making it unsuitable for rapid screening.

[0007] Furthermore, CN115612725A discloses a method for detecting hemoglobin gene mutations using real-time quantitative polymerase chain reaction (RT-qPCR) and TaqMan probes. This method boasts high accuracy and specificity, but requires specialized training, dedicated equipment, and a stable power supply, making it difficult to apply in resource-constrained environments.

[0008] In summary, existing technical solutions for blood typing and SCD testing suffer from problems such as complex operation, insufficient accuracy, unstable reagents, or limited applicability. There is an urgent need for a simple, rapid, accurate testing method that is applicable to various clinical environments. Utility Model Content

[0009] The purpose of this invention is to provide a rapid detection card for ABO, RhD blood type and sickle cell anemia.

[0010] The technical solution of this utility model is as follows:

[0011] A rapid test card for ABO, RhD blood typing and sickle cell anemia includes a card holder, a card cover, an ABD test strip, and an SCD test strip. The ABD and SCD test strips are housed in the card holder, and the card cover is affixed to the card holder.

[0012] The ABD test strip includes an ABD substrate and an ABD washing pad, an ABD reaction pad, and an ABD absorption pad disposed on the ABD substrate and sequentially contacted along the chromatography direction. The ABD reaction pad has Ctl point, A point, B point, and D point. Ctl point is coated with anti-RBC antibody, A point is coated with anti-A antibody, B point is coated with anti-B antibody, and D point is coated with anti-D antibody.

[0013] The SCD test strip includes an SCD substrate and an SCD washing pad, an SCD labeling pad, an SCD lysis pad, an SCD reaction pad, and an SCD absorption pad disposed on the SCD substrate and sequentially contacted along the chromatography direction. The SCD labeling pad is pre-coated with tracer-labeled anti-Hb antibody, the SCD lysis pad is pre-coated with dried saponin, and the SCD reaction pad has Ctl, A, S, and C points. The Ctl point is labeled with a protein that can bind to the tracer-labeled anti-Hb antibody, the A point is coated with anti-HbA antibody, the S point is coated with anti-HbS antibody, and the C point is coated with anti-HbC antibody.

[0014] In a preferred embodiment of this utility model, the card holder is provided with an ABD card slot and an SCD card slot that are parallel to each other, respectively adapted to install the ABD test strip and the SCD test strip.

[0015] More preferably, the card cover is provided with an ABD detection area corresponding to the ABD test strip and an SCD detection area corresponding to the SCD test strip. The ABD detection area is provided with an ABD rinsing hole corresponding to the ABD rinsing pad and an ABD sample addition and result display hole corresponding to the ABD reaction pad. The SCD detection area is provided with an SCD rinsing hole corresponding to the SCD rinsing pad and an SCD sample addition and result display hole corresponding to the SCD reaction pad.

[0016] More preferably, the ABD detection area is marked with the letters Ctl, A, B and D corresponding to the arrangement of Ctl, A, B and D points in the ABD reaction pad, and the SCD detection area is marked with the letters Ctl, A, S and C corresponding to the arrangement of Ctl, A, S and C points in the SCD reaction pad.

[0017] In a preferred embodiment of this invention, the tracer is a nano-metal particle or a colored latex particle.

[0018] More preferably, the tracer is nanocolloidal gold.

[0019] In a preferred embodiment of this utility model, the ABD reaction pad is an ABD reaction pad made of nitrocellulose, and the SCD reaction pad is an SCD reaction pad made of nitrocellulose.

[0020] In a preferred embodiment of this utility model, the ABD reaction pad is provided with Ctl points, A points, B points and D points arranged in a spatial rhombus shape, and the SCD reaction pad is provided with Ctl points, A points, S points and C points arranged in a spatial rhombus shape.

[0021] The beneficial effects of this utility model are:

[0022] 1. This invention uses a washing buffer containing surfactant to dilute the sample, ensuring uniform distribution of red blood cells (RBCs) on the reaction membrane, accelerating migration, and effectively reducing background reddening and control line tailing. Compared to existing technologies (such as CN210626494U), it significantly improves the accuracy of RhD detection, especially in samples with high hematocrit or high viscosity.

[0023] 2. By introducing a lysis pad into the SCD test strip, this invention eliminates the need for pre-lysis of blood samples, simplifies the operation process, and reduces the risk of operational errors. Compared with existing technologies (such as US20160116489A1), it reduces the complexity of detection and improves user-friendliness.

[0024] 3. This invention employs a reactive membrane sampling method, avoiding competition between bound and unbound hemoglobin in glass fiber sampling, thus lowering the detection limit and reducing the risk of misdiagnosis. Furthermore, the detection points are arranged in a diamond shape, minimizing signal cross-interference and ensuring that each detection point provides an independent and clear signal, thereby improving the reliability and specificity of SCD detection.

[0025] 4. This invention simultaneously detects ABO / RhD blood type and SCD using a single test card, significantly saving testing time, sample volume, and cost. Compared to existing technologies (such as CN106796215A and WO / 2017 / 106334), this method is more efficient and particularly suitable for resource-constrained clinical environments.

[0026] 5. The design of this utility model does not require complex equipment or professional training, is suitable for point-of-care testing, and can be widely used in various clinical environments (including resource-limited areas), providing a practical and reliable diagnostic solution. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the card cover of Embodiment 1 of this utility model.

[0028] Figure 2 This is a schematic diagram of the card holder in Embodiment 1 of this utility model.

[0029] Figure 3 This is an exploded three-dimensional structural diagram of the ABD test strip of Embodiment 1 of this utility model.

[0030] Figure 4 This is an exploded three-dimensional structural diagram of the SCD test strip of Embodiment 1 of this utility model. Detailed Implementation

[0031] The technical solution of this utility model will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0032] like Figures 1 to 4 As shown, a rapid test card for ABO, RhD blood typing and sickle cell anemia includes a card holder 2, a card cover 1, an ABD test strip 3, and an SCD test strip 4. The ABD test strip 3 and the SCD test strip 4 are installed in the card holder 2, and the card cover 1 is placed on the card holder 2.

[0033] like Figure 3 As shown, the ABD test strip 3 includes an ABD substrate 34 and an ABD rinsing pad 31 (made of rolled glass fiber), an ABD reaction pad 32 (made of nitrocellulose) and an ABD absorption pad 33 disposed on the ABD substrate 34 and sequentially contacted along the chromatography direction. The ABD reaction pad 32 has Ctl points 324, A points 321, B points 323 and D points 322 arranged in a spatial diamond shape. Ctl point 324 is coated with anti-RBC antibody, A point 321 is coated with anti-A antibody, B point 323 is coated with anti-B antibody, and D point 322 is coated with anti-D antibody.

[0034] like Figure 4 As shown, the SCD test strip 4 includes an SCD substrate 46 and an SCD rinsing pad 41 (rolled glass fiber material), an SCD labeling pad 42 (sheet glass fiber material), an SCD lysis pad 43, an SCD reaction pad 44 (nitrocellulose material) and an SCD absorption pad 45 disposed on the SCD substrate 46 and sequentially contacted along the chromatography direction. The SCD labeling pad 42 is pre-coated with tracer-labeled anti-Hb antibody. The SCD lysis pad 43 is pre-coated with dried saponin. The SCD reaction pad 44 has Ctl points 444, A points 442, S points 441 and C points 443 arranged in a spatial rhomboid shape. Ctl point 444 is labeled with a protein that can bind to the tracer-labeled anti-Hb antibody. A point 442 is coated with anti-HbA antibody. S point 441 is coated with anti-HbS antibody. C point 443 is coated with anti-HbC antibody. The tracer is a nano-metal particle or a colored latex particle, and in this embodiment, it is preferably a nano-colloidal gold.

[0035] Specifically:

[0036] The preparation method of SCD-labeled pad 42 includes: rapidly adding anti-human hemoglobin monoclonal antibody to a colloidal silicon-gold dispersion with a particle size of 40-60 nm and a concentration of 0.03-0.05 wt% at a ratio of 15-50 μg / mL, rapidly mixing, reacting for 4-15 min, then rapidly adding blocking buffer at a ratio of 10-15 μL / mL, reacting for 4-15 min, centrifuging at 8000-10000 rpm for 5-10 min, discarding the supernatant to obtain the precipitate, and then adding it at a ratio of 100-400 μL / mL. Add a reconstitution solution (3.9325g Trizmabase, 2.86g sodium citrate, 1.739ml concentrated hydrochloric acid, 5g casein, 1g polyvinylpyrrolidone, 60g sucrose, 5g bovine serum albumin, 1g sodium azide, and bring the volume to 1L with ultrapure water) to the precipitate in the specified proportions, and sonicate to reconstitute the solution to obtain an anti-human Hb antibody-colloidal gold complex. Spread the anti-human Hb antibody-colloidal gold complex evenly onto a sheet of glass fiber pad and dry it at 25-40℃ for 2-6 hours to obtain SCD-labeled pad 42.

[0037] SCD pyrolysis pad 43 has a thickness of 0.2-0.5mm and a basis weight of 60-90g / m³. 2 A hydrophilic glass fiber pad, pre-coated with dried saponin. The preparation method of this SCD lysis pad 43 includes: uniformly coating a 4-8 g / L saponin solution onto the hydrophilic glass fiber pad, with a volume-to-area ratio of approximately 0.5-0.8 mL / cm². 2 Dry at 25-45℃ for 2-6 hours to obtain SCD pyrolysis pad 43.

[0038] The preparation method of SCD reaction pad 44 includes: diluting anti-HbA antibody, anti-HbS antibody and anti-HbC antibody to 0.25-2 mg / mL with coating buffer and coating them onto point A442, point S441 and point C443 of nitrocellulose membrane, and then drying them at a temperature of 20-32℃ and a humidity of <30% for 40 min-2 h; diluting goat anti-mouse IgG antibody to 1-3 mg / mL with coating buffer and coating it onto point C444 of nitrocellulose membrane, and then drying it at a temperature of 20-32℃ and a humidity of <30% for 40 min-2 h.

[0039] The antibodies mentioned above are specifically shown in Tables 1 and 2 below:

[0040] Table 1

[0041]

[0042] Table 2

[0043]

[0044]

[0045] like Figure 1 and 2As shown, the card holder 2 has an ABD card slot 21 and an SCD card slot 22 that are parallel to each other, respectively adapted to install the ABD test strip 3 and the SCD test strip 4; the card cover 1 has an ABD detection area 11 corresponding to the ABD test strip 3 and an SCD detection area 12 corresponding to the SCD test strip 4. The ABD detection area 11 has an ABD label hole 111, an ABD rinsing hole 113 corresponding to the ABD rinsing pad 31, and an ABD sample addition & result display hole 112 corresponding to the ABD reaction pad 32. The SCD detection area 12 has an SCD label hole 111. The D label hole 121, the SCD rinsing hole 123 corresponding to the SCD rinsing pad 41, and the SCD sample addition & result display hole 122 corresponding to the SCD reaction pad 44; the ABD detection area 11 is marked with the letters Ctl, A, B and D corresponding to the arrangement of Ctl points 324, A points 321, B points 323 and D points 322 in the ABD reaction pad 32; the SCD detection area 12 is marked with the letters Ctl, A, S and C corresponding to the arrangement of Ctl points 444, A points 442, S points 441 and C points 443 in the SCD reaction pad 44.

[0046] The specific method of using this utility model is as follows:

[0047] The procedure for using this invention to detect blood group A antigen, B antigen, RhD antigen, and Hb A, Hb S, and Hb C in human whole blood samples is as follows:

[0048] (1) Sample dilution: Use a dropper to draw 1 drop of the whole blood sample of the subject and place it in a U-shaped cup. Add 1 drop of washing solution (using 10mM PBS as solvent and containing 3% Tween 20) and mix well.

[0049] (2) Sample addition: Add 1 drop of diluted sample to well 112 of ABD sample addition & result display and well 122 of SCD sample addition & result display to ensure that the sample covers each detection point and Ctl point;

[0050] (3) Add rinsing solution: Add 3-5 drops of rinsing solution to ABD rinsing hole 113 and SCD rinsing hole 123 respectively (as above). The rinsing solution flows from ABD rinsing pad 31 and SCD rinsing pad 41 along their respective chromatography directions through ABD reaction pad 32 and SCD reaction pad 44, and finally enters ABD absorption pad 33 and SCD absorption pad 45 respectively.

[0051] (4) Result Interpretation: Wait 2-5 minutes and observe whether the detection points in wells 112 (ABD sample addition & result display) and 122 (SCD sample addition & result display) show color to determine the analytes contained in the sample. Interpretations exceeding 15 minutes are invalid. Specific interpretation rules are as follows:

[0052] ABD system:

[0053] Color development No color development Color development Color development <![CDATA[A RhD + ]]> No color development Color development Color development Color development <![CDATA[B RhD + ]]> No color development No color development Color development Color development <![CDATA[O RhD + ]]> Color development Color development Color development Color development <![CDATA[AB RhD + ]]> Color development No color development No color development Color development <![CDATA[A RhD - ]]> No color development Color development No color development Color development <![CDATA[B RhD - ]]> No color development No color development No color development Color development <![CDATA[O RhD - ]]> Color development Color development No color development Color development <![CDATA[AB RhD - ]]> / / / No color development invalid

[0054] SCD system:

[0055] Color development No color development No color development Color development Hb AA Color development Color development No color development Color development Hb AS Color development No color development Color development Color development Hb AC No color development Color development No color development Color development Hb SS No color development Color development Color development Color development Hb SC No color development No color development Color development Color development Hb CC Color development Color development Color development Color development invalid / / / No color development invalid

[0056] Interpretation of test results:

[0057] A positive result indicates the presence of the analyte in the sample. For ABD test strip 3, the analytes are blood group A antigen (corresponding to point A 321), B antigen (corresponding to point B 323), and RhD antigen (corresponding to point D 322). For SCD test strip 4, the analytes are Hb A (corresponding to point A 442), Hb S (corresponding to point S 441), and Hb C (corresponding to point C 443).

[0058] If the test site does not show color, it indicates a negative result, meaning that the sample does not contain this type of analyte.

[0059] Validity: Regardless of the result, Ctl points 444 and 324 must develop color; otherwise, the result is invalid. In particular, for SCD test strip 4, if points A 442, S 441, and C 443 develop color simultaneously, the result is still invalid even if Ctl point 444 develops color.

[0060] The ABD detection zone 11 works on the principle of solid-phase immunochromatography. Anti-A, anti-B, anti-D, and anti-RBC antibodies are immobilized on a reaction membrane. If red blood cells in the sample undergo an immune binding reaction with the immobilized antibodies, the red blood cells will be trapped at the detection point, which will appear red, indicating a positive reaction. If no antigen-antibody immune reaction occurs, the red blood cells cannot be trapped at the detection point, which will appear white, indicating a negative reaction. Regardless of whether the test result is positive or negative, the Ctl point must show color; otherwise, the test is invalid.

[0061] The SCD detection zone 12 works on the following principle: Using the immunochromatographic colloidal gold method, immune complexes (complexes consisting of anti-human Hb antibody-colloidal gold complex-hemoglobin antigen-hemoglobin antibody) are formed at points A (442), S (441), C (443), and Ctl (444), leading to color development. Specifically, color development at point A (442) indicates the presence of HbA in the sample; at point S (441), it indicates the presence of HbS; and at point C (443), it indicates the presence of HbC. Point Ctl (444) serves as a control point and must develop color; otherwise, the test strip is invalid.

[0062] When the antigen concentration is too high, excessive hemoglobin may not bind to the anti-human Hb antibody-colloidal gold complex and instead directly bind to the hemoglobin antibody on the SCD reaction pad 44 along the chromatographic direction. This results in the hemoglobin antibody binding sites on the SCD reaction pad 44 being occupied, preventing further binding to the hemoglobin-anti-human Hb antibody-colloidal gold complex, thus leading to low sensitivity. In this embodiment, the sample to be tested is directly added to the SCD reaction pad 44. The washing solution containing Tween 20 passes sequentially through the SCD washing pad 41, SCD labeling pad 42, and SCD lysis pad 43, dissolving and mixing the anti-human Hb antibody-colloidal gold complex from the SCD labeling pad 42 and the saponin from the SCD lysis pad 43. Upon reaching the SCD reaction pad 44, the red blood cells in the sample lyse under the synergistic effect of saponin and Tween 20, releasing hemoglobin. Different hemoglobins bind to the goat anti-mouse IgG antibody labeled at point C444 and the antibody coated at point A442, respectively. Anti-HbA antibody, anti-HbS antibody coated at S point 441, and anti-HbC antibody coated at C point 443 bind to form corresponding immune complexes. These immune complexes then combine with the anti-human Hb antibody-colloidal gold complex from the SCD labeling pad 42 to form different anti-human Hb antibody-colloidal gold complex-hemoglobin antigen complexes. Excess unbound different hemoglobins will simultaneously chromatographically enter the SCD absorption pad 45. This solves the problem that excess antigen in the test sample first binds to the antibody on the SCD reaction pad 44, resulting in weak or no color development, thus improving the detection sensitivity.

[0063] The above description is only a preferred embodiment of the present utility model, and therefore cannot be used to limit the scope of the present utility model. All equivalent changes and modifications made in accordance with the scope of the present utility model patent and the contents of the specification should still fall within the scope of the present utility model.

Claims

1. A rapid test card for ABO, RhD blood typing and sickle cell anemia, characterized in that: The system includes a card holder, a card cover, an ABD test strip, and an SCD test strip. The ABD and SCD test strips are installed in the card holder, and the card cover is placed on the card holder. The ABD test strip includes an ABD substrate and an ABD washing pad, an ABD reaction pad, and an ABD absorption pad disposed on the ABD substrate and sequentially contacted along the chromatography direction. The ABD reaction pad has Ctl point, A point, B point, and D point. Ctl point is coated with anti-RBC antibody, A point is coated with anti-A antibody, B point is coated with anti-B antibody, and D point is coated with anti-D antibody. The SCD test strip includes an SCD substrate and an SCD washing pad, an SCD labeling pad, an SCD lysis pad, an SCD reaction pad, and an SCD absorption pad disposed on the SCD substrate and sequentially contacted along the chromatography direction. The SCD labeling pad is pre-coated with tracer-labeled anti-Hb antibody, the SCD lysis pad is pre-coated with dried saponin, and the SCD reaction pad has Ctl, A, S, and C points. The Ctl point is labeled with a protein that can bind to the tracer-labeled anti-Hb antibody, the A point is coated with anti-HbA antibody, the S point is coated with anti-HbS antibody, and the C point is coated with anti-HbC antibody.

2. The rapid test card for ABO, RhD blood type and sickle cell anemia as described in claim 1, characterized in that: The card holder is provided with an ABD card slot and an SCD card slot that are parallel to each other, respectively adapted to install the ABD test strip and the SCD test strip.

3. The rapid test card for ABO, RhD blood type and sickle cell anemia as described in claim 2, characterized in that: The card cover is provided with an ABD detection area corresponding to the ABD test strip and an SCD detection area corresponding to the SCD test strip. The ABD detection area is provided with an ABD rinsing hole corresponding to the ABD rinsing pad and an ABD sample addition and result display hole corresponding to the ABD reaction pad. The SCD detection area is provided with an SCD rinsing hole corresponding to the SCD rinsing pad and an SCD sample addition and result display hole corresponding to the SCD reaction pad.

4. The rapid test card for ABO, RhD blood type and sickle cell anemia as described in claim 3, characterized in that: The ABD detection area is marked with the letters Ctl, A, B and D corresponding to the arrangement of Ctl, A, B and D points in the ABD reaction pad, and the SCD detection area is marked with the letters Ctl, A, S and C corresponding to the arrangement of Ctl, A, S and C points in the SCD reaction pad.

5. A rapid test card for ABO, RhD blood typing and sickle cell anemia as described in any one of claims 1 to 4, characterized in that: The tracer is a nano-metal particle or a colored latex particle.

6. The rapid test card for ABO, RhD blood type and sickle cell anemia as described in claim 5, characterized in that: The tracer is nanocolloidal gold.

7. A rapid test card for ABO, RhD blood typing and sickle cell anemia as described in any one of claims 1 to 4, characterized in that: The ABD reaction pad is an ABD reaction pad made of nitrocellulose, and the SCD reaction pad is an SCD reaction pad made of nitrocellulose.

8. A rapid test card for ABO, RhD blood typing and sickle cell anemia as described in any one of claims 1 to 4, characterized in that: The ABD reaction pad has Ctl points, A points, B points and D points arranged in a spatial rhombus shape, and the SCD reaction pad has Ctl points, A points, S points and C points arranged in a spatial rhombus shape.

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