Methods for detecting platelet aggregation rate and their applications, and automated blood testing lines

Abstract

This invention relates to the field of platelet aggregation rate detection technology, specifically disclosing a method for detecting platelet aggregation rate and its application, as well as a blood testing automated line. The detection method includes the following steps: adding an anticoagulant to a whole blood sample and mixing thoroughly to obtain a sample solution; adding platelet staining solution to the sample solution to stain it, obtaining a first test sample; taking a portion of the first test sample for platelet detection, obtaining a scatter plot of cell particles and platelet concentration in the first test sample; adding an aggregation-inducing solution to the remaining first test sample to obtain a second test sample; taking a portion of the second test sample at regular intervals for platelet detection, obtaining a scatter plot of cell particles and platelet concentration for the second test sample at each time point; and calculating the platelet aggregation rate based on the above detection results. The detection method of this invention is simple, highly efficient, and provides reliable detection results.

Description

[Technical Field]

[0001] This invention relates to the field of platelet aggregation rate detection technology, and in particular to a method for detecting platelet aggregation rate, its application, and a blood testing automated line. [Background Technology]

[0002] Platelet aggregation rate can be used for medical diagnosis. For example, a decreased platelet aggregation rate can be used to assist in the diagnosis of uremia, cirrhosis, primary thrombocytopenic purpura, acute leukemia, and MDS; while an increased platelet aggregation rate can be used to assist in the diagnosis of myocardial infarction, angina pectoris, cerebral infarction, diabetes, deep vein thrombosis, hyperβ-lipoproteinemia, and early stage of disseminated intravascular coagulation. Therefore, obtaining platelet aggregation rate has significant clinical guiding value.

[0003] Currently, platelet aggregation rate is generally measured using either phototurbidimetry or impedance spectroscopy. Phototurbidimetry first involves low-speed centrifugation of a whole blood sample, then collecting the supernatant as platelet-rich plasma (PRP). The remaining sample is then centrifuged at high speed, and the supernatant is collected as platelet-poor plasma (PPP). PRP is considered to be rich in platelets, while PPP, due to high-speed centrifugation causing platelets to settle to the lower layer, is considered to be platelet-free. Therefore, although phototurbidimetry can reflect platelet aggregation function to some extent, it requires centrifugation to remove interference from red and white blood cells, resulting in a testing time exceeding 30 minutes. Furthermore, PRP after removing red and white blood cells does not accurately reflect the true state of platelets. Impedance spectroscopy, on the other hand, involves passing consecutive individual blood cell particles through a narrow resistance testing port. Different volumes of blood cell particles will produce different resistance peaks when passing through this port. Detecting this process allows the determination of the impedance peak position of each cell particle, thus determining the volume and number of cell particles based on the peak value and the number of peak positions. This method can directly test whole blood samples. The platelet aggregation rate at the corresponding testing time point is calculated by measuring the changes in platelet volume and number before and after platelet aggregation. However, because the impedance method directly tests whole blood samples, it is susceptible to interference from red and white blood cells in platelet volume measurement and counting, resulting in a relatively large error. [Summary of the Invention]

[0004] One of the objectives of this invention is to provide a method for detecting platelet aggregation rate, in order to solve the problems of low efficiency and poor reliability of existing platelet aggregation rate detection methods.

[0005] To achieve the above technical objectives, the following technical solution is adopted:

[0006] A method for detecting platelet aggregation rate includes the following steps:

[0007] Add anticoagulant to the whole blood sample and mix well to obtain the sample solution;

[0008] Platelet staining solution is added to the sample solution to stain the sample solution, thereby obtaining the first test sample;

[0009] A portion of the first test sample was taken for platelet detection to obtain a scatter plot of cell particles and platelet concentration in the first test sample;

[0010] Add polymerizing solution to the remaining first test sample to obtain a second test sample. Take a portion of the second test sample at regular intervals for platelet detection to obtain a scatter plot of cell particles and platelet concentration of the second test sample at each time point.

[0011] Based on the above test results, the platelet aggregation rate was calculated.

[0012] Preferably, the preset calculation formula for calculating the platelet aggregation rate is:

[0013]

[0014] Where P represents platelet aggregation rate, A0 represents the platelet concentration detection value corresponding to the first test sample, and A n This represents the platelet concentration value detected in the nth test corresponding to the second test sample, where n is a natural number greater than or equal to 3.

[0015] Preferably, the platelet concentration value corresponding to the nth detection is the number of platelet concentrations detected in the nth detection when the platelet concentrations in the (n-2), (n-1), and nth detections are the same or nearly the same. Preferably, the preset calculation formula is:

[0016]

[0017] Where P represents the platelet aggregation rate detection value, A0 represents the platelet concentration detection value corresponding to the first detection, and A x This represents the average value of the platelet concentration values ​​from the (n-2), (n-1), and (n-n)th tests when the platelet concentration values ​​are the same or approach the same.

[0018] Preferably, the platelet staining solution contains a platelet staining agent with the following general structural formula I:

[0019]

[0020] In the formula, R1, R2, R6, R7, R8, and R9 are selected from hydrogen atoms, C1-C atoms, and C2-C2 atoms. 18 Alkyl, C1-C18 Alkyl COOR 10 C1-C 18 Alkyl COR 10 C1-C 18 Alkyl CNR 10 R 10 C1-C 18 Alkyl OR 10 Any one of them; R3, R4, R5, R 10 Selected from any one of C1-C8 alkyl groups;

[0021] X - Selected from any one of halide ions, sulfate ions, phosphate ions, and boron halide ions; A is selected from Cl-C 18 At least one of alkyl, CH2O, and CH2CH2O; n is selected from any value from 2 to 20.

[0022] Preferably, the platelet staining solution further includes a buffer and an osmotic pressure regulator;

[0023] The buffer includes at least one of tris(hydroxymethyl)aminomethane buffer, tris(hydroxymethyl)methylglycine buffer, imidazole, phosphate, and citrate buffer.

[0024] The osmotic pressure regulator includes at least one of the following: halogen salts, sulfates, carbonates, alkali metal salts of organic acids, glycerol, mannitol, and glucose.

[0025] Preferably, the platelet staining solution may further contain a red blood cell nucleic acid staining agent, wherein the red blood cell nucleic acid staining agent has the following general formula II:

[0026]

[0027] Among them, R 11 R 12 These represent alkyl groups with 1 to 3 carbon atoms respectively; Y represents chloride or sulfate ions.

[0028] Preferably, the polymerization solution comprises an active agent, a salt, and a third solvent;

[0029] The active agent includes at least one of adenosine diphosphate, collagen, arachidonic acid, thrombin, adrenaline, prostaglandin G2, prostaglandin H2, and thromboxane A2.

[0030] The concentration of the surfactant in the polymerization solution is 0.3 mmol / L to 3.5 mmol / L;

[0031] The amount of the active agent added per milliliter of the test solution is 2.425 × 10⁻⁶. -6 mmol ~ 3.5 × 10-5 mmol.

[0032] Compared with the prior art, the platelet aggregation rate detection method provided by the embodiments of the present invention directly detects whole blood samples without centrifugation, separation and other steps. This not only effectively ensures the true morphology of platelets, but also prevents red blood cells and white blood cells from interfering with the volume or number of platelets during the detection process. Furthermore, the detection can be completed within 10 minutes. Therefore, the detection method of the embodiments of the present invention has the characteristics of high detection efficiency and good reliability of detection results.

[0033] A second objective of this invention is to provide the application of the above-mentioned platelet aggregation rate detection method in a hematology analyzer, wherein the hematology analyzer includes a sampling module, a reagent addition module, a reaction module, a delivery module, a detection module, and a controller.

[0034] The sampling module is used to collect the sample solution and distribute it to the reaction module;

[0035] The reagent addition module is used to add platelet staining solution to the reaction module for staining to obtain the first test sample;

[0036] The delivery module is used to send a portion of the first sample to be tested into the detection module for detection;

[0037] The reagent addition module is further configured to add the polymerization solution to the remaining first test sample in the reaction module to obtain the second test sample;

[0038] The delivery module is also used to send a portion of the second sample to be tested into the detection module for detection at a preset time.

[0039] The detection module includes a flow chamber and an optical unit. The flow chamber is connected to the delivery module. The delivery module is used to inject a portion of the first test sample and a portion of the second test sample into the flow chamber at preset times, so that the portion of the first test sample and the portion of the second test sample form corresponding sample streams in the flow chamber. The optical unit is used to perform optical detection on the sample streams to output a detection signal, which is a scatter plot of cell particles.

[0040] The first controller acquires the detection signal and calculates the platelet aggregation rate based on the detection signal.

[0041] In some embodiments, the blood cell analyzer further includes an information output module; the first controller transmits the acquired detection signal to the information output module, and the information output module outputs a platelet concentration change curve, wherein the vertical axis of the platelet concentration change curve is the platelet concentration value and the horizontal axis is time.

[0042] In some implementations, when the platelet concentration tends to stabilize, the hematology analyzer stops injecting the second sample into the flow chamber.

[0043] In some embodiments, the blood cell analyzer includes a routine blood count (RBC) testing mode and a platelet aggregation rate (PAC) testing mode. When the first controller receives the RBC testing mode information, the first controller controls the reagent addition module to add platelet staining solution to the reaction module for staining, obtaining the first test sample. The delivery module delivers a portion of the first test sample to the detection module for testing to obtain the RBC test result. When the first controller receives the PAC testing module information, the first controller controls the reagent addition module to add the aggregation-inducing solution to the remaining first test sample, obtaining the second test sample. The delivery module delivers a portion of the second test sample to the detection module for testing at regular intervals to obtain the test result corresponding to each time point and calculate the platelet aggregation rate.

[0044] In some implementations, when the platelet aggregation rate is not in the range of 30% to 70%, the first controller outputs abnormal detection information.

[0045] The third objective of this invention is to provide the above-mentioned platelet aggregation rate detection method for the auxiliary diagnosis of uremia, cirrhosis, primary thrombocytopenic purpura, acute leukemia, MDS, myocardial infarction, angina pectoris, cerebral infarction, diabetes, deep vein thrombosis, hyperβ-lipoproteinemia, and early stage of disseminated intravascular coagulation.

[0046] The fourth objective of this invention is to provide a blood testing automated line, including a blood cell analyzer, a coagulation analyzer, a platelet aggregation track, a display, and a second controller as described above; when the platelet aggregation rate calculated by the blood cell analyzer is not within the range of 30% to 70%, the display outputs abnormal detection information; the second controller controls the sample on the platelet aggregation track to be transported to the coagulation analyzer for platelet function testing based on the abnormal detection information. [Attached Image Description]

[0047] Figure 1 A simplified flowchart illustrating the platelet aggregation rate detection method provided in this embodiment of the invention;

[0048] Figure 2 A simplified structural block diagram of a blood cell analyzer provided in an embodiment of the present invention;

[0049] Figure 3 This is a three-dimensional structural diagram of a blood cell analyzer provided in an embodiment of the present invention;

[0050] Figure 4 A simplified structural block diagram of a blood testing assembly line provided in an embodiment of the present invention;

[0051] Figure 5 The scatter plot of cell particles over time is obtained by the platelet aggregation rate detection method provided in Embodiment 1 of the present invention.

[0052] Figure 6 The platelet concentration change curve over time obtained by the platelet aggregation rate detection method provided in Embodiment 1 of the present invention;

[0053] Figure 7 The platelet aggregation rate detection method provided in Embodiment 1 of the present invention yields a platelet aggregation rate change curve over time.

[0054] Figure 8 The platelet aggregation rate detection method provided in Embodiment 1 of the present invention and the platelet concentration scatter plot and linear fitting curve obtained by impedance counting;

[0055] Figure 9 The platelet aggregation rate detection method provided in Embodiment 1 of the present invention, along with the platelet concentration scatter plot and linear fitting curve obtained by microscopic counting;

[0056] Figure 10 The platelet aggregation rate detection method provided in Embodiment 1 of the present invention, along with the scatter plot and linear fitting curve of immature platelet concentration obtained by microscopic counting;

[0057] Figure 11 The scatter plot of cell particles over time is obtained by the platelet aggregation rate detection method provided in Embodiment 2 of the present invention.

[0058] Figure 12 The platelet concentration change curve over time obtained by the platelet aggregation rate detection method provided in Embodiment 2 of the present invention;

[0059] Figure 13 The platelet aggregation rate detection method provided in Embodiment 2 of the present invention yields a platelet aggregation rate change curve over time.

[0060] Figure 14 The scatter plot of cell particles over time is obtained by the platelet aggregation rate detection method provided in Embodiment 3 of the present invention.

[0061] Figure 15 The platelet concentration change curve over time obtained by the platelet aggregation rate detection method provided in Embodiment 3 of the present invention;

[0062] Figure 16 The platelet aggregation rate detection method provided in Embodiment 3 of the present invention yields a platelet aggregation rate change curve over time.

[0063] Figure 17 The scatter plot of cell particles over time is obtained by the platelet aggregation rate detection method provided in Embodiment 4 of the present invention.

[0064] Figure 18 The platelet concentration change curve over time obtained by the platelet aggregation rate detection method provided in Embodiment 4 of the present invention;

[0065] Figure 19 The platelet aggregation rate detection method provided in Embodiment 4 of the present invention yields a platelet aggregation rate change curve over time.

[0066] Figure 20 The scatter plot of cell particles over time is obtained by the platelet aggregation rate detection method provided in Embodiment 5 of the present invention.

[0067] Figure 21 The platelet concentration change curve over time obtained by the platelet aggregation rate detection method provided in Embodiment 5 of the present invention;

[0068] Figure 22 The platelet aggregation rate detection method provided in Embodiment 5 of the present invention yields a platelet aggregation rate change curve over time.

[0069] Figure label:

[0070] 10. Blood cell analyzer; 11. Sampling module; 12. Reagent addition module; 13. Reaction module; 14. Delivery module; 15. Detection module; 151. Flow chamber; 152. Optical unit; 16. First controller; 17. Information output module; 20. Blood testing production line; 21. Coagulation analyzer; 22. Assembly track; 23. Second controller; 24. Display.

Detailed Implementation Methods

[0071] The present invention will be further described below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0072] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0073] Please see Figure 1 , Figure 2 and Figure 3 This invention provides a method for detecting platelet aggregation rate, specifically including the following steps:

[0074] S01. Add anticoagulant to whole blood sample, mix well, and obtain sample solution.

[0075] In step S01, whole blood samples can be collected from the subject, specifically by collecting venous blood. The amount of anticoagulant added to the whole blood sample is sufficient to prevent aggregation during the test; the specific amount of anticoagulant added can be adjusted based on the amount of whole blood sample used. Since it is not necessary to test platelet aggregation function, all anticoagulants except sodium citrate would have an irreversible impact on this function; therefore, sodium citrate is selected as the anticoagulant.

[0076] S02. Add platelet staining solution to the sample solution obtained in step S01 to stain the sample solution and obtain the first test sample.

[0077] In step S02, the platelet staining solution is used to dilute the sample solution and stain at least the nucleic acids of the platelets. In some embodiments, the platelet staining solution contains a platelet staining agent to achieve staining of platelet nucleic acids.

[0078] In some embodiments, the platelet staining agent comprises at least one platelet staining agent represented by general formula I:

[0079]

[0080] In the formula, R1, R2, R6, R7, R8, and R9 are selected from hydrogen atoms, C1-C atoms, and C2-C2 atoms. 18 Alkyl, C1-C 18 Alkyl COOR 10 C1-C 18 Alkyl COR 10 C1-C 18 Alkyl CNR 10 R10 C1-C 18 Alkyl OR 10 Any one of them; R3, R4, R5, R 10 Selected from any one of C1-C8 alkyl groups; X - A is selected from any one of halide ions, sulfate ions, phosphate ions, and boron halide ions; A is selected from Cl-C. 18 At least one of alkyl, CH2O, and CH2CH2O; n is selected from any value from 2 to 20. In this embodiment, the platelet staining solution includes at least one of the above-mentioned platelet staining agents. Platelet staining agents with the structure shown in general formula I have good cell membrane permeability and can penetrate the platelet membrane to stain the nucleic acids inside the platelets, but they do not easily penetrate the cell membranes of red blood cells and white blood cells, thus failing to stain them. Based on this, more accurate staining is achieved, and the process does not require the removal of red blood cells and white blood cells, thereby effectively improving detection efficiency. At the same time, the scattered distribution of platelets is screened by the fluorescence signal after staining, resulting in low false negative and false positive rates and improving the reliability of the detection results. In some embodiments, the content of the platelet staining agent in the platelet staining solution is 1 mg / L to 5 mg / L. In some embodiments, the mixing ratio of the platelet staining solution and the sample solution obtained in step S01 is 50:1 to 100:1.

[0081] In some embodiments, the platelet staining solution also includes buffers and osmotic pressure regulators.

[0082] In some embodiments, the buffer includes at least one of tris(hydroxymethyl)aminomethane (Tris) buffer, tris(hydroxymethyl)methylglycine (Tricine) buffer, imidazole, phosphate, and citrate buffer. The buffer is used to maintain pH levels, preventing excessive acidity or alkalinity from completely destroying the morphology, structure, and physiological characteristics of blood cells. Furthermore, the buffer can also promote spheroidization of blood cells for easier identification and statistical analysis.

[0083] In some embodiments, the osmotic pressure regulator includes at least one selected from halogen salts, sulfates, carbonates, alkali metal salts of organic acids, glycerol, mannitol, and glucose.

[0084] In some embodiments, the platelet staining solution further includes a first solvent for dissolving the buffer and osmotic pressure regulator. Specifically, the first solvent includes deionized water or double-distilled water.

[0085] In some embodiments, the content of each component in the platelet staining solution is as follows: the buffer is 3 g / L to 20 g / L, and the osmotic pressure regulator is 0.02 g / L to 0.5 g / L. In some embodiments, the pH of the platelet staining solution is 8 to 10, and the osmotic pressure is 160 mOsm / kg to 230 mOsm / kg. Maintaining the pH of the platelet staining solution between 8 and 10 can effectively maintain the morphology, structure, and physiological characteristics of blood cells within a suitable detection range, while maintaining the osmotic pressure in the sample solution below the osmotic pressure of blood cells under normal conditions (300 mOsm / kg). This forces flattened red blood cells and originally scattered platelets to absorb water and swell into spherical or near-spherical shapes without bursting, thus facilitating identification and statistical analysis.

[0086] In some embodiments, the platelet staining solution also contains a red blood cell nucleic acid staining agent, which has a structure as shown in general formula II, thereby allowing for the staining of platelets and red blood cells separately. Since different cells produce different fluorescence intensities after staining, it is convenient to detect and count the status of red blood cells when detecting platelets.

[0087]

[0088] Among them, R 11 R 12 These represent alkyl groups with 1 to 3 carbon atoms respectively; Y represents chloride or sulfate ions.

[0089] S03. Take a portion of the first test sample obtained in step S02 and perform platelet detection to obtain a scatter plot of cell particles and platelet concentration in the first test sample.

[0090] In step S03, when performing platelet testing, a blood cell analyzer 10 is used for the test.

[0091] Specifically, the hematology analyzer 10 includes a sampling module 11, a reagent addition module 12, a reaction module 13, a delivery module 14, a detection module 15, and a first controller 16. The sampling module 11 collects sample solution and distributes it to the reaction module 13. The reagent addition module 12 adds platelet staining solution to the reaction module 13 to obtain a first test sample. The detection module 15 includes a flow chamber 151 and an optical unit 152. The flow chamber 151 is connected to the delivery module 14, which delivers a portion of the first test sample into the flow chamber 151 of the detection module 15 to form a corresponding sample stream. The optical module performs optical detection on the sample stream formed by the first test sample and outputs the detection signal obtained from the optical detection. The first controller 16 receives the detection signal, which is a scatter plot of cell particles. The blood cell analyzer 10 provided in this embodiment of the invention includes a routine blood test mode and a platelet aggregation rate test mode. When the first controller 16 receives information from the routine blood test mode, the first controller 16 controls the reagent addition module 12 to add platelet staining solution to the reaction module 13 for staining to obtain a first test sample. The delivery module 14 delivers a portion of the first test sample to the detection module 15 for detection to obtain the routine blood test result. When the first controller 16 receives information from the platelet aggregation rate detection module 15, the first controller 16 controls the reagent addition module 12 to add aggregation solution to the remaining first test sample to obtain a second test sample. The delivery module 14 delivers a portion of the second test sample to the detection module 15 for detection at regular intervals to obtain the test result corresponding to each time point and calculate the platelet aggregation rate.

[0092] S04. Add polymerization solution to the remaining first test sample to obtain the second test sample. Take a portion of the second test sample at regular intervals for platelet detection to obtain the scatter plot of cell particles and platelet concentration of the second test sample at each time point.

[0093] In step S04, the aggregation-inducing solution is used to induce cell aggregation in the remaining cells of the first test sample. In some embodiments, the aggregation-inducing solution includes an active agent, a salt, and a second solvent. In some embodiments, the active agent includes at least one of adenosine diphosphate (ADP), collagen, arachidonic acid, thrombin, adrenaline, prostaglandin G2, prostaglandin H2, and thromboxane A2; the salt includes sodium chloride, etc.; and the second solvent includes at least one of deionized water, double-distilled water, etc. In some embodiments, the concentration of the active agent in the aggregation-inducing solution is 0.3 mmol / L to 3.5 mmol / L. The concentration of the active agent should not be too high. If the concentration is too high, when mixed with the sample solution, platelets will directly aggregate and reach the maximum aggregation rate, making it impossible to observe and obtain the aggregation rate at various time points during the aggregation process.

[0094] In some embodiments, the amount of the active agent added per milliliter of the first test sample is 2.25 × 10⁻⁶. - 6 mmol ~ 3.5 × 10 -5 mmol. Excessive addition of the active agent can easily lead to the attainment of the maximum aggregation rate, making it impossible to observe the platelet aggregation process and hindering the analysis of clinical information from whole blood samples based on the aggregation rate.

[0095] In step S04, the reagent addition module 12 of the cell analyzer adds a polymerization agent to the remaining first test sample in the reaction module 13, causing the polymerization agent to mix with the remaining first test sample to obtain a second test sample. Subsequently, at a preset time interval, the delivery module 14 delivers a portion of the second test sample into the detection module 15 for detection. The delivery module 14 injects the second test sample into the flow chamber 151 at preset times each time, so that the delivered second test sample forms a corresponding sample stream in the flow chamber 151. The optical unit 152 is used to perform optical detection on the sample stream to output a detection signal, which is a scatter plot of cell particles. The first controller 16 acquires the detection signal. In some embodiments, the preset time interval between two consecutive detections of the second test sample is 10s to 120s.

[0096] S05. Calculate the platelet aggregation rate based on the detection results of steps S03 and S04.

[0097] In step S05, based on the detection signal of the first sample to be tested obtained in step S03 and the platelet concentration corresponding to the detection signals at different time points obtained in step S04, a curve showing the change of platelet concentration over time is plotted. The aggregation rate and maximum aggregation rate of platelets at each time point can then be calculated or read from the curve. Furthermore, based on the detection results at different time points, a scatter plot of cell particles at the corresponding time points is obtained. In some embodiments, the blood cell analyzer 10 also includes an information output module 17; the first controller 16 transmits the acquired detection signal to the information output module 17, and the information output module 17 outputs a platelet concentration change curve, where the vertical axis of the platelet concentration change curve represents the platelet concentration value, and the horizontal axis represents time.

[0098] In some implementations, the platelet aggregation rate is calculated according to the following preset formula:

[0099]

[0100] Where P represents platelet aggregation rate, A0 represents the platelet concentration detection value corresponding to the first test sample, and A nThis represents the platelet concentration detection value corresponding to the nth test for the second sample to be tested, where n is a natural number greater than or equal to 3. In some embodiments, the platelet concentration detection value corresponding to the nth test is determined based on the following result: when the (n-2), (n-1), and nth tests are performed, the platelet concentration detection values ​​are the same or tend to be the same, which is the platelet concentration detection value of the nth test, that is, the delivery module 14 can be stopped from delivering the second sample to the flow chamber 151.

[0101] In some implementations, the platelet aggregation rate is calculated according to the following preset formula:

[0102]

[0103] Where P represents the platelet aggregation rate detection value, A0 represents the platelet concentration detection value corresponding to the first detection, and A x This represents the average of the platelet concentration values ​​from the (n-2), (n-1), and (n-n)th tests when the values ​​are the same or approach the same, i.e., A. x The value is determined by averaging the platelet concentration values ​​from the (n-2), (n-1), and nth tests. When the platelet concentration values ​​from the (n-2), (n-1), and nth tests are the same or approach the same, the average value of the platelet concentration values ​​from the (n-2), (n-1), and nth tests is considered A. x The value.

[0104] In some implementations, when the platelet concentration detection value of the first test sample and the platelet concentration detection values ​​of the second test sample corresponding to multiple time points detected at a preset time are plotted as a curve, and the curve shows a trend of approaching horizontality, the delivery module 14 stops delivering the second test sample from the reaction module 13 to the detection module 15, and determines the maximum platelet aggregation rate based on the point where the curve approaches horizontality.

[0105] Based on the above test results, when the obtained platelet aggregation rate is not within the range of 30% to 70%, the first controller 16 outputs abnormal detection information. In some embodiments, when the platelet aggregation rate is not within the range of 35% to 65%, the first controller 16 outputs abnormal detection information. Furthermore, it is necessary to test the platelet coagulation function of the whole blood sample for further medical analysis.

[0106] The platelet aggregation rate detection method provided in this invention has high detection efficiency and reliable accuracy, and therefore can be used to assist in the diagnosis of uremia, cirrhosis, primary thrombocytopenic purpura, acute leukemia, MDS, myocardial infarction, angina pectoris, cerebral infarction, diabetes, deep vein thrombosis, hyperβ-lipoproteinemia, and early stage of disseminated intravascular coagulation. In specific application, a whole blood sample is obtained from the subject through venous blood collection, and then the test is performed according to steps S01 to S05. Based on the test results, a medical analyzer is used to assist in the medical diagnosis of whether the subject has the aforementioned conditions.

[0107] Please see Figures 1 to 3 as well as Figure 4 Based on the above-mentioned platelet aggregation rate detection method, this embodiment of the invention also provides a blood testing line 20. Specifically, the blood testing line 20 includes a blood cell analyzer 10, a coagulation analyzer 21, a platelet aggregation track 22, a display 24, and a second controller 23. The blood cell analyzer 10 has a routine blood test mode and a platelet aggregation rate detection mode. When the platelet aggregation rate calculated by the blood cell analyzer 10 exceeds 30%–70%, the display 24 outputs abnormal detection information. The second controller 23 controls the sample transport on the platelet aggregation track 22 to the coagulation analyzer 21 for platelet function testing based on the abnormal detection information, and the display 24 outputs the platelet function test results for medical analysis.

[0108] To better illustrate the technical solutions of the embodiments of the present invention, further explanations and descriptions are provided below through multiple examples.

[0109] Example 1

[0110] A method for detecting platelet aggregation rate includes the following steps:

[0111] (11) Obtain venous blood from the subject as a whole blood sample. Add sodium citrate to the whole blood sample and mix them well to obtain a sample solution for later use.

[0112] (12) Take 200 μL of the sample solution from step (11), add 15 mL of platelet staining solution, mix thoroughly to dilute and stain the sample solution, and obtain the first sample to be tested.

[0113] The platelet staining solution contains 3.6 g / L imidazole (buffer), 14 g / L trisodium citrate (buffer), and 2 mg / L platelet staining agent as shown in the following chemical formula:

[0114]

[0115] After adding platelet staining solution, the pH of the first test sample was approximately 8.5; the solvent in the platelet staining solution was deionized water.

[0116] (13) Take 1 mL of the first test sample obtained in step (12) and perform platelet detection to obtain the scatter plot of cell particles and platelet concentration in the first test sample.

[0117] (14) Add the polymerization solution to the remaining first test sample from step (12), mix well to obtain the second test sample, and take 1 mL of the second test sample every 60 s for platelet detection. Obtain the platelet concentration and scatter plot of cell particles at different time points. The results are shown in Table 1 and Figure 5 As shown.

[0118] The polymerization solution is a 3.28 mmol / L ADP-xNa saline solution; and the amount of polymerization solution used is 1 μL of polymerization solution added to every 2 mL of the first test sample in step (12).

[0119] Table 1

[0120]

[0121] (15) Based on the data in Table 1, plot the curves showing the changes in platelet concentration and platelet aggregation rate over time. The results are as follows: Figure 6 and Figure 7 As shown.

[0122] From Table 1 and Figures 5 to 7 As can be seen, platelets did not aggregate without the addition of the aggregation-inducing solution, but rapidly aggregated after the addition of the aggregation-inducing solution. The aggregation rate reached half within 90 seconds, and exceeded 80% in just 390 seconds, after which it remained stable. This indicates that in this embodiment, the maximum platelet aggregation rate can be obtained in less than 6.5 minutes, and the platelet aggregation process can be clearly observed, which is beneficial for obtaining clinical information from whole blood samples.

[0123] Platelet counting (PLT) of the samples from Example 1 was performed using the electrical impedance method, specifically using a BF8001 instrument. The platelet concentrations obtained by the electrical impedance method and the platelet concentrations obtained in Example 1 were plotted as a scatter plot and linearly fitted. The results are as follows: Figure 8 As shown. Simultaneously, platelet and immature platelet (IPF) counts were performed on the samples from Example 1 using microscopic counting. The platelet concentrations obtained from microscopic counting and those obtained from Example 1 were plotted as a scatter plot and linearly fitted. The results are shown below. Figure 9As shown; the concentrations of immature platelets obtained by microscopic counting and Example 1 were plotted as scatter plots and linearly fitted, and the results are as follows. Figure 10 As shown.

[0124] from Figure 8 It can be seen that the platelet aggregation rate detection method provided in Embodiment 1 of the present invention has a high degree of overlap with the platelet count results obtained by the impedance method, and shows a good linear relationship. This indicates that the embodiment of the present invention not only has a simple detection process and high detection efficiency, but also the detection results are comparable to those obtained by the impedance method.

[0125] from Figure 9 and Figure 10 It can be seen that the platelet aggregation rate detection method provided in Embodiment 1 of the present invention yields platelet count results with a high degree of overlap with microscopic platelet counts and immature platelet counts, and exhibits a good linear relationship. This indicates that the embodiment of the present invention not only has a simple detection process and high detection efficiency, but also that the detection results are comparable to those of the electrical impedance method. Furthermore, it demonstrates that the platelet aggregation rate detection method provided in the embodiment of the present invention can detect more blood parameters than the electrical impedance method.

[0126] Example 2

[0127] A method for detecting platelet aggregation rate includes the following steps:

[0128] (21) Obtain venous blood from the subject as a whole blood sample. Add sodium citrate to the whole blood sample, mix well, and obtain a sample solution for later use.

[0129] (22) Take 200 μL of the sample solution from step (21) and add it to 20 mL of platelet staining solution. Mix thoroughly to dilute and stain the sample solution, obtaining the test solution. The platelet staining solution contains 3.6 g / L imidazole, 10 g / L sodium carbonate, and 2 mg / L platelet staining agent. The chemical formula of the platelet staining agent is:

[0130]

[0131] After processing the platelet staining solution, the pH of the first test sample was approximately 9.5.

[0132] The solvent in the platelet staining solution is deionized water.

[0133] (23) Take 1 mL of the first test sample obtained in step (22) and perform platelet detection to obtain the scatter plot of cell particles and platelet concentration in the first test sample.

[0134] (24) Add the polymerization solution to the remaining first test sample from step (22), mix well to obtain the second test sample, and take 1 mL of the second test sample every 120 s for platelet detection to obtain platelet concentration and scatter plots of cell particles at different time points. The detection results are shown in Table 2 and Figure 11 As shown.

[0135] The polymerization solution is a 3.28 mmol / L ADP-xNa saline solution; and the amount of polymerization solution used is 7.4 μL added to each 2 mL of the first test sample in step (22).

[0136] Table 2

[0137] Time s 0 120 240 360 480 600 720 <![CDATA[Platelet concentration 10 9 / L]]> 269 200 173 150 132 124 121 Platelet aggregation rate % 0 26 36 44 51 54 55

[0138] (25) Based on the data in Table 2, plot the curves of platelet concentration versus time and platelet aggregation rate versus time. The results are as follows: Figure 12 and Figure 13 As shown.

[0139] From Table 2 and Figures 11 to 13 As can be seen, platelets did not aggregate when no aggregation-inducing solution was added, but platelets began to aggregate after the addition of the aggregation-inducing solution. The aggregation rate reached half within 240 seconds and more than 50% within 480 seconds, and then remained stable. This shows that in this embodiment, the maximum platelet aggregation rate can be obtained in only about 8 minutes, and the platelet aggregation process can also be clearly observed, which is beneficial for obtaining clinical information from whole blood samples.

[0140] Example 3

[0141] A method for detecting platelet aggregation rate includes the following steps:

[0142] (31) Obtain venous blood from the subject as a whole blood sample. Add sodium citrate to the whole blood sample, mix well, and obtain a sample solution for later use.

[0143] (32) Take 200 μL of the sample solution from step (31) and add 30 mL of platelet staining solution.

[0144] The platelet staining solution contains 3.6 g / L glycine, 13 g / L sodium citrate, and 3 mg / L platelet staining agent, and the chemical formula of the platelet staining agent is:

[0145]

[0146] After adding the platelet staining solution, the pH of the first test sample was approximately 10; the solvent in the platelet staining solution was deionized water.

[0147] (33) Take 1 mL of the test solution obtained in step (32) and perform platelet detection to obtain a scatter plot of cell particles and platelet concentration in the test solution.

[0148] (34) Add the polymerization agent to the remaining test solution from step (32), mix well, and perform platelet detection every 60 seconds to obtain scatter plots of platelet concentration and cell particles at different time points. The detection results are shown in Table 3 and Figure 14 As shown.

[0149] The polymerization solution is a 3.28 mmol / L ADP-xNa saline solution; and the amount of polymerization solution used is 10 μL of polymerization solution added to every 2 mL of the remaining first test sample in step (32).

[0150] Table 3

[0151] Time s 0 60 120 180 240 300 360 420 <![CDATA[Platelet concentration 10 9 / L]]> 104 77 59 45 37 37 32 30 Platelet aggregation rate % 0 26 43 57 64 64 69 71

[0152] Based on the data in Table 3, curves showing the changes in platelet concentration and platelet aggregation rate over time were plotted. The results are as follows: Figure 15 and Figure 16 As shown.

[0153] From Table 3 and Figure 14 and Figure 16 As can be seen, platelets did not aggregate without the addition of the aggregation-inducing solution, but rapidly aggregated after the addition of the aggregation-inducing solution. The aggregation rate reached half within 120 seconds and over 64% within 300 seconds, and then remained stable. This indicates that in this embodiment, the maximum platelet aggregation rate can be obtained in just 5 minutes, and the platelet aggregation process can be clearly observed, which is beneficial for obtaining clinical information from whole blood samples.

[0154] Example 4

[0155] A method for detecting platelet aggregation rate includes the following steps:

[0156] (41) Obtain venous blood from the subject as a whole blood sample. Add sodium citrate to the whole blood sample, mix well, and obtain a sample solution for later use.

[0157] (42) Take 200 μL of the sample solution from step (41) and 1 mL of platelet staining solution, add them to 20 mL of platelet diluent, mix thoroughly to obtain the first sample to be tested.

[0158] The platelet diluent contains 5 g / L Tricine and 15 g / L sodium citrate, and the solvent in the platelet diluent is deionized water; the solvent in the platelet staining solution is ethanol, the concentration of the platelet staining agent in the platelet staining solution is 2 mg / L, and the chemical formula of the platelet staining agent is:

[0159]

[0160] The pH of the platelet diluent is approximately 8.5. After adding the platelet diluent and platelet staining solution, the pH of the first test sample is approximately 8.5.

[0161] (43) Take 1 mL of the first test sample obtained in step (42) and perform platelet detection to obtain the scatter plot of cell particles and platelet concentration in the test solution.

[0162] (44) Add the polymerization solution to the remaining first test sample from step (42), mix well to obtain the second test sample, and take 1 mL of the second test sample every 60 s for platelet detection to obtain platelet concentration and scatter plot of cell particles at different time points. The detection results are shown in Table 4 and Figure 17 As shown.

[0163] The polymerization solution is a 3.28 mmol / L ADP-xNa saline solution; and the amount of polymerization solution used is 15 μL of polymerization solution added to every 2 mL of the remaining first test sample in step (42).

[0164] Table 4

[0165] Time s 0 60 120 180 240 300 360 420 <![CDATA[Platelet concentration 10 9 / L]]> 104 77 59 45 37 37 37 37 Platelet aggregation rate % 0 26 43 57 64 64 64 64

[0166] (45) Based on the data in Table 4, plot the curves showing the changes in platelet concentration and platelet aggregation rate over time. The results are as follows: Figure 18 and Figure 19 As shown.

[0167] From Table 4 and Figure 17 and Figure 19 As can be seen, platelets did not aggregate without the addition of the aggregation-inducing solution, but rapidly aggregated after the addition of the aggregation-inducing solution. The aggregation rate reached half between 60 and 120 seconds, and 64% at 240 seconds, after which it remained stable. This indicates that in this embodiment, the maximum platelet aggregation rate can be obtained in just 4 minutes, and the platelet aggregation process can be clearly observed, which is beneficial for obtaining clinical information from whole blood samples.

[0168] Example 5

[0169] A method for detecting platelet aggregation rate includes the following steps:

[0170] (51) Obtain venous blood from the subject as a whole blood sample. Add sodium citrate to the whole blood sample, mix well, and obtain a sample solution for later use.

[0171] (52) Take 200 μL of the sample solution from step (51) and 1 mL of platelet staining solution, add them to 15 mL of platelet diluent, mix thoroughly to obtain the first sample to be tested.

[0172] The platelet diluent contained 5 g / L Tricine and 15 g / L sodium citrate. After adding the platelet diluent and platelet staining solution, the pH of the first test sample was approximately 8.5.

[0173] The platelet staining solution contains 5 mg / L of platelet staining agent and 5 mg / L of erythrocyte nucleic acid dye, and the chemical formula of the platelet staining agent is:

[0174]

[0175] The chemical formula of the erythrocyte nucleic acid dye is:

[0176]

[0177] The solvent in the platelet diluent is deionized water. The solvent in the platelet staining solution is a mixture of methanol and ethanol, with a mass ratio of methanol to ethanol of 7:93.

[0178] (53) Take 1 mL of the first sample obtained in step (52) for platelet detection, and obtain the scatter plot of cell particles and platelet concentration in the test solution.

[0179] (54) Add the polymerization solution to the remaining first test sample from step (52), mix well to obtain the second test sample, and take 1 mL of the second test sample every 60 s for platelet detection to obtain scatter plots of platelet concentration and cell particles at different time points. The detection results are shown in Table 5 and Figure 20 As shown.

[0180] The polymerization solution is a 65.6 mmol / L ADP-xNa saline solution; and the amount of polymerization solution used is 10 μL of polymerization solution added to every 2 mL of the remaining first test sample in step (52).

[0181] Table 5

[0182] Time s 0 60 120 180 <![CDATA[Platelet concentration 10 9 / L]]> 216 14 14 10 Platelet aggregation rate % 0 94 94 95

[0183] (55) Based on the data in Table 5, plot the curves of platelet concentration versus time and platelet aggregation rate versus time. The results are as follows: Figure 21 and Figure 22 As shown.

[0184] From Table 5 and Figures 20 to 22 As can be seen, platelets did not aggregate without the addition of the aggregation-inducing solution, but after the addition of the aggregation-inducing solution, platelets aggregated rapidly, with an aggregation rate of over 90% within 60 seconds, and then maintained a stable state. This indicates that in this embodiment, the maximum aggregation rate of platelets can be obtained in less than 1 minute. However, the aggregation rate is too fast, making it impossible to effectively observe the platelet aggregation process, which is not conducive to obtaining clinical information from whole blood samples.

[0185] In summary, as can be seen from Examples 1 to 5 above, the platelet aggregation rate detection method provided by the embodiments of the present invention can not only obtain the maximum platelet aggregation rate within 10 minutes, but also effectively observe the change process of platelet aggregation rate during the detection process, thereby providing effective clinical data for clinical medicine.

[0186] The above description is merely an embodiment of the present invention. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of the present invention, but these improvements all fall within the protection scope of the present invention.

Claims

1. A method for detecting platelet aggregation rate, characterized in that, The steps include the following: Add anticoagulant to the whole blood sample and mix well to obtain the sample solution; Platelet staining solution is added to the sample solution to stain the sample solution, thereby obtaining the first test sample; A portion of the first test sample was taken for platelet detection to obtain a scatter plot of cell particles and platelet concentration in the first test sample; Add polymerizing solution to the remaining first test sample to obtain a second test sample. Take a portion of the second test sample at regular intervals for platelet detection to obtain a scatter plot of cell particles and platelet concentration of the second test sample at each time point. Calculate the platelet aggregation rate based on the test results; The platelet staining solution contains a platelet staining agent, which is used to penetrate the platelet membrane to stain the nucleic acids inside the platelets, but does not penetrate the cell membranes of red blood cells and white blood cells and therefore does not stain the red blood cells and white blood cells; The general structural formula I of the platelet staining agent is: ……（I）； In the formula, R1, R2, R6, R7, R8, and R9 are selected from hydrogen atoms, C1-C atoms, and C2-C2 atoms. 18 Alkyl, C1-C 18 Alkyl COOR 10 C1-C 18 Alkyl COR 10 C1-C 18 Alkyl CNR 10 R 10 C1-C 18 Alkyl OR 10 Any one of them; R3, R4, R5, R 10 Selected from any one of C1-C8 alkyl groups; X - It is selected from any one of halide ions, sulfate ions, phosphate ions, and boron halide ions; A is selected from C1-C 18 At least one of alkyl, CH2O, and CH2CH2O; n is any value between 2 and 20.

2. The method for detecting platelet aggregation rate as described in claim 1, characterized in that, The preset formula for calculating the platelet aggregation rate is as follows: ； Where P represents platelet aggregation rate, A0 represents the platelet concentration detection value corresponding to the first test sample, and A n This represents the platelet concentration value detected in the nth test corresponding to the second test sample, where n is a natural number greater than or equal to 3.

3. The method for detecting platelet aggregation rate as described in claim 2, characterized in that, The platelet concentration value corresponding to the nth test is the number of platelet concentrations detected in the nth test when the platelet concentrations detected in the n-2nd, n-1st, and nth tests are the same or tend to be the same.

4. The method for detecting platelet aggregation rate as described in claim 2, characterized in that, The preset calculation formula is: ； Where P represents the platelet aggregation rate detection value, A0 represents the platelet concentration detection value corresponding to the first test, and A x This represents the average value of the platelet concentration values ​​from the (n-2), (n-1), and (n-n)th tests when the platelet concentration values ​​are the same or approach the same.

5. The method for detecting platelet aggregation rate as described in claim 1, characterized in that, The platelet staining solution also includes buffers and osmotic pressure regulators; The buffer includes at least one of tris(hydroxymethyl)aminomethane buffer, tris(hydroxymethyl)methylglycine buffer, imidazole, phosphate, and citrate buffer. The osmotic pressure regulator includes at least one of the following: halogen salts, sulfates, carbonates, alkali metal salts of organic acids, glycerol, mannitol, and glucose.

6. The method for detecting platelet aggregation rate as described in claim 1, characterized in that, The platelet staining solution may also contain a red blood cell nucleic acid staining agent, the red blood cell nucleic acid staining agent having a general structural formula II as follows: ……(II); Among them, R 11 R 12 These represent alkyl groups with 1 to 3 carbon atoms respectively; Y represents chloride or sulfate ions.

7. The method for detecting platelet aggregation rate as described in any one of claims 1 to 4, characterized in that, The polymerization liquid includes an active agent, a salt, and a third solvent; The active agent includes at least one selected from adenosine diphosphate, collagen, arachidonic acid, thrombin, adrenaline, prostaglandin G2, prostaglandin H2, and thromboxane A2; the concentration of the active agent in the polymerization solution is 0.3 mmol / L to 3.5 mmol / L; the amount of the active agent added per milliliter of the test solution is 2.425 × 10⁻⁶. -6 mmol ~ 3.5 × 10 -5 mmol.

8. The application of the platelet aggregation rate detection method according to any one of claims 1-7 in a hematology analyzer, characterized in that, The blood cell analyzer includes a sampling module, a reagent addition module, a reaction module, a delivery module, a detection module, and a first controller. The sampling module is used to collect the sample solution and distribute it to the reaction module; The reagent addition module is used to add platelet staining solution to the reaction module for staining to obtain the first test sample; The delivery module is used to send a portion of the first sample to be tested into the detection module for detection; The reagent addition module is further configured to add the polymerization solution to the remaining first test sample in the reaction module to obtain the second test sample; The delivery module is also used to send a portion of the second sample to be tested into the detection module for detection at a preset time. The detection module includes a flow chamber and an optical unit. The flow chamber is connected to the delivery module. The delivery module is used to inject a portion of the first test sample and a portion of the second test sample into the flow chamber at preset times, so that the portion of the first test sample and the portion of the second test sample form corresponding sample streams in the flow chamber. The optical unit is used to perform optical detection on the sample streams to output a detection signal, which is a scatter plot of cell particles. The first controller acquires the detection signal and calculates the platelet aggregation rate based on the detection signal.

9. The application of the platelet aggregation rate detection method as described in claim 8 in a hematology analyzer, characterized in that, The blood cell analyzer also includes an information output module; the first controller transmits the acquired detection signal to the information output module, and the information output module outputs a platelet concentration change curve, wherein the vertical axis of the platelet concentration change curve is the platelet concentration value and the horizontal axis is time.

10. The application of the platelet aggregation rate detection method as described in claim 9 in a hematology analyzer, characterized in that, When the platelet concentration stabilizes, the blood cell analyzer stops injecting the second sample into the flow chamber.

11. The application of the platelet aggregation rate detection method as described in claim 8 in a hematology analyzer, characterized in that, The blood cell analyzer includes a routine blood test mode and a platelet aggregation rate test mode. When the first controller receives the routine blood test mode information, the first controller controls the reagent addition module to add platelet staining solution to the reaction module for staining to obtain the first test sample. The delivery module delivers a portion of the first test sample to the detection module for detection to obtain the routine blood test result. When the first controller receives information from the platelet aggregation rate detection module, the first controller controls the reagent addition module to add the aggregation solution to the remaining first test sample to obtain the second test sample. The delivery module delivers a portion of the second test sample to the detection module for detection at regular intervals to obtain the detection results corresponding to each time point and calculate the platelet aggregation rate.

12. The application of the platelet aggregation rate detection method as described in claim 8 in a hematology analyzer, characterized in that, When the platelet aggregation rate is not within the range of 30% to 70%, the first controller outputs abnormal detection information.

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