HIT functional detection method and device, storage medium and medical experiment diagnosis platform

Abstract

The invention discloses an HIT functional detection method and device, a storage medium and a medical experiment diagnosis platform, and the method comprises the following steps: preparing standard platelet-rich plasma and platelet-deficient plasma to be detected, and preparing heparin working solutions with different concentrations; a reaction system is built according to the standard platelet-rich plasma, the platelet-deficient plasma to be detected and the heparin working solutions with different concentrations, and the reaction system comprises a heparin-free group, a low-heparin-concentration group and a high-heparin-concentration group; respectively adding a fluorescently labeled CD41 / CD61 antibody, a fluorescently labeled CD62P antibody and Annexin V into the heparin-free group, the low-heparin-concentration group and the high-heparin-concentration group, and obtaining procoagulant platelet proportions corresponding to the heparin-free group, the low-heparin-concentration group and the high-heparin-concentration group through flow cytometry; and generating an HIT detection result according to the procoagulant platelet proportions corresponding to the heparin-free group, the low-heparin-concentration group and the high-heparin-concentration group and a preset proportion threshold. Therefore, radioactive substance application is avoided, the operation process is simple and convenient, the detection precision is high, and clinical application is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical experiment diagnosis, and in particular to a HIT functional detection method, a computer readable storage medium, a HIT functional detection device and a medical experiment diagnosis platform. BACKGROUND

[0002] HIT (Heparin-Induced Thrombocytopenia) is an antibody-mediated prothrombotic adverse reaction characterized by platelet-activating antibodies against platelet factor 4 (PF4)-heparin complexes, which manifests clinically as a decrease in platelet count and a high thrombotic risk. Thrombosis caused by HIT can occur in arteries or veins, and in severe cases, it can progress to pulmonary embolism, limb necrosis, etc., which is life-threatening. Therefore, timely and accurate diagnosis is crucial for the management of HIT, which can avoid the imbalance of treatment strategies caused by relying on empirical management before diagnosis.

[0003] However, the clinical diagnosis of HIT is complex. The current gold standard functional detection, such as the serotonin-release assay (SRA) and the heparin-induced platelet activation (HIPA), lacks a finished kit, the experimental process is complex, and the requirements for instruments and experimental techniques are high. The SRA method also has radioactive hazards, etc. Therefore, it is difficult for domestic hospital coagulation laboratories to routinely carry out HIT laboratory diagnosis, and there is still a large gap in this field. Therefore, there is an urgent need for a safe, simple, repeatable, and clinically applicable HIT functional detection method. SUMMARY

[0004] The present application aims to at least partially solve one of the technical problems in the related art. To this end, the first object of the present application is to provide a HIT functional detection method that can avoid the use of radioactive substances, has a simple operation process and high detection accuracy, and has clinical application value.

[0005] The second object of the present application is to provide a computer readable storage medium.

[0006] The third object of the present application is to provide a HIT functional detection device.

[0007] The fourth object of the present application is to provide a medical experiment diagnosis platform.

[0008] To achieve the above object, the HIT functional detection method according to the first aspect of the present application comprises: preparing standard platelet-rich plasma and to-be-tested platelet-poor plasma, and preparing heparin working solution with different concentrations; building a reaction system according to the standard platelet-rich plasma, the to-be-tested platelet-poor plasma and the heparin working solution with different concentrations, wherein the reaction system comprises a no-heparin group, a low heparin concentration group and a high heparin concentration group; adding fluorescently labeled CD41 / CD61 antibody, CD62P antibody and Annexin V into the no-heparin group, the low heparin concentration group and the high heparin concentration group respectively, and obtaining the corresponding coagulation-promoting platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry; and generating a HIT detection result according to the coagulation-promoting platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group and a preset ratio threshold.

[0009] The HIT functional detection method according to the embodiments of the present application can avoid the use of radioactive substances, has a simple operation process and high detection precision, and has clinical application value.

[0010] In addition, the HIT functional detection method according to the above embodiments of the present application can have the following additional technical features: According to one embodiment of the present application, the no-heparin group comprises: 90 standard platelet-rich plasma, 40 to-be-tested platelet-poor plasma and 70 calcium-containing buffer solution; the low heparin concentration group comprises: 90 standard platelet-rich plasma, 40 to-be-tested platelet-poor plasma, 30 heparin working solution with a first concentration and 40 calcium-containing buffer solution; and the high heparin concentration group comprises: 90 standard platelet-rich plasma, 40 to-be-tested platelet-poor plasma, 30 heparin working solution with a second concentration and 40 calcium-containing buffer solution.

[0011] According to one embodiment of the present application, the no heparin group corresponds to a heparin concentration of 0 U / ml, the low heparin concentration group corresponds to a heparin concentration of 0.5 U / ml, and the high heparin concentration group corresponds to a heparin concentration of 100 U / ml.

[0012] According to one embodiment of the present application, the method for obtaining the proportion of procoagulant platelets corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry comprises: obtaining the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry, respectively; obtaining the CD62P and PS double positive platelet subpopulation corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry from the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group, respectively; and obtaining the proportion of procoagulant platelets corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group according to the proportion of the CD62P and PS double positive platelet subpopulation in the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group.

[0013] According to one embodiment of the present application, the method for generating the HIT detection result according to the proportion of procoagulant platelets corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group and the preset proportion threshold value comprises: if the proportion of procoagulant platelets corresponding to the no heparin group is less than the preset proportion threshold value, the proportion of procoagulant platelets corresponding to the low heparin concentration group is greater than the preset proportion threshold value, and the inhibition degree of the high heparin concentration group exceeds the preset percentage threshold value, determining that the HIT detection result is positive.

[0014] According to one embodiment of the present application, the preset proportion threshold value is the mean value of the proportion of procoagulant platelets detected under low heparin conditions of at least a plurality of standard platelet-rich plasma of healthy donors plus 2 times the standard deviation.

[0015] According to one embodiment of the present application, the method further comprises: after the reaction system is built, the no heparin group, the low heparin concentration group and the high heparin concentration group are respectively shaken and incubated at room temperature for a preset first time period; and after the fluorescently labeled CD41 / CD61 antibody, CD62P antibody and Annexin V are added to the no heparin group, the low heparin concentration group and the high heparin concentration group, respectively, the no heparin group, the low heparin concentration group and the high heparin concentration group are respectively incubated in the dark for a preset second time period.

[0016] To achieve the above object, the computer readable storage medium according to the second aspect of the present application stores a HIT functional detection program, and the HIT functional detection program is executed by a processor to implement the HIT functional detection method according to the above embodiment of the present application.

[0017] The computer readable storage medium according to the embodiment of the present application can avoid the use of radioactive substances, has a simple operation process and high detection accuracy, and has clinical application value by executing the HIT functional detection program stored thereon.

[0018] To achieve the above object, the HIT functional detection device according to the third aspect of the present application comprises: a test material preparation module configured to prepare standard platelet-rich plasma and to-be-detected platelet-poor plasma, and to prepare heparin working solutions with different concentrations; a reaction system building module configured to build a reaction system according to the standard platelet-rich plasma, the to-be-detected platelet-poor plasma and the heparin working solutions with different concentrations, wherein the reaction system comprises a no-heparin group, a low heparin concentration group and a high heparin concentration group; a flow cytometry detection module configured to add fluorescently labeled CD41 / CD61 antibodies, CD62P antibodies and Annexin V into the no-heparin group, the low heparin concentration group and the high heparin concentration group respectively, and to obtain the corresponding coagulation-promoting platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry; and a HIT detection module configured to generate a HIT detection result according to the coagulation-promoting platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group and a preset ratio threshold.

[0019] The HIT functional detection device according to the embodiment of the present application can avoid the use of radioactive substances, has a simple operation process and high detection accuracy, and has clinical application value by mixing and incubating the standard platelet-rich plasma and the to-be-detected platelet-poor plasma in different heparin concentration environments, then adding fluorescently labeled CD41 / CD61 antibodies, CD62P antibodies and Annexin V, detecting the coagulation-promoting platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry, and generating a HIT detection result according to the coagulation-promoting platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group and a preset ratio threshold.

[0020] To achieve the above object, the medical experiment diagnosis platform according to the fourth aspect of the present application comprises the HIT functional detection device according to the above embodiment of the present application.

[0021] The medical experiment diagnosis platform according to the embodiment of the present application can avoid the use of radioactive substances, has a simple operation process and high detection accuracy, and has clinical application value by using the aforementioned HIT functional detection device.

[0022] Additional aspects and advantages of the present application will be given in part in the following description, become apparent from the following description, or be learned by the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a flowchart of a HIT functional detection method according to an embodiment of the present application; Figure 2 is a flowchart of a HIT functional detection method according to an embodiment of the present application; Figure 3 is a PCP ratio diagram of a positive control sample according to an embodiment of the present application; Figure 4 is a PCP ratio diagram of a negative control sample according to an embodiment of the present application; Figure 5 is a flowchart of a HIT functional detection method according to a specific embodiment of the present application; Figure 6 is a block diagram of a HIT functional detection device according to an embodiment of the present application; Figure 7 is a block diagram of a medical experiment diagnosis platform according to an embodiment of the present application. DETAILED DESCRIPTION

[0024] Embodiments of the present application are described in detail below with reference to the accompanying drawings, wherein the same or similar notations represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by way of example with reference to the accompanying drawings are illustrative and are intended to explain the present application, and are not to be understood as limiting the present application.

[0025] Embodiments of a HIT functional detection method, a computer readable storage medium, a HIT functional detection device and a medical experiment diagnosis platform of the present application are described below with reference to the accompanying drawings.

[0026] Figure 1 is a flowchart of a HIT functional detection method according to an embodiment of the present application.

[0027] Specifically, in some embodiments of the present application, as shown in Figure 1 the HIT functional detection method comprises: S101, preparing standard platelet-rich plasma and platelet-poor plasma to be tested, and preparing heparin working solution of different concentrations.

[0028] It can be understood that in this embodiment of the present application, the preparation process of standard platelet-rich plasma (PRP) and platelet-poor plasma (PPP) to be tested, and the preparation process of heparin working solution of different concentrations are as follows: Standard platelet-rich plasma (PRP): (1) Collect 3 mL of fresh sodium citrate anticoagulant whole blood from a healthy donor, and stand at room temperature for 20 minutes; (2) centrifuge at 120 g for 15 minutes, collect the upper PRP, and stand for 20 minutes for standby.

[0029] Platelet-poor plasma (PPP) to be tested: (1) Collect 3 mL of fresh sodium citrate anticoagulant whole blood from a patient, centrifuge at 2000 g for 15 minutes, and take the supernatant PPP; (2) incubate in a 56°C water bath for 30 minutes, and cool down for standby.

[0030] Heparin working solution: According to the division of the no-heparin group, the low-heparin concentration group and the high-heparin concentration group, the corresponding concentration of heparin working solution is configured.

[0031] S102, according to the standard platelet-rich plasma, the platelet-poor plasma to be tested and the different concentrations of heparin working solution, a reaction system is built, wherein the reaction system includes a no-heparin group, a low-heparin concentration group and a high-heparin concentration group.

[0032] It can be understood that in this embodiment of the present application, the standard platelet-rich plasma (PRP) prepared from fresh sodium citrate anticoagulant whole blood of a healthy donor is mixed with the platelet-poor plasma (PPP) to be tested prepared from fresh sodium citrate anticoagulant whole blood of a patient and a calcium-containing buffer, and different concentrations of heparin working solution are added respectively to form the no-heparin group, the low-heparin concentration group and the high-heparin concentration group in the reaction system.

[0033] S103, fluorescently labeled CD41 / CD61 antibody, CD62P antibody and Annexin V are added respectively in the no-heparin group, the low-heparin concentration group and the high-heparin concentration group, and the corresponding pro-coagulation platelet ratios of the no-heparin group, the low-heparin concentration group and the high-heparin concentration group are obtained by flow cytometry.

[0034] It can be understood that in this embodiment of the present application, by adding fluorescently labeled CD41 / CD61 antibody (for example, FITC-CD41 / CD61), CD62P antibody (for example, PE-CD62P) and Annexin V (for example, APC-Annexin V antibody) respectively in the no-heparin group, the low-heparin concentration group and the high-heparin concentration group, the antibodies with fluorescent labeling are combined with the platelet population, and then the corresponding pro-coagulation platelet ratios of the no-heparin group, the low-heparin concentration group and the high-heparin concentration group are counted by flow cytometry.

[0035] S104, according to the corresponding pro-coagulation platelet ratios of the no-heparin group, the low-heparin concentration group and the high-heparin concentration group and the preset ratio threshold, a HIT detection result is generated.

[0036] It can be understood that in this embodiment of the application, the accurate HIT functional detection is realized by comparing the differences between the coagulation-promoting platelet ratios corresponding to the no-heparin group, the low heparin concentration group and the high heparin concentration group and the preset ratio threshold value, and the corresponding HIT detection result is generated.

[0037] Further, in some embodiments of the application, the no-heparin group comprises 90 standard platelet-rich plasma, 40 the to-be-tested platelet-poor plasma, and 70 calcium-containing buffer solution; the low heparin concentration group comprises 90 standard platelet-rich plasma, 40 the to-be-tested platelet-poor plasma, 30 the first concentration heparin working solution, and 40 calcium-containing buffer solution; the high heparin concentration group comprises 90 standard platelet-rich plasma, 40 the to-be-tested platelet-poor plasma, 30 the second concentration heparin working solution, and 40 calcium-containing buffer solution.

[0038] It can be understood that in this embodiment of the application, the total amount of the reaction system is 200 , wherein the no-heparin group is mixed by 90 standard platelet-rich plasma, 40 the to-be-tested platelet-poor plasma, and 70 calcium-containing buffer solution (instead of heparin solution), the low heparin concentration group is mixed by 90 standard platelet-rich plasma, 40 the to-be-tested platelet-poor plasma, 30 the first concentration heparin working solution, and 40 calcium-containing buffer solution, and the high heparin concentration group is mixed by 90 standard platelet-rich plasma, 40 the to-be-tested platelet-poor plasma, 30 the second concentration heparin working solution, and 40 calcium-containing buffer solution.

[0039] Further, in some embodiments of the application, the no-heparin group corresponds to a heparin concentration of 0 U / ml, the low heparin concentration group corresponds to a heparin concentration of 0.5 U / ml, and the high heparin concentration group corresponds to a heparin concentration of 100 U / ml.

[0040] It can be understood that, in this embodiment of the present application, after adding different concentrations of heparin working solution in the no heparin group, the low heparin concentration group and the high heparin concentration group respectively, the no heparin group, the low heparin concentration group and the high heparin concentration group will reach the corresponding heparin concentration, specifically, the no heparin group (adding calcium-containing buffer) corresponds to the heparin concentration of 0 U / ml, the low heparin concentration group (adding the first concentration heparin working solution) corresponds to the heparin concentration of 0.5 U / ml, and the high heparin concentration group (adding the second concentration heparin working solution) corresponds to the heparin concentration of 100 U / ml.

[0041] Further, in some embodiments of the present application, as shown in Figure 2 the no heparin group, the low heparin concentration group and the high heparin concentration group correspond to the proportion of procoagulant platelets, including: S201, acquiring the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry respectively.

[0042] It can be understood that, in this embodiment of the present application, the flow cytometer is used to detect the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group.

[0043] S202, acquiring the CD62P and PS double positive platelet subpopulation corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group from the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry respectively.

[0044] It can be understood that, in this embodiment of the present application, after screening the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group, the flow cytometer is also used to detect the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group from the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group.

[0045] S203, acquiring the proportion of procoagulant platelets corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group according to the proportion of CD62P and PS double positive platelet subpopulation in the CD41 / CD61 positive platelet population corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group.

[0046] It can be understood that in this embodiment of the present application, after screening the platelet population expressing CD41 / CD61 positive and the platelet subpopulation expressing CD62P and PS double positive (procoagulant platelets) in the no heparin group, the low heparin concentration group and the high heparin concentration group, the proportion of the number of platelet subpopulation to the number of total platelet population, that is, the proportion of procoagulant platelets (PCP) corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group.

[0047] Further, in some embodiments of the present application, according to the proportion of procoagulant platelets corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group and the preset proportion threshold, the HIT detection result is generated, including: if the proportion of procoagulant platelets corresponding to the no heparin group is less than the preset proportion threshold, the proportion of procoagulant platelets corresponding to the low heparin concentration group is greater than the preset proportion threshold, and the inhibition degree of the high heparin concentration group exceeds the preset percentage threshold, it is determined that the HIT detection result is positive.

[0048] It can be understood that when the proportion of procoagulant platelets corresponding to the no heparin group is less than the preset proportion threshold, the proportion of procoagulant platelets corresponding to the low heparin concentration group is greater than the preset proportion threshold, and the inhibition degree of the high heparin concentration group (i.e. the PCP proportion of the high heparin concentration group decreases by a preset percentage threshold compared with the PCP proportion of the low heparin concentration group) exceeds the preset percentage threshold, it can be judged that the pathological characteristics of the platelet-poor plasma to be tested meet the HIT disease characteristics, at this time, the HIT detection result of the platelet-poor plasma to be tested is determined to be positive.

[0049] Specifically, Figure 3 is the PCP result of flow partition screening based on the positive control sample, wherein blue represents the low heparin concentration group, yellow represents the high heparin concentration group, and red represents the no heparin concentration group. It can be seen that under the condition that the total number of original platelets is similar, the PCP proportion of the low heparin concentration group is obviously increased, and the PCP proportion of the high heparin concentration group is obviously decreased, and then combined with the preset proportion threshold, the HIT detection result can be determined to be positive, and Figure 4 is the PCP result of flow partition screening based on the negative control sample, wherein blue represents the low heparin concentration group, yellow represents the high heparin concentration group, and red represents the no heparin concentration group. It can be seen that under the condition that the total number of original platelets is similar, the PCP proportion of the no heparin concentration group, the low heparin concentration group and the high heparin concentration group has little difference, and the PCP proportion is obviously reduced compared with the positive control sample, and then combined with the preset proportion threshold, the HIT detection result can be determined to be negative. Thus, by accurately distinguishing the positive sample and the negative sample, the HIT detection can be more accurately realized.

[0050] Optionally, in the above-mentioned embodiments of the present application, the preset percentage threshold can be set according to actual testing requirements, for example, the preset percentage threshold can be 50%.

[0051] Further, in some embodiments of the present application, the preset ratio threshold is the mean value of the pro-coagulant platelet ratio detected in the standard platelet-rich plasma of at least a plurality of healthy donors under low heparin conditions plus 2 times the standard deviation.

[0052] It can be understood that, in this embodiment of the present application, the mean value of the pro-coagulant platelet ratio detected in the standard platelet-rich plasma of at least a plurality of healthy donors under low heparin conditions plus 2 times the standard deviation can be used as the preset ratio threshold.

[0053] Further, in some embodiments of the present application, the method further comprises: after the reaction system is built, the no-heparin group, the low heparin concentration group and the high heparin concentration group are respectively shaken and incubated at room temperature for a preset first time length; and after the fluorescently labeled CD41 / CD61 antibody, the CD62P antibody and the Annexin V are respectively added to the no-heparin group, the low heparin concentration group and the high heparin concentration group, the no-heparin group, the low heparin concentration group and the high heparin concentration group are respectively incubated in the dark for two preset time lengths.

[0054] It can be understood that, in this embodiment of the present application, after the reaction system is built, the no-heparin group, the low heparin concentration group and the high heparin concentration group are respectively shaken and incubated at room temperature for a preset first time length, so that the heparin and the platelet population can fully bind and react; and after the fluorescently labeled CD41 / CD61 antibody, the CD62P antibody and the Annexin V are respectively added to the no-heparin group, the low heparin concentration group and the high heparin concentration group, the no-heparin group, the low heparin concentration group and the high heparin concentration group are respectively incubated in the dark for two preset time lengths, so that the antibody and the platelet population can fully bind and react.

[0055] Optionally, in the above-mentioned embodiments of the present application, the first preset time length and the second preset time length can be set according to actual testing requirements, for example, the first preset time length can be 1 hour and the second preset time length can be 15 minutes.

[0056] The specific detection process of the HIT function detection method of the embodiments of the present application will be described below in combination with Figure 5 and specific embodiments of the present application. Figure 5 As shown in S1, fresh sodium citrate anticoagulated whole blood of a healthy donor is used to prepare platelet-rich plasma (PRP), which is mixed with platelet-poor plasma (PPP) of a patient to be tested and calcium-containing buffer, and is incubated under conditions of no heparin (0 U / mL), low heparin concentration (0.5 U / mL) and high heparin concentration (100 U / mL).

[0057] S2, adding fluorescently labeled CD41 / CD61 antibody, CD62P antibody and Annexin V to detect platelet surface markers S3, screening CD41 / CD61 positive platelet population by flow cytometry, and calculating the proportion of CD62P (P-selectin) and PS (Phosphatidylserine) double positive procoagulant platelets (PCP).

[0058] S4, taking the mean value of the PCP proportion detected in the low heparin condition of at least 10 healthy donor PPPs plus 2 times the standard deviation as the threshold, wherein when the PCP proportion in the no heparin group is lower than the threshold, the low heparin group is higher than the threshold, and the inhibition degree of the high heparin group exceeds 50%, it is determined that the functional detection of HIT is positive.

[0059] It should be noted that in some embodiments of the present application, other sources of platelets and plasma, similar platelet activation or apoptosis markers, antibodies carrying fluorescent labels, different incubation conditions and detection platforms can also be used to achieve the purpose of functional detection of HIT.

[0060] I. Platelet source substitution Not limited to healthy donor PRP, but also can use: (1) PRP of relatively healthy patients without related interference factors (2) Platelet suspension prepared by platelet concentrates.

[0061] II. Plasma source substitution The sample to be tested is not limited to PPP, but can also use: (1) Serum treated to replace PPP; (2) Commercially available reference plasma as a control or mixed matrix; III. Detection index substitution In addition to CD41 / CD61 screening platelets, PCP determination in addition to CD62P+PS double positive, other platelet activation markers such as CD63, CD40L, PAC-1, etc. can also be used; IV. Determination standard substitution Not limited to "healthy donor threshold + 2SD", but also can use: (1) The optimal cutoff value determined by ROC curve; (2) Taking the control IgG group / substitute antibody group as the background signal control; (3) Using machine learning or discriminant function to calculate comprehensive score, instead of single threshold.

[0062] V. Incubation system replacement The total amount of the system can be different (such as 100 , 150 ), the addition amount of each part can be different, the proportion is reasonable, and it meets the instrument detection requirement; The incubation condition is not limited to room temperature shaking, and the following can also be used: (1) constant temperature water bath with slight shaking; (2) microplate shaker incubation; (3) incubator (5% , 37°C).

[0063] VI. Different fluorescently labeled antibody replacement It is not limited to using the following labeled antibody combination: FITC-CD41 / CD61, PE-CD62P, APC-AnnexinV, as long as the antibody for detecting several related markers can also be used.

[0064] VII. Heparin concentration setting replacement It is not limited to 0 / 0.5 / 100 U / mL, and can be replaced by: (1) a certain range of changes representing different heparin concentration environments of "no heparin concentration", "low heparin concentration" and "high heparin concentration"; (2) gradient concentration (such as 0.1, 0.5, 1, 10, 100 U / mL, etc.); (3) depending on different specifications of commercial heparin sodium injection.

[0065] It should be understood that, based on the above-mentioned HIT functional detection method of the embodiments of the present application, compared with the prior art: (1) both specificity and sensitivity are higher, overcoming the problem of high false positive rate of immunological detection, which is helpful for clinical diagnosis of H IT. (2) No radioactivity labeling, high safety. (3) The method can be standardized, suitable for routine development in clinical laboratory. (4) It can replace the existing gold standard functional method, significantly improving the accessibility of H IT detection.

[0066] In summary, according to the HIT functional detection method of the embodiment of the present application, the standard platelet-rich plasma and the platelet-poor plasma to be detected are mixed and incubated under different heparin concentration environments, then the fluorescently labeled CD41 / CD61 antibody, CD62P antibody and Annexin V are added, and the flow cytometry is used to detect the corresponding procoagulant platelet proportion of the no heparin group, low heparin concentration group and high heparin concentration group, and the HIT detection result is generated according to the procoagulant platelet proportion of the no heparin group, low heparin concentration group and high heparin concentration group and the preset proportion threshold. Thus, the application of radioactive substances is avoided, the operation process is simple and the detection precision is high, and the method has clinical application value.

[0067] Based on the HIT functional detection method of the aforementioned embodiment of the present application, the present embodiment further provides a computer readable storage medium having a HIT functional detection program stored thereon, and the HIT functional detection program is executed by a processor to realize the HIT functional detection method of the aforementioned embodiment of the present application.

[0068] It should be understood that the specific implementation of the computer readable storage medium of the embodiment of the present application can refer to the specific implementation of the HIT functional detection method of the aforementioned embodiment of the present application, and to reduce redundancy, it will not be described here.

[0069] In summary, according to the computer readable storage medium of the embodiment of the present application, by executing the HIT functional detection program stored thereon, the application of radioactive substances can be avoided, the operation process is simple and the detection precision is high, and the method has clinical application value.

[0070] Figure 6 is a block schematic diagram of the HIT functional detection device according to the embodiment of the present application.

[0071] Specifically, in some embodiments of the present application, as shown in Figure 6 the HIT functional detection device 100 comprises a test material preparation module 10, a reaction system building module 20, a flow cytometry detection module 30 and a HIT detection module 40.

[0072] The test material preparation module 10 is configured to prepare standard platelet-rich plasma and to-be-tested platelet-poor plasma, and to prepare heparin working solutions with different concentrations; the reaction system building module 20 is configured to build a reaction system according to the standard platelet-rich plasma, the to-be-tested platelet-poor plasma and the heparin working solutions with different concentrations, wherein the reaction system includes a no-heparin group, a low heparin concentration group and a high heparin concentration group; the flow cytometry detection module 30 is configured to add fluorescently labeled CD41 / CD61 antibodies, CD62P antibodies and Annexin V to the no-heparin group, the low heparin concentration group and the high heparin concentration group respectively, and to obtain the procoagulant platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry; and the HIT detection module 40 is configured to generate a HIT detection result according to the procoagulant platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group and a preset ratio threshold.

[0073] Further, in some embodiments of the present application, the no-heparin group includes: 90 standard platelet-rich plasma, 40 to-be-tested platelet-poor plasma and 70 calcium-containing buffer; the low heparin concentration group includes: 90 standard platelet-rich plasma, 40 to-be-tested platelet-poor plasma, 30 heparin working solution with a first concentration and 40 calcium-containing buffer; and the high heparin concentration group includes: 90 standard platelet-rich plasma, 40 to-be-tested platelet-poor plasma, 30 heparin working solution with a second concentration and 40 calcium-containing buffer.

[0074] Further, in some embodiments of the present application, the no-heparin group corresponds to a heparin concentration of 0 U / ml, the low heparin concentration group corresponds to a heparin concentration of 0.5 U / ml, and the high heparin concentration group corresponds to a heparin concentration of 100 U / ml.

[0075] Further, in some embodiments of the present application, the flow cytometry detection module 30 is further configured to obtain CD41 / CD61 positive platelet populations and CD62P and PS double positive platelet subpopulations of the no-heparin group, the low heparin concentration group and the high heparin concentration group by flow cytometry respectively; and to obtain the procoagulant platelet ratios of the no-heparin group, the low heparin concentration group and the high heparin concentration group according to the CD41 / CD61 positive platelet populations and the CD62P and PS double positive platelet subpopulations of the no-heparin group, the low heparin concentration group and the high heparin concentration group.

[0076] Further, in some embodiments of the present application, the HIT detection module 40 is further configured to determine the HIT detection result as positive if the procoagulant platelet ratio corresponding to the low heparin concentration group is greater than the preset ratio threshold, and the inhibition degree of the high heparin concentration group exceeds the preset percentage threshold.

[0077] Further, in some embodiments of the present application, the preset ratio threshold is the mean value of the procoagulant platelet ratio of the standard platelet-rich plasma of at least a plurality of healthy donors detected under the low heparin condition plus 2 times the standard deviation.

[0078] Further, in some embodiments of the present application, after the reaction system is built, the no heparin group, the low heparin concentration group and the high heparin concentration group are shaken and incubated at room temperature for a preset first time period, and after the fluorescently labeled CD41 / CD61 antibody, the CD62P antibody and the Annexin V are added to the no heparin group, the low heparin concentration group and the high heparin concentration group respectively, the no heparin group, the low heparin concentration group and the high heparin concentration group are incubated in the dark for two preset time periods respectively.

[0079] It should be understood that the specific implementation of the HIT functional detection device of the embodiments of the present application corresponds to the specific implementation of the HIT functional detection method of the embodiments of the present application described above, and to reduce redundancy, it will not be described here.

[0080] In summary, according to the HIT functional detection device of the embodiments of the present application, by mixing and incubating the standard platelet-rich plasma and the platelet-poor plasma to be tested in different heparin concentration environments, then adding the fluorescently labeled CD41 / CD61 antibody, the CD62P antibody and the Annexin V, and using flow cytometry to detect the procoagulant platelet ratio corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group, and generating the HIT detection result according to the procoagulant platelet ratio corresponding to the no heparin group, the low heparin concentration group and the high heparin concentration group and the preset ratio threshold. Thus, the use of radioactive substances is avoided, the operation process is simple and the detection accuracy is high, which has clinical application value.

[0081] Figure 7 is a block schematic diagram of a medical experiment diagnosis platform according to an embodiment of the present application.

[0082] Specifically, in some embodiments of the present application, as shown in Figure 7 the medical experiment diagnosis platform 1000 includes the HIT functional detection device 100 of the embodiments of the present application described above.

[0083] It should be understood that the specific implementation of the medical experiment diagnosis platform 1000 of the embodiments of the present application can refer to the specific implementation of the aforementioned HIT function detection method of the embodiments of the present application. In order to reduce redundancy, it will not be repeated here.

[0084] In summary, according to the medical experiment diagnosis platform of the embodiments of the present application, by using the aforementioned HIT function detection device, the application of radioactive substances can be avoided, the operation process is simple and the detection precision is high, and the medical experiment diagnosis platform has clinical application value.

[0085] It should be noted that the logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a list of executable instructions for implementing logical functions, which can be embodied in any computer-readable medium for use by or in connection with an instruction execution system, apparatus or device, such as a computer-based system, a system including a processor or other system that can fetch the instructions from an instruction execution system, apparatus or device and execute the instructions, or in conjunction with these instruction execution systems, apparatus or devices. For the purpose of this specification, "computer-readable medium" can be any device that can contain, store, communicate, propagate or transport programs for use by or in connection with an instruction execution system, apparatus or device, or in conjunction with these instruction execution systems, apparatus or devices. More specific examples (non-exhaustive list) of computer-readable medium include the following: electrical connections having one or more wires (electronic devices), portable computer diskette (magnetic devices), random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), fiber optic devices, and portable compact disc read-only memory (CD ROM). In addition, the computer-readable medium can even be paper or other suitable medium on which the program can be printed, because the program can be electronically obtained, for example, by optical scanning of the paper or other medium, followed by editing, interpretation or processing, if necessary, in other suitable ways, and then stored in the computer memory.

[0086] It should be understood that parts of the present application can be realized by hardware, software, firmware or a combination thereof. In the above-mentioned embodiments, a plurality of steps or methods can be realized by software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if realized by hardware, and as in another embodiment, any one or a combination of the following technologies known in the art can be used: discrete logic circuit with logic gate circuit for implementing logical functions on data signals, application specific integrated circuit with suitable combination of logic gate circuit, programmable gate array (PGA), field programmable gate array (FPGA) and the like.

[0087] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the description of the present application, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0088] In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like is based on the orientation or positional relationship shown in the drawings, and is only for the purpose of facilitating the description of the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation of the present application.

[0089] In addition, the terms "first", "second" are only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise specifically limited.

[0090] In the present application, unless otherwise specifically defined and limited, the terms "mounting", "connection", "connection", "fixing" and the like should be understood in a broad sense, for example, it can be fixed connection, or detachable connection, or integral; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, it can be the internal communication of two elements or the interaction relationship between two elements, unless otherwise specifically limited. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0091] In the present application, unless otherwise explicitly specified and limited, the first feature is "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature is "above", "over" and "on" the second feature can be that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is horizontally higher than the second feature. The first feature is "below", "under" and "under" the second feature can be that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is horizontally lower than the second feature. Although the embodiments of the present application have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-mentioned embodiments within the scope of the present application.

Claims

1. A HIT functional testing method, characterized in that, The method includes: Standard platelet-rich plasma and platelet-poor plasma to be tested were prepared, and working solutions of different concentrations of heparin were prepared. A reaction system was constructed based on the standard platelet-rich plasma, the platelet-poor plasma to be tested, and the working solutions of different concentrations of heparin, wherein the reaction system included a heparin-free group, a low-heparin concentration group, and a high-heparin concentration group. Fluorescently labeled CD41 / CD61 antibody, CD62P antibody, and Annexin V were added to the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group, respectively. The procoagulant platelet ratios of the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group were obtained by flow cytometry. HIT test results are generated based on the procoagulant platelet ratios and preset ratio thresholds corresponding to the heparin-free group, the low heparin concentration group, and the high heparin concentration group.

2. The HIT functional testing method according to claim 1, characterized in that, The heparin-free group includes: 90 Standard platelet-rich plasma, 40 Platelet-poor plasma and 70 Calcium-containing buffer; the low heparin concentration group includes: 90 Standard platelet-rich plasma, 40 Platelet-poor plasma to be tested, 30 First concentration heparin working solution and 40 Calcium-containing buffer; the high heparin concentration group includes: 90 Standard platelet-rich plasma, 40 Platelet-poor plasma to be tested, 30 Second concentration heparin working solution and 40 Calcium-containing buffer solution.

3. The HIT functional testing method according to claim 1, characterized in that, The heparin-free group corresponds to a heparin concentration of 0 U / ml, the low heparin concentration group corresponds to a heparin concentration of 0.5 U / ml, and the high heparin concentration group corresponds to a heparin concentration of 100 U / ml.

4. The HIT functional testing method according to claim 1, characterized in that, The step of obtaining the procoagulant platelet ratios corresponding to the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group by flow cytometry includes: The CD41 / CD61 positive platelet populations corresponding to the heparin-free group, the low heparin concentration group, and the high heparin concentration group were obtained by flow cytometry, respectively. The CD62P and PS double-positive platelet subsets were obtained from the CD41 / CD61 positive platelet populations corresponding to the heparin-free group, the low heparin concentration group, and the high heparin concentration group by flow cytometry. The proportion of procoagulant platelets in the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group is obtained based on the proportion of CD62P and PS double-positive platelet subsets in the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group relative to the CD41 / CD61 positive platelet population in the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group.

5. The HIT functional testing method according to claim 1, characterized in that, The process of generating HIT test results based on the procoagulant platelet ratios and preset ratio thresholds corresponding to the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group includes: If the proportion of procoagulant platelets in the heparin-free group is less than the preset proportion threshold, the proportion of procoagulant platelets in the low heparin concentration group is greater than the preset proportion threshold, and the inhibition degree in the high heparin concentration group exceeds the preset percentage threshold, then the HIT test result is determined to be positive.

6. The HIT functional testing method according to claim 5, characterized in that, The preset ratio threshold is the mean plus twice the standard deviation of the procoagulant platelet ratio detected under low heparin conditions from at least a plurality of healthy donors' standard platelet-rich plasma.

7. The HIT functional testing method according to claim 1, characterized in that, The method further includes: After the reaction system is set up, the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group are incubated at room temperature with shaking for a preset first time. After fluorescently labeled CD41 / CD61 antibody, CD62P antibody, and Annexin V are added to the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group, the heparin-free group, the low-heparin concentration group, and the high-heparin concentration group are incubated in the dark for two preset times.

8. A computer-readable storage medium storing a HIT functional testing program thereon, wherein the HIT functional testing program, when executed by a processor, implements the HIT functional testing method as described in any one of claims 1-7.

9. A HIT functional testing device, characterized in that, The device includes: The test material preparation module is used to prepare standard platelet-rich plasma and platelet-poor plasma to be tested, and to prepare working solutions of different concentrations of heparin. The reaction system construction module is used to construct a reaction system based on the standard platelet-rich plasma, the platelet-poor plasma to be tested, and the working solutions of different concentrations of heparin, wherein the reaction system includes a heparin-free group, a low heparin concentration group, and a high heparin concentration group. The flow cytometry detection module is used to add fluorescently labeled CD41 / CD61 antibody, CD62P antibody and Annexin V to the heparin-free group, the low heparin concentration group and the high heparin concentration group respectively, and obtain the procoagulant platelet ratio corresponding to the heparin-free group, the low heparin concentration group and the high heparin concentration group by flow cytometry. The HIT detection module is used to generate HIT detection results based on the procoagulant platelet ratio and preset ratio threshold corresponding to the heparin-free group, the low heparin concentration group and the high heparin concentration group.

10. A medical laboratory diagnostic platform, characterized in that, The medical experimental diagnostic platform includes the HIT functional testing device as described in claim 9.

Images