A multi-parameter flow cytometry-based platelet detection kit and methods of use thereof

Abstract

The application discloses a platelet detection kit based on multi-parameter flow cytometry and a use method thereof, relates to the technical field of in-vitro diagnosis, and covers the core pathological links of thrombosis and downstream key events of thrombosis by simultaneously detecting the proportion of PNH clone cells, the platelet activation state and the proportion of platelet-leukocyte aggregates (PLAs), thereby efficiently and accurately evaluating the thrombosis risk of PNH patients. The application directly detects cell surface markers and cell-cell interaction by using multi-color flow cytometry, can early and sensitively identify abnormal activation signals, and avoids the limitations of traditional coagulation indexes (such as D-dimer) in PNH thrombosis early warning.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically a platelet detection kit based on multiparameter flow cytometry and its usage method. Background Technology

[0002] Phlegmonous nephropathy (PNH) is a clonal blood disorder caused by a mutation in the PIG-A gene, resulting in a deficiency in the synthesis of the glycosylphosphatidylinositol (GPI) anchor. The absence of the GPI anchor leads to a lack of complement regulatory proteins such as CD55 and CD59 on the surface of cells like erythrocytes and platelets, causing hemolysis and thrombosis. Thrombosis is the leading cause of death in PNH patients, and its pathogenesis is complex, involving complement activation, abnormal platelet activation, and leukocyte interactions. Currently, there is a lack of sensitive and specific tools for assessing the risk of thrombosis in PNH. Traditional coagulation markers (such as D-dimer) have limited value in predicting PNH-specific thrombosis. The mechanism linking CD59 deficiency, CD62p expression, and PLA (platelet-leukocyte aggregate) formation: Platelets of the PNH clone (CD59) are more easily activated by complement (such as C5b) due to the lack of CD59 protection, resulting in high expression of CD62p on the platelet surface. The highly expressed CD62p binds to the PSGL-1 receptor on the surface of leukocytes to form stable PLAs. These PLAs not only promote leukocyte activation and release of procoagulant substances, but also directly participate in microvascular thrombosis.

[0003] Therefore, simultaneously detecting PNH clone size (degree of CD59 deletion), platelet activation status (CD62p expression), and PLA levels is essential to comprehensively reflect the core pathophysiological process of thrombosis in PNH patients. Furthermore, in current flow cytometry platforms, PNH and PLA detection are performed independently, which is not convenient for integrated research. Summary of the Invention

[0004] The purpose of this invention is to provide a platelet detection kit based on multi-parameter flow cytometry and its usage method, in order to solve the problem in the prior art that it is impossible to simultaneously detect PNH clone size (CD59 deletion degree), platelet activation status (CD62p expression) and PLA levels, and that PNH and PLA detection on the flow cytometry platform are independent.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A platelet detection kit based on multiparameter flow cytometry, the kit comprising a fluorescently labeled antibody combination, lysis buffer, buffer solution, fixative solution and analytical control; The fluorescently labeled antibody combination consists of anti-CD59 fluorescently labeled antibody, anti-CD62p fluorescently labeled antibody, anti-CD45 fluorescently labeled antibody, and anti-CD41 fluorescently labeled antibody or anti-CD61 fluorescently labeled antibody.

[0006] As a preferred technical solution, the fluorescent label is any one of FITC, PE, APC-Cy7, APC, PerCP, and PE-Cy7; As a preferred technical solution, the lysis buffer is hemolysin; the buffer solution is PBS buffer; and the fixative solution is platelet incubation solution.

[0007] As a preferred technical solution, the analytical control is an isotype control antibody.

[0008] A method for using a platelet detection kit based on multiparameter flow cytometry, characterized by comprising the following steps: Step S1: Collect peripheral venous blood from PNH patients using sodium citrate anticoagulant tubes to obtain anticoagulated blood; Step S2: Add the fluorescently labeled antibody to the flow cytometry tube and premix. Take an appropriate amount of the anticoagulated blood obtained in step S1 and add it to the premixed fluorescently labeled antibody mixture. Incubate at room temperature in the dark. Step S3: After incubation, add lysis buffer to the flow cytometry tube, mix well, and centrifuge to remove the supernatant; then add buffer solution to the flow cytometry tube, mix well, and centrifuge to remove the supernatant; finally, add fixative to the flow cytometry tube, vortex to mix, and obtain the test sample. Step S4: Add the sample to the flow cytometer for detection and obtain a flow cytogram. Based on the FSC / SSC characteristics, delineate the white blood cell population and platelet population in the flow cytogram for analysis. Platelet-leukocyte aggregates were screened within the leukocyte population based on the expression of CD45 and CD41 / CD61, and the percentage of platelet-leukocyte aggregates in the total leukocyte population or specific leukocyte subsets was calculated. Normal platelets and PNH clone platelets were initially distinguished within the platelet population based on CD59 expression, and the proportion of PNH clone platelets within the platelet population was calculated. Further analysis of the activation levels of normal platelets and PNH clone platelets within the platelet population was conducted based on CD62p expression and mean fluorescence intensity. Step S5: Based on three indicators—the percentage of platelet-leukocyte aggregates in the total white blood cell population or a specific white blood cell subset, the proportion of PNH clone platelets, and the degree of activation of normal platelets and PNH clone platelets within the platelet population—the patient's thrombosis status is determined.

[0009] As a preferred technical solution, in step S2, the volume ratio of anticoagulated blood to fluorescently labeled antibody is 1:5-10; and the incubation time at room temperature in the dark is 20-40 minutes.

[0010] As a preferred technical solution, in step S4, CD45 is positive for leukocytes, and CD41 or CD61 is positive for platelets. The cell population that simultaneously expresses CD45 positivity and CD41 / CD61 positivity is the platelet-leukocyte aggregate.

[0011] As a preferred technical solution, in step S4, CD59 is expressed positively for normal platelets and negatively for PNH clone platelets.

[0012] As a preferred technical solution, in step S4, CD62p is positive for both normal platelets and PNH clone platelets. The proportion of positive CD62p and the average fluorescence intensity reflect the degree of activation of PNH clone platelets and residual normal platelets.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for using a platelet detection kit based on multi-parameter flow cytometry. Through multi-parameter combined, mechanism-driven approaches, it integrates the traditionally separate detection of PNH clones and PLAs into a single kit and detection process. It simultaneously detects the proportion of PNH clones, platelet activation status, and the proportion of platelet-leukocyte aggregates (PLAs), covering the core pathological steps of thrombosis and key downstream thrombosis events, providing a more comprehensive assessment and clearer mechanism. This invention uses multicolor flow cytometry to directly detect cell surface markers and intercellular interactions, enabling early and sensitive identification of abnormal activation signals, avoiding the limitations of traditional coagulation indicators (such as D-dimer) in PNH thrombosis early warning. This invention is suitable for regular testing, dynamically tracking changes in patient thrombosis risk and evaluating treatment effectiveness, providing objective evidence for further treatment adjustments. Furthermore, through comprehensive multi-indicator assessment, it can accurately identify high-risk thrombosis patients, guiding prophylactic anticoagulation or anticomplement therapy; while avoiding overtreatment of low-risk patients, achieving personalized and optimized treatment strategies. Attached Figure Description

[0014] Fig. 1 This is a scatter plot of flow cytometry cells in this invention; Fig. 2 This is a diagram showing the proportion of PNH clone platelets in this invention. Fig. 3 This is an analysis diagram of platelet-leukocyte aggregates (PLAs) in this invention; Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] This invention provides a platelet detection kit based on multiparameter flow cytometry, comprising a fluorescently labeled antibody combination, lysis buffer, fixative, and isotype control antibody; the fluorescently labeled antibody combination consists of anti-CD59 fluorescently labeled antibody, anti-CD62p fluorescently labeled antibody, anti-CD45 fluorescently labeled antibody, and anti-CD41 fluorescently labeled antibody or anti-CD61 fluorescently labeled antibody. The fluorescent label includes, but is not limited to, any one of FITC, PE, APC-Cy7, APC, PerCP, and PE-Cy7.

[0017] Specifically, in this invention, CD59 is fluorescently labeled as FITC; CD62p is fluorescently labeled as PE; CD45 is fluorescently labeled as antibody APC-Cy7; and CD41 or CD61 is fluorescently labeled as APC.

[0018] Specifically, in this invention, the lysis buffer is hemolysin, the fixative is platelet incubation solution, and the analytical control is isotype control antibody; This invention also provides a method for using a platelet detection kit based on multiparameter flow cytometry, comprising the following steps: Step S1: Collect peripheral venous blood from PNH patients using sodium citrate anticoagulant tubes to obtain anticoagulated blood; Step S2: Add the fluorescently labeled antibody to the flow cytometry tube and premix. Take an appropriate amount of the anticoagulated blood obtained in step S1 and add it to the premixed fluorescently labeled antibody mixture. Incubate at room temperature in the dark. Specifically, in this step, 5 μL of anticoagulated blood is added, and the total volume of fluorescently labeled antibody added to the flow cytometry tube is 25–40 μL. The tube is then incubated at room temperature in the dark for 20–40 min. Step S3: After incubation, add lysis buffer to the flow cytometry tube, mix well, and centrifuge to remove the supernatant; then add dilution buffer to the flow cytometry tube, mix well, and centrifuge to remove the supernatant; finally, add fixative, vortex to mix, and obtain the test sample; Specifically, in this step, 1 ml of lysis buffer, 1 ml of buffer solution, and 1 ml of fixative solution are added. Step S4: Add the sample to the flow cytometer for detection and obtain a flow cytogram. Based on the FSC / SSC characteristics, delineate the white blood cell population and platelet population in the flow cytogram for analysis. Platelet-leukocyte aggregates were screened within the leukocyte population based on the expression of CD45 and CD41 / CD61, and the percentage of platelet-leukocyte aggregates in the total leukocyte population or specific leukocyte subsets was calculated. Normal platelets and PNH clone platelets were initially distinguished within the platelet population based on CD59 expression, and the proportion of PNH clone platelets within the platelet population was calculated. Further analysis of the activation levels of normal platelets and PNH clone platelets within the platelet population was conducted based on CD62p expression and mean fluorescence intensity. Specifically, in this step, CD45 is expressed positively for leukocytes, while CD41 or CD61 is expressed positively for platelets. Cell populations expressing both CD45 and CD41 / CD61 are platelet-leukocyte aggregates. CD59 is expressed positively for normal platelets but negatively for PNH clone platelets. CD62p is expressed positively for both normal and PNH clone platelets. The proportion of positive CD62p and the average fluorescence intensity reflect the activation level of PNH clone platelets and residual normal platelets. Step S5: Based on three indicators—the percentage of platelet-leukocyte aggregates in the total white blood cell population or a specific white blood cell subset, the proportion of PNH clone platelets, and the degree of activation of normal platelets and PNH clone platelets within the platelet population—the patient's thrombosis status is determined.

[0019] Example 1 This embodiment describes the detection of a specific sample using the aforementioned detection kit. The method of using the detection kit is the same as described above, and the detection steps S1-S5 are as described. The detection results are as follows. Figs. 1-3 As shown; The left panel of Figure 1 shows the platelet population delineated by SSC-A, and the percentage of CD61-positive platelets. This represents the proportion of CD61-labeled platelets in the sample, with 98.4% of the cells being CD61-positive platelets.

[0020] The left panel of Figure 1 shows the percentage of CD62p-positive activated platelets within the platelet population, representing the degree of platelet activation in the sample. The percentage of CD62p-positive activated platelets at 1.49% indicates a low level of platelet activation in this sample.

[0021] Figure 2 shows the proportion of PNH clone platelets in platelets (CD61+). The analysis of the proportion of CD59 positive normal platelets in the platelet population (shown as 98.3% in the figure) leads to the conclusion that the proportion of PNH clone platelets (CD59 negative) is only about 1.7%, indicating that the proportion of PNH clones in this sample is low.

[0022] Figure 3 represents the platelet-leukocyte aggregate (PLAs) analysis. The definition of platelet-leukocyte aggregate (PLAs) is a cell population that simultaneously expresses CD45 positive (leukocytes) and CD41 positive (platelets). The figure shows the percentage of PLAs in different leukocyte subsets (e.g., 7.48, 3.54, 2.45), which represents the level of PLAs in the sample. These values ​​show that the percentage of PLAs in this sample is relatively low.

[0023] Summary of thrombosis in this sample Based on three indicators: the proportion of PNH clonal platelets (approximately 1.7%) is extremely low; the degree of platelet activation (CD62p+ accounts for 1.49%) is low; and the proportion of platelet-leukocyte aggregates (PLAs) is low; therefore, the PNH patient corresponding to this test sample has a low risk of thrombosis.

[0024] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A platelet detection kit based on multi-parameter flow cytometry, characterized in that, The detection kit includes a fluorescently labeled antibody combination, lysis buffer, buffer solution, fixative solution, and analytical control. The fluorescently labeled antibody combination consists of anti-CD59 fluorescently labeled antibody, anti-CD62p fluorescently labeled antibody, anti-CD45 fluorescently labeled antibody, and anti-CD41 fluorescently labeled antibody or anti-CD61 fluorescently labeled antibody.

2. The platelet detection kit based on multi-parameter flow cytometry according to claim 1, characterized in that, The fluorescent label is any one of FITC, PE, APC-Cy7, APC, PerCP, and PE-Cy7.

3. The platelet detection kit based on multi-parameter flow cytometry according to claim 1, characterized in that, The lysis buffer is hemolysin; the buffer solution is PBS buffer; and the fixative solution is platelet incubation solution.

4. The platelet detection kit based on multi-parameter flow cytometry according to claim 1, characterized in that, The analytical control standard is an isotype control antibody.

5. A method of using a platelet detection kit based on multiparameter flow cytometry according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step S1: Collect peripheral venous blood from PNH patients using sodium citrate anticoagulant tubes to obtain anticoagulated blood; Step S2: Add the fluorescently labeled antibody to the flow cytometry tube and premix. Take an appropriate amount of the anticoagulated blood obtained in step S1 and add it to the premixed fluorescently labeled antibody mixture. Incubate at room temperature in the dark. Step S3: After incubation, add lysis buffer to the flow cytometry tube, mix well, and centrifuge to remove the supernatant; then add buffer solution to the flow cytometry tube, mix well, and centrifuge to remove the supernatant; finally, add fixative to the flow cytometry tube, vortex to mix, and obtain the test sample. Step S4: Add the sample to the flow cytometer for detection and obtain a flow cytogram. Based on the FSC / SSC characteristics, delineate the white blood cell population and platelet population in the flow cytogram for analysis. Platelet-leukocyte aggregates were screened within the leukocyte population based on the expression of CD45 and CD41 / CD61, and the percentage of platelet-leukocyte aggregates in the total leukocyte population or specific leukocyte subsets was calculated. Normal platelets and PNH clone platelets were initially distinguished within the platelet population based on CD59 expression, and the proportion of PNH clone platelets within the platelet population was calculated. Further analysis of the activation levels of normal platelets and PNH clone platelets within the platelet population was conducted based on CD62p expression and mean fluorescence intensity. Step S5: Based on three indicators—the percentage of platelet-leukocyte aggregates in the total white blood cell population or a specific white blood cell subset, the proportion of PNH clone platelets, and the degree of activation of normal platelets and PNH clone platelets within the platelet population—the patient's thrombosis status is determined.

6. The method of using the platelet detection kit based on multi-parameter flow cytometry according to claim 5, characterized in that, In step S2, the volume ratio of anticoagulated blood to fluorescently labeled antibody is 1:5-10; the incubation time at room temperature in the dark is 20-40 min.

7. The method of using the platelet detection kit based on multi-parameter flow cytometry according to claim 5, characterized in that, In step S4, CD45 is positive for leukocytes, and CD41 or CD61 is positive for platelets. The cell population that simultaneously expresses CD45 and CD41 / CD61 is the platelet-leukocyte aggregate.

8. The method of using the platelet detection kit based on multi-parameter flow cytometry according to claim 5, characterized in that, In step S4, CD59 is expressed positively in normal platelets and negatively in PNH clone platelets.

9. The method of using the platelet detection kit based on multi-parameter flow cytometry according to claim 5, characterized in that, In step S4, CD62p is positive for both normal platelets and PNH clone platelets. The proportion of positive CD62p and the average fluorescence intensity reflect the degree of activation of PNH clone platelets and residual normal platelets.

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