Kit for heparin-induced platelet flow cytometry

Abstract

The invention discloses a heparin-induced platelet flow cytometry kit, and relates to the technical field of in vitro diagnostic reagents, the kit comprises a donor platelet preparation module, a heparin gradient excitation module, a bifluorescent antibody labeling module and a quality control reference module, the heparin gradient excitation module comprises ungraded heparin solutions with three concentration gradients of 0 IU / mL, 0.3 IU / mL and 100 IU / mL, the bifluorescent antibody labeling module comprises an anti-CD41 antibody labeled by PE and an anti-CD62P antibody labeled by FITC, the HEPLA index is calculated by detecting the expression change of CD62P after donor platelets are activated by patient serum under different heparin concentration conditions, and the heparin concentration of the patient is detected to be detected. The heparin-induced thrombocytopenia functional confirmation and diagnosis method does not need to use radioactive isotopes, is simple, convenient and rapid to operate and accurate and reliable in detection result, and has good clinical application value.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, specifically to a reagent kit for heparin-induced platelet flow cytometry. Background Technology

[0002] Heparin-induced thrombocytopenia (HIT) is a serious, immune-mediated adverse drug reaction that occurs in patients receiving heparin-based anticoagulant therapy. The pathogenesis involves the production of IgG antibodies against platelet factor 4 (PF4) and the heparin complex. These pathological antibodies bind to the FcγRIIa receptor on the platelet surface, leading to platelet activation, aggregation, and consumption, clinically manifested as a decreased platelet count and a high risk of thrombosis.

[0003] Laboratory diagnostic strategies for heparin-dependent platelet-activating immunoassay (HIT) fall into two main categories: immunological and functional testing. Immunological testing methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, latex immunoturbidimetry, and particle immunofiltration. These methods diagnose HIT by detecting anti-PF4 / heparin complex antibodies in serum. Immunological tests have high sensitivity but relatively low specificity because not all patients producing anti-PF4 / heparin antibodies will develop clinical HIT; a significant proportion of antibodies are non-pathological. Functional testing methods, on the other hand, confirm the diagnosis by assessing whether the antibodies in the patient's serum have the ability to heparin-dependently activate platelets, and have higher clinical specificity.

[0004] The traditional gold standard for functional testing is C-serotonin release assay The C-serotonin release assay (SRA) quantitatively assesses platelet activation by detecting the release of radiolabeled serotonin from activated platelets. While SRA boasts high sensitivity and specificity, its application is limited by several factors: First, it requires the use of radioactive isotopes, placing stringent demands on laboratory facilities and personnel qualifications; second, the experimental procedure is complex, requiring pre-labeling of donor platelets with radioactive isotopes, resulting in a high technical barrier; third, the turnaround time is long; and finally, only a few reference laboratories are capable of performing this test, leading to poor clinical accessibility. Heparin-induced platelet activation assay (HIPA) and light transmission aggregation assay (LTA) are other alternative functional assays, but they also suffer from low standardization, cumbersome procedures, and the need for specialized equipment.

[0005] Flow cytometry assesses platelet activation status by detecting the expression of P-selectin (CD62P) on the surface of activated platelets, offering advantages such as speed, simplicity, and the absence of radioactive isotopes. However, existing flow cytometry HIT detection methods lack standardized reagent preparation, operating procedures, and interpretation criteria, resulting in poor comparability of results between different laboratories and limiting their clinical application and promotion.

[0006] Chinese patent CN109709292A discloses a heparin detection kit. This kit utilizes the property of heparinase I to degrade heparin and assesses the residual heparin in the blood by detecting the R value using a thromboelastography instrument. This technology is mainly used to assess the residual level of heparin and the anticoagulant effect during anticoagulation therapy. Its technical principle is to neutralize the anticoagulant activity of heparin through an enzymatic reaction, which is completely different from the immunological pathogenesis and functional detection principle of HIT.

[0007] Given the shortcomings of the existing technologies, there is an urgent need in this field to develop a standardized, rapid, and accurate functional confirmatory test kit and detection method for HIT to meet the clinical needs for early diagnosis and confirmatory diagnosis of HIT. Summary of the Invention

[0008] The primary objective of this invention is to provide a heparin-induced platelet flow cytometry kit that integrates a donor platelet preparation module, a heparin gradient excitation module, a dual-fluorescent antibody labeling module, and a quality control reference module to standardize and regulate HIT functional confirmatory testing, thereby improving the accuracy and clinical accessibility of the test.

[0009] This invention is implemented as follows: This invention provides a heparin-induced platelet flow cytometry kit, comprising four functional units: a donor platelet preparation module, a heparin gradient excitation module, a dual-fluorescent antibody labeling module, and a quality control reference module.

[0010] The donor platelet preparation module is designed based on the physiological characteristics of platelets. Platelets are enucleated disc-shaped cells, 2-4 μm in diameter, with a density of approximately 1.04-1.07 g / mL. They can be separated from red blood cells and white blood cells in whole blood by differential centrifugation. The donor platelet preparation module of this invention employs a low-speed centrifugation parameter setting component. The centrifugal force is set to 80-120 g, preferably 100 g, which is sufficient to cause red blood cells to settle while retaining suspended platelets. The centrifugation time is set to 4-6 minutes, preferably 5 minutes, within which a platelet-rich supernatant can be obtained. The braking force is set to zero, meaning the rotor is allowed to decelerate naturally after centrifugation. This setting avoids surface disturbance and resuspension of settled cells caused by sudden braking, ensuring the purity of platelet-rich plasma (PRP). The yellow liquid obtained after centrifugation is PRP, whose platelet concentration is typically 2-5 times that of whole blood, providing sufficient target cells for subsequent platelet activation detection.

[0011] The design of the heparin gradient activation module is based on the unique heparin dependence of pathological HIT antibodies. Studies have shown that the epitope recognized by HIT antibodies is a novel conformational epitope exposed after PF4 binds to heparin. In the presence of low concentrations of heparin (typically 0.1-1 IU / mL), heparin and PF4 form an optimal complex ratio, exposing the most antibody recognition epitopes. At this point, the binding of HIT antibodies to the PF4 / heparin complex is most complete, thereby activating platelets through the FcγRIIa receptor-mediated signaling pathway. However, in the presence of high concentrations of heparin (typically greater than 50 IU / mL), excess heparin dissociates the PF4 / heparin complex, disrupting the conformation of the antibody recognition epitopes and inhibiting HIT antibody-mediated platelet activation. This unique dose-response relationship is the core principle of HIT functional assays and is key to distinguishing pathological HIT antibodies from non-pathological antibodies.

[0012] The heparin gradient activation module of this invention comprises three concentration gradients of unfractionated heparin (UFH) solution components. The basal concentration component has a heparin concentration of 0 IU / mL, i.e., no exogenous heparin is added, serving as a reference for the basal level of heparin that may remain in the patient's serum. The low-concentration activation component has a heparin concentration of 0.2-0.5 IU / mL, preferably 0.3 IU / mL. This concentration maximizes the formation of the PF4 / heparin complex and the binding of HIT antibodies, achieving optimal activation of HIT pathological antibody-mediated platelet activation. The high-concentration inhibitory component has a heparin concentration of 80-150 IU / mL, preferably 100 IU / mL. This concentration of excess heparin competitively inhibits the formation of the PF4 / heparin complex, thereby inhibiting HIT antibody-mediated platelet activation. By comparing the differences in platelet activation levels under the three concentration conditions, specific recognition of HIT pathological antibodies can be achieved.

[0013] The dual-fluorescent antibody labeling module uses two fluorescein-labeled monoclonal antibodies to achieve specific platelet recognition and assessment of activation status. The platelet-specific labeling antibody is a phycoerythrin (PE)-labeled anti-CD41 monoclonal antibody. CD41 is platelet membrane glycoprotein II. b (GPII) b ), and GPIII a Together they form the fibrinogen receptor complex GPII b / GPIII a Polyoxin is a unique surface marker of platelets, expressed in both resting and activated platelets, making it suitable for platelet population delineation. Polyoxin exhibits high quantum yield and bright orange-red fluorescence (emission peak at 578 nm), which is well-matched to the FL2 channel (565-605 nm) of flow cytometers.

[0014] Platelet activation marker antibodies were selected from anti-CD62P monoclonal antibodies labeled with fluorescein isothiocyanate (FITC) or allophycocyanin (APC). CD62P, or P-selectin, is a platelet α-granule membrane protein located on the inner membrane of the α-granule in resting platelets. When platelets are activated, the α-granule fuses with the platelet plasma membrane and releases its contents; P-selectin then translocates to the platelet surface, becoming a specific marker of platelet activation. FITC fluorescein emits green fluorescence (emission peak at 519 nm), matching the FL1 channel (515-545 nm); APC fluorescein emits red fluorescence (emission peak at 660 nm), matching the FL4 channel (645-680 nm). Both fluoresceins achieve good spectral separation from PE, avoiding fluorescence overflow interference.

[0015] The quality control reference module includes negative and positive control reagents to monitor the system performance and result reliability of each batch of tests. The negative control uses a mixture of donor PRP and phosphate-buffered saline (PBFS) to represent the resting platelet state without any activation stimulation, with a CD62P positivity rate of less than 2%. The positive control contains thrombin receptor agonist peptide (TRAP), preferably TRAP-6 (amino acid sequence SFLLRN), which can induce strong platelet activation by activating protease-activated receptor-1 (PAR-1) on the platelet surface, with a CD62P positivity rate of greater than 80%. The positive control is provided in lyophilized powder form and needs to be dissolved in 150-250 μL of 65%-75% ethanol for 15-30 minutes before use. After dissolution, it can be used directly or stored at -20°C for later use.

[0016] The kit of this invention also includes a buffer module providing 10-fold concentrated phosphate-buffered saline (PBS) with a pH of 7.2-7.6, which is diluted to a working concentration before use. PBS is used to dilute serum samples, prepare heparin working solutions, and dilute samples before flow cytometry.

[0017] The present invention has the following beneficial effects: First, the kit provided by this invention achieves standardized and modular design for HIT functional confirmatory assays. By integrating four key steps—donor platelet preparation, heparin gradient activation, dual-fluorescent antibody labeling, and quality control—into independent functional modules, each with clear parameter specifications and operating guidelines, the variability of experimental procedures is effectively reduced, and the repeatability and comparability of test results are improved.

[0018] Secondly, the three-concentration gradient heparin stimulation system used in this invention fully utilizes the unique heparin-dependent characteristics of pathological HIT antibodies. By setting three test conditions—0 IU / mL basal concentration, 0.3 IU / mL low-concentration stimulation, and 100 IU / mL high-concentration inhibition—it can effectively distinguish between pathological HIT antibodies with platelet activation ability and non-pathological antibodies without activation ability, significantly improving the clinical specificity of the detection.

[0019] Third, this invention employs a dual-fluorescent antibody labeling strategy using CD41 and CD62P. CD41 is used to specifically delineate platelet populations, while CD62P is used to assess platelet activation status; the two are complementary in function. This dual-labeling strategy avoids false positive or false negative results that may result from single labeling, thus improving the accuracy and reliability of the detection.

[0020] Fourth, this invention uses a flow cytometry platform for detection, compared to traditional methods. The C-SRA method has significant advantages: it does not require the use of radioactive isotopes, avoiding radiation hazards and complex waste disposal procedures; it has high throughput, with a single test capable of analyzing thousands to tens of thousands of platelet events, resulting in stronger statistical power; it has short turnaround time, with reports issued within 2-3 hours from sample receipt; and it has high equipment availability, with most hospital laboratories equipped with flow cytometers, facilitating its clinical application.

[0021] Fifth, the HEPLA index calculation method established in this invention provides an objective and quantitative standard for result interpretation. By calculating the ratio of the difference in platelet activation under low-concentration activation conditions to the sum under high-concentration inhibition conditions, the influence of differences in platelet reactivity among different donors is eliminated, making the test results more stable and reliable, and facilitating result comparison between different laboratories. Attached Figure Description

[0022] Figure 1Scatter plot of flow cytometry results for HIT-negative patient 1.

[0023] Figure 2 Scatter plot of flow cytometry results for HIT-negative patient 2. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0026] Example 1: Composition of the Heparin-Induced Platelet Flow Cytometry Kit This embodiment provides a heparin-induced platelet flow cytometry kit, which includes the following four functional modules: Donor Platelet Preparation Module: This module provides a standardized parameter guide card for preparing platelet-rich plasma, including operating procedures for setting the centrifugal force to 100 g, the centrifugation time to 5 minutes, and the braking force to zero (braking level 0). The module also includes two EDTA anticoagulant vacuum blood collection tubes (purple-tipped tubes) for collecting venous blood from healthy donors.

[0027] Heparin gradient activation module: This module contains three concentrations of unfractionated heparin (UFH) solution: basal concentration component (0 IU / mL, i.e., heparin-free control), 1 tube × 1 mL; low-concentration activation component (0.3 IU / mL), 1 tube × 1 mL; and high-concentration inhibition component (100 IU / mL), 1 tube × 1 mL. The heparin solution is prepared with phosphate buffer and stored at 2–8°C. Shelf life is 12 months.

[0028] Dual-fluorescent antibody labeling module: This module contains two fluorescently labeled monoclonal antibodies: PE-labeled anti-human CD41 monoclonal antibody (1 tube × 100 tests, 10 μL per test); and FITC-labeled anti-human CD62P monoclonal antibody (1 tube × 100 tests, 5 μL per test). Antibodies should be stored at 2–8°C protected from light and have a shelf life of 18 months.

[0029] Quality Control Reference Module: This module contains the positive control reagent TRAP-6 lyophilized powder, 1 tube × 100 μg. The amino acid sequence of TRAP-6 is SFLLRN, and its molecular weight is approximately 747 Da. Before use, dissolve in 200 μL of 70% ethanol and allow to stand at room temperature for 15-30 minutes until completely dissolved. The concentration of the dissolved TRAP-6 working solution is approximately 670 μM. Dilute to a final concentration of approximately 33 μM at a ratio of 1:20 before use. The lyophilized powder should be stored at -20℃ for 24 months; the dissolved working solution should be stored at -20℃ for 3 months.

[0030] Buffer Module: This module contains one 10x concentrated PBS buffer vial (100 mL), pH 7.4. Dilute with deionized water at a ratio of 1:9 to the working concentration before use. Store at room temperature. Shelf life is 24 months.

[0031] Example 2: Preparation method of donor platelet-rich plasma This embodiment provides a standardized method for preparing donor platelet-rich plasma (PRP).

[0032] Donor screening: Healthy volunteers were selected as platelet donors. They were required to have not taken antiplatelet drugs such as aspirin or clopidogrel within the past two weeks, not taken nonsteroidal anti-inflammatory drugs within the past week, have no history of platelet dysfunction, and have a platelet count between 150-400 × 10⁻⁶. 9 / L is within the normal range. Donors with blood type O are preferred to reduce the potential impact of ABO blood group antigens on platelet activation.

[0033] Sample Collection: Collect 4-5 mL of donor venous blood using an EDTA anticoagulant vacuum blood collection tube. Use a 21G or larger inner diameter needle during blood collection to avoid platelet activation due to shear force. After blood collection, gently invert the tube 3-5 times to ensure thorough mixing of the anticoagulant and blood.

[0034] Centrifugation: Place the anticoagulated whole blood in a horizontal rotor centrifuge and set the centrifugation parameters to 100 g, 5 minutes, and zero braking (braking level 0). After centrifugation, let it stand for 1 minute to allow the liquid level to stabilize.

[0035] PRP Acquisition: Carefully aspirate the upper pale yellow liquid (approximately 40%-50% of the whole blood volume) using a plastic transfer pipette or micropipette, avoiding contact with the middle white membrane layer. Transfer the acquired PRP to a new plastic centrifuge tube and store at room temperature (20-25℃) until use. PRP should be used within 4 hours of collection.

[0036] Quality control: Platelet count in PRP is measured using a fully automated hematology analyzer; the normal range should be 200-600 × 10⁻⁶. 9 / L. Simultaneously detect white blood cell and red blood cell contamination in PRP; white blood cell count should be <0.5×10⁻⁶. 9 / L, red blood cell count should be <0.1×10 12 / L.

[0037] The PRP platelet concentration prepared in this embodiment was 450 × 10⁻⁶. 9 / L, white blood cell count was 0.2×10 9 / L, red blood cell count was 0.05×10 12 / L, meets quality requirements.

[0038] Example 3: Method for preparing patient serum samples This embodiment provides a standardized method for preparing patient serum samples.

[0039] Sample Collection: Collect 3-5 mL of venous blood from suspected HIT patients into additive-free vacuum blood collection tubes (red-head tubes). For patients who have discontinued heparin, it is recommended to collect samples within 24-48 hours after discontinuation, at which time HIT antibody titers are usually at a high level. If additive-free tubes cannot be used, citrate anticoagulant tubes can be used, but subsequent processing needs to be adjusted accordingly.

[0040] Blood clot formation: Place the blood collection tube vertically in a room temperature (20-25℃) environment and let it stand for 30 minutes to allow the blood to coagulate naturally and form a blood clot. Centrifuging too early may result in fibrin residues remaining in the serum, affecting subsequent tests, while centrifuging too late may lead to blood cell lysis.

[0041] Centrifugation: Centrifuge the coagulated blood sample at 1000 g for 20 minutes. After centrifugation, the serum will be on the top layer as a pale yellow, transparent liquid, while the blood clot will be at the bottom.

[0042] Serum Acquisition: Carefully aspirate the supernatant serum using a plastic transfer pipette or micropipette and transfer it to a new microcentrifuge tube. Avoid aspirating the red blood cell layer on the surface of the blood clot. For mildly hemolyzed samples, centrifuge at 8000g for 8 minutes before use to remove cell debris.

[0043] Sample Preservation: Fresh serum should be tested within 12 hours of collection. If immediate testing is not possible, the serum should be aliquoted and stored at -80°C. Frozen samples can be stored for more than 6 months. Thaw at room temperature before use. Thawed samples should be tested within 4 hours to avoid repeated freeze-thaw cycles.

[0044] The patient serum prepared in this embodiment is a pale yellow transparent liquid with no hemolysis, no chyle, and no obvious turbidity, which meets the testing requirements.

[0045] Example 4: Method for constructing the reaction system This embodiment provides a standardized method for constructing a HIT confirmatory test reaction system.

[0046] Reagent preparation: Equilibrate all reagents to room temperature (20-25℃) before use. Dilute 10x concentrated PBS with deionized water at a ratio of 1:9 to prepare PBS at the working concentration. Dissolve TRAP-6 lyophilized powder in 200 μL of 70% ethanol and let stand at room temperature for 20 minutes.

[0047] EP tube numbering: Prepare 5 1.5 mL microcentrifuge tubes and label them as I, II, III, IV, and V, corresponding to negative control, positive control, 0 IU / mL heparin group, 0.3 IU / mL heparin group, and 100 IU / mL heparin group, respectively.

[0048] Reaction system preparation: Add reagents to each EP tube sequentially according to the components and volumes shown in Table 1. Use a calibrated micropipette for sample addition to ensure accuracy. The order of addition is PBS buffer, PRP, patient serum / quality control reagent, and heparin solution.

[0049] Table 1. Reaction System Preparation Table Mixing and Incubation: After adding samples to each tube, gently mix by pipetting up and down 5-8 times with a micropipette, taking care to avoid generating air bubbles. Incubate the mixed reaction system at room temperature (20-25℃) in the dark for 1 hour (60 minutes). During incubation, the EP tubes can be placed in a dark box or drawer to avoid non-specific platelet activation caused by light exposure.

[0050] The total volume of the reaction system prepared in this embodiment is 50 μL, of which PRP accounts for 20% (10 μL), patient serum accounts for 20% (10 μL), heparin / quality control reagent accounts for 10% (5 μL), and PBS buffer accounts for 50%-70% (25-35 μL). This ratio has been optimized to obtain the best signal-to-noise ratio.

[0051] Example 5 Antibody staining and flow cytometry detection method This embodiment provides a standardized method for antibody staining and flow cytometry detection.

[0052] Flow cytometry tube preparation: Prepare 5 flow cytometry tubes (5 mL polystyrene round-bottom tubes), labeled I, II, III, IV, and V respectively, corresponding one-to-one with the EP tube numbers in Example 4.

[0053] Antibody staining solution preparation: Add 10 μL of PE-antiCD41 antibody, 5 μL of FITC-antiCD62P antibody, and 35 μL of 1×PBS buffer to each flow cytometry tube, mix well and set aside.

[0054] Sample transfer and staining: After incubation, aspirate 5 μL of the reaction mixture from each EP tube and add it to the corresponding numbered flow cytometry tube. Gently mix the sample and antibody staining solution using a micropipette. After mixing, incubate at room temperature (20-25℃) in the dark for 15 minutes.

[0055] Sample dilution: After staining, add 450 μL of 1×PBS buffer to each flow cytometry tube for dilution, mix gently, and then proceed with the analysis. Diluted samples should be analyzed within 30 minutes to avoid fluorescence quenching or platelet aggregation caused by prolonged storage.

[0056] Flow cytometry setup: This example uses a BD FACSCanto II flow cytometer for detection. Prior to detection, Spherotech 8-peak calibration microspheres were used for routine instrument quality control according to the manufacturer's instructions. Forward scattered light (FSC) and side scattered light (SSC) were set to logarithmic mode, and the photomultiplier tube (PMT) voltage was adjusted so that platelet colonies were located in the central region of the scatter plot. The FL1 channel (FITC) and FL2 channel (PE) were set to logarithmic mode. Fluorescence compensation settings were performed to correct for FITC overflow into the PE channel (approximately 17%) and PE overflow into the FITC channel (approximately 2%).

[0057] Gating strategy: First, a scatter plot of SSC-Log versus FL2-Log (PE-CD41) is constructed to delineate platelet populations in the CD41-positive region (Gate 1), excluding cell debris (CD41-negative) and potentially contaminating leukocytes (larger cells). Then, based on Gate 1, a histogram is constructed using FL1-Log (FITC-CD62P), and an activation threshold cursor is set. The threshold is determined by overlaying the FL1 histograms of the negative control (tube I) and the positive control (tube II), with the cursor positioned at the intersection of the two curves.

[0058] Data acquisition: 10,000 CD41 positive events were collected for each sample. The percentage of events in each sample that are located to the right of the activation threshold (i.e., CD62P positive) was recorded and expressed as %R or %CD62P+.

[0059] The test results of this embodiment are shown in Table 2.

[0060] Table 2 Flow cytometry results This embodiment provides a standardized method for interpreting HIT confirmatory test results.

[0061] Quality control verification: Before interpreting patient results, the quality control of this batch of tests must be verified to ensure it is up to standard. The passing criteria are: negative control (tube I) CD62P positivity rate <5%; positive control (tube II) CD62P positivity rate >70%. If the quality control fails, the results of this batch of tests are invalid, and the cause must be investigated and the tests repeated.

[0062] In this embodiment, the positive rate of the negative control CD62P was 0.92%, and the positive rate of the positive control CD62P was 88.5%, both of which met the qualification standards.

[0063] Qualitative Interpretation Method 1 (Three-Criterion Method): A positive HIT confirmatory test requires the simultaneous fulfillment of the following three conditions: Condition A, CD62P positivity rate >5% in the 0 IU / mL heparin group (tube III); Condition B, CD62P positivity rate increases in the 0.3 IU / mL heparin group (tube IV) compared to the 0 IU / mL group; Condition C, CD62P positivity rate decreases in the 100 IU / mL heparin group (tube V) compared to the 0.3 IU / mL group. When all three conditions are met, the HIT confirmatory test is considered positive; if any condition is not met, the HIT confirmatory test is considered negative.

[0064] In this embodiment: Condition A, the CD62P positivity rate in tube III is 0.81%, <5%, therefore not met; Condition B, the CD62P positivity rate in tube IV (1.01%) is higher than that in tube III (0.81%), therefore met; Condition C, the CD62P positivity rate in tube V (1.43%) is higher than that in tube IV (1.01%), therefore not met. Overall judgment: HIT confirmatory test negative.

[0065] Quantitative interpretation method two (HEPLA index method): Calculate the heparin platelet activation (HEPLA) index, the formula is: HEPLA index = [(A 0.3 -A 100 ) / ((A 0.3 +A 100 )]×100% Among them, A 0.3 The positive rate of CD62P in the 0.3 IU / mL heparin group, A 100 The positive rate of CD62P in the 100 IU / mL heparin group.

[0066] A HEPLA index ≥ 14% is considered a positive HIT confirmatory test; a HEPLA index < 14% is considered a negative HIT confirmatory test.

[0067] In this embodiment: HEPLA index = [(1.01-1.43) / (1.01+1.43)]×100% = -17.2%. The HEPLA index is negative (<14%), which indicates a negative HIT confirmatory test.

[0068] Results report: Combining the two interpretation methods, the HIT confirmatory test result of the patient sample in this embodiment is negative (-).

[0069] Example 7: Detection of HIT-positive patient samples This example provides the test results of a HIT-positive patient sample.

[0070] Patient Information: The patient is a 61-year-old male who was admitted to the ICU due to acute myocardial infarction. After receiving low molecular weight heparin anticoagulation therapy for 5 days, his platelet count progressively decreased from 185×10⁻⁶ on admission. 9 / L decreased to 72×10 9 / L, 4Ts score of 6 (medium to high probability), submitted for HIT confirmatory test.

[0071] Sample processing and testing: Donor PRP preparation, patient serum preparation, reaction system construction, antibody staining, and flow cytometry were performed according to the methods described in Examples 2-5. Parallel testing was conducted using PRP from four healthy O-type donors.

[0072] The test results are shown in Table 3.

[0073] Table 3. Flow cytometry results of HIT-positive patients Result interpretation: Three-condition method: Condition A, the CD62P positivity rate of all four donors in the 0 IU / mL group is >5%, which is met; Condition B, the CD62P positivity rate of all four donors in the 0.3 IU / mL group is significantly higher than that in the 0 IU / mL group, which is met; Condition C, the CD62P positivity rate of all four donors in the 100 IU / mL group is significantly lower than that in the 0.3 IU / mL group, which is met. Overall judgment: HIT confirmatory test positive.

[0074] HEPLA index method: The HEPLA indices of the four donors were 57.5%, 59.8%, 54.1%, and 55.2%, respectively, all ≥14%. Overall judgment: HIT confirmatory test positive.

[0075] Results report: The HIT confirmatory test result of this patient was positive (+). It is recommended to immediately discontinue all heparin drugs and switch to non-heparin anticoagulants (such as argatroban, bivalirudin, etc.), and closely monitor the recovery of platelet count.

[0076] Example 8 Reagent Kit Performance Validation – Precision Evaluation This embodiment provides precision performance verification data for the reagent kit of the present invention.

[0077] Intra-batch precision: Select one HIT-positive serum sample and one HIT-negative serum sample, and perform 10 tests in the same batch to calculate the coefficient of variation (CV) of CD62P positivity rate and HEPLA index.

[0078] Inter-batch precision: Select the same positive and negative sera, test twice a day for 5 consecutive days, and calculate the inter-batch CV.

[0079] The results are shown in Table 4.

[0080] Table 4 Precision Validation Results Conclusion: The intra-assay CVs of all indicators in positive samples were <10%, and the inter-assay CVs were <15%, meeting the precision requirements for quantitative indicators in clinical testing. Negative samples had relatively higher CVs due to lower signal values, but the interpretation results of the Hepla index were stable and consistent (all negative), not affecting clinical diagnosis.

[0081] Example 9 Reagent Kit Performance Validation – Comparison with SRA Method This embodiment provides the reagent kit of the present invention and Methodological comparison data of the gold standard C-serotonin release assay (SRA).

[0082] Sample collection: Serum samples were collected from 42 clinically suspected HIT patients. All samples were simultaneously sent for testing using the flow cytometry kit of this invention (FCA) and SRA testing in a reference laboratory.

[0083] Results interpretation: The FCA method is considered positive if the HEPLA index is ≥14%, and the SRA method is considered positive if the radioactive release rate is ≥20%.

[0084] The comparison results are shown in Table 5.

[0085] Table 5 Comparison results of FCA method and SRA method Diagnostic performance calculation: Sensitivity = 18 / 19 × 100% = 94.7%; Specificity = 21 / 23 × 100% = 91.3%; Positive predictive value = 18 / 20 × 100% = 90.0%; Negative predictive value = 21 / 22 × 100% = 95.5%; Overall accuracy = (18+21) / 42 × 100% = 92.9%; Kappa coefficient = 0.86.

[0086] The flow cytometry kit of this invention has a concordance rate of 92.9% with the SRA gold standard, and a Kappa coefficient of 0.86, indicating a high degree of consistency. This demonstrates that the kit has good clinical diagnostic performance and can be used as a reliable alternative to the HIT functional confirmatory test.

[0087] Example 10: Optimization and Comparison of Different Heparin Concentration Gradients This embodiment provides optimized experimental data for each concentration parameter in the heparin gradient excitation module of the present invention.

[0088] Experimental design: Five serum samples from confirmed HIT-positive cases were selected, and the platelet activation was tested under different low-concentration heparin (0.1, 0.2, 0.3, 0.5, 1.0 IU / mL) and high-concentration heparin (50, 100, 150, 200 IU / mL) conditions to determine the optimal concentration parameters.

[0089] The optimization results of the low-concentration excitation component are shown in Table 6.

[0090] Table 6 Optimization results of low-concentration heparin Conclusion: The highest platelet activation rate was achieved at a low concentration of 0.3 IU / mL, which is the optimal concentration for PF4 / heparin complex formation and HIT antibody binding. Therefore, this invention selects 0.3 IU / mL as the preferred concentration of the low-concentration activating component.

[0091] The optimization results of the high-concentration inhibitory components are shown in Table 7.

[0092] Table 7 Optimization results of high-concentration heparin Conclusion: The inhibitory effect of high-concentration heparin reaches a good level at 100 IU / mL. Further increasing the concentration to 150-200 IU / mL yields limited increase in inhibitory effect, but may increase reagent costs and potential non-specific interference. This invention selects 100 IU / mL as the preferred concentration of the high-concentration inhibitory component.

[0093] Example 11 Comparison of Optimized Antibody Staining Protocols This embodiment provides optimized experimental data on antibody dosage and incubation time in a dual-fluorescent antibody labeling module.

[0094] CD41 antibody dosage optimization: With a fixed CD62P-FITC antibody dosage of 5 μL, the effect of PE-CD41 antibody dosage ranging from 2 μL to 20 μL on platelet depletion was tested. The median fluorescence intensity and coefficient of variation of CD41-positive events were used as evaluation indicators.

[0095] The results showed that stable platelet fluorescent labeling could be obtained with PE-CD41 dosage in the range of 8-12 μL, with a median fluorescence intensity of approximately 10. 3.5 -10 4 The fluorescence intensity was <10%. When the dosage was <8 μL, the fluorescence intensity was low, which could make it difficult to distinguish between platelets and fragments; when the dosage was >15 μL, the increase in fluorescence intensity was not significant, resulting in reagent waste. Therefore, this invention selects 10 μL as the preferred dosage of PE-CD41 antibody.

[0096] CD62P antibody dosage optimization: With a fixed PE-CD41 antibody dosage of 10 μL, the effect of FITC-CD62P antibody dosage ranging from 1 μL to 10 μL on the signal-to-noise ratio (SNR) of platelet activation detection was tested. The ratio of CD62P positivity rates between positive and negative controls (i.e., SNR) was used as the evaluation index.

[0097] The results showed that a good signal-to-noise ratio (>20) was obtained when the FITC-CD62P dosage was in the range of 3-8 μL. When the dosage was <3 μL, the activation detection rate of the positive control decreased; when the dosage was >8 μL, the background fluorescence of the negative control increased slightly, and the signal-to-noise ratio decreased instead of increasing. Therefore, this invention selected 5 μL as the preferred dosage of FITC-CD62P antibody.

[0098] Optimization of staining incubation time: The impact of antibody staining incubation time from 5 minutes to 30 minutes on the test results.

[0099] The results showed that the detection results were stable within the incubation time range of 10-20 minutes, with a CV of CD62P positivity rate of <5%. Incubation times <10 minutes may result in incomplete antibody binding and low signal intensity; incubation times >25 minutes may lead to platelet aggregation, affecting single-cell resolution in flow cytometry. This invention selected 15 minutes as the preferred incubation time for antibody staining.

[0100] Example 12: Stability Verification of the Reagent Kit This embodiment provides stability verification data for each component of the reagent kit of the present invention.

[0101] Accelerated stability test: Each component of the reagent kit was placed in a 37°C incubator for accelerated aging, and samples were taken at 0, 7, 14, 21, and 28 days to evaluate the changes in reagent performance.

[0102] Real-time stability test: Store the kit according to the conditions specified in the instructions (antibody 2-8℃ protected from light, heparin solution 2-8℃, TRAP-6 lyophilized powder -20℃, PBS room temperature), and take samples at 0, 3, 6, 9, and 12 months to evaluate the changes in reagent performance.

[0103] Evaluation indicators: Using the same batch of HIT positive and negative control sera, the CD62P positivity rate and HEPLA index were measured, and the percentage deviation from the 0 time point was calculated. The acceptable standard was a deviation of <15%.

[0104] Accelerated stability testing showed that after 28 days of accelerated aging at 37℃, the deviations for the heparin solution component were <8%, the antibody component <12%, and the TRAP-6 component <10%, all within acceptable limits. Based on this, it is estimated that the kit's shelf life can reach over 12 months under normal storage conditions.

[0105] Real-time stability results showed that during the 12-month real-time stability monitoring period, the deviation of the detection results of each component was <10%, indicating that the kit has good stability under the specified storage conditions and a shelf life of up to 12 months.

[0106] Example 13 Clinical Application Case 1 This embodiment provides a clinical application example of HIT screening after cardiac surgery.

[0107] Clinical Data: The patient was a 68-year-old male who underwent coronary artery bypass grafting (CABG) for coronary atherosclerotic heart disease and received low molecular weight heparin anticoagulation therapy postoperatively. On postoperative day 6, his platelet count increased from 156 × 10⁻⁶ on postoperative day 1. 9 / L decreased to 68×10 9 / L, a decrease of 56%, clinical suspicion of HIT, and samples were sent for HIT antibody testing and HIT confirmatory testing.

[0108] Test results: HIT antibody ELISA test was positive (OD value 1.85, positive threshold 0.4); HIT confirmatory test results of the kit of this invention: CD62P positive rate of 0 IU / mL group was 7.2%, CD62P positive rate of 0.3 IU / mL group was 52.8%, CD62P positive rate of 100 IU / mL group was 11.5%, HEPLA index was 64.3%, and it was judged as positive.

[0109] Clinical management: Upon diagnosis of HIT, low molecular weight heparin was immediately discontinued and replaced with argatroban via continuous intravenous infusion for anticoagulation. On postoperative day 10, the platelet count recovered to 102 × 10⁻⁶. 9 / L, recovered to 168×10 on the 14th day after surgery. 9 / L, the patient improved and was discharged.

[0110] Clinical significance: In this case, after the patient tested positive for ELISA, the functional confirmatory test using the kit of this invention confirmed the diagnosis of HIT, allowing for a timely change in the anticoagulation regimen and avoiding serious thrombotic complications that could have been caused by continued use of heparin.

[0111] Example 14: Clinical Application Case Two This example provides a clinical application case of ELISA false positive exclusion.

[0112] Clinical data: The patient was a 55-year-old female who was receiving unfractionated heparin anticoagulation therapy for deep vein thrombosis. On the 8th day of treatment, her platelet count decreased from 198 × 10⁻⁶ on admission. 9 / L decreased to 142×10 9 / L, a decrease of 28%, 4Ts score of 4 (moderate possibility), sent for HIT-related testing.

[0113] Test results: HIT antibody ELISA test was positive (OD value 0.68, positive threshold 0.4); HIT confirmatory test results of the kit of this invention: CD62P positive rate 1.2% in 0 IU / mL group, CD62P positive rate 2.5% in 0.3 IU / mL group, CD62P positive rate 1.8% in 100 IU / mL group, HEPLA index 16.3%, judged as negative.

[0114] Clinical Management: Based on the combined results of immunological and functional confirmatory tests, the patient's ELISA positivity was considered to be due to non-pathological antibodies and did not meet the diagnostic criteria for HIT. Heparin anticoagulation therapy was continued, while other causes of thrombocytopenia (such as infection, medications, etc.) were investigated. The platelet count subsequently stabilized at 130-150 × 10⁻⁶. 9 Within the range of / L, no new thrombotic events occurred.

[0115] Clinical significance: In this case, the patient was positive for ELISA but negative for functional confirmatory tests, indicating that the antibody was a non-pathological antibody without platelet-activating ability. The kit of this invention helped rule out the diagnosis of HIT, avoiding unnecessary changes in anticoagulation regimens and the resulting bleeding risks and increased medical costs.

[0116] Example 15: Clinical Application Case 3 This embodiment demonstrates according to Figure 1 and Figure 2 The specific method for interpreting the results of the flow cytometry detection spectrum shown.

[0117] Figure 1The chart shows scatter plots under three UFH concentration conditions, with the X-axis representing the FITC fluorescence channel (CD62P) and the Y-axis representing the PE fluorescence channel (CD41). The R3 gate delineates the proportion of CD62P-positive platelets within the CD41-positive platelet population. From left to right: R3 = 0.92% for UFH 0.0 IU / mL; R3 = 1.03% for UFH 0.3 IU / mL; and R3 = 0.65% for UFH 100 IU / mL. Interpretation of results: The CD62P positivity rate in the 0 IU / mL group (0.92%) is <5%, not meeting positivity condition A; the 0.3 IU / mL group (1.03%) shows a slight increase compared to the 0 IU / mL group (0.92%), but the increase is minimal; the 100 IU / mL group (0.65%) shows a decrease compared to the 0.3 IU / mL group (1.03%). HEPLA index = [(1.03-0.65) / (1.03+0.65)]×100%=22.6%. Although the HEPLA index is ≥14%, condition A is not met, and the absolute activation rate of all three groups is <2%, which is at the normal background level. Therefore, the overall judgment is that the HIT confirmatory test is negative.

[0118] Figure 2 The results showed that R3 was 0.81% at UFH 0.0 IU / mL, 1.01% at UFH 0.3 IU / mL, and 1.43% at UFH 100 IU / mL. Interpretation: The CD62P positivity rate in the 0 IU / mL group (0.81%) was <5%, failing to meet positivity condition A; the 100 IU / mL group (1.43%) showed an increase compared to the 0.3 IU / mL group (1.01%), failing to meet positivity condition C. The HEPLA index = [(1.01-1.43) / (1.01+1.43)] × 100% = -17.2% < 14%. Overall, the HIT confirmatory test was negative.

[0119] The two negative results above demonstrate that the kit of the present invention can effectively exclude non-HIT patients and avoid misdiagnosis.

[0120] The working principle of this invention's kit is based on the immunopathogenesis of HIT and the specific heparin dependence of pathological antibodies. During HIT pathogenesis, heparin binds to PF4 released from platelet α-granules to form an immunogenic complex, inducing the body to produce IgG antibodies. These pathological antibodies have a dual function: their Fab fragment recognizes and binds to a novel conformational epitope on the PF4 / heparin complex, while their Fc fragment binds to the FcγRIIa receptor on the platelet surface. When the antibody binds to both simultaneously, it triggers an intraplatelet signal transduction cascade, leading to platelet activation, α-granule release (P-selectin eversion to the surface), procoagulant microparticle formation, and platelet aggregation. This immune activation process is heparin concentration-dependent: under optimal low-concentration heparin conditions (approximately 0.3 IU / mL), the formation of the PF4 / heparin complex and antibody binding efficiency are highest, resulting in the strongest platelet activation. However, under high-concentration heparin conditions (approximately 100 IU / mL), excess heparin competitively binds to the heparin binding site on PF4, disrupting the stability of the complex and the conformation of the antibody epitope, thereby significantly inhibiting HIT antibody-mediated platelet activation. The kit of this invention utilizes this specific dose-response curve, employing a three-concentration gradient heparin activation system and HEPLA index quantification to achieve highly specific detection of HIT pathological antibodies.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A heparin-induced platelet flow cytometry kit characterized in that, It comprises: a donor platelet preparation module, which comprises a low-speed centrifugation parameter setting assembly for preparing platelet-rich plasma, the centrifugal force of the low-speed centrifugation parameter setting assembly is set to 80-120g, the centrifugation time is set to 4-6 minutes, and the brake force is set to zero braking; a heparin gradient excitation module, which comprises three concentration gradient unfractionated heparin solution components, namely a basic concentration component, a low concentration excitation component and a high concentration inhibition component, wherein the heparin concentration of the basic concentration component is 0 IU / mL, the heparin concentration of the low concentration excitation component is 0.2-0.5 IU / mL, and the heparin concentration of the high concentration inhibition component is 80-150 IU / mL; a double-fluorescent antibody labeling module, which comprises a platelet-specific labeling antibody and a platelet activation labeling antibody, the platelet-specific labeling antibody is a phycoerythrin-labeled anti-CD41 monoclonal antibody, and the platelet activation labeling antibody is a fluorescein isothiocyanate-labeled or allophycocyanin-labeled anti-CD62P monoclonal antibody; a quality control reference module, which comprises a negative quality control reagent and a positive quality control reagent, and the positive quality control reagent comprises a thrombin receptor agonistic peptide.

2. The heparin-induced platelet flow cytometry kit according to claim 1, wherein, The heparin concentration of the low concentration excitation component is 0.3 IU / mL, and the heparin concentration of the high concentration inhibition component is 100 IU / mL.

3. The heparin-induced platelet flow cytometry kit according to claim 1, wherein, The use amount of the phycoerythrin-labeled anti-CD41 monoclonal antibody is 8-12 μL / test, and the use amount of the anti-CD62P monoclonal antibody is 3-8 μL / test.

4. The heparin-induced platelet flow cytometry kit of claim 1, wherein, The thrombin receptor agonistic peptide is TRAP-6, which has an amino acid sequence of SFLLRN, and the use concentration is 10-50 μM.

5. The heparin-induced platelet flow cytometry kit of claim 1, wherein, It further comprises a buffer module, which comprises a 10-fold concentrated phosphate buffer, the pH value of the phosphate buffer is 7.2-7.6, and the 10-fold concentrated phosphate buffer is diluted to 1-fold working concentration when used.

6. The heparin-induced platelet flow cytometry kit of claim 1, wherein, The positive quality control reagent is in the form of a freeze-dried powder, which needs to be dissolved in 150-250 μL of ethanol with a volume fraction of 65%-75% before use, and the dissolution time is 15-30 minutes.

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