A homogeneous chemiluminescent kit for detecting platelet-monocyte aggregates and a method for detecting the same

Abstract

The application belongs to the field of biological medicine, and particularly relates to a homogeneous chemiluminescence kit for detecting platelet-monocyte aggregates and a detection method thereof. The kit comprises: R1 reagent: CD41 antibody-DNA1 conjugate; R2 reagent: CD14 antibody-DNA2 conjugate; R3 reagent: DNA3 labeled with acridinium ester; and R4 reagent: antioxidant combined with graphene oxide. The application realizes direct whole blood detection of platelet-monocyte aggregates in 5-10 minutes through homogeneous chemiluminescence+orthogonal DNA hybridization+special sample stabilizer, greatly simplifies the process, reduces the cost, and enables the sample to be stable at room temperature for 6 hours. The detection result is highly correlated with flow cytometry, and has good diagnostic efficiency for sepsis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates and its detection method. Background Technology

[0002] Platelet-monocyte aggregates (PMA) are atypical cell aggregates formed by platelets and monocytes after platelet activation, and their formation is mainly mediated by P-selectin (CD62P) on the platelet surface. PMA is a sensitive marker of platelet activation and plays an important role in the pathophysiology of various diseases.

[0003] Studies have shown that platelets (PMA) have good clinical reference value in the diagnosis of sepsis and can serve as a biomarker for sepsis diagnosis. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Although there have been significant advancements in the diagnosis and treatment of sepsis in recent years, the mortality rate of sepsis remains as high as 20%–40%. The pathogenesis of sepsis is complex, involving excessive inflammatory response, coagulation disorders, immune dysregulation, and tissue damage. In the early stages of sepsis, platelets are at the crossroads between the immune and coagulation systems, playing a crucial role in hemostasis, coagulation, and maintaining endothelial integrity.

[0004] Currently, PMA detection mainly employs flow cytometry. Its basic principle involves staining whole blood samples with fluorescently labeled antibodies (CD41-FITC-labeled platelets, CD14-PE-labeled monocytes), and then detecting the proportion of double-positive cells (CD41+CD14+) using a flow cytometer. However, flow cytometry for PMA detection has the following limitations: 1) Complex operation: It requires multiple pretreatment steps such as erythrocyte lysis, centrifugation, and washing, which can easily lead to in vitro platelet activation; 2) Long detection time: The entire detection process typically takes 30-60 minutes; 3) Expensive equipment: It requires specialized flow cytometers, resulting in high costs; 4) High technical requirements: It requires professional technicians for operation and data analysis; 5) High sample processing requirements: Samples must be tested as soon as possible after collection, otherwise PMA levels will change. Other detection methods, such as ADAM imaging cytometers, while relatively simpler to operate, still suffer from low throughput and high costs. Summary of the Invention

[0005] Technical issues

[0006] Existing PMA detection methods suffer from drawbacks such as complex operation, long testing time, expensive equipment, and high technical requirements, failing to meet the practical needs of rapid clinical diagnosis of sepsis. Therefore, there is an urgent need to develop a PMA detection method that is simple to operate, rapid, and cost-effective.

[0007] Technical solution

[0008] The first aspect of this invention provides a homogeneous chemiluminescence kit for detecting platelet-monocyte aggregates, the kit comprising: R1 reagent: CD41 antibody-DNA1 conjugate; R2 reagent: CD14 antibody-DNA2 conjugate; R3 reagent: DNA3 labeled with acrid ester; R4 reagent: an antioxidant bound to graphene oxide; wherein the DNA1 contains a pairing sequence 1 and a pairing sequence 2; wherein the 3' end of the DNA1 is modified with NH2C7; the pairing sequence 1 is GCTGAGTT; the pairing sequence 2 is CAACGAC; the base sequence of the DNA1 is as shown in SEQ ID NO.1; the DNA2 has a pairing sequence 3 and a pairing sequence 4; wherein the 5' end of the DNA2 is modified with NH2C6; the pairing sequence 3 is complementary to the pairing sequence 2; the pairing sequence 3 is GTCGTTG; the pairing sequence 4 is GCTGAGAT; the base sequence of the DNA2 is as shown in SEQ ID NO.1. As shown in NO.2; the DNA3 contains pairing sequence 5 and pairing sequence 6; wherein, the 5' end of the DNA3 is modified with NH2C6 and coupled to acridinium ester through NH2C6; pairing sequence 5 is complementary to pairing sequence 4, and pairing sequence 6 is complementary to pairing sequence 1; pairing sequence 5 is ATCTCAGC; pairing sequence 6 is AACTCAGC; the base sequence of the DNA3 is shown in SEQ ID NO.3; during detection, the CD41 antibody specifically binds to the antigenic epitope of platelets, and the CD14 antibody specifically binds to the antigenic epitope of monocytes, forming an orthotopic complex: DNA1-CD41 antibody-platelet-monocyte-CD14 antibody-DNA2, and the orthotopic complex can hybridize with DNA3; wherein, the clone number of the CD41 antibody is SZ22, and the clone number of the CD14 antibody is M5E2.

[0009] The specific details of the complementary pairing of DNA3 with DNA1 and DNA2 are as follows: each pairing sequence 2 and pairing sequence 3 consists of 7 bases; each pairing sequence 1, pairing sequence 4, pairing sequence 5 and pairing sequence 6 consists of 8 bases; pairing sequence 1 and pairing sequence 2 are separated by 2 bases; pairing sequence 3 and pairing sequence 4 are directly linked; and pairing sequence 5 and pairing sequence 6 are directly linked. Specifically, the pairing sequence 1 consists of bases from the 3rd to 10th base position of the single-stranded DNA 1 starting from the 5' end; the pairing sequence 2 consists of bases from the 13th to 19th base position of the single-stranded DNA 1 starting from the 5' end; the pairing sequence 3 consists of bases from the 11th to 17th base position of the single-stranded DNA 2 starting from the 3' end; the pairing sequence 4 consists of bases from the 3rd to 10th base position of the single-stranded DNA 2 starting from the 3' end; the pairing sequence 5 consists of bases from the 3rd to 10th base position of the single-stranded DNA 3 starting from the 5' end; and the pairing sequence 6 consists of bases from the 5th to 12th base position of the single-stranded DNA 3 starting from the 3' end.

[0010] The detection principle of PMA is as follows: Chemiluminescence Resonance Energy Transfer (CRET) is a homogeneous chemiluminescence detection technique based on the principle of adjacent hybridization. This technique utilizes the conjugation of DNA molecules with antibodies. When two antibodies simultaneously bind to different epitopes of the target molecule, the DNA molecules form adjacent complexes, which hybridize with DNA probes labeled with fluorescent molecules, generating chemiluminescent signals. Specifically, in this invention, when PMA is present in the sample, the conjugates of DNA1 and CD41 antibody 1, and DNA2 and CD14 antibody, bind to antigenic epitopes on the platelet membrane surface and the monocyte membrane surface, respectively, forming DNA1-CD41 antibody-platelet-monocyte-CD14 antibody-DNA2 complexes. This ensures that DNA1 and DNA2 are sufficiently close to form adjacent complexes, which can hybridize with DNA3. The complexes are hardly adsorbed by graphene oxide, and luminescence is achieved upon the addition of a chemiluminescent substrate. In the absence of PMA in the sample, the DNA1-CD41 antibody-platelet-monocyte-CD14 antibody-DNA2 complex cannot be formed. Therefore, DNA3 is adsorbed onto the graphene oxide surface via π-π stacking. Upon addition of the chemiluminescent substrate, the acridinium ester (AE) terminally labeled with DNA3 cannot oxidize and emit light due to the presence of antioxidants. Even the small amount of chemiluminescence is quenched by chemiluminescent resonance energy transfer (CRET). A chemiluminescent (CL) signal can be obtained through automatic incubation using a portable HSCL-5000 chemiluminescence analyzer. The reason why the luminescent signal originates from activated platelet-monocyte aggregates, rather than from random proximity of free platelets and free monocytes, is as follows: 1) The order-of-magnitude gap in effective interaction distance: The stable effective interaction distance for adjacent DNA hybridization is only <100 nm (usually <50 nm). Only when platelets and monocytes form a stable PMA through physiological adhesion can CD41 antibody-DNA1 and CD14 antibody-DNA2, respectively bound to the two cell membranes, be firmly fixed within this nanometer-scale spatial range, forming a continuous and stable adjacent complex. Even if free platelets (diameter 2 μm~4 μm) and free monocytes (diameter 10 μm~30 μm) randomly collide and approach each other, the distance between their cell membranes is still in the micrometer range (1 μm = 1000 nm), which is two orders of magnitude smaller than the effective hybridization distance. This makes it impossible for DNA1 and DNA2 to enter the spatial range where stable hybridization can occur. 2) The transient nature of random collisions cannot meet the time requirements of hybridization reactions. Random collisions of free cells are instantaneous contacts caused by Brownian motion. They will separate immediately after the collision and cannot maintain the adjacent state of DNA1 and DNA2 within the 5 min to 10 min incubation period of the kit.The formation and stability of DNA hybrid chains require continuous spatial proximity. Instantaneous proximity cannot complete the hybridization reaction, let alone accumulate to produce detectable luminescent signals.

[0011] In some embodiments, the homogeneous chemiluminescence kit further includes R5 reagent: a chemiluminescent substrate; the chemiluminescent substrate is an alkaline solution of hydrogen peroxide; wherein the pH of the alkaline solution is 8.5~9.5, preferably 9.0, and the concentration of the hydrogen peroxide is 2%v / v~5%v / v, preferably 3%v / v.

[0012] In some embodiments, the kit further includes: a stabilizer blood collection tube; the stabilizer blood collection tube is prepared by the following steps: adding a sample stabilizer of 2.2% v / v to 2.8% v / v, or 2.5% v / v of blood collection volume, to a sterile vacuum blood collection tube, drying it with sterile hot air, evacuating it to the appropriate blood collection volume, and sealing it.

[0013] In some embodiments, drying is performed by sterile hot air at 35°C to 39°C, preferably 37°C.

[0014] In some embodiments, the stabilizer blood collection tube is stored at 3°C ​​to 5°C, preferably 4°C, away from light, and has a shelf life of 6 months.

[0015] In some embodiments, the sample stabilizer includes a basic anticoagulant, an activation inhibitor, a reversible fixation component, a protective buffer component, and a solvent; wherein the basic anticoagulant is EDTA-K2; the activation inhibitor includes PGE1 and modified CTAD, the modified CTAD including sodium citrate, theophylline, adenosine, and dipyridamole; the reversible fixation component includes paraformaldehyde and glutaraldehyde; the protective buffer component includes glycine and BSA; and the solvent is sterile PBS.

[0016] In some embodiments, the modified CTAD comprises 9 mmol / L to 11 mmol / L sodium citrate, 2 mmol / L to 2.5 mmol / L theophylline, 3.2 mmol / L to 3.8 mmol / L adenosine, and 0.0018 mmol / L to 0.0022 mmol / L dipyridamole. Preferably, it comprises 10 mmol / L sodium citrate, 2.2 mmol / L theophylline, 3.5 mmol / L adenosine, and 0.002 mmol / L dipyridamole.

[0017] In some embodiments, the sample stabilizer contains 1.6 mg / mL to 2 mg / mL EDTA-K2, 0.9 μmol / L to 1.1 μmol / L PGE1, 9 mmol / L to 11 mmol / L sodium citrate, 2 mmol / L to 2.5 mmol / L theophylline, 3.2 mmol / L to 3.8 mmol / L adenosine, 0.0018 mmol / L to 0.0022 mmol / L dipyridamole, 0.45% v / v to 0.55% v / v paraformaldehyde, 0.045% v / v to 0.055% v / v glutaraldehyde, 45 mmol / L to 55 mmol / L glycine, and 0.18% m / v to 0.22% m / v BSA. The preferred formulation includes 1.8 mg / mL EDTA-K2, 1 μmol / L PGE1, 10 mmol / L sodium citrate, 2.2 mmol / L theophylline, 3.5 mmol / L adenosine, 0.002 mmol / L dipyridamole, 0.5% v / v paraformaldehyde, 0.05% v / v glutaraldehyde, 50 mmol / L glycine, and 0.2% m / v BSA.

[0018] In some embodiments, the sample stabilizer is prepared by the following steps: adding EDTA-K2, PGE1 stock solution, modified CTAD stock solution, paraformaldehyde + glutaraldehyde composite fixative stock solution, glycine and BSA to sterile PBS, adjusting the pH to 7.2±0.1, and sterilizing through a filter membrane to obtain the sample stabilizer.

[0019] In some embodiments, the PGE1 stock solution is a 9 mmol / L to 11 mmol / L PGE1 solution prepared with anhydrous ethanol; the modified CTAD stock solution comprises: 90 mmol / L to 110 mmol / L sodium citrate, 20 mmol / L to 25 mmol / L theophylline, 32 mmol / L to 38 mmol / L adenosine, and 0.018 mmol / L to 0.022 mmol / L dipyridamole, with sterile PBS as the solvent; the paraformaldehyde + glutaraldehyde composite fixative stock solution comprises: 9% v / v to 11% v / v paraformaldehyde and 0.9% v / v to 1.1% v / v glutaraldehyde.

[0020] In some embodiments, the filter membrane has a size of 0.22 μm.

[0021] In some embodiments, the sample stabilizer must be prepared and used immediately.

[0022] In some embodiments, DNA1 / DNA2 are each coupled to CD41 antibody / CD14 antibody via a coupling agent; wherein, DNA1 / DNA2 are each covalently bound to the amino group on CD41 antibody / CD14 antibody via a coupling agent, and the coupling agent is sodium bis(succinimide) octanoate.

[0023] In some embodiments, the CD41 antibody-DNA1 conjugate (R1 reagent) or CD14 antibody-DNA2 conjugate (R2 reagent) is prepared by the following steps: 3 μL of 9 g / L~11 g / L bis(succinimide) octanoate sodium salt (BS3) solution is added to every 1 mg of antibody, followed by 6.5 μL of DNA1 or DNA2 at 18 mM / L~22 mM / L, and the mixture is thoroughly mixed and incubated at 35℃~37℃ for 25 min~35 min.

[0024] In some embodiments, the acridine ester-labeled DNA3 (R3 reagent) is prepared by the following steps: 10 μL of 18 mM / L to 22 mM / L DNA3 solution is added to every 1 mg of acridine ester solution (3.6 g / L to 4.4 g / L), mixed thoroughly, and incubated at 35°C to 37°C for 25 min to 35 min.

[0025] In some embodiments, the antioxidant is selected from one or more of vitamin C, vitamin E, tea polyphenols, or glutathione.

[0026] The second aspect of this invention provides a detection method for the homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates as described in any one of the above claims, comprising the following steps: S1. Mixing reagents R1, R2, R3, and R4 to obtain a detection solution; S2. Mixing the sample to be tested with the detection solution to obtain a mixed solution; then incubating at 35℃~37℃ for 5 min~10 min; S3. Adding reagent R5 to the mixed solution, and acquiring the light signal using a chemiluminescence detector to obtain the chemiluminescence value of the sample to be tested; S4. The instrument automatically retrieves the calibration curve, and by substituting the chemiluminescence value into the calibration curve, the percentage of platelet-monocyte aggregates in the sample to be tested (the proportion of platelet-bound monocytes to all monocytes) can be reported.

[0027] In some embodiments, the test sample is a whole blood sample contained in a stabilizer blood collection tube; the test sample contains 1.6 mg / mL~2 mg / mL EDTA-K2, 0.9 μmol / L~1.1 μmol / L PGE1, 9 mmol / L~11 mmol / L sodium citrate, 2 mmol / L~2.5 mmol / L theophylline, 3.2 mmol / L~3.8 mmol / L adenosine, 0.0018 mmol / L~0.0022 mmol / L dipyridamole, 0.45%v / v~0.55%v / v paraformaldehyde, 0.045%v / v~0.055%v / v glutaraldehyde; 45 mmol / L~55 mmol / L glycine and 0.18%m / v~0.22%m / v BSA.

[0028] In some embodiments, the test sample remains stable even after being placed at room temperature for 6 hours.

[0029] In some embodiments, the final concentration of reagent R1 in the detection solution in step S1 is 1 nM to 20 nM, preferably 10 nM; the final concentration of reagent R2 is 1 nM to 20 nM, preferably 10 nM; the final concentration of reagent R3 is 0.05 μM to 0.2 μM, preferably 0.15 μM; the final concentration of reagent R4 is 20 μg / mL to 25 μg / mL, preferably 20 μg / mL; and the concentration ratio of reagent R1, reagent R2 and reagent R3 in the detection solution is 1:1 to 1.5:10 to 20, preferably 1:1:15.

[0030] In some embodiments, the volume ratio of the sample to be tested in the mixed solution to the detection solution in step S2 is 1:10~20, preferably 1:20; the volume ratio of the R5 reagent to the detection solution in the mixed solution in step S3 is 0.8~1:1, preferably 1:1.

[0031] Technical effect

[0032] 1. Direct detection of whole blood: The kit of this invention uses homogeneous chemiluminescence to directly detect whole blood samples without the need for complex pretreatment steps such as red blood cell lysis, centrifugation and washing, which greatly simplifies the detection process and shortens the detection time. At the same time, it avoids the platelet activation that may occur in in vitro samples and can reflect the true state of PMA in vivo.

[0033] 2. Fast detection speed: The detection process of the kit for PMA in this invention takes a very short time, generally within 5 to 10 minutes, which is very suitable for clinical environments that require rapid diagnosis and quick treatment decisions, especially for the emergency diagnosis of sepsis.

[0034] 3. Simple Operation: The reagent kit of this invention has a simple operation procedure for detecting PMA, making it easy to implement in different medical environments, including those with limited resources. No professional technicians are required; ordinary laboratory personnel can complete the test.

[0035] 4. Low cost: Since the complex separation and washing steps are eliminated and expensive flow cytometers are not required, the cost of detecting PMA using the kit of this invention is relatively low, making large-scale detection more economical and more practical.

[0036] 5. High accuracy: The kit of this invention has a good correlation with the results of flow cytometry detection of PMA (R>0.90), and the detection accuracy is high, which can meet the needs of clinical diagnosis.

[0037] 6. Applicable to sepsis diagnosis: The kit of this invention is particularly suitable for initial risk screening, rapid diagnosis and disease monitoring of sepsis, and can provide clinicians with timely diagnostic information, which helps to improve the prognosis of sepsis patients.

[0038] 7. No immediate testing required: The sample stabilizer of this invention (PGE1 + modified CTAD + reversible fixation component + protective buffer component) can effectively inhibit the spontaneous activation of platelets in whole blood samples in vitro. At the same time, it is fully compatible with homogeneous chemiluminescence detection systems, with no matrix interference. The low concentration of fixation component it contains has good reversibility, does not affect antibody-antigen binding, can fix the formed PMA structure and prevent its dissociation, and the detection precision and accuracy are not significantly different from fresh samples, solving the clinical limitation that "samples need to be tested as soon as possible, otherwise the PMA level will change". Attached Figure Description

[0039] Figure 1 A schematic diagram of complementary pairing of DNA sequences: showing the complementary pairing relationships between DNA1, DNA2, and DNA3;

[0040] Figure 2 A schematic diagram illustrating the principle of PMA detection: showing the principle of CRET technology for detecting PMA;

[0041] Figure 3 The standard curve plot shows the linear relationship between the percentage of PMA and the chemiluminescence emission level (RLU).

[0042] Figure 4 A scatter plot showing the correlation between homogeneous chemiluminescence and flow cytometry;

[0043] Figure 5 Bland-Altman analysis plot: showing the consistency analysis results of the two methods;

[0044] Figure 6A comparison chart of PMA levels in each group: showing the distribution of PMA levels in the healthy control group, the infection group, and the sepsis group;

[0045] Figure 7 ROC curve: The ROC curve for PMA diagnosis of sepsis (AUC=0.847) is shown. Detailed Implementation

[0046] To facilitate the implementation of the technical solution applied for, the terms and expressions involved in this invention will first be explained and defined in general terms below.

[0047] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0048] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0049] The following provides a further description of the homogeneous chemiluminescence reagent kit and detection method for detecting platelet-monocyte aggregates provided by the present invention.

[0050] Example 1: Preparation of R3 reagent (DNA3 coupled with AE)

[0051] Step 1: Prepare DNA3 solution: Take 20 μM DNA3 (purchased from Genscript; sequence: 5'-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3', where the base sequence is shown in SEQ ID NO.3), add 1 mL of purified water to dissolve;

[0052] Step 2, Prepare AE (acridone ester) solution: Weigh 4 mg AE (name: NSP-DMAE-NHS, manufacturer: purchased from Suzhou Yake Technology Co., Ltd., CAS No.: 194357-64-7) and dissolve it in 1 mL of purified water;

[0053] Step 3, Coupling: Add 10 μL of DNA3 solution to each 1 mg AE solution, mix well, and incubate at 37°C for 30 min;

[0054] Step 4, Dialysis: Aspirate the coupled DNA3 from the EP tube and transfer it to a dialysis bag (5 kDa). Place the sealed dialysis bag into a beaker containing 2-3 L of TE solution (10 mM Tris, 1 mM EDTA, pH=8.0) and perform dialysis. Change the dialysis solution every 2-3 hours, for a total of three dialysis cycles. After dialysis, collect the liquid from the dialysis bag and transfer it to a centrifuge tube to obtain the prepared R3 reagent (DNA3 labeled with AE). Store at 2℃-8℃ for later use.

[0055] Example 2: Preparation of R1 or R2 reagent (antibody-labeled DNA1 or DNA2)

[0056] Step 1: Prepare BS3 solution: Weigh 10 mg of bis(succinimide) octanoate sodium salt (BS3) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number S304724) and dissolve it in 1 mL of purified water.

[0057] Step 2: Prepare DNA1 / DNA2 solution: Take 20 μM DNA1 / DNA2 (purchased from GenScript; DNA1 sequence: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3', where the base sequence is shown in SEQ ID NO.1; DNA2 sequence: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3', where the base sequence is shown in SEQ ID NO.2; the complementary pairing relationship between DNA1, DNA2, and DNA3 is as follows... Figure 1 (As shown), add 1 mL of purified water to dissolve.

[0058] Step 3: Antibody conjugation: Take out the aliquoted CD41 antibody or CD14 antibody, thaw it, and centrifuge to mix. Add 3 μL of BS3 solution and 6.5 μL of DNA1 or DNA2 to each 1 mg of antibody, mix well, and incubate at 37°C for 30 min.

[0059] Step 4, Dialysis: Aspirate the conjugated antibody from the EP tube and place it into a dialysis bag (100 kDa). Seal the dialysis bag and place it in a beaker containing 2-3 L of PBS solution for dialysis. Change the dialysis solution every 2-3 hours, for a total of three dialysis cycles. After dialysis, collect the liquid from the dialysis bag into a centrifuge tube to obtain the prepared R1 reagent (CD41 antibody-DNA1 conjugate) or R2 reagent (CD14 antibody-DNA2 conjugate), and store it at 2℃-8℃ for later use.

[0060] Example 3: Detection of PMA in whole blood

[0061] Step 1: Prepare the detection reagents: Mix reagents R1 (CD41 antibody-DNA1 conjugate), R2 (CD14 antibody-DNA2 conjugate), R3 (DNA3 labeled with AE), and R4 (an antioxidant (vitamin C) bound to graphene oxide; purchased from Xianfeng Nano, catalog number XF248) to obtain a mixed detection solution. The final concentrations of reagents R1, R2, R3, and R4 in the detection solution are 10 nM, 10 nM, 0.15 μM, and 20 μg / mL, respectively.

[0062] Step 2, Incubation: Mix 10 μL of different percentages of PMA calibration solution (0%, 5%, 10%, 20%, 40%, 60%, 80%, and 100%) or 10 μL of whole blood sample with 200 μL of the above detection solution, place the mixture in the rotor detection port of the HSCL-5000 chemiluminescence analyzer, and start the incubation at 37°C for 10 min. After incubation, rotate the detection port to the detection position, aligning it with the light source module.

[0063] Step 3, Detection: After incubation, add 200 μL of chemiluminescent substrate (hydrogen peroxide in PBS buffer solution, pH=9.0, H2O2 concentration 3% v / v, PBS concentration 10 mM) to the HSCL-5000 chemiluminescence analyzer, and immediately detect the chemiluminescence signal of the solution using a photomultiplier tube (PMT) (see schematic diagram of PMA detection principle). Figure 2 As shown in the figure, the detection time is 3 s, and the calibration curve of PMA and the chemiluminescence value (RLU) of PMA in whole blood samples are obtained.

[0064] Step 4, Calculation: The instrument automatically calls up the calibration curve. Substitute the RLU value of the sample to be tested into the calibration curve, and the percentage of PMA in the sample will be reported.

[0065] Example 4 Antibody Pairing Screening

[0066] To screen for the optimal antibody pairings, we tested multiple pairs of CD41 and CD14 antibodies to evaluate their performance in detecting PMA. The candidate antibody information is shown in Table 1, with a total of 6 pairs.

[0067] 1) Preparation of antibody conjugates: According to Example 3 above, CD41 antibodies were labeled with DNA1 and CD14 antibodies were labeled with DNA2 to prepare reagent R1 (CD41 antibody-DNA1 conjugate) and reagent R2 (CD14 antibody-DNA2 conjugate).

[0068] 2) Test method: Whole blood samples from healthy individuals and whole blood samples activated with ADP (5 μM) were used as controls. Then, the chemiluminescence signals of each antibody pair combination were detected according to the method in Example 3. Finally, the signal ratio (S1 / S0) and background signal level were calculated. The results are shown in Table 1.

[0069] Table 1. Candidate Antibody Information

[0070]

[0071] 3) Results: When CD41 antibody with clone number SZ22 (purchased from BD Biosciences) and CD14 antibody with clone number M5E2 (purchased from BD Biosciences, clone number M5E2) were selected, the best detection performance was observed, with the highest signal-to-weight ratio (18.4) and the lowest background signal. Therefore, this pairing combination was selected for subsequent experiments.

[0072] Example 5: Methodological Evaluation Experiment

[0073] 1) Linear range determination

[0074] Method: Samples with different PMA percentages (0%, 5%, 10%, 20%, 40%, 60%, 80%, 100%) were prepared and tested using the kit of this invention. Each concentration point was measured three times and the average value was calculated.

[0075] Table 2 Linear Range Determination

[0076]

[0077] Results: As shown in Table 2 and Figure 3 As shown, the linear regression equation is obtained: y = 1172.4x + 4212.2, R0 2 = 0.9976. This indicates that the reagent kit of the present invention has a good linear relationship in the range of 0% to 100%, with a correlation coefficient R of 0.9976. 2 >0.99.

[0078] 2) Precision determination

[0079] Methods: For intra-batch precision, samples at low, medium, and high concentration levels were measured 20 times within the same batch, and the CV value was calculated. For inter-batch precision, samples at low, medium, and high concentration levels were measured twice daily for 10 consecutive days, and the CV value was calculated.

[0080] Table 3 Precision Measurement

[0081]

[0082] Results: As shown in Table 3, the kit of the present invention has an intra-batch CV of <5% and an inter-batch CV of <6%, indicating good precision.

[0083] 3) Detection limit determination

[0084] Method: The blank sample was measured 20 times repeatedly, and the mean and standard deviation were calculated. The concentration corresponding to the mean plus twice the standard deviation was taken as the limit of detection.

[0085] Results: The limit of detection was 2.1% PMA.

[0086] 4) Stability testing

[0087] Methods: The reagent kit was stored at 2℃~8℃ and removed at 0, 1, 2, 3, 6 and 12 months to test the same batch of samples and evaluate the stability of the reagent.

[0088] Results: The kit showed good stability with a detection deviation of <10% when stored at 2℃~8℃ for 12 months.

[0089] Example 6: Methodological Comparison Experiment with Flow Cytometry

[0090] 1) Experimental Design

[0091] 107 clinical samples were collected, including: a healthy control group (n=30), an infection group (non-sepsis, n=37), and a sepsis group (n=40). The percentage of PMA in each sample was simultaneously detected using the homogeneous photochemical reagent kit of this invention and flow cytometry (Beckman CoulterNavios). The flow cytometry procedure was as follows: 50 μL of whole blood was collected, and 5 μL each of CD41-FITC and CD14-PE antibodies were added and labeled in the dark for 20 min; then 500 μL of erythrocyte lysis buffer was added and incubated in the dark for 10 min; then 2 mL of physiological saline was added, vortexed, and centrifuged at 250×g for 5 min, and the supernatant was discarded; finally, the cells were resuspended in 500 μL of physiological saline and analyzed using the flow cytometry instrument, counting 100,000 cells. The percentage of PMA was obtained by analysis using Kaluza software.

[0092] 2) Results

[0093] The homogeneous chemiluminescence immunoassay results and flow cytometry results for the three groups are shown in Tables 4-6.

[0094] Table 4. Detailed test data of 30 healthy control group members.

[0095]

[0096] Table 5. Detailed test data of 37 cases in the infection group.

[0097]

[0098] Table 6. Detailed test data of 40 cases in the sepsis group

[0099]

[0100] 3) Comparative Analysis

[0101] ① Correlation analysis

[0102] Correlation analysis was performed between homogeneous chemiluminescence immunoassay results and flow cytometry results for 107 samples. The results are shown in Table 7 and... Figure 4 As shown, the homogeneous photochemical reagent kit of the present invention has a good correlation (R=0.977) with the flow cytometry detection results, and the consistency is good.

[0103] Table 7 Correlation Analysis

[0104]

[0105] ②Bland-Altman analysis

[0106] Bland-Altman analysis was performed on the homogeneous chemiluminescence immunoassay results and flow cytometry results of 107 samples. The results are shown in Table 8. Figure 5 As shown, the difference between the two methods falls within the consistency limit 95% of the time, indicating good consistency.

[0107] Table 8 Bland-Altman Analysis

[0108]

[0109] ③ Comparison of PMA levels among groups

[0110] The results of homogeneous chemiluminescence immunoassay and flow cytometry in the three groups were statistically analyzed, and the results are shown in Table 9. Figure 6 As shown, there were statistically significant differences in PMA levels among the three groups (P<0.001); among them, the PMA level in the sepsis group was significantly higher than that in the infection group (P<0.01), and the PMA level in the infection group was significantly higher than that in the healthy control group (P<0.001).

[0111] Table 9 Comparison of healthy control group, infection group and sepsis group

[0112]

[0113] Example 7: Evaluation of diagnostic efficacy for sepsis

[0114] 1) Experimental Design

[0115] After detecting the PMA levels in 107 samples (30 healthy controls, 37 infected cases, and 40 cases of sepsis) using the homogeneous photoluminescence reagent kit of this invention, ROC curves were plotted to evaluate the diagnostic efficacy of PMA for sepsis.

[0116] 2) Diagnostic efficacy results

[0117] The ROC curve analysis of PMA in diagnosing sepsis and the results of its comparison with inflammatory markers are shown in Tables 10 and 11, respectively: PMA has better diagnostic efficacy for sepsis than traditional inflammatory markers (PCT, CRP, platelet count), as shown in Tables 10 and 11. Figure 7 As shown, the AUC reached 0.847, indicating good clinical application value.

[0118] Table 10 ROC Curve Analysis

[0119]

[0120] Table 11 Comparison of Inflammatory Markers

[0121]

[0122] 3) Prognostic assessment of septic shock

[0123] Forty patients with sepsis were divided into two groups based on whether they experienced shock: a shock subgroup (n=14) and a non-shock subgroup (n=26). The results are shown in Table 12. There was a statistically significant difference in PMA levels between the two groups (P<0.001). PMA levels are associated with the severity of sepsis and can be used to assess the prognosis of patients with sepsis.

[0124] Table 12 Prognostic Assessment Results

[0125]

[0126] Example 8: Formulation, preparation, and preparation of the sample stabilizer and blood collection tubes.

[0127] The samples used in the above embodiments were all ordinary EDTA tubes (containing only EDTA-K2 (final concentration 1.8 mg / mL whole blood), without other stabilizer components), and required immediate testing. To address the clinical limitation of abnormal fluctuations in PMA levels after samples are left at room temperature and the need for immediate testing, this invention further investigates a sample stabilizer.

[0128] 1) Final concentration of core formula (for 2 mL whole blood collection tubes)

[0129] The sample stabilizer of this invention uses EDTA-K2 as the base anticoagulant and combines three major functional components. The final concentration (the final concentration after dilution of whole blood samples) is shown in Table 13.

[0130] Table 13 Sample stabilizer components and their functions

[0131]

[0132] Note: The concentrations of each component in the working solution of the sample stabilizer are 40 times those in Table 13.

[0133] 2) Preparation of sample stabilizer

[0134] PGE1 stock solution: Accurately weigh PGE1 powder and prepare a 10 mmol / L stock solution with anhydrous ethanol. Store at -20℃ protected from light in separate containers. Shelf life is 3 months.

[0135] Modified CTAD stock solution: Dissolve citric acid, theophylline, adenosine, and dipyridamole in sterile PBS to prepare a 10× concentrated stock solution, sterilize with a 0.22 μm filter membrane, store at 4℃, and have a shelf life of 1 month.

[0136] Paraformaldehyde + glutaraldehyde composite fixative stock solution: Prepare 10% paraformaldehyde + 1% glutaraldehyde stock solution in sterile PBS, store at 4°C protected from light, shelf life 2 weeks.

[0137] Preparation of sample stabilizer working solution: According to the final concentration ratio in Table 13, add EDTA-K2, the above-mentioned PGE1 stock solution, modified CTAD stock solution, paraformaldehyde + glutaraldehyde composite fixative stock solution, glycine, and BSA to sterile PBS, adjust the pH to 7.2±0.1, and sterilize through a 0.22 μm filter membrane to obtain the sample stabilizer working solution (prepare and use immediately).

[0138] 3) Preparation of blood collection tubes

[0139] ① Preparation of stabilizer blood collection tubes

[0140] Under aseptic conditions, take a sterile vacuum blood collection tube with a volume of 2 mL, add 50 μL of sample stabilizer working solution to each tube, dry it with sterile hot air at 37°C (to avoid sample dilution errors caused by liquid residue), then vacuum it to a volume of 2 mL whole blood, seal it, and store it at 4°C in the dark. It is valid for 6 months.

[0141] ② Preparation of control blood collection tubes

[0142] Blank control tubes (ordinary EDTA tubes): containing only EDTA-K2 (final concentration 1.8 mg / mL whole blood), without other stabilizer components, prepared by the same method;

[0143] Control tube 1 (standard CTAD tube): containing commercially available standard CTAD formula with EDTA-K2+ (final whole blood concentration: sodium citrate 0.109 mol / L, theophylline 3.7 mmol / L, adenosine 7.1 mmol / L, dipyridamole 0.0055 mmol / L), prepared by the same method;

[0144] Control 2 tubes (PGE1 single-component tubes): Whole blood containing EDTA-K2+1 μmol / L PGE1, without other stabilizer components, prepared by the same method;

[0145] Control 3 tubes (fixative single-component tubes): Whole blood containing EDTA-K2 + 0.5% paraformaldehyde + 0.05% glutaraldehyde, without other activation inhibitors, prepared by the same method.

[0146] Example 9: Verification of the stabilizing effect of the sample stabilizer

[0147] This embodiment aims to verify the stabilizing effect of the sample stabilizer prepared in Example 8 on PMA in whole blood samples (to verify the reversibility and non-interference of the sample stabilizer, and to ensure that it does not affect the accuracy, specificity and precision of the homogeneous chemiluminescence detection system), and to clarify the stability duration and performance boundaries of the sample stabilizer in healthy human samples, in vitro activated samples and sepsis clinical samples.

[0148] 1) Enrolled sample

[0149] The experiment included 50 samples, which matched the sample type of the clinical validation in Example 6, as detailed below:

[0150] Whole blood samples from healthy individuals: 20 cases, from health check-up volunteers, aged 18 to 60 years, with no infection, thrombosis, or blood system diseases, and who had not taken antiplatelet or anticoagulant drugs in the past 2 weeks;

[0151] ADP-activated whole blood samples: 10 cases from the above-mentioned healthy volunteers. A final concentration of 5 μmol / LADP was added to the whole blood samples, and the samples were incubated at 37°C for 10 min to prepare activated samples, simulating the highly activated state of platelets in vivo.

[0152] Clinical sample of sepsis patients: 20 patients diagnosed with sepsis in the hospital ICU, meeting the Sepsis-3.0 diagnostic criteria, consistent with the inclusion criteria in Example 6.

[0153] 2) Sample processing flow

[0154] Step 1, Blood Collection: All subjects underwent venous blood collection using a 21G lancet. The first 2 mL of blood was discarded to avoid platelet activation in vitro due to puncture. Subsequently, 2 mL of whole blood was collected into each of the five types of blood collection tubes (stabilizer tube, blank control tube, control tube 1, control tube 2, and control tube 3). After blood collection, the tubes were gently inverted and mixed 8 times immediately.

[0155] Step 2, Room temperature placement: All blood collection tubes were placed at room temperature (25℃±2℃), protected from light, and left to stand. Five testing time points were set: testing immediately after blood collection (0 h, baseline value), 2 h, 4 h, 6 h, and 8 h.

[0156] Step 3: Parallel testing: At each time point, each sample was simultaneously tested for PMA using a homogeneous photoluminescence reagent kit and the gold standard method of flow cytometry. Each sample was tested three times, and the average value was taken.

[0157] 3) Detection methods

[0158] ① Homogeneous chemiluminescence immunoassay for PMA detection

[0159] The steps in Example 3 were strictly followed, and the test solution formulation, incubation conditions, luminescent substrate, and instrument parameters were completely consistent.

[0160] ② Flow cytometry detection of PMA

[0161] The procedure was strictly followed in Example 6, with identical parameters for red blood cell lysis, centrifugation and washing, antibody incubation, and instrumental testing. 100,000 cells were counted to obtain the percentage of CD41+CD14+ double-positive cells (PMA percentage).

[0162] 4) Evaluation Indicators

[0163] relative deviation rate

[0164] The calculation formula is: Relative deviation rate (%) = (Detection value after placement - 0 h baseline value) / 0 h baseline value × 100%. The smaller the absolute value of the deviation, the better the stabilization effect.

[0165] Precision

[0166] Calculate the coefficient of variation (CV) (%) for repeated testing of the same sample;

[0167] antibody binding rate

[0168] The binding rate of CD41 and CD14 antibodies to positive cells was detected by flow cytometry to verify the reversibility of the sample stabilizer.

[0169] Matrix interference rate

[0170] The interference rate (%) of the sample stabilizer on the luminescence signal of the homogeneous chemiluminescence detection system is calculated using the following formula: Interference rate (%) = (Detection value of calibrator with sample stabilizer - Detection value of calibrator without sample stabilizer) / Detection value of calibrator without sample stabilizer × 100%.

[0171] 5) Experimental Results

[0172] ① Verification of the reversibility and non-interference of the sample stabilizer

[0173] The results of the sample stabilizer reversibility are shown in Table 14.

[0174] Table 14 Effect of sample stabilizers on antibody-antigen binding rate (n=20, healthy human samples)

[0175]

[0176] Conclusion: The low-concentration reversible fixative in the sample stabilizer of this invention can be completely neutralized by glycine in the stabilizer system under the detection incubation condition of 37°C, and has no significant effect on the binding of CD41 and CD14 antibodies to antigens, with a binding rate deviation of ≤1.2%, which meets the requirements of clinical testing.

[0177] The results of the interference verification are shown in Table 15.

[0178] Table 15. Verification of the interference of sample stabilizers on the homogeneous chemiluminescence detection system.

[0179]

[0180] Conclusion: At the final concentration of the detection system, the sample stabilizer of this invention does not significantly interfere with the chemiluminescence signal and linear range of the homogeneous chemiluminescence detection system, with an absolute interference rate of ≤1.7% and intra-batch CV <5%, and is fully compatible with the homogeneous chemiluminescence detection system.

[0181] ② Validation of room temperature stability of whole blood samples from healthy individuals

[0182] The results of room temperature stability verification of whole blood samples from healthy individuals are shown in Table 16.

[0183] Table 16. Relative deviation rate of PMA detection values ​​of whole blood samples from healthy individuals in different groups after being placed at room temperature for different times (homogeneous chemiluminescence method, n=20, M±SD)

[0184]

[0185] Note: Compared with the blank control tube, P<0.001, and there were extremely significant statistical differences at all time points; the flow cytometry results were completely consistent with the above trend, with a correlation coefficient R=0.951.

[0186] Key results: After 2 hours of storage at room temperature, the average PMA detection value of the ordinary EDTA tube group decreased by 30.2%, while the deviation of the stabilizer blood collection tube group was only -3.2%, with an absolute value of <5%. After 6 hours of storage at room temperature, the absolute value of the deviation of the stabilizer blood collection tube group was <15%, which fully meets the needs of clinical sample transportation and batch testing.

[0187] ③ Validation of room temperature stability of ADP-activated whole blood samples

[0188] The stabilization effect was verified in extreme scenarios where platelets are highly activated and PMA is more easily dissociated. The results are shown in Table 17.

[0189] Table 17 Relative deviation rate of PMA detection values ​​of ADP-activated whole blood samples from different groups after being placed at room temperature for different times (homogeneous chemiluminescence method, n=10, M±SD)

[0190]

[0191] Note: Compared with the blank control tube, P<0.001, and there were highly statistically significant differences at all time points.

[0192] Key results: Even for highly activated platelet samples, the absolute deviation of the stabilizer blood collection tubes of this invention was <5% after 2 hours at room temperature and <15% after 6 hours, which is far superior to single-component (control tubes 2 and 3) and commercially available standard CTAD tubes (control tube 1), solving the core pain point of rapid PMA dissociation in activated samples.

[0193] ④ Validation of room temperature stability of clinical samples from sepsis patients

[0194] The clinical samples used in the core application scenarios of this invention were used to verify the actual application effect, and the results are shown in Table 18.

[0195] Table 18. Relative deviation rate of PMA detection values ​​of whole blood samples from sepsis patients in different groups after being placed at room temperature for different times (homogeneous chemiluminescence method, n=20, M±SD).

[0196]

[0197] Note: Compared with the blank control tube, P<0.001, all time points showed extremely significant statistical differences; Bland-Altman analysis with the 0h baseline showed that 96.7% of the sample differences fell within the 95% agreement limit, indicating good agreement.

[0198] Key results: When sepsis clinical samples were left at room temperature for 2 hours, the absolute deviation of the samples in the stabilizer blood collection tube group of this invention was <5%, and the absolute deviation was <15% after 6 hours. This fully meets the needs of clinical samples for transportation from the ward to the laboratory, emergency batch testing, and external testing, and solves the clinical limitation of "samples needing to be tested immediately".

[0199] ⑤ Experimental Conclusion

[0200] 1. The sample stabilizer of the present invention (PGE1 + modified CTAD + reversible fixation component + protective buffer component) can effectively inhibit the spontaneous activation of platelets in whole blood samples in vitro, while fixing the already formed PMA structure and preventing its dissociation, thus solving the clinical limitation that "samples need to be tested as soon as possible, otherwise the PMA level will change".

[0201] 2. Performance indicators: When placed at room temperature (25℃) for 2 hours, the absolute value of the relative deviation of the sample PMA detection value is <5%; when placed for 4 hours, the deviation is <10%; when placed for 6 hours, the deviation is <15%. The stability time is more than 3 times that of ordinary EDTA tubes, which fully meets the needs of clinical sample transportation, batch testing, and emergency testing.

[0202] 3. Compatibility: The sample stabilizer is fully compatible with the homogeneous chemiluminescence detection system, with no matrix interference. Its low-concentration immobilization components have good reversibility and do not affect antibody-antigen binding. The detection precision and accuracy are not significantly different from those of fresh samples.

[0203] 4. Clinical applicability: It showed stable effects in healthy human samples, platelet-activated samples, and sepsis clinical samples, making it suitable for the core clinical application scenarios of the kit and significantly improving the clinical applicability of the kit.

[0204] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates, characterized in that, The kit includes: R1 reagent: CD41 antibody-DNA1 conjugate; R2 reagent: CD14 antibody-DNA2 conjugate; R3 reagent: DNA3 labeled with acridinium ester; R4 reagent: an antioxidant that binds to graphene oxide; The DNA1 contains a pairing sequence 1 and a pairing sequence 2; wherein the 3' end of the DNA1 is modified with NH2C7; the pairing sequence 1 is GCTGAGTT; the pairing sequence 2 is CAACGAC; the base sequence of the DNA1 is shown in SEQ ID NO.1; The DNA2 has a pairing sequence 3 and a pairing sequence 4; wherein the 5' end of the DNA2 is modified with NH2C6; the pairing sequence 3 is complementary to the pairing sequence 2; the pairing sequence 3 is GTCGTTG; the pairing sequence 4 is GCTGAGAT; the base sequence of the DNA2 is shown in SEQ ID NO.2; The DNA3 contains pairing sequence 5 and pairing sequence 6; wherein the 5' end of the DNA3 is modified with NH2C6 and coupled to acridinium ester via NH2C6; pairing sequence 5 is complementary to pairing sequence 4, and pairing sequence 6 is complementary to pairing sequence 1; pairing sequence 5 is ATCTCAGC; pairing sequence 6 is AACTCAGC; the base sequence of the DNA3 is shown in SEQ ID NO. 3; During detection, the CD41 antibody specifically binds to the antigenic epitope of platelets, and the CD14 antibody specifically binds to the antigenic epitope of monocytes, forming an orthotopic complex: DNA1-CD41 antibody-platelet-monocyte-CD14 antibody-DNA2. The orthotopic complex can hybridize with DNA3. The clone number of the CD41 antibody is SZ22, and the clone number of the CD14 antibody is M5E2.

2. The homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates according to claim 1, characterized in that, The homogeneous chemiluminescence reagent kit also includes R5 reagent: a chemiluminescent substrate; the chemiluminescent substrate is an alkaline solution of hydrogen peroxide; wherein the pH of the alkaline solution is 8.5~9.5, and the concentration of the hydrogen peroxide is 2%v / v~5%v / v.

3. The homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates according to claim 1, characterized in that, The kit also includes: a stabilizer blood collection tube; the stabilizer blood collection tube is prepared by the following steps: adding a sample stabilizer of 2.2% v / v to 2.8% v / v of blood collection volume to a sterile vacuum blood collection tube, drying it with sterile hot air, evacuating it to the appropriate blood collection volume, and sealing it.

4. The homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates according to claim 3, characterized in that, The sample stabilizer comprises a basic anticoagulant, an activation inhibitor, a reversible fixation component, a protective buffer component, and a solvent; wherein the basic anticoagulant is EDTA-K2; the activation inhibitor comprises PGE1 and modified CTAD, the modified CTAD comprising sodium citrate, theophylline, adenosine, and dipyridamole; the reversible fixation component comprises paraformaldehyde and glutaraldehyde; the protective buffer component comprises glycine and BSA; and the solvent is sterile PBS.

5. The homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates according to claim 1, characterized in that, Each of the DNA1 and DNA2 is conjugated to the CD41 antibody / CD14 antibody via a conjugating agent; wherein the conjugating agent is sodium bis(succinimide) octanoate.

6. The homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates according to claim 1, characterized in that, The antioxidant is selected from one or more of vitamin C, vitamin E, tea polyphenols, or glutathione.

7. The detection method of the homogeneous chemiluminescence reagent kit for detecting platelet-monocyte aggregates according to any one of claims 2-6, characterized in that, Includes the following steps: S1. Mix reagents R1, R2, R3, and R4 to obtain the detection solution; S2. Mix the sample to be tested with the detection solution to obtain a mixed solution; then incubate at 35℃~37℃ for 5 min~10 min. S3. Add reagent R5 to the mixed solution, and collect the light signal using a chemiluminescence detector to obtain the chemiluminescence value of the sample to be tested; S4. The instrument automatically retrieves the calibration curve. Substituting the chemiluminescence value into the calibration curve will report the percentage of platelet-monocyte aggregates in the sample to be tested.

8. The method according to claim 7, characterized in that, The test sample is a whole blood sample contained in a stabilizer collection tube; the test sample contains 1.6 mg / mL~2 mg / mL EDTA-K2, 0.9 μmol / L~1.1 μmol / L LPGE1, 9 mmol / L~11 mmol / L sodium citrate, 2 mmol / L~2.5 mmol / L theophylline, 3.2 mmol / L~3.8 mmol / L adenosine, 0.0018 mmol / L~0.0022 mmol / L dipyridamole, 0.45%v / v~0.55%v / v paraformaldehyde, 0.045%v / v~0.055%v / v glutaraldehyde; 45 mmol / L~55 mmol / L glycine and 0.18%m / v~0.22%m / v BSA.

9. The method according to claim 7, characterized in that, In step S1, the final concentration of reagent R1 in the detection solution is 1 nM to 20 nM; the final concentration of reagent R2 is 1 nM to 20 nM; the final concentration of reagent R3 is 0.05 μM to 0.2 μM; and the final concentration of reagent R4 is 20 μg / mL to 25 μg / mL. The concentration ratio of reagent R1, reagent R2, and reagent R3 in the detection solution is 1:1 to 1.5:10 to 20.

10. The method according to claim 7, characterized in that, In step S2, the volume ratio of the sample to be tested in the mixed solution to the detection solution is 1:10~20; in step S3, the volume ratio of the R5 reagent to the detection solution in the mixed solution is 0.8~1:1.

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