Platelet ultrastructure imaging detection sample processing reagent, kit and application

Abstract

The present invention discloses a platelet ultrastructure imaging detection sample processing reagent, comprising a diluent; the ratio of the diluent, blood preservation solution and benchtop solution is 1:7 to 12. The present invention also provides a platelet ultrastructure imaging detection sample processing kit, comprising the sample processing reagent of the present invention, preferably also including a fixative, the fixative comprising 4% to 8% paraformaldehyde by volume, and the solvent comprising a PHEM buffer solution with a pH of 6.8 to 7.2, wherein the platelet microtubule structure fixative preferably comprises 5.5% to 6.6% paraformaldehyde, 0.05% to 0.15% glutaraldehyde and 0.1% to 0.2% TritonX-100 by volume. In addition, the present invention also provides a platelet ultrastructure imaging detection sample processing reagent for use in preparing imaging detection samples, wherein the diluent is first added to the platelet plasma obtained by centrifugation, and the platelets are then fixed after being allowed to stand for recovery after dilution, thereby obtaining unactivated platelets, so that the test results are closer to the true condition of platelets under physiological conditions.

Description

Technical Field

[0001] The present invention belongs to the technical field of platelet structure detection, and more specifically, relates to a platelet ultrastructure imaging detection sample processing reagent, a kit and applications. Background Art

[0002] Super-resolution fluorescence microscopy technology has broken through the optical diffraction limit and achieved nanoscale optical detection within cells, providing a new means for the detection of intracellular subcellular structures (such as microfilaments, microtubules, mitochondria, etc.). However, the application of super-resolution fluorescence microscopy technology in the field of platelet detection is very rare.

[0003] Compared to ordinary cells, platelets are extremely sensitive and easily activated. Activation alters the subcellular structure of platelets, making the platelets studied difficult to represent the true state of circulating platelets under physiological conditions. Therefore, imaging of platelet ultrastructure must be performed without activating the platelets, placing high demands on experimental procedures that are often difficult to achieve with existing detection methods.

[0004] Currently, super-resolution fluorescence imaging of platelet ultrastructure is only performed on fixed dead cells. However, before fixing the platelets, they need to be separated and purified from whole blood. This process can easily lead to platelet activation for unknown reasons, and no solutions to this problem have been reported. Summary of the Invention

[0005] In response to the above-mentioned defects or improvement needs of the prior art, the present invention provides a platelet ultrastructure imaging detection sample processing reagent, kit and application, the purpose of which is to significantly reduce the concentration of anticoagulant in the plasma after centrifugation by adding a diluent prepared by adding blood preservation fluid and benchtop fluid to dilute the plasma sample after centrifugation, thereby obtaining unactivated platelets, thereby solving the technical problem that the platelets obtained by existing separation and purification are usually activated platelets and are difficult to represent the true state of circulating platelets under physiological conditions.

[0006] To achieve the above objectives, according to one aspect of the present invention, a platelet ultrastructure imaging detection sample processing reagent is provided, wherein the sample processing reagent includes a diluent; the diluent includes a blood preservation solution and a benchtop solution, wherein the volume ratio of the blood preservation solution to the benchtop solution is 1:7 to 12.

[0007] Preferably, in the platelet ultrastructure imaging detection sample processing reagent, the diluent has a ratio of blood preservation solution to benchtop solution of 1:9.

[0008] Preferably, the platelet ultrastructure imaging detection sample processing reagent, the benchtop liquid includes 7.5-8.5g / L sodium chloride, 0.18-0.25g / L potassium chloride, 0.18-0.25g / L magnesium chloride hexahydrate, 0.58-0.72g / L sodium dihydrogen phosphate dihydrate, 0.90-1.15g / L sodium bicarbonate, 0.85-1.05g / L glucose, and 2.15-2.50g / L 4-hydroxyethylpiperazineethanesulfonic acid, the solvent is water, and the pH is 7.0-7.5.

[0009] According to another aspect of the present invention, there is also provided a use of the reagent according to the present invention in preparing a sample for platelet ultrastructure imaging detection.

[0010] Preferably, the platelet ultrastructure imaging detection sample processing reagent is used in the preparation of imaging detection samples, which uses the platelet ultrastructure imaging detection processing reagent to first dilute the platelet sample mixed with the anticoagulant after centrifugation, and then fix and label it to obtain the sample to be imaged and detected; the dilution is performed according to the volume ratio of the centrifuged platelet sample to the diluent of 1:4 to 10 to protect the platelets, and the anticoagulant includes an EDTA salt type anticoagulant.

[0011] Preferably, the use of the platelet ultrastructure imaging detection sample processing reagent in preparing an imaging detection sample comprises the following steps:

[0012] (1) Whole blood centrifugation to obtain platelet-rich plasma: Whole blood is collected by blood collection tube and centrifuged to obtain platelet-rich plasma, wherein the platelet-rich plasma is mixed with an EDTA salt type anticoagulant;

[0013] (2) Platelet pre-protection treatment: dilute the platelet plasma obtained in step (1) with the diluent at a volume ratio of 1:4 to 10, and allow to stand for recovery;

[0014] (3) Platelet fixation: adding a fixative to the platelets after standing and recovering in step (2), fixing them, removing the supernatant by centrifugation to retain the platelet precipitate, resuspending the platelet precipitate in phosphate buffer, and centrifuging to remove the supernatant. Resuspending the fixed platelets in phosphate buffer to obtain a platelet suspension;

[0015] (4) Preparation of test samples: Based on the platelet count of the sample, the platelet suspension in step (3) is taken, diluted and spread onto a culture dish, and allowed to settle for more than 1 hour. The platelets are then immunofluorescently stained to obtain the sample to be imaged.

[0016] Preferably, the use of the platelet ultrastructure imaging detection sample processing reagent in the preparation of imaging detection samples, wherein the fixative in step (3), wherein the fixative for platelet microtubule structure, comprises 5.5% to 6.6% paraformaldehyde, 0.05% to 0.15% glutaraldehyde and 0.1% to 0.2% Triton X-100 by volume, and the solvent comprises PHEM buffer with a pH of 6.8 to 7.2.

[0017] According to another aspect of the present invention, a kit for processing platelet ultrastructure imaging detection samples is provided, wherein the kit comprises the platelet ultrastructure imaging detection sample processing reagent according to the present invention.

[0018] Preferably, the kit for processing sample for platelet ultrastructure imaging detection further comprises a fixative and a labeling reagent; the fixative comprises 4% to 8% paraformaldehyde by volume, and the solvent comprises a PHEM buffer with a pH of 6.8 to 7.2; the labeling reagent comprises a primary antibody dilution solution and a fluorescent secondary antibody dilution solution for labeling one or more combinations of platelet microtubules, dense granules, mitochondria, and α-granules.

[0019] Preferably, the kit for processing sample for platelet ultrastructure imaging detection, wherein the fixative for platelet microtubule structure comprises 5.5% to 6.6% paraformaldehyde, 0.05% to 0.15% glutaraldehyde and 0.1% to 0.2% Triton X-100 in volume percentage.

[0020] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0021] The platelet ultrastructure imaging detection sample processing reagent provided by the present invention includes a diluent, wherein the diluent includes a blood preservation solution and a benchtop solution. The use of the two in a ratio can reduce the probability of platelets separated by centrifugation being activated during the static recovery process. Preferably, the volume ratio of the blood preservation solution to the benchtop solution is 1:7 to 12. The diluent at this ratio can well protect the platelets from activation, making the detection results of the platelet ultrastructure closer to the in vivo situation. It can be seen that the platelet ultrastructure imaging detection sample processing reagent provided by the present invention can obtain non-activated platelet test samples.

[0022] The present invention also provides an application of a platelet ultrastructure imaging detection sample processing reagent in the preparation of imaging detection samples. The platelet ultrastructure imaging detection sample processing reagent described in the present invention is used to improve the existing method. In this method, after centrifugation to obtain platelet plasma, a diluent is first added to dilute it and then allowed to stand for recovery. After standing for recovery, the platelets are fixed, and the platelet ultrastructure is detected after immunofluorescence staining; after centrifugation, the diluent described in the present invention is first added to dilute it and then the platelets are fixed, which can significantly reduce the number of activated platelets; and this method can perform detection when the platelets are not activated, and the detection results obtained are closer to the actual state of circulating platelets under physiological conditions, which is of great significance for drug or disease research.

[0023] In addition, the platelet ultrastructure imaging detection sample processing kit provided by the present invention includes the sample processing reagent of the present invention, and preferably also includes a fixative, wherein the fixative for the platelet microtubule structure includes paraformaldehyde, glutaraldehyde (GA), and Triton X-100. The three are used in combination to reduce the change of the fixative reagent on the platelet microtubule structure, especially including a microtubule fixative with a volume percentage of 5.5% to 6.6% paraformaldehyde (PFA), 0.05% to 0.15% glutaraldehyde (GA), and 0.1% to 0.2% Triton X-100. After the platelets are fixed, their microtubule structure is almost unchanged. The kit provided by the present invention has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a photograph of platelet microtubules;

[0025] Figure 2 This is a photograph of dense platelet granules;

[0026] Figure 3 This is a picture of platelet mitochondria;

[0027] Figure 4 This is a photograph of platelet alpha granules;

[0028] Figure 5 This is a comparison of microtubule structures taken after samples were prepared using three sample treatment reagents (with different dilutions);

[0029] Figure 6 This is a comparison of microtubule structures taken after samples were prepared using three sample treatment reagents (with different fixatives). DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0031] Blood samples from regular hospital blood collection tubes (purple-top tubes) were collected using centrifugal separation methods. Subsequent super-resolution imaging revealed many changes in the microtubule structure of platelets. However, such changes rarely occurred in platelets separated using gel columns. Analysis suggests that many platelets obtained using centrifugal separation were activated, while platelets obtained using gel columns were rarely activated. Comparing the two methods, further analysis revealed that plasma samples mixed with EDTA salt-type anticoagulants require a long period of static recovery after centrifugation. During this static recovery process, the anticoagulant concentration in the purple-top blood collection tubes is high and coexists with the platelets for a long time. Therefore, it is speculated that the activation of platelets may be caused by the long-term coexistence of the high-concentration anticoagulant with the platelets.

[0032] Compared with the centrifugal separation method, the number of platelets obtained by the gel separation method is too small, which is not convenient for the collection and statistics of subsequent imaging detection data. Therefore, when performing large-scale super-resolution imaging, detection and statistics, the centrifugal separation method is better. However, when centrifugal separation is used to process samples, the platelets to be tested are activated, resulting in a large difference between the test results and the physiological state of circulating platelets under physiological conditions. After further analysis of our experiments, we found that the activation of platelets is caused by high concentrations of anticoagulants.

[0033] Therefore, the present invention proposes adding a diluent to the platelet plasma obtained after centrifugation, and then performing static recovery and super-resolution imaging detection; experimental results have confirmed that the phenomenon of platelet activation is significantly reduced when the test sample obtained by this method is tested.

[0034] The present invention provides a platelet ultrastructure imaging detection sample processing reagent, which includes a diluent; the diluent includes a blood preservation solution and a benchtop solution, preferably the volume ratio of the blood preservation solution to the benchtop solution is 1:7-12. The diluent with this ratio can better protect platelets from activation; more preferably the diluent has a blood preservation solution to benchtop solution volume ratio of 1:9;

[0035] The benchtop solution comprises 7.5-8.5 g / L sodium chloride (NaCl), 0.18-0.25 g / L potassium chloride (KCl), 0.18-0.25 g / L magnesium chloride hexahydrate (MgCl2·6H2O), 0.58-0.72 g / L sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), 0.90-1.15 g / L sodium bicarbonate (NaHCO3), 0.85-1.05 g / L glucose, and 2.15-2.50 g / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES). The solvent is water, and the pH is 7.0-7.5.

[0036] The blood preservation solution refers to citric acid-sodium citrate-dextrose (abbreviated as ACD solution), with a pH value between 4.5 and 5.5, such as blood preservation solution (I), blood preservation solution II, and blood preservation solution III.

[0037] In addition, the present invention also provides a use of the platelet ultrastructure imaging detection sample processing reagent according to the present invention in preparing platelet ultrastructure imaging detection samples.

[0038] The application comprises the following steps:

[0039] (1) Whole blood centrifugation to obtain platelet-rich plasma: Whole blood is collected using a blood collection tube (purple-top tube) and centrifuged to obtain platelet-rich plasma, in which the platelet-rich plasma is mixed with a high concentration of EDTA salt type anticoagulant, such as EDTA-K2 / EDTA-K3 anticoagulant;

[0040] (2) Platelet pre-protection treatment: diluting the platelet plasma obtained in step (1) with a diluent according to a preset ratio and allowing to stand for recovery; the preset ratio is preferably platelet plasma: diluent = 1:4-10; the platelet plasma obtained after whole blood centrifugation is first added with a diluent, diluted, and then allowed to stand for recovery, which is beneficial for protecting platelets from being activated by high concentrations of anticoagulants;

[0041] The diluents include blood preservation solution and benchtop solution. Experimental results show that using 100% blood preservation solution (ACD) or benchtop solution alone as the diluent does not significantly reduce platelet activation. This may be because the blood preservation solution contains a sterile aqueous solution of sodium citrate, citric acid, and glucose, with a pH of 4.5 to 5.5. The acidic diluent easily causes platelet activation. Although benchtop solution has the functions of maintaining osmotic pressure, controlling acid-base balance, and supplying energy and inorganic salts necessary for cell metabolism and survival, when used alone as a diluent, platelets are activated and their microtubule structure shows significant diffusion. However, when both blood preservation solution and benchtop solution are used together, the diffusion of platelet microtubule structure is extremely small, indicating that their protective effect on platelets is significantly better than using either blood preservation solution or benchtop solution alone. The preferred volume ratio of blood preservation solution to benchtop solution is 1:7 to 12. The diluent with this ratio can better protect platelets from activation, providing the most realistic state for subsequent research on platelet ultrastructure.

[0042] (3) Platelet fixation: adding a fixative of the same volume as the diluent in step (2) to the platelets obtained in step (2) after being allowed to recover, fixing the platelets, and centrifuging to remove the supernatant and retain the platelet precipitate to obtain fixed platelets; resuspending the precipitate with phosphate buffered saline (PBS) and centrifuging to remove the supernatant, and resuspending the fixed platelets with PBS solution to obtain a platelet suspension.

[0043] The fixative for platelet microtubule structure is a combination of paraformaldehyde (PFA) with glutaraldehyde (GA) and Triton X-100. This is because the platelet microtubule structure is a very sensitive and easily changeable structure. The structure imaged after fixation with paraformaldehyde (PFA) alone is prone to change. However, the combination of PFA with glutaraldehyde (GA) and Triton X-100 has less effect on the change of the microtubule structure. Preferably, the combination includes 5.5% to 6.6% paraformaldehyde (PFA), 0.05% to 0.15% glutaraldehyde (GA), and 0.1% to 0.2% Triton X-100 by volume. Under this ratio, the imaged microtubule structure after fixation is clearer and the microtubule structure is almost unaffected. Fixation with other commonly used fixatives can cause significant blurring of the microtubule structure.

[0044] (4) Preparation of test samples: Based on the platelet count of the sample, the platelet suspension in step (3) is taken and diluted. The diluted platelet solution is spread on a glass-bottomed culture dish and allowed to settle for more than 1 hour. The platelets are immunofluorescently stained to obtain the test sample for immunofluorescence staining.

[0045] When the above-mentioned test samples are used for platelet ultrastructure detection, super-resolution fluorescence imaging is used to detect the platelet ultrastructure; and information on changes in the platelet ultrastructure can be obtained by analyzing and statistically analyzing changes in the platelet ultrastructure morphology.

[0046] The present invention also provides a platelet ultrastructure imaging detection sample processing kit, which comprises the platelet ultrastructure imaging detection sample processing reagent of the present invention, and preferably also includes a fixative;

[0047] The fixative comprises 4% to 8% paraformaldehyde (PFA) by volume, and the solvent comprises PHEM buffer, wherein the pH of the PHEM buffer is 6.8 to 7.2; wherein the fixative for platelet microtubule structure further comprises glutaraldehyde (GA) and Triton X-100, preferably comprising 5.5% to 6.6% paraformaldehyde (PFA), 0.05% to 0.15% glutaraldehyde (GA) and 0.1% to 0.2% Triton X-100 by volume. Under these conditions, the imaging structure of the microtubules after fixation is clearer, while fixation with other commonly used fixatives will cause the microtubule structure to be significantly diffused; the fixative for the platelet microtubule structure more preferably comprises 6% paraformaldehyde (PFA), 0.1% glutaraldehyde (GA) and 0.6% Triton X-100 by volume, and the solvent comprises PHEM buffer solution.

[0048] The PHEM buffer preferably includes 17.5-18.5 g / L 1,4-piperazinediethanesulfonic acid (PIPES), 4.8-7.2 g / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 2.2-3.6 g / L ethylene glycol-bis-(2-aminoethyl)tetraacetic acid (EGTA), 0.35-0.55 g / L magnesium chloride hexahydrate (MgCl2.6H2O), the solvent is water, and the pH is 6.8-7.2.

[0049] The kit more preferably further comprises a labeling reagent, which comprises a cell perforation solution, a blocking solution, a washing solution, a primary antibody dilution solution for labeling one or more combinations of platelet microtubules, dense granules, mitochondria, and α-granules, and a fluorescent secondary antibody dilution solution.

[0050] The following are examples:

[0051] Example 1 Platelet ultrastructure (microtubule structure) sample processing reagent 1

[0052] The platelet ultrastructure imaging detection sample processing reagent 1 includes a diluent, a fixative and a labeling reagent; wherein the diluent comprises 10% blood preservation solution (I) and 90% benchtop solution; the benchtop solution comprises 8.064g / L sodium chloride (NaCl), 0.216g / L potassium chloride (KCl), 0.203g / L magnesium chloride hexahydrate (MgCl2·6H2O), 0.6552g / L sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), 1.0092g / L sodium bicarbonate (NaHCO3), 0.9908g / L glucose, 2.384g / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), the solvent is water, and the pH is 7.2;

[0053] The fixing solution includes 6% paraformaldehyde (PFA), 0.1% glutaraldehyde (GA) and 0.15% Triton X-100 by volume, and the solvent is a PHEM buffer solution; the PHEM buffer solution includes 18.147g / L 1,4-piperazinediethanesulfonic acid (PIPES), 5.956g / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 2.922g / L ethylene glycol-bis-(2-aminoethyl)tetraacetic acid (EGTA), and 0.406g / L magnesium chloride hexahydrate (MgCl2·6H2O), the solvent is water, and the pH is 7.0;

[0054] The labeling reagent includes a cell perforation solution, a blocking solution, a cleaning solution, a primary antibody dilution solution for labeling platelet microtubules, and a fluorescent secondary antibody dilution solution; wherein the cell perforation solution is a PBS solution containing 0.2% Triton X-100, the blocking solution is a PBS solution containing 10% goat serum (NAS), 1% bovine serum albumin (BSA), 0.05% Triton X-100, and 0.05% antibacterial preservative Proclean 300, the cleaning solution is a PBS solution containing 0.1% Tween 20, the platelet microtubule primary and secondary antibody dilution solutions are mouse α-tubulin monoclonal antibody (primary antibody) diluted 1:500 in the blocking solution; and anti-mouse Alexa Fluor 488 antibody (secondary antibody) is diluted 1:500 in the blocking solution.

[0055] Example 2 Platelet Ultrastructure (Dense Granules) Sample Processing Reagent 2

[0056] The platelet ultrastructure imaging detection sample processing reagent 2 includes a diluent, a fixative, and a labeling reagent; wherein the diluent comprises a blood preservation solution and a benchtop solution in a volume ratio of 1:9; the fixative comprises 8% paraformaldehyde (PFA) by volume, and the solvent is PHEM buffer (same as in Example 1);

[0057] The labeling reagent includes a cell punching solution, a blocking solution, and a washing solution (same as in Example 1), as well as a primary antibody dilution solution for labeling dense particles and a fluorescent secondary antibody dilution solution;

[0058] The primary and secondary antibody dilution solutions for dense granules were mouse CD63 monoclonal antibody (primary antibody) diluted 1:100 in blocking buffer, and anti-mouse Alexa Fluor 488 antibody (secondary antibody) diluted 1:500 in blocking buffer.

[0059] Example 3 Platelet Ultrastructure (Mitochondria) Sample Processing Reagent 3

[0060] The platelet ultrastructure imaging detection sample processing reagent 3 includes a diluent, a fixative and a labeling reagent; wherein the diluent comprises a blood preservation solution and a benchtop solution in a volume ratio of 1:7; the fixative comprises 8% paraformaldehyde (PFA) by volume, and the solvent is PHEM buffer (same as in Example 1);

[0061] The labeling reagents include cell perforation solution, blocking solution and washing solution (same as in Example 1), as well as a dilution solution of a primary antibody for labeling mitochondria and a dilution solution of a fluorescent secondary antibody;

[0062] The mitochondrial primary and secondary antibody dilution solutions were rabbit TOMM20 monoclonal antibody (primary antibody) diluted 1:250 in blocking buffer, and anti-rabbit Alexa Fluor 488 antibody (secondary antibody) diluted 1:500 in blocking buffer.

[0063] Example 4 Platelet Ultrastructure (α Granules) Sample Processing Reagent 4

[0064] The platelet ultrastructure imaging detection sample processing reagent 4 includes a diluent, a fixative and a labeling reagent; wherein the diluent comprises a blood preservation solution and a benchtop solution at a volume ratio of 1:12; the fixative comprises 8% paraformaldehyde (PFA) by volume, and the solvent is PHEM buffer (same as in Example 1);

[0065] The labeling reagents include cell perforation solution, blocking solution and washing solution (same as in Example 1), as well as a primary antibody dilution solution for labeling α particles and a fluorescent secondary antibody dilution solution;

[0066] The α-granule antibody primary and secondary antibody dilution solutions were rabbit VWF monoclonal antibody (primary antibody) diluted 1:1000 in blocking solution, and anti-rabbit Alexa Fluor 488 antibody (secondary antibody) diluted 1:500 in blocking solution.

[0067] Example 5 Platelet Ultrastructure Detection

[0068] After preparing test samples using the sample processing reagents in Examples 1-4 according to the treatment methods provided by the present invention, detection was performed using super-resolution fluorescence imaging technology, specifically comprising the following steps:

[0069] (1) Whole blood separation to obtain platelets: Whole blood obtained from a blood collection tube is centrifuged at a centrifugal force of 200 g for 12 minutes to obtain platelet-rich plasma.

[0070] (2) Platelet pre-protection treatment: Platelet-rich plasma was diluted with diluent at a ratio of 1:4 to 10, and then placed in a 37°C, 5% CO2 incubator for two hours to recover.

[0071] (3) Platelet fixation: Add an equal volume of fixative to the platelet suspension after recovery. After 30 minutes of fixation, centrifuge at 1500g for 3 minutes. Remove the supernatant and retain the platelet pellet. Resuspend the pellet with 1 mL of PBS solution and centrifuge again at 1500g for 3 minutes. Remove the supernatant and resuspend the fixed platelets with 5 mL of PBS solution.

[0072] (4) Platelet plating: Dilute an appropriate amount of platelet suspension according to the platelet count of the sample, spread 200 μL of the diluted platelet solution onto a glass-bottomed culture dish, and let it settle for at least 1 hour.

[0073] (5) Platelet immunofluorescence staining: Platelet punching: Add 200uL of cell punching solution to the dish and let it stand for 10 minutes, then discard the liquid. Platelet blocking: Add 200uL of blocking solution to the dish and let it stand for 1 hour, then discard the liquid. Primary antibody incubation: Add 150uL of primary antibody dilution solution to the dish, incubate the antibody and platelets at 4°C for 12h to 16h, discard the liquid after the incubation, and wash with cleaning solution 5 times, 5 minutes each time. Secondary antibody incubation: Add 150uL of secondary antibody dilution solution to the dish, incubate the antibody and platelets at room temperature for 1 hour, discard the liquid after the incubation, and wash with cleaning solution 5 times, 5 minutes each time. Post-treatment: Add 200uL of 4% paraformaldehyde (PFA) to the dish and fix for 10 minutes. After fixation, discard the liquid, wash 3 times with PBS, and store the dish at 4°C.

[0074] The samples prepared above were subjected to super-resolution fluorescence imaging detection, and the results are shown in Figures 1 to 4 ,in Figure 1 This is the result of photographing platelet microtubules. Figure 2 This is the result of photographing dense platelet granules. Figure 3 This is the result of photographing platelet mitochondria. Figure 4 This is the photographic result of platelet α granules.

[0075] Comparative Example 1 Platelet Ultrastructure (Microtubule Structure) Sample Processing Reagent 5

[0076] The platelet ultrastructure imaging detection sample processing reagent includes a diluent, a fixative, and a labeling reagent; the diluent is 100% blood preservation solution (ACD), and the fixative is the same as that in Example 1.

[0077] Comparative Example 2 Platelet Ultrastructure (Microtubule Structure) Sample Processing Reagent 6

[0078] The platelet ultrastructure imaging detection sample processing reagent includes a diluent, a fixative, and a labeling reagent; the diluent is a 100% benchtop solution, and the fixative is the same as that in Example 1.

[0079] Since the changes in microtubule structure are most obvious in platelets, the test results are more representative. The following samples were processed and tested to detect microtubule structure, as follows:

[0080] The sample treatment reagents in Example 1, Comparative Example 1 and Comparative Example 2 were used to prepare the test sample (same as Example 5) according to the treatment method provided by the present invention, and the super-resolution fluorescence imaging technology was used for detection. The results are shown in FIG. Figure 5 .

[0081] Comparative Example 3 Platelet Ultrastructure (Microtubule Structure) Sample Processing Reagent 7

[0082] The platelet ultrastructure imaging detection sample processing reagent 7 includes a diluent, a fixative, and a labeling reagent; wherein the diluent comprises a blood preservation solution and a benchtop solution in a ratio of 1:9; the fixative is 8% paraformaldehyde (PFA), and the solvent is PHEM buffer (same as in Example 1).

[0083] Comparative Example 4 Platelet Ultrastructure (Microtubule Structure) Sample Processing Reagent 8

[0084] The platelet ultrastructure imaging detection sample processing reagent 7 includes a diluent, a fixative, and a labeling reagent; wherein the diluent comprises a blood preservation solution and a benchtop solution in a ratio of 1:9; the fixative is 4% glutaraldehyde (GA), and the solvent is PHEM buffer (same as in Example 1).

[0085] The sample treatment reagents in Example 1, Comparative Example 3 and Comparative Example 4 were used to prepare the test sample (same as Example 5) according to the treatment method provided by the present invention. The live cells were used as the control and the super-resolution fluorescence imaging technology was used for detection. The results are shown in FIG. Figure 6 .

[0086] Depend on Figure 6It can be seen that when using 8% or 4% glutaraldehyde as the fixative, the platelet microtubule structure showed a diffusion phenomenon, among which the diffusion phenomenon was more obvious after treatment with the reagent of Comparative Example 4. However, when the sample was treated with the sample treatment reagent of the present invention, there was no obvious difference in the platelet microtubule structure compared with that in living cells, indicating that the platelets were hardly activated when the sample was treated with the sample treatment reagent provided by the present invention.

[0087] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A platelet ultrastructure imaging detection sample processing reagent, characterized in that: The sample processing reagent includes a diluent and a fixative for inhibiting platelet activation; the diluent includes a blood preservation solution and a benchtop solution, and the pH value of the blood preservation solution is 4.5-5.5; When the platelet ultrastructure is microtubules, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:9; the fixative comprises 5.5% to 6.6% paraformaldehyde, 0.05% to 0.15% glutaraldehyde, and 0.1% to 0.2% Triton X-100 by volume, and the solvent comprises PHEM buffer with a pH of 6.8 to 7.2; When the platelet ultrastructure is dense granules, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:9; the fixative comprises 4% to 8% paraformaldehyde by volume, and the solvent comprises a PHEM buffer solution with a pH of 6.8 to 7.2; When the platelet ultrastructure is mitochondria, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:7; the fixative comprises 4% to 8% paraformaldehyde by volume, and the solvent comprises a PHEM buffer solution with a pH of 6.8 to 7.2; When the platelet ultrastructure is α particles, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:12; the fixative includes 4% to 8% paraformaldehyde by volume, and the solvent includes a PHEM buffer solution with a pH of 6.8 to 7.

2.

2. The platelet ultrastructure imaging detection sample processing reagent according to claim 1, characterized in that: The benchtop solution includes 7.5-8.5 g / L sodium chloride, 0.18-0.25 g / L potassium chloride, 0.18-0.25 g / L magnesium chloride hexahydrate, 0.58-0.72 g / L sodium dihydrogen phosphate dihydrate, 0.90-1.15 g / L sodium bicarbonate, 0.85-1.05 g / L glucose, and 2.15-2.50 g / L 4-hydroxyethylpiperazineethanesulfonic acid. The solvent is water, and the pH is 7.0-7.

5.

3. A use of the platelet ultrastructure imaging detection sample processing reagent according to any one of claims 1 or 2 in preparing platelet ultrastructure imaging detection samples, characterized in that: The platelet sample mixed with anticoagulant obtained by centrifugation is first diluted with a diluent and then fixed with a fixative.

4. The use according to claim 3, characterized in that The platelet ultrastructure imaging detection sample processing reagent is used to dilute the platelet sample mixed with the anticoagulant after centrifugation with a diluent, and then fix and label it with a fixative after standing to recover, to obtain a sample to be imaged and detected; the dilution is performed according to a volume ratio of 1:4 to 10 between the centrifuged platelet sample and the diluent to protect the platelets, and the anticoagulant includes an EDTA salt type anticoagulant; When the platelet ultrastructure is microtubules, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:9; the fixative comprises 5.5% to 6.6% paraformaldehyde, 0.05% to 0.15% glutaraldehyde, and 0.1% to 0.2% Triton X-100 by volume, and the solvent comprises PHEM buffer with a pH of 6.8 to 7.2; When the platelet ultrastructure is dense granules, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:9; the fixative comprises 4% to 8% paraformaldehyde by volume, and the solvent comprises a PHEM buffer solution with a pH of 6.8 to 7.2; When the platelet ultrastructure is mitochondria, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:7; the fixative comprises 4% to 8% paraformaldehyde by volume, and the solvent comprises a PHEM buffer solution with a pH of 6.8 to 7.2; When the platelet ultrastructure is α particles, the volume ratio of the blood preservation solution to the benchtop solution in the diluent is 1:12; the fixative includes 4% to 8% paraformaldehyde by volume, and the solvent includes a PHEM buffer solution with a pH of 6.8 to 7.

2.

5. A kit for platelet ultrastructure imaging detection sample processing, characterized in that: The kit comprises the platelet ultrastructure imaging detection sample processing reagent according to any one of claims 1 or 2.

6. The platelet ultrastructure imaging detection sample processing kit according to claim 5, characterized in that: The kit further comprises a labeling reagent; the labeling reagent comprises a primary antibody dilution solution and a fluorescent secondary antibody dilution solution for labeling one or more combinations of platelet microtubules, dense granules, mitochondria, and α granules.

Images