A leukocyte extract combination and its application

Abstract

The present invention relates to a leukocyte extract solution combination and its application, wherein the leukocyte extract solution combination comprises a fixing working solution, a stopping working solution and a lysis working solution; the fixing working solution comprises formaldehyde and PBS; the stopping working solution comprises Tris and PBS; the lysis working solution comprises TritonX100 and PBS. The leukocyte extract solution combination of the present invention can prolong the storage time of blood, and the extracted leukocytes can be used for proteomics detection, and the information is preserved completely.

Description

Technical Field

[0001] The invention belongs to the technical field of cell extraction and relates to a leukocyte extract combination and application thereof. Background Art

[0002] White blood cells are a type of colorless, spherical, nucleated blood cells. As immune cells of the human body, changes in the biological and physical properties of their components are closely related to the occurrence, development and prognosis of diseases. However, white blood cells not only account for a small volume of blood cells, but also account for an extremely low number, about one thousandth of red blood cells, and are low-abundance components. Therefore, the analysis of white blood cells and their subpopulations requires the removal of most of the red blood cells in the whole blood to extract the white blood cell components.

[0003] The time from clinical blood collection to blood analysis is uncontrollable. If the blood is transported or stored for too long, the stability of the blood components may be affected, resulting in the in vitro analysis results not accurately reflecting the true state of the blood in the body. Therefore, there is an urgent need for a leukocyte extraction solution that is simple to operate and can extend the blood storage time. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a leukocyte extract combination and application thereof.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a leukocyte extract solution combination, the leukocyte extract solution combination comprises a fixation working solution, a stop working solution and a lysis working solution;

[0007] The fixing working solution includes formaldehyde and PBS;

[0008] The stop working solution includes Tris and PBS;

[0009] The lysis working solution includes TritonX100 and PBS.

[0010] The present invention provides a leukocyte extraction solution combination, which includes a fixing working solution for storage, a suspending working solution for suspending fixation, and a lysis working solution for extraction, and proposes a method for leukocyte extraction, which solves the problem that the blood is out of the body for too long and the blood leukocyte protein and protein phosphorylation signals change. The extraction solution combination of the present application is used in combination, and the proportion of extracted leukocytes is high, and it can be used to extract leukocytes from blood that has been stored for a long time (within 2 weeks), which preserves the information of the blood to the greatest extent, and the obtained leukocytes have good results for omics detection.

[0011] Preferably, the fixing working solution comprises formaldehyde 0.25-2% and PBS 98-99.75% in terms of mass percentage.

[0012] The mass percentage of the formaldehyde can be selected from 0.25%, 0.4%, 0.5%, 0.75%, 0.8%, 1%, 1.25%, 1.5%, 1.6%, 1.75%, 2% and the like; the mass percentage of PBS can be selected from 98%, 98.25%, 98.5%, 98.75%, 99%, 99.25%, 99.5%, 99.75% and the like; other specific point values ​​within the above numerical range can be selected, which will not be described one by one here.

[0013] Preferably, the stop working solution comprises Tris 1.5-24.2% and PBS 75.8-98.5% in terms of mass percentage.

[0014] The mass percentage of Tris can be selected from 1.5%, 3%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 24%, 24.2%, etc., and the mass percentage of PBS can be selected from 75.8%, 77%, 79%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98%, 98.5%, etc. Other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0015] Preferably, the lysis working solution comprises TritonX1000 0.05-0.1% and PBS 99.9-99.95% in terms of mass percentage.

[0016] The mass percentage of TritonX100 can be selected from 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1% and the like; the mass percentage of PBS can be selected from 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95% and the like. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0017] Preferably, the pH value of the suspension working solution is 7.35-7.45, for example, 7.35, 7.36, 7.37, 7.38, 7.39, 7.4, 7.41, 7.42, 7.43, 7.44, 7.45, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described one by one here.

[0018] The pH of the working solution was adjusted using HCl.

[0019] In a second aspect, the present invention provides a use of the leukocyte extract combination according to the first aspect in leukocyte extraction.

[0020] In a third aspect, the present invention provides a method for extracting leukocytes, the method comprising:

[0021] (1) mixing blood with a fixing working solution and allowing the mixture to stand to obtain a first mixed solution;

[0022] (2) mixing the first mixed solution with the suspended working solution to obtain a second mixed solution;

[0023] (3) centrifuging the second mixed solution, mixing the precipitate with the lysis working solution, lysing, and centrifuging after lysis;

[0024] (4) Mix the precipitate obtained in step (3) with PBS solution, centrifuge, and resuspend the precipitate.

[0025] The blood needs to be fully mixed with the anticoagulant in the blood collection tube after leaving the body, the blood and the fixative need to be mixed within 30 minutes after the blood is removed from the body, the stop solution and the first mixed solution need to be mixed within 10 minutes after the blood and the fixative are allowed to stand, and the second mixed solution needs to be centrifuged and mixed with the lysis working solution within 48 hours after the blood and the fixative are mixed.

[0026] Preferably, the volume ratio of blood to fixative working solution is 1:(2-5), wherein the specific point values ​​in (2-5) can be selected from 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, etc. Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0027] Preferably, the volume ratio of the first mixed liquid to the suspension working liquid is 1:(0.8-1), wherein the specific point values ​​in (0.8-1) can be selected from 0.8, 0.82, 0.84, 0.86, 0.88, 0.9, 0.92, 0.94, 0.96, 0.98, 1, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0028] Preferably, the standing time in step (1) is 10-15 min, for example, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc. Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0029] Preferably, the mixing time in step (2) is 10 min-14 days, for example, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here one by one.

[0030] Preferably, the centrifugal speed in step (3) is independently 500-600 rpm, and the time is 8-15 min.

[0031] The rotation speed can be selected as 500rpm, 510rpm, 520rpm, 530rpm, 540rpm, 550rpm, 560rpm, 570rpm, 580rpm, 590rpm, 600rpm, etc., and the time can be selected as 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, 12.5min, 13min, 13.5min, 14min, 14.5min, 15min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0032] Preferably, the centrifugation speed in step (4) is 200-300 rpm and the time is 10-15 min.

[0033] The rotation speed can be selected as 200rpm, 210rpm, 220rpm, 230rpm, 240rpm, 250rpm, 260rpm, 270rpm, 280rpm, 290rpm, 300rpm, etc., and the time can be selected as 10min, 11min, 12min, 13min, 14min, 15min, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described here one by one.

[0034] Preferably, the volume of blood to lysis working solution is 1:(8-10), wherein the specific point values ​​in (8-10) can be selected from 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8, 10, etc. Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0035] In a fourth aspect, the present invention provides white blood cells extracted according to the white blood cell extraction method described in the third aspect.

[0036] In a fifth aspect, the present invention provides a use of the leukocytes according to the fourth aspect in proteomics analysis.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides a leukocyte extracting liquid combination and proposes a leukocyte extracting method, which solves the problem that the leukocyte protein and protein phosphorylation signal in the blood change when the blood is out of the body for too long. The extracting liquid combination of the present application is used in combination, and the extracted leukocytes have a high proportion, and can be used to extract leukocytes from blood that has been stored for a long time (within 2 weeks), preserving the information of the blood to the greatest extent, and the obtained leukocytes have good results for omics detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the result of mixing with the stop solution in Example 3 and leaving it for 48 hours.

[0040] Figure 2 This is a graph showing the result of centrifuging the supernatant after mixing with the stop solution in Example 4.

[0041] Figure 3 This is the ALB horizontal flow test result diagram. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0043] Preparation Example 1

[0044] This preparation example provides a leukocyte extract solution combination, which includes a fixation working solution, a stop working solution and a lysis working solution.

[0045] The fixing working solution comprises 1.6% formaldehyde and the solvent is PBS;

[0046] The stop working solution includes Tris 24.2%, the solvent is PBS, and the pH is adjusted to 7.4 using HCl;

[0047] The lysis working solution includes TritonX10000.1% and the solvent is PBS.

[0048] Unless otherwise specified, the working solutions used in the following examples are all the working solutions described in Preparation Example 1.

[0049] Example 1

[0050] The method of leukocyte extraction is as follows:

[0051] (1) Mix 500 μL of blood with 1 mL of fixation working solution, gently invert up and down 10 times to mix, and let stand at room temperature for 10 minutes. This is considered the end of fixation. The CTL group did not perform this step and directly proceeded to step (2).

[0052] (2) Mix the fixed blood and centrifuge at 500g for 10 min. Use a pipette to remove the supernatant, avoiding blowing away the cell pellet. Collect the pellet. Transfer the supernatant to a new 50 mL centrifuge tube and centrifuge again at 500g for 10 min. Discard the supernatant and collect the pellet.

[0053] (3) The cell pellets obtained by two centrifugations in step (2) were combined and lysed by removing red blood cells. The amount of lysate added was 5 mL, and the pellets were allowed to stand at room temperature for 30 min for lysis. The pellets were inverted several times during the lysis process. In the CTL group, 8 volumes of commercially available red blood cell lysis buffer were added according to the amount of pellet, and the pellets were lysed at 37°C for 10 min. The pellets were gently mixed for full lysis.

[0054] (4) Centrifuge the product of step (3) at 500 g for 8 min, aspirate the supernatant with a pipette, being careful not to blow up the precipitate, and observe the color of the precipitate.

[0055] (5) If the precipitate is reddish, repeat the red blood cell lysis procedure. If the precipitate is normal in color, proceed directly to the next step.

[0056] (6) Mix the precipitate with PBS and centrifuge for 10 min. The specific centrifugation speed is shown in Table 1. Use a pipette to aspirate the supernatant to avoid blowing away the precipitate.

[0057] (7) Resuspend the cell pellet in 500 μL PBS and transfer to a 1.5 mL centrifuge tube. Count and detect the components by flow cytometry.

[0058] The lysis working solution used in the CTL group in step (3) is a commercially available product purchased from Bio-Tech, the product name is red blood cell lysis solution, the product number is C3702-120mL, and the lysis working solution and fixation working solution used in the PFA group are the products prepared in Preparation Example 1.

[0059] Table 1

[0060] name fixed Red blood cell lysis Platelet removal centrifuge speed CTL-H / Commercially available products 300g CTL-L / Commercially available products 200g PFA-H Preparation Example 1 Preparation Example 1 300g PFA-L Preparation Example 1 Preparation Example 1 200g

[0061] The obtained product was tested for components by flow cytometry, and the results of the percentage of each cell in the total number of cells are shown in Table 2, wherein granulocytes are part of white blood cells, and platelets and red blood cells are part of non-immune cells.

[0062] Table 2

[0063]

[0064] It can be seen from the data in the table that after the cells are fixed with the fixing working solution, the proportion of white blood cells and granulocytes is higher, and the proportion of other cells is lower, which meets the requirements of this application.

[0065] Example 2

[0066] The method of leukocyte extraction is as follows:

[0067] (1) Mix 1 mL of blood with 2 mL of fixation working solution, gently invert upside down 10 times to mix, and let stand at room temperature for 10 minutes. This marks the end of fixation.

[0068] (2) Mix the fixed blood and centrifuge at 500g for 10 min. Use a pipette to remove the supernatant, avoiding blowing away the cell pellet. Transfer the supernatant to a new 50 mL centrifuge tube and centrifuge again at 500g for 10 min.

[0069] (3) The cell pellets obtained by two centrifugations in step (2) were combined and mixed with 10 mL of lysis working solution to remove red blood cells and lyse. The pellets were allowed to stand at room temperature for 30 min for lysis, and were inverted several times during the lysis process. In the CTL group, 8 volumes of commercially available red blood cell lysis solution were added according to the amount of pellet, and the pellets were lysed at 37°C for 10 min, with gentle mixing for complete lysis.

[0070] (4) Centrifuge the product of step (3) at 500 g for 8 min, aspirate the supernatant with a pipette, being careful not to blow up the precipitate, and observe the color of the precipitate.

[0071] (5) If the precipitate is reddish, repeat the red blood cell lysis procedure. If the precipitate is normal in color, proceed directly to the next step.

[0072] (6) Mix the product of step (5) with PBS, centrifuge at 200 g for 10 min, and remove the supernatant with a pipette to avoid blowing away the precipitate.

[0073] (7) Add 500 μL PBS to resuspend the cells, transfer them to a 1.5 mL centrifuge tube after pipetting, and detect the cell components by flow cytometry antibody staining.

[0074] The obtained products were fixed, lysed and detected at different times. The CTL group was not fixed, and red blood cells were lysed using a commercial product, which was Biyuntian Red Blood Cell Lysis Solution: C3702-120mL. The PFA group used the fixation working solution prepared in Preparation Example 1 of this application, and the BD group used a commercial product for fixation and lysis, without the need to remove red blood cells (i.e., steps 4 and 5 were not performed), and the product was Phosflow TM Lyse / Fix Buffer 5X, catalog number 558049, specific time is shown in Table 3, and the results of the percentage of each cell in the total number of cells are shown in Table 4, where granulocytes are part of white blood cells, and platelets and red blood cells are part of non-immune cells.

[0075] Table 3

[0076]

[0077]

[0078] Table 4

[0079] Grouping name leukocyte(%) Granulocytes (%) Non-immune cells (%) Platelets (%) Red blood cells (%) 1 CTL 54.13 24.38 28.32 4.21 14.97 2 PFA-10min 95.59 60.11 3.47 3.06 0.12 3 PFA-6H 83.02 50.82 16.62 15.56 0.03 4 PFA-24H 79.65 47.73 19.90 17.94 0.60 5 PFA-48H 26.76 16.33 71.80 0.02 65.05 6 BD-10min 89.52 55.82 8.092 6.812 1.26 7 BD-6H 86.04 53.01 9.898 8.728 0.99 8 BD-24H 82.57 50.87 15.288 14.228 1.05 9 BD-48H 82.16 49.91 14.424 11.554 2.87

[0080] The combination of fixation working solution and lysis working solution can lyse blood with a fixation time of less than 24 hours. If the fixation time exceeds 24 hours, the number of white blood cells will drop sharply, and the longer the fixation time, the worse the red blood cell lysis. The absolute number of granulocytes is not affected, but platelets cannot be removed by low-speed centrifugation.

[0081] Example 3

[0082] The method of leukocyte extraction is as follows, and the test results are as follows Figure 1 shown.

[0083] (1) 500 μL of blood was mixed with 2 mL of fixation working solution, gently inverted up and down 10 times to mix, and allowed to stand at room temperature for 10 minutes, which was considered the end of fixation. The blood was then mixed with 2.5 mL of stop working solution and allowed to stand for 48 hours after mixing with the stop solution. The formaldehyde concentrations in the fixation solution and the Tris-HCl concentrations in the stop solution of different groups are shown in Table 5 (the group without stop solution was supplemented with an equal amount of PBS).

[0084] (2) Mix the stopped blood, centrifuge at 500g for 10 min, take the supernatant with a pipette to avoid blowing up the cell pellet, transfer the supernatant to a new 50mL centrifuge tube and centrifuge again at 500g for 10 min.

[0085] (3) The cell pellets obtained by two centrifugations in step (2) were combined, mixed with the lysis working solution to remove red blood cells and lysed, and allowed to stand at room temperature for 30 minutes for lysis, and inverted several times during the lysis process.

[0086] (4) Centrifuge the product of step (3) at 500 g for 8 min, aspirate the supernatant with a pipette, being careful not to blow up the precipitate, and observe the color of the precipitate.

[0087] (5) If the precipitate is reddish, repeat the red blood cell lysis procedure. If the precipitate is normal in color, proceed directly to the next step.

[0088] (6) Mix the product of step (5) with PBS, centrifuge at 200 g for 10 min, and remove the supernatant with a pipette to avoid blowing away the precipitate.

[0089] (7) The cells were resuspended in 500 μL PBS and transferred to a 1.5 mL centrifuge tube. The cells were counted and stained with flow cytometry antibodies to detect cell components.

[0090] The lysis working solution is the lysis working solution prepared in Preparation Example 1, the concentrations of the solvents in the fixative and the stop solution are shown in Table 5, and the solvents are all PBS solutions.

[0091] Table 5

[0092]

[0093] After the addition of the stop solution, the lysis was carried out 48 hours later. Figure 1 The results showed that A2-1, A2-2, A3-1, and A3-2 failed to lyse red blood cells, indicating that the concentration of the stop solution is very important for the subsequent red blood cell lysis effect. If the amount added is too low, red blood cell lysis will fail. Therefore, excessive stop solution is added for subsequent development.

[0094] Example 4

[0095] The method of leukocyte extraction is consistent with that in Example 3. Except for the control group without the addition of the fixative, an excess of the stop solution is added. The only difference is the time of adding the stop solution. The specific grouping treatment is shown in Table 6, where L1, L2, and L4 are delayed extraction groups after 2 weeks of fixation with different concentrations of fixatives. The results are shown in Table 7. The lysis working solution is the working solution prepared in Preparation Example 1. The concentrations of the stop working solutions in different groups are shown in Table 6. The concentration of the fixative working solution used in Group C1 is 0.4%, the concentration of the fixative working solution used in Group C2 is 0.8%, and the concentration of the fixative working solution used in Group C4 is 1.6%. In Table 6, 0.4%, 0.8%, and 1.6% all refer to the mass percentage of formaldehyde in the fixative working solution, and the solvents are all PBS.

[0096] Table 6

[0097]

[0098]

[0099] Table 7

[0100]

[0101] Note: The data of albumin levels are derived from the median of the MFI mean fluorescence intensity after compensation adjustment. For ease of understanding and display, the negative data are normalized and the baseline value is adjusted to 0 as the lowest reference point.

[0102] Figure 2 This is the supernatant after centrifugation of the second mixed solution, which can show the level of red blood cell hemolysis. Figure 2From left to right in the figure are C0 without fixation, C1-C4 with fixation without termination, groups 1-6 at a PFA concentration of 0.4%, groups 7-12 at a PFA concentration of 0.8%, and groups 13-18 at a PFA concentration of 1.6. The level of red blood cell hemolysis can be seen from the figure. Fixation with different concentrations of fixative working solutions showed a similar trend in site signal preservation. For the specific detection sites after fixation, the red blood cell contamination was low. And the blood can still lyse red blood cells after being placed for 2 weeks. If the fixation is not terminated in time, the longer the placement time, the higher the concentration of the fixative working solution, and the more serious the platelet contamination. By comparing the values ​​of ALB (albumin) MFI, the contamination caused by cross-linking of high-abundance plasma proteins with white blood cells was evaluated. The results are as follows. Figure 3 As shown, Figure 3 It can show the level of plasma protein cross-linking. If fixation is not stopped in time, the longer the storage time, the higher the PFA concentration and the more serious the ALB plasma contamination.

[0103] Example 5

[0104] Preservation of phosphorylation signals

[0105] (1) PBMCs were purchased from Miaoshun Biotechnology. Healthy human PBMC samples were stored in cryopreserved tubes. After PBMCs were revived, they were evenly divided into 14 portions, with 2×10 cells in each portion. 7 Incubate in the incubator for 1 hour.

[0106] (2) Use culture medium to prepare 300 ng / mL IL2 solution, which is an activator of the JAK-STAT signaling pathway.

[0107] (3) The group without IL2 was the negative control, i.e., group C0. All other groups were added with IL2 (IL2 was added to the culture medium to a working concentration of 50 ng / mL), incubated in the incubator for 15 min, and PBS was added to terminate the reaction.

[0108] (4) Wash once with PBS, centrifuge at 600g for 15min to collect the cell pellet. The pellet of group C0 was collected immediately and frozen at -80℃. The pellet of group C1 was collected and placed at 4℃ for 1 hour to simulate the loss of phosphorylation signal required for conventional blood PBMC extraction, and then transferred to -80℃ for freezing. The cells of the other groups were treated with different concentrations of fixative and stop solutions according to the table below (except for the step where the lysis working solution was not required, the other steps were the same as the blood fixation operation), placed at 4℃ for 24h to simulate signal loss, and then transferred to -80℃ for freezing.

[0109] (5) The obtained cell precipitate is lysed, peptides are extracted, tyrosine is enriched with the same starting peptide amount, and then the tyrosine phosphorylation site is detected by mass spectrometry.

[0110] The specific fixed working solution concentrations, treatment time, and test results of different groups are shown in Table 8, and the site detection results are shown in Table 9.

[0111] Table 8

[0112]

[0113] Note: Total amount of peptide extraction: refers to the total amount of peptides obtained after protein enzymatic hydrolysis of the sample, in micrograms (μg), reflecting the overall protein enzymatic hydrolysis efficiency of the sample.

[0114] Phosphorylation enrichment starting peptide amount: refers to the amount of starting peptide used for enrichment in phosphorylation modification enrichment experiments. In order to ensure the comparability of data in each group, a uniform adjustment is made based on the lowest total amount in the group.

[0115] Number of identified phosphorylation sites: refers to the number of phosphorylation sites identified in a sample by mass spectrometry analysis, reflecting the level of detectable phosphorylation modification in the sample. The number of sites under different experimental conditions is used to evaluate the efficiency of sample processing, enrichment, and identification.

[0116] After the phosphoproteomics was taken off the machine, based on the downstream signaling pathways activated by IL2, we focused on whether the phosphorylation sites of the JAK-STAT pathway, the activation target of IL2, were detected. Group C0 represents the state of fresh cells without IL-2 treatment, reflecting the original level of the unactivated JAK-STAT pathway; Group C1 is fresh cells treated with IL-2, which were placed at 4°C for 1 hour before being put on the machine, simulating the attenuation of the signal under non-fixation conditions. Groups 1-12 were treated with IL2, fixed with different PFA concentrations and then put on the mass spectrometer, showing the most detected IL2 activation signals, and all 12 treatment groups detected the phosphorylation sites in Table 9, some of which were new phosphorylation sites that had not been reported in the literature, further proving the effectiveness of the fixation method for signal retention.

[0117] Table 9

[0118]

[0119]

[0120] Different concentrations of fixative working solution showed a similar trend in site signal preservation, and different fixation times showed a similar trend in site signal preservation. Sample No. 3 had an error and was not included in the analysis. The 1.6% PFA concentration fixation group had the largest number of detected sites and the smallest intra-group difference.

[0121] Example 6

[0122] Large volume fixed stop ratio test

[0123] Test method:

[0124] In order to meet the requirements of proteomics on cell quantity, we increased the blood reaction system from 500μL to 5mL and tested the effects of different blood fixation ratios on blood components.

[0125] First, take three groups of 5mL blood. The ratio of blood in group 1 to the fixative is 1:2. The ratio of blood in group 2 to the fixative is 1:5, and the fixative is the fixative prepared in the preparation example. After fixation for 10 minutes, add 1.3M Tris stop solution (mass concentration is 24.2%), of which the 1:2 fixation group adds a stop working solution with a volume ratio of 1:1, and the 1:5 group adds a stop working solution with a volume ratio of 5:4. After standing at 4℃ for 48h, lyse with 1:8 lysis solution. Group C0 is not fixed. After the blood is removed from the body, immediately add red blood cell lysis solution for lysis at the ratio recommended in the instructions (the product is Biyuntian red blood cell lysis solution: C3702-120mL).

[0126] The proportion of different cell components was detected by flow cytometry. It can be seen that different fixed ratios have little effect on the cell proportion, but a large ratio has a lower ALB plasma protein level, indicating lower plasma protein contamination. Subsequent large-volume experiments were all conducted using a fixed ratio of 1:5.

[0127] Table 10

[0128]

[0129] Note: The data of albumin levels are derived from the median of the mean fluorescence intensity (MFI) after compensation adjustment.

[0130] Example 7

[0131] Mass spectrometry test results

[0132] This is a demonstration of real patient blood proteomics on the machine, and it tests the lysis of blood after fixation and long-distance transportation.

[0133] Two samples were taken from each group, including a healthy control group and patients with scleroderma (autoimmune disease), and 5 mL of blood was taken from each group. The samples were fixed at a fixed ratio of 1:5. After 10 minutes, a 1.3 M Tris stop solution (with a mass concentration of 24.2%) with a volume ratio of 5:4 was added. The samples were allowed to stand for 10 minutes after the fixation was stopped. Both the fixation working solution and the stop working solution were prepared in Preparation Example 1.

[0134] The samples were transported with ice packs for less than 48 hours. The lysis experiment was performed using a 1:10 lysis working solution. The platelets were centrifuged and the cell pellets were frozen in a -80 degree refrigerator.

[0135] The cell precipitate obtained by the above operation was lysed, peptides were extracted, and the whole protein (A), serine and threonine phosphorylated peptides (P), and tyrosine phosphorylated peptides (Y) were enriched with the same starting peptide amount, and then the mass spectrometer was used to detect the number of sites identified by proteomics. The biological correlation between the differential sites between the disease and healthy groups and the disease was analyzed.

[0136] Table 11

[0137]

[0138]

[0139] Table 12

[0140]

[0141] Table 12 shows the detection results of other autoimmune disease proteomics sites recorded in historical documents. This experiment confirmed that the blood fixation technology can be used for proteomics detection of blood samples sent remotely, and the number of identifications is more than that recorded in historical documents.

[0142] The applicant declares that the present invention illustrates a leukocyte extract combination and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0143] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0144] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A leukocyte extract composition, characterized in that: The leukocyte extraction solution combination includes a fixing working solution, a stopping working solution and a lysis working solution; The fixing working solution includes formaldehyde and PBS; The stop working solution includes Tris and PBS; The lysis working solution includes TritonX100 and PBS.

2. The leukocyte extract composition according to claim 1, characterized in that: The fixing working solution comprises formaldehyde 0.25-2% and PBS 98-99.75% by mass percentage; Preferably, the stop working solution comprises Tris 1.5-24.2% and PBS 75.8-98.5% in terms of mass percentage; Preferably, the lysis working solution comprises TritonX1000 0.05-0.1% and PBS 99.9-99.95% in terms of mass percentage; Preferably, the pH of the stop working solution is 7.35-7.

45.

3. Use of the leukocyte extracting solution combination according to claim 1 or 2 in leukocyte extraction.

4. A method for extracting leukocytes, characterized in that: The method comprises: (1) mixing blood with a fixing working solution and allowing the mixture to stand to obtain a first mixed solution; (2) mixing the first mixed solution with the suspended working solution to obtain a second mixed solution; (3) centrifuging the second mixed solution, mixing the precipitate with the lysis working solution, lysing and centrifuging after lysis; (4) Mix the precipitate obtained in step (3) with PBS solution, centrifuge, and resuspend the precipitate.

5. The method for extracting leukocytes according to claim 4, characterized in that: The volume ratio of blood to fixative working solution is 1:(2-5).

6. The method for extracting leukocytes according to claim 4 or 5, characterized in that: The volume ratio of the first mixed liquid to the suspension working liquid is 1:(0.8-1).

7. The method for extracting leukocytes according to any one of claims 4 to 6, characterized in that: The standing time in step (1) is 10-15 minutes; Preferably, the mixing time in step (2) is 10 minutes to 14 days.

8. The method for extracting leukocytes according to any one of claims 4 to 7, characterized in that: The centrifugation speed in step (3) is independently 500-600 rpm and the time is 8-15 min; Preferably, the centrifugation speed in step (4) is 200-300 rpm and the time is 10-15 min; Preferably, the volume ratio of blood to lysis working solution is 1:(8-10).

9. Leukocytes extracted by the leukocyte extraction method according to any one of claims 4 to 8.

10. Use of the leukocyte according to claim 9 in proteomic analysis.

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