An analysis method for detecting the H3K27me3 level of each immune cell group in human whole blood by flow cytometry

Abstract

This invention discloses an analytical method for detecting the H3K27me3 level of various immune cell populations in human whole blood using flow cytometry. The method involves a one-step pretreatment process of lysing erythrocytes and simultaneously fixing leukocytes in a human whole blood sample. Fluorescently labeled antibodies (BV785-CD3, BV650-CD19, BV605-CD14, AF488-HLA-DR, AF647-H3K27, Pacific Blue-H3) are used to stain the lysed and fixed leukocytes. After antibody staining, the sample is subjected to flow cytometry for gating analysis. The H3K27me3 / H3 level in each immune cell population—T cells (CD3+CD19-), B cells (CD3-CD19+), monocytes (CD14+HLADR+), and granulocytes (CD14-HLADR-)—is calculated using a formula. The whole blood sample, after the one-step lysing and fixing process, can be stored at -60 to -90°C for 5 days. The method exhibits good accuracy, reliability, and stability.

Description

Technical Field

[0001] This invention relates to a method for analyzing H3K27me3 levels, specifically a method for detecting H3K27me3 levels in various immune cell populations in human whole blood using flow cytometry. Background Technology

[0002] Flow cytometry (FCM) is a comprehensive detection technology that integrates laser technology, fluid dynamics, computer image processing, and fluorescent labeling. It can perform qualitative or quantitative analysis on the physical, biochemical, immunological, genetic, molecular biological characteristics, and functional status of cells, magnetic beads, or other biological particles in flowing liquids. Its main features are: (1) Fast detection speed, capable of counting tens of thousands of cells or single-molecule microspheres in a short time. (2) Multi-parameter detection and analysis of the same molecule, including molecular size, morphology, and protein expression level. (3) Automated high-throughput detection, capable of simultaneously and automatically detecting a large number of samples without human intervention. (4) Accurate and reliable detection results, possessing powerful image processing and data analysis capabilities, resulting in objective and reliable detection results. Currently, FCM has been widely applied in various fields of basic research and clinical practice, covering cell biology, immunology, oncology, pharmacology, genetics, and clinical testing. Among these applications, immune cell phenotype analysis is one of the most frequently used areas of flow cytometry in clinical practice. It primarily involves identifying and characterizing immune cell populations by detecting surface or intracellular markers. These immune cells include T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, platelets, and granulocytes. The analysis also allows for precise detection of histone methylation levels in different immune cell populations.

[0003] Histones are a major component of chromatin, primarily composed of five proteins: H1, H3, H2A, H2B, and H4. They are basic due to their high content of basic arginine and lysine. Simultaneously, the amino-terminal amino acid residues can be covalently modified, altering chromatin conformation and leading to transcriptional activation or silencing of downstream genes, thus participating in cell division, apoptosis, memory formation, and even influencing the immune system and inflammatory responses. Studies have shown that histones undergo different post-translational modifications under the action of different enzymes, mainly including methylation, acetylation, phosphorylation, ubiquitination, and glycosylation. Specifically, under the catalysis of methyltransferases, the N-termini of arginine and lysine in histones can be methylated; lysine can be mono-, di-, or tri-methylated, and arginine can be mono- or di-methylated. Histone methylation is a reversible biological process regulated by various intracellular histone methyltransferases and demethylases. Methyltransferases catalyze methylation, while histone demethylation is catalyzed by histone demethylases. The regulatory effect of histone methylation on gene transcriptional state depends on the methylated residues and the degree of methylation.

[0004] Trimethylation of histone H3 at lysine 27 (H3K27me3) is one of the most common histone modifications, mediated by the EZH2 subunit of PRC2. H3K27 methylation is a key mediator of gene transcription repression and participates in several important biological processes. Currently, histone methylation detection mainly relies on several common methods, such as chromatin immunoprecipitation (ChIP), Western blotting, protein microarrays, and mass spectrometry. ChIP can provide high-resolution gene localization information, but it is complex and time-consuming; Western blotting is simple to perform, but it can only detect methylated histones at the whole-cell level; protein microarrays and mass spectrometry offer high throughput and automation, but are expensive. Because these methods cannot separate different cell populations, they cannot accurately obtain the methylation levels of different immune cell populations simultaneously in whole blood.

[0005] To date, no patents have been published regarding analytical methods for detecting H3K27me3 levels of various immune cell populations in human whole blood using flow cytometry. Therefore, this study developed an analytical method for detecting H3K27me3 levels of various immune cell populations in human whole blood using flow cytometry. Summary of the Invention

[0006] Purpose of the invention: To address the shortcomings of existing detection technologies, this invention provides an accurate, reliable, and stable analytical method for detecting the H3K27me3 levels of various immune cell populations in human whole blood using flow cytometry.

[0007] Technical solution: The present invention describes an analytical method for detecting the H3K27me3 level of various immune cell populations in human whole blood using flow cytometry. The method involves first staining with surface antibodies, followed by staining with intranuclear antibodies after cell membrane rupture and fixation; or staining with both surface and intranuclear antibodies simultaneously after cell membrane rupture and fixation.

[0008] Further, the surface antibody is a fluorescently labeled anti-CD3, CD19, CD14, and HLA-DR antibody; the intracellular antibody is a fluorescently labeled anti-Tri-Methyl-Histone H3 (Lys27) antibody and a fluorescently labeled anti-Histone H3 antibody.

[0009] Furthermore, the concentrations of the anti-CD3 (hereinafter referred to as BV785-CD3), CD19 (hereinafter referred to as BV650-CD19), CD14 (hereinafter referred to as BV605-CD14), and HLA-DR (hereinafter referred to as BV605-CD14) antibodies are 50 μg / mL, 50 μg / mL, 150 μg / mL, and 200 μg / mL, respectively.

[0010] The concentrations of the anti-Tri-Methyl-Histone H3 (Lys27) antibody (hereinafter referred to as AF647-H3K27) and the fluorescently labeled anti-Histone H3 antibody (hereinafter referred to as Pacific Blue-H3) were 100 μg / mL and 50 μg / mL, respectively.

[0011] Furthermore, a one-step method was used to lyse red blood cells and fix white blood cells to preprocess whole blood samples before staining.

[0012] Further, 20 times the sample volume of 1×Lyse / Fix Buffer (1×Lyse / Fix Buffer needs to be preheated in a 37°C water bath for 30 min before use) was added to the whole blood sample, and the red blood cells were lysed and the white blood cells were fixed in a 37°C water bath for 10 min.

[0013] Furthermore, Tri-Methyl-Histone H3 (Lys27) and Total Histone H3 were detected simultaneously, and the ratio of H3K27me3 to total H3 protein was used as the histone methylation level of each immune cell subset.

[0014] Furthermore, the formula for calculating the H3K27me3 / H3 level is as follows:

[0015] Formula for calculating H3K27me3 / H3 (%):

[0016] ;

[0017] in:

[0018] Sample (AF647-H3K27 Median MFI): Median fluorescence value of H3K27 Alexa Fluor in the sample tube.

[0019] Sample (Pacific Blue-H3 Median MFI): Median fluorescence value of H3 Pacific Blue in the sample tube.

[0020] Furthermore, FMO was used as a control.

[0021] A one-step pretreatment method was used to lyse red blood cells and fix white blood cells in human whole blood samples. Fluorescently labeled antibodies (BV785-CD3, BV650-CD19, BV605-CD14, AF488-HLA-DR, AF647-H3K27, PacificBlue-H3) were used to stain the lysed and fixed white blood cells. After antibody staining, flow cytometry was used to perform gating analysis on the samples. The H3K27me3 / H3 level in each immune cell population, namely T cells (CD3+CD19-), B-cells (CD3-CD19+), monocytes (CD14+HLADR+), and granulocytes (CD14-HLADR-), was calculated using a formula.

[0022] The research approach of this method is as follows:

[0023] 1. This method is for analyzing the H3K27me3 levels of various immune subpopulations in clinical human whole blood samples. Because trimethylated histone H3 at position 27 (H3K27me3) is extremely unstable, the H3K27me3 levels of various immune subpopulations in whole blood change during transport to the testing center, thus failing to accurately reflect changes in H3K27me3 levels in whole blood after clinical drug administration. This method addresses the stability issue of clinical whole blood samples during transport. Currently, there are very few reports and patents regarding methods for detecting H3K27me3 levels of various immune subpopulations in whole blood, especially those supporting clinical biosample analysis.

[0024] 2. In the initial exploration of the method, a conventional approach was used: whole blood was collected into ordinary EDTA-K2, Cyto-ChexBCT flow cytometry whole blood sample preservation tubes, or Cell-Free DNA Streck™BCT blood collection tubes. When the whole blood was directly stored at room temperature or 2–8°C, the H3K27me3 levels in various immune subpopulations in the whole blood changed significantly compared to baseline. Conventional methods for detecting H3K27me3 levels in various immune subpopulations in whole blood resulted in poor pre-staining stability of the whole blood samples, failing to meet the stability requirements for clinical biosample analysis. Therefore, further optimization and exploration of whole blood processing were conducted. The final method developed uses a one-step method of lysing red blood cells and fixing white blood cells to preprocess freshly collected whole blood samples. The initial exploration of the method also included exploring the amount of 1×Lyse / Fix Buffer used, whether preheating was necessary, and the water bath duration, resulting in a method with good stability.

[0025] 3. This method detects the H3K27me3 levels of various immune cell subsets in human whole blood, including T cells, B cells, monocytes, and granulocytes. In the initial exploration of this method, surface antibody staining was performed first after erythrocyte lysis and leukocyte fixation, followed by nuclear antibody staining after cell membrane rupture and fixation. The surface antibodies used were fluorescently labeled anti-CD3, CD19, CD14, and HLA-DR antibodies; the nuclear antibodies were fluorescently labeled anti-Tri-Methyl-HistoneH3 (Lys27) antibody and fluorescently labeled anti-Histone H3 antibody. Later, to simplify the experimental procedure, the method was optimized so that surface and nuclear antibodies can be stained simultaneously after cell membrane rupture and fixation, saving experimental time.

[0026] 4. This method simultaneously detected Tri-Methyl-Histone H3 (Lys27) and Total Histone H3, and the ratio of H3K27me3 to total H3 protein was used as the histone methylation level of each immune cell subset.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The method of the present invention uses a one-step method to lyse red blood cells and fix white blood cells to preprocess freshly collected whole blood samples, so that the activity of H3K27 methyltransferase and H3K27me3 demethylase in cells can be fixed immediately after whole blood collection, and the histone methylation level of each immune cell subset is kept stable.

[0029] 2. The method of this invention first performs surface antibody staining, followed by nuclear antibody staining after cell membrane rupture and fixation. The surface antibodies used are fluorescently labeled anti-CD3, CD19, CD14, and HLA-DR antibodies; the nuclear antibodies are fluorescently labeled anti-Tri-Methyl-Histone H3 (Lys27) antibody and fluorescently labeled anti-Histone H3 antibody. Alternatively, surface and nuclear antibodies can be stained simultaneously after cell membrane rupture and fixation, simplifying the experimental procedure and saving experimental time.

[0030] 3. This method uses FMO as a control, which facilitates the determination of the location of granulocyte populations. Attached Figure Description

[0031] Figure 1 The flow cytometry analysis gate diagram (6 colors) for the sample is generated based on the expression of six proteins. Six fluorescently labeled antibodies (BV785-CD3, BV650-CD19, BV605-CD14, AF488-HLA-DR, AF647-H3K27, and Pacific Blue-H3) mentioned in this invention are selected. Under this analysis template, four immune cell populations can be simultaneously circled: T cells (CD3+CD19-), B-cells (CD3-CD19+), monocytes (CD14+HLADR+), and granulocytes (CD14-HLADR-). The expression of Tri-Methyl-Histone H3 (Lys27) and TotalHistone H3 in the four immune cell populations can be visually displayed. The H3K27 Alexa Fluor and H3K27 Alexa Fluor in the sample tube can be obtained from the analysis diagram. The median fluorescence value of PacificBlue can be used to calculate the H3K27me3 / H3 levels of four immune cell populations using the formula for calculating H3K27me3 / H3 levels. Detailed Implementation Plan

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0033] Whole blood pretreatment:

[0034] One-step method (-60~-90℃ cracking and solidification)

[0035] Whole blood was collected in Cell-Free DNA Streck™ BCT blood collection tubes. 20 times the sample volume of 1×Lyse / Fix Buffer was added to the whole blood sample (preheated in a 37°C water bath for 30 min), and the blood was incubated in a 37°C water bath for 10 min to perform one-step lysis of red blood cells and fixation of white blood cells. The blood was then immediately frozen at -60~-90°C.

[0036] Detailed steps for sample analysis:

[0037] 1. Samples stored at -60~-90℃ should be thawed immediately in a 37℃ water bath after removal. Proceed to the next step after complete thawing.

[0038] 2. Prepare two centrifuge tubes, labeled “Sample” and “FMO” respectively. Add 10 mL of the processed whole blood sample to each tube and centrifuge at 500 g for 8 min at room temperature.

[0039] 3. Discard the supernatant, add 6 mL of PBS, resuspend the cells, and centrifuge at 500 g at room temperature for 8 min;

[0040] 4. Discard the supernatant, add 6 mL of PBS, resuspend the cells, and centrifuge at 500 g at room temperature for 8 min;

[0041] 5. Discard the supernatant, add 100 µL of Stain Buffer to each tube, and resuspend the cells;

[0042] 6. Add 1 mL of freshly prepared 1× fixation / permeabilization buffer to each tube, mix well, and incubate at 2-8°C in the dark for approximately 40±5 min.

[0043] 7. Add 2 mL of 1×FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8℃, and discard the supernatant;

[0044] 8. Add 2 mL of 1×FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8℃, and discard the supernatant;

[0045] 9. Add 100 µL of 1×FOXP3 permeabilization buffer to each tube and resuspend the cells;

[0046] 10. Add 5 μL BV785-CD3 (50 μg / mL), 5 μL AF488-HLA-DR (200 μg / mL), 5 μL BV605-CD14 (150 μg / mL), 5 μL BV650-CD19 (50 μg / mL), 2 μL LAF647-H3K27 (100 μg / mL), and 2 μL Pacific Blue-H3 (50 μg / mL) to the sample tubes. Add 5 μL BV785-CD3 (50 μg / mL), 5 μL BV605-CD14 (150 μg / mL), 5 μL BV650-CD19 (50 μg / mL), 2 μL LAF647-H3K27 (100 μg / mL), and 2 μL Pacific Blue-H3 to the FMO tubes. Mix Blue-H3 (50 μg / mL) thoroughly and incubate at 2-8°C in the dark for 30 min ± 5 min.

[0047] 11. Add 2 mL of 1×FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8℃, and discard the supernatant.

[0048] 12. Add 2 mL of 1×FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8℃, and discard the supernatant.

[0049] 13. Add 350 μL of Stain Buffer to each tube to resuspend the cells. Store at 2–8°C until analysis.

[0050] 14. Flow Cytometry Detection: Turn on the flow cytometer and perform equipment calibration: According to the instrument's built-in program, use CSTSetup Beads to calibrate the fluorescence channels. In the initial experiment, the voltage and compensation were determined using single-labeled tubes and Blank tubes for 6 fluorescence channels and saved as templates. 10,000 All Events cells were collected in the single-labeled tubes, and the number of cells collected for each sample was 5,000 CD14+HLA-DR+ cells or the entire cell suspension volume.

[0051] 15. After the flow cytometry analysis is complete, use BD FACS Diva software to set the analysis template obtained by gate selection. For gate selection strategies, see [link to relevant documentation]. Figure 1 Use FSC-A / SSC-A to circle the All cells population; from the All cells, use FSC-A / FSC-H to circle single cells (Singlets), excluding adhesions; from the Singlelets, use FSC-A / SSC-A to circle the LYM cell population and the Non-LYM cell population; from the LYM cell population, use CD3 BV786-A / CD19 BV650-A to circle CD3+CD19- cells (T cells) and CD3-CD19+ cells (B cells); from the Non-LYM cell population, use CD14 BV605-A / HLA-DR-AF488-A to circle CD14+HLA-DR+ cells (monocytes) and CD14-HLA-DR- cells (granulocytes). The median fluorescence intensity (Median MFI) of AF647-H3K27 and Pacific Blue-H3 was obtained in monocytes, granulocytes, B cells, and T cells, respectively, and the data were processed by Microsoft Excel 2016.

[0052] The formula for calculating the H3K27me3 / H3 level is as follows:

[0053] Formula for calculating 27me3 / H3 (%):

[0054] ;

[0055] In addition, the stability of whole blood samples before staining was verified using this method, and the results are shown in Table 1.

[0056] Table 1. Stability of one-step method (-60~-90℃) before staining

[0057] Time (Day) T-cell (CD3+CD19-) H3K27 / H3% CV% B-cell (CD3-CD19+)H3K27 / H3% CV% Monocytes (CD14+HLADR+) H3K27 / H3% CV% Granulocytes (CD14-HLADR-) H3K27 / H3% CV% 0d 115.0 NA 306.9 NA 22.3 NA 71.1 NA 60 ~ -90℃1d 123.0 4.8 390.5 16.9 19.7 8.5 58.4 13.9 60 ~ -90℃5d 128.7 7.9 406.0 19.7 22.9 1.8 64.0 7.5

[0058] Whole blood samples were pretreated immediately after collection using a one-step method (-60~-90℃ cracking and solidification). Baseline samples after cracking and solidification were analyzed immediately (see detailed sample analysis steps 1~15). Other samples after cracking and solidification were stored at -60~90℃ for 1 day and 5 days, and then analyzed (see detailed sample analysis steps 1~15). After flow cytometry analysis, data were acquired, and the H3K27me3 / H3 levels of T cells (CD3+CD19-), B-cells (CD3-CD19+), monocytes (CD14+HLADR+), and granulocytes (CD14-HLADR-) were calculated in the baseline (0d), -60~90℃ stored for 1d, and 5d samples. The results are shown in Table 1. The results indicate that the CV% of H3K27me3 / H3 levels in T cells (CD3+CD19-) stored at -60~90℃ for 1 day and 5 days compared to baseline (0 days) was 4.8% and 7.9%, respectively; the CV% of H3K27me3 / H3 levels in B-cells (CD3-CD19+) stored at -60~90℃ for 1 day and 5 days compared to baseline (0 days) was 16.9% and 19.7%, respectively; the CV% of H3K27me3 / H3 levels in monocytes (CD14+HLADR+) stored at -60~90℃ for 1 day and 5 days compared to baseline (0 days) was 8.5% and 1.8%, respectively; and the CV% of H3K27me3 / H3 levels in monocytes (CD14+HLADR+) stored at -60~90℃ for 1 day and 5 days compared to baseline (0 days) was 8.5% and 1.8%, respectively. The CV% of H3K27me3 / H3 level of granulocytes (CD14-HLADR-) in the sample was 13.9% and 7.5% respectively compared with baseline (0d). In summary, whole blood samples can be stored at -60~-90℃ for 5 days after one-step fission solidification, and the method can still obtain relatively accurate results (CV% of H3K27me3 / H3 (%) ≤ 20% compared with Day 0).

[0059] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. An analysis method for detecting the level of H3K27me3 in each immune cell population in human whole blood by flow cytometry, characterized in that, The method comprises the following steps: (a) after collecting a human whole blood sample, immediately adding 20 times the sample volume of 1x Lyse / Fix Buffer, preheating the 1x Lyse / Fix Buffer in a 37℃ water bath for 30 minutes, and then treating the whole blood sample in a 37℃ water bath for 10 minutes to complete the red blood cell lysis and white blood cell fixation in one step; (b) storing the sample treated in step (a) at -60~-90℃ for 1 to 5 days; (c) after the sample is recovered, performing a fixed membrane breaking treatment, and then simultaneously adding surface antibodies and intranuclear antibodies for incubation and staining; (d) detecting the sample after staining by using flow cytometry; The method makes the variation coefficient (CV) of the H3K27me3 / H3 ratio of T cells, B cells, monocytes and granulocytes after the whole blood sample is treated in step (a), stored at -60~-90℃ for 5 days, and detected in steps (c) and (d) all not more than 20%.

2. The method according to claim 1, wherein the method is characterized in that: The surface antibodies are fluorescently labeled anti-CD3, CD19, CD14 and HLA-DR antibodies; and the intranuclear antibodies are fluorescently labeled anti-Tri-Methyl-Histone H3 (Lys27) and Histone H3 antibodies.

3. The method according to claim 2, wherein the method is characterized in that: The concentrations of the anti-CD3, CD19, CD14 and HLA-DR antibodies are 50 μg / mL, 50 μg / mL, 150 μg / mL and 200 μg / mL respectively. The concentrations of the anti-Tri-Methyl-Histone H3 (Lys27) and fluorescently labeled anti-Histone H3 antibodies are 100 μg / mL and 50 μg / mL respectively.

4. The method according to claim 1, wherein the method is characterized in that: Tri-Methyl-Histone H3 (Lys27) and Total Histone H3 are detected simultaneously, and the ratio of H3K27me3 to H3 total protein is used as the histone methylation level of each immune cell subpopulation.

5. The method according to claim 4, wherein the method is used to detect the level of H3K27me3 in each immune cell population in human whole blood by flow cytometry. The calculation formula of the H3K27me3 / H3 level is as follows: The calculation formula of H3K27me3 / H3 (%) is as follows: ； Wherein: Sample (AF647-H3K27 Median MFI): H3K27 Alexa Fluor median fluorescence value of the sample tube Sample (Pacific Blue-H3 Median MFI): H3 Pacific Blue median fluorescence value of the sample tube.

6. The method according to claim 1, wherein the method is used to detect the level of H3K27me3 in each immune cell population in human whole blood by flow cytometry. FMO is used as a control.

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