EDTA anticoagulation blood collection tube for stabilizing monocyte hla-dr expression and method of making

Abstract

The application belongs to the field of blood detection, and particularly relates to an EDTA anticoagulation blood collection tube for stabilizing HLA-DR expression of mononuclear cells and a preparation method thereof. The blood collection tube comprises a blood collection tube body, a sealing rubber plug, an aluminum cover and a protective agent. The protective agent is prepositioned in the form of a freeze-dried cake at the bottom of the blood collection tube, and the blood after blood collection will re-dissolve the freeze-dried cake and uniformly mix with the freeze-dried cake. The protective agent comprises an anticoagulant, a membrane stabilizer, a metabolic inhibitor, a buffer salt and a freeze-dried excipient. The anticoagulant is EDTA-K2, the membrane stabilizer is CHS, the metabolic inhibitor comprises 2-DG and NaN3, and the buffer salt is HEPES. CHS, 2-DG and NaN3 in the protective agent of the blood collection tube can maintain HLA-DR in whole blood stable for up to 72 hours at room temperature through a three-layer synergistic mechanism, and the protective agent can be completely eluted, so that the natural conformation of HLA-DR is restored to nearly 100%, there is no epitope shielding, and the blood collection tube can be directly compatible with the existing EDTA threshold system.

Description

Technical Field

[0001] This invention belongs to the field of blood testing, specifically relating to EDTA anticoagulant blood collection tubes with stable monocyte HLA-DR expression and their preparation method. Background Technology

[0002] Monocyte HLA-DR expression (mHLA-DR) is a key biomarker for assessing the state of the body's innate immune function, especially valuable in the clinical monitoring of patients with sepsis, major trauma, and immunosuppression. Flow cytometry-based quantitative detection of mHLA-DR typically employs the BD Quantibrite technology system, which uses anti-CD14 antibodies to identify monocytes and anti-HLA-DR-PE antibodies to quantify antigen expression, reporting results as antibody binding capacity per cell (ABC).

[0003] However, mHLA-DR detection faces a severe challenge in pretreatment stability. Studies have shown that when whole blood is stored at room temperature in EDTA (ethylenediaminetetraethyl) anticoagulant tubes, mHLA-DR expression increases significantly and falsely over time. Monneret et al. (Bicentric evaluation of stabilizing sampling tubes for assessment of monocyte HLA-DR expression in clinical samples. 2021) reported that after EDTA tubes were stored at room temperature for 48-72 hours, the ABC value of mHLA-DR increased by more than 100%, seriously interfering with the accurate assessment of immunosuppression. The mechanism involves: (1) EDTA chelates Ca 2+ Leading to cell membrane calcium 2+ (1) Gradient collapse, abnormally increased membrane fluidity; (2) Imbalance in the endocytosis-circulation pathway, shortened HLA-DR recovery cycle; (3) Ca 2+ The -calmodulin-calmodulin-NFAT pathway is blocked, downregulating the expression of E3 ubiquitin ligase MARCH1 (membrane-associated RING-CH type protein 1), reducing HLA-DR lysosomal degradation; (4) the activity of calcium-dependent metalloproteinase (ADAM17) is reduced, and the shedding of HLA-DR extracellular domain is reduced. The above cascade reaction leads to the continuous accumulation of HLA-DR on the surface of monocytes.

[0004] To overcome the instability of EDTA tubes, existing technologies primarily employ chemical fixation strategies. For example, Streck developed the Cyto-Chex BCT tube, which chemically fixes leukocyte surface antigens using an internal formaldehyde releaser, achieving stability at room temperature for 14 days. However, this technology has inherent limitations: formaldehyde covalently cross-links protein amino groups, permanently altering the three-dimensional conformation of HLA-DR, leading to reduced antibody accessibility and a systematic underestimation of approximately 30% in detection results (Monneret et al., 2021). This means that the Cyto-Chex BCT tube cannot accurately reflect the patient's true mHLA-DR expression level, and clinical decision thresholds (such as the sepsis immune paralysis threshold 8000ABC) need to be recalibrated, increasing the complexity of clinical applications.

[0005] Therefore, there is an urgent clinical need for a new type of whole blood collection tube that combines the antigen authenticity of EDTA tubes (no epitope masking) with the stability of Cyto-Chex BCT tubes (long-term storage at room temperature). Summary of the Invention

[0006] Technical issues The purpose of this invention is to provide an EDTA anticoagulated whole blood collection tube that has been systematically screened and validated. By incorporating a three-layer synergistic protective agent composition, it inhibits the compensatory activation cascade of monocytes triggered by EDTA chelation of calcium ions without affecting the native conformation of HLA-DR and antibody binding ability, thereby maintaining the stability of mHLA-DR surface expression during 72h room temperature storage.

[0007] This invention provides a novel EDTA anticoagulated whole blood collection tube that has been systematically screened and validated. Through a three-layer synergistic mechanism of cholesterol hemisuccinate (stabilizing membrane structure), 2-deoxyglucose (inhibiting glycolysis / glycosylation), and sodium azide (inhibiting oxidative phosphorylation), it achieves stable mHLA-DR expression at room temperature for 72 hours (%Delta≤10%) without masking the HLA-DR epitope.

[0008] Technical solution The first aspect of this invention provides an EDTA anticoagulant blood collection tube for stabilizing HLA-DR expression in monocytes, comprising a blood collection tube body, a sealing rubber stopper, an aluminum cap, and a protective agent; the protective agent is pre-placed at the bottom of the blood collection tube in the form of a lyophilized cake, and after blood collection, the blood reconstitutes the lyophilized cake and mixes it uniformly with the protective agent; the protective agent comprises an anticoagulant, a membrane stabilizer, a metabolic inhibitor, a buffer salt, and a lyophilization excipient; wherein, the anticoagulant is EDTA-K2, with a reconstituted concentration of 1.2 mg / mL-2.5 mg / mL; the membrane stabilizer is CHS, with a reconstituted concentration of 0.15 mM-0.25 mM; the metabolic inhibitor comprises 2-DG and NaN3, with reconstituted concentrations of 8 mM-18 mM and 0.015% w / v-0.04% w / v, respectively; the buffer salt is HEPES, with a reconstituted concentration of 15 mM-25 mM; and the lyophilization excipient is selected from trehalose, mannitol, or sucrose.

[0009] In some embodiments, the concentrations of each component after reconstitution of the protective agent are: EDTA-K 21.5 mg / mL-2.2 mg / mL, CHS 0.15 mM-0.25 mM, 2-DG 10 mM-15 mM, NaN 30.02% w / v-0.03% w / v, and HEPES 15 mM-25 mM.

[0010] In some embodiments, the concentrations of each component of the protective agent after reconstitution are: EDTA-K 21.5 mg / mL-2.0 mg / mL, CHS 0.15 mM-0.25 mM, 2-DG 10 mM-15 mM, NaN 30.02% w / v-0.03% w / v, HEPES 18 mM-22 mM.

[0011] In some embodiments, the lyophilization excipient is trehalose, and the concentration after reconstitution is 0.5 mg / mL-1.5 mg / mL.

[0012] In some embodiments, the pH of the system is maintained at 7.2-7.4 after the freeze-dried cake is reconstituted.

[0013] In some embodiments, the moisture content of the freeze-dried cake is ≤3% by mass.

[0014] In some embodiments, the blood collection tube is a sterile vacuum blood collection tube; the sealing stopper is a butyl rubber stopper.

[0015] In some embodiments, after whole blood samples collected by the EDTA anticoagulant blood collection tube are stored at room temperature for 72 hours, the change rate of mHLA-DR ABC value is ≤10%, and the epitope retention rate is >95%.

[0016] In some embodiments, epitope retention rate (%) = standard process ABC / enhanced wash ABC × 100%.

[0017] In some embodiments, the EDTA anticoagulant blood collection tubes with stable monocyte HLA-DR expression can be stored at room temperature for 10-12 months.

[0018] The second aspect of the present invention provides a method for preparing EDTA anticoagulant blood collection tubes with stable HLA-DR expression in monocytes as described in any of the above claims, comprising the following steps: S1. Preparing a protective agent stock solution: HEPES, EDTA-K2·2H2O, 2-DG and trehalose are stirred and dissolved, CHS and NaN3 are added and mixed, the pH is adjusted to 7.2-7.4, and the solution is filtered for sterilization; S2. Preparing a freeze-dried cake: The protective agent stock solution is dispensed and freeze-dried, and after freeze-drying, nitrogen gas is used for protection and the tubes are sealed; S3. Assembling the blood collection tubes: The freeze-dried cake is transferred to the bottom of the blood collection tubes, a sealing rubber stopper and an aluminum cap are installed, vacuum is applied, and the tubes are sterilized by irradiation.

[0019] In some embodiments, the process parameters for freeze drying in step S2 are as follows: cooling to -45°C to -35°C at a rate of 1°C / min and holding for 3.5h to 4.5h; heating to -26°C to -18°C at a rate of 0.5°C / min to 1.0°C / min and drying at a vacuum degree ≥99.99% for 10h to 15h; and heating to 18°C ​​to 22°C at a rate of 0.3°C / min and drying at a vacuum degree ≥99.995% for 4h to 8h.

[0020] A third aspect of the present invention provides the use of EDTA anticoagulation blood collection tubes expressing HLA-DR in stable monocytes as described in any of the preceding claims in the preparation of an in vitro diagnostic kit for the diagnosis of sepsis-induced immune paralysis.

[0021] Technical effect 1) This invention, through systematic screening targeting the specific biological needs of HLA-DR, discovered a synergistic combination of CHS, 2-DG, and NaN3 from candidate compounds. This combination simultaneously achieves 72-hour stability, preservation of native conformation, and direct compatibility with existing thresholds. Furthermore, this invention, through response surface methodology optimization, determined the reasonable concentration range of each component and provided sufficient experimental data support. In addition, this invention directly confirmed the mechanism of action and synergistic principle of each component through experiments such as Western blotting, laser confocal microscopy, flow cytometry endocytosis tracking, and ATP bioluminescence assay. The specific mechanism is explained as follows: First layer of defense (stabilizing membrane structure): EDTA chelates Ca... 2+ Subsequently, the cell membrane lipid raft structure loses Ca2+. 2+ Dependent anchoring and abnormally increased mobility trigger rapid HLA-DR endocytosis. CHS, as a core component of the membrane lipid raft, precisely inserts into the phospholipid bilayer through hydrophobic interactions and the water-lipid interface affinity of the hemisuccinate groups, replacing Ca²⁺. 2+The structural stabilizing function of 2-DG maintains the lateral distribution stability of HLA-DR in the membrane microdomain. Laser confocal microscopy experiments confirmed that after adding CHS to the EDTA-treated model, the co-localization coefficient (Manders M1) of HLA-DR and the lipid raft marker Flotillin-1 recovered from 0.42±0.08 to 0.78±0.05 (close to the physiological level of 0.82±0.04), confirming that CHS directly restored the membrane microdomain localization of HLA-DR. The second layer of defense (conformation locking): 2-DG competitively inhibits hexokinase to block glycolysis and, as an N-linked glycosylation analog, interferes with the glycosylation modification of the nascent HLA-DR α / β chains. Since the surface expression of HLA-DR strictly depends on the complete glycosylation of the endoplasmic reticulum-Golgi pathway, 2-DG effectively blocks the transport of newly synthesized HLA-DR to the cell membrane. Western blot experiments directly confirmed that after 24 h of treatment with 2-DG (12 mM), the glycosylated form of the HLA-DR β chain (approximately 34 kDa) was significantly reduced, while the unglycosylated precursor (approximately 29 kDa) accumulated. After digestion with endoglycosidase H (Endo H), the band positions of the treated and control groups tended to be consistent, confirming that 2-DG specifically blocked high-mannose glycosylation modification. This effect was reversible and could be completely removed by washing with PBS or standard lysis steps before detection. After washing, cells restored normal glycosylation levels and HLA-DR surface expression. The third layer of defense (freezing energy metabolism): NaN3 inhibited mitochondrial cytochrome c oxidase (Complex IV), blocking oxidative phosphorylation. In synergy with 2-DG, it achieved dual-target energy deprivation of glycolysis and oxidative phosphorylation, reducing the ATP level of monocytes to 3.8% ± 1.2% of the basal level (ATP bioluminescence assay). Flow cytometry endocytosis tracking experiments confirmed that, under NaN3 treatment, the endocytosis rate of HLA-DR-PE antibody after incubation at 37°C for 30 min decreased from 62.3%±8.1% in the control group to 4.7%±2.3%, confirming that the vesicle transport system was effectively frozen. Dynamic observation using laser confocal microscopy showed that the intracellular motility of HLA-DR-positive vesicles decreased from 0.42 μm / s in the control group to below 0.03 μm / s, approaching complete quiescence. A three-layer synergistic effect was observed: CHS inhibited the endocytosis and loss of existing HLA-DR, 2-DG blocked the replenishment of newly synthesized HLA-DR, and NaN3 froze vesicle transport dynamics. These three factors synergistically achieved stock stabilization, rather than chemical fixation by Cyto-Chex BCT tubes. Therefore, after removing the protective agent using standard washing steps before detection, the HLA-DR epitopes almost completely recovered their native conformation, with no systematic underestimation.It is particularly important to emphasize that this synergistic effect is highly selective: the correction magnitude of the composition for each marker is gradient (approximately 97% for HLA-DR, approximately 42% for CD80, approximately 37% for CD86, approximately 34% for CD163, and no drift or correction required for CD14), consistent with its mechanism dependence on EDTA-induced cascade reactions. This confirms that its effect is not simply cell death or total freezing, but rather a selective intervention targeting the biological pathways of HLA-DR (glycosylation-secretion, lipid raft anchoring, endocytosis-circulation).

[0022] 2) Under 72-hour storage conditions, the ABC value increase rate of mHLA-DR in conventional EDTA tubes exceeds 100%, while the ABC value change rate of the blood collection tubes of this invention is ≤10%, and the 8000-15000 ABC threshold system established by existing EDTA tubes can be directly used without recalibration. This advantage stems from the complete preservation of the natural conformation of HLA-DR (eptopy retention rate >95%), rather than chemical modification of the detection signal. Therefore, it is fully compatible with the BD Quantibrite kit and standard lysis and washing procedures, without requiring changes to existing laboratory operating procedures. In contrast, the formaldehyde fixation method (Cyto-Chex BCT tubes) results in approximately 30% systematic underestimation due to irreversible epitope masking (masking rate approximately 25%), requiring calibration of the detection values ​​before use, and thus failing to achieve the direct compatibility of this invention.

[0023] 3) No epitope masking and completely reversible conformation: Epitope retention rate >95% as determined by enhanced wash-recovery method, no correction factor required. This is fundamentally different from the irreversible epitope masking (approximately 25%) of the formaldehyde fixation scheme (Cyto-Chex BCT tube). The protective agent of this invention can be completely removed by standard washing steps, and the HLA-DR natural conformation is 100% restored.

[0024] 4) Specific stability rather than general cytotoxicity: While achieving HLA-DR stability, the monocyte survival rate remained above 85% at 72h (trypan blue exclusion method), confirming that the protocol is not a simple cell killing or general metabolic freeze, but a precise regulation of the HLA-DR specific pathway.

[0025] 5) Controllable cost and secure supply chain: The protective agent components are all conventional biochemical reagents, eliminating the need for imported special blood collection tubes. The cost per tube is only about 1 / 5 of that of Cyto-Chex BCT tubes, significantly reducing medical costs.

[0026] 6) Anti-interference ability: When common clinical interference factors (such as hyperlipidemia and hemolyzed samples) are present, the coefficient of variation (CV) of the mHLA-DR test results of the blood collection tube of the present invention is still less than 8%, which is significantly better than conventional EDTA tubes (CV>15%). Detailed Implementation

[0027] To facilitate the implementation of the technical solutions applied for, the terms and expressions involved in this invention will first be explained and defined in general terms and expressions below.

[0028] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0029] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0030] The following provides a further description of the EDTA anticoagulant blood collection tube with stable monocyte HLA-DR expression and its preparation method provided by the present invention.

[0031] Example 1: Preparation and Packaging of Protective Agent Composition 1.1 Reagents and Materials Table 1. Reagents and Materials

[0032] 1.2 Preparation of Protectant Solution Table 2. Components for preparing mother liquor (10× concentrate, 500 μL per tube)

[0033] Preparation steps: (1) Take a clean beaker and add about 35 mL of ultrapure water; (2) Add 2.383g HEPES, 0.9g EDTA-K2·2H2O, 0.985g 2-DG and 0.5g trehalose in sequence, and stir magnetically at room temperature for 10 minutes until completely dissolved; (3) 48.7 mg CHS was first dissolved in 0.5 mL of anhydrous DMSO (ultrasonic treatment for 2 min, power 100 W), and then slowly added dropwise to the above aqueous solution while stirring. The final volume of DMSO was <1% (about 0.5% v / v), which is non-toxic to cells. (4) Add 0.125g of NaN3 and stir until completely dissolved; (5) Fine-tune the pH to 7.25±0.05 using 1M NaOH or 1M HCl; (6) Adjust the volume to 50 mL and filter through a 0.22 μm sterile filter for sterilization; (7) Dispense into clean vials, 500 μL per vial, for a total of 100 vials.

[0034] 1.3 Freeze-drying Using standard freeze-drying procedures (Christ Alpha 2-4 LSCplus freeze dryer): (1) Pre-freezing: Cool to -40℃ at a rate of 1℃ / min and hold for 4 hours; (2) First drying: Heat to -20℃ at 0.5℃ / min for 12 hours, with a vacuum degree ≥99.99%; (3) Second drying: Heat to +20℃ at 0.3℃ / min, for 6 hours, with a vacuum degree ≥99.995%; (4) Sealing: After the freeze-drying is completed, high-purity nitrogen (99.999%) is used for protection, and the rubber stopper is used for sealing.

[0035] After freeze-drying, the freeze-dried cake is white and loose, with residual moisture ≤3% (determined by Karl Fischer method), and reconstitution time ≤30s (complete reconstitution is achieved by infusion of 5mL of whole blood).

[0036] 1.4 Blood collection tube assembly (1) Transfer the freeze-dried cake to the bottom of a 5mL vacuum blood collection tube, ensuring that the freeze-dried cake is centered at the bottom of the tube; (2) Install the butyl rubber stopper and press-seal the aluminum cap; (3) Vacuum each tube to the preset blood collection volume of 5 mL; (4) Sterilization by Co-60 irradiation (25 kGy); (5) Label, package, and store in a dry environment at room temperature.

[0037] Example 2: Systematic Screening Experiment for a Single Component 2.1 Screening Experiment of Membrane Stabilizer System 1) Experimental Objective EDTA chelates Ca 2+ In an in vitro model, the effects of 10 candidate membrane stabilizers on the integrity of lipid rafts in monocyte membranes and HLA-DR expression were systematically compared to screen for the optimal membrane stabilizer.

[0038] 2) Experimental materials Candidate membrane stabilizers: Cholesterol, phosphatidylserine, sphingomyelin, cholesterol sulfate, cholesterol hemisuccinate (CHS), phosphatidylcholine, ceramide, dihydrosphingosine, phytosterol, 7-ketocholesterol.

[0039] 3) Experimental methods Whole blood was collected from healthy donors using standard EDTA tubes and divided into 11 groups (10 candidate membrane stabilizers + blank control). Immediately after blood collection, the corresponding membrane stabilizer (final concentration 0.20 mM) was added to each group, and the mixture was stored at room temperature for 72 hours before flow cytometry analysis. Lipid raft integrity was evaluated using the colocalization coefficient of HLA-DR and Flotillin-1 (Manders M1, laser confocal microscopy). The 72-hour mHLA-DR change rate was determined using the BD Quantibrite standard procedure, with each group's own 0-hour ABC as 100% reference: 72-hour mHLA-DR change rate (%) = [(mHLA-DR...]...] ABC72h -mHLA-DR ABC0h ) / mHLA-DR ABC0h The mHLA-DR epitope retention rate was independently determined using the enhanced wash-recovery method: each 72-hour sample was divided into two parts. One part was processed according to the BD Quantibrite standard lysis-wash procedure, and the other part was washed twice with PBS after lysis (2 mL PBS resuspended each time, centrifuged at 200×g for 5 min). The epitope retention rate (%) = standard procedure ABC / enhanced wash ABC × 100%. This indicator reflects epitope accessibility; a retention rate <95% indicates epitope occlusion. The above two indicators correspond to the two independent effects of changes in total antigen amount and epitope accessibility under EDTA conditions, respectively, avoiding the inability to interpret values ​​due to the mixing of two effects by a single formula. The Manders colocalization coefficient M1 was calculated using z-stack sequences (0.5 μm step, 20 layers) acquired by a Zeiss LSM 880 laser confocal microscope and the Fiji plugin of ImageJ software (the threshold was automatically set, and the proportion of colocalized pixels was calculated after background subtraction). Cell viability was determined using the trypan blue exclusion method combined with a Countstar automated cell counter (final trypan blue concentration 0.4%, 500 cells / sample). Cell viability (%) = (number of viable cells / total number of cells) × 100%.

[0040] 4) Experimental Results Table 3. Screening results of membrane stabilizers (n=6, mean ± SD)

[0041] As shown in Table 3, CHS performed best among the 10 candidate membrane stabilizers: the 72h mHLA-DR change rate was +42.5%, significantly lower than other candidate membrane stabilizers (all >45%), the lipid raft colocalization coefficient recovered to 0.78 (close to the physiological level of 0.82), and the cell viability was 92.7%. However, CHS alone cannot meet the clinical application standard of %Delta≤10%, suggesting that stabilizing the membrane structure alone is insufficient to block the surface accumulation of HLA-DR, and it is necessary to combine it with metabolic inhibitors to block its new synthesis and endocytosis pathways. The epitope retention rates of each group were between 94% and 99% (enhanced wash-recovery method), confirming that none of the candidate membrane stabilizers had formaldehyde-like epitope masking, and the difference in their stabilizing effect stemmed from the actual antigen accumulation inhibition ability rather than the difference in epitope masking; the epitope retention rate of the blank control group was 98.6%, confirming that the increase in ABC in the conventional EDTA tubes after 72h was due to the actual accumulation of surface antigens, rather than changes in the number or conformation of epitopes.

[0042] 2.2 Screening Experiments for Metabolic Inhibitor Systems 1) Experimental Objective Based on the glycosylation-dependent characteristics of HLA-DR, we systematically compared the effects of 12 candidate metabolic inhibitors on the new synthesis and surface expression of HLA-DR, and screened the optimal glycolysis / glycosylation inhibitor.

[0043] 2) Experimental materials Candidate metabolic inhibitors: 2-DG, oligomycin, rotenone, antimycin A, fluorodeoxyuridine, 6-mercaptopurine, sirolimus, actinomycin (CHX), 3-bromopyruvic acid (3-BrPA), sodium oxalate, dichloroacetic acid, niclosamide.

[0044] 3) Experimental methods Healthy donor EDTA-anticoagulated whole blood was used and divided into 13 groups (12 candidate inhibitors + blank control). Each group received the corresponding effective concentration of a candidate inhibitor immediately after blood collection (due to significant differences in potency among different inhibitors, such as 2-DG's inhibition of ECG glycosylation). 50 Approximately 5 mM, while the IC50 of niclosamide is... 50Since the concentration is only about 0.5 μM, a uniform concentration was not used. Instead, the generally accepted effective concentrations were selected as the final concentrations for screening to reflect the true dose-response relationship. The mHLA-DR change rate after 72 h at room temperature was detected using the BD Quantibrite standard procedure. The HLA-DR glycosylation blocking rate was evaluated by Western blotting (anti-HLA-DR α chain antibody, Santa Cruz) and digestion experiments with endonuclease H (Endo H, New England Biolabs). The apoptosis rate and cell necrosis rate were detected by Annexin V-FITC / PI double staining. The ATP content was determined by CellTiter-Glo bioluminescence assay.

[0045] 4) Experimental Results Table 4. Screening results of metabolic inhibitors (n=6, mean ± SD)

[0046] Results Analysis: As shown in Table 4, 2-DG exhibited unique characteristics among the 12 candidate inhibitors: its glycosylation blocking rate reached 78.3%, while the apoptosis rate was only 5.8% and the necrosis rate was 1.2%, confirming its highly specific and low-cytotoxic blocking effect on HLA-DR neosynthesis. Although CHX could completely inhibit protein synthesis, it caused cellular stress response due to the simultaneous inhibition of all protein synthesis, and was not HLA-DR specific; moreover, after CHX inhibited protein synthesis, HLA-DR on the cell surface still accumulated due to reduced endocytosis (72h mHLA-DR change rate +45.2%), and could not achieve stabilization on its own. Although oligomycin, rotenone, and antimycin A could significantly reduce ATP, their glycosylation blocking rate was <15%, and they caused significant apoptosis (>18%), indicating that simple energy deprivation was insufficient to block HLA-DR neosynthesis. These results reveal that for glycoproteins like HLA-DR, it is necessary to directly block their glycosylation-secretion pathway rather than just inhibiting energy metabolism, a finding that exceeds the conventional understanding of those skilled in the art.

[0047] 2.3 Screening and Co-validation Experiments of Oxidative Phosphorylation Inhibitors 1) Experimental Objective Based on the determination of CHS and 2-DG, the optimal oxidative phosphorylation inhibitor was screened, and the synergistic effect of the three components was verified.

[0048] The reason why oxidative phosphorylation inhibitors need to be screened based on the established CHS and 2-DG is that membrane stabilizers act directly on the cell membrane structure and exert their stabilizing effect without relying on other components, thus requiring independent screening. Metabolic inhibitors (2-DG) primarily block the HLA-DR glycosylation-secretion pathway, which is independent of membrane lipid raft integrity and can theoretically be independently evaluated in the absence of membrane stabilizers. Oxidative phosphorylation inhibitors (NaN3), on the other hand, inhibit ATP generation to freeze vesicle transport. Their effectiveness is influenced by baseline energy metabolism levels and must be evaluated based on a defined formulation of CHS (maintaining membrane structure) and 2-DG (reducing new synthetic load) to accurately reflect their freezing effect on existing HLA-DR endocytosis and cycling.

[0049] 2) Experimental materials Candidate inhibitors of oxidative phosphorylation: NaN3, oligomycin, 4-trifluoromethoxyphenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), BAM15, mitoxazine, metformin.

[0050] 3) Experimental methods In a basic formulation containing CHS (0.20 mM) and 2-DG (12 mM), the above seven oxidative phosphorylation inhibitors were used in combination (final concentrations referencing commonly used effective concentrations in the literature), and the cells were stored at room temperature for 72 h before analysis. Vesicle transport dynamics (vesicle movement velocity) were evaluated using laser confocal microscopy live-cell imaging (HLA-DR-GFP transfected THP-1 mononuclear cell line); mitochondrial membrane potential was detected by JC-1 staining; cell viability was determined by trypan blue exclusion method combined with a Countstar automated cell counter (final trypan blue concentration 0.4%, count 500 cells / sample). PE (phycoerythrin) fluorescence interference was detected by setting up negative control tubes treated with the same concentration of inhibitor but without fluorescently labeled antibodies: using the same flow cytometer PE fluorescence channel (488nm excitation, 585 / 42nm bandpass filter) as the BD Quantibrite standard procedure, the autofluorescence intensity (MFI) of unstained cells after inhibitor treatment was measured. With the unstained cells without inhibitor as the baseline, the autofluorescence increase (%) was calculated as (inhibitor group MFI - baseline MFI) / baseline MFI × 100%. An increase of ≤2% was considered as no interference.

[0051] 4) Experimental Results Table 5. Screening results of oxidative phosphorylation inhibitors (n=6, mean ± SD)

[0052] Results analysis: NaN3 at a concentration of 0.025% w / v showed the best performance, reducing the 72-hour mHLA-DR change rate to +4.2% and vesicle motility to 0.03 μm / s (near complete quiescence), while maintaining cell viability at 92.3% and without interfering with PE fluorescence channels. While oligomycin also showed some effect, cell viability significantly decreased to 75.2%. FCCP and DNP induced apoptosis by disrupting mitochondrial membrane potential, and FCCP interfered with PE fluorescence (+8.5%). These results confirm the unique advantages of NaN3 as an inhibitor of oxidative phosphorylation.

[0053] Example 3: Validation of Synergistic Effect Whole blood from healthy donors was collected using standard EDTA tubes. A 2×2×2 total factor assay was used (CHS: 0 / 0.20 mM; 2-DG: 0 / 12 mM; NaN3: 0 / 0.025% w / v). The corresponding concentrations of candidate preservatives were added immediately after blood collection. Eight groups of samples were stored at room temperature for 72 hours before testing. The results are shown in Table 6. Table 6. Validation of the synergistic effect of the three components (n=8, mean ± SD)

[0054] Note: "-" represents 0 nM, and "+" represents the respective concentrations (CHS: 0.20 mM; 2-DG: 12 mM; NaN3: 0.025% w / v).

[0055] Results analysis: The mHLA-DR change rate of the three-component combination (4.2%) was lower than that of any pairwise or single-component combination, confirming a significant superadditive synergistic effect. This synergistic effect could not be predicted by simply adding the known functions of each component.

[0056] Example 4: Optimization experiment of formulation concentration range (Box-Behnken design) 4.1 Experimental Design Based on the confirmed synergistic effect of CHS, 2-DG, and NaN3, the concentrations of the three components were optimized using a Box-Behnken response surface design (BBD). The 72-h mHLA-DR change rate (Y1, %) and epitope retention rate (Y2, %) (enhanced wash-recovery method) were used as response values. Each factor was set with 3 levels (-1, 0, +1), as shown in Table 7, for a total of 17 experimental points (N=2×3×(3-1)+5; including 5 center points).

[0057] Table 7. Three-component factors and levels

[0058] 4.2 Experimental Results Response surface methodology was performed using Design-Expert 13 software, resulting in the following quadratic regression equation: Y1=4.85-1.26A+0.85B-2.15C+0.42AB+0.18AC-0.25BC+2.35A2+1.82B2+1.56C2; Y2=96.2+1.85A-0.62B+2.42C-0.35AB+0.28AC-0.15BC-1.92A2-1.45B2-1.68C2.

[0059] The model's R² = 0.968, Adj R² = 0.935, and Pred R² = 0.812 indicate that the model fits well and has predictive value.

[0060] Response surface analysis shows that: (1) 2-DG had a significant effect on both Y1 and Y2 in the range of 8-18 mM (p<0.01), with the optimal range being 10-15 mM. Below 8 mM, glycolysis inhibition was insufficient, with Y1>15%; above 18 mM, cytotoxicity increased, with Y2<90%.

[0061] (2) NaN3 had a significant negative effect on Y1 in the range of 0.015%-0.04% (p<0.05), with the optimal range being 0.02%-0.03%. Below 0.015%, oxidative phosphorylation was not completely inhibited, and Y1>12%; above 0.04%, cell viability was <70%, and Y2<85%.

[0062] (3) CHS had a significant effect on both Y1 and Y2 in the range of 0.10-0.30 mM (p<0.01), with the optimal range being 0.15-0.25 mM. Below 0.10 mM, the membrane stabilization effect was insufficient, with Y1>20%; above 0.30 mM, excessive hydrophobic insertion led to a reverse decrease in membrane fluidity, restricting HLA-DR lateral diffusion and reducing antibody binding efficiency, with Y2<92%.

[0063] (4) The interaction terms AB (2-DG×NaN3) and AC (2-DG×CHS) are both positive, indicating that 2-DG and NaN3, and 2-DG and CHS have a synergistic enhancement effect; while BC (NaN3×CHS) is negative but has a small absolute value, indicating that the interaction between the two is weak.

[0064] 4.3 Verification Experiment Six verification points were randomly selected from the predicted optimal region, with six donors in each group. The relative deviations between the experimentally measured values ​​and the model predictions were all <8%, confirming the reliability of the model's predictions. The final concentration ranges for this invention were determined as follows: 2-DG 8mM-18mM (preferably 10mM-15mM), NaN 0.015%-0.04% (preferably 0.02%-0.03%), and CHS 0.10mM-0.30mM (preferably 0.15mM-0.25mM).

[0065] Example 5: Stability verification of the final optimized formulation (healthy donor) 5.1 Experimental Methods Table 8. Experimental Materials

[0066] Note: Final optimized formulation (12mM 2-DG + 0.025%w / v NaN3 + 0.20mM CHS + 20mM HEPES + 1.8mg / mL EDTA-K2, pH 7.25).

[0067] The experimental materials are shown in Table 8. 15 mL of venous blood was collected from each donor and evenly distributed into three types of blood collection tubes (5 mL per tube): conventional EDTA tubes (control), Cyto-Chex BCT tubes (commercially available blood collection tubes), and the blood collection tubes of this invention (final optimized formula). The tubes were stored at room temperature (22±2℃) protected from light. 50 μL of whole blood was collected at 0 h, 24 h, 48 h, and 72 h, and processed according to the BD Quantibrite standard procedure, with three replicates per tube at each time point.

[0068] 5.2 Experimental Results Table 9. Results of mHLA-DR ABC stability validation in healthy donors (mean ± SD, n = 10 cases × 3 replicates)

[0069] Results analysis: 1) Conventional EDTA tubes: The average rise rate over 72 hours relative to 0 hours was 103.5% (range: 90.2%-115.3%). 2) Cyto-Chex BCT tubes: The average change rate of 72h relative to 0h is -2.0% (time dimension, based on its own 0h baseline), showing excellent stability during storage; however, its absolute value is systematically 27.3% lower than that of conventional EDTA tubes from 0h onwards (15433 vs 21248) (accuracy dimension, based on the true baseline of EDTA tubes at 0h). This lower value is due to the formaldehyde cross-linking fixation that is completed immediately after blood collection, which remains constant at each time point. 3) The blood collection tube of this invention: the average increase rate of 72h relative to 0h is only 2.8% (range: -1.8% to +8.6%), which meets the preset acceptance criteria (mean %Delta≤10%), and the absolute value of 0h (20657) is not statistically different from that of conventional EDTA tube (21248) (paired t test, p=0.312), confirming that there is no baseline epitope occlusion.

[0070] 5.3 Specificity Validation The 72-hour change rates of other mononuclear cell markers (CD14, CD86, CD80, CD163) were simultaneously detected in the same sample, and the results are shown in Table 10: Table 10. 72-hour variation rate of CD14, CD86, CD80, and CD163

[0071] Note: HLA-DR data are from Table 9.

[0072] Results Analysis: The blood collection tubes of this invention exhibited high selectivity and stability for HLA-DR. It should be noted that selectivity was evaluated based on the correction margin for EDTA-induced drift, rather than a direct comparison of the absolute values ​​of the changes in each marker: CD14 is a GPI-anchored protein, and its surface turnover does not depend on the MARCH1-regulated endocytosis-circulation pathway. It itself showed no significant drift in conventional EDTA tubes (-2.3%), and there were no statistically significant differences among the three types of blood collection tubes (p>0.05), classifying it as a background-stable marker, confirming that the protective agent does not alter the detection baseline of the naturally stable antigen. HLA-DR drifted most dramatically in conventional EDTA tubes (+103.5%), which was corrected to +2.8% in the blood collection tubes of this invention, a correction margin of approximately 97%. CD86 and CD80 showed moderate drift (+45.2% and +38.6%), which were corrected to +28.6% and +22.4%, respectively, with correction margins of approximately 37% and 42%. CD163 showed relatively small drift (+12.5%), corrected to +8.3%. The corrected magnitude gradients of each marker (HLA-DR >> CD80 > CD86 > CD163 > CD14 ≈ 0) and their effects on the EDTA-induced cascade reaction (Ca 2+ The mechanistic dependence gradient of chelation-MARCH1 downregulation-accelerated endocytosis cycle is completely consistent. CD86 and CD80 are both glycosylated membrane proteins, and their surface expression is partially dependent on glycosylation and vesicle transport. The slight influence of 2-DG / NaN3 is consistent with the expected mechanism. This result confirms that this invention is not a simple complete cell freezing or cell death fixation (if it were complete freezing, all membrane markers should show uniform changes similar to Cyto-Chex BCT tubes), but a precise intervention targeting the highly selective biological pathways of HLA-DR (glycosylation-secretion, lipid raft anchoring, endocytosis-circulation).

[0073] Example 6: Reversibility Verification (Epitope Masking / Recovery Experiment) 6.1 Experimental Principle The formaldehyde-releasing agent in Cyto-Chex BCT tubes permanently alters the three-dimensional conformation of HLA-DR by covalently cross-linking protein amino groups, leading to reduced antibody accessibility. This masking effect is irreversible in standard washing steps. This invention employs a reversible metabolic inhibition and membrane stabilization strategy; theoretically, thorough washing with PBS before detection can remove the protective agent and restore the native conformation of HLA-DR.

[0074] 6.2 Experimental Methods Six healthy donor samples were collected in conventional EDTA tubes, Cyto-Chex BCT tubes, and the blood collection tubes of this invention, respectively. After being stored at room temperature for 72 hours, three parallel treatments were performed: (1) Standard process group: Process once according to the BD Quantibrite standard pyrolysis-washing process; (2) Enhanced washing group: After lysis, 2 more PBS washes were added (3 washes in total), each time with 2 mL PBS resuspended and centrifuged at 200×g for 5 min; (3) Super-enhanced washing group: After lysis, 4 more PBS washes were added (5 washes in total), each time 2 mL PBS was used for resuspending, and centrifugation was performed at 200×g for 5 min.

[0075] Compare the mHLA-DR ABC values ​​of each group and calculate the recovery rate = enhanced washing group / standard process group × 100%.

[0076] 6.3 Experimental Results Table 11. Comparison of mHLA-DR ABC before and after washing (n=6, after 72h room temperature storage)

[0077] Results analysis: 1) Standard EDTA tubing: average recovery rate 99.5% ± 2.3%, confirming that the standard washing process itself does not cause HLA-DR loss; 2) Cyto-Chex BCT tubes: The average recovery rate was 133.1% ± 5.9%, confirming significant epitope shading (shading rate of approximately 24.8%, i.e., 1-1 / 1.331). After enhanced washing, partial recovery was achieved, but the level still did not reach EDTA levels. After super-enhanced washing, the recovery rate was 133.8%, which was not significantly different from that after enhanced washing, confirming that formaldehyde crosslinking is an irreversible modification. 3) The blood collection tubes of this invention: the average recovery rate was 99.5% ± 3.2%, which was not statistically different from EDTA tubes (p = 0.987), confirming that there was no irreversible epitope masking. The recovery rate after super-enhanced washing was 98.9%, further confirming that the protective agent could be completely removed.

[0078] Example 7: Clinical Sample Validation (Sepsis Patients) 7.1 Experimental Materials Patients with sepsis: 12 cases from the ICU, meeting the Sepsis-3 diagnostic criteria, with APACHE II scores of 15-28. Venous blood was collected in three types of blood collection tubes, stored at room temperature for 72 hours, and then centrally tested.

[0079] 7.2 Experimental Results Table 12. Stability validation of sepsis patient samples over 72 hours (ABC values)

[0080] Results analysis: 1) EDTA tube: average increase of 103.7% over 72 hours; Cyto-Chex BCT tube: average change of -28.4% over 72 hours; blood collection tube of this invention: average change of +3.4% over 72 hours. All 12 patients met the acceptance criteria of %Delta≤10%.

[0081] 2) Clinical decision impact analysis: Taking the diagnostic threshold of 8000ABC for sepsis immune paralysis as an example: 72 hours after EDTA tube, 3 out of 12 patients (P05, P08, P12) were misdiagnosed as having recovered immune function due to false elevation of immune paralysis; 4 patients (P03, P05, P08, P12) were misdiagnosed as having immune paralysis due to systemic hypotension of immune function; the blood collection tube of this invention does not have such misdiagnosis and can directly use the clinical threshold established by existing EDTA tubes.

[0082] Example 8: Methodological Evaluation 8.1 Precision Verification Five whole blood samples were collected from healthy donors using the blood collection tubes of this invention. After being stored at room temperature for 24 hours (simulating a clinical transportation scenario), each sample underwent three independent staining-lysis-washing-testing cycles to assess intra-batch reproducibility. The results are shown in Table 13.

[0083] Table 13. Precision Validation Data

[0084] Results analysis: The average intra-assay CV was 3.97% (range 2.93%-4.59%), which meets the precision requirements for flow cytometry immunophenotyping (typically accepted standard CV ≤ 20%).

[0085] 8.2 Verification of Interference Factors Simulated hyperlipidemia samples (triglycerides added to a final concentration of 5 mmol / L), hemolyzed samples (mechanical hemolysis, final hemoglobin concentration of 2 g / L), and high bilirubin samples (bilirubin concentration of 200 μmol / L) were prepared separately, collected in the blood collection tubes of this invention, and tested after being stored at room temperature for 72 hours.

[0086] Table 14. Validation data of interference factors

[0087] Results Analysis: Under the three interference conditions, the 72-hour mHLA-DR change rates of the blood collection tubes of this invention were +5.2%±2.1%, +6.8%±2.5%, and +5.5%±2.3%, respectively, with CVs of 6.2%, 7.8%, and 6.5%, respectively, all meeting the acceptance criteria of %Delta≤10% and CV≤20%. In contrast, the 72-hour change rates of conventional EDTA tubes under the same interference conditions were +112.5%±15.3%, +118.2%±16.5%, and +108.6%±14.2%, respectively, with CVs of 18.5%, 21.2%, and 17.8%, respectively, indicating that the blood collection tubes of this invention have significant anti-interference advantages.

[0088] Example 9: Direct Verification Experiment of Mechanism of Action 9.1 The inhibitory effect of 2-DG on HLA-DR glycosylation (Western Blot) Whole blood was collected from healthy donors and incubated with 2-DG (12 mM), sodium oxalate (20 mM, as a glycolysis inhibition control), or PBS (control). Mononuclear cells were then isolated (CD14+ magnetic bead sorting). Total protein was extracted and subjected to SDS-PAGE and Western blotting (anti-HLA-DR α chain antibody, 1:1000). Some samples were digested with endoglucosidase H (Endo H, 500 U, 37°C, 1 h) and analyzed.

[0089] Results analysis: The control group showed typical HLA-DR α-chain glycosylation bands (approximately 34 kDa) and unglycosylated precursors (approximately 29 kDa); the 2-DG treatment group showed a significant reduction in glycosylation bands and accumulation of unglycosylated precursors; after Endo H digestion, the glycosylation band in the control group shifted to the same position as the unglycosylated precursor, while the band position in the 2-DG treatment group showed no significant change, confirming that 2-DG had pre-blocked high-mannose glycosylation modification. The glycosylation bands in the sodium oxalate treatment group showed no significant difference from those in the control group, confirming that simple glycolysis inhibition (without blocking glycosylation) is insufficient to affect the glycosylation state of HLA-DR. This result directly confirms the mechanism by which 2-DG inhibits the surface expression of HLA-DR by blocking its glycosylation.

[0090] 9.2 The protective effect of CHS on the integrity of membrane lipid rafts (laser confocal microscopy) THP-1 cell lines were used and treated for 2 hours with EDTA (5 mM, simulating the EDTA environment after blood collection), EDTA+CHS (0.20 mM), or PBS (control). Immunofluorescence staining was performed after fixation: anti-HLA-DR-FITC (green) and anti-Flotillin-1-PE (red, lipid raft marker). Manders colocalization coefficient M1 was observed and calculated using a laser confocal microscope (Zeiss LSM 880).

[0091] Results analysis: In the control group, HLA-DR and Flotillin-1 showed significant colocalization (M1=0.82±0.04); in the EDTA-treated group, colocalization was significantly reduced (M1=0.42±0.08), and HLA-DR showed intracellular aggregation; in the EDTA+CHS-treated group, colocalization recovered to near control levels (M1=0.78±0.05), and intracellular aggregation was significantly reduced. These results directly confirm the mechanism by which CHS protects HLA-DR membrane localization by maintaining lipid raft integrity.

[0092] 9.3 The freezing effect of NaN3 on vesicle transport (flow cytometry endocytosis tracking) Whole blood was collected from healthy donors and pre-incubated at 37°C for 30 min with NaN3 (0.025% w / v), oligomycin (5 μM), or PBS (control). Anti-HLA-DR-PE antibody (clone L243, BD Biosciences) was added and incubated at 37°C for 30 min (allowing for endocytosis). Endocytosis was terminated with ice-cold PBS. For some samples, surface-bound antibodies were removed using acidic washing buffer (pH 2.5, 0.5 M NaCl + 0.1 M glycine), retaining only the endocytic antibodies. Flow cytometry was used to detect the endocytosis rate.

[0093] Results analysis: The endocytosis rate in the control group was 62.3% ± 8.1%; the endocytosis rate in the NaN3-treated group decreased to 4.7% ± 2.3%; and the endocytosis rate in the oligomycin-treated group was 15.6% ± 4.2%. These results directly confirm that NaN3 effectively freezes the transport of HLA-DR endocytic vesicles by inhibiting oxidative phosphorylation, and its effect is superior to other oxidative phosphorylation inhibitors.

[0094] 9.4 ATP Level Detection The ATP content of monocytes after treatment with each formulation was detected using the ATP bioluminescence assay (CellTiter-Glo).

[0095] Results analysis: After 24 h of treatment with the blood collection tubes of this invention (0.20 mM CHS + 12 mM 2-DG + 0.025% w / v NaN3), ATP levels decreased to 3.8% ± 1.2% of the baseline level; 2-DG treatment alone resulted in 45.2% ± 5.8%; NaN3 treatment alone resulted in 22.4% ± 3.5%; and the baseline formulation (CHS + 2-DG) resulted in 18.5% ± 2.8%. These results confirm the mechanism by which the three components synergistically achieve deep energy deprivation.

[0096] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A stable EDTA anticoagulant blood collection tube expressing HLA-DR in monocytes, characterized in that, The product includes a blood collection tube body, a sealing rubber stopper, an aluminum cap, and a protective agent; the protective agent is pre-placed at the bottom of the blood collection tube in the form of a freeze-dried cake, and after blood collection, the blood will re-dissolve the freeze-dried cake and mix it evenly with the product. The protective agent comprises an anticoagulant, a membrane stabilizer, a metabolic inhibitor, a buffer salt, and a lyophilization excipient; wherein the anticoagulant is EDTA-K2, with a reconstituted concentration of 1.2 mg / mL-2.5 mg / mL; the membrane stabilizer is CHS, with a reconstituted concentration of 0.15 mM-0.25 mM; the metabolic inhibitors include 2-DG and NaN3, with reconstituted concentrations of 8 mM-18 mM and 0.015% w / v-0.04% w / v, respectively; the buffer salt is HEPES, with a reconstituted concentration of 15 mM-25 mM; and the lyophilization excipient is selected from trehalose, mannitol, or sucrose.

2. The EDTA anticoagulant blood collection tube with stable monocyte HLA-DR expression according to claim 1, characterized in that, After reconstitution of the protective agent, the concentrations of each component are as follows: EDTA-K2 1.5 mg / mL - 2.2 mg / mL, CHS 0.15 mM - 0.25 mM, 2-DG 10 mM - 15 mM, NaN 3 0.02% w / v - 0.03% w / v, HEPES 15 mM - 25 mM.

3. The EDTA anticoagulant blood collection tube with stable monocyte HLA-DR expression according to claim 1, characterized in that, The lyophilization excipient is trehalose, with a concentration of 0.5 mg / mL to 1.5 mg / mL after reconstitution.

4. The EDTA anticoagulant blood collection tube with stable monocyte HLA-DR expression according to claim 1, characterized in that, After the freeze-dried cake was reconstituted, the pH of the system was maintained at 7.2-7.

4.

5. The EDTA anticoagulant blood collection tube with stable monocyte HLA-DR expression according to claim 1, characterized in that, The moisture content of the freeze-dried cake is ≤3% by mass.

6. The EDTA anticoagulant blood collection tube with stable monocyte HLA-DR expression according to claim 1, characterized in that, The blood collection tube is a sterile vacuum blood collection tube; the sealing stopper is a butyl rubber stopper.

7. The EDTA anticoagulant blood collection tube with stable monocyte HLA-DR expression according to claim 1, characterized in that, The whole blood samples collected from the EDTA anticoagulant blood collection tubes, after being stored at room temperature for 72 hours, showed a change rate of ≤10% in the ABC value of mHLA-DR and an epitope retention rate >95%.

8. A method for preparing an EDTA anticoagulant blood collection tube expressing HLA-DR in stable monocytes as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Preparation of the protective agent stock solution: Dissolve HEPES, EDTA-K2·2H2O, 2-DG and trehalose by stirring, add CHS and NaN3 and mix well, adjust the pH to 7.2-7.4, and filter to sterilize; S2. Preparation of freeze-dried cake: After the mother liquor of the preservative is dispensed into portions, it is freeze-dried, and then protected with nitrogen and sealed after completion; S3. Blood collection tube assembly: Transfer the freeze-dried cake to the bottom of the blood collection tube, install the sealing rubber stopper and aluminum cap, vacuum, and sterilize by irradiation.

9. The method for preparing EDTA anticoagulant blood collection tubes with stable monocyte HLA-DR expression according to claim 8, characterized in that, The freeze-drying process parameters in step S2 are as follows: cooling to -45℃ to -35℃ at 1℃ / min and holding for 3.5h to 4.5h; heating to -26℃ to -18℃ at 0.5℃ / min to 1.0℃ / min and drying at a vacuum degree ≥99.99% for 10h to 15h; and heating to 18℃ to 22℃ at 0.3℃ / min and drying at a vacuum degree ≥99.995% for 4h to 8h.

10. Use of the EDTA anticoagulation blood collection tube expressing HLA-DR in stable monocytes according to any one of claims 1-7 in the preparation of an in vitro diagnostic kit for the diagnosis of sepsis-induced immune paralysis.

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