CN108303548B

CN108303548B - Calibration method for improving consistency of detection result of C-reactive protein

Info

Publication number: CN108303548B
Application number: CN201810128691.1A
Authority: CN
Inventors: 吴春颖; 王清涛; 张顺利; 马怀安; 赵婷婷; 张瑞; 岳育红
Original assignee: Beijing Center For Clinical Laboratory; Beijing Chaoyang Hospital
Current assignee: Beijing Center For Clinical Laboratory; Beijing Chaoyang Hospital
Priority date: 2018-02-08
Filing date: 2018-02-08
Publication date: 2021-03-30
Anticipated expiration: 2038-02-08
Also published as: CN108303548A

Abstract

The invention discloses a calibration method for improving consistency of detection results of C-reactive protein. The invention provides a C-reactive protein calibrator, which is prepared by the following steps: collecting the in vitro serum of different individuals, mixing to obtain 2 serum solutions with different concentrations of C-reactive protein, and obtaining the following 2 concentrations of C-reactive protein calibrator after setting values: 1) high value sample: the result of the fixed value of the C-reactive protein concentration is 109.9 +/-9.1 mg/L serum solution; 2) low value sample: the result of the C-reactive protein concentration fixed value is 27.1 +/-2.3 mg/L serum solution. Experiments prove that the CRP special calibrator developed by the invention is suitable for calibrating clinical conventional CRP detection reagents. The calibration method can improve the consistency and comparability of CRP detection results of individual patients, has good application prospect and is worthy of popularization.

Description

Calibration method for improving consistency of detection result of C-reactive protein

Technical Field

The invention relates to the field of clinical detection, in particular to a calibration method for improving consistency of detection results of C-reactive protein.

Background

C-reactive protein (CRP) is an acute inflammatory positive phase reactive protein synthesized by the liver. CRP concentrations in normal human blood are low, synthesis rapidly increases when the body encounters stress, tissue trauma and various inflammatory stimuli, and is secreted from hepatocytes into the blood, with high levels of CRP being detectable 12-18 hours after infection. CRP, elevated 12-14 days after infection, may drop to baseline levels. Therefore, the method is one of indexes for evaluating inflammatory diseases for years, and the increase amplitude is related to the degree of infection. CRP has been widely used in clinical applications as one of the important markers for diagnosing bacterial infections. CRP is also a clinically important indicator for assessing heart disease incidence, recurrence and mortality. In recent years, research shows that inflammation plays an important role in the occurrence and development processes of atherosclerosis and tumors. The accuracy of measurement of serum CRP has received extensive attention in view of its important role. Currently, international traceability union (JCTLM) is beginning to advance the traceability and standardization work of CRP, and the work and research in this aspect are less in China.

The common methods for detecting CRP are various, including nephelometry, turbidimetry, radioimmunoassay, chemiluminescence, ELISA, and point-of-care CRP detection (POCT). At present, the method for measuring CRP in serum in clinical laboratories is mainly an immune turbidimetry, including a latex-enhanced transmission turbidimetry and a rate scattering turbidimetry, which are mainly used for an automatic analysis system, the rate scattering turbidimetry is used for a closed detection system in the immune detection field, and the latex-enhanced transmission turbidimetry is used for an open detection system in the biochemical detection field.

At present, the consistency of the detection results of domestic CRP is not ideal, more CRP detection systems are adopted in laboratories, the difference between the results is larger, and the CV% difference of different laboratory detection systems is larger. 2016 CRP second indoor evaluation data issued by clinical examination centers of Ministry of health indicate that the mean value difference of different groups of the same concentration measurement is large (23.64-28.63 mg/L), the precision reproducibility difference between laboratories of different detection methods is large (CV% is 5.42-12.35%), three laboratories adopt a chemiluminescence method to measure the concentration to reach 10389.33mg/L, CV% also reaches 172.85%, and foreign Roberts and other researches also prove that the detection numerical value difference of different detection systems to the sample with the same concentration is large.

The important way for realizing the consistency of the detection result is the standardization of the detection method, and the key is to ensure the traceability of the detection result. In order to standardize the CRP assay, in 1987, the WHO developed the first CRP International Standard substance WHO IS 85/506, which was a pure substance at a concentration of 98 mg/L. In 1989, the International society for clinical chemistry (IFCC) plasma protein Committee (C-PP) began to develop a universal reference substance. The reference substance comprises 15 serum proteins including CRP. In 1993, this generic Reference substance was issued by the European Community institute of standards (BCR) certification as CRM 470, calibrated for CRP using the international standard WHO IS 85/506. Then formally named ERM DA-470. After the release of ERM-DA470, in vitro diagnostic reagent manufacturers began assigning values to their calibrators using this standard substance, and the inter-laboratory measurements differed significantly less between the various laboratories.

With the depletion of ERM-DA470, the reference substance and measurement Institute (IRMM) in conjunction with the international association of clinical chemistry (IFCC) began to develop a new standard substance, which was formally released in 2008 under the name ERM-DA470 k. This material was pre-tested prior to preparation. The pre-experimental data showed that the addition of purified CRP, lyophilization and reconstitution resulted in CRP with variable assay values, with about 20% variation. The final new reference substance ERM-DA470k/IFCC was not added to the CRP rating. In 2009, ERM-DA472/IFCC was developed by IRMM in combination with IFCC. ERM-DA472/IFCC and ERM-DA470k/IFCC use the same serum pool, and ERM-DA472/IFCC directly liquid freezes the serum, given that lyophilization will affect the results of the CRP assay. In 2011, they developed ERM-DA 474/IFCC. The development process is substantially the same as ERM-DA 472/IFCC. ERM-DA474/IFCC is widely used for quantity value tracing of CRP diagnostic reagent calibrators of manufacturers at present.

At present, the CRP standard substance without human serum matrix in China cannot be used for the accuracy verification of indoor quality evaluation mechanisms due to the reasons that the international standard substance is expensive, the transportation period is long, the transportation conditions cannot be guaranteed and the like, and cannot meet the requirement of increasing national enterprises on CRP high-order standard substances.

Disclosure of Invention

In order to solve the above problems, the present invention provides a C-reactive protein calibrator and a calibration method for improving the consistency of the detection results of C-reactive protein.

The C-reactive protein calibrator provided by the invention is prepared by the following steps: collecting the in vitro serum of different individuals, mixing to obtain 2 serum solutions with different concentrations of C-reactive protein, and then respectively carrying out value determination on each serum solution in the 2 serum solutions with different concentrations of C-reactive protein to obtain the following 2 concentrations of C-reactive protein calibrators:

1) high value sample: the result of the fixed value of the C-reactive protein concentration is 109.9 +/-9.1 mg/L serum solution;

2) low value sample: the result of the C-reactive protein concentration fixed value is 27.1 +/-2.3 mg/L serum solution.

The serum is clear and transparent in appearance, except for abnormal characters such as jaundice, hemolysis, lipemia and chyle, and except for human serum infected by viruses such as HIV, hepatitis A, hepatitis B and hepatitis C.

Wherein, the method for performing the value determination on each serum solution in the 2 serum solutions with different C-reactive protein concentrations comprises the following steps: and (3) adopting a C-reactive protein international standard substance to carry out quantitative value transfer to the candidate C-reactive protein standard substance to carry out value determination.

Further, the method for "performing the value determination on each serum solution of the 2 serum solutions with different concentrations of C-reactive protein" may specifically comprise the following steps:

(a1) diluting the international standard substance of the C-reactive protein to obtain a series of diluted solutions of the international standard substance of the C-reactive protein with different concentrations, and calculating to obtain the theoretical concentration of the C-reactive protein in each diluted solution.

(a2) Determining the concentration of the C-reactive protein in each of the diluted solutions obtained in the step (a1) using N quantitative detection systems for C-reactive protein.

(a3) For each quantitative detection system of the C-reactive protein in the N quantitative detection systems of the C-reactive protein, performing linear fitting by adopting the theoretical concentration of the C-reactive protein in each diluted solution obtained in the step (a1) and the measured concentration of the C-reactive protein in each diluted solution obtained in the step (a2) to obtain a C-reactive protein fixed value standard curve; namely, N C-reactive protein constant value standard curves are obtained by N C-reactive protein quantitative detection systems.

(a4) And respectively determining the concentration of the C-reactive protein in the candidate C-reactive protein standard substance by adopting the N quantitative detection systems for the C-reactive protein.

(a5) Substituting the measured concentration of the C-reactive protein in the candidate C-reactive protein standard substance obtained in step (a4) into the equation of the corresponding C-reactive protein fixed value standard curve obtained in step (a3) for each of the N C-reactive protein quantitative detection systems, thereby obtaining the concentration of the C-reactive protein in the candidate C-reactive protein standard substance.

Substituting the concentration value of the C-reactive protein in the candidate C-reactive protein standard substance, measured by a certain C-reactive protein quantitative detection system in the step (a4), into the equation of the C-reactive protein fixed value standard curve corresponding to the C-reactive protein quantitative detection system obtained in the step (a3), so as to obtain the concentration value of the C-reactive protein in the candidate C-reactive protein standard substance, wherein the concentration value is the fixed value result of the C-reactive protein concentration in the candidate C-reactive protein standard substance by the C-reactive protein quantitative detection system. N quantitative results are obtained by aiming at N C-reactive protein quantitative detection systems.

Wherein, the abscissa (X) of the C-reactive protein constant value standard curve is theoretical concentration, and the ordinate (Y) is measured concentration. And (5) substituting the measured concentration of the C-reactive protein in the candidate C-reactive protein standard substance into the Y value of the C-reactive protein constant value standard curve equation, and calculating to obtain the X value as the concentration of the C-reactive protein in the candidate C-reactive protein standard substance.

(a6) Averaging the concentrations of the C-reactive proteins in all the candidate C-reactive protein standard substances of the N quantitative C-reactive protein detection systems obtained in the step (a5), wherein the obtained average value is the final fixed value result of the concentrations of the C-reactive proteins in the candidate C-reactive protein standard substances.

The N in the method may be a natural number of 2 or more, specifically, N is a natural number of 3 to 20, and more specifically, N is 10.

Further, the step (a1) may be specifically implemented by a method including the following steps:

(A) preparing a series of diluted solutions of the C-reactive protein international standard substances with different concentrations: mixing the diluent and the C-reactive protein international standard substance according to different volume ratios, thereby obtaining a series of diluent solutions of the C-reactive protein international standard substance with different concentrations; in the process of preparing each diluted solution of the international standard substance for C-reactive protein, the diluted solution and the international standard substance for C-reactive protein, which constitute the diluted solution of the international standard substance for C-reactive protein, are respectively weighed.

(B) Density determination of dilution solutions of the international standard substance for C-reactive protein in series of different concentrations: measuring the respective densities of the series of diluted solutions of the international standard substance for C-reactive protein at different concentrations prepared in step (a) separately.

(C) Obtaining theoretical concentration of the C-reactive protein in dilution solutions of the international standard substance of the C-reactive protein with a series of different concentrations: calculating the theoretical concentration of the C-reactive protein in each diluted solution of the series of diluted solutions of the international standard substance for C-reactive protein with different concentrations according to the following formula,

wherein C1 represents the concentration of C-reactive protein in the international standard substance for C-reactive protein; ρ 1 represents the density of the international standard substance for C-reactive protein; m1 represents the mass of the C-reactive protein international standard substance in the diluted solution of the C-reactive protein international standard substance; c2 represents the concentration of C-reactive protein in the dilution; ρ 2 represents the density of the diluent; m2 represents the mass of the dilution in the dilution of the international standard substance for C-reactive protein; ρ 3 represents the density of a diluted solution of the international standard substance for C-reactive protein; c represents the theoretical concentration of the diluted solution of the international standard substance of the C-reactive protein.

More specifically, in the step (B), the density of the diluted solution of the international standard substance for C-reactive protein may be measured according to a method comprising the steps of:

(b1) weighing the mass of the container and the gun head of the sample adding gun, and recording the mass as m 1;

(b2) sucking a sample to be tested with the volume of constant v by using the tip of the sample adding gun in the step (b1), placing the sample to be tested into the container in the step (b1) (the step comprises the step of wiping off the sample to be tested adhered to the outside of the tip of the sample adding gun), weighing the sample to be tested integrally, and marking the sample as m 2;

(b3) calculating the density of the sample to be detected according to the following formula: rho_{To be measured}＝(m2-m1)/v。

Wherein, the steps (b1) and (b2) can be repeated for 3 to 5 times according to the needs. Accordingly, the parameters m1 and m2 to the right of the equation equal sign in step (b3) are averaged for 3-5 repetitions.

In the invention, the C-reactive protein international standard substance is specifically ERM-DA 474/IFCC.

In the present invention, in the step (a1), when the international standard substance for C-reactive protein is diluted, the diluent used is specifically low-value serum; the low value serum is more specifically serum containing C-reactive protein at a concentration of 0.27mg/L (the concentration of 0.27mg/L is determined by the hypersensitivity method).

Before the formal test, the quantitative detection system for the C-reactive protein is required to pass performance verification of a series of measurement systems such as precision, accuracy, system linearity and carried pollution. And after the performance verification is passed, performing formal fixed value experiments within one week.

The application of the C-reactive protein calibrator described in the foregoing in any one of the following applications also falls within the scope of the present invention:

(i) calibrating the C-reactive protein detection reagent;

(ii) improving the consistency and/or comparability of the CRP detection results of the multiple systems to individual patients.

The "system" herein includes an apparatus for quantitatively detecting C-reactive protein and reagents used. Such as a clinical laboratory C-reactive protein detection system.

The calibration method for improving the consistency of the detection result of the C-reactive protein provided by the invention specifically comprises the following steps:

(c1) preparing N parts of C-reactive protein calibrator solutions with different concentrations: one is the high value sample in the C-reactive protein calibrator, one is the low value sample in the C-reactive protein calibrator, and the other N-2 is a series of mixed samples formed by mixing the high value sample and the low value sample according to different volume ratios, and the theoretical concentration of the C-reactive protein in each mixed sample is calculated;

(c2) and (C1) calibrating the quantitative detection system for C-reactive protein by using the N prepared C-reactive protein calibrator solutions with different concentrations (namely, setting a reference, namely, a so-called true value corresponding to a certain signal), and then detecting the concentration of the C-reactive protein in the serum sample to be detected.

In the step (C1), in the process of preparing the series of mixed samples, for each mixed sample in the series of mixed samples, the low value sample and the high value sample which constitute the mixed sample are respectively weighed, the density of the mixed sample is measured, then the volume of the mixed sample is obtained according to the density of the mixed sample and the mass sum of the low value sample and the high value sample which constitute the mixed sample, and further the theoretical concentration of the C-reactive protein in each mixed sample is obtained through calculation.

The invention utilizes IFCC/ERM DA-474 to trace the quantity value and develop CRP candidate standard substance of 2-level human serum matrix, and the definite value and the uncertainty of the candidate standard substance are 109.9 +/-9.1 mg/L and 27.1 +/-2.3 mg/L respectively. The special calibrator prepared by the invention is derived from fresh frozen mixed human serum, so that the special calibrator has good interchangeability. The current literature shows that the consistency of the detection results of various laboratories is obviously improved when the compatible calibrator is used for detecting human mixed serum. By adopting the special calibrator, individual serum trays of a single patient representing clinical actual detection conditions are detected, and the result shows that the degree of systematical consistency after calibration is obviously improved, the coefficient of variation among systems is changed into 7.03%, and the optimal CV requirement derived from biogenetic variation is met. The proportional system deviation and the fixed system deviation are both obviously reduced, and the correlation is obviously improved. The accuracy of 3 detection systems after calibration is also obviously improved. In conclusion, the CRP special calibrator developed by the invention is suitable for calibrating clinical conventional CRP detection reagents. The calibration method can improve the consistency and comparability of CRP detection results of individual patients, has good application prospect and is worthy of popularization.

Drawings

FIG. 1 shows an enhanced turbidimetry calibration curve (8 systems).

FIG. 2 is a standard curve (2 systems) for rate nephelometry.

Fig. 3 shows the CRP accuracy verification results for high concentration (L1) samples.

Fig. 4 shows the CRP accuracy verification results for low concentration (L2) samples.

Fig. 5 shows evaluation results of CRP accuracy verification survey in 2015. Wherein the target value is (27.1, 109.9) mg/L; the evaluation criterion was 12.5% of the allowable deviation (1/2 TEA); the "meter" shaped symbols in the figures are the coordinates of the identification target values.

FIG. 6 is a correlation between Texas and other system specific calibrators before and after calibration.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation, quantification and verification of C-reactive protein calibrator

The general preparation process of the calibration sample of C-reactive protein obtained in this example is as follows: collecting the in vitro serum of different individuals, mixing to obtain 2 serum solutions with different concentrations of C-reactive protein, and then respectively carrying out value determination on each serum solution in the 2 serum solutions with different concentrations of C-reactive protein to obtain the following 2 concentrations of C-reactive protein calibrators:

Materials and methods

1. Candidate standard substance preparation material and method

1.1. Material

1.1.1. Serum

Collected in the department of Hospital clinical laboratory of Beijing from 2016 (6 months to 2016 (8 months)). According to the international organization for standardization (ISO) guideline 35 and the World Health Organization (WHO) guideline for collecting serum raw materials for preparing standard substances, the serum of healthy examiners and patients in hospital is collected and placed in 50ml of polyethylene sealed centrifuge tubes, the appearance is clear and transparent, except the abnormal characters such as jaundice, hemolysis, lipemia and chyle, except the serum infected by viruses such as HIV, hepatitis A, hepatitis B, hepatitis C and the like, and no additive or preservative is added. The serum of the patients with CRP in the high (80.1-200mg/L) and low (10.1-80mg/L) concentration ranges is collected respectively, and each concentration range collects about 800 ml.

1.1.2. Collecting pipe

50ml screw cap polyethylene clean plastic tube for serum collection. 1ml polyethylene tube with screw cap, and sterilized by ethylene oxide to be used as serum subpackage tube.

1.1.3. The Beckmann Immage800 full-automatic immunity analyzer (SN:9372) and a matched reagent calibration product, wherein the batch number is M310472. Two indoor quality control products with concentration levels of berle liquid protein, the batch number is 52482-52483, and the reagent, the calibration product and the quality control product are all used within the valid period range.

1.1.4. The filter device has filter membrane pore sizes of 0.45 μm and 0.20 μm, respectively. Equipped with a vacuum suction pump (HENGAO, usa).

2.1. Method of producing a composite material

2.1.1. Preparation and filtration packaging of candidate standard substance

Two concentration ranges of serum, each of about 800ml, will be collected. All sera were removed from the-80 ℃ freezer in two concentration ranges and thawed at room temperature. Then, the thawed sera from the two ranges were mixed well and about 0.5ml was taken out. The primary assays were each performed using a Beckman Immage800 full-automatic immunoassay analyzer. The two different CRP concentration levels after mixing were approximately as follows:

level 1(L1) was 800ml of the CRP high value sample, was a mixture of 503 serum samples, was free of any added substances, and was measured to have a CRP concentration of about 110.0mg/L using a Beckmann Immage800 analyzer.

Level 2(L2) was a 817ml sample of CRP low value, mixed with 524 samples of serum, without any added material, and measured using a Beckmann Immage800 analyzer to give a CRP concentration of about 25.3 mg/L.

The filtration and subpackage steps of the two concentrations of mixed serum are entrusted to Beijing Conchester Stent biotechnology limited for processing, and the filtration and the subpackage of the serum are strictly carried out according to the requirements of accepted Standard Operation Procedures (SOP); sealing each tube with 1 ml; 754 pieces and 743 pieces of standard substances with high and low concentrations are prepared, and are respectively distinguished by tube caps with different colors, namely a green cap (high level L1) and a white cap (low level L2). The candidate standard substances are placed in a freezing box and stored in a refrigerator at the temperature of-80 ℃, and the temperature condition of the refrigerator is monitored and recorded every day.

2. Materials, methods and Experimental procedures for Multi-System Joint constant value experiments

2.1. Material

2.1.1. Detection system

According to data information of CRP laboratory evaluations of serology in 2015-2016 years of sanitation and Beijing city, 10 CRP serological detection systems in 2 laboratories are selected from a main measuring instrument and a brand reagent for CRP measurement as a constant value system of CRP candidate targets, and the method specifically comprises the following steps:

a total of 5 detection systems, 4 transmission turbidimetry and 1 scattering turbidimetry at the clinical laboratory of the Beijing Chaoyang Hospital (Table 1):

A. the Siemens ADVIA 2400 full-automatic biochemical analyzer is matched with reagents, calibration products and quality control products of Germany Siemens original factories;

B. preparing a German;

C. the Siemens ADVIA 2400 full-automatic biochemical analyzer is matched with a domestic Ledman reagent, a calibration material and a quality control material;

D. preparing a Siemens ADIVA 2400 full-automatic biochemical instrument with a German Roche reagent, a calibrator and a quality control product;

E. the Beckman Immage800 full-automatic immunity analyzer is matched with reagents, calibration products and Berkeley quality control products of a factory.

A total of 5 measurement systems of 4 transmission turbidimetry and 1 scattering turbidimetry in the university of Beijing Luhe Hospital (Table 2):

F. the Beckman AU5821 full-automatic biochemical analyzer is matched with a Beckman original factory reagent, a calibrator and a quality control product;

G. the Beckman AU5821 full-automatic biochemical analyzer is matched with Beijing nine-strength reagent, calibrator and quality control material;

H. the Beckman AU5821 full-automatic biochemical analyzer is matched with a Japanese ponding reagent, a calibrator and a quality control product;

I. the Beckman AU5821 full-automatic biochemical instrument is matched with Ningbo Meikang reagent, a calibrator and a quality control product;

J. siemens BN II full-automatic immunity analyzer is matched with reagents, calibration products and quality control products of a stock house.

TABLE 1 Beijing Chaoyang Hospital clinical laboratory 5 detection system reagents, calibrators, quality control lot numbers and goods numbers

TABLE 2 Beijing Luhe Hospital clinical laboratory 5 detection System reagents, calibrator, quality control lot and cargo number

2.1.2. Detection material and sample

2.1.2.1. Reagent: all assay system reagents and calibrators and indoor quality controls were used during the expiration time.

2.1.2.2. Sample and consumable: 1ml of each sample is filled with candidate CRP standard substances with high and low concentrations and good uniformity and stability detection (step 1); ERM-DA474 International Standard substance, purchased from IFCC, under the batch number: 003095, respectively; the dilution is CRP low-value mixed human serum (hereinafter referred to as low-value serum) which is detected to be 0.27mg/L by a hypersensitivity method. And auxiliary materials such as an analytical balance, a sample adding gun, a beaker, a gun head and the like which are calibrated and within the valid period range.

2.2. Method of producing a composite material

2.2.1. The principle is as follows: after a series of preliminary experiments, the final decision is to adopt 10 valuing systems of two clinical laboratory in Beijing city's third-class hospital to jointly valuate the candidate standard substance with two concentrations of CRP high and low, and carry out quantity value transmission by using ERM-DA474 international standard substance of IFCC. Weighing low-value serum and an international standard substance ERM-DA474 by a gravimetric method, accurately preparing a series of standard working solutions with known concentration of ERM-DA474 by using the low-value serum, diluting a high-value candidate standard substance by using the method to prepare a high-value target working solution, and detecting samples of the ERM-DA474 standard working solution and the candidate standard substance in the same batch by using 10 detection systems which pass performance verification respectively. And (3) drawing a standard curve by using the CRP actual detection concentration of the ERM-DA474 standard working solution and the corresponding theoretical value. Serum CRP concentrations in the candidate target samples at both high and low concentrations were calculated from the standard curve.

2.3. Experimental procedure

2.3.1. Preparation before setting value of measurement system

a) Skilled instrumentation; and the technical support personnel of the original factory are accompanied and guided in the whole process and participate in the detection on the experiment day, so that the measurement error and the emergency situation are reduced to the maximum extent.

b) And (3) performance verification: all detection systems participating in joint setting are required to pass performance verification of a series of measurement systems such as precision, accuracy, system linearity and carried pollution before formal tests. And after the performance verification is passed, performing formal fixed value experiments within one week.

2.3.2. Dilution of International Standard substances

According to ERM-DA474 International Standard substance Specification for IFCC. The serum CRP concentration of ERM-DA474 in each bottle was 41.2 + -2.5 mg/L. 6 ERM-DA474 frozen human serum standard substances are taken, and redissolved and mixed strictly according to the instruction. After mixing uniformly, sampling by using a sample adding gun, taking low-value serum (CRP concentration is 0.27mg/L) as a diluent, measuring weight by using an analytical balance weighing method, and accurately preparing a working curve analysis solution; ERM-DA474 was finally diluted to 4 concentration levels by weighing, as shown in Table 3, and labeled as W1-W4, and frozen at-80 ℃ for use.

TABLE 3 dilution of ERM-DA474 international standard substance by weighing

Note: 1. sharing 4940 μ l of ERM-DA474 international standard, and calculating the actual theoretical concentration by density; 2, 2400 mu l of each W1-W4 is prepared into 4 pieces, 600 mu l of each piece is frozen at minus 80 ℃ for standby, and one piece is taken from each laboratory every day for detection.

The actual theoretical concentration in table 3 is calculated specifically as follows:

firstly, using low-value serum (CRP concentration is 0.27mg/L) as a diluent, diluting ERM-DA474 into four diluted solutions of W1-W4 in Table 1, and accurately measuring the specific weights of the diluent and ERM-DA474 when preparing the four diluted solutions of W1-W4 by using an analytical balance weighing method;

second, density measurement of four diluted solutions W1-W4

(1) The mass of the vial and the tip of the loading gun was weighed out first using a certified balance and recorded as m 1.

(2) The tip of the sample application gun was carefully removed from the vial using sterile rubber gloves.

(3) Accurately sucking 200 mul of sample to be detected by adopting a calibrated sample adding gun, wiping off liquid on the outer surface of a gun head of the sample adding gun, placing the gun head of the sample adding gun containing the sample to be detected in a small bottle, and weighing the whole sample with the mass of m 2.

(4) This was repeated 3 times and recorded in detail.

(5) The density was averaged according to the formula of calculation of the density of the liquid, ρ ═ m2-m1)/v1, where v1 was 200 μ l.

(6) The densities of four diluted solutions W1-W4 were calculated according to the procedures (1) to (5).

Thirdly, calculating the theoretical concentration of four diluted solutions W1-W4

The theoretical concentrations of the four dilutions W1-W4 were calculated based on the concentration of CRP in the low serum (0.27mg/L) as a dilution and the concentration of CRP in ERM-DA474 (41.2. + -. 2.5 mg/L). If the concentration of ERM-DA474 is C1g/L, the density is rho 1, the weighed mass is M1, the concentration of low-value serum is C2g/L, the density is rho 2, the weighed mass is M2 and the density of a certain tube is rho 3 for a certain tube W1 diluted solution, the theoretical concentration of W1 is obtained by the formula:

2.3.3. preparation of serum samples of CRP candidate standards

The level 2(L2) candidate target physical theoretical value (about 25mg/L) is within the linear range of the working curve, so no dilution is required, and U2 is the L2 candidate standard substance; the level 1(L1) candidate target physical theory value (about 110mg/L) is beyond the linear range of the working curve, and in consideration of the actual use and the accuracy of assignment of the high-value candidate target, the L1 target is diluted by low-value serum into two concentrations, namely U3 and U4, and then assignment is carried out, the same low-value serum (0.27mg/L) is adopted for the preparation of U3 and U4 and the dilution liquid (W1-W4) of ERM-DA474, the specific preparation is shown in Table 4, 18000 mu L of each co-preparation of U2-U4 is adopted, each average is 12, each 1500 mu L is put into a freezing storage at-80 ℃ for standby.

TABLE 4 dilution of candidate standards by gravimetric method

Note: u2 is L2 undiluted; u3 and U4 are L1 in a volume ratio of 1: 4 and 1: 3 diluted preparation, using weighing method to calculate (see the above "actual theoretical concentration calculation method in table 3" specifically).

2.3.4. Density determination of all fixed value samples

Because the densities of various working fluids are required in the accurate assignment calculation process of CRP, the densities of various working fluids are accurately measured by a weighing method before the preparation is finished and the split charging and the freezing storage are carried out: the formula: p is m/v, 200. mu.l is aspirated, the assay is weighed on a balance and calculated after three measurements are made, and the average value is calculated, see Table 5.

TABLE 5 gravimetric method for determining sample density

2.3.5.CRP candidate Standard valuing experiments

2.3.5.1. Indoor quality control

The fixed value is respectively carried out on 10 measurement systems which pass the performance verification in two laboratories, all the measurement is completed within one week after the performance verification is passed, all the systems are maintained and calibrated on the measurement day, then the indoor quality control of two levels is within the range of 2SD, and then the sample measurement is carried out.

2.3.5.2. Measurement operation

In the multisystem combined setting, each laboratory is divided into two days, samples to be measured which are subpackaged in advance are taken out from a refrigerator at minus 80 ℃ one by one before measurement every day, ice bags are adopted for conveying among laboratories, the samples are conveyed to the setting laboratory within one hour, and the samples are placed at room temperature and started to be detected after being completely re-melted. 10 measurement systems are adopted in two laboratories, 1 in each of W1-W4 and 3 in each of U2-U4 are taken in each laboratory every day, and the two laboratories are re-melted at room temperature. Each test system W1-W4 was tested 3 times per day in each laboratory, and each test system U2-U4 was tested 6 times x 3-18 times per day. Before measurement every day, after the indoor quality control of each measurement system reaches the specified standard (namely the quality control is qualified, the instrument can work normally), measurement is started for W1-W4 and U2-U3 in sequence, the stability of indoor temperature and humidity is noticed in the measurement process, and detection samples in the indoor quality control and system precision performance verification are additionally made in the measurement process and the later period of all systems, so that the stability of the measurement system is ensured.

2.3.5.3. Constant value statistical method

And (4) using a straight line fitting method to determine the value of the candidate standard substance. The theoretical concentration and the actual measured concentration after dilution with the international standard substance were used, and each system made a straight line. Substituting the average value of the actual numerical values measured by the candidate standard substance sample into a linear equation to obtain an X value which is the concentration of the candidate standard substance working solution, obtaining accurate fixed value data of each measuring system to the candidate standard substances L1 and L2 after operation, and then averaging the fixed value results of 10 systems to obtain the final concentration of the candidate standard substance.

2.3.5.4. Statistical method of uncertainty

According to the ISO guideline 35 file, the total uncertainty is synthesized from the uncertainty introduced by the fixed value measurement, homogeneity, long term stability of the candidate standard substance and the measurement tools such as analytical balance, etc., the formula is calculated:

wherein u2ver in the formula is the uncertainty of measurement, and the uncertainty comprises a standard curve, the repeatability of measurement and ERM-DA 474; u2bb uncertainty introduced for inhomogeneity; u2lts is the uncertainty introduced by the long-term stability, u2grav is the uncertainty introduced by the analytical balance equivalent measurement tool. The specification for the international standard substance ERM-DA474 gives an extended uncertainty of 2.5mg/L and should therefore be taken into account in the calculation. The analytical operation includes class a and class B uncertainties, and in this experiment, in addition to class a, class B uncertainties of analytical balances are also considered.

3. Candidate standard substance accuracy verification

3.1. Laboratory and detection system

3.1.1. Laboratory

42 medical institutional clinical laboratories, Beijing.

3.1.2. Detection system

Conventional CRP serological immunodetection system or biochemical detection system, such as Hitachi, Siemens, Roche, Beckman series analyzer and its assorted reagent, calibrator and quality control product.

3.2. Method for verifying accuracy

3.2.1. Distributing samples

Two concentration levels of CRP candidate standards, 2 for each concentration level, were distributed to 42 clinical laboratories in the beijing area.

3.2.2. Measurement method

Respectively measuring 1 count in 2 days (2 days apart), standing the sample at room temperature for 30 minutes on the day of measurement, and after the sample is completely melted and is balanced to room temperature, slightly reversing the sample up and down for 10 times and mixing the sample to avoid generating bubbles. Each sample CRP is continuously measured for 3 times in the existing CRP serological detection system of each laboratory, and the stability of the detection system and the indoor quality control reaching the standard (namely the quality control is qualified, and the instrument can normally work) are ensured before measurement.

3.2.3. Transportation of

During transportation, the sample is placed in a heat-insulating container with an ice bag to ensure that the sample is in a frozen state. If the sample can not be detected in time, the sample is stored in a refrigerator at 2-8 ℃ for refrigeration, the detection is required to be completed within one week, and the data is reported to a mail box of a temporary inspection center in Beijing.

3.2.4. Culling outliers

And (3) rejecting outliers by using a proper method, namely a Grubbs method recommended in the national standard GBT 4883-2008 data statistical treatment and normal sample outlier judgment and treatment.

3.2.5. Data processing

Calculating the mean value and standard deviation of results of 6 times of each laboratory by using Excel 2007 software, drawing a schematic diagram of accuracy verification by taking the assigned result as a target value and the total allowable error (1/2TEA) of the clinical test center 1/2 of the department of health as an allowable limit; removing outliers from the mean value results of 42 laboratories by adopting the national standard (GBT 4883-(sd1, equivalent to U1) and standard substance identification (x2) and uncertainty (U2) using the potency function

And verifying the accuracy of the fixed value.

3.2.6. Statistical method for inter-room investigation

Bias (%) - (hospital clinical laboratory result-constant)/constant X100%

Second, result in

1. Multisystem constant value result

Fixed value standard curve of 1.1.10 detection systems

As shown in fig. 1 and 2. As can be seen from the figure: the linear relation of each system is good, and the fixed value of the candidate standard substance is in the fixed value range of the standard curve.

Systematic valuation results for CRP candidate Standard substance 10

The results of systematic assignment of CRP candidate standard 10 are shown in Table 6. As can be seen from the table: the average values of all detection systems are very close, namely U2, U3 and U4 are respectively 26.24-27.64 mg/L, 21.45-22.53 mg/L and 27.41-28.6 mg/L.

TABLE 6 CRP candidate Standard substance 10 systematic assessment results

Multi-system quantitative results for CRP candidate standard substances

The final definite value of the candidate standard object is the mean value of the definite values of 10 systems, and the mean value of 10 total uncertainties of the systems is measured according to the uncertainty in the definite value process. Specific results are shown in Table 7. As can be seen from the table: the fixed value and the uncertainty of the candidate standard substances at the two levels are 109.9 +/-9.1 mg/L and 27.1 +/-2.3 mg/L respectively.

TABLE 7 CRP candidate Standard substance multisystem constant results

Level of	Constant concentration (mg/L)	Definite value uncertainty Uc (mg/L)	Relative degree of uncertainty Uc%
				L1	109.9	9.1	8.25
L2	27.1	2.3	8.58

1.4. Total uncertainty evaluation and final certainty results

The specific results are shown in Table 8. As can be seen from the table: the uncertainty of the CRP candidate standard was fitted with the error of the valuing process, homogeneity, stability and balance weighing and extended (spreading factor k 2) with a final uncertainty of 9.1mg/L and 2.3mg/L for both levels.

TABLE 8 CRP candidate Standard substance Total uncertainty evaluation and Final valuing results

In conclusion, the CRP frozen mixed human serum candidate standard substance is subjected to ERM-DA474 international standard substance mass value transmission, and is subjected to combined value determination by 2 laboratories and 10 mainstream detection systems, and the final value determination result is as follows: level 1 (L1): 109.9 +/-9.1 mg/L; level 2 (L2): 27.1 +/-2.3 mg/L, and the uncertainty level is equivalent to the international standard substance ERM-DA 474.

2. Accuracy verification result

2.1. Accuracy verification

The CRP candidate standard substance samples are distributed to 46 laboratories of Beijing city, 42 laboratories report results, wherein 30 hospitals of the third-level hospitals and 12 hospitals of the second-level hospitals.

2.2 CRP candidate Standard substance level 1(L1) accuracy verification results are processed

Of the 42 results reported back at L1, the overall mean was 114.19mg/L, the standard deviation was 19.10, the median: 109.58 with a maximum of 162.07mg/L and a minimum of 70.1 mg/L. All results were 1 outlier (33.37mg/L) according to Grubbs' method, the post-culling results are shown in FIG. 3, the tolerance for the evaluation of the quality of the CRP project from the room is. + -. 25%, with the upper and lower limits. + -. 12.5% (1/2 TEA). From FIG. 3, it can be seen that the test results of the laboratory L1 are qualified when the upper limit is higher than 8 laboratories, the lower limit is lower than 3 laboratories, and 73.8% of the laboratory L1 in Beijing is qualified.

2.3 CRP candidate Standard substance level 2(L2) accuracy verification results are processed

Of the 42 results reported at L2, all methods had a mean of 26.51mg/L, a standard deviation of 3.23, a median: 25.65, a maximum of 33.90mg/L and a minimum of 20.02 mg/L. According to the Grubbs method, all results were free of outliers. As shown in fig. 4. The CRP project was assessed as being within + -25% of the mean-room quality with an upper and lower limit of + -12.5% (1/2 TEA). As can be seen from FIG. 4, the test results of the laboratory L2 are qualified 76.1% above the upper limit and 5 below the lower limit.

The laboratory-reported data for L1 and L2 were normally distributed by SPSS17.0 Kolmogorov-Smirnov Z test with efficacy functions of 0.18 and 0.20, both less than 1.

2.4. Clinical laboratory accuracy verification result evaluation example

Taking a hospital as an example, the CRP sample is measured by using a Beckman Immage800 full-automatic immunoassay analyzer, the mean value and the fixed value bias of the detection results of 2 horizontal samples dispensed at this time are respectively 5.49 percent and 2.45 percent, the measurement results can be considered to be under the evaluation standard that the bias is less than or equal to +/-12.5 percent, the CRP measurement results of the conventional measurement system are correct, and the results are shown in Table 9.

TABLE 9 CRP accuracy evaluation results in Beijing Hospital

Sample batch number	Mean value	Target value	Biased for all	Standard deviation of	Coefficient of variation%	Instrument for measuring the position of a moving object	Method
								2015L1	109.67	109.9	5.49	0.99	3.85	Beckman (Beckman)	Rate nephelometry
2015L2	25.67	27.1	-2.45	3.33	3.03	Beckman (Beckman)	Rate nephelometry

The Uton graph of the CRP accuracy verification survey evaluation result in 2.5.2015 is shown in FIG. 5. The number of laboratories within acceptable limits is 30, accounting for 71.4% of all reported results. It can be seen from the figure that points at the lower left and upper right corners have negative and positive biases, respectively, but a point at the upper left outside the box in the figure has the problem that the low value is too low and the high value is too high, which may be related to the cause of the methodology itself or the misoperation of the laboratory.

Example 2 establishment of a calibration method to improve the consistency of the assay results for C-reactive protein

A method of material

1. Serum pan collection

After-test serum (concentration range 7.1-123.0 mg/L, Beckman 5821 Japan hydrops reagent) of 21 patients with different CRP high, medium and low concentrations is collected from the affiliated Luhe hospital of capital medical university from 19 to 23 in 2017, 6 and 19 months, 500 mul each is frozen at-80C, and the time from serum collection to freezing storage is not more than 30 hours. Samples of hemolysis, jaundice and lipemia were excluded from the collection.

2. Detection system

4 transmission turbidimetry detection systems were selected in the department of laboratory, Beijing rising Yang Hospital, affiliated with the university of capital medical sciences:

B. the Siemens ADVIA 2400 full-automatic biochemical analyzer is matched with a Beijing Lidemann reagent, a calibrator and a quality control product;

C. the Siemens ADVIA 2400 full-automatic biochemical analyzer is matched with German Roche reagent, calibrator and quality control material;

D. the Siemens ADVIA 2400 full-automatic biochemical analyzer is matched with German.

3. Experimental protocol

3.1 preparation of Special calibrators

The low value sample (L: 27.1mg/L) and the high value sample (H: 109.9mg/L)27.1 in the C-reactive protein calibrator prepared in example 1 were prepared into calibration spots 1-5 having a total volume of 500. mu.l by volume ratio of L, 4L +1H, 3L +2H, 1L +4H, and H according to the weighing method, and the theoretical concentrations thereof were calculated as shown in Table 10.

TABLE 10 formulation and concentration of calibration points

The theoretical concentrations in table 10 were calculated specifically as follows:

a first step of preparing a calibration point 1-5 having a total volume of 500. mu.l from the low value sample (L: 27.1mg/L) and the high value sample (H: 109.9mg/L) in the C-reactive protein calibrator prepared in example 1 in a volume ratio of L, 4L +1H, 3L +2H, 1L +4H, during which the specific weights of the low value sample (L: 27.1mg/L) and the high value sample (H: 109.9mg/L) used in the preparation of each solution are accurately measured by an analytical balance weighing method;

second step, Density measurement of solutions

(4) This was repeated 3 times and recorded in detail.

(6) The density of each solution was calculated according to the procedures (1) to (5).

Thirdly, calculating the theoretical concentration of each solution

The theoretical concentration of each solution was calculated from the CRP concentration in the low value sample (L: 27.1mg/L) and the high value sample (H: 109.9 mg/L). For a certain tube of solution, the concentration of CRP in a high-value sample is C1g/L, the density is rho 1, the weighed mass is M1, the concentration of CRP in low-value serum is C2g/L, the density is rho 2, the weighed mass is M2, the density of a certain tube per se is rho 3, and the theoretical concentration of the certain tube is obtained by using the formula:

3.2 detection and statistical methods

First, the reagent manufacturers of A, B, C and system D calibrated the instruments with their respective calibrators and then tested their respective quality controls. After the quality control product is controlled (namely the quality control is qualified, the instrument can work normally), 21 patient serums are respectively detected for 2 times, the average coefficient of variation (CV1) of 4 systems is calculated, and the international standard substance ERM-DA474/IFCC is detected for 2 times only by adopting the systems A, B and C.

Then, after 4 systems were calibrated respectively using the CRP-specific calibrator prepared above, 21 patient samples were retested 2 times, and the evaluation coefficient of variation (CV2) was calculated, and the international standard substance ERM-DA474/IFCC was tested 2 times using only the a, B, and C systems.

Finally, CV1 and CV2 before and after calibration of the special calibrator are respectively compared with an optimal coefficient of variation CV (CV is 0.25 CV) derived based on the biological variation degree_I，CV_IIs the degree of biological variation in an individual), and the uniformity improvement effect is judged. Meanwhile, the bias of ERM-DA474/IFCC and the authentication value (41.2mg/L) before and after the calibration of the special calibrator is calculated, and the calibration is judgedWhether the post-bias is within its uncertainty (2.5 mg/L).

Second, experimental results

Before calibration, the average Coefficient of Variation (CV) between dessely, ridemann, siemens and roche reagents was 18.64% (table 11), with dessely as the comparative detection system and the other detection systems as the evaluation systems, the resulting slope ranged from 0.90 to 1.09 (table 13, fig. 6), and the correlation coefficient ranged from 0.996 to 0.998; when ERM-DA474/IFCC is used as a correct verification substance, the relative deviation of the mean value of 2 detections of 3 systems is 7.46 to-26.86 percent, the absolute deviation is 3.08 to-11.07 mg/L, and the relative deviation exceeds the uncertainty (2.5mg/L) range of ERM-DA474/IFCC (Table 14).

The mean Coefficient of Variation (CV) between dessely, ridmann, siemens and roche reagents after calibration was 7.03% (table 12), less than the CV derived based on biological variability (CV ═ 10.6%). The Desai is taken as a comparison detection system, other detection systems are taken as evaluation systems, the obtained slope range is 0.93-0.96 (table 13, figure 6), and the correlation coefficient is 0.997-0.999; when ERM-DA474/IFCC is used as a correct verification substance, the relative deviation of the mean value of 2 times of detection of 3 systems is 2.31 to-7.21 percent, the absolute deviation is 0.95 to-2.97 mg/L, and the uncertainty (2.5mg/L) of the ERM-DA474/IFCC of other systems except the detection system of the Roche reagent is within the range (Table 14).

TABLE 11 comparability of the Pre-calibration 4 systems for the dedicated calibrators

TABLE 12 comparability of post-calibration 4 systems with dedicated calibrators

TABLE 13 comparability and correlation before and after calibration of Texas and other systems

TABLE 14 accuracy of ERM-DA474/IFCC detection by each system before and after calibration

The accuracy of the test result can provide scientific basis for disease diagnosis and treatment monitoring of patients. CRP is a marker which is commonly used in clinic for judging infection and inflammation, and has important significance for identifying bacterial or viral infection. The standardization of CRP dates back to the 80's of the 20 th century. At present, reagents and calibrators of various manufacturers claim to be sourced to the international standard substance ERM-DA 474/IFCC. The invention prepares the CRP special calibrator with 2 concentration levels, and adopts 10 clinical common CRP detection systems to transfer the value of ERM-DA474/IFCC to the special calibrator, namely to obtain the fixed value of the special calibrator.

The special calibrator prepared by the invention is derived from fresh frozen mixed human serum, so that the special calibrator has good interchangeability. The current literature shows that the consistency of the detection results of various laboratories is obviously improved when the compatible calibrator is used for detecting human mixed serum. The invention adopts the special calibrator to detect individual serum trays of single patients which can represent clinical actual detection conditions, and the result shows that the degree of systematical consistency after calibration obviously improves the coefficient of variation among systems to 7.03 percent, thereby meeting the optimal CV requirement derived from biogenetic variation. The proportional system deviation and the fixed system deviation are both obviously reduced, and the correlation is obviously improved. The accuracy of 3 detection systems after calibration is also obviously improved, and besides the Roche detection system, the detection deviation of the Lidman and the Siemens is less than the uncertainty of the fixed value of the ERM-DA 474/IFCC.

In conclusion, the CRP special calibrator developed by the invention is suitable for calibrating clinical conventional CRP detection reagents. The calibration method can improve the consistency and comparability of CRP detection results of individual patients, has good application prospect and is worthy of popularization.

Claims

1. A C-reactive protein calibrator is prepared by the following steps: collecting the in vitro serum of different individuals, mixing to obtain 2 serum solutions with different concentrations of C-reactive protein, and then respectively carrying out value determination on each serum solution in the 2 serum solutions with different concentrations of C-reactive protein to obtain the following 2 concentrations of C-reactive protein calibrators:

2) low value sample: the result of the fixed value of the C-reactive protein concentration is 27.1 +/-2.3 mg/L serum solution;

the method for valuing each serum solution in the 2 serum solutions with different concentrations of C-reactive protein comprises the following steps: the candidate C-reactive protein standard substance is subjected to value determination by adopting a quantity value transmission mode of the C-reactive protein international standard substance;

the method for valuing each serum solution in the 2 serum solutions with different concentrations of C-reactive protein comprises the following steps:

(a1) diluting the international standard substance of the C-reactive protein to obtain a series of diluted solutions of the international standard substance of the C-reactive protein with different concentrations, and calculating to obtain the theoretical concentration of the C-reactive protein in each diluted solution;

(a2) respectively measuring the concentration of the C-reactive protein in each diluted solution obtained in the step (a1) by adopting N quantitative detection systems of the C-reactive protein;

(a3) for each quantitative detection system of the C-reactive protein in the N quantitative detection systems of the C-reactive protein, performing linear fitting by adopting the theoretical concentration of the C-reactive protein in each diluted solution obtained in the step (a1) and the measured concentration of the C-reactive protein in each diluted solution obtained in the step (a2) to obtain a C-reactive protein fixed value standard curve;

(a4) respectively measuring the concentration of C-reactive protein in the candidate C-reactive protein standard substance by adopting the N quantitative detection systems for C-reactive protein;

(a5) substituting the measured concentration of C-reactive protein in the candidate C-reactive protein standard substance obtained in step (a4) into the equation of the corresponding C-reactive protein fixed value standard curve obtained in step (a3) for each of the N C-reactive protein quantitative detection systems, thereby obtaining the concentration of C-reactive protein in the candidate C-reactive protein standard substance;

(a6) averaging the concentrations of C-reactive protein in all the candidate C-reactive protein standard substances of the N quantitative C-reactive protein detection systems obtained in the step (a5), wherein the obtained average value is the final fixed value result of the concentration of C-reactive protein in the candidate C-reactive protein standard substances;

n is a natural number of more than 2;

the step (a1) is realized according to a method comprising the following steps:

(A) preparing a series of diluted solutions of the C-reactive protein international standard substances with different concentrations: mixing the diluent and the C-reactive protein international standard substance according to different volume ratios, thereby obtaining a series of diluent solutions of the C-reactive protein international standard substance with different concentrations; in the process of preparing each part of the diluted solution of the C-reactive protein international standard substance, weighing the diluted solution and the C-reactive protein international standard substance which form the diluted solution of the C-reactive protein international standard substance respectively;

(B) density determination of dilution solutions of the international standard substance for C-reactive protein in series of different concentrations: measuring respective densities of the dilution solutions of the international standard substance for C-reactive protein prepared in the step (a) in series of different concentrations, respectively;

wherein C1 represents the concentration of C-reactive protein in the international standard substance for C-reactive protein; ρ 1 represents the density of the international standard substance for C-reactive protein; m1 represents the mass of the C-reactive protein international standard substance in the diluted solution of the C-reactive protein international standard substance; c2 represents the concentration of C-reactive protein in the dilution; ρ 2 represents the density of the diluent; m2 represents the mass of the dilution in the dilution of the international standard substance for C-reactive protein; ρ 3 represents the density of a diluted solution of the international standard substance for C-reactive protein; c represents the theoretical concentration of the diluted solution of the international standard substance of the C-reactive protein;

the C-reactive protein international standard substance is ERM-DA 474/IFCC;

in the step (a1), when the international standard substance of the C-reactive protein is diluted, the adopted diluent is low-value serum; the low value serum is serum containing C-reactive protein with the concentration of 0.27 mg/L.

2. The C-reactive protein calibrator according to claim 1, wherein: in the step (B), the density of the diluted solution of the international standard substance for C-reactive protein is measured according to a method comprising the following steps:

(b2) sucking a sample to be tested with the volume of constant v by using the gun head of the sample adding gun in the step (b1), then placing the sample to be tested into the container in the step (b1), and weighing the sample to be tested as m 2;

3. The C-reactive protein calibrator according to claim 2, wherein: steps (b1) and (b2) were repeated 3-5 times, and the parameters m1 and m2 to the right of the equation equal sign in step (b3) were averaged for 3-5 times of repetition.

4. Use of a C-reactive protein calibrator according to any one of claims 1 to 3 for any one of:

(i) calibrating the C-reactive protein detection reagent;

5. A calibration method for improving consistency of detection results of C-reactive protein comprises the following steps:

(c1) preparing N parts of C-reactive protein calibrator solutions with different concentrations: one part is the high value sample in the C-reactive protein calibrator of any one of claims 1-3, one part is the low value sample in the C-reactive protein calibrator of any one of claims 1-3, and N-2 parts are serial mixed samples formed by mixing the high value sample and the low value sample according to different volume ratios, and the theoretical concentration of the C-reactive protein in each mixed sample is calculated;

(c2) calibrating the C-reactive protein quantitative detection system by adopting the N parts of C-reactive protein calibrator solutions with different concentrations prepared in the step (C1), and then detecting the concentration of the C-reactive protein in the serum sample to be detected;

and N is a natural number of more than 3.