CN112684189A - Calibration method for improving TSH detection result consistency, TSH candidate standard substance and preparation method thereof - Google Patents
Calibration method for improving TSH detection result consistency, TSH candidate standard substance and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a calibration method for improving the consistency of TSH detection results, a TSH candidate standard substance and a preparation method thereof. The preparation method of the TSH candidate standard substance for improving the consistency of the TSH detection result comprises the following steps: collecting the in vitro serum of different individuals, mixing to obtain 11 serum solutions with different TSH concentrations, and carrying out definite value on the TSH concentrations in the 11 serum solutions with different TSH concentrations to obtain the TSH candidate standard substance with 11 concentrations. For different manufacturers, the candidate standard substance is used for preparing a TSH special calibrator with the concentration equal to that of the original calibrator to replace the original calibrator, and the consistency of the individual serum TSH detection results is greatly improved compared with the results obtained before and after the TSH special calibrator is adopted. The calibration method for improving the TSH detection result consistency is suitable for the calibration of clinical conventional TSH detection reagents, and can improve the consistency and comparability of TSH detection results of different manufacturers.
Description
Technical Field
The invention relates to the technical field of clinical detection, in particular to a calibration method for improving TSH detection result consistency, a TSH candidate standard substance and a preparation method thereof.
Background
Thyroid Stimulating Hormone (TSH) is a glycoprotein Hormone secreted by adenohypophysis basophils and consists of 211 amino acids, and its molecular structure consists of noncovalent α, β subunit heterodimers, wherein the Hormone is specific for biological and immunological functions and is β subunit, and α subunit is substantially identical to α subunit of Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH) and Human Chorionic Gonadotropin (HCG). TSH can promote secretion of T3 (triiodothyronine) and T4 (thyroxine), and is the most sensitive index for judging thyroid gland function under negative feedback of T3 and T4, and the level of the index should be closely monitored in the course of thyroid gland disease treatment.
Currently, immunoassays of TSH mainly include Radioimmunoassay (RIA), immunoradiometric Immunoassay (IRMA), Chemiluminescence Immunoassay (CLIA), Electrochemiluminescence Immunoassay (ECLIA), Time-resolved Fluoroimmunoassay (trafia), and Chemiluminescence Enzyme Immunoassay (CLEIA). TSH manufacturers have large detection results, and Beckmann Access 2 is about 20 times higher than Siemens Immunite 2000 in some TSH detection values with low concentration. Recent studies have shown that TSH varies less between methods at normal concentrations and more at abnormal concentrations. The comparison research of 6 TSH detection methods by domestic Malong et al discovers that the methods have different differences, and discovers that the correlation between the radioimmunoassay and the radioimmunoassay with an automatic detection system is poor (r is 0.38-0.41).
An important way to achieve consistency in test results is the standardization of the test method, published by the 1993 international association of clinical chemistry (IFCC), the european community's standard substance office (BCR) and the american pathologist association (CAP) in combination with the serum protein-certified reference substance CRM 470, which was shown to assign values to calibrators using this standard substance, resulting in significantly smaller inter-laboratory measurement differences between the various laboratories. However, there has been no practical study on TSH, which is a test item using the chemiluminescence principle, to reduce the difference in the detection results between detection systems. The invention adopts a plurality of candidate standard substances with fixed values to prepare special calibrators of various manufacturers to replace user terminal calibrators of the manufacturers, thereby realizing the consistency of the inspection results.
Disclosure of Invention
Aiming at the technical problems, the invention provides a calibration method for improving the consistency of TSH detection results, a TSH candidate standard substance and a preparation method thereof, which can overcome the defects in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a method for preparing a TSH candidate standard substance for improving the consistency of TSH detection results comprises the following steps:
s1: collecting the in vitro serum of different individuals, and mixing to obtain 11 serum solutions with different TSH concentrations;
s2: and (3) respectively determining the TSH concentration of each serum solution in the 11 serum solutions with different TSH concentrations to obtain 11 TSH candidate standard substances with different concentrations.
Further, the step S2 uses TSH international standard substance to measure and deliver TSH values to determine TSH concentrations in the 11 serum solutions (candidate TSH standard substances) with different TSH concentrations.
Further, the method for determining the TSH concentration in the serum solution with 11 different TSH concentrations specifically comprises the following steps:
s2.1: diluting the TSH international standard substance to obtain diluted solutions of the TSH international standard substance with different concentrations, and calculating the TSH authentication concentration in each diluted solution;
s2.2: respectively measuring the concentration of TSH in each diluted solution obtained in the step S2.1 by adopting N TSH quantitative detection systems;
s2.3: for each TSH quantitative detection system in the N TSH quantitative detection systems, performing linear fitting by adopting the authentication concentration of TSH in each diluted solution obtained in the step S2.1 and the determination concentration of TSH in each diluted solution obtained in the step S2.2 to obtain a TSH fixed value standard curve, namely obtaining N TSH fixed value standard curves by the N TSH quantitative detection systems;
s2.4: respectively measuring the TSH concentration in the TSH candidate standard substance by adopting the N TSH quantitative detection systems;
s2.5: for each TSH quantitative detection system in the N TSH quantitative detection systems, substituting the measured concentration of TSH in the TSH candidate standard substance obtained in step S2.4 into the equation of the corresponding TSH fixed-value standard curve obtained in step S2.3 to obtain the concentration of TSH in the TSH candidate standard substance, that is, substituting the concentration value of TSH in the TSH candidate standard substance measured by a certain TSH quantitative detection system in step S2.4 into the equation of the TSH fixed-value standard curve corresponding to the TSH quantitative detection system obtained in step S2.3 to obtain the concentration value of TSH in the TSH candidate standard substance, which is the fixed-value result of the TSH quantitative detection system on the concentration of TSH in the TSH candidate standard substance, and obtaining N fixed-value results for the N TSH quantitative detection systems; wherein, the abscissa (X) of the TSH fixed value standard curve is the authentication concentration, and the ordinate (Y) is the measurement concentration; in the step S2.5, substituting the measured concentration of TSH in the candidate TSH standard substance into the Y value of the TSH fixed value standard curve equation, and calculating the obtained X value as the concentration of TSH in the candidate TSH standard substance;
s2.6: averaging the concentrations of TSH in all the candidate TSH standard substances of the N TSH quantitative detection systems obtained in the step S2.5, wherein the obtained average value is the final fixed value result of the TSH concentration in the candidate TSH standard substances;
and N is a natural number more than or equal to 2.
Further, the N is a natural number of 3-20.
Further, N is 10.
Further, said step S2.1 comprises the steps of:
S2.1.1A series of diluted solutions of the TSH international standard substance at different concentrations were prepared: mixing a diluent and the TSH international standard substance stock solution according to different volume ratios, thereby obtaining a series of diluted solutions of the TSH international standard substance with different concentrations; in the process of preparing each diluted solution of the TSH international standard substance, weighing the diluted solution and the stock solution of the TSH international standard substance which form the diluted solution of the TSH international standard substance respectively;
s2.1.2 series density determination of diluted solutions of the TSH international standard substance at different concentrations: separately measuring the densities of the series of diluted solutions of the international standard substance for TSH formulated in step S2.1.1 at different concentrations;
s2.1.3, obtaining the certified concentration of TSH in a series of different concentrations of diluted solutions of the TSH international standard substance: calculating the certified concentration of TSH in each diluted solution of the series of diluted solutions of the international standard substance for TSH at different concentrations according to the following formula:
wherein C1 represents the concentration of TSH in the TSH international standard substance stock solution, ρ 1 represents the density of the TSH international standard substance stock solution, M1 represents the mass of the TSH international standard substance stock solution of the TSH international standard substance, M2 represents the mass of the diluent in the diluted solution of the TSH international standard substance, ρ 2 represents the density of the diluted solution of the TSH international standard substance, and C represents the certified concentration of the TSH international standard substance standard series.
Further, in the step S2.1, the dilution used for diluting the TSH international standard substance is mixed human serum or Hanks solution containing Bovine Serum Albumin (BSA), and the mixed human serum and Hanks solution containing BSA are solutions with a TSH concentration of 0 μ IU/mL; the serum is clear and transparent in appearance, except for abnormal characters such as jaundice, hemolysis, lipemia, chyle and the like.
According to another aspect of the present invention, there is provided a TSH candidate standard substance prepared by the above method, wherein the TSH candidate standard substance is a serum solution of 11 different TSH concentrations, and the fixed values of the serum solutions of 11 different TSH concentrations are 0.191 μ IU/mL (L1), 0.500 μ IU/mL (L2), 2.094 μ IU/mL (L3), 4.118 μ IU/mL (L4), 4.929 μ IU/mL (L5), 6.748 μ IU/mL (L6), 8.245 μ IU/mL (L7), 12.604 μ IU/mL (L8), 16.401 μ IU/mL (L9), 23.880 μ IU/mL (L10), and 44.985 μ IU/mL (L11), respectively.
Further, the TSH candidate standard substance also includes a very high value sample (L12).
Since the product calibrators from manufacturers generally have high values higher than L11, the serum collected after the test with TSH concentration greater than 150 μ IU/mL was mixed into L12, the system of values used is shown in Table 6, the values used are L5-L11, the dilution times of L12 by weight are 5.801, 9.734 and 20.080, respectively, and the dilution used is mixed human serum (L) with TSH concentration of 0 μ IU/mL is shown in Table 7. The final L12 rating results are shown in Table 8.
The density measurement of the L12 solution and the dilution point comprises the following specific steps:
(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) The formula is calculated according to the density of the liquid:
ρH＝(m2-m1)/v1
(5) repeating the steps (1) - (4) for 2 times.
The densities of L12 were measured to be 1.025 and 1.022 with an average of 1.024.
(6) The density ρ of each mixed solution was obtained in the same mannerMixing ofSee table 7.
The dilution factor is calculated by the formula:
wherein N represents the dilution factor, WHDenotes the mass of L12, WLDenotes the mass of L, ρMixing ofDenotes the density, ρ, of the mixed liquidHThe density of L12 was expressed, the basic information of the L12 constant value detection system is shown in Table 6, and the L12 constant value dilution factor is shown in Table 7Shown in the figure.
L12 was evaluated using the above method and 5 different TSH detection systems as shown in table 8.
The invention provides a calibration method for improving TSH detection result consistency, which comprises the following steps:
(1) preparing M TSH calibrator solutions of different concentrations: one part is from the high value sample in the TSH candidate standard substance, one part is from the low value sample in the TSH candidate standard substance, and the other M-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 concentration of the TSH obtained by mixing is from the concentration of each manufacturer user terminal calibrator;
(2) after calibrating TSH detection reagents of various manufacturers by adopting calibrators of various manufacturers, detecting the TSH concentration in serum of a certain number of individuals, and detecting each individual serum for 2 times;
(3) calibrating the TSH quantitative detection system by adopting M TSH calibrator solutions with different concentrations prepared in the step (1) (namely setting a standard and a corresponding so-called 'true value' of a certain signal), and then detecting the concentration of TSH in the individual serum sample for 2 times;
and M is a natural number which is more than or equal to 2 (M is 1 under special conditions).
Further, in the step (1), 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 constituting 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, the mass sum of the low value sample and the high value sample constituting the mixed sample, and further the authentication concentration of the TSH in each mixed sample is obtained through calculation.
The invention has the beneficial effects that: the TSH candidate standard substance prepared by the invention is derived from mixed human serum, so that the interchangeability is good, and the fixed value is derived from international standard substances, so that the consistency and the accuracy of the detection result of each laboratory can be improved; the TSH candidate standard substance is adopted to detect individual serum trays of a single patient which can represent clinical actual detection conditions, and the result shows that the degree of uniformity among systems after calibration is obviously improved, the median of TSH detection value variation coefficients among systems is changed from 17.94% to 9.56%, the median of TSH detection value variation coefficients of mixed human serum is changed from 17.10% to 5.52%, and the requirement (9.65%) of variation Coefficient (CV) derived from biological variation is met; the TSH candidate standard substance is suitable for the calibration of a clinical conventional TSH detection reagent; the calibration method of the invention can improve the consistency and comparability of the TSH detection result of individual patients.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the interchangeability of various substrate-diluted TSH international standards between the ADVIA CentaurXP and Immulite2000 systems (A is 4 substrates and B is 2 substrates used for the same), wherein the judgment criteria for interchangeability is that the TSH international standards are within two 95% confidence lines (dotted lines) above and below the Deming regression line drawn by the individual serum TSH detection values.
FIG. 2 shows the interchangeability of various substrate-diluted TSH international standards between the ADVIA CentaurXP and Architect i2000sr systems (A is 4 substrates and B is 2 substrates used for the same), wherein the judgment criteria for interchangeability is that the TSH international standard is within two 95% confidence lines (dotted lines) above and below the Deming regression line drawn by the individual serum TSH detection values.
FIG. 3 shows the interchangeability of the TSH international standards diluted in various matrices between the ADVIA CentaurXP and DXI800 systems (A is 4 matrices, B is 2 matrices used for the same values), wherein the judgment criteria for interchangeability is that the TSH international standards are within two 95% confidence lines (dotted lines) above and below the Deming regression line drawn by the TSH detection value of the individual serum.
FIG. 4 shows the interchangeability of various substrate-diluted TSH international standards between the ADVIA CentaurXP and Liaison XL systems (A is 4 substrates and B is 2 substrates used for the determination), wherein the criterion of interchangeability is that the TSH international standards are within two 95% confidence lines (dotted lines) above and below the Deming regression line drawn by the individual serum TSH detection values.
FIG. 5 shows the interchangeability of the ADVIA CentaurXP and CI 1000 systems for various substrate dilutions of TSH international standards (A for 4 substrates and B for 2 substrates used for the same), where the criterion for interchangeability is that the TSH international standards are within the two 95% confidence lines (dotted lines) above and below the Deming regression line drawn for individual serum TSH measurements.
FIG. 6 shows the interchangeability of various substrate-diluted TSH international standards between ADVIA CentaurXP and Cobas 601 systems (A is 4 substrates and B is 2 substrates used for the fixed values), wherein the judgment criteria for interchangeability is that the TSH international standards are within two 95% confidence lines (dotted lines) above and below the Deming regression line drawn by the TSH detection value of individual serum.
FIG. 7 shows the interchangeability of the TSH international standard substance diluted with various matrices between the ADVIA CentaurXP and the Autolumo A2000plus system (A is 4 matrices, B is 2 matrices used for the fixed values), wherein the judgment criteria for interchangeability is that the TSH international standard substance is within the two 95% confidence lines (dotted lines) above and below the Deming regression line drawn by the TSH detection value of the individual serum.
FIG. 8 is a TSH international standard substance (L1 and L2) assignment curve (A is mixed human serum with TSH concentration of 0 μ IU/mL, B is Hanks liquid containing bovine serum albumin) at different concentrations diluted with DXI800 in two matrixes;
FIG. 9 shows the assignment curves of different concentrations of TSH international standard substance (L3 and L4) diluted with DXI800 in two matrices (A is mixed human serum with TSH concentration of 0 μ IU/mL, and B is Hanks solution containing bovine serum albumin);
FIG. 10 is a TSH international standard substance (L5, L6 and L7) assignment curve (A is mixed human serum with TSH concentration of 0 μ IU/mL, B is Hanks solution containing bovine serum albumin) at different concentrations diluted with DXI800 in two matrices;
FIG. 11 is a set of curves assigned to different concentrations of TSH international standards (L8, L9 and L10) diluted with DXI800 in two matrices (A is human serum cocktail with TSH concentration of 0 μ IU/mL, B is Hanks' solution with bovine serum albumin);
FIG. 12 is a TSH international standard substance (L11) assignment curve (A is mixed human serum with TSH concentration of 0 μ IU/mL, B is Hanks solution containing bovine serum albumin) at different concentrations diluted with DXI800 in two matrices;
FIG. 13 is a set of TSH international standard substance (L1 and L2) assignment curves at different concentrations diluted with Cobas 601 in two matrices (matrix A is mixed human serum with TSH concentration of 0 μ IU/mL, and B is Hanks solution containing bovine serum albumin);
FIG. 14 shows assignment curves of different concentrations of TSH international standard substance (L3 and L4) diluted with Cobas 601 in two matrices (A is mixed human serum with TSH concentration of 0 μ IU/mL, and B is Hanks solution containing bovine serum albumin);
FIG. 15 shows assignment curves of different concentrations of TSH international standard substance (L5, L6 and L7) diluted with Cobas 601 in two matrices (A is mixed human serum with TSH concentration of 0 μ IU/mL, and B is Hanks solution containing bovine serum albumin);
FIG. 16 shows assignment curves of different concentrations of TSH international standard substance (L8, L9 and L10) diluted with Cobas 601 in two matrices (A is mixed human serum with TSH concentration of 0 μ IU/mL, and B is Hanks solution containing bovine serum albumin);
FIG. 17 is a set of TSH international standard substance (L11) assignment curves (A is mixed human serum with TSH concentration of 0 μ IU/mL, B is Hanks solution containing bovine serum albumin) at different concentrations diluted with Cobas 601 in two matrices;
fig. 18 is a comparison graph of the improved effect of the TSH detection result consistency before and after calibration of the dedicated calibrator (a is the intersystem comparability before calibration of the dedicated calibrator, and B is the intersystem comparability after calibration of the dedicated calibrator).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Step one (TSH candidate standard substance fixed value)
A preparation method of a special calibration product for a TSH candidate standard substance for improving the consistency of TSH detection results comprises the following steps:
s1: collecting the in vitro serum of different individuals, and mixing to obtain 11 serum solutions with different TSH concentrations;
s2: and (3) respectively determining the TSH concentration in the 11 serum solutions with different TSH concentrations to obtain 11 TSH candidate standard substances with different concentrations.
In the step S2, the TSH concentration in the 11 serum solutions (candidate TSH standard substances) with different TSH concentrations is determined by means of quantitative delivery of the TSH international standard substance NIBSC 81/565, interchangeability of TSH international standard substances diluted by multiple substrates among multiple systems is shown in fig. 1-7, and basic information of TSH detection systems is shown in table 1 (see table 2 for dilution).
The judgment standard for interchangeability of the TSH international standard substances between the two systems is that the TSH international standard substances are in two 95% confidence line ranges above and below a Deming regression line drawn by individual serum TSH detection values. The specific implementation step is that serum of 29 patients with different TSH concentrations in high, medium and low levels (ethical approval 2018-2-26-1) is collected from the Beijing Kogyang Hospital affiliated to the university of capital medicine. The serum of each sample was divided into 8 portions according to the manufacturer's requirements and tested 3 times in 8 different systems (see table 1 for 8 test systems except IS 1200 and magumi 2000 plus). Samples were required to be free of hemolysis, lipemia and jaundice in appearance, and initial concentrations of TSH were measured using a Siemens ADVIA CentauerXP immunoassay to range from 0.09. mu.IU/mL to 84.03. mu.IU/mL. The reason why the compatibility evaluation of the TSH international standard substances is carried out by taking the ADVIA CentaurXP as a comparison system is that the system is an instrument which is used by the laboratory conventionally, and the laboratory passes the double recognition of the International organization for standardization (ISO)15189 and the American society of pathologists (CAP), so that the detection quality is guaranteed.
TABLE 1 TSH detection System basic information
AMR, analyzing the measuring range; RR, reference range; ECL, electrochemiluminescence; CL, chemiluminescence; CMIA, chemiluminescent microparticle immunoassay.
TABLE 2 basic information on TSH International Standard substance dilutions
IS, TSH international standard substance; HSP0, TSH concentration 0 μ IU/mL human serum mixture; BSA, bovine serum albumin (Sigma, B2064, concentration 46 g/L).
From FIGS. 1-7, it can be seen that in addition to ADVIA CentauerXP and Cobas 601, mixed human serum (HSP0) with a TSH concentration of 0. mu.IU/mL allows interchangeability of the diluted international standard for TSH across the systems. Whereas the BSA containing Hanks solution enables the interchangeability of the diluted TSH international standard between the ADVIA CentauerXP and DXI 800/Cobas 601/Autolumo A2000plus systems. Therefore, the two matrixes are selected as the dilution of the TSH international standard substance to draw a calibration curve to determine the value of the TSH candidate standard substance, and finally, the value obtained by each value system is required to be within the range of plus/minus 3 times of standard deviation of the overall mean value.
Furthermore, in order to explore the importance of the interchangeability of TSH international standard substances to multi-system valuing in the field of in vitro diagnosis quantity value tracing, the invention firstly defines two problems: (1) different dilutions allow different interchangeability of TSH international standard (figures 1-7). (2) Only if TSH international standard substances are made to have interchangeability, the fixed value of TSH candidate standard substances can be consistent among/in multiple systems, and the fixed value of the multiple systems can be successful.
For example, when TSH international standard substance is diluted with HSP0 and BSA-containing Hanks solution, respectively, and the fixed value curves obtained by DXI800 system are shown in fig. 8-12, it can be seen that the slopes of the left 5 fixed value curves and the slopes of the right 5 fixed value curves are close to each other, the fixed values are consistent (2 fixed value samples are used for each concentration level), and the median of the bias is smaller than the maximum allowable bias (11.7%) derived based on biological variation; however, when TSH international standard substance was diluted with HSP0 and BSA-containing Hanks solution, and the fixed value curves obtained using the Cobas 601 system are shown in fig. 13-17, it can be seen that the slopes of the left 5 fixed value curves and the right 5 fixed value curves are very different, the fixed values are not consistent (2 fixed value samples are used for each concentration level), and the median value of the bias is larger than the maximum allowable bias (11.7%) derived based on the biological variation, as shown in table 3. Therefore, we concluded that in valuing a candidate TSH standard, a substrate-drawn fixed value curve that allows interchangeability of TSH international standard was selected as much as possible for valuing. When using interchangeable matrices (see FIG. 3B), such as TSH international standards diluted with HSP0 and BSA containing Hanks solution, TSH candidate standards were scored at DXI800 system, both matrices were used and the results compared to other test systems scored to meet the requirement of within plus/minus 3 standard deviations of the overall mean (tables 3-5). When a substrate without interchangeability (see fig. 6B) is used, for example, a TSH international standard substance is diluted with HSP0, the TSH candidate standard substance is rated in the Cobas 601 system, and the rating result is greatly different from that of the rating curve obtained by other systems or a substrate with interchangeability of the TSH international standard substance, and cannot meet the requirement of adding/subtracting 3 times of the standard deviation from the overall mean value (table 3, table 5). Therefore, when multi-system valuing is carried out on the TSH candidate standard substance, relevant interchangeability research needs to be carried out firstly when TSH international standard substance diluent is selected, a substrate which enables the international standard substance to have interchangeability is selected as far as possible for valuing, and the consistency and the success rate of the valuing are improved.
Further, in order to verify the wide suitability of HSP0 and BSA-containing Hanks solution for TSH candidate standard substance valuing, the present invention additionally selects 2 TSH mainstream detection systems (IS 1200 and magumi 2000plus, see table 1), serial dilutions of TSH international standard substances are performed, a fixed value curve IS drawn as TSH candidate standard substance fixed value, finally, it IS found that the TSH international standard substances are diluted with HSP0 and BSA-containing Hanks solution, the fixed value result obtained with the IS 1200 system IS close to the overall mean value, the TSH international standard substances are diluted with HSP0, the fixed value result obtained with the magumi 2000plus system IS close to the overall mean value, see table 4.
Finally, the results of the 13-value-setting methods of the 10 detection systems are shown in Table 5.
TABLE 3 bias of same assay system for TSH candidate Standard quantitation using different substrates
TABLE 5 Final rating of TSH candidate Standard substance
The method for performing the value determination in the step S2 specifically includes the following steps:
s2.1: diluting the TSH international standard substance by using a diluent (hereinafter referred to as an appropriate diluent) which enables the TSH international standard substance to have interchangeability among systems to obtain diluted solutions of the TSH international standard substance with different concentrations, namely a fixed value curve, and calculating the TSH authentication concentration in each diluted solution;
s2.2: adopting 10 TSH quantitative detection systems (13 quantitative methods, wherein ADVIA CentaurXP, DXI800 and IS 1200 adopt 2 diluents to dilute the international standard substances to respectively obtain 10 quantitative curves, and the other 7 quantitative systems adopt 1 diluent to dilute the international standard substances to respectively obtain 5 quantitative curves) to respectively determine the concentration of TSH in each dilution solution obtained in the step S2.1, wherein the TSH quantitative detection systems are required to pass performance verification of a series of measurement systems such as precision, accuracy, system linearity and carried pollution before formal test, and performing formal quantitative test after the performance verification passes;
s2.3: for each of the 10 quantitative TSH detection systems, the TSH international standard substance is diluted with the suitable diluent obtained in step S2.1 to obtain the certified concentration of TSH, and then linear fitting is performed with the measured concentration of TSH in each diluted solution obtained in step S2.2 to obtain a TSH quantitative value standard curve. Because the TSH concentration ranges of the standard curve and the TSH candidate standard substance are wide, a plurality of fixed value curves are adopted for the TSH candidate standard substance. Namely, 65 TSH fixed value standard curves are obtained by 13 TSH fixed value methods;
s2.4: respectively measuring the concentration of TSH in the TSH candidate standard substance by adopting the 13 TSH value determination methods;
s2.5: for each TSH quantitative determination system in the 13 TSH quantitative determination methods, substituting the measured concentration of TSH in the candidate TSH standard substance obtained in step S2.4 into the equation of the corresponding TSH quantitative determination standard curve obtained in step S2.3 to obtain the concentration of TSH in the candidate TSH standard substance, that is, substituting the concentration value of TSH in the candidate TSH standard substance measured by a certain TSH quantitative determination system in step S2.4 into the equation of the TSH quantitative determination standard curve corresponding to the TSH quantitative determination system obtained in step S2.3 to obtain the concentration value of TSH in the candidate TSH standard substance, that is, the concentration value result of TSH in the candidate TSH standard substance by the TSH quantitative determination system, and selecting 2 samples for each concentration level in the 13 TSH quantitative determination methods to obtain 26 fixed value results; wherein, the abscissa (X) of the TSH fixed value standard curve is the authentication concentration, and the ordinate (Y) is the measurement concentration; in step S2.5, the measured concentration of TSH in the TSH candidate standard substance is substituted into the Y value of the TSH fixed-value standard curve equation, and the calculated X value is the concentration of TSH in the TSH candidate standard substance, as shown in table 4;
s2.6: averaging the TSH concentrations in all the candidate TSH standard substances obtained in step S2.5 for the 13 TSH valuing methods, and obtaining the average value, which is the final valuing result of the TSH concentrations in the candidate TSH standard substances, as shown in table 5.
Said step S2.1 comprises the steps of:
S2.1.1A series of diluted solutions of the TSH international standard substance at different concentrations were prepared: mixing a diluent and the TSH international standard substance stock solution according to different volume ratios, thereby obtaining a series of diluted solutions of the TSH international standard substance with different concentrations; in the process of preparing each diluted solution of the TSH international standard substance, weighing the diluted solution and the stock solution of the TSH international standard substance which form the diluted solution of the TSH international standard substance respectively;
s2.1.2 series density determination of diluted solutions of the TSH international standard substance at different concentrations: separately measuring the densities of the series of diluted solutions of the international standard substance for TSH formulated in step S2.1.1 at different concentrations;
s2.1.3, obtaining the certified concentration of TSH in a series of different concentrations of diluted solutions of the TSH international standard substance: calculating the certified concentration of TSH in each diluted solution of the series of diluted solutions of the international standard substance for TSH at different concentrations according to the following formula:
wherein C1 represents the concentration of TSH in the TSH international standard substance stock solution, ρ 1 represents the density of the TSH international standard substance stock solution, M1 represents the mass of the TSH international standard substance stock solution of the TSH international standard substance, M2 represents the mass of the diluent in the diluted solution of the TSH international standard substance, ρ 2 represents the density of the diluted solution of the TSH international standard substance, and C represents the certified concentration of the TSH international standard substance standard series.
In the step S2.1, the dilution liquid used for diluting the TSH international standard substance is mixed human serum or Hanks liquid containing BSA, and the serum and the Hanks liquid containing BSA are serum with TSH concentration of 0 mu IU/mL; the serum is clear and transparent in appearance, except for abnormal characters such as jaundice, hemolysis, lipemia, chyle and the like.
The TSH concentration in the TSH international standard substance stock solution in the step S2.1.3 is obtained by adopting a weighing method of Phosphate Buffer Solution (PBS) solution containing bovine serum albumin to dilute the TSH international standard substance (WHO NIBSC 81/565), and the concentration is 1181.356 mu IU/mL.
The calibrator prepared by the method is 11 serum solutions (L1-L11) with different TSH concentrations, and the fixed values of the 11 serum solutions with different TSH concentrations are shown in Table 5.
Step two (constant value of extreme high value sample L12)
Since the product calibrators from manufacturers generally have high values higher than L11, the serum collected after the test with TSH concentration greater than 150 μ IU/mL was mixed into L12, the system of values used is shown in Table 6, the values used are L5-L11, the dilution times of L12 by weight are 5.801, 9.734 and 20.080, respectively, and the dilution used is mixed human serum (L) with TSH concentration of 0 μ IU/mL is shown in Table 7. The final L12 rating results are shown in Table 8.
The density measurement of the L12 solution and the dilution point comprises the following specific steps:
(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) The formula is calculated according to the density of the liquid:
ρH＝(m2-m1)/v1
(5) repeating the steps (1) - (4) for 2 times.
The densities of L12 were measured to be 1.025 and 1.022 with an average of 1.024.
(6) The density ρ of each mixed solution was obtained in the same mannerMixing ofSee table 7.
The dilution factor is calculated by the formula:
wherein N represents the dilution factor, WHDenotes the mass of L12, WLDenotes the mass of L, ρ mix denotes the density of the mixed liquid, ρHThe density of L12 is shown, and the basic information of the constant value detection system of L12 is shown in Table 6:
TABLE 6 TSH very high sample L12 constant value detection System basic information
The fixed dilution factor for L12 is shown in table 7:
TABLE 7 TSH very high sample L12 fixed dilution multiple (weighing method)
The L12 was rated using the above method and 5 different TSH detection systems as shown in table 8:
TABLE 85 System valuate TSH extreme sample L12
Step three (calibration method for improving TSH detection result consistency)
1. Detecting system basic information
The improvement degree of the consistency of the detection results between systems before and after calibration is compared by adopting 6 detection systems (shown in table 9) which are mainstream in the in vitro diagnosis market and adopting a prepared special calibration product for TSH.
TABLE 9 TSH reconciled detection System basic information
2. Preparation of special calibrator for TSH of each detection system
As shown in Table 10, each calibrator was prepared by the weighing method using a constant value of L1-L12 TSH candidate standard substance in accordance with the concentration of the calibrator as a product of a manufacturer, and the volume of each calibrator was 1ml, and each calibrator was prepared using two concentration values of adjacent high (H) and low (L) values. As calculated by the following formula,
wherein C isTargetFor the concentration of the manufacturer calibrator, V is the volume of the special calibrator (1ml), WHIs the mass of the high-value sample, pHDensity of high value sample, CHIs the concentration of the high value sample, pMixing ofThe density of the mixture is taken as the average of the high and low value samples, pLDensity of low-value sample, CLIs the concentration of the low value sample. Finally calculating the required high-value sample quality (W)H) Calculating the mass (W) of the low-value sample according to a formulaL) Is WL＝V×ρMixing of-WH。
TABLE 10 TSH Special calibrator formulation (weighing method, g)
3. Serum pan collection
Serum (the TSH concentration range is 0.13-84.3 mu IU/mL, Siemens ADVIA CentaurrXP detection system) which is collected by 22 patients with different TSH high, middle and low concentrations and is detected and finished in 22 cases at the Beijing Korean Hospital affiliated to the university of capital medical science is divided into 6 parts according to 4 detection amounts of 6 manufacturers, frozen at-80C, and frozen for no more than 48 hours from the collection of the serum to the completion of the freezing. Samples of hemolysis, jaundice and lipemia were excluded from the collection.
4. The mixed human serum is the TSH candidate standard substance with 11 concentration levels.
The instrument is subjected to necessary inspection and maintenance before the experiment, and the indoor quality control product is detected after the instrument is calibrated by adopting a primary factory calibration product. After passing the quality control, the 33 samples were mixed well and each sample was tested 2 times. And then, after the calibration is carried out by adopting the prepared TSH special calibrator, the 33 samples are detected again, and each sample is detected for 2 times.
Finally, the Coefficient of Variation (CV) before and after calibration of the TSH-specific calibrator is compared with the optimal CV (CV is 0.25 × CVI, CVI is the biological variation in an individual) derived based on the biological variation, and the uniformity improvement effect is judged. Meanwhile, the ADVIA CentaurXP is used as a comparison system, the Passing-Bablok regression is carried out by adopting MedCalc statistical software, and the change of a regression line before and after calibration is compared.
Before and after calibration, the median of the TSH detection value (CV) of the 6 systems is changed from 17.94 percent to 9.56 percent, and the median of the TSH detection value (CV) of the mixed human serum is changed from 17.10 percent to 5.52 percent, so that the medium requirement (9.65 percent) of CV derived from biological variation is met. The ADVIA CentaurXP is used as a comparison detection system, other detection systems are used as evaluation systems, the slope range obtained before calibration is 0.738-1.128, and the correlation coefficient is 0.989-0.999 as shown in Table 11; the slope obtained after calibration ranges from 0.954 to 1.053, and the correlation coefficient ranges from 0.986 to 0.999.
The calibration product calibration front-to-back consistency improvement effect pair is shown in fig. 18.
TABLE 11 ADVIA CentaurXP before and after calibration for TSH Special calibrators comparability and correlation with other test systems
The application of the calibrator in any one of the following applications also belongs to the protection scope of the invention:
(i) calibrating the TSH detection reagent by using a special calibrator;
(ii) the special calibrator is adopted to improve the consistency and/or comparability of the TSH detection results of the multiple systems to individual patients.
The system includes an instrument for quantitative detection of TSH and reagents used, such as a clinical laboratory TSH detection system.
In conclusion, by means of the technical scheme of the invention, the TSH candidate standard substance (TSH special calibrator) prepared by the invention is derived from fresh frozen mixed human serum, so that the TSH candidate standard substance has good interchangeability. After the calibration product is adopted to calibrate a plurality of TSH detection systems, when the TSH concentration of individual human serum or human mixed serum is detected, the consistency of the detection results of laboratories is obviously improved. The accuracy of the test result can provide scientific basis for disease diagnosis and treatment monitoring of patients. The invention adopts the calibrator to detect individual serum discs of single patients which can represent clinical actual detection conditions, and the result shows that the degree of consistency among systems after calibration is obviously improved, the median of the TSH detection value variation coefficient among systems is changed from 17.94% to 9.56%, the median of the TSH detection value variation coefficient of mixed human serum is changed from 17.10% to 5.52%, and the CV medium requirement (9.65%) derived from biological variation is met. In a word, the TSH special calibrator and the uniformity application embodiment thereof developed by the invention are suitable for the calibration of clinical conventional TSH detection reagents, so that the consistency and comparability of TSH detection results of individual patients are improved, and the TSH special calibrator has a good application prospect and is worthy of popularization.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A preparation method of a TSH candidate standard substance for improving the consistency of TSH detection results is characterized by comprising the following steps:
s1: collecting the in vitro serum of different individuals, and mixing to obtain 11 serum solutions with different TSH concentrations;
s2: and (3) respectively determining the TSH concentration of each serum solution in the 11 serum solutions with different TSH concentrations to obtain 11 TSH candidate standard substances with different concentrations.
2. The method for preparing a TSH candidate standard substance according to claim 1, wherein the step S2 is performed by quantifying TSH concentrations in the 11 serum solutions with different TSH concentrations by means of TSH international standard substance.
3. The method for preparing a TSH candidate standard substance according to claim 2, wherein the method of quantifying the TSH concentration in the serum solutions of 11 different TSH concentrations specifically comprises the steps of:
s2.1: diluting the TSH international standard substance to obtain diluted solutions of the TSH international standard substance with different concentrations, and calculating the TSH authentication concentration in each diluted solution;
s2.2: respectively measuring the concentration of TSH in each diluted solution obtained in the step S2.1 by adopting N TSH quantitative detection systems;
s2.3: for each TSH quantitative detection system in the N TSH quantitative detection systems, performing linear fitting by adopting the authentication concentration of the TSH in each diluted solution obtained in the step S2.1 and the determination concentration of the TSH in each diluted solution obtained in the step S2.2 to obtain a TSH fixed value standard curve;
s2.4: respectively measuring the TSH concentration in the TSH candidate standard substance by adopting the N TSH quantitative detection systems;
s2.5: for each of the N TSH quantitative determination systems, substituting the measured concentration of TSH in the TSH candidate standard substance obtained in step S2.4 into the equation of the corresponding TSH fixed-value standard curve obtained in step S2.3, thereby obtaining the concentration of TSH in the TSH candidate standard substance;
s2.6: averaging the concentrations of TSH in all the candidate TSH standard substances of the N TSH quantitative detection systems obtained in the step S2.5, wherein the obtained average value is the final fixed value result of the TSH concentration in the candidate TSH standard substances;
and N is a natural number more than or equal to 2.
4. The method of preparing a TSH candidate standard substance according to claim 3, wherein the step S2.1 comprises the steps of:
s2.1.1 mixing the diluted solution and the TSH international standard substance according to different volume ratios, thereby obtaining a series of diluted solutions of the TSH international standard substance with different concentrations; in the process of preparing each part of the diluted solution of the TSH international standard substance, weighing the diluted solution of the TSH international standard substance and the stock solution of the TSH international standard substance respectively;
s2.1.2 measuring the density of the diluted solutions of the TSH international standard substance prepared in step S2.1.1 in series of different concentrations, respectively;
s2.1.3 the certified concentration of TSH in each of the series of dilutions of the international standard substance for TSH was calculated according to the following formula:
wherein C1 represents the TSH concentration in the TSH international standard substance stock solution, ρ 1 represents the density of the TSH international standard substance stock solution, M1 represents the mass of the TSH international standard substance stock solution, M2 represents the mass of the diluent, ρ 2 represents the density of the diluent, and C represents the certified concentration of the TSH international standard substance standard series.
5. The method for preparing TSH candidate standard substance according to claim 3, wherein the TSH international standard substance is diluted in step S2.1 by using human serum cocktail or Bovine Serum Albumin (BSA) -containing Hanks solution, wherein the TSH concentration in the human serum cocktail and the BSA-containing Hanks solution is 0 μ IU/mL.
6. A TSH candidate standard substance prepared by the method according to any one of claims 1 to 5, which comprises L1 with a fixed value of TSH concentration of 0.191 μ IU/mL, L2 with a fixed value of TSH concentration of 0.500 μ IU/mL, L3 with a fixed value of TSH concentration of 2.094 μ IU/mL, L4 with a fixed value of TSH concentration of 4.118 μ IU/mL, L5 with a fixed value of TSH concentration of 4.929 μ IU/mL, L6 with a fixed value of TSH concentration of 6.748 μ IU/mL, L7 with a fixed value of TSH concentration of 8.245 μ IU/mL, L8 with a fixed value of TSH concentration of 12.604 μ IU/mL, L9 with a fixed value of TSH concentration of 16.401 μ IU/mL, L10 with a fixed value of TSH concentration of 23.880 μ IU/mL, and L11 with a fixed value of TSH concentration of 44.985 μ IU/mL.
7. The TSH candidate standard substance according to claim 6, further comprising a very high value sample, wherein the very high value sample is L12, the L12 is prepared by mixing serum after test with TSH concentration of more than 150 μ IU/mL, an N-rating system is adopted, L5-L11 is used as rating, dilution times are calculated by weighing for L12 as 5.801, 9.734 and 20.080 times, respectively, the used dilution is mixed human serum with TSH concentration of 0 μ IU/mL, the TSH concentration of L12 is 171.123 μ IU/mL,
and N is a natural number more than or equal to 3.
8. A calibration method for improving TSH detection result consistency is characterized by comprising the following steps:
(1) preparing M TSH calibrator solutions of different concentrations: a sample of high value derived from the TSH candidate standard according to claims 6 and 7, a sample of low value derived from the TSH candidate standard according to claim 6, and M-2 samples of a series of proprietary calibrators prepared by mixing the high value sample and the low value sample at different volume ratios, wherein the TSH concentration requirement is equivalent to the manufacturer's working calibrators concentration;
(2) firstly, calibrating a detection system by adopting a calibrator of a manufacturer, detecting a series of individual serum of fresh frozen patients after passing quality control, calibrating the TSH quantitative detection system by using M TSH calibrator solutions with different concentrations prepared in the step (1), and then detecting the concentration of TSH in a serum sample to be detected; the manufacturers are N manufacturers, and the manufacturers are different,
and M and N are natural numbers more than or equal to 2.
9. The method of claim 8, wherein in step (1), during the process of preparing the special calibrator samples, the density of the low value sample and the density of the high value sample are measured according to the calibrator concentration of the manufacturer, so as to calculate the required mass, and the samples are weighed accurately, so as to ensure that the prepared special calibrator concentration is equal to the calibrator concentration of the manufacturer.
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| CN115831382A (en) * | 2022-12-26 | 2023-03-21 | 北京大学第三医院（北京大学第三临床医学院） | Conversion method and system of hormone detection data |
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