CN117538549A - Prediction system for evaluating clinical efficacy of plasma exchange and DSA removal - Google Patents
Prediction system for evaluating clinical efficacy of plasma exchange and DSA removal Download PDFInfo
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Abstract
The invention provides a prediction system for evaluating clinical effects of Plasma Exchange (PE) on clearing serum DSA, and provides an accurate detection system containing homemade DSA weak positive quality control substances and a treatment effect evaluation system for PE on clearing serum DSA for patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT). The invention optimizes the current DSA detection method, self-makes weak positive quality control product, and improves the accuracy and reliability of extremely low MFI value in DSA detection system. On the basis, the invention firstly proposes that the dilution serum DSA (dilution ratio, 1:16) before allo-HSCT treatment can predict the MFI value of the serum DSA after PE treatment; and by comparing the MFI differences between pre-treatment diluted and undiluted serum DSA, predicting the clinical efficacy of PE to clear serum DSA, the physician is assisted in selecting a personalized treatment regimen more appropriate for the patient.
Description
Technical Field
The invention belongs to the technical field of biological diagnosis, and particularly relates to a self-made HLA antibody weak positive quality control product, a detection system containing the self-made DSA weak positive quality control product and a curative effect evaluation system for removing serum DSA by plasma exchange.
Background
Currently, allogeneic hematopoietic stem cell transplantation (allogeneic hematopoietic stem cell transplantation, allo-HSCT) is a potential cure for a variety of hematological disorders. With the comprehensive development of techniques such as HLA haploid donor transplantation, HLA-mismatched unrelated donor transplantation, umbilical blood transplantation, etc., graft Failure (GF) is also becoming an independent risk factor for influencing hematopoietic reconstitution after transplantation and reducing overall survival rate. In recent years, several studies have reported that donor-specific HLA antibodies (donor specific antibody, DSA) as a primary barrier against successful implantation of donor cells are important factors leading to implantation failure and poor prognosis [1-3] . DSA positivity not only results in ineffective platelet infusion and failure of hematopoietic reconstitution, but also significantly increases the incidence and mortality of graft versus host disease (graft versus host disease, GVHD) [4] . Injury of donor cells by NK cell and T cell mediated cytotoxicity in patients is one of the important mechanisms leading to implantation failure, and antibody mediated apoptosis is also involved. In addition, antibodies or complements further cause damage to endothelial progenitor cells, mesenchymal cells in the bone marrow microenvironment, ultimately affecting hematopoietic stem cell survival and proliferation. Thus, removal of serum DSA in vivo prior to patient implantation can be achievedSo as to improve the implantation success rate of the transplantation and reduce the death rate related to the transplantation.
Currently, there is no international clinical guideline for serum-free DSA treatment regimens. Common strategies include (1) therapeutic Plasmapheresis (PE) removal of DSA; (2) monoclonal antibodies directed against the B cell surface CD20 antigen and proteasome inhibitors directed against plasma cells kill B cells and plasma cells, preventing or inhibiting antibody production; (3) inhibition of complement response [5,6] . In recent years, DSA desensitization treatment regimen consisting of plasmapheresis in combination with toximab proposed by the MD Anderson cancer center is being widely tried [7] However, this treatment is effective only on a part of patients, and the treatment effect is complex and variable. How to predict the efficacy of a therapeutic regimen has become a central issue in reducing the mortality associated with transplantation in patients.
Disclosure of Invention
The invention is based on the above study, and provides a method for predicting clinical effect of removing serum DSA by plasma replacement by studying average fluorescence intensity (MFI) difference between undiluted serum DSA and diluted serum DSA (dilution ratio, 1:16) before PE treatment of patients transplanted with allogeneic hematopoietic stem cells: by comparing the decrease ratio of the serum MFI values described above, the clinical response of patients receiving PE treatment is predicted: serum DSA before and after dilution is characterized by being divided into complete response group (CR group, MFI value decrease ratio > 70%), partial response group (PR group, MFI value decrease ratio between 30-70%) and no response group (NR group, MFI value decrease ratio < 30%).
Analysis of Kaplan-Meier survival showed significant differences in survival between the above groups in patients receiving allo-HSCT (P < 0.01). Results of retrospective study data based on CR group patients showed that patients did not differ in the time of implantation of the myeloid versus megakaryotic lines after bone marrow transplantation, whether standard or intensive treatment regimens were used. This suggests: it is economical, safe and effective to employ standard treatment regimens for patients predicted to have a therapeutic effect of CR; however, the results of retrospective study data based on NR groups of patients demonstrate that patients with more aggressive intensive therapies (including propidium, rituximab, bortezomib, and spleen zone irradiation, etc.) achieved longer survival times with significantly shorter implantation times of megakaryocytes during transplantation than patients treated with standard protocols.
Based on the experimental results, the invention further perfects the current DSA detection method so as to meet the accuracy and reliability of the MFI value with extremely low titer in the DSA pre-evaluation system. DSA detection requires HLA antibody specific detection and is known to be donor HLA typing, HLA antibody specific detection reagents comprise positive quality control microspheres (002-microsphere) coated with IgG antibodies capable of binding to secondary antibodies (goat anti-human IgG conjugates), but this quality control can only control DSA detection in undiluted serum samples, cannot control DSA detection in diluted serum samples, and the differences between different analysis batches cannot be effectively monitored.
Based on the method, the DSA detection system is optimized, and the control of weak positive properties of the HLA antibody is prepared and detected along with analysis batch: firstly, the quality control can be carried out on the combination condition of the antigen and the antibody before the secondary antibody incubation, and the control rule of the weak positive quality control product represents the control of the whole process of the batch of experiments; and secondly, after the analysis of the weak positive quality control product is added, checking whether deviation of +/-13 s exists or not by recording the highest MFI value of the positive quality control every day, so that the stability of the whole experiment can be judged.
In a first aspect of the invention, an HLA antibody weak-positive property control product is provided, and the preparation method comprises the following steps: classifying according to the malignant blood disease, screening weak positive serum sample with DSA highest MFI value in 1000-2000 in fresh sample, centrifuging at 10000rpm for 10min, discarding upper white polymer and lower precipitate, and collecting middle clear serum.
Wherein the preservation volume of the weak positive serum sample is required to be not less than 2ml. When split charging is carried out, split charging is carried out in an EP tube of 0.2ml according to the specification of 30 mu L/tube, the quality control number is written after the tube cover is tightly covered, then the EP tube is placed in a 96-hole freezing box, and the freezing box cover is frozen below-20 ℃ after the split charging date, the quality control number and the batch number of quality control products are written.
In the invention, the quality evaluation method of the self-made HLA antibody weak-positive quality control product comprises the following steps:
(1) The quality control standard of the HLA antibody weak positive quality control product is as follows: and (3) judging that the result is positive, recording the highest MFI value of the positive quality control product every day, and importing the highest MFI value into an L-J quality control chart. Common quality control rules include: 12s is a warning rule, which indicates that a horizontal quality control value exceeds X+/-2 s, and sends out a warning; 13s is a runaway rule, which indicates that a level quality control value exceeds X+/-3 s, indicating that the random error is increased to cause runaway. If 13s does not appear, the quality control result is controlled;
(2) Uniformity: when the number of the split charging tubes is less than or equal to 100 tubes, randomly extracting 5 samples for carrying out the same batch detection; when the number of the split charging tubes is more than 100, 10 samples are randomly extracted for carrying out the same batch detection; counting the cost difference percentage of the MFI with the highest detection result, and if the cost difference percentage is less than or equal to 20%, conforming to the requirement on the uniformity of the quality control product;
(3) Stability evaluation method: after the quality control product is continuously detected for 20 days, the mean value and the variance of the highest MFI value are counted through a retrospective analysis method, the mean value and the variance are used as an L-J quality control chart to monitor subsequent quality control data, and when the highest MFI value occurs for 13 seconds, the stability of the quality control product is suspected after other factors are eliminated. If the detection result of the prepared quality control product is still controlled after the quality control product is used for 6 months, the stability period of the quality control product is at least 6 months, and the validity period can be used when the quality control product is prepared again.
In a second aspect, the invention provides a clinical application of the HLA antibody weak positive quality control in a DSA detection system.
In a third aspect of the invention, an optimized DSA detection system is provided, comprising an HLA-specific antibody detection kit, an HLA antibody weak positive quality control, a negative quality control, and a secondary antibody label.
The control of weak yang of the HLA antibody is the control of weak yang of the HLA antibody in the first aspect;
the positive quality control product is positive quality control microsphere (002 # microsphere), and the coated IgG antibody can be combined with secondary antibody (goat anti-human IgG conjugate).
The detection principle of the DSA detection system is as follows: the method uses immunomagnetic bead flow liquid phase chip technology (Luminex method) to detect HLA specific IgG antibodies in serum. The test serum was first incubated with magnetic beads to bind each HLA antibody to the antigen, and then added to a goat anti-human IgG conjugate label labeled with R-phycoerythrin. The flow cytometer detects the fluorescence intensity of each PE-labeled magnetic bead and collects data in real time. The reaction intensity of the serum is detected, and the specificity of the PRA and HLA is represented by comparing the reaction intensity with the set reaction intensity of the serum.
In a fourth aspect of the present invention, there is provided a therapeutic efficacy pre-evaluation system for serum DSA removal by plasmapheresis, comprising:
the input display module is used for inputting basic information of a patient, and MFI values of undiluted serum and diluted serum DSA before treatment;
the calculation module is used for calculating the reduction ratio of the serum MFI values of the two;
the judging module is used for judging the patient as a complete response group, a partial response group and a no response group based on a preset rule and transmitting a judgment result to the input display module;
the storage module is used for storing various information in the operation process of the DSA treatment effect evaluation system;
the control module is used for controlling the input display module, the calculation module and the judgment module to normally operate;
the judging module judges based on the following rules: a complete response group was judged when the MFI value of pre-treatment diluted serum DSA decreased by >70% compared to undiluted serum; a partial response group is judged when the decrease ratio of the MFI values of the two is between 30% and 70%; a non-responsive group was determined when the decrease ratio of the MFI values of both was < 30%.
In a fifth aspect of the present invention, there is provided a non-transitory computer readable storage medium having stored thereon a computer program for execution by a processor of the steps of implementing a serum-free DSA efficacy assessment system. The computer program can be implanted into different control systems according to requirements to realize the processing and analysis of serum DSA data.
In a sixth aspect of the present invention, there is provided a method for detecting DSA antibody titer in a serum sample, wherein the efficacy evaluation system analyzes the detection result, comprising the steps of:
(1) The procoagulant supernatant serum or anticoagulated plasma 600. Mu.L supernatant was pipetted into a 1.5ml EP tube and centrifuged at 10000rpm for 10min.
(2) Transferring 400 mu L of the supernatant to a new EP tube, centrifuging at 10000rpm for 10min, taking 20 mu L of centrifuged serum, and adding the serum to the bottom of a 96-well plate; meanwhile, another part of serum obtained after centrifugation is diluted by Phosphate Buffer Solution (PBS) according to the ratio of 1:16, and the final volume is 20ul and added to the bottom of the 96-well plate. Adding a negative quality control product: 20ul of negative control serum (LS-NC) was added to the bottom of the 96-well plate. Adding a positive quality control product: taking out the self-made HLA antibody weak-positive property control product which is frozen and stored in a refrigerator at the temperature of minus 20 ℃ and adding 20ul of the self-made HLA antibody weak-positive property control product to the bottom of a 96-well plate after re-melting.
(3) And taking out the various HLA specific antibody detection kits, gently shaking for 20s, or repeatedly blowing for several times to fully and uniformly mix.
(4) According to the category of the detection antibody, 5 mu L of corresponding fluorescent microsphere reagent is respectively absorbed.
(5) Sealing the sealing film, oscillating and mixing for 10s, and centrifuging the fluorescent microsphere reagent to the bottom of the plate. Incubate in a shaking table at room temperature for 30min. At this time, a washing solution was prepared, and 10 Xwashing buffer (LSPWABF) was diluted with distilled water to 1 Xworking solution.
(6) After incubation, 150 μl of 1×wash buffer was added to each well, the sealing film was sealed and not reusable, mixed well, and centrifuged at 3700rpm for 5min.
(7) The wash liquid in the 96-well plate was thrown off and the plate was back-fastened to absorbent paper. 200. Mu.L of 1 Xwashing buffer was added to each well, the sealing film was sealed, and the mixture was homogenized and centrifuged at 3700rpm for 5min.
(8) The wash solution (intensity and direction of attention) was discarded and the plate was back-fastened to absorbent paper.
(8) Repeating the steps 7 and 8 once.
(10) Secondary antibody labeling: the PE-goat anti-human IgG conjugate (LS-AB 2) was diluted into a 1 Xantibody working solution, i.e., a "secondary antibody working solution" was prepared in a ratio of 1. Mu.L LS-AB2 to 99. Mu.L of 1 Xwash buffer. And adding 100 mu L of secondary antibody working solution into each hole, sealing the sealing film, and vibrating and uniformly mixing for 10s. Incubate in a shaking table at room temperature for 30min.
(11) After the incubation was completed, centrifugation was carried out at 3,700rpm for 5min. The supernatant in the 96-well plate is thrown off, and the plate is reversely buckled on the absorbent paper.
(12) The steps 7 and 8 are repeated twice.
(13) 80. Mu.L of 1 XPBS (pH 7.4) was added to each well, and after air-blow mixing, the mixture was transferred to an ELISA plate, read on a machine-readable plate, and analyzed by software.
The invention has the beneficial effects that:
(1) The invention firstly proposes to predict the clinical effect of PE on clearing serum DSA by comparing the average fluorescence intensity difference of undiluted serum DSA and diluted serum DSA before treatment. Analysis of Kaplan-Meier survival showed significant differences in survival time between different predicted treatment efficacy groups for allo-HSCT patients. This is of great importance for the selection of a subsequent treatment regimen for a patient.
(2) Based on the experimental results, the invention continuously optimizes the serum DSA detection method, self-prepares HLA antibody weak positive quality control products, can control the quality of DSA detection in undiluted serum samples, can control the quality of DSA detection in diluted serum samples, and can effectively monitor the difference between different analysis batches so as to adapt to the accuracy and reliability of the ultra-low titer MFI value in a DSA removal curative effect evaluation system.
Drawings
Fig. 1 is a diagram of HLA antibody specific assay quality control.
FIG. 2 is an analysis of the correlation of the pre-treatment diluted serum MFI values with the post-treatment serum MFI values according to the present invention. Wherein the abscissa represents the pre-treatment diluted serum MFI values and the ordinate represents post-treatment serum MFI values.
FIG. 3 is a survival curve analysis of PE clear serum DSA antibody titers, wherein blue groups, complete response CR groups; yellow group, partial response PR group; red group, no response NR group.
FIG. 4 is a graph showing the prediction of the time of implantation of the granule and macrosystem after bone marrow transplantation in the CR group, wherein the treatment regimen is performed in the yellow group; blue group, standard treatment protocol was used.
FIG. 5 is a graph of predicted bone marrow engraftment post-granular and macro implantation times for the NR group, wherein the yellow group, using an intensive treatment regimen; blue group, standard treatment protocol was used.
FIG. 6 is an analysis of survival curves for patients in the predictive NR group, wherein the yellow group employed an intensive treatment regimen; blue group, standard treatment protocol was used.
Fig. 7 shows a block diagram of the DSA antibody pre-evaluation detection system.
Fig. 8 shows a decision flow chart of the DSA antibody pre-evaluation detection system.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
1. Experimental sample
Of 437 patients who were serially transplanted between 1 in 2021 and 31 in 2023, 26 received a regrind DSA clearance regimen including PE treatment, while closely monitoring DSA antibody titers before and after treatment.
2. DSA antibody detection
1. Principle of detection
The HLA-specific IgG antibodies in serum were detected using immunomagnetic bead flow liquid phase chip technology (Luminex method). The test serum was first incubated with magnetic beads to bind each HLA antibody to the antigen, and then labeled with an added goat anti-human IgG conjugate labeled with R-phycoerythrin. The flow cytometer detects the fluorescence intensity of each PE-labeled magnetic bead and collects data in real time. The reaction intensity of the detected serum is compared with the reaction intensity of the set serum to reflect the PRA pattern and HLA specificity.
2. Instrument and reagent
The instrumentation used in the detection process is shown in table 1 below:
table 1 summary of the instrumentation used in the detection process
| Name of the name | Manufacturer' s | Model number |
| Plate type low-temperature centrifugal machine | Thermo | Thermomicro17 |
| Liquid phase chip system | Luminex | Luminex200 |
| High-speed centrifuge | Thermo | ThermoPico17Centrifuge |
| Cradle | Thermo | 4625-1CEM |
TABLE 2 summary of reagents used in the detection procedure
3. Preparation of HLA antibody weak positive quality control product
3.1 preparation method
(1) Selecting a weak positive sample with the highest MFI value within the range of 1000-2000 after detection, wherein the requirement of the serum quantity separated from the sample is more than or equal to 2ml; (2) Centrifuging a sample meeting the requirements at 10000rpm for 10 minutes, discarding the upper white polymer and the lower sediment, and reserving clear serum in the middle layer; (3) Subpackaging into 0.2ml EP tubes according to 30 mu L/tube specification, covering a tube cover, and writing a quality control number on the tube cover; (4) Placing the EP tube in a 96-hole freezing box, writing quality control product split charging date, quality control number and batch number on the freezing box cover, and freezing below-20 ℃; and (5) filling a quality control product preparation and split charging record list.
3.2 quality assessment
(1) The uniformity evaluation method comprises the following steps: after the quality control product is packaged, randomly extracting a plurality of samples from the whole body for uniformity inspection, and randomly extracting 5 samples for same batch detection when the number of packaged tubes is less than or equal to 100 tubes; when the number of split charging tubes is more than 100, 10 samples are randomly extracted for the same batch detection. The abnormal value appearing in the detection should not be removed at will before the cause is not ascertained; and counting CV% value of the highest MFI of the detection result, and if CV% value is less than or equal to 20%, the uniformity of the quality control product meets the requirement.
(2) Stability evaluation method: after the quality control product is continuously detected for 20 days, the mean value and the variance of the highest MFI value are counted through a retrospective analysis method, the mean value and the variance are used as an L-J quality control chart to monitor subsequent quality control data, and when the highest MFI value occurs for 13 seconds, the stability of the quality control product is suspected after other factors are eliminated. If the detection result of the prepared quality control product is still controlled after the quality control product is used for 6 months, the stability period of the quality control product is at least 6 months, and the validity period can be used when the quality control product is prepared again.
(3) Homemade HLA antibody weak positive quality control standard: the positive quality control product is judged, the highest MFI value of the positive quality control product is recorded every day and is imported into an L-J quality control chart, and the condition that 13s does not appear represents that the quality control result is under control. FIG. 1 illustrates a runaway condition: the batch of samples will be considered for re-experiments when the highest MFI value exceeds ± 13 s.
4. Detection step
(1) The procoagulant supernatant serum or anticoagulated plasma 600. Mu.L supernatant was pipetted into a 1.5ml EP tube and centrifuged at 10000rpm for 10min.
(2) Transferring 400 mu L of the supernatant to a new EP tube, centrifuging at 10000rpm for 10min, taking 20 mu L of centrifuged serum, and adding the serum to the bottom of a 96-well plate; meanwhile, another part of serum obtained after centrifugation is diluted by Phosphate Buffer Solution (PBS) according to the ratio of 1:16, and the final volume is 20ul and added to the bottom of the 96-well plate. Adding a negative quality control product: 20ul of negative control serum (LS-NC) was added to the bottom of the 96-well plate. Adding a positive quality control product: taking out the self-made HLA antibody weak-positive property control product which is frozen and stored in a refrigerator at the temperature of minus 20 ℃ and adding 20ul of the self-made HLA antibody weak-positive property control product to the bottom of a 96-well plate after re-melting.
(3) And taking out the various HLA specific antibody detection kits, gently shaking for 20s, or repeatedly blowing for several times to fully and uniformly mix.
(4) According to the category of the detection antibody, 5 mu L of corresponding fluorescent microsphere reagent is respectively absorbed.
(5) Sealing the sealing film, oscillating and mixing for 10s, and centrifuging the fluorescent microsphere reagent to the bottom of the plate. Incubate in a shaking table at room temperature for 30min. At this time, a washing solution was prepared, and 10 Xwashing buffer (LSPWABF) was diluted with distilled water to 1 Xworking solution.
(6) After incubation, 150 μl of 1×wash buffer was added to each well, the sealing film was sealed and not reusable, mixed well, and centrifuged at 3700rpm for 5min.
(7) The wash liquid in the 96-well plate was thrown off and the plate was back-fastened to absorbent paper. 200. Mu.L of 1 Xwashing buffer was added to each well, the sealing film was sealed, and the mixture was homogenized and centrifuged at 3700rpm for 5min.
(8) The wash solution (intensity and direction of attention) was discarded and the plate was back-fastened to absorbent paper.
(8) Repeating the steps 7 and 8 once.
(10) Secondary antibody labeling: the PE-goat anti-human IgG conjugate (LS-AB 2) was diluted into a 1 Xantibody working solution, i.e., a "secondary antibody working solution" was prepared in a ratio of 1. Mu.L LS-AB2 to 99. Mu.L of 1 Xwash buffer. And adding 100 mu L of secondary antibody working solution into each hole, sealing the sealing film, and vibrating and uniformly mixing for 10s. Incubate in a shaking table at room temperature for 30min.
(11) After the incubation was completed, centrifugation was carried out at 3,700rpm for 5min. The supernatant in the 96-well plate is thrown off, and the plate is reversely buckled on the absorbent paper.
(12) The steps 7 and 8 are repeated twice.
(13) 80. Mu.L of 1 XPBS (pH 7.4) was added to each well, and after air-blow mixing, the mixture was transferred to an ELISA plate, read on a machine-readable plate, and analyzed by software.
5. Performance analysis of predictive methods
Specificity and sensitivity: the specificity and sensitivity of the LABScreen assay are comparable to that of the Flow method, which allows clear detection of anti-HLA antibodies in undiluted serum, even in serum samples that may be negative or near-threshold in CDC assays.
Repeatability: the correlation coefficient of the repeatability between the same batches is 98%, and the repeatability is good.
6. Determination of the ratio of diluted serum before treatment
To test the ideal degree of serum dilution, we selected five dilution ratios of 1:4,1:8,1:16,1:32,1:64, respectively, serum samples were taken before DSA positive patients received PE treatment, and dilution was performed in five ratios; serum samples were again taken after the patient had completed 2 PE treatments. The 7 serum samples were simultaneously run to check the MFI value of DSA. As a result, it was found that the MFI values of the pre-treatment 1:16 diluted serum and the post-treatment serum were most similar. Thus, we selected DSA of pre-treatment 1:16 diluted serum to predict MFI values of post-PE treatment serum DSA.
3. Analysis of results
1. Correlation analysis of serum DSA predictive value and actual value
The predicted value of serum DSA of the patient before treatment and the actual value of serum DSA after treatment were examined and correlation analysis was performed for both, and the results are shown in table 3 and fig. 2 below.
TABLE 3 serum DSA predictive value and actual value summary for patients
Note that: class I antigens, human leukocyte antigen-I; class II antigens, human leukocyte antigen-II
The patients were classified into complete response groups (CR group, DSA decrease rate > 70%), partial response groups (PR group, DSA decrease rate between 30-70%) and no response groups (NR group, DSA decrease rate < 30%) according to their characteristics of actual and predicted DSA antibody titers.
2. Different sets of survival curve analysis
Survival analysis was performed using Kaplan-Meier, see table 4 below and fig. 3:
TABLE 4 survival results of different efficacy groupings
The results suggest that there is a significant difference in patient survival between the above groups, with the survival rate of the complete non-responsive group (NR group) being significantly lower than that of the partial responsive group (PR group) and the complete responsive group (CR group).
3. Guiding selection of a treatment regimen
Standard treatment protocols for clearing serum DSA include: two courses of PE treatment (double frozen plasma replacement) followed by a single dose of rituximab (375 mg/m) on the second day after the last PE treatment 2 ) And 1000mg/kg IVIG; the intensive treatment regimen for clearing serum DSA included: four courses of PE procedure (frozen plasma with double the amount of replacement) followed by a single dose of rituximab (375 mg/m) on the second day after the last PE treatment 2 ) And 1000mg/kg IVIG; four doses of bortezomib (1-1.3 mg/m) were administered every 72 hours starting from the first PE 2 ). Patients with the three different clinical responses were divided into two subgroups, one subgroup using the intensive treatment regimen and the other subgroup using the standard treatment regimen. The time of implantation of the granulosa and megakaryotic lines during the implantation of the patient was recorded and finally survival analysis was performed between the subgroups of the different groups, the results being given in table 5 below:
table 5 relevant index for three groups of patients receiving serum-free DSA treatment regimen
Results of the time for implantation of the granulocytes and megakaryocytes after bone marrow transplantation in the CR group are shown in FIG. 4; results of the bone marrow transplantation post-granular and megakaryotic implantation times of the NR group are shown in FIG. 5, and results of survival curve analysis are shown in FIG. 6.
Results of retrospective study data based on CR-group patients showed that patients had no significant differences in the time of implantation of the myeloid versus megakaryotic lines after bone marrow transplantation, whether standard or intensive treatment regimens were used. This suggests: it is economical, safe, and effective to employ standard treatment regimens for patients with predicted efficacy of CR (fig. 4); however, retrospective study data results based on NR groups of patients showed that the implantation time of megakaryotic after transplantation was significantly shortened compared to patients treated with standard protocols and a longer survival time was obtained (fig. 6).
DSA curative effect evaluation system for implementing secondary serum elimination
The embodiment provides the application of the study, and the study result is formed into a serum-removed DSA efficacy evaluation system, so that data processing and analysis are automatically realized. The diagnosis system can be installed on a computer terminal or integrated with a DSA detection device, and after the serum sample is detected, the MFI data of the antibody is directly analyzed, and the curative effect evaluation result is displayed.
According to fig. 7, the serum-free DSA efficacy evaluation system of the present invention includes an input display module 1, a calculation module 2, a determination module 3, a storage module 4, a communication module 5, and a control module 6.
The input display module 1 is used for inputting basic information of a patient, an undiluted serum before treatment and an MFI value of a diluted serum DSA before treatment, or receiving target data transmitted by a detection device through the communication module 5 and displaying a diagnosis result;
the calculating module 2 is used for calculating the MFI value reduction ratio of the diluted serum DSA before treatment compared with the undiluted serum DSA;
the determination module 3 determines the patient as a complete response group (CR group), a partial response group (PR group), and a no response group (NR group) based on a preset rule, and transmits the determination result to the input display module 1. The preset rule is as follows: a complete response group was judged when the MFI value of the pre-treatment diluted serum DSA decreased by > 70%; a partial response group is judged when the MFI value of pre-treatment diluted serum DSA decreases by between 30% -70%; a non-responsive group was judged when the MFI value of pre-treatment diluted serum DSA decreased by < 30%;
the storage module 4 stores various information in the operation process of the serum-removed DSA curative effect evaluation system; the communication module 5 is used for information transmission between different modules and information transmission between the curative effect evaluation system and external equipment, such as communication connection between the detection device and a doctor terminal system; the control module 6 controls the normal operation of the above modules.
The diagnosis flow of the serum-eliminating DSA efficacy evaluation system in the invention is shown in fig. 8, and the steps are as follows:
s1, inputting basic information of a patient through an input display module 1, inputting or receiving MFI values of undiluted serum and diluted serum DSA before treatment, and entering S2;
s2, calculating the MFI value reduction ratio of the diluted serum DSA before treatment by the calculation module 2, and entering S3;
s3, the judging module 3 groups the patients based on preset rules and transmits the results to the input display module 1.
References cited in the background of the invention are as follows:
1.Huang Y,Luo C,Wu G,et al.Effects of donor-specific antibodies on engraftment and long-term survival after allogeneic hematopoietic stem cell transplantation-A systematic review and meta-analysis.Bone Marrow Transplant.2023；10.1038/s41409-023-01932-6.
2.Bettinotti MP.Evolution of HLA testing for hematopoietic stem cell transplantation:Importance of the candidate's antibody profile for donor selection.Hum Immunol.2022；83(10):721-729.
3.Cao LQ,Lv M,Xu LP,et al.Prevalence and risk factors of having antibodies to class Iand II human leukocyte antigens in older haploidentical allograft candidates.Sci Rep.2020；10(1):2367.
4.Wang L,Ji K,Chen L,et al.Posttransplant de novo DSA and NDSA affect GvHD,OS,and DFS after haplo-HSCT in patients without pre-existing HLA antibodies of hematological malignancies.Front Immunol.2022；13:1047200.
5.File B,Huang Y,Peedin A,Gergis U.The impact of HLA donor-specific antibodies on engraftment and the evolving desensitization strategies.Bone Marrow Transplant.2022；57(4):526-531.
6.Yoshihara S,Maruya E,Taniguchi K,et al.Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT.Bone Marrow Transplant.
7.Kongtim P,Cao K,Ciurea SO.Donor Specific Anti-HLA Antibody and Risk of Graft Failure in Haploidentical Stem Cell Transplantation.Adv Hematol.2016；2016:4025073.
while the invention has been described in detail with reference to the embodiments, it will be apparent to those skilled in the art that the invention is not limited to the embodiments, and that the equivalent modifications and substitutions can be made without departing from the spirit of the invention, and are intended to be included within the scope of the appended claims.
Claims (10)
1. A control product for weak positive properties of HLA antibody is characterized in that weak positive serum samples with the highest MFI value of DSA in 1000-2000 in fresh samples are screened, centrifuged for 10 minutes by a low-temperature centrifuge at 10000rpm, upper white polymer and lower sediment are discarded, serum with a clear middle layer is reserved, and the serum is stored below-20 ℃ after split charging.
2. The HLA antibody weak cationic quality control according to claim 1, wherein:
monitoring blood lipids, bilirubin and immunoglobulins in serum samples ensures that they are in the normal range and do not interfere with the weak positive results of MFI, and the storage volume of the weak positive serum samples is required to be no less than 2ml.
3. The HLA antibody weak cationic quality control according to claim 1, wherein:
and (3) centrifuging the weak positive serum sample at a high speed at 1000rpm by a low-temperature centrifuge within 1 hour after sampling the sample, rapidly split charging the sample, split charging the sample into an EP tube of 0.2ml according to the specification of 30 mu L/tube, and writing a clear quality control number after closing a tube cover.
4. The HLA antibody weak cationic quality control according to claim 1, wherein:
the homogeneity evaluation method of the HLA antibody weak-positive quality control product comprises the following steps: when the number of the split charging tubes is less than or equal to 100 tubes, randomly extracting 5 samples for carrying out the same batch detection; when the number of the split charging tubes is more than 100, 10 samples are randomly extracted for carrying out the same batch detection; counting the cost difference percentage of the MFI with the highest detection result, and if the cost difference percentage is less than or equal to 20%, conforming to the requirement on the uniformity of the quality control product;
the quality control standard of the HLA antibody weak positive quality control product is as follows: and (3) judging that the result is positive, recording the highest MFI value of the positive quality control product every day, importing the value into an L-J quality control chart, and controlling the quality control result if 13s does not appear.
5. Use of the HLA antibody weak positive quality control according to any one of claims 1 to 4 in the preparation of a DSA antibody pre-evaluation detection system.
6. A DSA antibody detection system, comprising an HLA-specific antibody detection kit, an HLA-antibody weak-positive quality control, a negative quality control, and a secondary antibody marker, wherein the HLA-antibody weak-positive quality control is the HLA-antibody weak-positive quality control of any one of claims 1 to 4.
7. A efficacy pre-evaluation system for serum DSA removal by plasmapheresis, comprising:
the input display module is used for inputting basic information of a patient, undiluted serum before treatment and MFI value of diluted serum DSA;
the calculating module is used for calculating the MFI value reduction ratio of the diluted serum DSA before treatment compared with the undiluted serum DSA;
and the judging module judges the patient as a complete response group, a partial response group and a no response group based on preset rules and transmits the judging result to the input display module.
8. The efficacy pre-evaluation system for plasmapheresis clearing serum DSA of claim 7, further comprising:
the storage module is used for storing various information in the running process of the DSA antibody pre-evaluation system;
the control module is used for controlling the input display module, the calculation module and the judgment module to normally operate;
the judging module judges based on the following rules: a complete response group (CR group) was judged when the MFI value of pre-treatment diluted serum DSA decreased by >70% compared to undiluted serum DSA; a partial response group (PR group) is determined when the MFI value decrease ratio of the two is between 30% and 70%; a non-responsive group (NR group) was determined when the decrease ratio of MFI values of both was < 30%.
9. The efficacy pre-evaluation system for plasmapheresis clearing serum DSA according to claim 7, wherein:
MFI values of undiluted serum and diluted serum DSA before treatment were measured by the DSA antibody detection system of claim 6.
10. A non-transitory computer readable storage medium, on which a computer program is stored, characterized in that the computer program, when being executed by a processor, implements the steps of claim 8 or 9.
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