CN103743907A - Kit, system and method for evaluating marrow microcirculation environment of patient after hematopoiesis stem cell transplantation - Google Patents

Kit, system and method for evaluating marrow microcirculation environment of patient after hematopoiesis stem cell transplantation Download PDF

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CN103743907A
CN103743907A CN201310314467.9A CN201310314467A CN103743907A CN 103743907 A CN103743907 A CN 103743907A CN 201310314467 A CN201310314467 A CN 201310314467A CN 103743907 A CN103743907 A CN 103743907A
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bone marrow
patient
microcirculation
stem cell
vegfr2
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CN103743907B (en
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黄晓军
孔圆
常英军
王亚哲
袁晓英
胡玥
王昱
刘代红
许兰平
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Shanghai Tissuebank Medical Laboratory Co ltd
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Peking University Peoples Hospital
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Abstract

The invention discloses a kit, a system and a method for evaluating marrow microcirculation environment of patient after hematopoiesis stem cell transplantation, and the kit, the system and the method are applied based on a flow cytometer. The kit comprises following fluorescent labeled monoclonal antibody reagents: CD34-FITC, VEGFR2-PE, CD45-PerCP and CD133-APC. The system is provided with a master control computer unit and a memory cell for storing data and graph for perspective research, and the configuration of the hardware is an important guarantee for realization of determination. The invention firstly employs an expression proportion of CD45-CD34+VEGFR2+ in bone marrow mononuclear cells as one of the indexes for evaluating marrow microcirculation environment quality of patient with blood disease after hematopoiesis stem cell transplantation, and has a guidance meaning for predicting generation of adverse symptoms of patient blood disease after hematopoiesis stem cell transplantation, and has an important reference value for formulating clinic treatment schemes.

Description

Kit, the system and method for the evaluation and test of patient's Microcirculation in Bone Marrow environment after mensuration hematopoietic stem cell transplantation
Technical field
The present invention relates to cell phenotype dedicated kit and the system in a kind of application circumstances, used, be specifically related to a kind of for measuring kit, the mensuration system of patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation and realizing the method for qualitative and quantitative analysis.
Background technology
Allogeneic Hematopoietic Stem Cell Transplantation is one of effective means for the treatment of Hematological Malignancies, but after transplanting, implant bad (English name Poor graft function, abbreviation PGF) incidence between 5 – 27%, remain one of major complications after Allogeneic Hematopoietic Stem Cell Transplantation, when serious, affect transplant patient's long term survival.Although bibliographical information, implant bad may with transplant before the CD34 of pre-service dosage, immunological rejection, graft versus host disease(GVH disease), virus infections, transplanting +the factors such as cell number are relevant, but implant bad pathogenesis after transplanting, not yet illustrate, and whether Microcirculation in Bone Marrow environment has with late complication the result of study that important inner link is not yet clear and definite under specific implantation condition.
Adopting long-term bone marrow culture experiment and colony to form experiment is the common method that in alternative body, inpatient with haematological diseases marrow stromal cell Hematopoiesis Support affect is assessed in research.The past research is by means of above-mentioned research method confirmation, and the inpatient with haematological diseases marrow stromal cell of accepting long-term chemotherapy is impaired, and the patient who accepts autologous hematopoietic stem cell transplantation can detect with transplanting rear 1 that every year marrow stromal cell is impaired before transplanting.We infer that Microcirculation in Bone Marrow environmental damage also may participate in implanting after hematopoietic stem cell transplantation bad generation thus.But long-term bone marrow culture experiment and colony form experiment has certain limitation: its cell population of interest comprises the marrow stromal cell component of one group of heterogeneity such as fibroblast, adipocyte and endothelial cell.Particularly early stage after hematopoietic stem cell transplantation, the marrow stromal cell quantity of rebuilding in patient body is little, has seriously limited applying of above-mentioned research method.Therefore, new in the urgent need to setting up, conveniently detection method to hematopoietic stem cell transplantation after the Microcirculation in Bone Marrow set of circumstances of inpatient with haematological diseases divide and carry out efficiency assessment.Whether more can this relate to and implant bad prediction in time and formulation science, the efficient and individuation of clinical treatment after hematopoietic stem cell transplantation.
For inpatient with haematological diseases, it was exactly very difficult thing originally that suitable Marrow donation person is found in marrow pairing, if because valuable social resources have been wasted in treatment and recovery that certain factor has been incured loss through delay the later stage, be the grief of medical circle.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of for measuring kit, the system and method for inpatient with haematological diseases Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation, utilize in this kit through Accurate Calibration and quantitative special agent, supporting device systems and technology control method, can measure quickly and accurately CD45 -cD34 +vEGFR2 +proportion in BMNC, thereby draw the quantitative and qualitative analysis conclusion of inpatient with haematological diseases Microcirculation in Bone Marrow environmental quality after hematopoietic stem cell transplantation, thereby after can predicting quickly and accurately hematopoietic stem cell transplantation, inpatient with haematological diseases is implanted bad generation, and the formulation of clinical treatment is had to important directive significance.
For solving the problems of the technologies described above, the technical solution used in the present invention 1 is:
A kind of kit of measuring patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation is provided, the application of this kit, based on flow cytometer, has configured following fluorescently-labeled monoclonal antibody reagent: CD34-FITC, VEGFR2-PE, CD45-PerCP and CD133-APC in this kit.
Adopt mentioned reagent box and flow cytometer to be used in conjunction with, can be accurately, quantitative measurement CD45 -cD34 +vEGFR2 +expression ratio in BMNC, for the evaluation and test of Microcirculation in Bone Marrow environment is laid a good foundation.
The present invention also provides a kind of dedicated system based on mentioned reagent box, the structure of this system comprises flow cytometer and supporting data analysis module, in described system, also comprise main control computer unit, and be connected to the system management module that stores primary control program on main control computer unit matching interface, the display LCD of allocating cache device module, store the data of perspective study and the memory module of figure, working storage module, be arranged on the control panel on system operation bench, store the communication module of inside and outside data communication agreement and data requirement translative mode, printer and interface circuit, flow cytometer is connected with the data bus of main control computer unit by communication interface circuit, and the supporting dedicated kit being provided with for measuring patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation in flow cytometer structure, in described dedicated kit, configured following fluorescently-labeled monoclonal antibody reagent: CD34-FITC, VEGFR2-PE, CD45-PerCP and CD133-APC.
Gordian technique of the present invention is supporting by the perfect outfit of host computer system and the clear and definite control method of specific aim task and supporting soft and hardware, and crucial sample devices-flow cytometer is transformed into the special system equipment of inpatient with haematological diseases Microcirculation in Bone Marrow environment report of accessment and test after hematopoietic stem cell transplantation.The purposes of flow cytometer is to measure with fluorescently-labeled sample of bone marrow, records two-dimentional scatter diagram.But, the qualitative and further quantitative test of determined cell-specific intension, flow cytometer and supporting analysis software are helpless.The realization of particular task, depends on that whether the apolegamy of dye marker reasonable, the calibration separatrix value of perspective study choose whether there is ubiquity and whether method of operating can be verified by clinical result.
Invention key of the present invention is also to have designed the said system based on the present invention, the method for inpatient with haematological diseases Microcirculation in Bone Marrow environment after mensuration hematopoietic stem cell transplantation, and the method comprises the following steps:
A, adopt the reagent in dedicated kit in flow cytometer Special test tube, to carry out fluorescence labeling sample of bone marrow to be measured, make test sample book;
B, the Special test tube that test sample book is housed is inserted in flow cytometer, start flow cytometer and supporting analysis software, according to specific data acquisition conditions in memory module, gather related data, form scatter diagram, and carry out streaming and establish an analysis, transmission key words sorting deposit working storage module in;
C, the data that recall perspective study in memory module and figure, be presented at the upper half of the display LCD of high definition, at bottom half, recalls the corresponding collection of illustrative plates in working storage module, by cursor chi, contrasts quantitative test;
D, determine CD45 -cD34 +vEGFR2 +in BMNC, express the position of the separatrix value of ratio;
E, by measure CD45 -cD34 +vEGFR2 +it is good that expression ratio>=separatrix value is defined as Microcirculation in Bone Marrow environment; CD45 -cD34 +vEGFR2 +it is bad that expression ratio < separatrix value is defined as Microcirculation in Bone Marrow environment;
F, from empirical data module, recall patient's Microcirculation in Bone Marrow environment measuring account after hematopoietic stem cell transplantation, insert determination data, correlation graph, after comparison, " well ", " uncertain ", " bad " are selected to insert in evaluation and test conclusion hurdle, from laser printer, export.
By above method, can instruct the management software in the primary control program in establishment native system, form the equipment of full rotation type, with inpatient with haematological diseases Microcirculation in Bone Marrow environment after fast detecting, assessment hematopoietic stem cell transplantation.The measurement result of convection type cell instrument of the present invention is by hematoxylin-eosin (English name Hematoxylin-eosin staining, abbreviation HE) dyeing and CD34 SABC (IHC) dye the blood vessel in Microcirculation in Bone Marrow environment in Bone Marrow of Patients biopsy specimen is carried out in situ quantitation checking, proved the science of assay method of the present invention, research of the present invention laid a good foundation.
Adopt kit of the present invention, system and method to measure after hematopoietic stem cell transplantation CD45 in inpatient with haematological diseases anticoagulant heparin BMNC -cD34 +vEGFR2 +expression ratio, and further logical HE dyeing and CD34 SABC (IHC) dye the blood vessel in Microcirculation in Bone Marrow environment in Bone Marrow of Patients biopsy specimen are carried out in situ quantitation checking.By clinical testing and statistical analysis, result shows: after the Allogeneic Hematopoietic Stem Cell Transplantation detecting by four look flow cytometers, implant bad patient (n=19) CD45 -cD34 +vEGFR2 +expression ratio be starkly lower than inpatient with haematological diseases (n=38) and the health donors (n=15) (0.008% vs. 0.16% vs. 0.18%, P<0.0001) of implanting good (English name Good graft function, abbreviation GGF); According to operator's working curve (ROC curve), determine CD45 -cD34 +vEGFR2 +in BMNC, expression ratio=0.055% is cut off value, by CD45 -cD34 +vEGFR2 +expression ratio <0.055% is defined as Microcirculation in Bone Marrow environment bad (or abnormal), by CD45 -cD34 +vEGFR2 +expression ratio>=0.055% is defined as Microcirculation in Bone Marrow environment good (or normal); By HE and CD34 immunohistochemical staining, further in marrow original position, confirm to transplant rear PGF patient (n=19) marrow blood vessel quantity and be starkly lower than GGF patient (n=38) and health donors (n=15) (2 vs. 4 vs. 6/bone trabecula, P<0.05); Prognostic factors prompting CD45 -cD34 +vEGFR2 +expression ratio <0.055% is that after hematopoietic stem cell transplantation, bad independent hazard factor occurs to implant inpatient with haematological diseases.Although CD45 that the present invention measures -cD34 +vEGFR2 +expression ratio in BMNC can not directly draw diagnostic result or health status, but its as intermediate result, can be used as prediction hematopoietic stem cell transplantation after inpatient with haematological diseases implant bad generation and guidance system field planting enters one of reference information of bad patient's clinical treatment.
The beneficial effect that adopts technique scheme to produce is: (1) the present invention is first by CD45 -cD34 +vEGFR2 +expression ratio in BMNC is as evaluating one of index of inpatient with haematological diseases Microcirculation in Bone Marrow environmental quality after hematopoietic stem cell transplantation, and verify by lot of experiments, for inpatient with haematological diseases after prediction hematopoietic stem cell transplantation, implant the bad directive significance that has, and the clinical treatment that field planting enters bad patient for guidance system has important reference value; (2) adopt kit of the present invention, system and supporting method, can Fast Measurement BMNC in CD45 -cD34 +vEGFR2 +expression ratio, to the evaluation of Microcirculation in Bone Marrow environment provide a kind of fast, the approach of accurate evaluation, be that the strong of prior art supplements.
Accompanying drawing explanation
In Fig. 1, A1, A2, A3 are not health donors, implant good and implant bone marrow cell hyperplasia degree typical case's HE colored graph (× 10) of bad patient;
Fig. 2 carries out CD45 to 1 routine health donors bone marrow cell -cD34 +vEGFR2 +the typical streaming of measuring is established an analysis chart;
Fig. 3 A implants bad patient and implants the rear CD34 of transplanting between good patient +the difference of number;
Fig. 3 B and Fig. 3 C are respectively healthy persons, implant bad patient and implant good patient CD34 +cell and CD45 -cD34 +vEGFR2 +cell accounts for the correlation results of BMNC ratio;
In Fig. 4, A1-A3 is respectively health donors, implants good and implant the typical HE colored graph (× 40) of bad Bone Marrow of Patients blood vessel,
In Fig. 5, B1-B3 is respectively health donors, implants good and implant the typical CD34 colored graph (× 40) of bad Bone Marrow of Patients blood vessel SABC;
Fig. 6 is the structural representation of system of the present invention;
Wherein, 1 represents main control computer unit, 2 representatives store the system management module of primary control program, LCD represents the prison of allocating cache device module 13, control display, 3 represent empirical data and graphics memory module, 4 represent working storage module, 5 representatives are arranged on the control panel on system operation bench, in 6 representatives store, the communication module of outer data communication agreement and data requirement translative mode, 7 representative printing machine and interface circuits, 8 represent flow cytometer, this instrument is connected with the data bus of main control computer 1 by communication interface circuit 12, the supporting test window 9 that has pair sample to carry out laser scanning sampling in flow cytometer 8 structures, the 10th, dedicated specimen test tube, 11 represent dedicated kit.
Embodiment
Embodiment 1
The present embodiment provides a kind of kit of measuring patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation, the application of this kit, based on flow cytometer, has configured following fluorescently-labeled monoclonal antibody reagent: CD34-FITC, VEGFR2-PE, CD45-PerCP and CD133-APC in this kit.The fluorescence labeling of each monoclonal antibody and one-tenth grading information are referring to table 1.
Table 1 streaming monoclonal antibody information
Title Fluorescence labeling Clone Catalog number Company
CD34 FITC 8G12 348053 BD Biosciences
VEGFR2 PE 89106 580494 BD Biosciences
CD45 PerCP Nothing 347464 BD Biosciences
CD133 APC 293C3 130-090-854 Miltenyi Biotec
10 × PBS damping fluid that also to have configured blood cytolysate, pH in described kit be 7.2 ~ 7.4 and the calf serum of supporting metering.
Particularly, in described kit, every kind of fluorescently-labeled monoclonal antibody reagent volumetric standards unit is: the CD34-FITC of 5 μ L, the VEGFR2-PE of 5 μ L, the CD45-PerCP of 10 μ L and the CD133-APC of 3 μ L, supporting at least 2 of the flow cytometer Special test tubes that are not less than 3-5mL that are provided with.During test, directly with micro-suction gun, draw every kind of fluorescently-labeled monoclonal antibody reagent in kit, the reagent of employing said ratio, test result favorable reproducibility, test result are accurate.
The present embodiment also provides patient's Microcirculation in Bone Marrow environment evaluating system after a kind of mensuration hematopoietic stem cell transplantation, system architecture comprises flow cytometer and supporting data analysis module, in described system, also comprise main control computer unit 1, and be connected to the system management module that stores primary control program 2 on main control computer unit 1 matching interface, the display LCD of allocating cache device module 13, store the data of perspective study and the memory module of figure 3, working storage module 4, be arranged on the control panel 5 on system operation bench, store the communication module 6 of inside and outside data communication agreement and data requirement translative mode, printer and interface circuit 7, flow cytometer 8 is connected with the data bus of main control computer unit 1 by communication interface circuit 12, and the supporting dedicated kit 11 being provided with for measuring patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation in flow cytometer 8 structures, in described dedicated kit, configured following fluorescently-labeled monoclonal antibody reagent: CD34-FITC, VEGFR2-PE, CD45-PerCP and CD133-APC.
Above hardware setting has guaranteed the specificity of system, the integrality that has facilitated operating process and quick, accuracy rate.Particularly the data of perspective study and the memory module of figure sets up and the setting of supporting management program module, the whole gamut of experience that has comprised seminar and achievement, by complicated and loaded down with trivial details test and analytic process is converted into, data and figure simple, robotization are compared.Make the routine test project dyeing, qualitative, quantitative experiment chamber problem is converted into general hospital.
The data of described perspective study and figure comprise that according to age, sex be mark, the Microcirculation in Bone Marrow environment streaming data plot storehouse that the Healthy People indicating respectively, the marrow of implanting well and implant bad patient make sample; In described stream data picture library, storing the dyeing monoclonal antibody adopting in described dedicated kit 11 is that the sample of bone marrow that reagent is made is established the CD45 of an analysis chart and mensuration according to measured scatter diagram, the streaming of specific data acquisition conditions collection related data in memory module 3 -cD34 +vEGFR2 +cellular expression ratio and the relevant Clinical symptoms of this sample of bone marrow.When wherein specific data acquisition conditions refers to image data, the set sampling condition of flow cytometer, comprises voltage, establishes a position, size and shape, the information such as number of samples (such as volume, number of cells).
The analytical approach that streaming is established an analysis chart is: first set up FSC/SSC point diagram, divide living cells region into R1 district, to get rid of dead cell and cell fragment; If CD45/SSC point diagram, according to the intensity of each crowd of cell CD45 and SSC, draws CD45 -sSC low, i.e. CD45 -district, to CD45 -cD34 in district +vEGFR2 +cell is established an analysis, draws CD45 -cD34 +vEGFR2 +expression ratio in BMNC.
Display LCD by working storage module 13 be connected with the data bus of main control computer unit 1, the supporting touch type electronic mouse that arranges.Because a large amount of graphic processing data and analysis, require the working storage module 13 of this display in supporting and the supporting touch type electronic mouse that arranges to drag fast, compare analysis with what realize figure.
The outfit of the memory module of the hardware configuration of native system and the data that contain perspective study and figure is the basic condition of realizing the object of the invention.The assembly process of the management software of control and guidance concrete operations is to realize another key of goal of the invention.
The method comprises the following steps:
A, adopt the reagent in dedicated kit in flow cytometer Special test tube, to carry out fluorescence labeling sample of bone marrow to be measured, make test sample book;
B, the Special test tube that test sample book is housed is inserted in flow cytometer, start flow cytometer 8 and supporting analysis software, according to specific data acquisition conditions, gather related data, form scatter diagram, and carry out streaming and establish an analysis, transmission key words sorting deposit working storage module 4 in;
C, the data that recall perspective study in memory module 3 and figure, be presented at the upper half of the display LCD of high definition, at bottom half, recalls the corresponding collection of illustrative plates in working storage module 4, by cursor chi, contrasts quantitative test;
D, determine CD45 -cD34 +vEGFR2 +in BMNC, express the position of the separatrix value of ratio;
E, by measure CD45 -cD34 +vEGFR2 +it is good that expression ratio>=separatrix value is defined as Microcirculation in Bone Marrow environment; CD45 -cD34 +vEGFR2 +it is bad that expression ratio < separatrix value is defined as Microcirculation in Bone Marrow environment;
F, from empirical data module 3, recall patient's Microcirculation in Bone Marrow environment measuring account after hematopoietic stem cell transplantation, insert determination data, correlation graph, after comparison, " well ", " uncertain ", " bad " are selected to insert in evaluation and test conclusion hurdle, from laser printer, export.
The preparation process of test sample book described in steps A comprises:
A1, get the Special test tube of the supporting setting of kit, in test tube, add respectively the CD34-FITC of 5 μ L, the VEGFR2-PE of 5 μ L, the CD133-APC of the CD45-PerCP of 10 μ L and 3 μ L, then add be no less than 300 μ L sample of bone marrow to be measured and guarantee bone marrow cell 5 × 10 6-1 × 10 7individual, mix, room temperature lucifuge is placed and is hatched for 15 minutes;
A2, add with the blood cytolysate 2mL after 10 times of 1 × PBS dilutions, mix rear lucifuge, room temperature is placed 8 minutes;
Centrifugal 5 minutes of A3,1500 revs/min, abandon supernatant, adds 2mL to contain the PBS of 0.5wt.% ~ 2wt.% serum, mixes;
Centrifugal 5 minutes of A4,1500 revs/min, abandon supernatant, adds 0.3mL1 × PBS damping fluid, mixes, and makes test sample book.
Wherein, the compound method of 10 × PBS damping fluid: take Na 2hPO 412H 2o 26.3g, NaH 2pO 42H 2o 3.0g and NaCl 85.0g are in volumetric flask, and adding distil water is to 1000mL, and normal temperature is preserved.By 10 times of 10 × PBS buffer solution dilutions, be 1 × PBS.
Streaming described in step B is established an analysis and is comprised:
B1, first set up FSC/SSC point diagram, divide living cells region into R1 district, to get rid of dead cell and cell fragment;
B2, establish CD45/SSC point diagram, according to the intensity of each crowd of cell CD45 and SSC, draw CD45 -sSC low, i.e. CD45 -district;
B3, to CD45 -cD34 in district +vEGFR2 +cell is established an analysis, draws CD45 -cD34 +vEGFR2 +expression ratio in BMNC.As shown in Figure 2, be that 1 routine health donors bone marrow cell is carried out to CD45 -cD34 +vEGFR2 +the typical streaming of measuring is established an analysis chart.
According to ROC curve, determine CD45 -cD34 +vEGFR2 +in BMNC, expression ratio=0.055% is for separatrix value, by CD45 -cD34 +vEGFR2 +expression ratio <0.055% is defined as endothelial progenitor cell to be reduced, and Microcirculation in Bone Marrow environment is bad, by CD45 -cD34 +vEGFR2 +it is normal that expression ratio>=0.055% is defined as endothelial progenitor cell, and Microcirculation in Bone Marrow environment is bad.
First the science that checking adopts mentioned reagent box, system and method to study Microcirculation in Bone Marrow environment below.Respectively by verifying about the immunohistochemical staining of inpatient with haematological diseases Microcirculation in Bone Marrow environment component after the HE dyeing of inpatient with haematological diseases Microcirculation in Bone Marrow environment component after hematopoietic stem cell transplantation and hematopoietic stem cell transplantation in embodiment 2 and embodiment 3.
The HE of inpatient with haematological diseases Microcirculation in Bone Marrow environment component dyeing after embodiment 2, hematopoietic stem cell transplantation
1, fixing: to get myeloid tissue and be cut into small pieces, in 10% neutral formalin solution, fix 24 hours; 1 × PBS damping fluid is washed 5 minutes, 5 times;
2, dehydration: the tissue specimen fixing respectively 70% ethanol 1 hour, 80% ethanol 1 hour, 95% ethanol I 1 hour, 95% ethanol II 2 hours, 95% ethanol III 2 hours, 100% ethanol I 1 hour and 100% ethanol 2 hours;
3, transparent: sample after dehydration is placed 10 minutes and 20 minutes respectively in dimethylbenzene I and dimethylbenzene II;
4, waxdip: the paraffin that the tissue after transparent is put into fusing soaks into, 3 times repeatedly, each 0.5-1 hour;
5, embedding: pour the new paraffin of fusing in embedding device, put into rapidly tissue block before it solidifies, will distinguish each face of tissue before putting into, by required section down; While being embedded with cavity tissue, tissue is kept flat or stood up;
6, section: cut the section that thickness is 4 μ m on microtome, launch 37 ℃ of water-baths, and drag on the special slide of SABC; 60 ℃ of oven for baking 4 hours;
7, dewaxing: section is positioned over to dimethylbenzene I, II each 5 minutes;
8, water inlet: section is positioned over respectively in 100%, 95%, 85%, 75%, 50% ethanol to each 5 minutes, is positioned over tap water 5 minutes;
9, HE dyeing: section is put in haematine dye liquor to 5 minutes, 1% aqueous hydrochloric acid solution differentiation several seconds (3～4 seconds), flowing water is positioned in 0.5% Yihong ethanolic solution 1 minute after rinsing;
10, dehydration: section is positioned over to 85%, 95% each several seconds of ethanol, each 1 minute of 100% ethanol I, II;
11, in dimethylbenzene, place the several seconds transparent, adopt neutral gum mounting;
12, optical microphotograph Microscopic observation HE dyeing histotomy, as shown in Figure 1 and Figure 4, the blood vessel quantity in marrow hemopoiesis capacity and each bone trabecula of result of study discovery health donors is greater than the good patient of implantation and is greater than the bad patient of implantation.
The immunohistochemical staining of inpatient with haematological diseases Microcirculation in Bone Marrow environment component after embodiment 3 hematopoietic stem cell transplantation
1, application antihuman CD 34 monoclonal antibody is carried out SABC detection to the blood vessel in organic slice, with HE dyeing, equally first paraffin section is dewaxed and is intake;
2, antigen retrieval: the 0.01M citrate buffer solution (pH 6.0) that preheating is put in section boils 20 minutes, naturally cooling 20 minutes;
3, with 3% hydrogen peroxide at room temperature, hatch 20 minutes, to eliminate the activity of endogenous peroxydase;
4, the section of application distilled water flushing, soaks in PBS damping fluid 5 minutes;
5, remove PBS damping fluid, every section adds 1 or the normal nonimmune animal blood serum of 50ul, sealing, under room temperature, hatch 10 minutes, the serum deprivation that inclines, does not wash, the antihuman CD 34 monoclonal antibody (U.S. Abcam company) that drips 1:100 dilution, 4 degrees Celsius of wet boxes spend the night;
6, PBS liquid rushes liquid and washes section, and each 5 minutes, totally 3 times;
7, drip Envision working fluid, in room temperature, place 30 minutes
8, PBS liquid rushes liquid and washes section, and each 5 minutes, totally 3 times;
9, remove PBS damping fluid, every section adds 2 or the freshly prepared DAB solution of 100ul, micro-Microscopic observation 3-10 minute;
10, tap water rinses, and haematine dye liquor is redyed 1 minute, and tap water rinses and returns indigo plant;
11, section is positioned over to 85%, 95% each several seconds of ethanol, each 1 minute of 100% ethanol I, II, place the several seconds transparent in dimethylbenzene, adopt neutral gum mounting;
12, the quantity of CD34 positive vessels in each bone trabecula in the each section of optical microphotograph Microscopic observation; As shown in Figure 5, the quantity that result of study is found CD34 positive vessels in the each bone trabecula of health donors is more than implanting good patient more than implanting bad patient.
Embodiment 4
After adopting kit in embodiment 1, system and method to the Allogeneic Hematopoietic Stem Cell Transplantation of accepting for medical treatment in The People's Hospital of Peking University, blood disease research institute of Peking University on April 15,1 day ~ 2013 April in 2012, implant bad (poor graft function, PGF) and implant good (good graft function, GGF) inpatient with haematological diseases, according to 1:2 ratio, carry out perspective matched pair study, to adopting the method in embodiment 2 and 3 to verify the result of embodiment 1.For every routine PGF patient, all choose at random 2 examples at the age, transplant before chemotherapy treatment, morbid state and transplant the GGF patient who matches aspect the evaluation time of rear Microcirculation in Bone Marrow environment and include this research in while transplanting.GGF enters group standard: after hematopoietic stem cell transplantation+28 day, continue to keep successfully implantation (neutrophil leucocyte absolute value [ANC] >0.5 × 10 9/ L continues more than 3 days; Platelet count [PLT] >20 × 10 9/ L continues more than 7 days; Hemoglobin level [Hb] >70g/L also departs from red blood cell transfusion).PGF enters group standard: after hematopoietic stem cell transplantation+28 day, occur that 2-3 is that haemocyte reduces and continues more than 3 days (ANC≤0.5 × 10 9/ L continues more than 3 days; PLT≤20 × 10 9/ L; Or Hb≤70g/L), can not depart from blood product infusion, with low hyperplasia marrow, without serious graft versus host disease(GVH disease) and hematology recurrence.
All patients is all in the Acceptable criterion diagnosis of blood disease research institute of Peking University and Allogeneic Hematopoietic Stem Cell Transplantation treatment.Allogeneic Hematopoietic Stem Cell Transplantation scheme is carried out according to the transplanting routine of blood disease research institute of Peking University.This research is examined by Ethics Committee of The People's Hospital of Peking University, all enters to organize patient and has all signed Informed Consent Form.
Below to implanting bad after hematopoietic stem cell transplantation and implanting CD45 in the Clinical symptoms of good two groups of inpatient with haematological diseases and marrow blood vessel -cD34 +vEGFR2 +expression ratio is carried out statistical analysis.
1, clinical characters
It is to transplant latter 90 days (58-264 days) that this group patient implants bad meta time of origin.Implant bad group and implant good group Bone Marrow of Patients be detection time transplant latter 90 days ( p>0.05).DNA fingerprint figure detects all patients of prompting and is complete donor type.GGF and two groups of patients' of PGF Clinical symptoms is as shown in table 2.
Table 2 is implanted the good Clinical symptoms with implanting bad patient after transplanting
Patient characteristic Implant bad group (n=19) Implant good group (n=38) P-Value b
The Microcirculation in Bone Marrow environmental assessment time (number of days after transplanting) 90(58-264) 90(30-270) 0.61
Bone marrow cell hyperplasia degree (%) (scope) 10(5-15) 45(30-50) <0.0001
Microcirculation in Bone Marrow environment composition
CD45 -CD34 +VEGFR2 +Ratio (%) (scope) 0.008(0.001-0.14) 0.16(0.07-0.25) <0.0001
CD34 +Blood vessel quantity (each bone trabecula) (scope) 2(0-4) 4(2-6) <0.0001
Peripheral blood cells counting
Meta white blood cell count(WBC) (× 10 9/ L) (scope) 1.73(0.32-5.34) 5(3.10-7.60) <0.0001
Meta neutrophil count (× 10 9/ L) (scope) 1.10(0.16-6.50) 3.65(1.55-6.38) <0.0001
Meta hemoglobin level (g/L) (scope) 76(56-103) 120(78-151) <0.0001
Meta platelet count (× 10 9/ L) (scope) 17(9-142) 119(61-243) <0.0001
Age during transplanting (year, median, scope) 27(16-57) 35.5(16-55) 0.48
Sex (male/female) 9/10 23/15 0.40
Medical diagnosis on disease
Acute myelocytic leukemia 4 9 1.00
Acute lymphoblastic leukemia 9 17 1.00
Chronic myelocytic leukemia 2 4 1.00
Myelodysplastic syndrome 4 8 1.00
Morbid state during transplanting 0.11
Mark danger 16 25
High-risk 3 13
Make a definite diagnosis the median time (moon, scope) of transplanting 12(4-20) 10(2-30) 0.32
Source of human stem cell 1.00
Marrow and peripheral blood 17 34
Peripheral blood 2 4
The mononuclearcell number (× 10 of transplanting 8/ kilogram, median, scope) 8.06(5.34-15.34) 7.59(3.50-10.08) 0.12
As can be seen from Table 2, this research enters to organize 57 examples to accept the inpatient with haematological diseases of Allogeneic Hematopoietic Stem Cell Transplantation altogether, comprise that 19 examples are implanted bad patients and 38 examples are implanted good patients, mononuclearcell number and the CD34 of sex matching degree, transplanting between abo blood group matching degree, donor-recipient between HLA matching degree, donor-recipient between chemotherapy treatment and morbid state, source of human stem cell, donor-recipient before age between two groups of patients, sex, medical diagnosis on disease, transplanting +cell number, make a definite diagnosis time of transplanting, carry out the Clinical symptoms there was no significant differences such as the time, pretreating scheme, graft versus host disease(GVH disease) medical history, cytomegalovirus infection medical history of Microcirculation in Bone Marrow environment measuring and anti-giant cell treatment course for the treatment of after transplanting ( p>0.05).Implant bad group of peripheral blood in patients cell count (comprising ANC, Hb, PLT) and myelosis degree, CD45 -cD34 +vEGFR2 +ratio and CD34 +blood vessel quantity be starkly lower than implant good group patient ( p<0.0001).
Health donors, implant bone marrow cell hyperplasia degree typical case's HE colored graph (× 10) good and that implant bad patient and also confirms: the bone marrow cell hyperplasia degree of health donors is better than implanting good patient and the bad patient of implantation successively.
Fig. 3 shows, although implant bad and implant the CD34 transplanting between good two groups of patients +cell quantity is without significant difference, but bad group of patient's of implantation CD34 after transplanting +cell proportion and endothelial progenitor cell ratio are all starkly lower than implants good group patient, and implants between good group patient and health donors without above-mentioned cell proportion no difference of science of statistics.
The cell quantity analysis of 2, implanting bad patient and implanting good peripheral blood in patients and marrow
The peripheral blood cells counting of implanting bad group of patient while carrying out Microcirculation in Bone Marrow environment measuring is starkly lower than implants good group patient.Implant bad group of patient's meta white blood cell count(WBC) (1.73 × 10 9/ L vs. 5 × 10 9/ L, p<0.0001), neutrophil count (1.1 × 10 9/ L vs. 3.65 × 10 9/ L, p<0.0001), hemoglobin level (76 g/L vs. 120 g/L, p<0.0001) and platelet count (17 × 10 9/ L vs. 119 × 10 9/ L, p<0.0001) be all starkly lower than and implant good group patient.
Implant bad Bone Marrow of Patients pathological manifestations for making hypovolemia, adipocyte ratio increases.The hematopoiesis capacity of implanting bad group of patient be starkly lower than implant good group patient and health donors (10% vs. 45% vs. 45%, p<0.0001).
3, by flow cytometry, quantitatively detect marrow CD34 +cell and CD45 -cD34 +vEGFR2 +ratio
As shown in Figure 3, although implant bad group and the implantation CD34 that well group patient transplants +cell quantity, without obvious significant difference, is still implanted bad group of patient's marrow CD34 +cell proportion be starkly lower than implant good group patient and health donors (0.07% vs. 0.26% vs. 0.26%, p<0.0001).
Implant bad group of Bone Marrow of Patients CD45 –cD34 +vEGFR2 +ratio be starkly lower than implant good group patient and health donors (0.008% vs. 0.16% vs. 0.18%, p<0.0001).
4, implant bad risk factors
As shown in table 2, dependent variable is bad for occurring after transplanting to implant, and independent variable comprises between chemotherapy treatment and morbid state before age, age, sex, medical diagnosis on disease, transplanting of patient, source of human stem cell, donor-recipient between HLA matching degree, donor-recipient mononuclearcell number and the CD34 of sex matching degree, transplanting between abo blood group matching degree, donor-recipient +after cell number, the time of making a definite diagnosis transplanting, transplanting, carry out time, pretreating scheme, graft versus host disease(GVH disease) medical history, cytomegalovirus infection medical history and the anti-giant cell of Microcirculation in Bone Marrow environment measuring and treat the Clinical symptoms such as the course for the treatment of, marrow endothelial progenitor cell ratio.Single factor prognostic analysis result shows medical diagnosis on disease (myelodysplastic syndrome vs. leukaemia), graft versus host disease(GVH disease) medical history and marrow endothelial progenitor cell (CD45 –cD34 +vEGFR2 +expression ratio) (<0.055% vs.>=0.055%) be impact transplant after patient implant bad hazards.
Prognostic factors shows, medical diagnosis on disease (myelodysplastic syndrome vs. leukaemia) and marrow endothelial progenitor cell (<0.055% vs. >=0.055%) are for affecting the independent hazard factor of implanting bad generation after hematopoietic stem cell transplantation.
In sum, system and method for the present invention can be quantitatively, inpatient with haematological diseases Microcirculation in Bone Marrow environment after qualitative determination hematopoietic stem cell transplantation, thereby have important directive significance to transplanting rear patient's successive treatment.

Claims (10)

1. measure the kit of patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation for one kind, the application of this kit is based on flow cytometer, it is characterized in that having configured in this kit following fluorescently-labeled monoclonal antibody reagent: CD34-FITC, VEGFR2-PE, CD45-PerCP and CD133-APC.
2. the kit of patient's Microcirculation in Bone Marrow environment evaluation and test after mensuration hematopoietic stem cell transplantation according to claim 1,10 × PBS damping fluid that to have it is characterized in that being also equipped with in described kit blood cytolysate, pH be 7.2 ~ 7.4 and the calf serum of supporting metering.
3. the kit of patient's Microcirculation in Bone Marrow environment evaluation and test after mensuration hematopoietic stem cell transplantation according to claim 1, it is characterized in that in described kit, every kind of fluorescently-labeled monoclonal antibody reagent volumetric standards unit is: the CD34-FITC of 5 μ L, the CD45-PerCP of the VEGFR2-PE of 5 μ L, 10 μ L and the CD133-APC of 3 μ L, supporting at least 2 of the flow cytometer Special test tubes that are not less than 3-5mL that are provided with.
4. patient's Microcirculation in Bone Marrow environment evaluating system after a mensuration hematopoietic stem cell transplantation, system architecture comprises flow cytometer and supporting data analysis module, it is characterized in that also comprising in described system main control computer unit (1), and be connected to the system management module that stores primary control program (2) on main control computer unit (1) matching interface, the display (LCD) of allocating cache device module (13), store the data of perspective study and the memory module of figure (3), working storage module (4), be arranged on the control panel (5) on system operation bench, store the communication module (6) of inside and outside data communication agreement and data requirement translative mode, printer and interface circuit (7), flow cytometer (8) is connected with the data bus of main control computer unit (1) by communication interface circuit (12), and the supporting dedicated kit (11) being provided with for measuring patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation in flow cytometer (8) structure, in described dedicated kit, configured following fluorescently-labeled monoclonal antibody reagent: CD34-FITC, VEGFR2-PE, CD45-PerCP and CD133-APC.
5. patient's Microcirculation in Bone Marrow environment evaluating system after mensuration hematopoietic stem cell transplantation according to claim 4, the data and the figure that it is characterized in that described perspective study comprise that according to age, sex be mark, the Microcirculation in Bone Marrow environment streaming data plot storehouse that the Healthy People indicating respectively, the marrow of implanting well and implant bad patient make sample; In described stream data picture library, storing the dyeing monoclonal antibody adopting in described dedicated kit (11) is that the sample of bone marrow that reagent is made is established the CD45 of an analysis chart and mensuration according to the measured scatter diagram of specific data acquisition conditions, streaming -cD34 +vEGFR2 +cellular expression ratio and the relevant Clinical symptoms of this sample of bone marrow.
6. patient's Microcirculation in Bone Marrow environment evaluating system after mensuration hematopoietic stem cell transplantation according to claim 5, is characterized in that streaming establishes the analytical approach of an analysis chart and be: first set up FSC/SSC point diagram, divide living cells region into R1 district; If CD45/SSC point diagram, according to the intensity of each crowd of cell CD45 and SSC, draws CD45 -district, to CD45 -cD34 in district +vEGFR2 +cell is established an analysis, draws CD45 -cD34 +vEGFR2 +expression ratio in BMNC.
7. patient's Microcirculation in Bone Marrow environment evaluating system after mensuration hematopoietic stem cell transplantation according to claim 4, it is characterized in that display (LCD) by working storage module (13) be connected with the data bus of main control computer unit (1), the supporting touch type electronic mouse that arranges.
8. the method that realizes patient's Microcirculation in Bone Marrow environment evaluation and test after hematopoietic stem cell transplantation based on system claimed in claim 4, is characterized in that the method comprises the following steps:
A, adopt the reagent in dedicated kit in flow cytometer Special test tube, to carry out fluorescence labeling sample of bone marrow to be measured, make test sample book;
B, the Special test tube that test sample book is housed is inserted in flow cytometer, start flow cytometer (8) and supporting analysis software, according to specific data acquisition conditions in memory module (3), gather related data, form scatter diagram, and carry out streaming and establish an analysis, transmission key words sorting deposit working storage module (4) in;
C, recall data and the figure of perspective study in memory module (3), be presented at the upper half of the display (LCD) of high definition, at bottom half, recall the corresponding collection of illustrative plates in working storage module (4), by cursor chi, contrast quantitative test;
D, determine CD45 -cD34 +vEGFR2 +in BMNC, express the position of the separatrix value of ratio;
E, by measure CD45 -cD34 +vEGFR2 +it is good that expression ratio>=separatrix value is defined as Microcirculation in Bone Marrow environment; CD45 -cD34 +vEGFR2 +it is bad that expression ratio < separatrix value is defined as Microcirculation in Bone Marrow environment;
F, from empirical data module (3), recall patient's Microcirculation in Bone Marrow environment measuring account after hematopoietic stem cell transplantation, insert determination data, correlation graph, after comparison, " well ", " uncertain ", " bad " are selected to insert in evaluation and test conclusion hurdle, from laser printer, export.
9. inpatient with haematological diseases Microcirculation in Bone Marrow environment evaluating method after detection hematopoietic stem cell transplantation according to claim 8, is characterized in that the preparation process of test sample book described in steps A comprises:
A1, get the Special test tube of the supporting setting of kit, in test tube, add respectively the CD34-FITC of 5 μ L, the VEGFR2-PE of 5 μ L, the CD133-APC of the CD45-PerCP of 10 μ L and 3 μ L, then add be no less than 300 μ L sample of bone marrow to be measured and guarantee bone marrow cell 5 × 10 6-1 × 10 7individual, mix, room temperature lucifuge is placed and is hatched for 15 minutes;
A2, add with the blood cytolysate 2mL after 10 times of 1 × PBS dilutions, mix rear lucifuge, room temperature is placed 8 minutes;
Centrifugal 5 minutes of A3,1500 revs/min, abandon supernatant, adds 2mL to contain the PBS of 0.5wt.% ~ 2wt.% serum, mixes;
Centrifugal 5 minutes of A4,1500 revs/min, abandon supernatant, adds 0.3mL1 × PBS damping fluid, mixes, and makes test sample book.
10. the method for inpatient with haematological diseases Microcirculation in Bone Marrow environment after detection hematopoietic stem cell transplantation according to claim 8, is characterized in that streaming described in step B establishes the step of an analysis and comprise:
B1, first set up FSC/SSC point diagram, divide living cells region into R1 district;
B2, establish CD45/SSC point diagram, according to the intensity of each crowd of cell CD45 and SSC, draw CD45 -district;
B3, to CD45 -cD34 in district +vEGFR2 +cell is established an analysis, draws CD45 -cD34 +vEGFR2 +expression ratio in BMNC.
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