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A kit, a system and a method for determining patient marrow microenvironment after hematopoietic stem cell transplantation

A technology of hematopoietic stem cells and kits, applied in measuring devices, instruments, fluorescence/phosphorescence, etc., can solve the problems of limited popularization and application

Active Publication Date: 2014-04-23
SHANGHAI TISSUEBANK MEDICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long-term culture experiments and colony formation experiments of bone marrow cells have certain limitations: the target cell population includes a group of heterogeneous bone marrow stromal cell components such as fibroblasts, adipocytes, and endothelial cells.
Especially in the early stage after hematopoietic stem cell transplantation, the number of reconstituted bone marrow stromal cells in patients is very small, which seriously limits the popularization and application of the above research methods

Method used

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  • A kit, a system and a method for determining patient marrow microenvironment after hematopoietic stem cell transplantation
  • A kit, a system and a method for determining patient marrow microenvironment after hematopoietic stem cell transplantation
  • A kit, a system and a method for determining patient marrow microenvironment after hematopoietic stem cell transplantation

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Effect test

Embodiment 1

[0029] The invention provides a kit for measuring the bone marrow microenvironment of patients after hematopoietic stem cell transplantation. The application of the kit is based on flow cytometry, and the kit is configured with the following fluorescently labeled monoclonal antibody reagent: CD34-FITC , CD146-PE, CD45-PerCP and CD133-APC, see Table 1 for information on fluorescent labels and components of each monoclonal antibody.

[0030] name fluorescent label clone catalog number company CD34 FITC 8G12 348053 BD Biosciences CD146 PE 89106 550315 BD Biosciences CD45 PerCP none 347464 BD Biosciences CD133 APCs 293C3 130-090-854 Miltenyi Biotec

[0031] The kit is also configured with erythrocyte lysate, 10×PBS buffer solution with a pH of 7.2-7.4, and matching calf serum.

[0032] Specifically, the volume standard unit of each fluorescently labeled monoclonal antibody reagent in the kit is: 5 μL of CD34-FITC, ...

Embodiment 2

[0058] Example 2, HE staining of bone marrow microenvironment components in patients with hematological diseases after hematopoietic stem cell transplantation

[0059] 1. Fixation: take bone marrow tissue and cut into small pieces, fix in 10% neutral formalin solution for 24 hours; wash 5 times with 1×PBS buffer solution, 5 minutes each time;

[0060] 2. Dehydration: Put the fixed tissue samples in 70% ethanol for 1 hour, 80% ethanol for 1 hour, 95% ethanol I for 1 hour, 95% ethanol II for 2 hours, 95% ethanol III for 2 hours, 100% ethanol I for 1 hour hours and 100% ethanol II for 2 hours;

[0061] 3. Transparent: put the dehydrated specimen in xylene I for 10 minutes and xylene II for 20 minutes;

[0062] 4. Wax immersion: soak the transparent tissue in molten paraffin, repeat 3 times, 0.5-1 hour each time;

[0063] 5. Embedding: Pour melted new paraffin into the embedding device, put the tissue block quickly before it solidifies, and distinguish the various sides of the t...

Embodiment 3

[0071] Example 3 Immunohistochemical staining of bone marrow microenvironment components in patients with hematological diseases after hematopoietic stem cell transplantation

[0072] 1. Immunohistochemical detection of blood vessels in bone marrow tissue sections was performed with anti-human CD34 monoclonal antibody, and paraffin sections were first dewaxed and watered as in HE staining;

[0073] 2. Antigen retrieval: put slices into preheated 0.01M citrate buffer (pH 6.0) and boil for 20 minutes, then cool naturally for 20 minutes;

[0074] 3. Incubate with 3% hydrogen peroxide at room temperature for 20 minutes to eliminate the activity of endogenous peroxidase;

[0075] 4. Rinse the sections with distilled water and soak in PBS buffer for 5 minutes;

[0076] 5. Remove the PBS buffer, add 1 drop or 50ul of normal non-immune animal serum to each slice, block, incubate at room temperature for 10 minutes, pour off the serum, do not wash, add 1:100 diluted anti-human CD34 mon...

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Abstract

The invention discloses a kit, a system and a method for determining patient marrow microenvironment after hematopoietic stem cell transplantation, wherein the applications of the kit, the system and the method are based on a flow cytometer, and the following fluorescently-labeled monoclonal antibody reagents are arranged in the kit: CD34-FITC, CD146-PE, CD45-PerCP and CD133-APC. The system comprises a main control computer unit, and a storage unit for storing empirical data and diagram, and the configuration of the hardware is an important guarantee for implementing determination. An expression proportion of a CD45-CD34-CD146+ cell in a bone marrow mononuclear cell is firstly used as an index for evaluating the quality of the marrow microenvironment of a blood disease patient after hematopoietic stem cell transplantation, has guiding significance for predicting generation of transplantation badness of the blood disease patient after hematopoietic stem cell transplantation, and has important reference value for guidance of making a clinic treatment scheme for a transplantation badness patient.

Description

technical field [0001] The invention relates to a special kit and system for cell phenotype used in a special application environment, in particular to a kit, a measurement system and a method for qualitative and quantitative analysis for measuring the bone marrow microenvironment of patients after hematopoietic stem cell transplantation. Background technique [0002] Allogeneic hematopoietic stem cell transplantation is one of the effective ways to treat hematological malignancies, but the incidence of poor implantation (English name Poor Graft Function, abbreviated PGF) after transplantation is between 5-27%. One of the main complications after transplantation, which seriously affects the long-term survival of transplant patients. Although reported in the literature, poor engraftment may be related to pre-transplant conditioning dose, immune rejection, graft-versus-host disease, viral infection, CD34 + However, the pathogenesis of poor implantation after transplantation h...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/64
CPCG01N15/14G01N33/5073G01N33/533G01N33/577
Inventor 黄晓军孔圆常英军王亚哲袁晓英胡玥王昱刘代红许兰平
Owner SHANGHAI TISSUEBANK MEDICAL LAB CO LTD
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