Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection.
10.19746/j.cnki.issn.1009-2137.2022.03.038
- Author:
Hong-Yu SU
1
;
Yi SHU
1
;
Guo FU
1
;
Zi-Yang LIU
1
;
Dan ZHU
1
;
La-Mei ZENG
1
;
De-Yu MA
1
;
Lin ZOU
2
,
3
Author Information
1. Department of Clinical Molecular Medicine of Children's Hospital in Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders， Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China.
2. Department of Clinical Molecular Medicine of Children's Hospital in Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders， Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China；Clinical Research Unit of Children's Hospital in Shanghai Jiaotong University
3. Institute of Pediatric Infection, Immunity, and Critical Care Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200062, China,E-mail: zoulin74@126.com.
- Publication Type:Journal Article
- Keywords:
EBER;
EBV;
Flow-FISH;
differential diagnosis
- MeSH:
Epstein-Barr Virus Infections;
Flow Cytometry/methods*;
Herpesvirus 4, Human;
Humans;
In Situ Hybridization, Fluorescence/methods*;
Lymphocyte Subsets
- From:
Journal of Experimental Hematology
2022;30(3):897-907
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample.
METHODS:Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope.
RESULTS:It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully.
CONCLUSION:A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.
