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High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories


Summerauer, Andrea Maria; Colombo, Lorenzo; Ogwang, Rodney; Berger, Christoph; Fehr, Jan; Bürgler, Simone (2018). High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories. Malaria Journal, 17:395.

Abstract

BACKGROUND Malaria is still a major health problem in sub-Saharan Africa and south-east Asia, but research on malaria in low-income countries can be a challenge due to the lack of laboratory equipment. In addition, severe malaria mainly affects very young children, which limits the amount of blood available for research purposes. Thus, there is a need for protocols that yield a maximum of information from a minimum amount of blood, which are operable in basically equipped laboratories.
RESULTS A protocol for tandem B and T helper (Th) cell isolation directly from whole blood, and a freezer-independent sample preservation method compatible with the warm and humid climate of malaria regions was established and validated. The protocol thereby circumvents the need of high-technology centrifuges and unimpeachable power supply for peripheral blood mononuclear cell isolation. Both purity and yield are excellent. Depending on the expression level of the genes of interest, between 2 and 5 ml of blood are adequate for reliable qRT-PCR results from both B and Th cells of healthy paediatric donors as well as paediatric malaria patients.
CONCLUSION This protocol for high purity high yield B cell and Th cell isolation and sample storage for subsequent qRT-PCR analysis from a minimal amount of blood is contrivable with basic equipment and independent of continuous power supply. Thus, it is likely to be of avail for many scientists performing malaria research in rural institutes or hospitals, and thus in countries where malaria is most prevalent.

Abstract

BACKGROUND Malaria is still a major health problem in sub-Saharan Africa and south-east Asia, but research on malaria in low-income countries can be a challenge due to the lack of laboratory equipment. In addition, severe malaria mainly affects very young children, which limits the amount of blood available for research purposes. Thus, there is a need for protocols that yield a maximum of information from a minimum amount of blood, which are operable in basically equipped laboratories.
RESULTS A protocol for tandem B and T helper (Th) cell isolation directly from whole blood, and a freezer-independent sample preservation method compatible with the warm and humid climate of malaria regions was established and validated. The protocol thereby circumvents the need of high-technology centrifuges and unimpeachable power supply for peripheral blood mononuclear cell isolation. Both purity and yield are excellent. Depending on the expression level of the genes of interest, between 2 and 5 ml of blood are adequate for reliable qRT-PCR results from both B and Th cells of healthy paediatric donors as well as paediatric malaria patients.
CONCLUSION This protocol for high purity high yield B cell and Th cell isolation and sample storage for subsequent qRT-PCR analysis from a minimal amount of blood is contrivable with basic equipment and independent of continuous power supply. Thus, it is likely to be of avail for many scientists performing malaria research in rural institutes or hospitals, and thus in countries where malaria is most prevalent.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
04 Faculty of Medicine > Epidemiology, Biostatistics and Prevention Institute (EBPI)
04 Faculty of Medicine > University Hospital Zurich > Clinic for Infectious Diseases
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:29 October 2018
Deposited On:15 Jan 2019 15:49
Last Modified:29 Apr 2019 07:10
Publisher:BioMed Central
ISSN:1475-2875
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1186/s12936-018-2547-3
PubMed ID:30373593

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