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Microfluidic based immunoaffinity mononuclear leukocytes isolation from whole blood
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. karolinska institutet. (Aman Russom)
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.ORCID iD: 0000-0002-0242-358X
(English)Manuscript (preprint) (Other academic)
Abstract [en]

CD4+ T cells, monocyte/macrophages and natural killer cells are believed to be the main source for HIV-1 reservoirs in peripheral blood. However, despite the potential these subsets of providing a wealth of new information about immune function and host pathology, current HIV latency studies are often based on PBMCs or only CD4+ T cells, mainly due to the lack of appropriate cell subset isolation methods. We present here a microfluidic chip-based method to capture and enrich the three mononuclear cells sub-population peripheral leukocyte sub-populations: CD4+ lymphocytes, natural killer cells and monocytes; using a single source of whole blood (volume < 200 μL) on a single integrated platform, within a time frame of 20 min. The single step isolation method can be used for downstream proteomics and genomics analysis to study the aberrations in these cell types’ functions in critical diseases such as HIV.

Keywords [en]
microfluidic, mononuclear leukocytes isolation, HIV-1 reservoir
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-242234OAI: oai:DiVA.org:kth-242234DiVA, id: diva2:1283306
Note

QC 20190131

Available from: 2019-01-28 Created: 2019-01-28 Last updated: 2019-02-14Bibliographically approved
In thesis
1. Development of novel molecular and microfluidics tools for identification and characterization of latent HIV-1 reservoir
Open this publication in new window or tab >>Development of novel molecular and microfluidics tools for identification and characterization of latent HIV-1 reservoir
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The existence of latent HIV-1 reservoir (LR) in all HIV-1 infected patients serves as a major obstacle to completely cure HIV-1 infection. However, up to now there is still no available assay that provides an accurate measurement of the reservoir size. This thesis aims to address this challenge from different aspects with several novel technologies, using both molecular and microfluidics-based tools. To find a proper tool to identify the latent HIV-1 reservoir, in Paper I and II, LIPS assay, RNAflow, and RNAscope assay were optimized and evaluated for indirect and direct detection of latent HIV-1 reservoir. The results indicated the LIPS method might not be sufficient for latent HIV-1 reservoir detection, although it has been proposed to quantify the latent HIV-1 reservoir indirectly. Furthermore, the optimized RNAscope technique performed better than RNAflow for transcription and translation competent latent HIV-1 reservoir identification. The RNAscope was also found to be independent of the HIV-1 subtype and can be applied to patient samples at single cell level. As there are currently no available surface biomarkers for latent HIV-1 reservoir, in Paper III, transcriptomics and proteomics-based analysis method for high-throughput selection of potential biomarker were established and applied to different patient groups. Twelve membrane protein-coding genes were identified as downregulated in the patient group who were hypothesized to have lower latent reservoir. These proteins might have the potential to be used as surface biomarkers for latent HIV-1 reservoir. CD4+ T cells, monocyte/macrophages, and natural killer cells are believed to be the primary source for HIV-1 reservoirs in peripheral blood. In paper IV, a microfluidic chip was developed to simultaneously isolate these three mononuclear leukocyte cell types directly from whole blood. The microfluidic method reduces the sample volume requirement and is a promising tool for latent HIV-1 reservoir study. Together, though further improvement and clinical verification are necessary, the work in this thesis has contributed to the advancement of latent HIV-1 reservoir characterization and may facilitate future development of the latent HIV-1 reservoir targeting and clearance methods with the ultimate goal – to cure HIV-1 infection.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. p. 65
Series
TRITA-CBH-FOU ; 2019:8
Keywords
HIV-1, Latent HIV-1 reservoir, HIV-1 characterization, Microfludics, Molecular detection
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-242235 (URN)978-91-7873-089-6 (ISBN)
Public defence
2019-03-01, Air&Fire, Science for life lab, Tomtebodavägen 23a, 171 65 Solna, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20190129

Available from: 2019-01-29 Created: 2019-01-28 Last updated: 2019-04-12Bibliographically approved

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