Blood handling and leukocyte isolation methods impact the global transcriptome of immune cells

Goods, Brittany A.; Vahey, Jacqueline; Steinschneider, Arthur F.; Askenase, Michael H.; Sansing, Lauren H; Love, J. Christopher

doi:10.1186/s12865-018-0268-6

Cited by 20 publications

(21 citation statements)

References 21 publications

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“…RNA-Seq libraries were generated as previously described ( 27 ). Briefly, RNA was extracted using the NucleoSpin RNA XS Kit (Macherey-Nagel, 740902) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu

Goods¹,

Askenase²,

Markarian³

et al. 2021

JCI Insight

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Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here, we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Hemorrhage Evacuation trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-Seq (scRNA-Seq), this is the first report to our knowledge that characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution. Our analysis revealed shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic rebleed that our transcriptional data indicated occurred prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods such as scRNA-Seq would enable greater understanding of complex intracerebral events.

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Section: Methodsmentioning

confidence: 99%

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu

Goods¹,

Askenase²,

Markarian³

et al. 2021

JCI Insight

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“…However, in a study of gene expression in stabilised whole blood compared to PBMCs as tools to evaluate perioperative inflammatory response in patients with advanced heart failure, the molecular fingerprints were similar between the two sample materials 176 . Since the handling of blood samples prior to PBMC isolation can affect gene expressions 178 to exclude CD as a diagnosis, or symptoms. This is the population from which we need to separate CD cases, so it is well suited as a control population, but it would add to the project to characterise gene expression in cases without suspected CD, preferably with a CD-incompatible HLA type.…”

Section: Discussionmentioning

confidence: 99%

Biomarkers of Inflammation and Intestinal Mucosa Pathology in Celiac Disease

Bragde

2019

Linköping University Medical Dissertations

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Celiac disease (CD) is a chronic small intestinal immune-mediated enteropathy triggered by gluten. The only currently available treatment is complying with a lifelong gluten-free diet, which should not be commenced before a CD diagnosis has been established by diagnostic test results (including histopathologic assessment of small intestinal biopsies and CD-specific antibody levels). This makes diagnostic swiftness and accuracy important. In cases with low CD-specific antibody levels and/or low-grade intestinal injuries the diagnosis can be difficult to establish. The main objective of this thesis was to complement and improve CD diagnostics by identifying and implementing new biomarkers, mainly based on gene expression, in small intestinal biopsies and blood. In paper I, genes were selected to reflect villous height, crypt elongation, immune response, and epithelial integrity. The results showed that a subset of those genes could discriminate active CD mucosa from mucosa without CD-related changes and grade the intestinal injury. In paper III, an unbiased investigation of gene expression in CD mucosa was performed using transcriptome analysis. Active CD and non-CD mucosa showed differential expression in a subset of genes, and some were differentially expressed in CD mucosa before histopathologic assessment could confirm intestinal alterations compatible with a CD diagnosis. Gene set analysis revealed that there are many biological processes affected in CD mucosa, including those associated with immune response, microbial infection, phagocytosis, intestinal barrier function, metabolism, and transportation. In parallel, gene expression was investigated in stabilised whole blood. Blood is a more accessible sampling material than intestinal biopsies, and stabilised blood is suitable for routine diagnostics since transcript levels are preserved at sampling. In paper II, expressions from a selection of genes were quantified in stabilised whole blood (RNA) and/or plasma (protein). Three genes with differential expression in CD were identified. Compared to the CD-specific autoantibodies against tissue transglutaminase (anti-TG2) alone, the addition of the information from the new potential markers resulted in a non-significant contribution to the diagnostic capacity of anti-TG2. An unbiased investigation using transcriptome analysis (paper IV) showed that gene level expression differences in stabilised whole blood were small between CD and non-CD. However, expression differences on a gene set level could potentially be used in CD diagnostics. CD-associated biological processes suggested UBD ubiquitin D ULN upper limit of normal ZFR zinc finger RNA binding protein Contents ix Contents Introduction .

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“…In the MISTIE III trial, 2% of patients in the surgical arm had a symptomatic re-bleeding event, however 32% had asymptomatic re-bleeding. 5,9 Hematoma effluent volume drainage is an imperfect measurement of rebleeding, as some hematoma cavities have variable connections with the ventricular or subarachnoid space and, at times, volume may increase due to CSF drainage. This event is difficult to identify or predict at the bedside.…”

Section: Figure 4 Supplementalmentioning

confidence: 99%

“…RNA-sequencing libraries were generated as previously described. 5 Briefly, RNA was extracted using the NucleoSpin RNA XS Kit (Macherey-Nagel, #740902) according to the manufacturer's instructions. Smart-Seq2 cDNA synthesis was performed with the following modifications: 1) input RNA was normalized prior to cDNA generation by diluting to ~1,000 cells per reaction, 2) reverse transcription was performed with Superscript III (ThermoFisher, #18080-085) in place of Superscript II according to the manufacturer instructions.…”

Section: Data Analysis For Mass Cytometrymentioning

confidence: 99%

“…The trial found that surgery was safe and reduced mortality, but did not improve overall functional outcomes 365 days after ICH. 5 As part of a trial sub-study (ICHseq), daily samples of both hematoma effluent from the catheter and peripheral blood were collected. These samples provided a unique opportunity to study the immune response to ICH over time in living patients.…”

Section: Introductionmentioning

confidence: 99%

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Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu

Goods

Askenase

Markarian

et al. 2020

Preprint

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Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury, particularly over time, in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Haemorrhage Evacuation (MISTIEIII) trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-sequencing, we characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution for the first time. Our analysis revealed rapid shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic re-bleed (second local exposure to blood) that our transcriptional data indicated occurred more than 30 hours prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods like scRNA-seq can inform our understanding of complex intracerebral events.

show abstract

Blood handling and leukocyte isolation methods impact the global transcriptome of immune cells

Cited by 20 publications

References 21 publications

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu

Biomarkers of Inflammation and Intestinal Mucosa Pathology in Celiac Disease

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu

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