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Our group recently identified a panel of ten genes whose RNA expression levels in whole blood have utility for detection of stroke. The purpose of this study was to determine the mechanisms by which these genes become differentially expressed during stroke pathology. First, we assessed the transcriptional distribution of the ten genes across the peripheral immune system by measuring their expression levels on isolated neutrophils, monocytes, B-lymphocytes, CD-4+ T-lymphocytes, CD-8+ T-lymphocytes, and NK-cells generated from the blood of healthy donors (n = 3). Then, we examined the relationship between the whole-blood expression levels of the ten genes and white blood cell counts in a cohort of acute ischemic stroke patients (n = 36) and acute stroke mimics (n = 15) recruited at emergency department admission. All ten genes displayed strong patterns of lineage-specific expression in our analysis of isolated leukocytes, and their whole-blood expression levels were correlated with white blood cell differential across the total patient population, suggesting that many of them are likely differentially expressed in whole blood during stroke as an artifact of stroke-induced shifts in leukocyte counts. Specifically, factor analysis inferred that over 50% of the collective variance in their whole-blood expression levels across the patient population was driven by underlying variance in white blood cell counts alone. However, the cumulative expression levels of the ten genes displayed a superior ability to discriminate between stroke patients and stroke mimics relative to white blood cell differential, suggesting that additional less prominent factors influence their expression levels which add to their diagnostic utility. These findings not only provide insight regarding this particular panel of ten genes, but also into the results of prior stroke transcriptomics studies performed in whole blood.

Our group recently identified 16 genes whose peripheral blood expression levels are differentially regulated in acute ischemic stroke. The purpose of this study was to determine whether the early expression levels of any of these 16 genes are predictive for post-stroke blood brain barrier (BBB) disruption. Transcriptional expression levels of candidate genes were measured in peripheral blood sampled from ischemic stroke patients at emergency department admission, and BBB permeability was assessed at 24 hour follow up via perfusion-weighted imaging. Early heightened expression levels of AKAP7, a gene encoding a protein kinase A-binding scaffolding molecule, were significantly associated with BBB disruption 24 hours post-hospital admission. We then determined that AKAP7 is predominantly expressed by lymphocytes in peripheral blood, and strongly co-expressed with ITGA3, a gene encoding the adhesion molecule integrin alpha 3. Subsequent in vitro experiments revealed that heightened expression of AKAP7 and ITGA3 in primary human lymphocytes is associated with a highly adherent phenotype. Collectively, our results suggest that AKAP7 expression levels may have clinical utility as a prognostic biomarker for post-stroke BBB complications, and are likely elevated early in patients who later develop post-stroke BBB disruption due to the presence of an invasive lymphocyte population in the peripheral blood.

Objective: The identification of stroke-associated biomarkers represents a means by which prehospital triage could be expedited to increase the probability of successful intervention. Thus, the objective of this work was to use high-throughput transcriptomics in combination with basic machine learning techniques to identify a pattern of gene expression in peripheral whole blood which could be used to identify acute ischemic stroke (AIS) in the acute care setting. Methods: A two-stage study design was used which included a discovery cohort and an independent validation cohort. In the discovery cohort, peripheral whole blood samples were obtained from 39 AIS patients upon emergency department admission, and from 24 neurologically asymptomatic controls. Microarray was used to measure the expression of over 22,000 genes and a pattern recognition technique known as genetic algorithm k-nearest neighbors (GA/kNN) identified a pattern of gene expression that optimally discriminated between AIS and controls. In an independent validation cohort, the gene expression pattern was tested for its ability to discriminate between 39 AIS patients and each of two different control groups, one consisting of 30 neurologically asymptomatic controls, and the other consisting of 15 stroke mimics, with gene expression levels being assessed by qRT-PCR. Results: In the discovery cohort, GA/kNN identified ten transcripts (ANTXR2, STK3, PDK4, CD163, MAL, GRAP, ID3, CTSZ, KIF1B, and PLXDC2) whose coordinate pattern of expression correctly identified 98.4% of subjects (97.4% sensitive, 100% specific). In the validation cohort, the same 10 transcripts correctly identified 95.6% of subjects when comparing AIS patients to asymptomatic controls (92.3% sensitive, 100% specific), and 96.3% of subjects when comparing AIS patients to stroke mimics (97.4% specific, 93.3% sensitive). Conclusion: These results demonstrate that a highly accurate RNA-based companion diagnostic for AIS is plausible using a relatively small number of markers. The pattern of gene expression identified in this study shows strong diagnostic potential, and warrants further evaluation to determine true clinical efficacy.

Early and accurate diagnosis of stroke improves the probability of positive outcome. The objective of this study was to identify a pattern of gene expression in peripheral blood that could potentially be optimised to expedite the diagnosis of acute ischaemic stroke (AIS). A discovery cohort was recruited consisting of 39 AIS patients and 24 neurologically asymptomatic controls. Peripheral blood was sampled at emergency department admission, and genome-wide expression profiling was performed via microarray. A machine-learning technique known as genetic algorithm k-nearest neighbours (GA/kNN) was then used to identify a pattern of gene expression that could optimally discriminate between groups. This pattern of expression was then assessed via qRT-PCR in an independent validation cohort, where it was evaluated for its ability to discriminate between an additional 39 AIS patients and 30 neurologically asymptomatic controls, as well as 20 acute stroke mimics. GA/kNN identified 10 genes (ANTXR2, STK3, PDK4, CD163, MAL, GRAP, ID3, CTSZ, KIF1B and PLXDC2) whose coordinate pattern of expression was able to identify 98.4% of discovery cohort subjects correctly (97.4% sensitive, 100% specific). In the validation cohort, the expression levels of the same 10 genes were able to identify 95.6% of subjects correctly when comparing AIS patients to asymptomatic controls (92.3% sensitive, 100% specific), and 94.9% of subjects correctly when comparing AIS patients with stroke mimics (97.4% sensitive, 90.0% specific). The transcriptional pattern identified in this study shows strong diagnostic potential, and warrants further evaluation to determine its true clinical efficacy.