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Article ; Online: Ensuring Full Spectrum Flow Cytometry Data Quality for High-Dimensional Data Analysis.

Ferrer-Font, Laura / Kraker, Geoffrey / Hally, Kathryn E / Price, Kylie M

Current protocols

2023 Volume 3, Issue 2, Page(s) e657

Abstract: Full spectrum flow cytometry (FSFC) allows for the analysis of more than 40 parameters at the single-cell level. Compared to the practice of manual gating, high-dimensional data analysis can be used to fully explore single-cell datasets and reduce ... ...

Abstract	Full spectrum flow cytometry (FSFC) allows for the analysis of more than 40 parameters at the single-cell level. Compared to the practice of manual gating, high-dimensional data analysis can be used to fully explore single-cell datasets and reduce analysis time. As panel size and complexity increases so too does the detail and time required to prepare and validate the quality of the resulting data for use in downstream high-dimensional data analyses. To ensure data analysis algorithms can be used efficiently and to avoid artifacts, some important steps should be considered. These include data cleaning (such as eliminating variable signal change over time, removing cell doublets, and antibody aggregates), proper unmixing of full spectrum data, ensuring correct scale transformation, and correcting for batch effects. We have developed a methodical step-by-step protocol to prepare full spectrum high-dimensional data for use with high-dimensional data analyses, with a focus on visualizing the impact of each step of data preparation using dimensionality reduction algorithms. Application of our workflow will aid FSFC users in their efforts to apply quality control methods to their datasets for use in high-dimensional analysis, and help them to obtain valid and reproducible results. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Data cleaning Basic Protocol 2: Validating the quality of unmixing Basic Protocol 3: Data scaling Basic Protocol 4: Batch-to-batch normalization.
MeSH term(s)	Flow Cytometry/methods ; Data Accuracy ; Algorithms ; Antibodies
Chemical Substances	Antibodies
Language	English
Publishing date	2023-02-06
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.657
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Article ; Online: Neutrophil-Enriched Biomarkers and Long-Term Prognosis in Acute Coronary Syndrome: a Systematic Review and Meta-analysis.

Yiu, Jaquelina Y T / Hally, Kathryn E / Larsen, Peter D / Holley, Ana S

Journal of cardiovascular translational research

2023 Volume 17, Issue 2, Page(s) 426–447

Abstract: Activated neutrophils release a range of inflammatory products that represent potential biomarkers, and there is interest in the prognostic value of these in acute coronary syndrome (ACS) patients. We conducted a systematic review to examine neutrophil- ... ...

Abstract	Activated neutrophils release a range of inflammatory products that represent potential biomarkers, and there is interest in the prognostic value of these in acute coronary syndrome (ACS) patients. We conducted a systematic review to examine neutrophil-enriched biomarkers and the occurrence of major adverse cardiovascular events (MACE) in patients with ACS. We identified twenty-seven studies including 17,831 patients with ACS. The most studied biomarkers were neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase (MPO). Meta-analyses showed that elevated NGAL was associated with higher MACE rates (unadjusted risk ratio (RR) 1.52, 95% CI 1.12-2.06, p = 0.006) as were elevated MPO levels (unadjusted RR 1.61, 95% CI 1.22-2.13, p = 0.01). There was limited data suggesting that increased levels of calprotectin, proteinase-3 and double-stranded DNA were also associated with MACE. These results suggest that higher levels of neutrophil-enriched biomarkers may be predictive of MACE in patients with ACS, although higher-quality studies are needed to confirm these observations.
MeSH term(s)	Humans ; Acute Coronary Syndrome/blood ; Acute Coronary Syndrome/mortality ; Acute Coronary Syndrome/diagnosis ; Acute Coronary Syndrome/therapy ; Biomarkers/blood ; Neutrophils/immunology ; Prognosis ; Lipocalin-2/blood ; Peroxidase/blood ; Predictive Value of Tests ; Time Factors ; Risk Assessment ; Male ; Risk Factors ; Female ; Up-Regulation ; Neutrophil Activation ; Middle Aged ; Aged
Chemical Substances	Biomarkers ; Lipocalin-2 ; Peroxidase (EC 1.11.1.7) ; MPO protein, human (EC 1.11.1.7) ; LCN2 protein, human
Language	English
Publishing date	2023-08-18
Publishing country	United States
Document type	Systematic Review ; Meta-Analysis ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2422411-X
ISSN	1937-5395 ; 1937-5387
ISSN (online)	1937-5395
ISSN	1937-5387
DOI	10.1007/s12265-023-10425-2
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Article ; Online: OMIP 083: A 21-marker 18-color flow cytometry panel for in-depth phenotyping of human peripheral monocytes.

Hally, Kathryn E / Ferrer-Font, Laura / Pilkington, Katherine R / Larsen, Peter D

Cytometry. Part A : the journal of the International Society for Analytical Cytology

2022 Volume 101, Issue 5, Page(s) 374–379

MeSH term(s)	Color ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukocytes, Mononuclear ; Monocytes
Language	English
Publishing date	2022-03-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2099868-5
ISSN	1552-4930 ; 0196-4763 ; 1552-4922
ISSN (online)	1552-4930
ISSN	0196-4763 ; 1552-4922
DOI	10.1002/cyto.a.24545
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Article ; Online: Increased levels of low density neutrophils (LDNs) in myocardial infarction.

Yiu, Jacquelina Y T / Hally, Kathryn E / Larsen, Peter D / Holley, Ana S

Acta cardiologica

2022 Volume 78, Issue 1, Page(s) 47–54

Abstract: Background: Recent evidence suggests that neutrophils are highly plastic cells that can display heterogeneous phenotypes. Low-density neutrophils (LDNs) have been described in many inflammatory conditions, and are thought to represent an immature, ... ...

Abstract	Background: Recent evidence suggests that neutrophils are highly plastic cells that can display heterogeneous phenotypes. Low-density neutrophils (LDNs) have been described in many inflammatory conditions, and are thought to represent an immature, hyperactivated subtype of neutrophils. Neutrophils are significantly involved in the inflammatory response to myocardial infarction (MI), although we do not know the extent to which LDNs exist, or function, in MI. This study sought to determine the frequency and phenotype of LDNs in MI patients, compared to healthy subjects (HS). Methods: LDNs and normal-density neutrophils (NDNs) were isolated from the peripheral blood of MI subjects ( Results: We identified LDNs within the peripheral blood mononuclear cell (PBMC) fraction of blood, and this population is significantly enriched in MI patients (1.04 ± 0.75% of PBMCs), compared to HS (0.29 ± 0.24%, Conclusions: An increase in the frequency of hyperactivated, immature LDNs is an immunological feature of MI. We highlight a potential pathological role of LDNs in MI, which underscores the need to expand our current understanding of this subtype in MI and other cardiovascular diseases (CVDs).
MeSH term(s)	Humans ; Neutrophils/pathology ; Neutrophils/physiology ; Leukocytes, Mononuclear ; Myocardial Infarction/diagnosis
Language	English
Publishing date	2022-01-10
Publishing country	England
Document type	Journal Article
ZDB-ID	390197-x
ISSN	1784-973X ; 0001-5385
ISSN (online)	1784-973X
ISSN	0001-5385
DOI	10.1080/00015385.2021.2015145
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Article ; Online: Immunoglobulin G levels predicts risk of recurrent adverse cardiovascular events in myocardial infarction patients.

Hally, Kathryn E / Holley, Ana S / Kristono, Gisela A / Harding, Scott A / Larsen, Peter D

Acta cardiologica

2019 Volume 75, Issue 6, Page(s) 497–502

Abstract: Background: ...

Abstract	Background:
MeSH term(s)	Aged ; Biomarkers/blood ; C-Reactive Protein/metabolism ; Female ; Follow-Up Studies ; Humans ; Immunoglobulin G/blood ; Incidence ; Male ; Myocardial Infarction/blood ; Myocardial Infarction/epidemiology ; Myocardial Infarction/prevention & control ; New Zealand/epidemiology ; Prognosis ; Recurrence ; Retrospective Studies ; Risk Assessment/methods ; Risk Factors ; Secondary Prevention/methods
Chemical Substances	Biomarkers ; Immunoglobulin G ; C-Reactive Protein (9007-41-4)
Language	English
Publishing date	2019-05-10
Publishing country	England
Document type	Journal Article
ZDB-ID	390197-x
ISSN	1784-973X ; 0001-5385
ISSN (online)	1784-973X
ISSN	0001-5385
DOI	10.1080/00015385.2019.1611189
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Article ; Online: A combined biomarker approach for characterising extracellular matrix profiles in acute myocardial infarction.

Brunton-O'Sullivan, Morgane M / Holley, Ana S / Hally, Kathryn E / Kristono, Gisela A / Harding, Scott A / Larsen, Peter D

Scientific reports

2021 Volume 11, Issue 1, Page(s) 12705

Abstract: Extracellular matrix (ECM) biomarkers are useful for measuring underlying molecular activity associated with cardiac repair following acute myocardial infarction (AMI). The aim of this study was to conduct exploratory factor analysis (EFA) to examine the ...

Abstract	Extracellular matrix (ECM) biomarkers are useful for measuring underlying molecular activity associated with cardiac repair following acute myocardial infarction (AMI). The aim of this study was to conduct exploratory factor analysis (EFA) to examine the interrelationships between ECM biomarkers, and cluster analysis to identify if distinct ECM profiles could distinguish patient risk in AMI. Ten ECM biomarkers were measured from plasma in 140 AMI patients: MMP-2, -3, -8, -9, periostin, procollagen I N-Terminal propeptide, osteopontin, TGF-β1, TIMP-1 and -4. EFA grouped eight ECM biomarkers into a two-factor solution, which comprised three biomarkers in Factor 1 and five biomarkers in Factor 2. Notably, ECM biomarkers were not separated based on biological function. Cluster analysis grouped AMI patients into three distinct clusters. Cluster One (n = 54) had increased levels of MMP-8, MMP-9, and TGF-B1. Cluster Two (n = 43) had elevated levels of MMP-2, MMP-3, osteopontin, periostin and TIMP-1, and increased high-sensitivity troponin T and GRACE scores. Cluster Three (n = 43) had decreased levels of ECM biomarkers. Circulating ECM biomarkers demonstrated collinearity and entwined biological functions based on EFA analysis. Using cluster analysis, patients with similar clinical presentations could be separated into distinct ECM profiles that were associated with differential patient risk. Clinical significance remains to be determined.
MeSH term(s)	Biomarkers/blood ; Cell Adhesion Molecules/blood ; Extracellular Matrix/metabolism ; Female ; Heart Disease Risk Factors ; Humans ; Male ; Matrix Metalloproteinases/blood ; Middle Aged ; Myocardial Infarction/blood ; Myocardial Infarction/metabolism ; Osteopontin/blood ; Peptide Fragments/blood ; Procollagen/blood ; Tissue Inhibitor of Metalloproteinases/blood ; Transforming Growth Factor beta1/blood
Chemical Substances	Biomarkers ; Cell Adhesion Molecules ; POSTN protein, human ; Peptide Fragments ; Procollagen ; SPP1 protein, human ; TGFB1 protein, human ; Tissue Inhibitor of Metalloproteinases ; Transforming Growth Factor beta1 ; procollagen Type I N-terminal peptide ; Osteopontin (106441-73-0) ; Matrix Metalloproteinases (EC 3.4.24.-)
Language	English
Publishing date	2021-06-16
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-021-92108-z
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Article ; Online: Linking Neutrophil Extracellular Traps and Platelet Activation: A Composite Biomarker Score for Predicting Outcomes after Acute Myocardial Infarction.

Hally, Kathryn E / Parker, Olivia M / Brunton-O'Sullivan, Morgane M / Harding, Scott A / Larsen, Peter D

Thrombosis and haemostasis

2021 Volume 121, Issue 12, Page(s) 1637–1649

Abstract: Background: Activation of both platelets and neutrophils can contribute to the risk of major adverse cardiovascular events (MACE) following acute myocardial infarction (AMI). Neutrophil extracellular traps (NETs) are an important product of the ... ...

Abstract	Background: Activation of both platelets and neutrophils can contribute to the risk of major adverse cardiovascular events (MACE) following acute myocardial infarction (AMI). Neutrophil extracellular traps (NETs) are an important product of the platelet-neutrophil axis and exaggerate vascular damage in cardiovascular disease. Additionally, activated platelets can drive NETosis and are directly linked to thromboembolic risk. Investigating the combined effect of biomarkers for NETosis and platelet activation represents a novel approach to risk prediction post-AMI. Here, we examined the utility of a composite biomarker score, inclusive of both pathways, for predicting MACE post-AMI. Methods and results: In a case-control design, 100 case patients who experienced MACE within 1 year of index admission were matched in a 1:2 ratio with control patients. Serum levels of myeloperoxidase-DNA, neutrophil elastase-DNA, and citrullinated histone H3 were assayed by enzyme-linked immunosorbent assay (ELISA) as markers of NET burden. To measure platelet activation, soluble P-selectin was assayed by ELISA in parallel. Platelet and neutrophil counts were also recorded. Composite biomarker scores, inclusive of biomarkers for NETosis and platelet activation, were assessed using multivariate regression modeling. These composite biomarker scores were independent predictors of 1-year MACE. The strongest association with MACE was observed using a composite of platelet count, soluble P-selectin, and all NET markers (odds ratio: 1.94; 1.16-3.25). Conclusion: Here, we demonstrate the importance of combining biomarkers of NETosis and platelet activation for risk prediction in patients with AMI. Combining biomarkers from closely linked, but distinct, biological pathways was more effective than utilizing either type of biomarker alone.
MeSH term(s)	Aged ; Biomarkers/blood ; Blood Platelets/metabolism ; Case-Control Studies ; Enzyme-Linked Immunosorbent Assay ; Extracellular Traps/metabolism ; Female ; Humans ; Leukocyte Count ; Male ; Middle Aged ; Myocardial Infarction/blood ; Myocardial Infarction/diagnosis ; Neutrophils/metabolism ; P-Selectin/blood ; Platelet Activation ; Platelet Count ; Predictive Value of Tests ; Prognosis ; Registries ; Risk Assessment ; Risk Factors ; Time Factors
Chemical Substances	Biomarkers ; P-Selectin ; SELP protein, human
Language	English
Publishing date	2021-05-13
Publishing country	Germany
Document type	Journal Article
ZDB-ID	518294-3
ISSN	2567-689X ; 0340-6245
ISSN (online)	2567-689X
ISSN	0340-6245
DOI	10.1055/s-0041-1728763
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Article ; Online: Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

Hally, Kathryn E / Bird, Georgina K / La Flamme, Anne C / Harding, Scott A / Larsen, Peter D

PloS one

2019 Volume 14, Issue 10, Page(s) e0223444

Abstract: Introduction: In addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. ... ...

Abstract	Introduction: In addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. Together, platelets and neutrophils play a role in inflammation through Toll-like receptor (TLR) expression, although we do not fully understand how platelets shape neutrophil responses to TLR stimulation. Here, we aimed to determine the extent to which platelets can modulate neutrophil function in response to in vitro stimulation with TLR4, TLR2/1, and TLR2/6 agonists. Methods: Neutrophils from 10 healthy individuals were cultured alone or with autologous platelets. Neutrophils ± platelets were left unstimulated or were stimulated with 1 or 100 ng/mL lipopolysaccharide (LPS; a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist) and fibroblast-stimulating lipopeptide (FSL)-1 (a TLR2/6 agonist). Neutrophil activation and phagocytic activity were assessed by flow cytometry, and elastase and interleukin-8 secretion were assessed by ELISA. Results: The addition of platelets attenuated neutrophil CD66b and CD11b expression in response to various doses of Pam3CSK4 and FSL-1. Furthermore, platelet co-culture was associated with higher CD62L expression (indicating reduced CD62L shedding) in response to these TLR agonists. Platelets also reduced elastase secretion in unstimulated cultures and in response to low-dose TLR stimulation. Conversely, platelet co-culture increased neutrophil phagocytosis in unstimulated cultures and in response to low-dose Pam3CSK4 and FSL-1. Platelets also increased IL-8 secretion in response to low-dose LPS. Conclusion: Platelets are complex immunomodulators that can attenuate some, and simultaneously augment other, neutrophil functions. This modulation can occur both in the absence and presence of TLR stimulation.
MeSH term(s)	Biomarkers ; Blood Platelets/metabolism ; Cytokines/metabolism ; Flow Cytometry ; Humans ; Immunophenotyping ; Interleukin-8/metabolism ; Male ; Neutrophil Activation/immunology ; Neutrophils/immunology ; Neutrophils/metabolism ; Phagocytosis/immunology ; Toll-Like Receptors/agonists ; Toll-Like Receptors/metabolism ; Young Adult
Chemical Substances	Biomarkers ; CXCL8 protein, human ; Cytokines ; Interleukin-8 ; Toll-Like Receptors
Language	English
Publishing date	2019-10-03
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0223444
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Article ; Online: A combined biomarker approach for characterising extracellular matrix profiles in acute myocardial infarction

Morgane M. Brunton-O’Sullivan / Ana S. Holley / Kathryn E. Hally / Gisela A. Kristono / Scott A. Harding / Peter D. Larsen

Scientific Reports, Vol 11, Iss 1, Pp 1-

2021 Volume 12

Abstract: Abstract Extracellular matrix (ECM) biomarkers are useful for measuring underlying molecular activity associated with cardiac repair following acute myocardial infarction (AMI). The aim of this study was to conduct exploratory factor analysis (EFA) to ... ...

Abstract	Abstract Extracellular matrix (ECM) biomarkers are useful for measuring underlying molecular activity associated with cardiac repair following acute myocardial infarction (AMI). The aim of this study was to conduct exploratory factor analysis (EFA) to examine the interrelationships between ECM biomarkers, and cluster analysis to identify if distinct ECM profiles could distinguish patient risk in AMI. Ten ECM biomarkers were measured from plasma in 140 AMI patients: MMP-2, -3, -8, -9, periostin, procollagen I N-Terminal propeptide, osteopontin, TGF-β1, TIMP-1 and -4. EFA grouped eight ECM biomarkers into a two-factor solution, which comprised three biomarkers in Factor 1 and five biomarkers in Factor 2. Notably, ECM biomarkers were not separated based on biological function. Cluster analysis grouped AMI patients into three distinct clusters. Cluster One (n = 54) had increased levels of MMP-8, MMP-9, and TGF-B1. Cluster Two (n = 43) had elevated levels of MMP-2, MMP-3, osteopontin, periostin and TIMP-1, and increased high-sensitivity troponin T and GRACE scores. Cluster Three (n = 43) had decreased levels of ECM biomarkers. Circulating ECM biomarkers demonstrated collinearity and entwined biological functions based on EFA analysis. Using cluster analysis, patients with similar clinical presentations could be separated into distinct ECM profiles that were associated with differential patient risk. Clinical significance remains to be determined.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: An IL-6-IL-8 score derived from principal component analysis is predictive of adverse outcome in acute myocardial infarction.

Kristono, Gisela A / Holley, Ana S / Hally, Kathryn E / Brunton-O'Sullivan, Morgane M / Shi, Bijia / Harding, Scott A / Larsen, Peter D

Cytokine: X

2020 Volume 2, Issue 4, Page(s) 100037

Abstract: Introduction: Many studies have shown that elevated biomarkers of inflammation following acute myocardial infarction (AMI) are associated with major adverse cardiovascular events (MACE). However, the optimal way of measuring the complex inflammatory ... ...

Abstract	Introduction: Many studies have shown that elevated biomarkers of inflammation following acute myocardial infarction (AMI) are associated with major adverse cardiovascular events (MACE). However, the optimal way of measuring the complex inflammatory response following AMI has not been determined. In this study we explore the use of principal component analysis (PCA) utilising multiple inflammatory cytokines to generate a combined cytokine score that may be predictive of MACE post-AMI. Methods: Thirteen inflammatory cytokines were measured in plasma of 317 AMI patients, drawn 48-72 h following symptom onset. Patients were followed-up for one year to determine the incidence of MACE. PCA was used to generate a combined score using six cytokines that were detectable in the majority of patients (IL-1β, -6, -8, and -10; MCP-1; and RANTES), and using a subset of cytokines that were associated with MACE on univariate analysis. Multivariate models using baseline characteristics, elevated individual cytokines and PCA-derived scores determined independent predictors of MACE. Results: IL-6 and IL-8 were significantly associated with MACE on univariate analysis and were combined using PCA into an IL-6-IL-8 score. The combined cytokine score and IL-6-IL-8 PCA-derived score were both significantly associated with MACE on univariate analysis. In multivariate models IL-6-IL-8 scores (OR = 2.77, p = 0.007) and IL-6 levels (OR = 2.18, p = 0.035) were found to be independent predictors of MACE. Conclusion: An IL-6-IL-8 score derived from PCA was found to independently predict MACE at one year and was a stronger predictor than any individual cytokine, which suggests this may be an appropriate strategy to quantify inflammation post-AMI. Further investigation is required to determine the optimal set of cytokines to measure in this context.
Language	English
Publishing date	2020-10-08
Publishing country	Netherlands
Document type	Journal Article
ISSN	2590-1532
ISSN (online)	2590-1532
DOI	10.1016/j.cytox.2020.100037
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