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Article ; Online: Fabrication of Multilayer Microfluidic Arrays for Passive, Efficient DNA Trapping and Profiling.

O'Keefe, Christine M / Wang, Tza-Huei Jeff

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2679, Page(s) 315–322

Abstract: Trace amounts of cell-free DNA containing cancer-specific biomarkers can be found in blood plasma. Detection of these biomarkers holds tremendous potential for applications such as noninvasive cancer diagnostics and therapeutic monitoring. However, such ... ...

Abstract	Trace amounts of cell-free DNA containing cancer-specific biomarkers can be found in blood plasma. Detection of these biomarkers holds tremendous potential for applications such as noninvasive cancer diagnostics and therapeutic monitoring. However, such DNA molecules are extremely rare, and a typical patient blood sample may only contain a few copies. Here we describe the fabrication and operation of a microfluidic device to efficiently trap single DNA molecules into chambers for detection of tumor-specific biomarkers through a passive, geometric manipulation strategy.
MeSH term(s)	Humans ; Microfluidics ; DNA ; Biomarkers, Tumor ; Lab-On-A-Chip Devices ; Neoplasms ; Microfluidic Analytical Techniques
Chemical Substances	DNA (9007-49-2) ; Biomarkers, Tumor
Language	English
Publishing date	2023-06-10
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3271-0_22
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Article ; Online: Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

Zhao, Yang / O'Keefe, Christine M / Hsieh, Kuangwen / Cope, Leslie / Joyce, Sonali C / Pisanic, Thomas R / Herman, James G / Wang, Tza-Huei

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

2023 Volume 10, Issue 16, Page(s) e2206518

Abstract: There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis ... ...

Abstract	There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.
MeSH term(s)	Humans ; Lung Neoplasms/diagnosis ; Lung Neoplasms/genetics ; Carcinoma, Non-Small-Cell Lung/diagnosis ; Carcinoma, Non-Small-Cell Lung/genetics ; Early Detection of Cancer ; DNA Methylation/genetics ; Polymerase Chain Reaction
Language	English
Publishing date	2023-04-11
Publishing country	Germany
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2808093-2
ISSN	2198-3844 ; 2198-3844
ISSN (online)	2198-3844
ISSN	2198-3844
DOI	10.1002/advs.202206518
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Article ; Online: Highly Efficient Real-Time Droplet Analysis Platform for High-Throughput Interrogation of DNA Sequences by Melt.

O'Keefe, Christine M / Kaushik, Aniruddha M / Wang, Tza-Huei

Analytical chemistry

2019 Volume 91, Issue 17, Page(s) 11275–11282

Abstract: Droplet microfluidic platforms have greatly enhanced the throughput and sensitivity of single-molecule and single-cell analyses. However, real-time analyses of individual droplets remain challenging. Most droplet microfluidic platforms have fundamental ... ...

Abstract	Droplet microfluidic platforms have greatly enhanced the throughput and sensitivity of single-molecule and single-cell analyses. However, real-time analyses of individual droplets remain challenging. Most droplet microfluidic platforms have fundamental drawbacks that undermine their utility toward applications that rely on real-time monitoring to identify rare variants, such as bacterial persistence, drug discovery, antibody production, epigenetic biomarker analyses, etc. We present a platform for high-density droplet trapping and real-time analysis with 100% loading and trapping efficiency at a packing density of 110,000 droplets per in
MeSH term(s)	Cysteine Dioxygenase/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Microfluidic Analytical Techniques ; Particle Size ; Polymerase Chain Reaction ; Surface Properties ; Time Factors ; Transition Temperature
Chemical Substances	CDO1 protein, human (EC 1.13.11.20) ; Cysteine Dioxygenase (EC 1.13.11.20)
Language	English
Publishing date	2019-08-19
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.9b02346
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Article ; Online: Assessing Pediatric Cephalosporin Allergic Reactions Through Direct Graded Oral Challenges.

Sillcox, Carly / Gabrielli, Sofianne / O'Keefe, Andrew / McCusker, Christine / Abrams, Elissa M / Eiwegger, Thomas / Atkinson, Adelle / Kim, Vy / Copaescu, Ana-Maria / Ben-Shoshan, Moshe

The journal of allergy and clinical immunology. In practice

2023 Volume 12, Issue 1, Page(s) 156–164.e4

Abstract: Background: Cephalosporins, β-lactam antibiotics, commonly cause allergic reactions.: Objective: To assess the clinical characteristics and management of pediatric patients with suspected cephalosporin allergy using direct graded oral challenges ( ... ...

Abstract	Background: Cephalosporins, β-lactam antibiotics, commonly cause allergic reactions. Objective: To assess the clinical characteristics and management of pediatric patients with suspected cephalosporin allergy using direct graded oral challenges (GOCs). Methods: Children referred for suspected cephalosporin allergy at 4 Canadian clinics were recruited over 10 years. Data on demographics, clinical reaction characteristics, and management were collected through a questionnaire. Patients underwent a direct GOC (initially 10% of the treatment dose, then 90% after 20 min), and reactions were monitored 1 week postchallenge. Families were contacted annually for up to 5 years to detect subsequent antibiotic reactions. Logistic regression analysis identified factors associated with positive GOC reactions. Results: Among the 136 patients reporting cephalosporin allergy, 75 (55.1%) were males with a median age of 3.9 years (interquartile range 2.3-8.7). Cefprozil represented the most common cephalosporin linked to the index reaction (67.6% of cases). Of the 136 direct GOCs, 5.1% had an immediate and 4.4% a nonimmediate reaction, respectively. Positive GOCs conducted in children with a history of skin-limited nonsevere rashes were classified as mild, benign skin rashes. Positive GOCs were more likely in children with food allergies (adjusted odds ratio 1.14; 95% confidence interval [95% CI] 1.00-1.29). Conclusions: Direct GOCs are safe and effective for diagnosing pediatric cases that report nonvesicular skin-limited symptoms while being treated with cephalosporins.
MeSH term(s)	Male ; Humans ; Child ; Child, Preschool ; Female ; Cephalosporins/adverse effects ; Skin Tests/adverse effects ; Canada/epidemiology ; Anti-Bacterial Agents/adverse effects ; Drug Hypersensitivity/diagnosis ; Drug Hypersensitivity/epidemiology ; Drug Hypersensitivity/etiology ; Monobactams ; Hypersensitivity/complications ; Penicillins/adverse effects
Chemical Substances	Cephalosporins ; Anti-Bacterial Agents ; Monobactams ; Penicillins
Language	English
Publishing date	2023-10-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2843237-X
ISSN	2213-2201 ; 2213-2198
ISSN (online)	2213-2201
ISSN	2213-2198
DOI	10.1016/j.jaip.2023.10.009
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Article: Highly Efficient Real-Time Droplet Analysis Platform for High-Throughput Interrogation of DNA Sequences by Melt

O’Keefe, Christine M / Kaushik, Aniruddha M / Wang, Tza-Huei

Analytical chemistry. 2019 July 29, v. 91, no. 17

2019

Abstract	Droplet microfluidic platforms have greatly enhanced the throughput and sensitivity of single-molecule and single-cell analyses. However, real-time analyses of individual droplets remain challenging. Most droplet microfluidic platforms have fundamental drawbacks that undermine their utility toward applications that rely on real-time monitoring to identify rare variants, such as bacterial persistence, drug discovery, antibody production, epigenetic biomarker analyses, etc. We present a platform for high-density droplet trapping and real-time analysis with 100% loading and trapping efficiency at a packing density of 110,000 droplets per in². To demonstrate real-time analysis capabilities, we perform digital PCR and parallelized digital high-resolution melt curve acquisition on droplets to discriminate methylation levels of a tumor suppressor gene, CDO1, on a molecule-by-molecule basis. We hope that this platform, which is compatible with a large range of droplet sizes and generation technologies, may facilitate high-throughput real-time analyses on a molecule-by-molecule or cell-by-cell basis of heterogeneous populations.
Keywords	DNA ; antibody formation ; biomarkers ; droplets ; drugs ; epigenetics ; melting ; methylation ; monitoring ; nucleotide sequences ; polymerase chain reaction ; tumor suppressor genes
Language	English
Dates of publication	2019-0729
Size	p. 11275-11282.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.9b02346
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Clinical characteristics and management of pediatric egg-induced anaphylaxis: A cross-sectional study.

Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology

2024

Abstract: Background: Egg is the third most common food allergy in children; however, data on pediatric egg-induced anaphylaxis are sparse.: Objective: To describe the clinical characteristics, management, and outcomes of pediatric egg-induced anaphylaxis.: ... ...

Abstract	Background: Egg is the third most common food allergy in children; however, data on pediatric egg-induced anaphylaxis are sparse. Objective: To describe the clinical characteristics, management, and outcomes of pediatric egg-induced anaphylaxis. Methods: Children presenting with anaphylaxis were recruited from 13 emergency departments as part of the Cross-Canada Anaphylaxis Registry, from which data on anaphylaxis triggered by egg were extracted. Multivariate logistic regression was used to determine factors associated with prehospital epinephrine autoinjector (EAI) use and to compare anaphylaxis triggered by egg with other triggers of food-induced anaphylaxis (FIA). Results: We recruited 302 children with egg-induced anaphylaxis. The mean age was 2.6 years (SD = 3.6), and 55.3% were male. Only 39.4% had previously been diagnosed with an egg allergy. Prehospital EAI use was 32.1%, but this was not significantly lower than in other triggers of FIA (P = .26). Only 1.4% of patients required hospital admission. Relative to other triggers of FIA, patients with egg-induced anaphylaxis were significantly younger (P < .001) and exhibited more vomiting (P = .0053) and less throat tightness (P = .0015) and angioedema (P < .001). Conclusion: To the best of our knowledge, this is the largest published cohort of pediatric egg-induced anaphylaxis. In this cohort, prehospital EAI use was very low. In addition, we identified certain symptoms that distinguish egg-induced from other triggers of FIA. Taken together, high suspicion is crucial in identifying egg-induced anaphylaxis, given the younger patient demographic and frequent lack of FIA history.
Language	English
Publishing date	2024-03-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	1228189-x
ISSN	1534-4436 ; 0003-4738 ; 1081-1206
ISSN (online)	1534-4436
ISSN	0003-4738 ; 1081-1206
DOI	10.1016/j.anai.2024.03.008
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Article ; Online: Nanoarray Digital Polymerase Chain Reaction with High-Resolution Melt for Enabling Broad Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Test.

Athamanolap, Pornpat / Hsieh, Kuangwen / O'Keefe, Christine M / Zhang, Ye / Yang, Samuel / Wang, Tza-Huei

Analytical chemistry

2019 Volume 91, Issue 20, Page(s) 12784–12792

Abstract: Toward combating infectious diseases caused by pathogenic bacteria, there remains an unmet need for diagnostic tools that can broadly identify the causative bacteria and determine their antimicrobial susceptibilities from complex and even polymicrobial ... ...

Abstract	Toward combating infectious diseases caused by pathogenic bacteria, there remains an unmet need for diagnostic tools that can broadly identify the causative bacteria and determine their antimicrobial susceptibilities from complex and even polymicrobial samples in a timely manner. To address this need, a microfluidic and machine-learning-based platform that performs broad bacteria identification (ID) and rapid yet reliable antimicrobial susceptibility testing (AST) is developed. Specifically, this platform builds on "pheno-molecular AST", a strategy that transforms nucleic acid amplification tests (NAATs) into phenotypic AST through quantitative detection of bacterial genomic replication, and utilizes digital polymerase chain reaction (PCR) and digital high-resolution melt (HRM) to quantify and identify bacterial DNA molecules. Bacterial species are identified using integrated experiment-machine learning algorithm via HRM profiles. Digital DNA quantification allows for rapid growth measurement that reflects susceptibility profiles of each bacterial species within only 30 min of antibiotic exposure. As a demonstration, multiple bacterial species and their susceptibility profiles in a spiked-in polymicrobial urine specimen were correctly identified with a total turnaround time of ∼4 h. With further development and clinical validation, this platform holds the potential for improving clinical diagnostics and enabling targeted antibiotic treatments.
MeSH term(s)	Anti-Bacterial Agents/pharmacology ; Bacteria/drug effects ; Bacteria/genetics ; Bacteria/isolation & purification ; DNA, Bacterial/genetics ; DNA, Bacterial/metabolism ; Machine Learning ; Microarray Analysis ; Microbial Sensitivity Tests/methods ; Nanotechnology ; Phenotype ; Polymerase Chain Reaction
Chemical Substances	Anti-Bacterial Agents ; DNA, Bacterial
Language	English
Publishing date	2019-09-26
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.9b02344
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Article: Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Khire, Tejas Suresh / Gao, Wei / Bales, Brian / Hsieh, Kuangwen / Grossmann, Greg / Park, Dong Jin M / O'Keefe, Christine / Brown-Countess, Arnyah / Peterson, Sara / Chen, Fan-En / Lenigk, Ralf / Trick, Alex / Wang, Tza-Huei / Puleo, Christopher

Antibiotics (Basel, Switzerland)

2023 Volume 12, Issue 11

Abstract: Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated ... ...

Abstract	Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).
Language	English
Publishing date	2023-11-19
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2681345-2
ISSN	2079-6382
ISSN	2079-6382
DOI	10.3390/antibiotics12111641
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Article ; Online: Multiplex Digital Methylation‐Specific PCR for Noninvasive Screening of Lung Cancer

Yang Zhao / Christine M. O'Keefe / Kuangwen Hsieh / Leslie Cope / Sonali C. Joyce / Thomas R. Pisanic / James G. Herman / Tza‐Huei Wang

Advanced Science, Vol 10, Iss 16, Pp n/a-n/a (2023)

2023

Abstract: Abstract There remains tremendous interest in developing liquid biopsy assays for detection of cancer‐specific alterations, such as mutations and DNA methylation, in cell‐free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy ... ...

Abstract	Abstract There remains tremendous interest in developing liquid biopsy assays for detection of cancer‐specific alterations, such as mutations and DNA methylation, in cell‐free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer‐dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation‐specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid‐biopsy‐based detection of non‐small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low‐volume liquid biopsy specimens from patients with computed tomography (CT)‐scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early‐stage NSCLC.
Keywords	cancer diagnostics ; digital PCR ; DNA methylation ; epigenetics ; liquid biopsy ; lung cancer ; Science ; Q
Subject code	610
Language	English
Publishing date	2023-06-01T00:00:00Z
Publisher	Wiley
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Design and implementation of an Internet-Based cancer risk assessment tool: Use over 10 years.

LaRiviere, Michael J / O'Keefe, Ryan / Carpenter, Maribel / Chao, Hann-Hsiang / Amaniera, Isabella / Vachani, Carolyn / Hampshire, Margaret K / Bach, Christina / Arnold-Korzeniowski, Karen / Metz, James M / Hill-Kayser, Christine

Cancer medicine

2022 Volume 12, Issue 2, Page(s) 1744–1761

Abstract: Background: Prevention and early intervention can improve survival and quality of life across all cancers. Patient understanding of risk factors and associated actionable lifestyle changes and screening programs is not well understood by clinicians ... ...

Abstract	Background: Prevention and early intervention can improve survival and quality of life across all cancers. Patient understanding of risk factors and associated actionable lifestyle changes and screening programs is not well understood by clinicians METHODS: An Internet-based tool, Reduce My Risk, was created in 2009 and made available on oncolink.org. Users voluntarily completed a survey regarding demographics and cancer risk factors, and received information about their cancer risk RESULTS: Twenty eight thousand and one surveys were completed from 2009 to 2019. Median age was 26 years (18-101); 60% were females, 87% lived in North America, and 37% had at least a bachelor's degree. Users reported on behavioral/ modifiable risk factors: 13% were current smokers, 52% were current consumers of alcohol, and 8% of those had ≥14 drinks/week. Body mass index (BMI) was ≥30 in 19%; 74% of all surveys reported dietary risks and 36% reported infrequent exercise. Excess UV exposure was reported by 19%. Among women, 36% reported performing breast self-examinations monthly, and 50% reported receiving clinician breast examinations at least once every 3 years. Sixty seven percent of men 55-75 years reported screening prostate specific antigen testing, with 50% receiving annual digital rectal examinations. Nonmodifiable risk factors included family cancer history (64%), genetic syndrome (3%), and cancer-predisposing health conditions (26%) CONCLUSIONS: Ninety-seven percent of users reported modifiable risk factors, and 60% reported ≥4 of these risk factors. Understanding detailed characteristics of a large number of respondents has the potential to improve educational interventions to reduce cancer risk through behavioral modification and cancer screening across the general public.
MeSH term(s)	Male ; Humans ; Female ; Adult ; Quality of Life ; Risk Factors ; Neoplasms/diagnosis ; Neoplasms/epidemiology ; Neoplasms/etiology ; Diet ; Risk Assessment
Language	English
Publishing date	2022-06-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	2659751-2
ISSN	2045-7634 ; 2045-7634
ISSN (online)	2045-7634
ISSN	2045-7634
DOI	10.1002/cam4.4952
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