Umbilical cord as an analytical matrix – A technical note

Journal Pre-proof Umbilical cord as an analytical matrix – A technical note Hayley R. Price, Camron Chehroudi, Stuart J. Knight, Alexander D. Smith, Dickson Lai, Hugh Kim, Tricia E. Wright, Michael WH. Coughtrie, Abby C. Collier PII:

S0143-4004(19)30712-X

DOI:

https://doi.org/10.1016/j.placenta.2019.12.001

Reference:

YPLAC 4060

To appear in:

Placenta

Received Date: 3 September 2019 Revised Date:

26 November 2019

Accepted Date: 2 December 2019

Please cite this article as: Price HR, Chehroudi C, Knight SJ, Smith AD, Lai D, Kim H, Wright TE, Coughtrie MW, Collier AC, Umbilical cord as an analytical matrix – A technical note, Placenta (2020), doi: https://doi.org/10.1016/j.placenta.2019.12.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Umbilical cord as an analytical matrix – a technical note.

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Hayley R. Pricea, Camron Chehroudib,c,d, Stuart J Knighta, Alexander D. Smitha, Dickson Laia,

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Hugh Kimb,c,d, Tricia E. Wrighte, Michael WH Coughtriea and Abby C. Colliera*

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a

Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Wesbrook Mall, Vancouver, BC V6T 1Z3, CANADA

b

Centre for Blood Research, 2350 Health Sciences Mall, The University of British Columbia, Vancouver, BC V6T 1Z3, CANADA

c

Faculty of Dentistry, 2199 Wesbrook Mall, The University of British Columbia, Vancouver, BC V6T 1Z3, CANADA

d

Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC V6T 1Z3, CANADA e

Department of Obstetrics, Gynecology, and Reproductive Sciences; University of California, San Francisco, San Francisco, CA 94115, USA

*

Corresponding author: Abby C. Collier, Ph.D. Faculty of Pharmaceutical Sciences The University of British Columbia 2405 Wesbrook Mall Vancouver, BC V6T 1Z3 CANADA Phone: +1-604-827-2380 Fax: +1-604-822-3035 e-mail: [email protected]

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Abstract

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The umbilical cord (UC) connects the fetal blood supply to the placenta, so is exposed to all

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systemic endo- and xenobiotics. We have extensive experience using UC as an analytical matrix for

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detecting and/or quantitating drugs, chemicals and endogenous compounds. This technical note

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describes advantages (large amount available, ease of collection, small sample needed for use, rapid

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availability) and challenges (clinical relationships, processing difficulties, matrix effects on analytes

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and detection technologies) of UC as an analytical matrix in ELISA and LC/MS platforms, and

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provides guidance for successfully working with this tissue.

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Introduction

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The use of human umbilical cord (UC) for post-partum screening has gained popularity

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recently as this tissue is exposed to all fetal compounds [1-9]. The UC is attractive because it is

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normally discarded so considered “waste” tissue. UCs also provide a number of options for study:

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fetal blood collection, human umbilical vein endothelial cell (HUVEC) isolation, whole tissue

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paraffin embedding or flash-freezing for histology, and tissue lysing for use in assays. Based on 10

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years of experience with UC [2, 4, 5, 9-12], we describe methodological challenges for their use,

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and strategies for success.

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Methods

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Collection and Processing

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UC are collected as soon as possible after birth by clamping and cutting, preferably a 10 –

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15 cm portion, and washing in buffer (Ringer’s solution, phosphate-buffered saline, others). Cord

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blood is collected by clamping one end of the cord and rolling towards the opposite end, or

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extracting directly from the cord. The umbilical arteries contain blood coming directly from the

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fetus and is more difficult to collect given smaller volumes. The umbilical vein contains blood

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returning to the fetal circulation after exchange with the maternal blood in the intervillous space.

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Pieces of UC can be placed in paraformaldehyde or frozen in OCT medium for

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histopathological examination. To study UC tissues biochemically or detect proteins and chemicals,

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UC should be processed fresh within a few hours of collection, or snap-frozen in liquid nitrogen and

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stored at -80°C until processing. Thawed or fresh tissue pieces are homogenized in buffer

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containing protease inhibitor to yield lysate. Lysates can be further processed to subcellular

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fractions as required for the molecule(s) of interest. These preparations can be used fresh or frozen

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at -80°C until use.

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Detection of Analytes The most common detection methods are ELISA and LC-MS, but GC/MS, HPLC, and

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spectrophotometric/fluorimetric methods have been reported [13-19]. This technical note refers to

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best practice for ELISA and LC/MS only.

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Results and Discussion

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Collection and processing

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UC can be collected non-invasively with consent prior to birth, allowing rapid collection and

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processing. There are fewer ethical concerns with UC compared to drawing blood or collecting

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neonatal urine, although likely similar to collecting hair or meconium [20]. Challenges to collection

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include access to patients for consent, access to delivery suites for collection, and co-operation of

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clinicians and staff. Relationships with clinical colleagues must be built and maintained,

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recognizing that the health and wellbeing of the mother and child are paramount and that collection

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for research is a secondary consideration. Respectful discussions around access and integrated

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procedures for patient consent, and tissue collection that minimally interferes with clinical

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procedures, are required. Transport and storage of tissue is also of concern: after 8 hr post-delivery

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tissue should be discarded due to likely poor quality. Over-fixing in paraformaldehyde (more than

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~24 hours) should be avoided as it makes antibody-based detection difficult and tissues brittle [12].

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When processing to lysates, the ratio 1:3 (w:v) tissue:buffer works best in our hands and

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typically 1g or less (0.4”, 1 cm) of cord is required for analytical or screening studies. Producing

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UC lysates is more difficult than other tissues as UCs are more resistant to mechanical disruption

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than organs such as placenta or liver. Ideally, lysates should be prepared fresh and used

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immediately, but if this is not possible snap freezing in liquid nitrogen and storage at -80 °C is best

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practice to keep molecules and tissues stable. The effects of freeze-thaw and time in storage on

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tissue and analyte stability should be tested [21, 22].

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Detection and quantitation of molecules in UC with ELISA. For ELISA, validation studies are required to determine whether absolute or relative

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quantification can be achieved. In general, if samples spiked with known concentrations of pure

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standards yield the same concentration when assayed, absolute quantitation is possible. In practice

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when using umbilical cords (a solid tissue) it is the lysates that are spiked prior to detection with

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ELISA. If detected concentration is different than the spiked concentration, then only relative

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quantitation is possible. Relative quantitation is valid for case-control studies to show differences

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but is not generalizable to the population. In UC we have achieved absolute quantitation for cotinine

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and methamphetamine [2], relative quantitation for opioids [4], catecholamines [5], cytokines, and

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VEGFs [4, 10] and only qualitative (present/absent) data for amphetamine, cannabis

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(tetrahydrocannabinol), and cocaine where quantifiable standards were not available [2].

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Single analyte and multiplex commercial ELISAs are available, and differ in terms of use,

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cost, and data generated. Single analyte ELISA can be developed in-house or purchased

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commercially for approximately US$120 - $450 per plate. Costs per plate for multiplex platforms

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are expensive, around US$1000/plate, but generate substantially more data. On a per analyte basis

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multiplex may be equivalent to, or cheaper than, traditional ELISA if specific plate reading

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technology is available and large cohorts are analyzed. Both platforms are comparable in terms of

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ease of use, assay time and validation.

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Detection and quantitation of molecules in UC with LC/MS.

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Umbilical cord lysate requires extensive extraction for use on LC/MS systems. Our

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laboratory has successfully used a liquid-liquid extraction using methyl tert-butyl ether to detect

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NSAIDs [9] while others have used solid phase extraction, which may yield better results [6-8, 23,

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24].

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Challenges using UC lysate for LC/MS screening, including matrix effects, are common, so

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standard curves must usually be prepared in blank UC lysate. Blank lysate for many endogenous

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molecules is impossible to collect or generate, although for some endo- and xenobiotics stripping

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(charcoal or dextran) can produce a blank for standards. Additionally, the UC matrix causes rapid

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deterioration of analytical columns. UC matrices also promote compound instability, either in

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solution (e.g. aceclofenac, phenylbutazone) [9]or when freeze/thawing (e.g. ibuprofen, salicylic

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acid) [9]. In our experience, all methods developed in solvent and most in plasma are more sensitive

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than UC lysate methods.

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Pharmacokinetic Considerations The window of detection of licit and illicit drugs is dependent on their biological half lives

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as well as affinity for the tissue of interest. We have successfully detected and quantitated NSAIDs

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in UC lysate, several of which have half lives of 2hr, so a terminal biological fate of ~10 hr (five

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times the half life) [9]. This means that UC can be used to detect drugs with very short half lives if

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they are ingested close to birth, and that the window of ingestion can be estimated. On the other

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hand, we have also detected tetrahydrocannabinol (THC) [2] which has a long and variable half life

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of ~1 day in infrequent users, up to 13 days in frequent users [25], meaning that ingestion could

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have occurred any time from 5 days to 65 days prior to delivery and making the window of

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ingestion impossible to estimate. Similarly, when analysing UC lysate for drug detection, positive

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results could occur when drugs are administrated during later stages of labour or delivery. If data

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regarding drug administration during delivery are not available (e.g. either not charted, or the

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medical record is not available), incorrect inferences as to maternal vs. medical drug use could be

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made.

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Moreover, estimating fetal exposure levels based on umbilical levels of compounds is

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challenging. While many drugs and environmental compounds flow bi-directionally from the

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maternal to the fetal compartment, others do not. For example, the anti-HIV drug dolutegravir

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preferentially accumulates and is trapped in placental tissue, hence placental levels of dolutegravir

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do not reflect fetal exposure [26]. Trapping in umbilical cord tissue has not, to our knowledge, been

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studied. Umbilical tissue us made up of Wharton’s jelly: mucopolysaccharides comprising

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hyaluronic acid and chondroitin sulfate. Based on this physicochemical profile, water soluble drugs

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(weak acids and bases) may well find the umbilical cord a preferential compartment, but this is not

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established. For UC tissue lysates, we assume that detection of licit and illicit drugs represents some

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level of exposure to the fetus, but may not be directly quantitative, rather only proportional.

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In summary, the UC provides a versatile tissue for screening a wide range of endogenous

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and exogenous compounds. UC are available immediately following birth, and its proximity to both

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the maternal and fetal circulations can provide insight into substance use, the fetal environment, and

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pregnancy outcomes. Although UCs present some challenges, careful study design can mitigate and

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overcome these issues.

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Acknowledgements: Hayley Price is supported by a Canadian Institutes of Health Research

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(CIHR) Drug Safety Evaluation and Cross Disciplinary Training (DSECT) Stream 1 Fellowship

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grant [DSN-143585] and a CIHR Frederick Banting and Charles Best Canada Graduate

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Scholarships - Doctoral Award [GSD-167041] and Abby Collier is supported by CIHR funding for

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SEARCH-PREVENT [FRN-158302].

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Conflict of Interest

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The authors declare no conflicts of interest.

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Highlights Umbilical cord tissue can be used for screening drugs, nutrients, environmental chemicals, and endogenous compounds. Umbilical cord is easily collected with fewer ethical issues than invasive samples (e.g. blood) but require careful collection and transport. Umbilical cords are resistant to mechanical disruption and present challenges for use in analytical technologies. With careful study design, umbilical tissues present an attractive screening modality.

Declarations of Interest The authors declare no conflicts of interest.