Chiral molecules and their interactions are critical in a variety of chemical and biological processes. Circular dichroism (CD) is the most widely used optical technique to study chirality, often performed in a solution phase. However, CD has low-efficiency on the order of 0.01–1 Consequently, there is a handedness to the molecule. The left- and right-handed molecules are non-superimposable mirror images of each other, called enantiomers. They have identical physical and chemical properties making it difficult to differentiate them. Chiral discrimination requires an interaction between two chiral systems—a chiral reagent with known handedness that can induce enantio-selectivity in the second chiral system.Chiral molecules lack 2. Homochirality plays a pivotal role in our daily life by serving as a chiral reagent for enantio-selectivity. For example, olfactory receptors in our nose, responsible for the sense of smell, interact differently with the two enantiomers of a chiral system. For instance, the right-handed enantiomer of limonene has an orange odour while the left-handed enantiomer has a lemon odour3.In nature, bio-molecules such as amino acids that make up life on earth are homochiral, where one enantiomer exists predominantly over the other due to asymmetric interactions8. CD has low-efficiency on the order of 0.01–112. Moreover, CD measurements are mostly conducted in solution phase, making it difficult to isolate the optical response of the chiral molecule from the influence of solvent-solute interactions14. Complementary techniques, such as vibrational circular dichroism and Raman optical activity15, have been used to specifically target the molecules in liquid media by probing the fundamental vibrational bands. However, their use in gas-phase studies has been limited. So, there is a continued effort to develop new gas-phase techniques to enhance chiral sensitivity; cavity ring-down polarimetry is one such example16.In practice, enantio-selectivity can be achieved using circularly polarized light (CPL) as a chiral reagent. CD is one of the most widely used chiroptical techniques. It arises due to the coupling of the electric and magnetic dipole transitions, thereby resulting in a differential absorption of left- and right-circularly polarized light17, mass spectrometry21 or a combination of both22. CD measurement with laser ionization mass spectrometry often use Resonance Enhanced Multiphoton Ionization (REMPI)27. They rely on resonant electronic absorption of one photon, which gives rise to the CD effect and is specific to the molecule, followed by ionization with additional photons of either the same or different colours. There have been very few studies on non-resonant laser ionization mass spectrometry28. Since the underlying principle of CD in laser ionization mass spectrometry is similar to the conventional CD, the effect is still considerably small for most UV–Vis transitions in organic molecules.Most gas-phase CD experiments employ highly sensitive techniques based on multiphoton ionization of chiral molecules instead of conventional absorption of CPL. Electrons or ions resulting from the light-matter interaction are detected using either imaging techniques such as photoelectron angular distribution30 has recently emerged as an effective gas-phase chiroptical technique with an efficiency of one to two orders of magnitude larger than CD. In PECD, the interaction between CPL and a chiral molecule results in asymmetric photoelectron angular dispersion that is identical but opposite in sign for the two enantiomers. PECD arises due to pure electric dipole transitions, therefore, it is highly efficient compared to conventional CD.Photoelectron circular dichroism (PECD)32. The momentum vectors of the fragments are enantio-sensitive as they are of equal magnitude but of opposite sign due to the non-symmetric behaviour of their coulomb potential. This technique is capable of imaging the absolute configuration of chiral molecules. However, it cannot be extended to complicated molecules because (1) the detection efficiency decreases exponentially for the increasing number of fragments and (2) reconstruction of the molecular structure may not be possible from the kinematic properties of the fragments.Coulomb explosion imaging is another chiroptical technique in which the momentum of each fragment is recorded when the molecule undergoes fragmentation upon rapid ionization by an intense laser pulse33: 34. For randomly orientated molecules, the laser field will preferentially ionize a subset of molecules whose molecular axes are aligned with the field.Ionization of a chiral molecule by strong IR field in the tunneling regime defined by the Keldysh parameter 35.Laser-induced propagation and acceleration of the ionized electron whose trajectory is influenced by the electric and magnetic field of the light; the latter corresponds to the non-dipole regime. The maximum energy of the recolliding electron is Inelastic recollision of the liberated electron with the parent ion leading to direct ionization or excitation of the parent ion, depending on the kinetic energy of the recolliding electron.Using limonene, 36, where coherent radiation emitted as high order harmonics of the driving field was used to differentiate the enantiomers. However, poor conversion efficiency makes cHHG technically challenging.Here we introduce a new extreme nonlinear chiroptical technique based on strong-field ionization of chiral molecules combined with mass spectrometry. Left- and right-handed elliptically polarized driving laser fields produce differential yields of doubly charged parent ion or fragment enabling chiral discrimination. The basic principle of elliptical dichroism relies on recollision physics explained by the semi-classical three-step model38. The first excited cationic state is a 41. When irradiated with intense light pulses, limonene undergoes ionization and fragmentation.Limonene consists of a cyclohexene ring with a methyl group on one end and an isopropenyl group on the other end connected to a carbon atom that serves as a chiral centre. The highest occupied molecular orbital (HOMO) is the carbon double bond (C=C) in the cyclohexene ring. Figure entclass1pt{minimaentclass1pt{minimaChiral response to elliptically polarized light, which acts as a chiral reagent, is shown in Fig. The colour maps of Fig. The chiral response is commonly expressed in terms of circular dichroism value. Similarly, elliptical dichroism (ED) can be defined as the ratio of the difference of the double ionization yield to the average double ionization yield of the enantiomers at a set ellipticity  modulation of the chiral signal mirroring the principal vibrational features observed in the photoelectron spectrum, and (b) flipping of the forward–backwards asymmetry in the angular emission of electrons relative to the photon beam direction38. The wavelength effects of the chiral signal are attributed to the excitation of vibronic states of the cation and/or intermediate electronic states of the neutral molecule.Wavelength dependence of chiral signal has been observed in chiroptical techniques such as absorption CD and PECD52. However, circularly and linearly polarized laser pulses still lead to different PECDs because of the angular dependence of the excitation process, which induces different anisotropies.Although CD and PECD involve excitation of intermediate states, since PECD arises from pure electric dipole interaction, the chiral signal is several orders of magnitude greater than the absorption CD. Currently, understanding the role of the intermediate resonances is subject of experimental and theoretical investigations. Recently it was demonstrated, by decoupling bound–bound and bound–continuum transitions, that the helicity of the light pulse that drives the bound–bound transitions has a negligible effect on the PECDIn ED-REFLMS, where the photon energy was varied from 0.51 to 0.95 eV, all three mechanisms that can contribute to the chiral signal are wavelength dependent. However, mechanism 3 alone cannot give rise to the flipping of the chiral signal with laser wavelength as it does not involve any intermediate states of the cation. In mechanism 1, when an electron is promoted into the continuum, the laser field is still intense enough to induce vibronic and multiphoton electronic excitation of the cation For example, from 1,500 to 1,750 nm where R-limonene produced more double ionization signal than S-limonene, the HOMO-2 at 10.57 eV and HOMO-3 at 11.05 eV can be accessed by 3-photon and 4-photon excitation, respectively. Similarly, from 2,150 to 2,300 nm they can be accessed 4-photon and 5-photon excitation, respectively. In the wavelength range where S-limonene produced more double ionization signal than R-limonene, HOMO-3 can be accessed by 4-photon excitation over the entire range, whereas HOMO-2 can be accessed only from 1,750 to 1,830 nm by 3-photon excitation.In mechanism 2, the parent ion is promoted to a highly excited Rydberg-like state. At recollision time, the field is minimum, decreasing the probability of multi-photon absorption. The induced transitions are, therefore, most likely from 1-photon absorption to a dense manifold of vibrational Rydberg-like cationic states. In either of the above mechanisms, laser-induced transitions access different excited higher electronic and vibronic cationic states. The change of wavelength can affect the allowed molecular orbital transitions within an enantiomer. Small variation in the orientation of the E1 and M1 dipole transitions alters the coupling term which favours certain transition channels over others directly influencing the chiral signal detected by ED-REFLMS.53. The ionization energy of limonene is The similarities between the elliptical dichroism signal for double ionization and fragmentation can be understood by considering the complex potential energy surfaces of limonene. Upon photoionization, the molecular ions can undergo fragmentation through a multitude of dissociation pathways via intermediary states where several rearrangement reactions occur prior to dissociationIn mechanism 1, recollision takes place on excited cationic states. Under normal circumstances, these excited cationic states evolve in time leading to more fragmentation. However, because the recollision dynamics occur on a short time scale of 2.5–5.5 fs , much shorter than the dissociation time, the molecule undergoes a direct transition from excited cationic state to either ground di-cationic state (double ionization) or excited di-cationic state (fragmentation). In mechanism 2, soft recollision with the parent ion leads to excitation of Rydberg like cationic states. The remnant laser field interacting with highly excited cationic states can induce chiral sensitive transitions leading to the removal of second electron or fragmentation. Coincidence measurements can shed light on the relative influence of the two mechanisms and on the origin of the fragment ion from the dissociation of excited cationic or di-cationic states.ED-REFLMS is a highly efficient technique where the chiral response can be obtained from either recollision-induced fragment or doubly charged ion Fig. . The tecIn principle, ED-REFLMS does not require any ellipticity variation. The wavelength response of the chiral signal can be obtained at a fixed ellipticity Figs. b, 5. ThiIn the ED-REFLMS technique, near-infrared femtosecond laser pulses are focused onto a randomly orientated limonene molecule Both enantiomers of limonene (from Sigma Aldrich) have sufficient vapour pressure at room temperature to be introduced into the vacuum chamber in gas phase through a precision leak valve. Limonene underwent a freeze and thaw cycle to minimize contamination of the interaction region. The pressure inside the vacuum chamber was maintained at Since limonene has low ionization potential, experiments were performed in the wavelength range of 1,300–2,400 nm to ensure adiabatic ionization. These wavelengths were obtained from an optical parametric amplifier (TOPAS-C) pumped by a Ti:sapphire laser system  operating at 1 KHz repetition rate, producing A quarter-wave plate (QWP) on a motorized rotation stage (with a Doubly charged limonene yields were recorded for each enantiomer by varying the ellipticity from


Collection of biospecimens is a critical first step to understanding the impact of COVID-19 on pregnant women and newborns - vulnerable populations that are challenging to enroll and at risk of exclusion from research. We describe the establishment of a COVID-19 perinatal biorepository, the unique challenges imposed by the COVID-19 pandemic, and strategies used to overcome them.A transdisciplinary approach was developed to maximize the enrollment of pregnant women and their newborns into a COVID-19 prospective cohort and tissue biorepository, established on March 19, 2020 at Massachusetts General Hospital (MGH). The first SARS-CoV-2 positive pregnant woman was enrolled on April 2, and enrollment was expanded to SARS-CoV-2 negative controls on April 20. A unified enrollment strategy with a single consent process for pregnant women and newborns was implemented on May 4. SARS-CoV-2 status was determined by viral detection on RT-PCR of a nasopharyngeal swab. Wide-ranging and pregnancy-specific samples were collected from maternal participants during pregnancy and postpartum. Newborn samples were collected during the initial hospitalization.P < 0.0001, and from 7 to 9 samples, P < 0.01). The number of samples collected per woman was higher in SARS-CoV-2 negative than positive women . The highest sample yield was for placenta (96%), umbilical cord blood (93%), urine (99%), and maternal blood (91%). The lowest-yield sample types were maternal stool (30%) and breastmilk (22%). Of the 61 delivered women who also enrolled their newborns, fewer women agreed to neonatal blood compared to cord blood .Between April 2 and June 9, 100 women and 78 newborns were enrolled in the MGH COVID-19 biorepository. The rate of dyad enrollment and number of samples collected per woman significantly increased after changes to enrollment strategy (from 5 to over 8 dyads/week, Establishing a COVID-19 perinatal biorepository required patient advocacy, transdisciplinary collaboration and creative solutions to unique challenges. This biorepository is unique in its comprehensive sample collection and the inclusion of a control population. It serves as an important resource for research into the impact of COVID-19 on pregnant women and newborns and provides lessons for future biorepository efforts. Key questions about the impact of COVID-19 infection on the pregnant patient and the developing fetus include, among others: (1) How does pregnancy impact the immune response to COVID-19? (2) What are mechanisms underlying severe maternal morbidity? (3) Does vertical transmission of the virus occur? If so, how? (4) Is breastfeeding safe and does it provide protection for the neonate? (5) What is the impact of maternal immune response to COVID-19 on the developing fetus? The establishment of a COVID-19 biorepository of specimens from the maternal-neonatal dyad is a critical first step to answering these questions; new data suggesting that pregnant women are at increased risk for severe illness from COVID-19 compared to non-pregnant counterparts further demonstrates the importance of research in this unique population [In December 2019, the newly emerged coronavirus, SARS-CoV-2, led to a surge in cases of pneumonia in Wuhan, China and by March 11, 2020, the World Health Organization classified COVID-19 as a world-wide pandemic. As of July 1, more than 2.6 million people in the United States and 10.6 million worldwide have been diagnosed with COVID-19 . With apso, how?  Is breasOvercoming barriers to the establishment of prospective observational cohort studies during a pandemic requires strategic planning and multidisciplinary effort . AcademiReports concerning the underlying biology of COVID-19 in pregnancy have largely focused on maternal outcomes and the question of vertical transmission , 13 and The Partners Institutional Review Board (IRB) approved a prospective, observational cohort study and biorepository of adult patients affected by or at risk for COVID-19 infection at Massachusetts General Hospital . This study did not exclude pregnant women from enrollment but had no mechanism for enrolling pregnant women systematically, and did not include the collection of critical pregnancy-relevant specimens such as umbilical cord blood, placenta, breastmilk, or vaginal swabs. Due to a healthcare-system-wide mandate that all adult COVID-19 biospecimens research be conducted under the umbrella of the existing protocols, our group wrote an amendment to include the collection of pregnancy-specific specimens, and to enroll the key comparator group of women pregnant during the pandemic but SARS-CoV-2 negative. This amendment was approved on April 20, 2020. Simultaneously, a separate prospective, observational cohort study and biorepository of samples from pediatric patients with COVID-19 infection or at risk of exposure to COVID-19 was established April 1, 2020 (Partners IRB #2020P000955). Due to the healthcare-system-wide mandate prohibiting any additional adult or pediatric biospecimen-related protocols during the height of the pandemic, there were no competing studies during the study period for either adult or pediatric patients.Universal screening for SARS-CoV-2 in patients presenting to the Labor and Delivery Unit at MGH was initiated on April 16, 2020, due to the high prevalence of COVID-19 in the greater Boston community. Screening and testing in asymptomatic and symptomatic patients occurred via real-time reverse transcription PCR (RT-PCR) for the presence of SARS-CoV-2 on nasopharyngeal swab. CDC criteria were used to define a positive test . Thus, aPregnant women were eligible for inclusion if they met the following criteria: (1) 18 years of age or older, (2) able to provide informed consent or with a healthcare proxy able to do so, (3) diagnosed with, or at risk for SARS-CoV-2 virus infection. As described all participants were tested for the presence of SARS-CoV-2 by RT-PCR of nasopharyngeal swab. Given the study’s primary focus on COVID-19 in pregnancy, contemporaneous controls were enrolled as a convenience sample, from women presenting to Labor and Delivery for care on the same days as enrolled cases. Overnight enrollment was typically restricted to SARS-CoV-2 positive patients, as consent could be obtained only by study clinicians . Newborns born to women who tested positive or negative for COVID-19 were eligible for inclusion in this study. Samples were obtained from newborns in the neonatal intensive care unit (NICU) and the well-baby nursery. The results presented in this paper are reflective of the enrollment of pregnant women and newborns through June 8, 2020. The target enrollment was 200 participants.In the first phase of recruitment  and limit exposure of non-essential staff to patients, recruitment was primarily virtual, and occurred by calling the patient’s hospital room. Hospital research restrictions prohibited research assistants or clinical research coordinators from enrolling patients or collecting specimens in the inpatient setting, and virtual phone enrollment was initially limited to clinician investigators .In the second phase of recruitment  virtual weekly Town Hall meetings; 2) in-person in-services occurring during day and night shifts; 3) training obstetricians in sample collection in a standardized fashion using a brief video created by two of the authors (L.L.S. and A.G.E.) demonstrating best practices for key sample collection on Labor and Delivery; 4) information sheets with step-by-step photo instructions included in the sample collection kits  in conjunction with a Steering Committee. The adult and pediatric consent forms are provided as Additional files Once a participant was enrolled, samples could be collected at all or any of three timepoints: antenatal, during the delivery hospitalization, or up to 12 weeks postpartum. In addition, for recovered participants, samples could be collected at regular intervals for up to 2 years after enrollment. Participants could elect to provide all samples or could decline to provide specific samples. Mothers gave separate informed consent for their newborn to participate in the pediatric study. Maternal specimens were collected primarily by Obstetrics study team members upon enrollment or by the patient’s clinical team if enrollment or delivery occurred overnight. Sample collection bags were pre-assembled into kits and labeled with randomly-generated patient identifiers. Upon enrollment, patients were assigned a study kit, which were kept on Labor and Delivery. Collection kit components are listed in Additional file The per patient cost of the study was approximately $120 for consumables related to sample collection kits and processing. This cost did not include personnel salary costs. The most expensive components of specimen collection kits included PaxGene tubes, RNALater, and stool/urine hats. Foregoing blood collection in PaxGene tubes for later RNA sequencing and taking fewer placental biopsies to minimize RNALater use could be ways to reduce per-patient costs. The most expensive components of sample processing included the cost of personal protective equipment for laboratory processors, the cost of Ficoll and other reagents related to blood processing and PBMC  isolation, and printable freezer-safe labels and cryovials for long-term specimen storage. Hand-labeled tubes and less expensive storage vials might be an area in which cost-savings could be achieved. Of note, many expensive, necessary, and back-ordered supplies were generously donated by research laboratories throughout MGH in an effort to support the establishment of this biorepository during a critical window of time.Samples were extensively annotated using REDCap electronic data capture tools hosted at MGH . Data weP < 0.05 was considered statistically significant. Analyses were performed using GraphPad Prism .Simple linear regression models were used to predict the rate at which women were enrolled over time, and the differences in slopes during Phase 1 versus Phase 2 were compared. Demographic characteristics of the enrolled cohort were compared to the overall MGH delivery population during the study period using Mann-Whitney or Chi-square tests where appropriate. The median number of samples collected per maternal participant was calculated before and after key enrollment strategy changes, and for SARS-CoV-2 positive and SARS-CoV-2 negative groups. Differences between groups were assessed by the Mann-Whitney test. The proportion of women or newborns in which each sample type was collected were compared between SARS-CoV-2 positive and SARS-CoV-2 negative groups by Fisher’s exact test. For all analyses, n = 15); symptomatic women who tested positive (n = 23); symptomatic women who tested negative (n = 8); and asymptomatic women who screened negative (n = 54). Newborns were categorized by their mother’s SARS-CoV-2 status, as determined by these criteria. Thirty enrolled women declined newborn participation, and 8 women enrolled remote from delivery have not yet decided on newborn enrollment.A flow chart describing study participants is presented in Fig. P = < 0.0001). Contrary to expectations, the rate at which enrolled mothers chose to enroll their newborn in the pediatric study during Phase 1 compared to Phase 2 did not increase . However, the overall rate at which dyads were enrolled in the biorepository did increase from 5 dyads per week to over 8 dyads per week (P < 0.0001) from Phase 1 to Phase 2, reflective of the improved overall efficiency of the enrollment process.Prior to the direct involvement of the Obstetrics and Neonatology team on April 2, no pregnant women had been enrolled in the study. The cumulative number of enrolled pregnant women over time increased significantly after passage of the amendment allowing enrollment of all pregnant women at risk for COVID-19 and collection of pregnancy-specific samples , indicated in Fig. Demographic information for the first 100 participants enrolled in the COVID-19 perinatal biorepository cohort compared to the overall MGH delivery population during the study period are presented in Table P = 0.004, Fig. P = 0.0007, Fig. Women were eligible to give 12 different sample types during their delivery hospitalization; undelivered women could provide 9 . Compared to Phase 1, significantly more samples were successfully collected from participants during Phase 2  or umbilical cord blood .The highest sample yields were for placenta (96%), umbilical cord blood (93%), urine (99%), and maternal blood (91%). The least-donated sample types were maternal stool (30%) and breastmilk (22%). Of the 61 enrolled women who delivered enrolled newborns, significantly fewer women agreed to neonatal blood sampling, compared to maternal blood  and pediatric (Yonker) protocols to ensure the successful inclusion of pregnant women and their newborns. An amendment was submitted to the adult IRB protocol to include the collection of pregnancy-specific samples, and a team of Obstetrics and Neonatology clinicians trained to both enroll and collect patient samples was assembled. The first pregnant SARS-CoV-2 positive woman was enrolled on April 2. Between April 2 and April 20, ten symptomatic, SARS-CoV-2 positive women and the majority of their newborns (for those who delivered) were enrolled in the biorepository. After the amendment was passed on April 20 permitting collection of pregnancy-specific samples  and enrollment of all pregnant women at risk for COVID-19, including a control population of women screening negative for SARS-CoV-2 on hospital admission, enrollment increased substantially, as demonstrated in Fig. Compared to the general adult or pediatric populations, enrollment of pregnant or newly postpartum women and their newborns requires the clinical expertise and creative input of both the Obstetrics and Neonatology study teams. We found that most pregnant women approached for enrollment were experiencing added emotional stressors – including fear of testing positive during universal screening, fear of exposing themselves or their newborn to COVID-19 during the delivery admission, and anxiety over the unknown impact of a COVID-19 diagnosis on their pregnancy and newborn. The time, effort, and sensitivity required to discuss participation in COVID-19 research during an inherently stressful hospitalization – whether for COVID-19 illness or for labor and delivery – was significant, and there were time constraints inherent in obtaining consent as we hoped to consent women for the study before the initial clinical blood draw or IV placement to facilitate research sample collection for those participants who wished to give blood. In addition, the mandate for virtual rather than in-person consent, due to strict limitations on face-to-face encounters to protect patients and staff and conserve PPE, posed another challenge to consenting patients in a dynamic and sometimes stressful situation. These challenges further exacerbated the ethical conundrum around enrolling women in labor or in the time-period around obstetric challenges, when pain, anxiety, and stress due to the normal physiology of labor or an obstetric complication may complicate the recruitment and consent process , 25.It became clear that a second conversation with Neonatology to enroll the newborn added to logistical challenges and time burden for participants, study staff, and clinical care providers. Due to these observations, a unified Obstetrics/Neonatology enrollment strategy was initiated (Phase 2). The rate at which mother-newborn dyads were enrolled increased from 5 per week to over 8 per week after this change, and our sample collection yield increased significantly after this intervention as well. These data suggest that future efforts to establish pregnancy biorepositories with an aim of capturing the dyad would be best served by having a single IRB protocol encompassing both maternal and newborn populations, permitting a single consent form and approach- this was not possible in our case due to hospital mandates limiting new COVID-19-related research protocols.Cross-training both study teams on both the adult and pediatric protocols, developing scripts for the consent to standardize the process, and instituting an outpatient consent strategy to complement the inpatient enrollment improved overall enrollment and sample collection rates. Although women were not more likely to agree to newborn participation after this strategy was implemented, the rate at which mother-newborn dyads were enrolled increased overall, from 5 per week to over 8 per week. The increase in number of maternal samples collected per patient after the initiation of a collaborative, unified enrollment strategy reflects the ability of Obstetrics study team members to focus on facilitating maternal sample collection, particularly of time-sensitive maternal blood and delivery specimens. Improvements in collection procedures, familiarity and willingness of clinical team members to assist in sample collection, and pre-identification of women undergoing planned deliveries by cesarean section or induction of labor for enrollment to facilitate sample collection during daytime hours all likely contributed to higher rates of sample collection over time.Our data indicate that more samples overall were collected from SARS-CoV-2 negative than positive women. As SARS-CoV-2 negative participants were approached during daytime hours and were often undergoing scheduled induction of labor or cesarean delivery, the more predictable timing may have facilitated sample collection as well as participant willingness to donate particular samples for research. In contrast, many SARS-CoV-2 positive participants were enrolled after arriving to Labor and Delivery in labor or after an unexpected screen positive on universal screening during admission for delivery. The inherently more unpredictable nature of these admissions and events may have contributed both to reduced ability of study clinicians to obtain time sensitive samples prior to delivery , and reduced willingness of participants to provide certain samples in the setting of emotional stress. Supporting the latter point, we found respiratory samples were also more often collected from SARS-CoV-2 negative women. As these samples can be collected at any point during hospitalization and were less likely to be impacted by study staff availability or timing of delivery, differences in sample retrieval are more likely reflective of the subject’s willingness to provide these samples. In addition to possible increased stress of SARS-Co-V-2 positive participants, which is an important area for future study, there are several other potential explanations for reduced sample collection from SARS-CoV-2 positive participants. SARS-CoV-2 positive women may have had more fear of discomfort associated with the nasal and oropharyngeal sample collections, as many of these women had to undergo more than one clinical nasopharyngeal swab for SARS-CoV-2 prior to their enrollment. It is also possible that despite appropriate availability and training on usage of PPE, study staff discomfort with obtaining respiratory samples on known SARS-CoV-2 positive women may have contributed to decreased collection rates.Delivery specimens  were successfully collected on greater than 90% of women who delivered. Strategies that likely contributed to this high collection rate included outpatient enrollment of women presenting for timed deliveries (induction of labor or scheduled cesarean sections), providing in person in-services, as well as written and video instructions on sample collection procedures to the clinical teams, and recruitment of senior Labor and Delivery nurses to the study team to assist with intrapartum and postpartum sample collection. We noted low rates of collection of stool and breastmilk, possibly due to early maternal discharge protocols during the pandemic, which limited the number of women who had full return of bowel function and established breastmilk supply prior to discharge.Newborn sample collection rates were highest for stool and urine, specimens considered clinical waste that required no additional procedure for the newborn. Urine was initially collected via urine bag, which is a standard, non-invasive method to collect urine from newborns. Several parents were distressed by the urine bag adhesive on newborn skin or expressed frustration with urine leakage. We therefore changed to collecting urine-soaked cotton balls placed in the diaper. While extracting urine from the cotton balls was slightly more challenging, this change improved parental and nursing satisfaction with the study. Maternal consent for a research-only, neonatal venous blood draw was significantly lower than consent given for maternal blood draw or draw from the umbilical cord. One strategy that reduced parental discomfort with neonatal blood draw was to instead collect an additional small volume of blood from the heel-stick used for the metabolic screen performed on every newborn in the state of Massachusetts, thus eliminating the need for an additional/clinically unnecessary procedure.The reallocation of resources to the care of adult COVID-19 patients in our hospital included deploying pediatric respiratory therapists and NICU ventilators to the adult intensive care unit, and transferring out all newborns who required ventilatory support to other hospitals. This in turn affected admission of high risk and extremely preterm newborns. Furthermore, there was an unanticipated backorder of nasopharyngeal swabs from April 29 to May 12, likely due to increased clinical demand. Thus, both the opportunity and ability to obtain tracheal and nasopharyngeal neonatal samples were limited during this enrollment period.Strengths of our study include the prospective enrollment of both COVID-19 positive pregnant women and controls who were asymptomatic and SARS-CoV-2 negative by nasopharyngeal swab. Both cases and controls delivered during the peak of the COVID-19 pandemic in Massachusetts. Such controls permit the most accurate assessment of the impact of SARS-CoV-2 infection, versus maternal stress and other unmeasured factors, on pregnancy and neonatal outcomes and biological tissue and fluid changes during the pandemic. We enrolled pregnant women across the full spectrum of disease severity, from critically ill to asymptomatic. We successfully recruited and enrolled 38 SARS-CoV-2 positive women and 25 of their newborns in a narrow time frame, overcoming major institutional changes that impacted the delivery of clinical care and research activities across all departments. Strengths of our methodology in establishing this biorepository include the synergistic efforts between Obstetric and Neonatology departments founded on a shared desire to advocate for inclusion of our unique patient populations. We describe a combined maternal and newborn consent process that increased the rate of recruitment, allowed for efficient use of time and resources, and likely improved the experience of women approached for enrollment (an important area for future study). High rates of collection of matched maternal blood, placenta, umbilical cord blood, neonatal blood, and neonatal specimens from enrolled participants is also a strength. While initial sample collection in acutely ill and convalescent pregnant and postpartum participants is the focus of this manuscript describing the earliest biorepository efforts, the potential for serial sample collection for up to 2 years after recovery in this cohort will permit assessment of the risk of reinfection and potential duration of protection in a dyad, also a strength of our study methodology.Our study is not without limitations. Our experience in setting up a maternal-newborn biorepository was limited by state- and institution-wide mandates imposing restrictions on the activities of clinical and research personnel. Our ability to collect certain samples, particularly newborn nasopharyngeal swabs and maternal vaginal swabs, was significantly impacted by a national shortage of needed swab types during March and April. Although we experienced shortages of collection, processing and storage supplies, generous donations from other investigators whose research activities had been suspended allowed us to continue enrollment and sample collection. While the samples in the perinatal biorepository are annotated with clinical outcomes through delivery and the postpartum period, longer-term follow-up studies of both the maternal and pediatric cohorts are planned in collaboration with Pediatric, Cardiology, Psychiatry, and Neurology colleagues. These long-term outcomes can in turn be correlated with perinatal biology, providing the potential to enrich understanding of the biological basis for long-term outcomes after COVID-19.Although other institutions and collaboratives in the United States and internationally are developing COVID-19 pregnancy registries with (e.g. the PRIORITY study)  and withData from other institutions in areas highly impacted by COVID-19, such as New York City and urban areas in Europe, have demonstrated the importance of leveraging existing protocols and pipelines to successfully build a biorepository during this unprecedented pandemic , 30. By The COVID-19 mother-newborn dyad biorepository described here will serve as an invaluable resource for further research into the impact of COVID-19 on the mother and the developing fetus. The specimens collected during the peak of the pandemic from the mother-newborn dyad and the accompanying clinical annotation have the potential to dramatically impact biological understanding of maternal inflammation and immune activation, how such processes may lead to severe maternal morbidity, and mechanisms of feto-placental vulnerability to inflammation or infection as well as mechanisms of antibody-mediated protection. To date, our understanding of the biological consequences of COVID-19 in pregnancy and clinical counseling in this regard has been largely guided by data from case reports and large case series –20, 36. Additional file 1. Supplementary Tables S1-S3. Table S1. List of supplies with catalog numbers for maternal sample collection kit. Table S2. Samples collected from maternal subjects by COVID status. Table S3. Samples collected from newborn subjects by mother's COVID status.Additional file 2. Instructions for collecting cord blood and placental biopsies.Additional file 3. Instructions for best practices for COVID-19 specimen handling.Additional file 4. Informational flier posted in clinical areas to advertise the COVID-19 biorepository.Additional file 5. Consent form - adult protocol.Additional file 6. Consent form - pediatric protocol.Additional file 7. Instructions for assembly of maternal sample collection kit.Additional file 8. REDCap data fields.


The outbreak of coronavirus disease 2019 (COVID-19) exposes vulnerable patients to high risk of mortality. Patients with left ventricular assist device (LVAD) usually have symptoms such as cough, fever, and shortness of breath because of their cardiac condition and comorbidity, therefore these related symptoms challenge the correct diagnosis in time within the COVID-19 pandemic.We report two case studies of patients with LVAD in whom COVID-19 related symptoms were overlapped by their cardiac status and comorbidities. In the first case, the patient was admitted for suspicion of COVID-19 due to cough and shortness of breath for 1 month. The blood test evocated a high index of suspicion of COVID-19. The nasopharyngeal test for COVID-19 performed on admission and at Day 2 was inconclusive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the test obtained on Day 3 of admission was positive, whereas computed tomography confirmed the diagnosis of COVID-19. This patient developed acute respiratory distress syndrome (ARDS) and nasal epistaxis within 48 h during hospitalization. The ARDS was treated by non-invasive ventilation and probabilistic antibiotics for 3 days and resulted significant improvement. The nasal epistaxis due to international normalized ratio increase was treated by nasal packing and vitamin K antagonist was switched to parenteral heparin infusion. The patient was kept hospitalized for 1 month for further supportive treatment. In the second case, the patient was admitted for recurrent anaemia due to melaena, the patient was tested for COVID-19 because of new-onset symptoms of cough and rhinorrhoea. The first nasopharyngeal test was positive, and sudden increase of anticoagulation status was noted in the setting of gastrointestinal bleeding. The anticoagulation status was controlled by parenteral heparin infusion, and the melaena was disappeared at Day 3. The moderate dyspnoea of the patient was quickly improved with nasal oxygen delivery for 4 days. The patient was discharged at Day 5.COVID-19 specific symptoms are challenging to distinguish in patients with LVADs, although radiological evidence can be beneficial in the COVID-19 diagnosis. We also observed the need for precise anticoagulation control to avoid bleeding or thrombotic events in these patients. Left ventricular assist device (LVAD) patients need careful examination to distinguish coronavirus disease 2019 (COVID-19) related symptoms that can be masked by patients’ underlying cardiac condition and comorbidities.For patients with LVAD and suspected of COVID-19, radiological tests are indispensable in cases of negative nasopharyngeal tests.Vitamin K antagonist should be replaced by parenteral heparin in COVID-19 positive patients with LVAD, and anti-factor Xa activity rather than active partial thromboplastin time, should be taken into consideration for anticoagulation monitoring.The outbreak of coronavirus disease 2019 (COVID-19), illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has caused more than 294 190 deaths worldwide by 14 May 2020.®) implanted in 2017 because of ischaemic myocardiopathy. This patient is also known to have implantable cardioverter-defibrillator, chronic renal failure, diabetes mellitus, morbid obesity (body mass index 32.89 kg/m2), obstructive arteriopathy of the lower limbs, and chronic infection on the driveline exit site under control with oral antibiotics. The patient described a cough and shortness of breath for 1 month on a scheduled teleconsultation for her LVAD on 27 March 2020. Following the teleconsultation, the patient was admitted immediately to the department of cardiac surgery for suspicion of COVID-19. The patient was admitted to a single-patient room with contact, droplet, and airborne precautions, including eye protection. On admission, the physical examination revealed normal parameters . Pulmonary auscultation revealed bilateral end-respiratory cackles, cardiac auscultation in the context of LVAD pump did not reveal other distinguishable murmur. The patient was thus authorized to take her usual medications including oral vitamin K antagonist (VKA). The blood test evocated a high index of suspicion of COVID-19. The nasopharyngeal swab test for COVID-19 was performed on hospital Day 0 by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and the result was negative. The patient became dyspnoeic on hospital Day 1 warranting oxygen delivered by nasal cannula at 2 L/min. A chest radiograph was performed on Day 1, which was reported as showing bilateral consolidative pulmonary opacity . A computed tomography (CT) was then performed which confirmed the diagnosis of COVID-19 .The first case concerned a 74-year-old women with a LVAD  delivered by mask at 15 L/min to maintain her saturation at 92%. The patient’s respiratory rate increased from 24 to 32 breaths per minute within 4 h. Meanwhile, the patient presented with significant nasal epistaxis that required nasal packing. The patient’s international normalized ratio (INR) was noted at 4.5 after the epistaxis started, which was an increase from her previous day’s INR which was 3.0 with her usual dose of VKA. Other haemostatic markers were also found to be abnormal (Table 1). Given the changing clinical presentation and the patient requiring more advanced care, the patient was transferred to the intensive care unit. A second nasopharyngeal test was performed in order to have nucleic acid confirmation of COVID-19, which also came back negative. The patient was treated by non-invasive ventilation for her hypoxia (FiO2 70%). The VKA was switched to parenteral heparin infusion in order to better control the anticoagulation status. For the pneumonia based on clinical presentation and radiologic findings, treatment with cefotaxime , spiramycin  and oseltamivir  was initiated on hospital Day 2. Nevertheless, a third test for COVID-19 was performed on hospital Day 3, and it was positive for COVID-19. On Day 5, the patient’s clinical condition improved, non-invasive ventilation was discontinued, and supplemental oxygen was decreased back to 2 L/min delivered via nasal cannula with an oxygen saturation of 98%. The patient was then transferred to the department of infectious disease.On Day 2, the patient rapidly developed acute respiratory distress syndrome (ARDS), which was treated with high concentration oxygen (FiOTable 1). On Day 14, another nasopharyngeal PCR was performed and the results reported back as negative, and a chest X-ray demonstrated decreased ground glass opacity . The patient was asymptomatic apart from her cough, which was decreasing in severity. During the patient’s time in the hospital, her anticoagulation was monitored with regular anti-factor Xa activity (Anti-Xa) measurements and no bleeding or thrombotic event were observed following the nasal epistaxis on Day 3. However, the antihaemophilia factor A (factor VIII) and Von Willebrand factor remained elevated (Table 1), which reflects the inflammatory and prothrombotic status. The LVAD function remained stable during this hospitalization without any alarms. The patient remains hospitalized at the time of this report (1 month) and has improved physical tolerance.In the following days, the patient developed acute renal failure related to intensive antibiotic use and was treated accordingly (evolutionary laboratory results in ®) in 2012 because of ischaemic myocardiopathy and has constant shortness of breath due to COPD and recurrent anaemia due to gastrointestinal bleeding. Several previous endoscopic procedures have revealed arteriovenous malformations. The patient previously had two periods of LVAD thrombosis with subtherapeutic INR inferior to 2. On admission, the physical examination did not reveal any respiratory issues. The patient continued to take his usual medications except oral VKA. The patient stated new-onset symptoms of cough and rhinorrhoea for approximately 1 week. With these new-onset symptoms, a nasopharyngeal swab test for COVID-19 was performed which confirmed COVID-19 by a positive result on the following day. Because the exact onset of his COVID-19 course could not be determined, we decided to keep the patient in hospital for respiratory surveillance after the blood transfusion for his gastrointestinal bleed. A chest radiograph taken on Day 0 did not show opacity signs . The patient did not have fever during his hospital admission, and blood tests did not show any inflammatory signs. Supplemental oxygen was initiated on Day 2 because the oxygen saturation dipped to 90%, with oxygen delivery via nasal cannula at 1 L/min. Oxygen was discontinued on the morning of hospital Day 5.A 64-year-old man was admitted to our department for blood transfusion due to melaena from ongoing gastrointestinal bleeding on 20 March 2020. This patient was implanted with an LVAD  was at 3.2. VKA was stopped and parenteral heparin was initiated on Day 0. A total of 3 units red blood cells was transfused which restored haemoglobin from 7.7 to 10 g/dL. The melaena decreased progressively with precise control of anti-Xa under heparin infusion, with complete resolution on hospital Day 3. With the melaena resolved, VKA was reintroduced on hospital Day 3. During this hospitalization, the LVAD parameters remained stable without any alarms.® INRange system, and also continue to monitor respiratory parameters. We have scheduled a video-capsule gastrointestinal exam for the patient after the pandemic crisis resolves. The patient remains asymptomatic to date, although no chest radiograph in follow-up was performed in order to reduce transmission risk during the quarantined period.With the improvement of haemoglobin and resolution of melaena, as well as patient-reported resolution of shortness of breath, he was discharged home. As part of the discharge planning the patient remained in isolation with regular teleconsultation to better control his INR using a CoagucheckFigure 4 presents our proposed management strategy for LVAD patients.Our report of two cases of patients with LVADs and infected by COVID-19 illustrates several aspects of the influence of the COVID-19 pandemic on a specific population of vulnerable people. For LVAD patients, more regular teleconsultation is needed to discover COVID-19 related symptoms. The two patients in our case report both presented with new-onset symptoms related to COVID-19, but diagnosis was complicated as both patients were used to having these COVID-19 related symptoms as part of their underlying heart failure. In the case of the first patient, she stated to have a cough for 1 month, but she reported this new-onset symptom only on a regularly scheduled teleconsultation. The second patient was tested for COVID-19 because of shortness of breath that, although often present during his anaemic periods, was reported by the patient as accompanied by rhinorrhoea for 1 week prior. These epidemiologic histories indicate that patients with an LVAD can be less vigilant of COVID-19 since it is difficult for them to discover specific COVID-19 related symptoms due to their underlying cardiac condition-related symptoms. As SARS-CoV-2 infection can rapidly progress to ARDS and lead to poor outcomes,,The impact of COVID-19 on LVAD patients can vary from mild respiratory symptoms to COVID-19 related ARDS. Our second patient presented with only a mild cough and rhinorrhoea related to COVID-19 and remained relatively stable throughout hospitalization. However, our first patient rapidly developed COVID-19 related ARDS within 4 h of presentation. The diagnosis and medical management were quite challenging for patient number 1 because of the first two negative nasopharyngeal COVID-19 tests. Current nasopharyngeal tests are reporting non-negligible false negative rates due to the quality of the swab, therefore CT images are now accepted as one of the diagnostic criteria of COVID-19,LVADs need well-controlled anticoagulation to prevent thrombosis, which could otherwise result in life-threatening dysfunction of the device. On the other hand, LVAD patients have higher risk of haemorrhage events due to anticoagulation therapy. It was reported that COVID-19 can alter the patients’ coagulation status, which can cause both thrombosis and haemorrhage.This report highlights the need to closely monitor patients with LVADs during this COVID-19 pandemic, as the correct diagnosis is challenging due to their underlying cardiac disease and associated symptoms. Radiological exams are indispensable in LVAD patients with suspected or confirmed COVID-19 diagnosis since they can guide the medical management. In cases of patients with LVAD where the INR is deranged in the context of COVID-19 infection, it may be appropriate to covert from a VKA to parenteral heparin infusion to better control anticoagulation and avoid thrombotic and bleeding events.Fabrice Vanhuyse, MD, PhD is a cardiac surgeon and Director of the VAD Program.European Heart Journal – Case Reports online.Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Consent: The author/s confirm that written consent for submission and publication of this case series including image(s) and associated text has been obtained from the patient in line with COPE guidance.Conflict of interest: none declared.Funding: The authors would like to thank the European Society of Cardiology for financially supporting this research.ytaa447_Supplementary_DataClick here for additional data file.


The COVID-19 pandemic has had a huge influence in almost all areas of life, affecting societies, economics, and health care systems worldwide. The paediatric cardiology community is no exception. As the challenging battle with COVID-19 continues, professionals from the Association for the European Paediatric and Congenital Cardiology receive many questions regarding COVID-19 in a Paediatric and Congenital Cardiology setting. The aim of this paper is to present the AEPC position on frequently asked questions based on the most recent scientific data, as well as to frame a discussion on how to take care of our patients during this unprecedented crisis. As the times are changing quickly and information regarding COVID-19 is very dynamic, continuous collection of evidence will help guide constructive decision-making. It is now more than a half year since the COVID-19 pandemic started to influence almost all areas of life, affecting societies, economics, and healthcare worldwide and it is likely that we will have to endure this for a long time ahead.Many questions have arisen regarding patients with CHD, with respect to increased susceptibility to the coronavirus, the risk of more severe disease in the presence of CHD, the value of testing, and regarding recent reports on the inflammatory disease with some Kawasaki features.The aim of this paper is to present the AEPC position on frequently asked questions regarding COVID-19 in a Paediatric and Congenital Cardiology setting, based on continuous monitoring and evaluation of scientific papers. It should be kept in mind that times are changing fast and this paper has been written with the best evidence available at the time of writing. The AEPC position will be updated in the future according to further evidence in the literature.Methods: This manuscript has been reviewed and approved by the AEPC Council for publication in Cardiology in the Young as an official position paper of AEPC.1 Nevertheless, and although not scientifically proven, most children and adults with CHD presenting with severe cyanosis, pulmonary hypertension, or heart failure should be monitored more closely during the SARS-Cov-2 pandemic.According to the current state of knowledge, COVID-19 infections in children are mostly mild and self-limiting. Children are at very low risk of being admitted to the ICU. However, children under the age of 1 year seem to have a higher risk for a more severe course of the infection. Whether children with underlying cardiac diseases are more susceptible to cardiac injury remains unknown. Three percent of patients from the United States of America cohort of 177 patients infected with SARS-Cov-2 had pre-existing cardiac disease and more frequent hospital admission compared with other underlying conditions.3 Some risk factors in the adult population for higher susceptibility to COVID-19 outcome are partly rebutted, like arterial hypertension. Deeper insights from the studies on the risk factors for mortality of COVID-19, did not prove evidence that hypertension contributes to poor outcome, even if prevalence is high.4As in adults, underlying cardiac diseases with a decreased cardiac reserve and enhanced metabolic demand contribute to increased severity of respiratory symptoms and worse outcome.5A comprehensive guidance document has been established that addresses all management aspects of cardiovascular diseases in adults during the COVID-19 pandemic.8Currently, there is only scarce data on COVID-19 in children with CHD, so that, no sound data is available for a detailed risk stratification.Despite the large number of cases with COVID-19, little is known about the risk and effects in children as well as adults with CHD. However, it seems that children have a substantially better prognosis than adults with cardiovascular risk factors.9 Data from China suggests the same tendency towards better survival and outcomes from critical illness in infants and children than reported for adult patients.11According to a recent multicentre cross-sectional study from 46 centres within The United States of America and Canada, the overall ICU mortality of COVID-19 in children is less than 5%, compared with published mortalities of 50–62% in adults admitted to the ICU. These conclusions were drawn from 48 critically ill children with COVID-19, of whom more than 80% had significant comorbidities including CHD.•Single-ventricle patients.•Infants under 1 year with unrepaired CHD requiring surgery or catheter intervention.•Patients with chronic cyanosis (oxygen saturations <85% persistently).•Patients with severe cardiomyopathies.•Patients with CHD on medication for heart failure.•Patients with pulmonary hypertension.•Patients after heart transplantation.•12Patients with CHD and significant comorbidities, e.g., chronic kidney disease, chronic lung disease.•13Grown-up CHD patients with Down’s syndrome have a 4fold increased risk to be admitted to hospital and a 10-fold increased risk for COVID-19-related death.The British Congenital Cardiac Association has published recommendations for precautions for some groups with other infections in CHD patients, based on existing knowledge from diseases, which have been extrapolated to the COVID-19 situation. According to the British Congenital Cardiac Association recommendations, the following CHD patient groups are estimated to be at high risk for a severe disease course with an increased risk for a negative outcome and should be particularly strict in following the social distancing and hygiene measures:14 Amongst several theories of protective mechanisms in the paediatric age group, the lower number and immaturity of angiotensin-converting enzyme 2 in children has been speculated to be one of the reasons for the milder nature of COVID-19.15To date, cardiac pathology in children has been described in very few cases beyond the systemic inflammatory disease, and in general cases, fatality is low. Tissue damage to lungs, heart, and other organs caused by SARS-CoV-2 is induced by binding of the virus with its surface spike glycoprotein to the host angiotensin-converting enzyme 2 receptor. In the human heart, the receptors are localised at the pericytes and their injury can lead to secondary capillary endothelial dysfunction, which is responsible for cardiac pathology.16 It remains obscure whether all cardiac pathologies are related to the primary viral attack on cardiomyocytes or whether it is the result of proinflammatory cytokines and systemic inflammatory response.Clinical symptoms of myocardial damage in adults affected with COVID-19 are rhythm disorders, hypotension, and cardiogenic shock. Biomarkers like troponin, creatine kinase, and lactate dehydrogenase are elevated. Acute myocarditis has been suspected in adults as myocardial interstitial oedema and diffuse late gadolinium enhancement could be documented.17 Together with a timely delayed presentation of symptoms (10–15 days), these findings might be interpreted in favour of inflammation-mediated myocardial injury rather than induced by the cytopathic effect of the virus.18Fulminant acute myocardial necrosis and mononuclear cell infiltration have been described, but only rarely have virus particles been demonstrated in myocardial tissue.19Hyperinflammation, imbalance of renin–angiotensin–aldosterone system, and a particular form of vasculopathy, thrombotic microangiopathy are reported to contribute to increased severity of COVID-19, not only in respiratory but also in cardiac failure.21 The special characteristics of the delayed immune response after COVID-19, named MIS-C are provided in question 4.In this context, impaired cardiac function, vasculitis, and septic shock as well as features of a Kawasaki disease shock syndrome have also been described in children, with distinct differences in inflammatory markers. While coronary involvement was variable, depressed left ventricular function, pericardial effusion, and arrhythmia were the main findings.22 The timeline of the symptoms, in that case, does not rule out completely that the patient was in the acute phase of COVID-19 rather than MIS-C when myocardial evidence of the virus was detected.It was recently published that SARS-CoV-2 could be isolated from cardiac tissue in a child that died within a COVID-19-related multisystem inflammatory syndrome.23 These reports came specifically from countries with a high number of SARS-CoV-2 infections, such as Italy, Spain, the United Kingdom, and the United States of America. Understanding of COVID-19 infection and multisystem inflammatory syndrome is evolving. Guidance has been issued and consistently renewed by the World Health Organization and the Centers for Disease Control and Prevention.25 An association to a SARS-CoV-2 infection or a secondary reaction has been discussed and according to the most recent literature, multisystem inflammatory syndrome manifests temporarily in association with SARS-CoV-2, appearing about 3–4 weeks after the acute infection.27 This may explain why many children had positive antibodies to SARS-CoV-2, but a negative reverse transcription-polymerase chain reaction on swab analysis at the time of multisystem inflammatory syndrome evaluation. Severe manifestation of COVID-19 in the paediatric age group is, in general, rare. The described severe cases do not present the first line with pulmonary symptoms, but with an untypical inflammatory reaction and gastrointestinal complaints. The cardiovascular system is affected by a vasculitis and in some cases by myocardial dysfunction. The clinical presentation shows overlapping features of a toxic shock syndrome and Kawasaki syndrome , and the criteria for Kawasaki disease do not apply fully for diagnosis.In the frame of the actual COVID-19 pandemic, an increasing number of children were admitted for clinical symptoms of the condition termed multisystem inflammatory syndrome .23 Some patients are described who have presented with a refractory vasodilatory shock syndrome with normal cardiac function. Not uncommonly children can present with septic or cardiogenic shock and impaired cardiac function (mainly left ventricular).Clinical data on multisystem inflammatory syndrome provide evidence that although multisystem inflammatory syndrome and Kawasaki disease seem to be based on an increased inflammatory reaction, multisystem inflammatory syndrome differs from other paediatric inflammatory syndromes.28Different organisations, such as the World Health Organization, the Centers for Disease Control and Prevention, and the Royal College of Paediatrics and Child Health have published definitions of the multisystem inflammatory syndrome, which are in slight variance from each other.As a reference, Table 25 and Centers for Disease Control and Prevention24 are summarised in this table. The CDC also comments that some individuals may fulfil full or partial criteria for Kawasaki disease, but should be reported if they meet the case definition for MIS-C. MIS-C should be considered in any paediatric death with evidence of SARS-CoV-2 infection.The definitions of multisystem inflammatory syndrome in children from the World Health Organization23It seems that important differences occur in clinical and laboratory findings, which distinguish multisystem inflammatory syndrome from other paediatric inflammatory syndromes, such as Kawasaki disease, Kawasaki disease shock syndrome, and toxic shock syndrome.29:•anaemia, lymphopenia,•elevated leucocytes and neutrophils,•normal or low platelets,•elevated fibrinogen and ferritin,•elevated troponin, N-terminal pro-b-type natriuretic peptide,•decreased albumin,•elevated interleukin 6, C-reactive protein,•lymphocytic myocarditis.The following major laboratory findings are described to characterise a potential multisystem inflammatory syndrome in comparison to Kawasaki disease28 Figure Patients with the suspected multisystem inflammatory syndrome should undergo recommended diagnostic blood, urine, virology, bacteriology, serology, echocardiography, and sonography tests at admission.Anti-inflammatory treatment: steroids, intravenous immunoglobulin, aspirin, and possibly interleukin 6 inhibitors.Shock: inotropes, management of cardiac dysfunction.Anti-platelet and anti-coagulation therapy: enoxaparin and others.Treatment strategies of the multisystem inflammatory syndrome are as follows:28The aetiology is not yet conclusive and might be in accordance with the inflammatory reaction seen with a delay in adults. Similar to the results of genetic analysis in Kawasaki research, the origin of the inflammation might be associated with genes that regulate the rate of production of T-cells or antibody responses or the clearing of antibody and immune complexes.30SARS-CoV-2 infection may occur in patients with channelopathies, e.g., congenital long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, and short QT syndrome, with a risk of pro-arrhythmia.5In the setting of long QT syndrome with COVID-19, the QT interval should be monitored closely because the combination of drugs (Hydroxychloroquine and Azithromycin) and stress factors (electrolyte disturbances and kidney dysfunction) may further prolong QT.31Fever-triggered malignant ventricular arrhythmia is the major concern in Brugada syndrome with COVID-19, therefore, fever should be aggressively treated with paracetamol.5In patients with catecholaminergic polymorphic ventricular tachycardia and COVID-19, beta-blockers and flecainide should be continued with monitoring of drug interactions with the above-mentioned drugs.The incidence of COVID-19 remains low in children. Numbers are quoted to be between 2 and 12% (depending on testing), with a generally milder course, but with variable symptoms that may mask the viral infection and cause a delay in making the diagnosis.32Despite common-sense judgement that pre-existing cardiac diseases should impose children with CHD to a higher risk, this has only rarely been demonstrated to date.Prioritisation of cardiac surgery, leaving the surgical activity to urgent and emergency procedures.Avoid the delay of urgent cardiac surgery.CHD patients planned for cardiac surgery if not an emergency should have tested negative for SARS-Cov-2 reverse transcription-polymerase chain reaction.Test- and symptom-based precautions to avoid transmission: separation of positive-tested COVID-19 patients from non-COVID-19 patients irrespective of symptoms. Staff working in rotations.Full personal protective equipment should be mandatory for staff who is performing a cardiac intervention for COVID-19-positive patients.In CHD, patients positive for SARS-CoV-2 (test or symptoms) surgery should be delayed until the test is negative or symptoms relieved  if clinically justifiable.Provide extensive logistics including anaesthetic and intensive care considerations to protect patients and medical staff.Continue transmission precautions at least 14 days after discharge from the hospital.The following strategies should minimise the risk of cardiac surgery during the COVID-19 pandemic:33 The subgroup of adults with CHD are likely to have the highest risk within patients with CHD6 as, at least in complex cases, premature ageing of the heart may be present together with limited cardiac reserve, thus lacking the advantage of children that are, in general, less susceptible to symptomatic SARS-CoV-2 infections.Paediatric heart catheterisation and intervention in COVID-19 patients have been required in exceptional circumstances. Elective cases have, in general, been postponed from the beginning of the pandemic. Meanwhile, as the numbers of affected children remain low, it seems reasonable to activate the programmes while continuing to use personal protective equipment and following institutional flow algorithms for COVID-19 patients, including switch to a negative pressure environment in the catheter laboratory if possible. A comprehensive guidance paper addresses most aspects of decision-making and resource allocation.34 Nevertheless, transmission precautions are of paramount importance and should be strictly followed until further knowledge of SARS-CoV-2 impact on the immune system is available.35Heart transplantation/immunosuppression: while immunosuppression, in general, enhances the risk for viral infections and increased severity, as has been described for younger children with influenza, there’s no supporting evidence in SARS-CoV-2. Data from solid organ transplantation do not show that immunosuppression during the COVID-19 pandemic leads to a less favourable outcome. Immunocompromised children, for example, those treated for cancer, only rarely needed modifications of their treatment. Accepting that not all patients are tested for COVID-19 at least symptomatic worsening has not been observed.36 In a few recent review articles on pregnant women with COVID-19, no cases of vertical transmission have been registered.39 A few newborn COVID-19 cases have been reported based on elevated immunoglobulin M levels and/or pneumonia on days 1 or 2 of life, with either negative or not performed COVID-19 testing on fetal blood, amniotic fluid, and placenta.43 However, it is not reliable to diagnose congenital COVID-19 based only on immunoglobulin M detection because immunoglobulin M assays can be false-positive and cross-reactivity may occur. The most likely cause of positive immunoglobulin M in many of those cases could be related to early infant infection due to postnatal contact with infected parents rather than fetal transmission.44Currently, criteria for vertical transmission have not been developed. Based on limited data, viremia rates in patients with COVID-19 appear to be low and transient, suggesting placental seeding and vertical transmission are unlikely.45 It is equally important to ensure that a neonate with critical CHD born to a mother with COVID-19 is managed in a timely manner and that testing for COVID-19 does not prolong or delay transfer to a tertiary centre with a neonatal CHD ICU able to deliver high quality and timely management of critical CHD.Even though there is no reliable data to confirm vertical COVID-19 transmission, the Centers for Disease Control and Prevention recommend that newborns born to mothers with known COVID-19 at the time of delivery should be considered to have suspected COVID-19 and should be tested and isolated from other healthy infants.47 Given the typically mild course of paediatric COVID-19, supportive care alone is suggested for the overwhelming majority of cases. Antiviral therapy generally should be reserved only for symptomatic children or those with underlying conditions with a high risk of disease progression.There is a lack of data supporting the efficacy of antiviral agents for the treatment of COVID-19 in children, though there are recommendations available from multicentre initial guidance on the use of antiviral agents in children.Remdesivir inhibits ribonucleic acid-dependent ribonucleic acid polymerase and has activity against coronaviruses. According to a multicentre panel, Remdesivir is preferred to other antiviral agents due to its better tolerance and beneficial effects in adults with COVID-19. Hence, the same assumption is being made for children, even though data regarding the benefits of Remdesivir for children with COVID-19 are lacking. On 22 October, 2020, the United States of America Food and Drug administration has approved Remdesivir to treat COVID-19 for use in adults and children 12 years of age and older and weighing at least 40 kg requiring hospitalisation.48Lopinavir/Ritonavir is not recommended for children due to the absence of efficacy and unfavourable pharmacodynamics and negative clinical trial data.49 Corticosteroids are reserved only for critically ill patients with COVID-19. There is insufficient evidence for or against systemic corticosteroids for mechanically ventilated patients with acute respiratory distress syndrome (CI)*, and for adults with COVID-19 and refractory shock. The National Institutes of Health recommends using low-dose corticosteroid therapy (BII)*.50The benefits and risks of immune modulators  and of convalescent plasma from recovered COVID-19 patients in the treatment of children with COVID-19 are uncertain. The use of these medications is referred only on a case-by-case basis according to disease severity. The routine use of COVID-19 patients is not recommended. The cytokine profiles of serum from some patients with moderate-to-severe COVID-19 overlap with those seen in macrophage activation syndrome and secondary hemophagocytic lymphohistiocytosis. Thus, interleukin 6 and interleukin 1 blockades and Janus kinase inhibition has been proposed as an approach to treat the systemic inflammation associated with severe COVID-19 illness.52The safety and effectiveness of dexamethasone for paediatric COVID-19 treatment have not been sufficiently evaluated as the Recovery trial did not include a significant number of children. According to the recent National Institutes of Health statement, dexamethasone is not generally recommended for children who require only low levels of oxygen support. On the other hand, dexamethasone may be beneficial in difficult cases where mechanical ventilation is required. Additional studies are needed to evaluate the use of steroids for the treatment of COVID-19 in children, including for multisystem inflammatory syndrome in children.53 Anticoagulation therapy should be coordinated with paediatric intensivists, haematologists, and paediatric interventional cardiologists.54Patients with MIS-C are at risk of experiencing thrombotic complications. Especially in the setting of severe LV dysfunction, there is a higher risk for apical LV thrombus. And those with Kawasaki disease who have large or giant CA aneurysms are at risk for myocardial infarction. Additionally, hypercoagulability is associated with COVID-19, thus patients may be at risk for venous thromboembolism. A minimum required treatment for Kawasaki disease includes low-dose aspirin. Systemic anticoagulation may be used for patients with moderate-to-severe LV dysfunction. VTE prophylaxis should be recommended for older children and adolescents hospitalised with moderate-to-severe MIS-C only with calculated low bleeding risk. There is evidence that low-molecular-weight heparins appear to be associated with better prognosis in patients with moderate-to-severe COVID-19-induced coagulopathies or elevated D-dimer levels. A weak recommendation of subcutaneous enoxaparin use in mild to high risk of VTE was recommended in COVID-19 PICU guidelines.55There is no available data on the use of vitamin A for COVID-19; however, it is recommended to be used as an adjunctive agent for COVID-19 patients due to its effect on decreased morbidity and mortality from measles-associated pneumonia.*Rating of Recommendations: A = Strong; B = Moderate; C = Optional.Rating of Evidence: I = One or more randomised trials with clinical outcomes and/or validated laboratory endpoints; II = One or more well-designed, non-randomised trials or observational cohort studies; III = Expert opinion.18The potential experimental therapies for COVID-19 have important cardiovascular side effects and toxicities. Data for these side effects are extrapolated from patients treated for autoimmune diseases , hepatitis , or human immunodeficiency virus infection (Lopinavir/Ritonavir).50 The QT interval should be monitored closely, especially in the setting of long QT syndrome with COVID-19.56 Other adverse cardiac events related to these drugs are less common, but include the following: ventricular hypertrophy, hypokinesia, heart failure, pulmonary arterial hypertension, and valvular dysfunction. Irreversible myocardial damage is seen in 12.9%, death in 30.8%, however, cardiac function normalises in the majority of patients (44.9%) upon withdrawal of Chloroquine and Hydroxychloroquine.57 On 17 June, 2020, the World Health Organization announced to stop the use of Hydroxychloroquine for COVID-19 patients, which was based on data from Solidarity and the Recovery trials. Both showed that Hydroxychloroquine does not result in the reduction of mortality of hospitalised COVID-19 patients, when compared with standard care.58Azithromycin, Hydroxychloroquine, and Lopinavir/Ritonavir can cause conduction disorders, especially QT prolongation. The combination of Hydroxychloroquine and Azithromycin is not recommended due to a synergistic effect increasing the risk of severe arrhythmia and deleterious impact on the cardiovascular system.59Lopinavir/Ritonavir may interact with antiplatelets, oral anticoagulants, digoxin, statins, and many others as it is a potent liver enzyme (CYP3A4) inhibitor.60 Interleukin 6 targeting has not been tested for secondary prevention in cardiovascular disease.Tocilizumab has been shown to influence lipid metabolism in rheumatoid arthritis patients. However recently, the ENTRACE clinical trial supported the cardiovascular safety of Tocilizumab in these patients.50 This applies to children, adolescents, and adults.There is no evidence that angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers/angiotensin receptor neprilysin inhibitors or low-dose acetylsalicylic acid worsen outcomes in patients with confirmed or suspected COVID-19 or increase susceptibility to COVID-19. COVID-19 is not an indication to stop angiotensin-converting enzyme inhibitors/Angiotensin II receptor blockers/angiotensin receptor neprilysin inhibitors or low-dose acetylsalicylic acid as stopping these medications may cause worsening of patient’s heart condition, unless symptomatic hypotension or shock, acute kidney injury, or hyperkalemia appears.56 Fever-triggered malignant ventricular arrhythmia is the major concern in Brugada syndrome with COVID-19 infection, therefore, fever should be aggressively treated with paracetamol.31According to the British Congenital Cardiac Association, the Canadian Cardiovascular Society, and other organisations, patients with heart failure and hypertension should preferentially choose acetaminophen over non-steroidal anti-inflammatory drugs for fever or pain to avoid decompensation of these cardiovascular conditions; however, there is yet no firm evidence.5In patients with catecholaminergic polymorphic ventricular tachycardia and COVID-19, beta-blockers and flecainide should be continued with monitoring of drug interactions with antiviral drugs.12The British Congenital Cardiac Association has suggested that adults and children with CHD, within the groups mentioned in the answer of question 1, should be considered more vulnerable to COVID-19 and other infections, and should therefore be particularly strict in following the social distancing and hygiene measures.Based on definitions of social distancing, this would exclude the attendance of such individuals at nurseries, school, college, or universities. Socialising with family, going to restaurants, and children’s parties should be avoided as well.12Children who do not fall into the mentioned groups should not be more likely to become infected with COVID-19 and there is no evidence of higher risk of complications, thus general rules of preventive measures should be applied.61On 22 September, 2020 the World Health Organization made an announcement of approximately 38 COVID-19 vaccines in clinical trials and 149 in preclinical evaluation.33 Hence, availability of catheterisation is limited in the vast majority of centres. Cardiac centres in regions with greater COVID-19 prevalence are more likely to delay urgent, but not emergency case types. The decision regarding urgent cardiac surgery is expected to be based on a multidisciplinary team decision and if possible, surgery should be delayed until the patient’s symptoms have improved and/or testing has been repeatedly negative.63Not only have non-invasive cardiological diagnostic procedures been limited to urgent and emergency situations, but also paediatric/congenital catheterisation laboratories have dramatically reduced case volumes by postponing elective procedures.The data regarding the COVID-19 crisis is being updated constantly. However, there is a scarce number of publications addressing the management of children with cardiac pathology. Hence, AEPC officers have been frequently asked various questions related to COVID-19 and patients with CHD. This led to the publication of FAQs and expert answers based on the most recent available literature sources. The goal of this document is to frame a discussion on how to take care of children with cardiac pathology in the setting of COVID-19 during this unprecedented crisis. As the times are changing quickly and information regarding COVID-19 is very dynamic, continuous collection of evidence will help guide constructive decision-making.


Stroke triggers neurogenesis in the striatum in mice, with new neurons deriving in part from the nearby subventricular zone and in part from parenchymal astrocytes. The initiation of neurogenesis by astrocytes within the striatum is triggered by reduced Notch-signaling, and blocking this signaling pathway by deletion of the gene encoding the obligate Notch coactivator Rbpj is sufficient to activate neurogenesis by striatal astrocytes in the absence of an injury. Here we report that blocking Notch-signaling in stroke increases the neurogenic response to stroke 3.5-fold in mice. Deletion of Rbpj results in the recruitment of a larger number of parenchymal astrocytes to neurogenesis and over larger areas of the striatum. These data suggest inhibition of Notch-signaling as a potential translational strategy to promote neuronal regeneration after stroke. Every year, 13.7 million people worldwide suffer a stroke, of whom 5.5 million die and as many suffer permanent loss of function with debilitating outcomes . In the Neurogenesis is abundant in the subventricular zone and in the dentate gyrus of the hippocampus in rodents and many other mammals. In the subventricular zone, neural stem cells differentiate into transit amplifying progenitor cells, which then mature into neuroblasts, identified through the markers Doublecortin (Dcx) and Polysyalilated Neuronal Cell Adhesion Molecule (PSA-NCAM). The neuroblasts migrate to the olfactory bulbs, where they differentiate into mature neurons. In the dentate gyrus, neural stem cells are largely quiescent, and 1-2% of them proliferate ,6,7. TheAfter an ischemic event, neuroblasts double-positive for the markers Dcx and PSA-NCAM, or mature neuronal markers like NeuN, emerge in the striatum ,4. It isThe presence of a regenerative program in the striatum poses the question of whether it is possible to enhance this process to promote recovery. The new neurons in the striatum derive, in roughly equal proportions, from the nearby neurogenic subventricular zone and from parenchymal astrocytes . ReducedRbpj specifically in astrocytes results in an approximate doubling of the number of cells that enter the neurogenic program after a striatal stroke, with a concomitant increase in neuroblasts. The neurogenic response is also more widespread in the stroke-afflicted striatum when Notch-signaling is blocked. This demonstrates that the neurogenic response after stroke can be substantially promoted by recruiting more parenchymal astrocytes to neurogenesis, identifying a new potential therapeutic target.In the present study, we assessed whether a further reduction of Notch-signaling in astrocytes could promote a stronger and more widespread neurogenic response to a striatal stroke. We report that deleting fl/fl; R26-tdTomato mice, at least two months old, were used for the experiments.All experimental procedures were carried out in compliance with Swedish laws and the ethical rules by the home institution . The ethical permit was reviewed and approved by Stockholms Norra Djurförsöksetiska Nämnd and the Malmö-Lund Ethical Committee. Mice were maintained on a 12 h light/dark cycle with access to food and water ad libitum. C57Bl6 Cx30-CreER; RbpjAnimals were administered 2 mg tamoxifen/day via intraperitoneal injection for five consecutive days. Tamoxifen was dissolved in 99% ethanol (1:10) in corn oil. Days were counted from the last day of injection.All experimental procedures were approved by the Malmö-Lund Ethical Committee. Middle cerebral artery occlusion was performed using the protocol by Andsbeg, Kokaia, and Lindvall, 2001 . Briefly3 in PBS). Following the blocking step, the sections were incubated with primary antibodies (diluted in blocking solution) overnight at 4 °C. The primary antibodies used were rabbit anti-Ascl1 , goat anti-Dcx , mouse anti-NeuN , rabbit anti-S100β  and rat anti-Ki67 . Subsequently, the sections were washed three times for 5 min in PBS before incubation with secondary antibodies (1:500 in blocking solution) against the species for the specific primary antibodies and conjugated with the fluorophores Alexa-488, Cy3 and Alexa-647. For Ascl1 detection we used a Tyramide Signal Amplification (TSA) protocol .Immunofluorescence was performed as previously described . BrieflyRbpj [Rbpj deletion and stroke could increase the generation of neuroblasts, or if the two conditions individually bring the neurogenic potential of striatal astrocytes to its full capacity.Astrocytes in the striatal parenchyma generate new neurons after stroke, as well as following ablation of Rbpj . We askefl/fl; R26-tdTomato mice, and a week later we induced an ischemic stroke using the middle cerebral artery occlusion model. Cx30-CreER allowed recombination specifically in astrocytes, and the reporter allele enabled identification of the recombined cells and their progeny [We induced conditional disruption of the gene for the obligate Notch coactivator Rbpj by administering tamoxifen to Cx30-CreER; Rbpj progeny . The mic+/Tomato+ cells were counted  compared with the contralateral uninjured side of the same animals  or with the stroke side from Rbpj+/+ animals . These findings demonstrated that the combination of stroke and blocking Notch-signaling increased the number of neuroblasts 2.5- and 3.5-fold, respectively, compared to either blocking Notch-signaling or stroke alone. Blocking Notch-signaling is thus an efficient way to promote neurogenesis after stroke.Neuroblasts are newly generated immature neurons, which can be identified by the expression of the marker Dcx. Tissue sections were stained for Dcx B, and Dc counted C. The nuClusters of neuroblasts appeared in more lateral positions within the striatum compared to the uninjured side in mice in which Notch-signaling had been blocked in astrocytes D. There Rbpj deletion [-/-Rbpj and stroke , the uninjured hemisphere of the same animals , and +/+Rbpj and stroke  compared to the contralateral uninjured striatum with Rbpj deletion only   A.p = 0.486) or in the medial striatum (p = 0.2), whereas the combination of stroke and Notch-signaling blockade led to a >40-fold increase in the number of Ascl1-expressing astrocytes in the lateral half of the striatum, with many of these cells residing in the stroke penumbra  compared to the uninjured side  (p = 0.18) (We quantified the number and size of Ascl1= 0.014) A. The di = 0.18) B.p = 0.34) or for neuroblasts   C,D.p = 0.022; 64 ± 40 vs. 21 ± 19 clustered cells/section; mean ± SD; p = 0.023). Single neuroblasts showed a significant increase in the stroke side compared to the contralateral  (p = 0.211). A possible explanation could be that neuroblasts, unlike transient amplifying progenitor cells, assumed a migratory phenotype during maturation and, in the presence of a stroke, detached from the cluster where they were generated.Finally, we compared the number of transient amplifying progenitor cells and neuroblasts that could be seen as singlets or in clusters in the injured hemisphere compared to the uninjured side. The numbers of transient amplifying Ascl1+/Tomato+ progenitor cells, both as single cells and clustered, were significantly increased in the stroke side  E, but thRbpj deletion recruits more astrocytes and increases neurogenesis approximately 3.5-fold in the striatum following stroke. After such blockade of Notch-signaling, more astrocytes in the striatum initiate the expression of the proneural transcription factor Ascl1 and give rise to a larger number of transit amplifying progenitor cell clusters and neuroblasts. Continuous Notch-signaling hinders astrocytes from entering the neurogenic trajectory in the healthy striatum. Stroke reduces Notch-signaling in astrocytes, but only a minority of astrocytes still initiates neurogenesis. Our finding that blocking Notch-signaling recruits substantially more astrocytes to neurogenesis following stroke suggests that the reduction in Notch-signaling caused by stroke is not of sufficient magnitude to recruit all striatal astrocytes competent to generate neurons.Ischemic stroke induces a limited neurogenic response in the striatum. Many of the generated neurons derive from local astrocytes, which initiate neurogenesis in response to the decreased Notch-signaling caused by the stroke. We report here that further reducing Notch-signaling by Rbpj deletion compared to Rbpj deletion alone also suggests that there are pathways other than Notch-signaling that may influence this process. Interestingly, whereas stroke or blocking Notch-signaling in isolation results in the induction of neurogenesis by astrocytes almost exclusively in the medial striatum, the combination results in substantial neurogenesis also in the lateral striatum. It seems possible that the medial striatum, with its proximity to the neurogenic niche in the subventricular zone, is a more neurogenesis-permissive environment with likely higher concentrations of mitogens. It is well known that brain injury results in increased concentrations of several mitogens and neurotrophic factors, which could contribute to the increased recruitment of astrocytes to the neurogenic lineage in the combination of stroke and blocked Notch-signaling. This is further supported by the finding that Rbpj deletion alone is insufficient to induce astrocytes in the somatosensory or occipital cortex to give rise to neurons, whereas the combination of inhibition of Notch signaling and a local injury triggers cortical neurogenesis by parenchymal astrocytes [The increased recruitment of astrocytes to neurogenesis by the combination of stroke and trocytes .There is currently no regenerative therapy to promote recovery after stroke, and the vast majority of neurons that die are never replaced. Therefore, the generation of new neurons from endogenous astrocytes represents an interesting therapeutic opportunity, in particular, since it may promote neuronal replacement without the need for intracerebral cell transplantation, which carries inherent risks. Our findings suggest that small molecules that inhibit Notch-signaling, which have been used in clinical trials for other indications, should be evaluated for a potential regenerative effect after stroke.