Two-color diffuse in vivo flow cytometer.

Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal. Aim: The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs. Approach: A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo. Results: In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice. Conclusions: We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.

Supratherapeutic Tacrolimus Concentrations With Nirmatrelvir/Ritonavir in Solid Organ Transplant Recipients Requiring Hospitalization: A Case Series Using Rifampin for Reversal.

Nirmatrelvir/ritonavir was recently granted emergency use authorization for mild to moderate coronavirus disease 2019. Drug-drug interactions between ritonavir and tacrolimus are underappreciated by nontransplant providers. We describe 2 solid organ transplant recipients prescribed nirmatrelvir/ritonavir for outpatient use who developed tacrolimus toxicity requiring hospitalization and were managed with rifampin for toxicity reversal.

Signal and measurement considerations for human translation of diffuse in vivo flow cytometry.

SIGNIFICANCE: "Diffuse in vivo flow cytometry" (DiFC) is an emerging technology for fluorescence detection of rare circulating cells directly in large deep-seated blood vessels in mice. Because DiFC uses highly scattered light, in principle, it could be translated to human use. However, an open question is whether fluorescent signals from single cells would be detectable in human-scale anatomies. AIM: Suitable blood vessels in a human wrist or forearm are at a depth of ∼2 to 4 mm. The aim of this work was to study the impact of DiFC instrument geometry and wavelength on the detected DiFC signal and on the maximum depth of detection of a moving cell. APPROACH: We used Monte Carlo simulations to compute fluorescence Jacobian (sensitivity) matrices for a range of source and detector separations (SDS) and tissue optical properties over the visible and near infrared spectrum. We performed experimental measurements with three available versions of DiFC (488, 640, and 780 nm), fluorescent microspheres, and tissue mimicking optical flow phantoms. We used both computational and experimental data to estimate the maximum depth of detection at each combination of settings. RESULTS: For the DiFC detection problem, our analysis showed that for deep-seated blood vessels, the maximum sensitivity was obtained with NIR light (780 nm) and 3-mm SDS. CONCLUSIONS: These results suggest that-in combination with a suitable molecularly targeted fluorescent probes-circulating cells and nanosensors could, in principle, be detectable in circulation in humans.

Short-Term Circulating Tumor Cell Dynamics in Mouse Xenograft Models and Implications for Liquid Biopsy.

MOTIVATION: Circulating tumor cells (CTCs) are widely studied using liquid biopsy methods that analyze fractionally-small peripheral blood (PB) samples. However, little is known about natural fluctuations in CTC numbers that may occur over short timescales in vivo, and how these may affect detection and enumeration of rare CTCs from small blood samples. METHODS: We recently developed an optical instrument called "diffuse in vivo flow cytometry" (DiFC) that uniquely allows continuous, non-invasive counting of rare, green fluorescent protein expressing CTCs in large blood vessels in mice. Here, we used DiFC to study short-term changes in CTC numbers in multiple myeloma and Lewis lung carcinoma xenograft models. We analyzed CTC detections in over 100 h of DiFC data, and considered intervals corresponding to approximately 1%, 5%, 10%, and 20% of the PB volume. In addition, we analyzed changes in CTC numbers over 24 h (diurnal) periods. RESULTS: For rare CTCs (fewer than 1 CTC per ml of blood), the use of short DiFC intervals (corresponding to small PB samples) frequently resulted in no detections. For more abundant CTCs, CTC numbers frequently varied by an order of magnitude or more over the time-scales considered. This variance in CTC detections far exceeded that expected by Poisson statistics or by instrument variability. Rather, the data were consistent with significant changes in mean numbers of CTCs on the timescales of minutes and hours. CONCLUSIONS: The observed temporal changes can be explained by known properties of CTCs, namely, the continuous shedding of CTCs from tumors and the short half-life of CTCs in blood. It follows that the number of cells in a blood sample are strongly impacted by the timing of the draw. The issue is likely to be compounded for multicellular CTC clusters or specific CTC subtypes, which are even more rare than single CTCs. However, we show that enumeration can in principle be improved by averaging multiple samples, analysis of larger volumes, or development of methods for enumeration of CTCs directly in vivo.

Use of remdesivir in the presence of elevated LFTs for the treatment of severe COVID-19 infection.

An 82-year-old man with an extensive medical history presented to the emergency room with complaints of generalised weakness and cough. He tested positive for COVID-19 10 days prior to presenting to the emergency room. Although his symptoms started a week prior to diagnosis, his weakness increased, warranting emergency response. A comprehensive metabolic panel was drawn from the patient on admission, indicating markedly high liver function tests (LFTs) ≥20 times above the upper limit of normal. On day 1 of admission, the decision was still made to start remdesivir (5-day course) due to decompensated acute respiratory failure as well as dexamethasone. The patient's LFTs significantly improved throughout his hospital stay. The patient made a full recovery and was discharged on day 10 of hospitalisation.

Castor bean organelle genome sequencing and worldwide genetic diversity analysis.

Castor bean is an important oil-producing plant in the Euphorbiaceae family. Its high-quality oil contains up to 90% of the unusual fatty acid ricinoleate, which has many industrial and medical applications. Castor bean seeds also contain ricin, a highly toxic Type 2 ribosome-inactivating protein, which has gained relevance in recent years due to biosafety concerns. In order to gain knowledge on global genetic diversity in castor bean and to ultimately help the development of breeding and forensic tools, we carried out an extensive chloroplast sequence diversity analysis. Taking advantage of the recently published genome sequence of castor bean, we assembled the chloroplast and mitochondrion genomes extracting selected reads from the available whole genome shotgun reads. Using the chloroplast reference genome we used the methylation filtration technique to readily obtain draft genome sequences of 7 geographically and genetically diverse castor bean accessions. These sequence data were used to identify single nucleotide polymorphism markers and phylogenetic analysis resulted in the identification of two major clades that were not apparent in previous population genetic studies using genetic markers derived from nuclear DNA. Two distinct sub-clades could be defined within each major clade and large-scale genotyping of castor bean populations worldwide confirmed previously observed low levels of genetic diversity and showed a broad geographic distribution of each sub-clade.

Team training in the neonatal resuscitation program for interns: teamwork and quality of resuscitations.

OBJECTIVE: Poor communication and teamwork may contribute to errors during neonatal resuscitation. Our objective was to evaluate whether interns who received a 2-hour teamwork training intervention with the Neonatal Resuscitation Program (NRP) demonstrated more teamwork and higher quality resuscitations than control subjects. METHODS: Participants were noncertified 2007 and 2008 incoming interns for pediatrics, combined pediatrics and internal medicine, family medicine, emergency medicine, and obstetrics and gynecology (n = 98). Pediatrics and combined pediatrics/internal medicine interns were eligible for 6-month follow-up (n = 34). A randomized trial was conducted in which half of the participants in the team training arm practiced NRP skills by using high-fidelity simulators; the remaining practiced with low-fidelity simulators, as did control subjects. Blinded, trained observers viewed video recordings of high-fidelity-simulated resuscitations for teamwork and resuscitation quality. RESULTS: High-fidelity training (HFT) group had higher teamwork frequency than did control subjects (12.8 vs 9.0 behaviors per minute; P < .001). Intervention groups maintained more workload management (control subjects: 89.3%; low-fidelity training [LFT] group: 98.0% [P < .001]; HFT group: 98.8%; HFT group versus control subjects [P < .001]) and completed resuscitations faster (control subjects: 10.6 minutes; LFT group: 8.6 minutes [P = .040]; HFT group: 7.4 minutes; HFT group versus control subjects [P < .001]). Overall, intervention teams completed the resuscitation an average of 2.6 minutes faster than did control subjects, a time reduction of 24% (95% confidence interval: 12%-37%). Intervention groups demonstrated more frequent teamwork during 6-month follow-up resuscitations (11.8 vs 10.0 behaviors per minute; P = .030). CONCLUSIONS: Trained participants exhibited more frequent teamwork behaviors (especially the HFT group) and better workload management and completed the resuscitation more quickly than did control subjects. The impact on team behaviors persisted for at least 6 months. Incorporating team training into the NRP curriculum is a feasible and effective way to teach interns teamwork skills. It also improves simulated resuscitation quality by shortening the duration.

Heart rate variability in response to pain stimulus in VLBW infants followed longitudinally during NICU stay.

The objective of this longitudinal study, conducted in a neonatal intensive care unit, was to characterize the response to pain of high-risk very low birth weight infants (<1,500 g) from 23 to 38 weeks post-menstrual age (PMA) by measuring heart rate variability (HRV). Heart period data were recorded before, during, and after a heel lanced or wrist venipunctured blood draw for routine clinical evaluation. Pain response to the blood draw procedure and age-related changes of HRV in low-frequency and high-frequency bands were modeled with linear mixed-effects models. HRV in both bands decreased during pain, followed by a recovery to near-baseline levels. Venipuncture and mechanical ventilation were factors that attenuated the HRV response to pain. HRV at the baseline increased with post-menstrual age but the growth rate of high-frequency power was reduced in mechanically ventilated infants. There was some evidence that low-frequency HRV response to pain improved with advancing PMA.

Changes in the PQRST intervals and heart rate variability associated with rewarming in two newborns undergoing hypothermia therapy.

BACKGROUND: Little is known about the effects of hypothermia therapy and subsequent rewarming on the PQRST intervals and heart rate variability (HRV) in term newborns with hypoxic-ischemic encephalopathy (HIE). OBJECTIVES: This study describes the changes in the PQRST intervals and HRV during rewarming to normal core body temperature of 2 newborns with HIE after hypothermia therapy. METHODS: Within 6 h after birth, 2 newborns with HIE were cooled to a core body temperature of 33.5 degrees C for 72 h using a cooling blanket, followed by gradual rewarming (0.5 degrees C per hour) until the body temperature reached 36.5 degrees C. Custom instrumentation recorded the electrocardiogram from the leads used for clinical monitoring of vital signs. Generalized linear mixed models were calculated to estimate temperature-related changes in PQRST intervals and HRV. RESULTS: For every 1 degrees C increase in body temperature, the heart rate increased by 9.2 bpm (95% CI 6.8-11.6), the QTc interval decreased by 21.6 ms (95% CI 17.3-25.9), and low and high frequency HRV decreased by 0.480 dB (95% CI 0.052-0.907) and 0.938 dB (95% CI 0.460-1.416), respectively. CONCLUSIONS: Hypothermia-induced changes in the electrocardiogram should be monitored carefully in future studies.

The behavioral pain response to heelstick in preterm neonates studied longitudinally: description, development, determinants, and components.

OBJECTIVES: Preterm infants often experience multiple painful procedures during their stay in neonatal intensive care units (NICUs). The objectives of this study were to evaluate behavioral responses to heelstick in preterm newborns, characterize developmental changes and the effects of other demographic and clinical variables on the pain response, and estimate the contributions of individual Neonatal Infant Pain Scale (NIPS) behaviors to the summary pain score. METHODS: A longitudinal study was conducted to evaluate the behavioral responses of 35 preterm newborns to multiple heelstick procedures during their stay in the NICU. Sixty-one video recordings of blood collection by heel lance were evaluated for behavioral pain response using the NIPS. Generalized linear mixed models were calculated to address the study objectives. RESULTS: The increases in NIPS scores from the baseline to the blood draw were highly significant (mean baseline score=3.34, mean blood draw score=5.45, p<0.001). The newborns' pain responses increased an average of 0.23 points on the NIPS scale each week (p=0.002). Lower NIPS scores during the heelstick procedure were associated with four clinical variables: younger post-menstrual age at birth, lower birthweight, mechanical ventilation, and longer length of stay in the NICU. Crying, arousal state, and facial grimace contributed more than 85% of the increase in NIPS scores during the heelstick procedure. DISCUSSION: While behavioral responses to pain are attenuated in young, severely ill preterm newborns, they can be reliably detected. The most robust pain behaviors are crying, changes in arousal state, and facial grimacing.

Noise and light exposures for extremely low birth weight newborns during their stay in the neonatal intensive care unit.

OBJECTIVES: The objectives of this study were to characterize noise and light levels for extremely low birth weight newborns throughout their stay in the NICU, evaluate factors influencing noise and light levels, and determine whether exposures meet recommendations from the American Academy of Pediatrics. METHODS: Sound and light were measured inside the beds of extremely low birth weight newborns (n = 22) from birth to discharge. Measurements were recorded for 20 consecutive hours weekly from birth until 36 weeks' postmenstrual age, biweekly until 40 weeks, and every 4 weeks thereafter. Clinical variables including bed type and method of respiratory support were recorded at each session. RESULTS: Age-related changes in respiratory support and bed type explained the weekly increase of 0.22 dB in sound level and 3.67 lux in light level. Old incubators were the noisiest bed types, and new incubators were the quietest. Light levels were significantly higher in open beds than in incubators. The variations in noise and light levels over time were greatest for open beds. Noise and light levels were much less affected by respiratory support in incubators compared with open beds. A typical extremely low birth weight neonate was exposed to noise levels averaging 56.44 dB(A) and light levels averaging 70.56 lux during their stay from 26 to 42 weeks' postmenstrual age in the NICU. Noise levels were rarely within American Academy of Pediatrics recommendations (5.51% of the time), whereas light levels almost always met recommendations (99.37% of the time). CONCLUSIONS: Bed type and respiratory support explained differences in noise and light levels that extremely low birth weight newborns experience during their hospital stay. Noise levels exceeded recommendations, although evidence supporting those recommendations is lacking. Well-designed intervention studies are needed to determine the effects of noise reduction on the development of extremely low birth weight newborns.

Spectral analysis of time series of events: effect of respiration on heart rate in neonates.

Certain types of biomedical processes such as the heart rate generator can be considered as signals that are sampled by the occurring events, i.e. QRS complexes. This sampling property generates problems for the evaluation of spectral parameters of such signals. First, the irregular occurrence of heart beats creates an unevenly sampled data set which must either be pre-processed (e.g. by using trace binning or interpolation) prior to spectral analysis, or analyzed with specialized methods (e.g. Lomb's algorithm). Second, the average occurrence of events determines the Nyquist limit for the sampled time series. Here we evaluate different types of spectral analysis of recordings of neonatal heart rate. Coupling between respiration and heart rate and the detection of heart rate itself are emphasized. We examine both standard and data adaptive frequency bands of heart rate signals generated by models of coupled oscillators and recorded data sets from neonates. We find that an important spectral artifact occurs due to a mirror effect around the Nyquist limit of half the average heart rate. Further we conclude that the presence of respiratory coupling can only be detected under low noise conditions and if a data-adaptive respiratory band is used.

Intensive care noise and mean arterial blood pressure in extremely low-birth-weight neonates.

Noise in neonatal intensive care units (NICUs) may impede growth and development for extremely low-birth-weight (ELBW, < 1000 g) newborns. We calculated correlations between NICU sound levels and ELBW neonates' heart rate and arterial blood pressure to evaluate whether this population experiences noise-induced stress. Sound levels inside the incubator, heart rate (HR), and arterial blood pressure recordings were simultaneously collected for eight ELBW neonates for 15 minutes during the first week of life. Cross-correlation functions were calculated for NICU noise, HR, and mean arterial blood pressure (MABP) recordings for each subject. ELBW neonates' HR and MABP were significantly correlated ( R = 0.16 at 2-second lag time), with stronger correlation apparent for higher-birth-weight ELBW newborns (0.22 versus 0.10). Lower-birth-weight newborns responded to increased noise with HR acceleration from 45 to 130 seconds after noise events, and higher-birth-weight infants initially responded with an HR deceleration at 25 to 60 seconds, then HR acceleration ~175 seconds after noise increased. MABP was not as strongly correlated with NICU sound levels, although some correlation coefficients were slightly outside the 95% confidence interval. Higher-birth-weight newborns' more mature neurological systems may be responsible for stronger correlations between HR and MABP. NICU noise influenced newborns' HR, indicating that these infants hear and respond to NICU sounds. ELBW newborns in the first week of life seem to maintain a relatively stable blood pressure in response to moderate NICU sound levels (50 to 60 dBA).

Effects of hypoxic-ischemic encephalopathy and whole-body hypothermia on neonatal auditory function: a pilot study.

We assessed the effects of hypoxic-ischemic encephalopathy (HIE) and whole-body hypothermia therapy on auditory brain stem evoked responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We performed serial assessments of ABRs and DPOAEs in newborns with moderate or severe HIE, randomized to hypothermia ( N = 4) or usual care ( N = 5). Participants were five boys and four girls with mean gestational age (standard deviation) of 38.9 (1.8) weeks. During the first week of life, peripheral auditory function, as measured by the DPOAEs, was disrupted in all nine subjects. ABRs were delayed but central transmission was intact, suggesting a peripheral rather than a central neural insult. By 3 weeks of age, peripheral auditory function normalized. Hypothermia temporarily prolonged the ABR, more so for waves generated higher in the brain stem but the effects reversed quickly on rewarming. Neonatal audiometric testing is feasible, noninvasive, and capable of enhancing our understanding of the effects of HIE and hypothermia on auditory function.

A comparison of fetal and neonatal heart rate variability at similar post-menstrual ages.

Substantial differences of heart rate variability (HRV) were found between fetuses and prematurely born neonates in the high-frequency band of the power spectrum. The range of post-menstrual ages of the fetuses and neonates were closely matched in this study. Growth of HRV was observed in low-frequency and high-frequency bands, reflecting maturation of the autonomic nervous system. The higher level of fetal HRV in the high-frequency band persisted even after accounting for age-related changes. Multiscale entropy was also higher in fetuses than in prematurely born neonates. These results suggest that the autonomic balance is poorer among neonates born prematurely than in fetuses of identical post-menstrual age.

Noise in contemporary neonatal intensive care.

Weekly sound surveys (n = 63) were collected, using 5 s sampling intervals, for two modern neonatal intensive care units (NICUs). Median weekly equivalent sound pressure levels (LEQ) for NICU A ranged from 61 to 63 dB (A weighted), depending on the level of care. NICU B L(EQ) measurements ranged from 55 to 60 dB (A weighted). NICU B was recently built with a focus on sound abatement, explaining much of the difference between the two NICUs. Sound levels exceeded 45 dB (A weighted), recommended by the American Academy of Pediatrics, more than 70% of the time for all levels of care. Hourly L(EQ)s below 50 dB (A weighted) and hourly L10s below 55 dB (A weighted), recommended by the Sound Study Group (SSG) of the National Resource Center, were also exceeded in more than 70% of recorded samples. A third SSG recommendation, that the 1 s L(MAX), should not exceed 70 dB (A weighted), was exceeded relatively infrequently (< 11% of the time). Peak impulse measurements exceeded 90 dB for 6.3% of 5 s samples recorded from NICU A and 2.8% of NICU B samples. Twenty-four h periodicities in sound levels as a function of regular staff activities were apparent, but short-term variability was considerable.

Longitudinal assessment of heart rate variability in very low birth weight infants during their NICU stay.

BACKGROUND: Maturation of the autonomic nervous system has not been studied in high-risk very low birth weight (VLBW) infants in the first few weeks of life. AIM: To characterize developmental changes in autonomic nervous system activity of high-risk VLBW infants from 23 to 38 weeks post-menstrual age by measuring heart rate variability (HRV). STUDY DESIGN AND SUBJECTS: In this prospective cohort study 38 infants admitted to Children's Memorial Hermann Hospital NICU were longitudinally followed weekly or biweekly. Heart period data were recorded while infants were resting in active sleep. OUTCOME MEASURES: Growth of spectral power of HRV in low-frequency (0.05-0.25 Hz) and high-frequency (0.25-1.00 Hz) bands was modeled with linear mixed-effects models. The high-frequency power provides a measure of respiratory sinus arrhythmia (RSA). RESULTS: Low-frequency power increases with post-menstrual age, and intubated infants have lower HRV. The increase in low-frequency power is faster (0.50+/-0.12 dB/week) than the increase in RSA (0.17+/-0.09 dB/week). CONCLUSION: This longitudinal data exhibits developmental maturation of the RSA and of the low-frequency power of HRV in high-risk VLBW infants.