Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Body weight-based iodinated contrast immersion timing for human fetal postmortem microfocus computed tomography.

Objectives: The aim of this study was to evaluate the length of time required to achieve full iodination using potassium tri-iodide as a contrast agent, prior to human fetal postmortem microfocus computed tomography (micro-CT) imaging. Methods: Prospective assessment of optimal contrast iodination was conducted across 157 human fetuses (postmortem weight range 2-298 g; gestational age range 12-37 weeks), following micro-CT imaging. Simple linear regression was conducted to analyse which fetal demographic factors could produce the most accurate estimate for optimal iodination time. Results: Postmortem body weight (r2 = 0.6435) was better correlated with iodination time than gestational age (r2 = 0.1384), producing a line of best fit, y = [0.0304 × body weight (g)] - 2.2103. This can be simplified for clinical use whereby immersion time (days) = [0.03 × body weight (g)] - 2.2. Using this formula, for example, a 100-g fetus would take 5.2 days to reach optimal contrast enhancement. Conclusions: The simplified equation can now be used to provide estimation times for fetal contrast preparation time prior to micro-CT imaging and can be used to manage service throughput and parental expectation for return of their fetus. Advances in knowledge: A simple equation from empirical data can now be used to estimate preparation time for human fetal postmortem micro-CT imaging.

An integrated single-cell analysis of human adrenal cortex development.

The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but many aspects of human adrenal gland development are not well understood. Here, we combined single-cell and bulk RNA sequencing, spatial transcriptomics, IHC, and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with more cell division in the outer definitive zone (DZ). Steroidogenic pathways favored androgen synthesis in the central fetal zone, but DZ capacity to synthesize cortisol and aldosterone developed with time. Core transcriptional regulators were identified, with localized expression of HOPX (also known as Hop homeobox/homeobox-only protein) in the DZ. Potential ligand-receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g., IGF2, PEG3). These findings reveal aspects of human adrenal development and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders, and neonatal stress.

Less-invasive autopsy for early pregnancy loss.

Autopsy investigations provide valuable information regarding fetal death that can assist in the parental bereavement process, and influence future pregnancies, but conventional autopsy is often declined by parents because of its invasive approach. This has led to the development of less-invasive autopsy investigations based on imaging technology to provide a more accessible and acceptable choice for parents when investigating their loss. Whilst the development and use of more conventional clinical imaging techniques (radiographs, CT, MRI, US) are well described in the literature for fetuses over 20 weeks of gestational age, these investigations have limited diagnostic accuracy in imaging smaller fetuses. Techniques such as ultra-high-field MRI (>3T) and micro-focus computed tomography have been shown to have higher diagnostic accuracy whilst still being acceptable to parents. By further developing and increasing the availability of these more innovative imaging techniques, parents will be provided with a greater choice of acceptable options to investigate their loss, which may in turn increase their uptake. We provide a narrative review focussing on the development of high-resolution, non-invasive imaging techniques to evaluate early gestational pregnancy loss.

Micro-CT Imaging of Pediatric Thyroglossal Duct Cysts: A Prospective Case Series.

Objectives: To determine the feasibility of micro-CT as a high-resolution 3D imaging tool for thyroglossal duct cysts and to evaluate its role augmenting traditional histopathological examination of resected specimens. Methods: A single centre, prospective case series of consecutive children undergoing excision of a thyroglossal duct cyst was performed at a quaternary paediatric referral hospital in the United Kingdom. Consecutive children listed for excision of a thyroglossal duct cyst whose parents agreed to participate were included and there were no exclusion criteria. Results: Surgically excised thyroglossal duct cyst or remnant specimens from five patients (two males, three females) were examined using micro-CT alongside traditional histopathological examination. In all cases, micro-CT imaging was able to demonstrate 3D imaging datasets of the specimens successfully and direct radio-pathological comparisons were made (Figures 1-5, Supplementary Video 1). Conclusions: The study has shown the feasibility and utility of post-operative micro-CT imaging of thyroglossal duct cysts specimens as a visual aid to traditional histopathological examination. It better informs the pathological specimen sectioning using multi-planar reconstruction and volume rendering tools without tissue destruction. In the complex, often arborised relationship between a thyroglossal duct cyst and the hyoid, micro-CT provides valuable image plane orientation and indicates proximity of the duct to the surgical margins. This is the first case series to explore the use of micro-CT imaging for pediatric thyroglossal duct specimens and it informs future work investigating the generalizability of micro-CT imaging methods for other lesions, particularly those from the head and neck region where precisely defining margins of excision may be challenging.

A pragmatic evidence-based approach to post-mortem perinatal imaging.

Post-mortem imaging has a high acceptance rate amongst parents and healthcare professionals as a non-invasive method for investigating perinatal deaths. Previously viewed as a 'niche' subspecialty, it is becoming increasingly requested, with general radiologists now more frequently asked to oversee and advise on appropriate imaging protocols. Much of the current literature to date has focussed on diagnostic accuracy and clinical experiences of individual centres and their imaging techniques (e.g. post-mortem CT, MRI, ultrasound and micro-CT), and pragmatic, evidence-based guidance for how to approach such referrals in real-world practice is lacking. In this review, we summarise the latest research and provide an approach and flowchart to aid decision-making for perinatal post-mortem imaging. We highlight key aspects of the maternal and antenatal history that radiologists should consider when protocolling studies (e.g. antenatal imaging findings and history), and emphasise important factors that could impact the diagnostic quality of post-mortem imaging examinations (e.g. post-mortem weight and time interval). Considerations regarding when ancillary post-mortem image-guided biopsy tests are beneficial are also addressed, and we provide key references for imaging protocols for a variety of cross-sectional imaging modalities.

Human fetal whole-body postmortem microfocus computed tomographic imaging.

Perinatal autopsy is the standard method for investigating fetal death; however, it requires dissection of the fetus. Human fetal microfocus computed tomography (micro-CT) provides a generally more acceptable and less invasive imaging alternative for bereaved parents to determine the cause of early pregnancy loss compared with conventional autopsy techniques. In this protocol, we describe the four main stages required to image fetuses using micro-CT. Preparation of the fetus includes staining with the contrast agent potassium triiodide and takes 3-19 d, depending on the size of the fetus and the time taken to obtain consent for the procedure. Setup for imaging requires appropriate positioning of the fetus and takes 1 h. The actual imaging takes, on average, 2 h 40 min and involves initial test scans followed by high-definition diagnostic scans. Postimaging, 3 d are required to postprocess the fetus, including removal of the stain, and also to undertake artifact recognition and data transfer. This procedure produces high-resolution isotropic datasets, allowing for radio-pathological interpretations to be made and long-term digital archiving for re-review and data sharing, where required. The protocol can be undertaken following appropriate training, which includes both the use of micro-CT techniques and handling of postmortem tissue.

Improving uptake of perinatal autopsy.

PURPOSE OF REVIEW: Uptake of perinatal autopsy has declined in the West over the past 30 years, largely because of reduced parental acceptance of a traditional invasive autopsy. Several studies have recently investigated the decline to identify the key factors and how they may be mitigated. RECENT FINDINGS: Three main themes were identified that have been found to improve uptake of perinatal autopsy: improved communication, in particular ensuring the consent process was conducted as a conversation with time spent talking through the procedure and allowing time for questions; health professional training to ensure staff discussing autopsy with parents have adequate understanding of the procedure and are able to convey confidence and empathy; and availability of less invasive autopsy, including noninvasive as well as minimally invasive options. These should be offered alongside standard autopsy, which some parents may still prefer. SUMMARY: This review highlights that the discussions that take place, and the options that are available to parents, can profoundly impact whether or not they consent to autopsy investigation. Further research should focus on the impact of offering less invasive options as well as evaluating the training and support materials that have recently been developed.

Postmortem microfocus computed tomography for noninvasive autopsies: experience in >250 human fetuses.

BACKGROUND: Noninvasive imaging autopsy alternatives for fetuses weighing <500 grams are limited. Microfocus computed tomography has been reported as a viable option in small case series with the potential to avoid an invasive autopsy. Implementation of postmortem microfocus computed tomography in a large cohort as part of routine clinical service has yet been unreported, and realistic "autopsy prevention rates" are unknown. OBJECTIVE: This study aimed to describe the range of abnormalities detectable on fetal microfocus computed tomography in a clinical setting and additional findings identified on the antenatal ultrasound and to estimate the invasive autopsy avoidance rate (ie, cases in which imaging was sufficient to deem autopsy unnecessary). STUDY DESIGN: A prospective observational case series of all fetuses referred for microfocus computed tomography imaging at a single institution was conducted for 3 years (2016-2019). Imaging was reported by 2 pediatric radiologists before autopsy, with "decision to proceed" based on the specialist perinatal pathologists' judgment and parental consent. Agreement rates between microfocus computed tomography and antenatal ultrasound were evaluated, and where feasible, diagnostic accuracy for microfocus computed tomography was calculated using autopsy as a reference standard. RESULTS: A total of 268 fetuses were included (2-350 grams body weight; 11-24 weeks' gestation), with cause for demise in 122 of 268 (45.5%). Of the 122 fetuses, 64 (52.5%) exhibited fetal anomalies. Although 221 of 268 (82.5%) fetuses had consent for invasive autopsy, only 29 of the 221 (13.1%) underwent this procedure, which implied an autopsy avoidance rate of 192 of 221 (86.9%). Complete agreement was present for all brain, thoracic, and abdominal pathologies, whereas sensitivity and specificity for cardiac anomalies were 66.7% and 91.7%, respectively. Microfocus computed tomography and antenatal ultrasound agreement was found in 219 of 266 cases (81.9%), with partial agreement in 21 of 266 (7.9%) and disagreement in 26 of 266 (10.5%), mostly because of additional cardiac, soft tissue, or genitourinary findings by microfocus computed tomography, which were not seen on the ultrasound. CONCLUSION: Fetal microfocus computed tomography imaging is a viable and useful tool for imaging early gestational fetuses and can avoid the need for invasive autopsy. Confirmation of antenatal diagnoses is achieved in most cases, and additional anomalies may also be detected.

Micro-computed tomography (micro-CT) for the assessment of myocardial disarray, fibrosis and ventricular mass in a feline model of hypertrophic cardiomyopathy.

Micro-computed tomography (micro-CT) is a high-resolution imaging modality that provides accurate tissue characterization. Hypertrophic cardiomyopathy (HCM) occurs as a spontaneous disease in cats, and is characterized by myocardial hypertrophy, disarray and fibrosis, as in humans. While hypertrophy/mass (LVM) can be objectively measured, fibrosis and myocyte disarray are difficult to assess. We evaluated the accuracy of micro-CT for detection and quantification of myocardial disarray and fibrosis by direct comparison with histopathology. 29 cat hearts (12 normal and 17 HCM hearts) underwent micro-CT and pathologic examination. Myocyte orientation was assessed using structure tensor analysis by determination of helical angle (HA), fractional anisotropy (FA) and myocardial disarray index (MDI). Fibrosis was segmented and quantified based on comparison of gray-scale values in normal and fibrotic myocardium. LVM was obtained by determining myocardial volume. Myocardial segments with low FA, low MDI and disruption of normal HA transmural profile on micro-CT were associated with myocardial disarray on histopathology. FA was consistently lower in HCM than normal hearts. Assessment of fibrosis on micro-CT closely matched the histopathologic evaluation. LVM determined by micro-CT was higher in HCM than normal hearts. Micro-CT can be used to detect and quantify myocardial disarray and fibrosis and determine myocardial mass in HCM.

Investigation of optimal sample preparation conditions with potassium triiodide and optimal imaging settings for microfocus computed tomography of excised cat hearts.

OBJECTIVE: To determine optimal sample preparation conditions with potassium triiodide (I2KI) and optimal imaging settings for microfocus CT (micro-CT) of excised cat hearts. SAMPLE: 7 excised hearts (weight range, 10 to 17.6 g) obtained from healthy adult cats after euthanasia by IV injection of pentobarbital sodium. PROCEDURES: Following excision, the hearts were preserved in 10% formaldehyde solution. Six hearts were immersed in 1.25% I2KI solution (n = 3) or 2.5% I2KI solution (3) for a 12-day period. Micro-CT images were acquired at time 0 (prior to iodination) then approximately every 24 and 48 hours thereafter to determine optimal sample preparation conditions (ie, immersion time and concentration of I2KI solution). Identified optimal conditions were then used to prepare the seventh heart for imaging; changes in voltage, current, exposure time, and gain on image quality were evaluated to determine optimal settings (ie, maximal signal-to-noise and contrast-to-noise ratios). Images were obtained at a voxel resolution of 30 µm. A detailed morphological assessment of the main cardiac structures of the seventh heart was then performed. RESULTS: Immersion in 2.5% I2KI solution for 48 hours was optimal for sample preparation. The optimal imaging conditions included a tube voltage of 100 kV, current of 150 µA, and exposure time of 354 milliseconds; scan duration was 12 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: Results provided an optimal micro-CT imaging protocol for excised cat hearts prepared with I2KI solution that could serve as a basis for future studies of micro-CT for high resolution 3-D imaging of cat hearts.

Assessment of the Spatial Heterogeneity of Breast Cancers: Associations Between Computed Tomography and Immunohistochemistry.

BACKGROUND: Tumour heterogeneity is considered an important mechanism of treatment failure. Imaging-based assessment of tumour heterogeneity is showing promise but the relationship between these mathematically derived measures and accepted 'gold standards' of tumour biology such as immunohistochemical measures is not established. METHODS: A total of 20 women with primary breast cancer underwent a research dynamic contrast-enhanced computed tomography prior to treatment with data being available for 15 of these. Texture analysis was performed of the primary tumours to extract 13 locoregional and global parameters. Immunohistochemical analysis associations were assessed by the Spearman rank correlation. RESULTS: Hypoxia-inducible factor-1α was correlated with first-order kurtosis (r = -0.533, P = .041) and higher order neighbourhood grey-tone difference matrix coarseness (r = 0.54, P = .038). Vascular maturity-related smooth muscle actin was correlated with higher order grey-level run-length long-run emphasis (r = -0.52, P = .047), fractal dimension (r = 0.613, P = .015), and lacunarity (r = -0.634, P = .011). Micro-vessel density, reflecting angiogenesis, was also associated with lacunarity (r = 0.547, P = .035). CONCLUSIONS: The associations suggest a biological basis for these image-based heterogeneity features and support the use of imaging, already part of standard care, for assessing intratumoural heterogeneity.

3D printing from microfocus computed tomography (micro-CT) in human specimens: education and future implications.

Microfocus CT (micro-CT) is an imaging method that provides three-dimensional digital data sets with comparable resolution to light microscopy. Although it has traditionally been used for non-destructive testing in engineering, aerospace industries and in preclinical animal studies, new applications are rapidly becoming available in the clinical setting including post-mortem fetal imaging and pathological specimen analysis. Printing three-dimensional models from imaging data sets for educational purposes is well established in the medical literature, but typically using low resolution (0.7 mm voxel size) data acquired from CT or MR examinations. With higher resolution imaging (voxel sizes below 1 micron, <0.001 mm) at micro-CT, smaller structures can be better characterised, and data sets post-processed to create accurate anatomical models for review and handling. In this review, we provide examples of how three-dimensional printing of micro-CT imaged specimens can provide insight into craniofacial surgical applications, developmental cardiac anatomy, placental imaging, archaeological remains and high-resolution bone imaging. We conclude with other potential future usages of this emerging technique.

Early clinical applications for imaging at microscopic detail: microfocus computed tomography (micro-CT).

Microfocus CT (micro-CT) has traditionally been used in industry and preclinical studies, although it may find new applicability in the routine clinical setting. It can provide high-resolution three-dimensional digital imaging data sets to the same level of detail as microscopic examination without the need for tissue dissection. Micro-CT is already enabling non-invasive detailed internal assessment of various tissue specimens, particularly in breast imaging and early gestational fetal autopsy, not previously possible from more conventional modalities such as MRI or CT. In this review, we discuss the technical aspects behind micro-CT image acquisition, how early work with small animal studies have informed our knowledge of human disease and the imaging performed so far on human tissue specimens. We conclude with potential future clinical applications of this novel and emerging technique.

Predicting response to neoadjuvant chemotherapy in primary breast cancer using volumetric helical perfusion computed tomography: a preliminary study.

OBJECTIVES: To investigate whether CT-derived vascular parameters in primary breast cancer predict complete pathological response (pCR) to neoadjuvant chemotherapy (NAC). METHODS: Twenty prospective patients with primary breast cancer due for NAC underwent volumetric helical perfusion CT to derive whole tumour regional blood flow (BF), blood volume (BV) and flow extraction product (FE) by deconvolution analysis. A pCR was achieved if no residual invasive cancer was detectable on pathological examination. Relationships between baseline BF, BV, FE, tumour size and volume, and pCR were examined using the Mann-Whitney U test. Receiver operating characteristic (ROC) curve analysis was performed to assess the parameter best able to predict response. Intra- and inter-observer variability was assessed using Bland-Altman statistics. RESULTS: Seventeen out of 20 patients completed NAC with four achieving a pCR. Baseline BF and FE were higher in patients who achieved a pCR compared with those who did not (P = 0.032); tumour size and volume were not significantly different (P > 0.05). ROC analysis revealed that BF and FE were able to identify responders effectively (AUC = 0.87; P = 0.03). There was good intra- and inter-observer agreement. CONCLUSIONS: Primary breast cancers which exhibited higher levels of perfusion before treatment were more likely to achieve a pCR to NAC.

Diffusion tensor imaging of the anal canal at 3 tesla: feasibility and reproducibility of anisotropy measures.

PURPOSE: To assess the feasibility and reproducibility of 3-tesla diffusion tensor imaging (DTI) of the anal canal. MATERIALS AND METHODS: DTI was performed in 25 men with no clinical history of anal canal disease undergoing MRI for prostate cancer. Analysis of fractional anisotropy (FA), relative anisotropy (RA), and apparent diffusion coefficient (ADC) were determined for the epithelial/subepithelial layer, internal sphincter, external sphincter, and puborectalis. The directionality of diffusion was recorded from color-coded tractography maps. Obturator internus and gluteus maximus served as reference muscles. Mean (SD) of values for FA, RA, and ADC were compared using analysis of variance. Intra and inter-rater agreement and test reproducibility (n = 5) was assessed by Bland-Altman statistics. RESULTS: Mean (SD) for the epithelial/subepithelial layer, internal, external sphincter, and puborectalis were as follows: FA: 0.283 (0.099); 0.337 (0.049); 0.415 (0.072); and 0.407 (0.062), respectively. RA: 0.241 (0.094); 0.292 (0.050); 0.371 (0.083); 0.361 (0.067), respectively; and ADC: 1.49 (0.23); 1.59 (0.19); 1.51 (0.28); and 1.54 (0.29) × 10(-3) mm(2) /s, respectively. Good overall intra and inter-rater agreement and test-retest reproducibility was noted (coefficient of variation of 4.8-19.4% and 5.9-12.9%, respectively). CONCLUSION: Anisotropy is evident in the anal canal with good inter-rater agreement and test reproducibility.