Ultrasound Obstet Gynecol 2014; 44: 600–609 Published online 12 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/uog.13330
Postmortem microcomputed tomography (micro-CT) of small fetuses and hearts C. M. LOMBARDI*#, V. ZAMBELLI†, G. BOTTA‡#, F. MOLTRASIO§, G. CATTORETTI¶**, V. LUCCHINI**∧, V. FESSLOVA†† and M. S. CUTTIN** *Department of Radiology-Studio Diagnostico Eco, Vimercate, Milan, Italy; †Department of Health Sciences, Universita´ di Milano-Bicocca, Milan, Italy; ‡Azienda Ospedaliera Citta` della Salute e delle Scienze di Torino, Presidio Ospedaliero OIRM-Sant’Anna, Turin, Italy; §Residency in Pathology, Universita´ degli Studi di Milano-Bicocca, Milan, Italy; ¶Department of Surgery and Interdisciplinary Medicine, Universita´ degli Studi di Milano-Bicocca, Milan, Italy; **Department of Pathology, AO San Gerardo, Monza, Italy; ††Pediatric Cardiology, IRCCS Policlinico San Donato, San Donato, Italy
K E Y W O R D S: congenital heart disease; fetal autopsy; first trimester; micro-CT; pregnancy; virtual autopsy
ABSTRACT Objective To assess the feasibility and utility of contrast-enhanced microcomputed tomography (microCT) for identifying structural anomalies in ex-vivo firstand second-trimester human fetuses and isolated fetal hearts. Methods Radiopaque iodine staining and micro-CT scanning protocols were first developed in rodent studies and then used to examine routinely fixed whole human fetuses (n = 7, weight 0.1–90 g, gestational age, 7–17 weeks) and isolated fetal hearts (n = 14, weight 0.1–5.2 g, gestational age, 11–22 weeks). Samples were scanned using an isotropic resolution of 18 (and, if necessary, 9 or 35) μm and findings were interpreted jointly by four fetal pathologists, a fetal cardiologist and a radiologist. Samples with gestational ages ≥ 13 weeks also underwent conventional autopsy or dissection. Results Micro-CT identified all anatomical structures and abnormalities documented by the macroscopic examination. In all seven cases involving fetuses ≤ 13 weeks (four fetuses, three isolated hearts), micro-CT excluded the presence of structural anomalies. In the remaining 14 cases, it provided all the information obtained with invasive autopsy or dissection and in seven of the 14 (two fetuses, five isolated hearts) it furnished additional diagnostic details. Conclusions This pilot study confirms the feasibility of postmortem contrast-enhanced micro-CT assessment of structural anomalies in whole small fetuses and fetal
hearts. Further study is needed to confirm our findings, particularly in whole fetuses, and to define the extent to which this virtual examination might be used instead of conventional invasive autopsy. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
INTRODUCTION Prenatal ultrasound-based diagnoses of structural anomalies (most involving the heart and great vessels) are being made at increasingly earlier gestational ages thanks to the ongoing technical refinement of equipment and the now widespread use of first-trimester aneuploidy screening1 . Experienced operators can identify most major structural2,3 and cardiac4 – 6 abnormalities during the first-trimester nuchal scan. Postmortem examination of the fetus and/or fetal organs results in modification or refinement of the prenatal diagnosis in roughly 30% of cases7 , but its feasibility and value are limited in very small fetuses. Parental objections to conventional autopsy have also contributed to a decline in the rate of perinatal autopsies8 – 12 . Several groups have proposed alternatives to postmortem dissection, including virtual autopsy based on imaging modalities such as magnetic resonance imaging (MRI). While the resolution offered by scanners used for conventional whole-body MRI in the clinical setting is too low for successful examination of first- and second-trimester fetuses, more promising results have been obtained with high-field (9.4-T) micro-MRI scanners designed for imaging small laboratory animals9,13 .
Correspondence to: Dr C. Lombardi, Department of Radiology-Studio Diagnostico Eco, Via Cremagnani 15, 20871 Vimercate, Milan, Italy (e-mail: [email protected]); Dr M. S. Cuttin, Department of Pathology, AO San Gerardo, Via Pergolesi 33, 20900 Monza, Italy (e-mail: [email protected]) #C.M.L. and G.B. contributed equally to this paper. ∧Deceased, 31 December 2013. Accepted: 31 January 2014
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ORIGINAL PAPER
Micro-CT of small fetuses and hearts Like all MRI-based approaches, however, their use in a clinical setting is limited by cost, time and space constraints. Computed tomography has been considered less suitable for virtual autopsy since complete depiction of the fetal anatomy requires impregnation of the body tissues with intravenously diffused contrast medium, which is not feasible after death (although direct injection of contrast agents into cardiac chambers and great vessels has been used for postmortem identification of certain cardiovascular anomalies in human fetuses14 ). However, micro-CT, has been used recently to examine fixed small-mammal (whole-body and isolated heart) specimens after their immersion in contrast media15,16 , and this method has displayed high accuracy in the identification of heart defects in neonatal and fetal mice17 . The present study was conducted to assess the feasibility and potential utility of this approach for identifying structural anomalies in ex-vivo first- and second-trimester human fetuses and isolated human fetal hearts (Videoclip S1).
METHODS
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pathologist (G.B., V.L. or M.S.C.) with full access to the results of the micro-CT examination. Our examination of the remaining 14 fetuses (gestational ages ranging from 11 to 22 weeks) was limited to the hearts, which had been identified as isolated specimens in the material submitted for postmortem examination (two cases) or were isolated en bloc with the great vessels by an experienced fetal pathologist (G.B., V.L. or M.S.C.). Hearts from fetuses aged ≥ 13 weeks were also subjected to conventional dissection by one of the fetal pathologists. In these cases, the order in which the two procedures were performed varied, and findings from the first examination were always made available to operators conducting the second examination. In six cases, in which prenatal ultrasound findings of a major cardiac defect were available, the fetal hearts underwent standard four-chamber sectioning first, and a full heart dissection was completed after the micro-CT study. Karyotypes were determined for all 21 human fetuses via analysis of amniotic fluid cells or fetal tissues, as described elsewhere4 .
Animal studies
Micro-CT scans
We examined the hearts or whole bodies of 53 ex-vivo rodents (42 male C57/BL6J mice weighing 22–25 g, 11 Sprague Dawley rats weighing 200–250 g) used in research conducted at the University of Milan-Bicocca with the approval of the Italian Ministry of Health and the University’s Animal Care Unit. Shortly after their death, 51 of the animals were dissected en bloc by laboratory personnel; the heart was removed from the chest, isolated from the lungs and blotted. The other two animals (mice weighing 24 and 30 g) were used for whole-body studies. Both had died spontaneously for reasons unrelated to the experimental protocol. The whole-body specimens (skinned to facilitate staining) and isolated hearts were fixed in 4% aqueous buffered formaldehyde, stored at room temperature, and imaged after 2–60 days.
Protocol development in animals
Ex-vivo human fetuses and fetal hearts All 21 of the ex-vivo human fetuses used for this study had been submitted (with parental consent) for postmortem examination in the Departments of Pathology of the University of Milan-Bicocca, San Gerardo Hospital or the University of Turin. The micro-CT studies we performed were considered part of the authorized autopsy by the institutional review boards. Sensitive data were anonymized in all cases. Upon receipt, each fetus was fixed in 4% buffered formaldehyde and stored at room temperature. The seven fetuses used for whole-body studies had succumbed to unexplained intrauterine death at gestational ages ranging from 7 to 17 weeks. Prenatal ultrasound findings were not available in any of these cases. The interval between miscarriage and micro-CT examination was 4–7 days. For fetuses aged ≥ 13 weeks, the virtual autopsy was followed by conventional autopsy/dissection. The latter was performed by an experienced fetal
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Staining and scanning protocols were developed in the animals, using as a reference the methods described by Degenhardt et al.15 for the study of mouse embryos. The protocol thus defined was then used to examine the human fetal specimens. All micro-CT scans were performed by an experienced radiologist (C.L.) with specific training in the use of micro-CT. Tissues were weighed and then stained by immersion in Lugol solution (10 g potassium iodide and 5 g iodine in 100 mL water), which was tested using different dilutions (25–100% in demineralized water), exposure times (48–100 h) and staining solution volume/specimen weight ratios (2–10 mL per g specimen weight). Immediately after staining, specimens were washed with alcohol to remove free iodine, blotted dry and subjected to micro-CT. All scans were performed with a Bruker Skyscan 1176 micro-CT scanner (Bruker, Brussels, Belgium) with a gantry diameter of 68 mm. Fixed acquisition times of 4, 6 and 25 min were used for resolutions at 35, 18 and 9 μm, respectively. The exposure time was 65, 300 and 1050 ms, respectively, the voltage was 80 kV in all cases, the current was 300, 313 and 300 μA, respectively, and the rotation step 0.70◦ , 0.50◦ and 0.30◦ , respectively. The projection data were corrected for distortion and reconstructed by adjusting, smoothing and correction of ring artifact and beam hardening. The reconstructed isotropic voxel size was 9, 18 and 35 μm3 , respectively. Images were reconstructed with the scanner software (NRecon 1.6.6.0, Skyscan, Brucker micro-CT, Belgium) and converted to DICOM format for analysis. The time for reconstruction ranged from 2 to 60 min, depending on the volume of the sample. Data obtained were analyzed on a Mac Pro computer (2.8 GHz Quad-Core, 16 GB memory, high-performance
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PCI Express graphics cards; Apple Inc., Cupertino, CA, USA) and examined using an OsiriX DICOM Viewer (64-bit format, version 5.8 OsiriX Foundation, Geneva, Switzerland). For external macroscopic examinations of the specimens, reconstruction was performed with surface rendering, an algorithm-based technique for extracting the surface components of the acquired volume. Volume rendering (a technique used to display a two-dimensional projection of a three-dimensional sampled dataset) was used for ‘virtual dissection’ of the specimen. Diagnostic imaging in human fetuses Fetuses and hearts were stained and scanned using specimen weight-specific protocols defined in our animal studies. Multiple scans were necessary when the specimen size exceeded 22 mm (the field of view of the micro-CT scanner in use). The technical quality of each scan was verified by the radiologist with a preliminary post-scan assessment. Findings were later examined jointly by all the specialists involved in the study (i.e. pathologists, fetal cardiologist, radiologist), with full access to any available data obtained during prenatal ultrasound studies or conventional autopsy/dissection. Analysis of the fetal anatomy depicted by the micro-CT scans included systematic examination of the following structures: skull and brain (skull integrity, lateral ventricles, thalami, cerebellum and cisterna magna); face (orbits, lenses and facial profile); full-length spine (vertebral alignment and overlying skin from cervical to sacral region); heart (four-chamber view of atria and ventricles, crux and atrioventricular valves; three-vessel cross-sectional view of pulmonary artery, aorta and superior vena cava); abdomen (stomach, abdominal wall, liver, spleen and umbilical cord insertion); kidneys and bladder; extremities (long bones, fingers and toes).
RESULTS Animal studies Iodine staining protocols for mouse samples were defined in studies of two whole ex-vivo mice (weight, 24 and 30 g), similar in size to a 14-week human fetus, and in 51 rat/mouse hearts (weight, 110–1159 mg), comparable in size to those of human fetuses at 12–16 weeks of gestation. Lugol solution concentrations, volumes and immersion times were adjusted to specimen size in order to obtain uniform, full-thickness staining. The final protocols that emerged from these experiments are shown in Table 1. The micro-CT equipment we used offered scan resolutions of 35, 18 and 9 μm. Comparison of images of the same specimen obtained at 9 vs 18 μm revealed no significant loss of diagnostic detail under the latter condition. Therefore, all specimens (regardless of size) were imaged at 18 μm, which required only 6 min per specimen as opposed to 24 min per specimen at 9 μm. If necessary, Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
Table 1 Staining protocols
Type A B C
Weight (g)
Lugol* (%)
Solution volume (mL)
Staining time (h)
2
25 50 50
10 10–20 20–900
48 48 72
*Lugol solution: 10 g potassium iodide and 5 g iodine in 100 mL water, diluted 25% or 50% in demineralized water.
details of small specimens (i.e. those weighing < 1 g) were re-examined at the higher resolution. For large specimens (> 2 g), which generally required two or more scans, pre-screening at 35 μm was followed by targeted imaging at 18 μm (when needed). Depending on specimen size and the level of resolution used, the volume of scan data for each specimen ranged from 0.8 to 2.5 gigabytes. As described previously15 , the stained samples displayed variable degrees of shrinkage caused by the high osmolarity of the Lugol solution. The loss was proportional to the solution concentration and the immersion time. These changes did not compromise scan readability. The duration of storage in 4% buffered formaldehyde (range, 4–60 days) had no effect on staining intensity or uniformity. Insufficient solution volumes were associated with weak, non-uniform contrast; no effects were observed with excessive volumes. Optimal results were obtained using approximately 10 mL staining solution for every gram of pre-stained specimen weight. The staining procedure had no effect on histological examination of the internal organs during subsequent conventional autopsy.
Human fetal studies Whole-body specimens Contrast-enhanced micro-CT was performed on seven whole fetuses (weight, 0.1–90 g) that had undergone unexplained intrauterine death at gestational ages of 7–17 weeks (Figure 1 and Videoclips S2–S4 and S6–S8). The characteristics of these fetuses are shown in Table 2. None presented any external signs of maceration and, at the time of the micro-CT scan, none had undergone any type of invasive procedure. Specimen preparation (rinsing with alcohol and drying) required < 5 min. Scan times varied depending on the resolution(s) used (6 min at a resolution of 18 μm). As shown in Table 2, all four of the fetuses aged ≤ 11 weeks (Fetuses 1–4) were judged to be structurally normal on the basis of micro-CT findings. Conventional autopsy was not attempted in any of these cases because of size constraints. In Fetus 5 (gestational age, 14 weeks), the micro-CT scan revealed clefts of both the lip and palate (Figure 2), the latter being unilateral and involving the entire length of the palate. On subsequent autopsy, only the lip involvement could be identified, and size restrictions made it impossible to assess the extent of the palate defect. Fetus 6 (Figure 1d and Videoclips S6 and S7) and
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Figure 1 Contrast-enhanced microcomputed tomography of whole (entire) fetuses from 7 to 15 weeks’ gestation, at resolution of 18 μm. Three-dimensional surface rendering is shown on the left, and the internal anatomical details obtained with volume sections are shown on the right. (a) Fetus 1 (Table 2), 7 weeks; (b) Fetus 3, 10 weeks; (c) Fetus 4, 11 weeks; (d) Fetus 6, 15 weeks. Scale bar in (a) is 1 mm and in (b,c,d) is 10 mm.
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Table 2 Contrast-enhanced microcomputed tomography (micro-CT) and conventional autopsy findings in the seven whole fetuses Fetal age (GW)
CRL (mm)
Weight (g)
1 2 3 4 5 6
7 9 10 11 14 15
7 22 28 46 77 100
7
17
130
Fetus
Staining type
Micro-CT findings
Conventional autopsy findings
Karyotype
0.1 0.2 0.4 2.6 7.5 28
A A A C C C
Not performed Not performed Not performed Not performed External examination: cleft lip Normal
Normal Normal Normal Normal Normal Normal
90
C
Normal Normal Normal Normal Cleft lip and palate Normal: detailed depiction of brain structure Normal fetus with periventricular hemorrhage
Normal fetus with germinal matrix bleeding
Normal
CRL, crown–rump length; GW, gestational weeks.
Figure 2 Fetus 5 (Table 2). (a) Contrast-enhanced microcomputed tomography scan surface rendering and external survey at 14 weeks revealed only a cleft lip (arrow). (b) The presence of a full-length, unilateral cleft palate (arrow) is documented in an axial plane. Scale bar: 10 mm.
Fetus 7 required three scans each, due to their size. Pre-screening at 35 μm and re-exploration of the regions of interest (brain, heart) at 18 μm revealed no structural abnormalities. In both cases, the scan provided excellent depiction of the fetal brain structures, including the posterior fossae. In Fetus 7, this was especially useful in detecting and characterizing a periventricular hemorrhage. The scan revealed involvement of the caudothalamic notch and glomi of choroid plexi (Figure 3 and Videoclip S8). On subsequent autopsy, the same lesion was defined only as ‘germinal matrix bleeding’. Isolated hearts Table 3 shows the characteristics of the 14 fetuses whose hearts were examined and the results of the micro-CT and conventional dissection examinations. Hearts from Cases
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1, 2 and 3 were included in the study because their size (0.1–0.2 g) precluded conventional dissection. In all three cases, the virtual micro-CT autopsy allowed us to exclude structural anomalies in the heart (Figure 4a). In hearts from Cases 4 (Videoclip S5), 5, 7, 9 and 10, the virtual autopsy was performed before conventional dissection or after an initial external examination of the specimen. The first four hearts in this subgroup were from fetuses with trisomy 21, and the pathologists asked that the micro-CT scan be carried out before the heart was dissected. In Case 10 the fetus had a normal karyotype, but external examination of the body had revealed multiple structural malformations and the macroscopic examination of the heart raised the suspicion of right isomerism. Both micro-CT and subsequent conventional dissection revealed normal cardiac anatomy in all five of
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Figure 3 Fetus 7 (Table 2). Longitudinal (a) and axial (b) study of the brain using contrast-enhanced microcomputed tomography at 17 weeks demonstrated the presence of hemorrhage (arrows). Scale bar: 10 mm. Table 3 Contrast-enhanced microcomputed tomography (micro-CT) and conventional autopsy findings in the 14 isolated fetal hearts Fetal age (GW)
Organ weight (g)
1 2 3 4
11 12 13 14
5 6 7 8 9 10 11 12 13 14
15 15 16 16 17 17 18 19 19 22
Case
Staining type
Micro-CT findings
Conventional autopsy findings
0.1 0.15 0.2 1.1
A A A B
1.6 1.6 2.3 2.4 3.3 3 4 4 4 5.2
B B C C C C C C C C
Normal Normal Normal Normal intracardiac findings, ARSA Normal Truncus arteriosus Normal AVSD balanced Normal Normal Right isomerism, AVSD Pulmonary atresia, hypoplastic RV Truncus arteriosus type 1 AVSD + TGA
Not performed Not performed Not performed Normal intracardiac findings, ARSA Normal Truncus arteriosus Normal Abnormality undefined Normal Normal Right isomerism Abnormal univentricular Truncus arteriosus or TOF AVSD + TGA or TOF
Karyotype Normal Normal Trisomy 18 Trisomy 21 Trisomy 21 Normal Trisomy 21 Normal Trisomy 21 Normal Normal Normal Normal Normal
ARSA, aberrant right subclavian artery; AVSD, atrioventricular septal defect; GW, gestational weeks; RV, right ventricle; TGA, transposition of great vessels; TOF, Tetralogy of Fallot.
these cases. In Case 4, both examinations also revealed an aberrant right subclavian artery (known to be associated with trisomy 21). In Cases 6, 8, 11, 12, 13 and 14, prenatal ultrasonography had indicated the presence of a major cardiac defect although no precise diagnosis had been made. In these cases, the virtual micro-CT autopsy was performed after external examination and standard four-chamber sectioning. After micro-CT, these six hearts were subjected to full dissection by the pathologists. In Case 6, both virtual autopsy and conventional dissection confirmed the presence of a truncus arteriosus, but no pulmonary artery or other vessels were seen arising at that level. In Case 8 (Figure 5), standard four-chamber sectioning indicated the presence of a major structural anomaly but failed to yield a precise diagnosis. The subsequent micro-CT scan clearly revealed an atrioventricular septal defect. In Case 11, both examinations disclosed right atrial isomerism, but the micro-CT examination also demonstrated the presence of an atrioventricular
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septal defect. In Case 12, the virtual autopsy (Figure 6) and preliminary dissection yielded clearly discordant results: the former indicated the presence of right ventricular hypoplasia and pulmonary atresia, the latter a univentricular heart. In Cases 13 and 14, the micro-CT diagnosis was largely concordant with that based on the preliminary dissection, but in Case 13 (Figure 7) it provided additional details, which allowed specification of the type of truncus arteriosus. In Case 14, the dissection findings were suggestive of either tetralogy of Fallot or transposition of the great vessels. The latter diagnosis was confirmed by the micro-CT study.
DISCUSSION In this study, we developed a contrast-enhanced micro-CT protocol for postmortem examination of whole-body specimens and isolated hearts from embryos or fetuses with gestational ages ranging from 7 to 22 weeks. This
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Figure 4 Contrast-enhanced microcomputed tomography of excised normal fetal hearts at 11 (a) and 15 (b,c) gestational weeks. (a) Heart 1 (Table 3). Volume rendering, showing a virtual dissection at the level of the great vessels: aorta (Ao) and pulmonary artery (PA) are visible, with a branch of the pulmonary artery with diameter 100 μm (arrow). (b) Heart 5 (Table 3). Macroscopic examination obtained with surface rendering. (c) Heart 5 (Table 3). Volume rendering section at the level of the crux. Arrow indicates insertion point of tricuspid septal leaflet. Scale bar: 1 mm.
Figure 5 Heart 8 (Table 3). Contrast-enhanced microcomputed tomography of excised fetal heart at 16 gestational weeks in a case of atrioventricular septal defect, showing evidence of a large defect between atria and ventricles and a common atrioventricular valve (curved arrow). Scale bar: 1 cm.
virtual autopsy yielded diagnoses in all seven cases (four whole fetuses, three hearts) in which conventional autopsy or dissection was precluded due to size restrictions. In seven other cases it provided all the information that could be obtained with conventional autopsy, and
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in the last seven (two whole fetuses and five isolated hearts), it also furnished additional details that improved diagnostic accuracy. Virtual fetal autopsy has been attempted with conventional whole-body MRI using a 1.5-T scanner, but the maximum resolution that could be achieved with this approach (800 μm) was too low11,18,19 . Somewhat better results have been obtained recently with a 3-T scanner with a maximum resolution of 300 μm20,21 . Thayyil et al.9 were the first to propose the study of small fetuses with micro-MRI, a well-established method for examining small laboratory animals22 – 26 . In the 17 fetuses (age, 11–22 weeks; weight, 5–400 g) that they studied with a 9.4-T micro-MRI scanner, the image resolution used (200 μm) was reportedly sufficient for detecting the structural abnormalities that could be identified with conventional autopsy and also for internal examination of the visceral organs. However, this high definition was at the expense of scan times greater than 90 min. The authors reported that their approach could also be carried out at a resolution of 18 μm (the level we achieved with micro-CT protocols), but it required scan times of over 18 h (compared with our time of 6 min using micro-CT). Prior to the present study, we carried out a preliminary series of experiments using a 7-T micro-MRI scanner (Pharmascan 70/16 US, Bruker Biospin, Karlsruhe, Germany) (results not shown) and observed similarly excessive scan times. We have shown that micro-CT analysis of iodine-stained tissues, thus far used only for small animal studies15,17 , can be used for postmortem examination of whole human fetuses with gestational ages of up to 17 weeks and for isolated fetal organs such as the heart. This approach offered several important advantages. First, it provided diagnoses in cases in which conventional autopsy or dissection was precluded by the
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Figure 6 Heart 12 (Table 3). Contrast-enhanced microcomputed tomography of excised fetal heart at 19 gestational weeks, with ventricles partially sectioned and shown surface-reconstructed (a) and digitally sectioned (b). The features of pulmonary atresia are evident: hypoplastic right ventricle (RV) with an imperforate hypoplastic pulmonary valve (arrow) and a large left ventricle (LV). Note the details of a cross-sectioned esophagus on the right in (b). Scale bar: (a) 1 cm; (b) 1 mm.
reduced dimensions of the fetus or the excised heart. It is important to note, however, that these diagnoses were not verified by other methods (an obvious limitation of the present study). Second, it furnished all information that could be obtained with autopsy or dissection and in many cases it provided important additional findings. Furthermore, it provided stable, detailed digital documentation of the postmortem findings that could be analyzed and interpreted by an interdisciplinary team of specialists (radiologist, pathologists, cardiologist), with potential gains in the accuracy of the diagnosis. The documented findings could also be archived, re-evaluated and used during remote consultation or for teaching purposes. Conventional perinatal autoptic confirmation of the fetal anatomy should be mandatory in cases of fetal demise27 , but there are obvious difficulties related to specimen size, especially in the early stages of gestation. Small specimens are not a limitation for virtual micro-CT autopsy, and it has the potential to provide greater diagnostic detail during the examination of small whole fetuses or excised organs. The cost and size of a commercial laboratory micro-CT scanner are similar to those of high-quality ultrasound equipment. Compared with high-field micro-MRI imaging, micro-CT scanners are characterized by much lower purchase and operating costs, much shorter scan times and markedly higher spatial resolution. These features make it a potentially valuable tool for pathologists, who are faced increasingly with requests for postmortem examination of fetuses that have died in utero during the early stages of gestation.
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It is important to note that while the advantages of iodine solutions to stain specimens (low cost, minimal toxicity and ease of preparation) have been documented in several studies on small mammals15 , these solutions also inevitably cause some degree of tissue shrinkage. This effect can alter the volume of the specimen, but not the relation between the structures. The resulting tissue distortion did not interfere with our examination of isolated fetal hearts, but it could produce artificial increases in the volume of body cavities, which might lead to erroneous suspicion of effusion. It should also be noted that our study was limited to whole fetuses aged ≤ 17 weeks. This decision was dictated in part by the bore diameter of the scanner we used, which was too small to accommodate the heads of older fetuses. However, there are already scanners on the market with larger bore diameters (e.g. the Siemens Inveon with a 12-cm internal bore and maximum resolution of 20 μm). A more serious potential limitation with larger fetuses is related to contrast-agent penetration of the internal organs, although the exact size and weight cut-offs have yet to be defined. (Our protocol ensured effective staining only of fetuses that could be examined with the scanner we used.) At the internal organ level, our attention was focused largely on examination of the fetal heart, which is the site of most of the congenital anomalies that can be detected by expert sonographers during early prenatal scans5 . We chose to examine isolated hearts because, when pregnancy is terminated early, the heart can usually be recovered by systematic examination of the material
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Figure 7 Heart 13 (Table 3). Contrast-enhanced microcomputed tomography of excised fetal heart at 19 gestational weeks, showing features of truncus arteriosus type I on surface-rendering (a) and digital sectioning (b): a large vessel overriding the ventricular septal defect and a small pulmonary artery (PA, arrow) originating from the truncus (T). Scale bar: 1 cm.
collected during uterine evacuation28 . Many features of first-trimester fetal heart anomalies that were previously unrecognized owing to the technical limitations of the postmortem examination can now be confirmed and clarified with contrast-enhanced micro-CT. Our experience in this pilot study requires confirmation in larger studies, but it suggests that postmortem micro-CT examination of small fetuses and fetal organs offers clear advantages over traditional autopsy and dissection, particularly for assessing anatomical and topographic features. Nonetheless, dissection and serial sectioning will continue to be necessary in some cases, for example those requiring collection of samples for histological and/or biomolecular studies.
ACKNOWLEDGMENTS This paper is dedicated to the late Anna Cappellini, M.D. (1943–2008), teacher and mentor for all of us. We wish to thank Dr Giacomo Bellani (Department of Health Sciences, Universita´ di Milano-Bicocca) for support and advice, Dr Sophie Lombardi (Residency in Radiology, Universita´ di Milano-Bicocca) for helping in the software setup and rendering, Dr Sonia Gorla (Department of Pathology, Ospedali di Desio-Vimercate) for providing diagnostic specimens, the San Gerardo Hospital Pharmacy for providing the Lugol solution and the laboratory technicians of the hospital for the diagnostic work. This study is self-financed with San Gerardo Hospital departmental funds and University of Milano-Bicocca startup funding.
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Micro-CT of small fetuses and hearts 10. Ben-Sasi K, Chitty LS, Franck LS, Thayyil S, Judge-Kronis L, Taylor AM, Sebire NJ. Acceptability of a minimally invasive perinatal/paediatric autopsy: healthcare professionals’ views and implications for practice. Prenat Diagn 2013; 33: 307–312. 11. Breeze ACG, Jessop FA, Set PAK, Whitehead AL, Cross JJ, Lomas DJ, Hackett GA, Joubert I, Lees CC. Minimally-invasive fetal autopsy using magnetic resonance imaging and percutaneous organ biopsies: clinical value and comparison to conventional autopsy. Ultrasound Obstet Gynecol 2011; 37: 317–323. 12. Cannie M, Votino C, Moerman P, Vanheste R, Segers V, Van Berkel K, Hanssens M, Kang X, Cos T, Kir M, Balepa L, Divano L, Foulon W, De Mey J, Jani J. Acceptance, reliability and confidence of diagnosis of fetal and neonatal virtuopsy compared with conventional autopsy: a prospective study. Ultrasound Obstet Gynecol 2012; 39: 659–665. 13. Votino C, Jani J, Verhoye M, Bessieres B, Fierens Y, Segers V, Vorsselmans A, Kang X, Cos T, Foulon W, De Mey J, Cannie M. Postmortem examination of human fetal hearts at or below 20 weeks’ gestation: a comparison of high-field MRI at 9.4 T with lower-field MRI magnets and stereomicroscopic autopsy. Ultrasound Obstet Gynecol 2012; 40: 437–444. 14. Votino C, Cannie M, Segers V, Dobrescu O, Dessy H, Gallo V, Cos T, Damry N, Jani J. Virtual autopsy by computed tomographic angiography of the fetal heart: a feasibility study. Ultrasound Obstet Gynecol 2012; 39: 679–684. 15. Degenhardt K, Wright AC, Horng D, Padmanabhan A, Epstein JA. Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining. Circ Cardiovasc Imaging 2010; 3: 314–322. 16. Metscher BD. MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions. Dev Dyn 2009; 238: 632–640. 17. Kim A J, Francis R, Liu X, Devine WA, Ramirez R, Anderton SJ, Wong LY, Faruque F, Gabriel GC, Leatherbury L, Tobita K, Lo CW. Microcomputed tomography provides high accuracy congenital heart disease diagnosis in neonatal and fetal mice. Circ Cardiovasc Imaging 2013; 6: 551–559. 18. Alderliesten ME, Peringa J, van der Hulst VPM, Blaauwgeers HLG, van Lith JMM. Perinatal mortality: clinical value
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SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Videoclip S1 Animation of preparation for microcomputed tomography (micro-CT). Fetal tissues are treated with contrast medium by placing the specimen into a flask of water into which iodine (Lugol) is added. By osmosis of the hypertonic solution, there is passive transfer of iodine into the specimen. The fetus is then placed in the micro-CT scanner, a small laboratory device which has been used thus far only in preclinical research on small animals. Videoclip S2 Fetus 1. Virtuoscopy (virtual autopsy) by sequential dorsal-to-ventral sagittal volume rendering in a 7-week fetus in which no abnormalities were identified. Scale bar: 67 mm. Videoclip S3 Fetus 4. Virtuoscopy in an 11-week fetus with crown–rump length of 4.6 cm. Videoclip S4 Fetus 5. Surface rendering of a 14-week fetus (crown–rump length, 7.7 cm) with cleft palate. Videoclip S5 Heart from Case 4. Virtuoscopy of isolated fetal heart at 14 weeks. Videoclip S6 Fetus 6. Virtuoscopy by sequential right-to-left and left-to-right sagittal volume rendering in a 15-week fetus with crown–rump length 10 cm. Videoclip S7 Fetus 6. Virtuoscopy by sequential head-to-toe transverse volume rendering in a 15-week fetus with crown–rump length 10 cm. Videoclip S8 Fetus 7. Vertex-to-base transverse virtuoscopy of the head of a 17-week fetus.
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Postmortem microcomputed tomography (micro-CT) of small fetuses and hearts C. M. LOMBARDI*#, V. ZAMBELLI†, G. BOTTA‡#, F. MOLTRASIO§, G. CATTORETTI¶**, V. LUCCHINI**∧, V. FESSLOVA†† and M. S. CUTTIN** *Department of Radiology-Studio Diagnostico Eco, Vimercate, Milan, Italy; †Department of Health Sciences, Universita´ di Milano-Bicocca, Milan, Italy; ‡Azienda Ospedaliera Citta` della Salute e delle Scienze di Torino, Presidio Ospedaliero OIRM-Sant’Anna, Turin, Italy; §Residency in Pathology, Universita´ degli Studi di Milano-Bicocca, Milan, Italy; ¶Department of Surgery and Interdisciplinary Medicine, Universita´ degli Studi di Milano-Bicocca, Milan, Italy; **Department of Pathology, AO San Gerardo, Monza, Italy; ††Pediatric Cardiology, IRCCS Policlinico San Donato, San Donato, Italy
K E Y W O R D S: congenital heart disease; fetal autopsy; first trimester; micro-CT; pregnancy; virtual autopsy
ABSTRACT Objective To assess the feasibility and utility of contrast-enhanced microcomputed tomography (microCT) for identifying structural anomalies in ex-vivo firstand second-trimester human fetuses and isolated fetal hearts. Methods Radiopaque iodine staining and micro-CT scanning protocols were first developed in rodent studies and then used to examine routinely fixed whole human fetuses (n = 7, weight 0.1–90 g, gestational age, 7–17 weeks) and isolated fetal hearts (n = 14, weight 0.1–5.2 g, gestational age, 11–22 weeks). Samples were scanned using an isotropic resolution of 18 (and, if necessary, 9 or 35) μm and findings were interpreted jointly by four fetal pathologists, a fetal cardiologist and a radiologist. Samples with gestational ages ≥ 13 weeks also underwent conventional autopsy or dissection. Results Micro-CT identified all anatomical structures and abnormalities documented by the macroscopic examination. In all seven cases involving fetuses ≤ 13 weeks (four fetuses, three isolated hearts), micro-CT excluded the presence of structural anomalies. In the remaining 14 cases, it provided all the information obtained with invasive autopsy or dissection and in seven of the 14 (two fetuses, five isolated hearts) it furnished additional diagnostic details. Conclusions This pilot study confirms the feasibility of postmortem contrast-enhanced micro-CT assessment of structural anomalies in whole small fetuses and fetal
hearts. Further study is needed to confirm our findings, particularly in whole fetuses, and to define the extent to which this virtual examination might be used instead of conventional invasive autopsy. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
INTRODUCTION Prenatal ultrasound-based diagnoses of structural anomalies (most involving the heart and great vessels) are being made at increasingly earlier gestational ages thanks to the ongoing technical refinement of equipment and the now widespread use of first-trimester aneuploidy screening1 . Experienced operators can identify most major structural2,3 and cardiac4 – 6 abnormalities during the first-trimester nuchal scan. Postmortem examination of the fetus and/or fetal organs results in modification or refinement of the prenatal diagnosis in roughly 30% of cases7 , but its feasibility and value are limited in very small fetuses. Parental objections to conventional autopsy have also contributed to a decline in the rate of perinatal autopsies8 – 12 . Several groups have proposed alternatives to postmortem dissection, including virtual autopsy based on imaging modalities such as magnetic resonance imaging (MRI). While the resolution offered by scanners used for conventional whole-body MRI in the clinical setting is too low for successful examination of first- and second-trimester fetuses, more promising results have been obtained with high-field (9.4-T) micro-MRI scanners designed for imaging small laboratory animals9,13 .
Correspondence to: Dr C. Lombardi, Department of Radiology-Studio Diagnostico Eco, Via Cremagnani 15, 20871 Vimercate, Milan, Italy (e-mail: [email protected]); Dr M. S. Cuttin, Department of Pathology, AO San Gerardo, Via Pergolesi 33, 20900 Monza, Italy (e-mail: [email protected]) #C.M.L. and G.B. contributed equally to this paper. ∧Deceased, 31 December 2013. Accepted: 31 January 2014
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ORIGINAL PAPER
Micro-CT of small fetuses and hearts Like all MRI-based approaches, however, their use in a clinical setting is limited by cost, time and space constraints. Computed tomography has been considered less suitable for virtual autopsy since complete depiction of the fetal anatomy requires impregnation of the body tissues with intravenously diffused contrast medium, which is not feasible after death (although direct injection of contrast agents into cardiac chambers and great vessels has been used for postmortem identification of certain cardiovascular anomalies in human fetuses14 ). However, micro-CT, has been used recently to examine fixed small-mammal (whole-body and isolated heart) specimens after their immersion in contrast media15,16 , and this method has displayed high accuracy in the identification of heart defects in neonatal and fetal mice17 . The present study was conducted to assess the feasibility and potential utility of this approach for identifying structural anomalies in ex-vivo first- and second-trimester human fetuses and isolated human fetal hearts (Videoclip S1).
METHODS
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pathologist (G.B., V.L. or M.S.C.) with full access to the results of the micro-CT examination. Our examination of the remaining 14 fetuses (gestational ages ranging from 11 to 22 weeks) was limited to the hearts, which had been identified as isolated specimens in the material submitted for postmortem examination (two cases) or were isolated en bloc with the great vessels by an experienced fetal pathologist (G.B., V.L. or M.S.C.). Hearts from fetuses aged ≥ 13 weeks were also subjected to conventional dissection by one of the fetal pathologists. In these cases, the order in which the two procedures were performed varied, and findings from the first examination were always made available to operators conducting the second examination. In six cases, in which prenatal ultrasound findings of a major cardiac defect were available, the fetal hearts underwent standard four-chamber sectioning first, and a full heart dissection was completed after the micro-CT study. Karyotypes were determined for all 21 human fetuses via analysis of amniotic fluid cells or fetal tissues, as described elsewhere4 .
Animal studies
Micro-CT scans
We examined the hearts or whole bodies of 53 ex-vivo rodents (42 male C57/BL6J mice weighing 22–25 g, 11 Sprague Dawley rats weighing 200–250 g) used in research conducted at the University of Milan-Bicocca with the approval of the Italian Ministry of Health and the University’s Animal Care Unit. Shortly after their death, 51 of the animals were dissected en bloc by laboratory personnel; the heart was removed from the chest, isolated from the lungs and blotted. The other two animals (mice weighing 24 and 30 g) were used for whole-body studies. Both had died spontaneously for reasons unrelated to the experimental protocol. The whole-body specimens (skinned to facilitate staining) and isolated hearts were fixed in 4% aqueous buffered formaldehyde, stored at room temperature, and imaged after 2–60 days.
Protocol development in animals
Ex-vivo human fetuses and fetal hearts All 21 of the ex-vivo human fetuses used for this study had been submitted (with parental consent) for postmortem examination in the Departments of Pathology of the University of Milan-Bicocca, San Gerardo Hospital or the University of Turin. The micro-CT studies we performed were considered part of the authorized autopsy by the institutional review boards. Sensitive data were anonymized in all cases. Upon receipt, each fetus was fixed in 4% buffered formaldehyde and stored at room temperature. The seven fetuses used for whole-body studies had succumbed to unexplained intrauterine death at gestational ages ranging from 7 to 17 weeks. Prenatal ultrasound findings were not available in any of these cases. The interval between miscarriage and micro-CT examination was 4–7 days. For fetuses aged ≥ 13 weeks, the virtual autopsy was followed by conventional autopsy/dissection. The latter was performed by an experienced fetal
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Staining and scanning protocols were developed in the animals, using as a reference the methods described by Degenhardt et al.15 for the study of mouse embryos. The protocol thus defined was then used to examine the human fetal specimens. All micro-CT scans were performed by an experienced radiologist (C.L.) with specific training in the use of micro-CT. Tissues were weighed and then stained by immersion in Lugol solution (10 g potassium iodide and 5 g iodine in 100 mL water), which was tested using different dilutions (25–100% in demineralized water), exposure times (48–100 h) and staining solution volume/specimen weight ratios (2–10 mL per g specimen weight). Immediately after staining, specimens were washed with alcohol to remove free iodine, blotted dry and subjected to micro-CT. All scans were performed with a Bruker Skyscan 1176 micro-CT scanner (Bruker, Brussels, Belgium) with a gantry diameter of 68 mm. Fixed acquisition times of 4, 6 and 25 min were used for resolutions at 35, 18 and 9 μm, respectively. The exposure time was 65, 300 and 1050 ms, respectively, the voltage was 80 kV in all cases, the current was 300, 313 and 300 μA, respectively, and the rotation step 0.70◦ , 0.50◦ and 0.30◦ , respectively. The projection data were corrected for distortion and reconstructed by adjusting, smoothing and correction of ring artifact and beam hardening. The reconstructed isotropic voxel size was 9, 18 and 35 μm3 , respectively. Images were reconstructed with the scanner software (NRecon 1.6.6.0, Skyscan, Brucker micro-CT, Belgium) and converted to DICOM format for analysis. The time for reconstruction ranged from 2 to 60 min, depending on the volume of the sample. Data obtained were analyzed on a Mac Pro computer (2.8 GHz Quad-Core, 16 GB memory, high-performance
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PCI Express graphics cards; Apple Inc., Cupertino, CA, USA) and examined using an OsiriX DICOM Viewer (64-bit format, version 5.8 OsiriX Foundation, Geneva, Switzerland). For external macroscopic examinations of the specimens, reconstruction was performed with surface rendering, an algorithm-based technique for extracting the surface components of the acquired volume. Volume rendering (a technique used to display a two-dimensional projection of a three-dimensional sampled dataset) was used for ‘virtual dissection’ of the specimen. Diagnostic imaging in human fetuses Fetuses and hearts were stained and scanned using specimen weight-specific protocols defined in our animal studies. Multiple scans were necessary when the specimen size exceeded 22 mm (the field of view of the micro-CT scanner in use). The technical quality of each scan was verified by the radiologist with a preliminary post-scan assessment. Findings were later examined jointly by all the specialists involved in the study (i.e. pathologists, fetal cardiologist, radiologist), with full access to any available data obtained during prenatal ultrasound studies or conventional autopsy/dissection. Analysis of the fetal anatomy depicted by the micro-CT scans included systematic examination of the following structures: skull and brain (skull integrity, lateral ventricles, thalami, cerebellum and cisterna magna); face (orbits, lenses and facial profile); full-length spine (vertebral alignment and overlying skin from cervical to sacral region); heart (four-chamber view of atria and ventricles, crux and atrioventricular valves; three-vessel cross-sectional view of pulmonary artery, aorta and superior vena cava); abdomen (stomach, abdominal wall, liver, spleen and umbilical cord insertion); kidneys and bladder; extremities (long bones, fingers and toes).
RESULTS Animal studies Iodine staining protocols for mouse samples were defined in studies of two whole ex-vivo mice (weight, 24 and 30 g), similar in size to a 14-week human fetus, and in 51 rat/mouse hearts (weight, 110–1159 mg), comparable in size to those of human fetuses at 12–16 weeks of gestation. Lugol solution concentrations, volumes and immersion times were adjusted to specimen size in order to obtain uniform, full-thickness staining. The final protocols that emerged from these experiments are shown in Table 1. The micro-CT equipment we used offered scan resolutions of 35, 18 and 9 μm. Comparison of images of the same specimen obtained at 9 vs 18 μm revealed no significant loss of diagnostic detail under the latter condition. Therefore, all specimens (regardless of size) were imaged at 18 μm, which required only 6 min per specimen as opposed to 24 min per specimen at 9 μm. If necessary, Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
Table 1 Staining protocols
Type A B C
Weight (g)
Lugol* (%)
Solution volume (mL)
Staining time (h)
2
25 50 50
10 10–20 20–900
48 48 72
*Lugol solution: 10 g potassium iodide and 5 g iodine in 100 mL water, diluted 25% or 50% in demineralized water.
details of small specimens (i.e. those weighing < 1 g) were re-examined at the higher resolution. For large specimens (> 2 g), which generally required two or more scans, pre-screening at 35 μm was followed by targeted imaging at 18 μm (when needed). Depending on specimen size and the level of resolution used, the volume of scan data for each specimen ranged from 0.8 to 2.5 gigabytes. As described previously15 , the stained samples displayed variable degrees of shrinkage caused by the high osmolarity of the Lugol solution. The loss was proportional to the solution concentration and the immersion time. These changes did not compromise scan readability. The duration of storage in 4% buffered formaldehyde (range, 4–60 days) had no effect on staining intensity or uniformity. Insufficient solution volumes were associated with weak, non-uniform contrast; no effects were observed with excessive volumes. Optimal results were obtained using approximately 10 mL staining solution for every gram of pre-stained specimen weight. The staining procedure had no effect on histological examination of the internal organs during subsequent conventional autopsy.
Human fetal studies Whole-body specimens Contrast-enhanced micro-CT was performed on seven whole fetuses (weight, 0.1–90 g) that had undergone unexplained intrauterine death at gestational ages of 7–17 weeks (Figure 1 and Videoclips S2–S4 and S6–S8). The characteristics of these fetuses are shown in Table 2. None presented any external signs of maceration and, at the time of the micro-CT scan, none had undergone any type of invasive procedure. Specimen preparation (rinsing with alcohol and drying) required < 5 min. Scan times varied depending on the resolution(s) used (6 min at a resolution of 18 μm). As shown in Table 2, all four of the fetuses aged ≤ 11 weeks (Fetuses 1–4) were judged to be structurally normal on the basis of micro-CT findings. Conventional autopsy was not attempted in any of these cases because of size constraints. In Fetus 5 (gestational age, 14 weeks), the micro-CT scan revealed clefts of both the lip and palate (Figure 2), the latter being unilateral and involving the entire length of the palate. On subsequent autopsy, only the lip involvement could be identified, and size restrictions made it impossible to assess the extent of the palate defect. Fetus 6 (Figure 1d and Videoclips S6 and S7) and
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Figure 1 Contrast-enhanced microcomputed tomography of whole (entire) fetuses from 7 to 15 weeks’ gestation, at resolution of 18 μm. Three-dimensional surface rendering is shown on the left, and the internal anatomical details obtained with volume sections are shown on the right. (a) Fetus 1 (Table 2), 7 weeks; (b) Fetus 3, 10 weeks; (c) Fetus 4, 11 weeks; (d) Fetus 6, 15 weeks. Scale bar in (a) is 1 mm and in (b,c,d) is 10 mm.
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Table 2 Contrast-enhanced microcomputed tomography (micro-CT) and conventional autopsy findings in the seven whole fetuses Fetal age (GW)
CRL (mm)
Weight (g)
1 2 3 4 5 6
7 9 10 11 14 15
7 22 28 46 77 100
7
17
130
Fetus
Staining type
Micro-CT findings
Conventional autopsy findings
Karyotype
0.1 0.2 0.4 2.6 7.5 28
A A A C C C
Not performed Not performed Not performed Not performed External examination: cleft lip Normal
Normal Normal Normal Normal Normal Normal
90
C
Normal Normal Normal Normal Cleft lip and palate Normal: detailed depiction of brain structure Normal fetus with periventricular hemorrhage
Normal fetus with germinal matrix bleeding
Normal
CRL, crown–rump length; GW, gestational weeks.
Figure 2 Fetus 5 (Table 2). (a) Contrast-enhanced microcomputed tomography scan surface rendering and external survey at 14 weeks revealed only a cleft lip (arrow). (b) The presence of a full-length, unilateral cleft palate (arrow) is documented in an axial plane. Scale bar: 10 mm.
Fetus 7 required three scans each, due to their size. Pre-screening at 35 μm and re-exploration of the regions of interest (brain, heart) at 18 μm revealed no structural abnormalities. In both cases, the scan provided excellent depiction of the fetal brain structures, including the posterior fossae. In Fetus 7, this was especially useful in detecting and characterizing a periventricular hemorrhage. The scan revealed involvement of the caudothalamic notch and glomi of choroid plexi (Figure 3 and Videoclip S8). On subsequent autopsy, the same lesion was defined only as ‘germinal matrix bleeding’. Isolated hearts Table 3 shows the characteristics of the 14 fetuses whose hearts were examined and the results of the micro-CT and conventional dissection examinations. Hearts from Cases
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1, 2 and 3 were included in the study because their size (0.1–0.2 g) precluded conventional dissection. In all three cases, the virtual micro-CT autopsy allowed us to exclude structural anomalies in the heart (Figure 4a). In hearts from Cases 4 (Videoclip S5), 5, 7, 9 and 10, the virtual autopsy was performed before conventional dissection or after an initial external examination of the specimen. The first four hearts in this subgroup were from fetuses with trisomy 21, and the pathologists asked that the micro-CT scan be carried out before the heart was dissected. In Case 10 the fetus had a normal karyotype, but external examination of the body had revealed multiple structural malformations and the macroscopic examination of the heart raised the suspicion of right isomerism. Both micro-CT and subsequent conventional dissection revealed normal cardiac anatomy in all five of
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Figure 3 Fetus 7 (Table 2). Longitudinal (a) and axial (b) study of the brain using contrast-enhanced microcomputed tomography at 17 weeks demonstrated the presence of hemorrhage (arrows). Scale bar: 10 mm. Table 3 Contrast-enhanced microcomputed tomography (micro-CT) and conventional autopsy findings in the 14 isolated fetal hearts Fetal age (GW)
Organ weight (g)
1 2 3 4
11 12 13 14
5 6 7 8 9 10 11 12 13 14
15 15 16 16 17 17 18 19 19 22
Case
Staining type
Micro-CT findings
Conventional autopsy findings
0.1 0.15 0.2 1.1
A A A B
1.6 1.6 2.3 2.4 3.3 3 4 4 4 5.2
B B C C C C C C C C
Normal Normal Normal Normal intracardiac findings, ARSA Normal Truncus arteriosus Normal AVSD balanced Normal Normal Right isomerism, AVSD Pulmonary atresia, hypoplastic RV Truncus arteriosus type 1 AVSD + TGA
Not performed Not performed Not performed Normal intracardiac findings, ARSA Normal Truncus arteriosus Normal Abnormality undefined Normal Normal Right isomerism Abnormal univentricular Truncus arteriosus or TOF AVSD + TGA or TOF
Karyotype Normal Normal Trisomy 18 Trisomy 21 Trisomy 21 Normal Trisomy 21 Normal Trisomy 21 Normal Normal Normal Normal Normal
ARSA, aberrant right subclavian artery; AVSD, atrioventricular septal defect; GW, gestational weeks; RV, right ventricle; TGA, transposition of great vessels; TOF, Tetralogy of Fallot.
these cases. In Case 4, both examinations also revealed an aberrant right subclavian artery (known to be associated with trisomy 21). In Cases 6, 8, 11, 12, 13 and 14, prenatal ultrasonography had indicated the presence of a major cardiac defect although no precise diagnosis had been made. In these cases, the virtual micro-CT autopsy was performed after external examination and standard four-chamber sectioning. After micro-CT, these six hearts were subjected to full dissection by the pathologists. In Case 6, both virtual autopsy and conventional dissection confirmed the presence of a truncus arteriosus, but no pulmonary artery or other vessels were seen arising at that level. In Case 8 (Figure 5), standard four-chamber sectioning indicated the presence of a major structural anomaly but failed to yield a precise diagnosis. The subsequent micro-CT scan clearly revealed an atrioventricular septal defect. In Case 11, both examinations disclosed right atrial isomerism, but the micro-CT examination also demonstrated the presence of an atrioventricular
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septal defect. In Case 12, the virtual autopsy (Figure 6) and preliminary dissection yielded clearly discordant results: the former indicated the presence of right ventricular hypoplasia and pulmonary atresia, the latter a univentricular heart. In Cases 13 and 14, the micro-CT diagnosis was largely concordant with that based on the preliminary dissection, but in Case 13 (Figure 7) it provided additional details, which allowed specification of the type of truncus arteriosus. In Case 14, the dissection findings were suggestive of either tetralogy of Fallot or transposition of the great vessels. The latter diagnosis was confirmed by the micro-CT study.
DISCUSSION In this study, we developed a contrast-enhanced micro-CT protocol for postmortem examination of whole-body specimens and isolated hearts from embryos or fetuses with gestational ages ranging from 7 to 22 weeks. This
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Figure 4 Contrast-enhanced microcomputed tomography of excised normal fetal hearts at 11 (a) and 15 (b,c) gestational weeks. (a) Heart 1 (Table 3). Volume rendering, showing a virtual dissection at the level of the great vessels: aorta (Ao) and pulmonary artery (PA) are visible, with a branch of the pulmonary artery with diameter 100 μm (arrow). (b) Heart 5 (Table 3). Macroscopic examination obtained with surface rendering. (c) Heart 5 (Table 3). Volume rendering section at the level of the crux. Arrow indicates insertion point of tricuspid septal leaflet. Scale bar: 1 mm.
Figure 5 Heart 8 (Table 3). Contrast-enhanced microcomputed tomography of excised fetal heart at 16 gestational weeks in a case of atrioventricular septal defect, showing evidence of a large defect between atria and ventricles and a common atrioventricular valve (curved arrow). Scale bar: 1 cm.
virtual autopsy yielded diagnoses in all seven cases (four whole fetuses, three hearts) in which conventional autopsy or dissection was precluded due to size restrictions. In seven other cases it provided all the information that could be obtained with conventional autopsy, and
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in the last seven (two whole fetuses and five isolated hearts), it also furnished additional details that improved diagnostic accuracy. Virtual fetal autopsy has been attempted with conventional whole-body MRI using a 1.5-T scanner, but the maximum resolution that could be achieved with this approach (800 μm) was too low11,18,19 . Somewhat better results have been obtained recently with a 3-T scanner with a maximum resolution of 300 μm20,21 . Thayyil et al.9 were the first to propose the study of small fetuses with micro-MRI, a well-established method for examining small laboratory animals22 – 26 . In the 17 fetuses (age, 11–22 weeks; weight, 5–400 g) that they studied with a 9.4-T micro-MRI scanner, the image resolution used (200 μm) was reportedly sufficient for detecting the structural abnormalities that could be identified with conventional autopsy and also for internal examination of the visceral organs. However, this high definition was at the expense of scan times greater than 90 min. The authors reported that their approach could also be carried out at a resolution of 18 μm (the level we achieved with micro-CT protocols), but it required scan times of over 18 h (compared with our time of 6 min using micro-CT). Prior to the present study, we carried out a preliminary series of experiments using a 7-T micro-MRI scanner (Pharmascan 70/16 US, Bruker Biospin, Karlsruhe, Germany) (results not shown) and observed similarly excessive scan times. We have shown that micro-CT analysis of iodine-stained tissues, thus far used only for small animal studies15,17 , can be used for postmortem examination of whole human fetuses with gestational ages of up to 17 weeks and for isolated fetal organs such as the heart. This approach offered several important advantages. First, it provided diagnoses in cases in which conventional autopsy or dissection was precluded by the
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Figure 6 Heart 12 (Table 3). Contrast-enhanced microcomputed tomography of excised fetal heart at 19 gestational weeks, with ventricles partially sectioned and shown surface-reconstructed (a) and digitally sectioned (b). The features of pulmonary atresia are evident: hypoplastic right ventricle (RV) with an imperforate hypoplastic pulmonary valve (arrow) and a large left ventricle (LV). Note the details of a cross-sectioned esophagus on the right in (b). Scale bar: (a) 1 cm; (b) 1 mm.
reduced dimensions of the fetus or the excised heart. It is important to note, however, that these diagnoses were not verified by other methods (an obvious limitation of the present study). Second, it furnished all information that could be obtained with autopsy or dissection and in many cases it provided important additional findings. Furthermore, it provided stable, detailed digital documentation of the postmortem findings that could be analyzed and interpreted by an interdisciplinary team of specialists (radiologist, pathologists, cardiologist), with potential gains in the accuracy of the diagnosis. The documented findings could also be archived, re-evaluated and used during remote consultation or for teaching purposes. Conventional perinatal autoptic confirmation of the fetal anatomy should be mandatory in cases of fetal demise27 , but there are obvious difficulties related to specimen size, especially in the early stages of gestation. Small specimens are not a limitation for virtual micro-CT autopsy, and it has the potential to provide greater diagnostic detail during the examination of small whole fetuses or excised organs. The cost and size of a commercial laboratory micro-CT scanner are similar to those of high-quality ultrasound equipment. Compared with high-field micro-MRI imaging, micro-CT scanners are characterized by much lower purchase and operating costs, much shorter scan times and markedly higher spatial resolution. These features make it a potentially valuable tool for pathologists, who are faced increasingly with requests for postmortem examination of fetuses that have died in utero during the early stages of gestation.
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
It is important to note that while the advantages of iodine solutions to stain specimens (low cost, minimal toxicity and ease of preparation) have been documented in several studies on small mammals15 , these solutions also inevitably cause some degree of tissue shrinkage. This effect can alter the volume of the specimen, but not the relation between the structures. The resulting tissue distortion did not interfere with our examination of isolated fetal hearts, but it could produce artificial increases in the volume of body cavities, which might lead to erroneous suspicion of effusion. It should also be noted that our study was limited to whole fetuses aged ≤ 17 weeks. This decision was dictated in part by the bore diameter of the scanner we used, which was too small to accommodate the heads of older fetuses. However, there are already scanners on the market with larger bore diameters (e.g. the Siemens Inveon with a 12-cm internal bore and maximum resolution of 20 μm). A more serious potential limitation with larger fetuses is related to contrast-agent penetration of the internal organs, although the exact size and weight cut-offs have yet to be defined. (Our protocol ensured effective staining only of fetuses that could be examined with the scanner we used.) At the internal organ level, our attention was focused largely on examination of the fetal heart, which is the site of most of the congenital anomalies that can be detected by expert sonographers during early prenatal scans5 . We chose to examine isolated hearts because, when pregnancy is terminated early, the heart can usually be recovered by systematic examination of the material
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Figure 7 Heart 13 (Table 3). Contrast-enhanced microcomputed tomography of excised fetal heart at 19 gestational weeks, showing features of truncus arteriosus type I on surface-rendering (a) and digital sectioning (b): a large vessel overriding the ventricular septal defect and a small pulmonary artery (PA, arrow) originating from the truncus (T). Scale bar: 1 cm.
collected during uterine evacuation28 . Many features of first-trimester fetal heart anomalies that were previously unrecognized owing to the technical limitations of the postmortem examination can now be confirmed and clarified with contrast-enhanced micro-CT. Our experience in this pilot study requires confirmation in larger studies, but it suggests that postmortem micro-CT examination of small fetuses and fetal organs offers clear advantages over traditional autopsy and dissection, particularly for assessing anatomical and topographic features. Nonetheless, dissection and serial sectioning will continue to be necessary in some cases, for example those requiring collection of samples for histological and/or biomolecular studies.
ACKNOWLEDGMENTS This paper is dedicated to the late Anna Cappellini, M.D. (1943–2008), teacher and mentor for all of us. We wish to thank Dr Giacomo Bellani (Department of Health Sciences, Universita´ di Milano-Bicocca) for support and advice, Dr Sophie Lombardi (Residency in Radiology, Universita´ di Milano-Bicocca) for helping in the software setup and rendering, Dr Sonia Gorla (Department of Pathology, Ospedali di Desio-Vimercate) for providing diagnostic specimens, the San Gerardo Hospital Pharmacy for providing the Lugol solution and the laboratory technicians of the hospital for the diagnostic work. This study is self-financed with San Gerardo Hospital departmental funds and University of Milano-Bicocca startup funding.
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
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SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Videoclip S1 Animation of preparation for microcomputed tomography (micro-CT). Fetal tissues are treated with contrast medium by placing the specimen into a flask of water into which iodine (Lugol) is added. By osmosis of the hypertonic solution, there is passive transfer of iodine into the specimen. The fetus is then placed in the micro-CT scanner, a small laboratory device which has been used thus far only in preclinical research on small animals. Videoclip S2 Fetus 1. Virtuoscopy (virtual autopsy) by sequential dorsal-to-ventral sagittal volume rendering in a 7-week fetus in which no abnormalities were identified. Scale bar: 67 mm. Videoclip S3 Fetus 4. Virtuoscopy in an 11-week fetus with crown–rump length of 4.6 cm. Videoclip S4 Fetus 5. Surface rendering of a 14-week fetus (crown–rump length, 7.7 cm) with cleft palate. Videoclip S5 Heart from Case 4. Virtuoscopy of isolated fetal heart at 14 weeks. Videoclip S6 Fetus 6. Virtuoscopy by sequential right-to-left and left-to-right sagittal volume rendering in a 15-week fetus with crown–rump length 10 cm. Videoclip S7 Fetus 6. Virtuoscopy by sequential head-to-toe transverse volume rendering in a 15-week fetus with crown–rump length 10 cm. Videoclip S8 Fetus 7. Vertex-to-base transverse virtuoscopy of the head of a 17-week fetus.
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Ultrasound Obstet Gynecol 2014; 44: 600–609.
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