Calcitonin and chromogranin A localization in medullary carcinoma of the thyroid by immunoelectron microscopy.

We used a post-embedding immunoelectron microscopy method, using protein A-gold, to detect calcitonin and chromogranin A immunoreactivity in three cases of human medullary thyroid carcinoma. Because the epoxy-embedded tissue had been fixed (glutaraldehyde or formaldehyde) and osmicated before embedment, the proteins were identified in optimally preserved tissue. Uranyl and lead staining was used after immunolabeling, so that the tissue was also optimally contrasted. The morphological advantage provided by osmication was tested by labeling rat thyroid gland C-cells for calcitonin. The protein A-gold technique allowed localization of both antigens to the contents of membrane-bound secretory granules in the tumor cells. In one case, labeling density for each antigen was measured over several intercellular compartments and the interstitium. Calcitonin, but not chromogranin A, reactivity was also identified in intracellular amyloid fibrils in two cases, showing that the constant region of calcitonin is preserved in amyloid deposits related to the tumor cells.

Immunoelectron microscopic labeling of immunoglobulin in plasma cells after osmium fixation and epoxy embedding.

Previous studies have found that immunoglobulin cannot be immunolabeled in tissues prepared for electron microscopy by usual methods. To test this conclusion, we used a protein A-gold postembedding immunolabeling method on tissues that were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epoxy resin; sections were pretreated with sodium metaperiodate. A variety of common fixation protocols were also used and the most suitable conditions for immunolabeling were determined. This technique permitted the ultrastructural localization of immunoglobulin light chains in optimally preserved and contrasted plasma cells from human tonsil, lymph nodes, plasmacytomas, and a renal biopsy. We were able to demonstrate multiple antigens in the same tissue and label antigens in tissues that had been stored for many years in epoxy resin. The technique allows quantitation of the gold label over plasma cell organelles and therefore gives information about the immunoglobulin secretory pathway in these cells. We found that the protein A-gold procedure compares favorably in technical ease with the immunoperoxidase, avidin-biotin peroxidase, and immunoglobulin-colloidal gold immunolabeling methods, and has added advantages in allowing precise localization and quantitation of the labeled antigen.

Calcification in porcine xenograft valves in children.

Calcification developed in the degenerating collagen of the cusps of three porcine xenograft heart valves implanted in children for less than 4 years. The morphologic features and effects of this calcification are presented. Calcification of porcine xenografts seems to occur more frequently and at an earlier stage after insertion in children than in adults. Host factors, possibly related to calcium homeostasis, may promote calcification; hence, these valves may not be appropriate for use in children.

Enzymatic isolation of human trophoblast and culture on various substrates: comparison of first trimester with term trophoblast.

A simple method is described for the isolation of trophoblast cells from both first trimester and term placenta. Trophoblast preparations were characterized by light microscopy, scanning and transmission election miscroscopy and immunohistochemistry to distinguish these cells from mesenchyme and endothelium. Trophoblast cells were cultured on various substrates and a comparison made of their ability to attach, proliferate and function. A collagen gel substrate produced by repolymerization of an acid soluble collagen fraction from chorionic villi allowed rapid attachment of trophoblast cells and maintainance of their original morphology. Term trophoblast cells were shown to become fully functional in short term (three day) cultures by virtue of their increased immunocytochemical staining for the presence of beta hCG, hPL and SPI. beta hCG increased significantly by day three thus demonstrating functional activation. Trophoblast cells from first trimester placenta formed proliferating colonies of hormone producing cells while those from term placenta reaggregated into clusters and closely resembled syncytiotrophoblast both morphologically and functionally. This short term culture system for term trophoblast will allow further studies into the biology of trophoblast polypeptide hormone synthesis and secretion.

Cyclopia and congenital cytomegalovirus infection.

We describe the first documented association of congenital cytomegalovirus (CMV) infection and cyclopia. A previous report has suggested that any infant with congenital ocular defects should be investigated for CMV infection [Frenkel et al, 1980]. Our case underlines this suggestion and questions a teratogenic role for CMV in cyclopiaholoprosencephaly. More documented cases may help clarify the relationship of transplacental CMV infection to the holoprosencephaly developmental field defect, including cyclopia.

Skeletal dysplasias with gracile bones: three new cases, including two offspring of a mother with a dwarfing condition.

We describe 3 new cases of a rare form of dwarfism (so-called "lethal skeletal dysplasia with gracile bones" or "osteocraniostenosis"), a condition characterized by thin, brittle bones and death in late gestation or early neonatal life. The first was a 37-week gestation female who died at delivery. She had facial anomalies and positional abnormalities of the hands and feet. The others were male stillborn sibs, who died in utero in the third trimester. Their mother had an undiagnosed dwarfing condition associated with body asymmetry, microcephaly, and unusual facial appearance. Both fetuses were documented by ultrasound to have short limbs and probable long bone fractures late in the second trimester. At autopsy, one fetus had no spleen and the other a hypoplastic spleen. Radiographically, all three cases had very thin diaphyses, diaphyseal fractures, and thin ribs and clavicles. In contrast to the first case who had a normally mineralized calvaria, the sibs had grossly deficient calvarial mineralization. Microscopically, endochondral ossification was qualitatively normal but quantitatively deficient in all three cases. The long bones, especially those of the sibs, lacked the well-defined outer cortex in the mid-shaft normally seen by the third trimester. This failure of organization into the cortex and medulla suggests a failure of bone remodelling. Given the variable presentation in these cases, "lethal skeletal dysplasia with gracile bones" is probably a heterogeneous disorder. The recurrence in one family suggests that the mother has somatic/germline mosaicism of a lethal gene, expressed clinically as growth failure and asymmetry.

Terminal deletion of the long arm of chromosome 3 [46,XX,del(3)(q27-->qter)].

We report on a terminal deletion of the long arm of chromosome 3 [46,XX,del(3)(q27-->qter)] in a female newborn infant who died 45 hours after delivery and had multiple congenital abnormalities including bilateral anophthalmia, congenital heart disease, and abnormal genitalia. The findings are compared to those of four previously reported cases with terminal del (3q).

Limb defects in homozygous alpha-thalassemia: report of three cases.

Homozygosity for the South-Asian alpha-thalassemia (--SEA/) deletion is a serious hematological condition that results, in most cases, in intrauterine or postnatal death due to anemia and severe hypoxia of prenatal onset. A relationship between congenital abnormalities and intra-uterine hypoxia has been postulated. However, since homozygosity for the (--SEA/) deletion is most common in underdeveloped countries where detailed autopsies are lacking, the incidence of congenital abnormalities among these babies has not been well delineated. We report on three newborn infants, homozygous for the (--SEA/) deletion, who were born with limb defects. We postulate that this combination is the result of prenatal hypoxia which may affect other fetal body organs. This should be taken into consideration when prenatal treatment of affected fetuses, with intrauterine blood transfusion, is suggested.

Partial tetrasomy with triplication of chromosome (5) (p14-p15.33) in a patient with severe multiple congenital anomalies.

We report on a newborn infant with a de novo triplication of the distal segment of 5p: 46,XX,trp(5) (pter-->p14::p14-->p15.33::p15.33--> qter) and multiple congenital anomalies consistent with triplication of 5p. Partial triplication was documented by fluorescence in situ hybridization with a cosmid probe specific for 5p15.2 and microdissected probes obtained from "5pter." Partial duplication of the short arm of chromosome 5 is associated with a specific phenotype that appears to be dependent on the chromosomal region duplicated. Duplication of 5p with breakpoints proximal to band p14 is generally associated with distinct craniofacial malformations, cardiac, renal, intestinal, and limb defects, and mental retardation, whereas duplications with breakpoints distal to 5p14 result in a milder phenotype characterized by minor facial anomalies, developmental delay, and seizures. The most proximal breakpoints of the partial triplication in this patient was estimated to be 5p14, suggesting that a more severe phenotype can occur with triplication of the more distal segment.

Inverted duplication of the distal short arm of chromosome 3 associated with lobar holoprosencephaly and lumbosacral meningomyelocele.

A fetus with lobar holoprosencephaly and lumbosacral meningomyelocele associated with duplication of the short arm of chromosome 3 is reported. The anomalies were detected on fetal ultrasound at 20 weeks' gestation and the autopsy findings correlated well with the prenatal findings. The fetal karyotype was 46,XY,der(3)del(3)(p26) dup(3)(p26p21.3). The association of holoprosencephaly with duplication 3p is well known, but to the best of our knowledge this is the first reported association of meningomyelocele with 3p duplication. These findings suggest that a gene or genes with a crucial role in central nervous system development are located on the short arm of chromosome 3.

Brachydactyly-short stature-hypertension (Bilginturan) syndrome: report on two families.

We report on two families with autosomal dominant brachydactyly of hands and feet and hypertension. All affected members of the first family had proportionate short stature. However, the propositus and the affected relatives in the second family were only short compared to unaffected relatives. The hypertension was medically responsive in all cases. The propositus in the second family had poor compliance and a striking generalized vasculopathy. All patients were of normal intelligence and had a normal facial appearance. The brachydactyly-short stature-hypertension syndrome was first reported by Bilginturan et al. [1973] in a Turkish family and the families reported by us are Caucasian and Hispanic. The gene causing this condition in the original Turkish family was recently mapped to 12p. Our report expands our existing knowledge and the ethnic diversity of this syndrome.

Diaphragmatic contraction band necrosis in a perinatal and infantile autopsy population.

A morphologic study of the anterolateral costal diaphragm in 125 newborns and infants who died suddenly showed that contraction band necrosis is a common finding. In cases that showed the most extensive lesions, acute asphyxia was the usual mode of death; within eight diagnostic categories birth asphyxia (11 of 26 cases) and sudden infant death syndrome (19 of 30 cases) had the highest frequency of lesions. It was more frequent than myocardial contraction band necrosis and myocardial coagulation necrosis among the cases studied. The morphologic age and, if present, the stage of healing in each case suggested that the diaphragmatic lesion commenced at or shortly before death or at the time of the cardiac arrest that led to death. Thus, the lesion appeared to represent a very early event after a lethal injury, but it had no specificity for the nature of the injury. Because skeletal muscle of the respiratory diaphragm structurally and functionally resembles myocardium, the pathogenesis of contraction band necrosis may be similar in the two muscle types.

Hemorrhagic endovasculitis-like lesion induced in placental organ culture.

In organ culture, human chorionic villi develop vascular changes that resemble so-called hemorrhagic endovasculitis. The latter is a morphologic finding more prevalent in placentas of stillborn infants but seen also in those of liveborn infants, in whom the lesion is localized rather than generalized. We compared the histologic vascular changes in short-term organ cultures of 15 placentas (10 term, 5 preterm) with the naturally occurring vascular lesion in 6 placentas (2 liveborn, 4 stillborn). All organ cultures of placentas from liveborn infants developed hemorrhagic endovasculitis-like lesions in the fetal stem arteries; these lesions were present as early as 1 day and persisted for 7 days in culture. A mechanism common to both the in vivo and in vitro systems depending on hypoxia and vascular smooth muscle contraction may explain both the naturally occurring and tissue culture-induced lesions.

Development of the respiratory diaphragm in childhood: diaphragmatic contraction band necrosis in sudden death.

The authors studied the respiratory diaphragm in 50 normally grown infants and children aged 1 to 16 years at the time of sudden death. By comparing the weights of both costal diaphragm and heart with age and height, the authors found that the diaphragm grows proportionately to the body as a whole and to the heart in particular. Diaphragmatic contraction band necrosis was found in 15 cases (30%). The incidence was similar in subjects dying of asphyxia (five of 21) to that in those dying of trauma (five of 20). It was present in two of 15 of those that died at once, and 13 of 35 of those who survived for varying periods with or without cardiopulmonary resuscitation. Myocardial contraction band necrosis was more common than diaphragmatic contraction band necrosis, being present in five of 11 of those who died at once, and 16 of 26 of those that survived for a period. Among individual subjects, the authors found no correlation of the presence of the diaphragmatic lesion with either cause or mode of death. Based on a comparison with the morphologically similar myocardial lesion, the etiopathogenesis of diaphragmatic contraction band necrosis may concern a local catecholamine effect.

Perinatal pulmonary hyperplasia due to laryngeal atresia.

A premature newborn boy, who died at birth because of cartilaginous laryngeal atresia, showed lung development that was far more advanced than normal for gestational age. The lungs, which were histologically normal, were three times the expected weight and showed a degree of alveolarization appropriate for 3 months' postnatal age. The lungs crowded the chest cavity so that the diaphragm was flattened and immobilized; the massive ascites, documented by ultrasound 5 weeks prior to delivery, appeared to be due to obstructed venous return. Thus, the pulmonary hyperplasia, a new finding in perinatal pathology, caused severe fetal ascites. Subsequently, the hyperplasia led to acute polyhydramnios that, ultrasonographically, was observed to develop 2 weeks prior to delivery. The observation that fetal lungs may be hyperplastic has a bearing on the known relationship between fetal lung growth and retention of lung fluid.

Morphologic and morphometric analyses of muscle in the neonatal myotonic dystrophy syndrome.

Autopsy studies of three premature siblings who died soon after birth with the neonatal myotonic dystrophy syndrome revealed pulmonary hypoplasia and congenital pleural effusions. Neither of these findings has been described previously in this condition. New ultrastructural findings include focal diaphragmatic myofiber degeneration and necrosis, which were attributed to over-stretching of the fetal diaphragm. In addition, abnormally small stores of free and intravesicular glycogen were observed in skeletal muscle fibers. The morphometric features of control fetal and neonatal skeletal muscle were recorded for comparison with muscle fiber measurements in the three infants. Fiber diameters in the latter were much smaller than expected for body weights. The morphologic and morphometric findings support the concept that fetal muscle maturation is severely retarded in this syndrome.

Oncocytic cardiomyopathy in an infant with oncocytosis in exocrine and endocrine glands.

An 11 week old female infant with congenitally malformed eyes died from intractable cardiac arrhythmia. The heart showed extensive oncocytic transformation of myocytes, and this distinctive cardiomyopathy affected the conduction system. Oncocytes were found also in endocrine (pituitary, thyroid) and exocrine (submandibular, sublingual, minor salivary) glands. There is morphologic evidence that the lesions were caused early in gestation, possibly by a viral infection such as rubella.

The morphology of the human newborn ductus arteriosus: a reappraisal of its structure and closure with special reference to prostaglandin E1 therapy.

The ductus arteriosus was examined in 103 fetuses and infants to define the normal structure, development, and morphologic features of the functional and anatomic phases of ductal closure. New contributions include ultrastructural observations and the light microscopic definition of the junctional regions of the ductus with the pulmonary artery and aorta. Observations relating to ductal closure include hyperemia of the ductal vasa vasorum, the presence of longitudinal muscle bands in the inner media, necrosis of the inner ductal wall, and organization of intraluminal thrombi. We found that a ductus arteriosus that fails to close normally is liable to show morphologic lesions, including intimal fibrinous deposits, medial hemorrhages, and dissecting aneurysms. The findings were used comparatively to investigate whether prostaglandin E1 infusion, given to maintain ductal patency in 7 infants with ductus dependent congenital heart disease, was associated with specific morphologic features. We could not delineate specific changes attributable to its use.

Fatal pulmonary arterial occlusive vascular disease following chemotherapy in a 9-month-old infant.

Fatal pulmonary hypertension developed in an infant during the 7-month period in which he received, via a central venous catheter, combination chemotherapy for stage IV neuroblastoma as well as intermittent parenteral feeding. In a lung biopsy and at autopsy, small pulmonary arteries showed diffuse medial hypertrophy and peripheral muscularization, very extensive concentric intimal fibrosis, and focal eccentric fibrosis evolving from organizing thrombi. Pulmonary veins were normal. Hypothetically, chemotherapeutic drug therapy (possibly potentiated either by the parenteral nutrition or simply by the vehicular fluids causing volume loading of the pulmonary circulation) could cause occlusive pulmonary arterial disease by several mechanisms, but the association has not been described previously, although use of such drugs has been reported with pulmonary veno-occlusive disease.

Fibrolamellar carcinoma of the liver in a patient with Fanconi anemia.

A 9-year-old boy with Fanconi anemia treated with oxymethalone, a synthetic androgen, died of intracerebral hemorrhage. At autopsy, the liver contained several adenomas and a large fibrolamellar hepatocellular carcinoma, as well as phlebectatic peliosis hepatis. The 11 previously reported cases of hepatocellular carcinoma in Fanconi anemia were not, apparently, of the fibrolamellar type, which has a better prognosis, occurs in children of both sexes, and generally is not associated with cirrhosis. The malignant potential of primary liver tumors associated with Fanconi anemia as well as the nature of their relationship to Fanconi anemia and to anabolic steroid therapy is discussed.