Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation.

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16-24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5-15 min. Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses. Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.     

Optimisation of postmortem tissue preservation and alternative protocol for serotonin transporter gene polymorphisms amplification in SIDS and SIUD cases

The major obstacle to genetic research in SIUD (sudden intrauterine unexplained death) and SIDS (sudden infant death syndrome) cases is the complex characteristics of the human anatomic samples available. In fact, in Italy autopsies are performed at least 24 h post-mortem and tissues can be left in formalin for long fixation times (> 4/5 days), thus compromising nucleic acids integrity. In this study we compared the quality of DNA and RNA extracted from tissues differently preserved. As expected, the DNA and RNA from formalin-fixed and paraffin-embedded tissues, formalin-acetic acid-alcohol tissues and ethanol tissues were of poor quality and not adequate for subsequent molecular analysis. The best results were obtained with RNAlater preserved tissues: this buffer was equivalent, if not superior, to freezing method for preservation of postmortem DNA and RNA.In addition, we introduce a new protocol for the amplification of the serotonin transporter gene promoter region (5-HTT) ideal to obtain the increase of specific product, avoiding artifacts formation.     

Classification of pulmonary alveolar proteinosis in newborns, infants, and children

Pulmonary alveolar proteinoses are rare pulmonary diseases characterised by an intraalveolar accumulation of surfactant protein A. Subtyping of alveolar proteinoses: Type I alveolar proteinoses: severe respiratory insufficiency in newborns, which will take a lethal course without lung transplant; hereditary SP-B deficiency and an intraalveolar accumulation of N-terminal incompletely processed SP-C. Type II alveolar proteinoses: occur in newborns and infants; often take a lethal course; show intraalveolar accumulation of precursors of SP-B and mature SP-B as well as an accompanying interstitial lung disease of variable severity. Type III alveolar proteinoses: in infants and children; do not generally take a lethal course; they are characterised by an intraalveolar accumulation of precursors of SP-B and mature SP-B without accompanying interstitial lung disease. "Cryptogenic" congenital, acquired (idiopathic), and secondary type III alveolar proteinoses can be distinguished.In newborns, infants, and children with pulmonary alveolar proteinosis, a detailed pathological-anatomical examination including immunohistochemical and molecular genetic analyses, should be performed in order to optimise the therapeutical management.     

Klassifikation der Alveolarproteinosen im Neugeborenen-, Säuglings- und Kindesalter

Pulmonary alveolar proteinoses are rare pulmonary diseases characterised by an intraalveolar accumulation of surfactant protein A. Subtyping of alveolar proteinoses: Type I alveolar proteinoses: severe respiratory insufficiency in newborns, which will take a lethal course without lung transplant; hereditary SP-B deficiency and an intraalveolar accumulation of N-terminal incompletely processed SP-C. Type II alveolar proteinoses: occur in newborns and infants; often take a lethal course; show intraalveolar accumulation of precursors of SP-B and mature SP-B as well as an accompanying interstitial lung disease of variable severity. Type III alveolar proteinoses: in infants and children; do not generally take a lethal course; they are characterised by an intraalveolar accumulation of precursors of SP-B and mature SP-B without accompanying interstitial lung disease. “Cryptogenic” congenital, acquired (idiopathic), and secondary type III alveolar proteinoses can be distinguished. In newborns, infants, and children with pulmonary alveolar proteinosis, a detailed pathological-anatomical examination including immunohistochemical and molecular genetic analyses, should be performed in order to optimise the therapeutical management.     

Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo

The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in the fetal lung level of SP-A protein and a threefold increase in fetal lung SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in fetal lung tissue obtained from glucocorticoid-treated pregnant does when compared to levels in fetuses of uninjected pregnant does. However, SP-B mRNA levels in fetal lung tissue from saline-injected controls were also significantly elevated, ～twofold, when compared to fetal lung SP-B mRNA levels in the uninjected control condition. SP-C mRNA levels in lung tissue of fetuses from both saline-injected and betamethasone-injected pregnant does were increased similarly, ～twofold, over SP-C mRNA levels in fetal lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B mRNA levels and that maternal stress alone can increase the expression of SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type II cells in fetuses of control and saline-injected does. However, SP-A mRNA was easily detected in both alveolar type II cells and bronchiolar epithelial cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type II cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-B mRNA levels in rabbit fetal lung tissue. SP-A mRNA was localized to both alveolar type II cells and in smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue. However, SP-B and SP-C mRNA were detected only in alveolar type II cells. © 1993 Wiley-Liss, Inc.     

Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury.

Surfactant protein-B (SP-B) is a small, hydrophobic peptide that plays a critical role in pulmonary function and surfactant homeostasis. To determine whether SP-B protects mice from oxygen-induced injury, heterozygous SP-B(+/-) gene-targeted mice and wild-type SP-B(+/+) littermates were exposed to hyperoxia (95% oxygen for 3 d) or room air. Although specific lung compliance in room air in SP-B(+/-) mice was slightly reduced as compared with that in SP-B(+/+) mice, it was reduced more markedly during hyperoxia (46% versus 25% decrease, respectively). The larger decrease in lung compliance in SP-B(+/-) mice was associated with increased severity of pulmonary edema, hemorrhage and inflammation, lung permeability and protein leakage into the alveolar space. Hyperoxia increased SP-B messenger RNA (mRNA) and total protein concentrations by 2-fold in SP-B(+/+) and SP-B(+/-) mice, but decreased the abundance of SP-B protein in lavage fluid relative to total protein only in SP-B(+/-) mice. Hyperoxia increased SP-B expression, but apparently not enough to maintain SP-B function and lung compliance in the presence of increased protein leakage in SP-B(+/-) mice. Increased alveolar-capillary leakage and relative deficiency of SP-B may therefore contribute to oxygen-induced pulmonary dysfunction in SP-B(+/-) mice. These data support the concept that SP-B plays an important protective role in the lung.     

Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity

Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.     

A time course study demonstrating RNA stability in postmortem skin

Knowledge of the factors regulating the rate of mRNA degradation, including postmortem delay, is important in determining the reliability of gene expression patterns in dermal tissue. Since RNA stability can be tissue dependent, this study evaluates the effect of postmortem interval on the integrity of total RNA or the levels of representative mRNA species in murine cutaneous tissue. Pieces of fresh skin tissue were excised for periods of 0-60 min from SKH-1 female hairless mice that were maintained at room temperature post-sacrifice. Total RNA was subsequently isolated and RNA integrity from each specimen was evaluated. Bioanalyzer profiles showed no apparent change in 28S/18S rRNA ratio or RNA integrity number at time points up to 60 min. Changes in mRNA expression levels of five selected genes were determined by real-time quantitative PCR. There were no statistical differences in the relative gene expressions of Ccnd1, Hif1alpha, cMyc and Cyr61 as a function of postmortem interval. Our data suggest that the molecular quality of cutaneous tissue is well preserved for at least 60 min after death, which can be regarded as important information for consideration of the order for tissue procurement in in vivo studies and acute ex vivo dermal studies.     

人脑组织库样本100例质量及影响因素分析

目的检测人脑组织库中保存的脑组织蛋白质、DNA及RNA质量,分析影响其质量的主要因素。方法选取中国医学科学院北京协和医学院人脑组织库保存的100例全脑组织样本,提取蛋白质、DNA及RNA,测定其浓度及吸光度,电泳检测完整性,用Western blotting及Rael-time PCR分别检测。根据不同年龄、死亡原因、取材延迟时间以及样本保存时间进行分组,统计学分析影响脑组织质量的因素。结果人脑组织库中大部分脑组织的DNA、RNA和蛋白质质量保存完好,不同年龄、死亡原因对其质量无显著影响。取材延迟时间超过24h者,其蛋白质浓度及RNA浓度及质量有显著下降。样本保存时间对蛋白质及DNA质量无显著影响,但保存1年以上可能对RNA浓度有影响。结论进行RNA相关分析的人脑组织标本取材延迟时间最好控制在8 h之内,-80℃环境下保存1年之内为佳。而DNA和蛋白质相关分析的时间可以适当延长。     

Significance of hemorrhagic endovasculitis in placentae from stillbirths

Hemorrhagic endovasculitis (HEV) is an alteration of fetal chorionic blood vessels within the placenta. This disruption of the integrity of the parietal blood vessels has been reported in association with poor pregnancy outcomes and perinatal complications. In stillbirths, its presence has been implicated as part of the mechanism of the cessation of fetal circulation in postmortem retention of the placenta.

The purpose of this study was to give an interpretation of this type of blood vessel damage: whether it is related to the arrest of blood circulation after stillbirth, or whether it constitutes an alteration of the integrity of placental chorionic circulation contributing to the death of the fetus.

From 1232 reviewed placentae, we analyzed 46 cases with HEV, 70% from livebirths and 30% from stillbirths. The most frequent lesions encountered in placentae were categorized microscopically. The distribution of these lesions in placentae from livebirth and stillbirth pregnancies was then described.

In association with HEV, villitis of unknown etiology and chorionic vessel thrombi occurred more frequently in placentae from livebirths. Fragmented erythrocytes and erythroblasts were more frequent in placentae from stillbirths.

Our study demonstrates that HEV is more consistently associated with other placental lesions in liveborn than in stillborn infants.     

Decreased surfactant protein-B expression and surfactant dysfunction in a murine model of acute lung injury

This study examines the relationships between inflammation, surfactant protein (SP) expression, surfactant function, and lung physiology in a murine model of acute lung injury (ALI). 129/J mice received aerosolized endotoxin lipopolysaccharide [LPS] daily for up to 96 h to simulate the cytokine release and acute inflammation of ALI. Lung elastance (E(L)) and resistance, lavage fluid cell counts, cytokine levels, phospholipid and protein content, and surfactant function were measured. Lavage and lung tissue SP content were determined by Western blot and immunohistochemistry, and tissue messenger RNA (mRNA) levels were assessed by Northern blot and in situ hybridization. Tumor necrosis factor-alpha and neutrophil counts in bronchoalveolar lavage fluid increased within 2 h of LPS exposure, followed by increases in total protein, interleukin (IL)-1beta, IL-6, and interferon-gamma. E(L) increased within 24 h of LPS exposure and remained abnormal up to 96 h. SP-B protein and mRNA levels were decreased at 24, 48, and 96 h. By contrast, SP-A protein and mRNA levels and SP-C mRNA levels were not reduced. Surfactant dysfunction occurred coincident with changes in SP-B levels. This study demonstrates that lung dysfunction in mice with LPS-ALI corresponds closely with abnormal surfactant function and reduced SP-B expression.     

Surfactant protein B inhibits endotoxin-induced lung inflammation

Transgenic mice, in which the level of surfactant protein (SP)-B mature peptide varied 5.6-fold between SP-B(+/-) and SP-B-overexpressing lines (SP-B+/+/+), were used to test the hypothesis that SP-B protects against endotoxin-induced lung inflammation. Intratracheal administration of endotoxin resulted in significantly lower concentration of SP-B mature peptide and elevated levels of total protein in bronchoalveolar lavage fluid of SP-B(+/-) mice compared with SP-B-overexpressing mice, indicating that endotoxin treatment leads to impairment of SP-B expression coincident with increased lung injury in SP-B(+/-) mice. Recruitment of inflammatory cells and elaboration of proinflammatory cytokines in bronchoalveolar lavage fluid were reduced in SP-B-overexpressing mice compared with SP-B(+/-) mice, suggesting that SP-B inhibited endotoxin-induced lung inflammation. Lung compliance and tissue damping were significantly decreased in SP-B(+/+) and SP-B(+/-) mice, but were not changed in SP-B(+/+/+) mice, consistent with a protective effect of SP-B. The minimum surface tension of large aggregate surfactant was significantly lower for surfactant isolated from SP-B-overexpressing mice, both in the absence and the presence of added plasma proteins. These data suggest that SP-B protected against endotoxin-induced lung inflammation by enhancing surfactant function, resulting in reduced lung injury, decreased influx of inflammatory cells, and lower cytokine levels; in contrast, levels of SP-B in SP-B(+/-) mice were further decreased by endotoxin treatment, likely exacerbating lung injury in this group.     

Surfactant-associated glycoproteins accumulate in alveolar cells and secretions during reparative stage of hyaline membrane disease

Surfactant-associated (SA) glycoproteins are lung-specific proteins produced in the human lung by alveolar type II cells and Clara cells. The distribution of these proteins was studied immunohistochemically in lung tissue obtained postmortem from 12 stillborn fetuses and 49 infants with hyaline membrane disease (HMD). By 21 weeks of gestation, SA glycoproteins were detected in the fetal alveolar epithelium and within Clara cells. The staining increased in intensity and extent with advancing gestational age. Infants with HMD who survived less than 48 hours did not generally exhibit stainable material either within type II cells or secretions, but staining was often noted in Clara cells as well as focally beneath hyaline membranes. In infants surviving more than 48 hours, intense staining of hyaline membranes, alveolar secretions, proliferating alveolar type II cells, and Clara cells was evident. Immunoreactivity was intense in hypertrophic type II cells that formed a continuous alveolar epithelial lining in lungs with bronchopulmonary dysplasia. Included in the population of infants with HMD were 15 infants with pulmonary hypoplasia. The lungs of these infants showed minimal staining for SA glycoproteins regardless of postnatal survival time. The results provide an immunomorphologic basis for defining normal and abnormal lung maturation. They also indicate that enhanced SA glycoprotein production is a sustained response of regenerating and hypertrophic type II cells in premature infants.     

Comparative genomic hybridization: a new tool for reproductive pathology

OBJECTIVE: To demonstrate the effectiveness of comparative genomic hybridization (CGH) for analysis of reproductive pathology specimens in clinical cytogenetics laboratories. DESIGN: A total of 856 CGH analyses were performed on various placental and fetal tissues derived from 368 specimens of spontaneous abortions and on placentas from 219 pregnancies with live-born infants. The live-born infants were clinically evaluated as normally developed, with either a normal birth weight or with intrauterine growth restriction; some live-born infants had an abnormal prenatal triple screen with normal cytogenetic results on amniotic fluid cell cultures. RESULTS: Comparative genomic hybridization analysis was successfully performed on 856 samples from spontaneously aborted specimens and term placentas. Failure of analysis occurred in 1.6% of samples and was due to an insufficient amount of tissue for DNA extraction. Comparative genomic hybridization identified aneuploidy in 53% of spontaneous abortion samples and 3.1% of term placentas. CONCLUSIONS: Comparative genomic hybridization analysis is a useful clinical tool for detection of aneuploidy in placental and fetal tissues. It provides a genome-wide screen while eliminating tissue culture failures, culture artifacts, and maternal cell contamination. We present practical guidelines for interpreting CGH profiles derived from human reproductive specimens.     

Ultrastructure of Lamellar Bodies in Congenital Surfactant Deficiency

Congenital surfactant deficiency (CSD) is a newly identified neonatal lung disorder associated with a variety of molecular defects affecting surfactant synthesis and secretion in alveolar type II cells. The authors present ultrastructural findings of abnormal lamellar bodies in lung biopsies from 4 infants with CSD. All were term infants presenting shortly after birth with severe respiratory failure that was unresponsive to conventional therapy and all died within the first month of life. Lung biopsies were performed between 8 and 25 days of age. Biochemical and molecular studies in 2 unrelated male infants identified SP-B deficiency, one case with 121 ins 2 mutation and the second with a 209 + 4 A > G mutaion. Light microscopy in both cases showed features of alveolar proteinosis. Ultrastructurally, alveolar type II cells lacked mature lamellar bodies, and their cytoplasm contained numerous pleomorphic inclusions with membranous and vesicular structures not seen in normal type II cells. The other 2 infants were a pair of siblings in whom molecular studies identified mutations in ABCA3 transporter gene. Light microscopy showed features of acinar dysplasia and desquamative interstitial pneumonitis. TEM studies revealed absence of mature lamellar bodies in type II cells and instead showed a mixture of cytoplsamic electron-dense inclusions with concentric membranes and distinctive electron dense aggregates. The ultrastructual changes in alveolar type II cells correlated well with specific gene defect. In SP-B deficiency, the absence of mature lamellar bodies is consistent with the postulated role for this protein in the formation of lamellar bodies. The lack of mature lamellar bodies in the ABCA3 gene mutations is due to the dysfunction of this endogenous lipid transporter that targets surfactant lipid moieties to the lamellar bodies. The findings demonstrate the importance of TEM studies of lung biopsies from infants with CSD as it is a critical adjunct in the diagnosis of neonatal lung disease and in defining the underlying cellular defects.