External validation of a simple risk score based on the ASPRE trial algorithm for preterm pre-eclampsia considering maternal characteristics in nulliparous pregnant women: a multicentre retrospective cohort study.

OBJECTIVE: To validate the performance of a first-trimester simple risk score based on the ASPRE trial algorithm for pre-eclampsia. DESIGN: Multicentre retrospective cohort analysis. SETTING: Four Italian hospitals. POPULATION: Unselected nulliparous women at 11-13 weeks of gestation from January 2014 through to January 2018. METHODS: Model performance was evaluated based on discrimination and calibration. MAIN OUTCOME MEASURES: Delivery before 37 weeks of gestation with a diagnosis of pre-eclampsia. RESULTS: Based on 73 preterm pre-eclampsia cases and 7546 controls (including 101 cases of late pre-eclampsia), the area under the receiver operating characteristics curve was 0.659 (95% CI 0.579-0.726). The sensitivity was 32.9% (95% CI 22.1-43.7) at a false-positive rate of 8.8%. The positive likelihood ratio was 3.74 (95% CI 2.67-5.23), the positive predictive value was 3.49% (95% CI 2.12-4.86%) and the negative predictive value was 99.3% (95% CI 99.1-99.5%). The sensitivity and positive likelihood ratio were approximately 40% lower than in the original study. The calibration analysis showed a good agreement between observed and expected risks (P = 0.037). Comparison with the Fetal Medicine Foundation (FMF) algorithm yielded a difference in the area under the curve of 0.084 (P = 0.007). CONCLUSIONS: In our Italian population, the simple risk score had a lower performance than expected for the prediction of preterm pre-eclampsia in nulliparous women. The FMF algorithm applied to the same data set resulted in a better prediction. TWEETABLE ABSTRACT: Simple risk score predicts preterm pre-eclampsia in Italy.

Multivariable evaluation of maternal hemodynamic profile in pregnancy complicated by fetal growth restriction: prospective study.

OBJECTIVE: To evaluate the association between fetal growth restriction (FGR) and maternal hemodynamic parameters using multivariable analysis, adjusting for major confounding factors, such as hypertensive disorders of pregnancy (pre-eclampsia and gestational hypertension). METHODS: A prospective cohort study was conducted between January 2013 and April 2016. Two cohorts of patients were recruited, between 24 and 39 weeks of gestation, in a high-risk outpatient setting. These cohorts comprised 49 appropriate-for-gestational-age singleton fetuses and 93 that were FGR (abdominal circumference (AC) at recruitment in the second half of pregnancy ≤ 10th percentile with a previous normal AC at 20-22 weeks). Maternal echocardiography was performed at the time of enrolment and included hemodynamic parameters of systolic and diastolic function and cardiac remodeling indices. Data were analyzed using a multivariable generalized linear model to estimate the association of FGR with maternal hemodynamic parameters after adjusting for significant confounding factors. RESULTS: In the multivariable analysis, after adjustment for hypertensive disorders of pregnancy and smoking, FGR was associated with a 14% increase in maternal total vascular resistance, 16% reduction in cardiac output, 13% reduction in left ventricular mass and 11% reduction in heart rate; similar results were observed for the corresponding indexed parameters. Hypertensive disorders of pregnancy in the absence of FGR were associated with a 25% increase in total vascular resistance, 16% increase in left ventricular mass and 14% reduction in diastolic function; similar results were observed for the corresponding indexed parameters. CONCLUSION: FGR is significantly and independently associated with several maternal hemodynamic parameters, even after adjustment for major confounding factors, such as hypertensive disorders of pregnancy. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Characterizing pearls structures using X-ray phase-contrast and neutron imaging: a pilot study.

Some cultured and natural pearls can be reliably distinguished by visual inspection and by the use of lens and microscope. However, assessing the origin of the pearls could be not straightforward since many different production techniques can now be found in the pearl market, for example in salt or freshwater environments, with or without a rigid nucleus. This wide range of products requires the use of new effective scientific techniques. Indeed, X-ray radiography has been used by gemologists since last century as the only safe and non-destructive way to visually inspect the interior of a pearl, and recently, also X-ray computed micro-tomography was used to better visualize the inner parts of the gems. In this study we analyzed samples of natural and cultured pearls by means of two non-destructive techniques: the X-ray Phase-Contrast Imaging (PCI) and the Neutron Imaging (NI). PCI and NI results will be combined for the first time, to better visualize the pearls internal morphology, thus giving relevant indications on the pearl formation process.

Maternal cardiac deceleration capacity: a novel insight into maternal autonomic function in pregnancies complicated by hypertensive disorders and intrauterine growth restriction.

OBJECTIVE: To explore maternal cardiac deceleration capacity (DC), a marker of autonomic function derived from electrocardiographic (ECG) signals, in pregnancies complicated by intrauterine growth restriction (IUGR) and hypertensive disorders of pregnancy (HDP) associated to IUGR (HDP-IUGR) or to appropriate for gestational age fetal growth (HDP-AGAf). METHODS: Prospective single center case-control study conducted at Buzzi Children's Hospital, Milan. Maternal ECGs were analyzed by Phase Rectified Signal Averaging (PRSA) method to obtain cardiac DC in women with: HDP-IUGR, HDP-AGAf, severe-IUGR, mild-IUGR and uncomplicated pregnancies. IUGR was defined as abdominal circumference <5th centile; severe-IUGR was associated with umbilical artery Doppler pulsatility index >2 standard deviations. Non-parametric tests were adopted. RESULTS: 269 women were recruited. Women with HDP-IUGR (n=35) showed significantly higher cardiac DC compared both to controls (n=141) (p=0.003) and women with HDP-AGAf (n=18) (p=0.01). Women with severe-IUGR (n=14) showed significantly higher DC than controls (p=0.01). Women with mild-IUGR (n=61) as well as women with HDP-AGAf showed no differences in DC compared to controls (both p=0.3). CONCLUSIONS: Women with pregnancy complicated by severe placental failure, such as HDP-IUGR and severe IUGR, show significant autonomic alterations, as indicated by elevated cardiac DC. On the contrary, pregnancy complications such as HDP-AGAf and mild IUGR show no impact on maternal autonomic balance. We present a new approach to explore maternal autonomic cardiovascular regulation that might reflect the severity of placental vascular insufficiency.

Clinical and epidemiological characteristics of infectious keratitis in Paraguay.

To describe the clinical and epidemiological characteristics of patients with severe infectious keratitis in Asunción, Paraguay between April 2009 and September 2011. All patients with the clinical diagnosis of severe keratitis (ulcer ≥2 mm in size and/or central location) were included. Empiric treatment consisted of topical antibiotics and antimycotics; in cases of advanced keratitis, fortified antibiotics were used. After microbiological analysis, treatment was changed if indicated. In total 48 patients (62.5 % males, 25 % farmers) were included in the analysis. A central ulcer was found in 81.3 % (n = 39). The median delay between onset of symptoms and time of first presentation at our institution was 7 days (range 1-30 days). Fungal keratitis was diagnosed in 64.5 % (n = 31) of patients, of which Fusarium sp. (n = 17) was the most common. Twenty-one patients (43.8 %) reported previous trauma to the eye. The globe could be preserved in all cases. While topical therapy only was sufficient in most patients, a conjunctival flap was necessary in six patients suffering from fungal keratitis. The high rate of fungal keratitis in this series is remarkable, and microbiological analysis provided valuable information for the appropriate treatment. In this setting, one has to be highly suspicious of fungal causes of infectious keratitis.

Nitazoxanide or CD3+/CD4+ lymphocytes for recovery from severe Cryptosporidium infection after allogeneic bone marrow transplant?

We describe a case of Cryptosporidium infection occurring in a child after allogeneic SCT for acute non-lymphoblastic leukemia. This patient presented an intestinal, biliar, and pancreatic Cryptosporidium disease associated with an intestinal aGvHD. The increase in CD3+/CD4+ cells secondary to the reduction of steroid therapy associated with the improvement of aGvHD and the use of antiparasitic treatments (especially nitazoxanide) improved the infection-related symptoms and led to a complete clearance of the Cryptosporidium.

DNA extraction from blood determination membrane card test.

Among the several techniques applied in our laboratory, in order to establish human nature of blood specimens we perform an immunodiagnostic analysis through a specific membrane test card, which is usually utilized as a fast screening test for human haemoglobin in diagnostic checks. This test works with a double-step method based upon solubilization of the sample in a proper buffer and its migration on a membrane monoclonal and polyclonal antibodies specific against haemoglobin are linked to. The complexes haemoglobin-antibodies migrate through the membrane and are revealed by an immunoreactive line turning reddish-coloured to show that the reaction took place and the system works. As this test can recognize both the whole haemoglobin molecule and the degraded one, it is usually exploited in forensic sciences to detect kind and species of blood specimens related to forensic case works. However, as a consequence of the small amounts of blood in the samples collected from the crime scene we often cannot perform this kind of analysis in order to save the sample and be able to genotype it. Then, we investigated the possibility to purify DNA from the blood specimens already dissolved in the extraction buffer and perform STR typing on it with several methods and after different storage conditions and time.

Single sperm cell isolation by laser microdissection.

In 1996, Van Oorschot and Jones firstly reported in scientific correspondence that short tandem repeat (STR) profiles could be obtained from cells left on different objects. Since then, forensic scientists have focused their efforts in isolating single cells as it can be extremely helpful in solving case works where sexual violence was concerned. Laser microdissection is a micromanipulation procedure allowing to cut off precisely the cells of interest from tissue samples or smears by a laser beam fitted with an optical microscope. We have harvested single sperm cells by laser microdissection using a Leica AS LMD (Leica Microsystems, Germany); laser setting, pulse laser intensity and laser alignment as well as recovery of the specimen have been properly fitted to the samples we were dealing with. Different tissue preservation, fixation, histological staining (Papanicolau, Nuclear Fast Red-Picroindigocarmine) methods and number of harvested cells for each sample have been evaluated as well. Finally, the genotype of sperm cells has been determined by STR typing, evaluating the sensibility of this forensic technique according to instrumental and biological above-mentioned variables.

Laser microdissection and DNA typing of cells from single hair follicles.

Isolation and identification of single cells from tissue samples or smears assume a great relevance in pathological and forensic applications; in this latter field, the possibility to identify a specific genetic profile can be obtained by short tandem repeat (STR) typing, allowing to achieve a scientific proof important in law courts. It is well known that DNA extraction may be performed from several tissue fragments, blood traces, spermatozoa as well as telogen hair. However, in the last case, few follicle cells are coupled to a great amount of keratin reducing the efficiency of DNA amplification. Recently, the introduction of laser microdissection technique has greatly improved the capability to select single cells without any cross-contamination. In the present report, we have performed a laser microdissection using a Leica AS LMD (Leica Microsystems, Germany), utilized on cutting the telogen hair in order to exclusively collect the lower part of the follicle and reduce keratin contamination. In this way we can accurately extract an adequate amount of DNA, successfully typed by STR profile.

Experimental procedures comparing the activity of different Taq polymerases.

Forensic investigations involve several scientific branches among which biological analyses are much more frequently requested as a consequence of their importance and great versatility towards most of the traces found on the crime scene. Biological analyses are lead in subsequent steps: extraction, amplification and STR typing of the specimens collected on the crime scene. All of these techniques have been modified from original protocols according to the kind of sample to process. A critical point in our analysis is trying to amplify small amounts of DNA extracted from decomposed tissues or objects, small biological traces have been left on, with high fidelity and account. That's why we have decided to settle on an experimental procedure aimed to find the best DNA polymerase according to our purposes. We have tested different Taq polymerases on the same known DNA sample at several dilutions and have compared quality and amount of amplified DNA in order to appreciate the amplifying capability of each enzyme. These data have been analyzed as a function of the technical properties of each engineered Taq polymerase and results are shown in details.

A novel approach in personal identification from tissue samples undergone different processes through STR typing.

Short tandem repeats (STRs) or microsatellites have been recognized worldwide as a powerful tool for human identification. They have become widely used in human identification especially in criminal cases and mass disasters. Police departments are often interested in cases where tissues are already decomposed and only do bones remain to let them perform laboratory analyses. Bone is the most resistant tissue in animal body to time depending degradation and putrefaction, but it is often hard to extract DNA from it because of its highly mineralized structure, which makes DNA extraction and/or amplification hard to carry out. We have performed human nuclear DNA extraction and STR typing in three different cases, on bones and bone fragments from long time dead persons found buried, in the sea, almost completely burnt and whose tissues were already decomposed. We report these caseworks as we would like to show how forensic scientists are improving their skill in identifying people whose corps have undergone several kinds of processes, even independently on the time passed and the level of putrefaction of their tissues.

Adenoviral-mediated gene transfer to the filtering bleb in rabbits.

PURPOSE: To determine if genes can be transferred to fibroblasts in the filtering bleb using adenoviral vectors. MATERIALS AND METHODS: Twelve New Zealand albino rabbits underwent bilateral full-thickness sclerostomies. On postoperative day 1 an adenoviral vector carrying a reporter gene (lacZ) was injected into the right-eye bleb and saline was injected into the left eye bleb of each rabbit. Three rabbits were euthanized on each of the after days (days 3, 7, 14, and 21). The eyes were enucleated and tissue samples from the filtering bleb were processed for beta-galactosidase activity (a marker for lacZ gene expression) and expression of vimentin (a fibroblast marker). The median number of cells per high-power field with both beta-galactosidase activity and vimentin expression on days 3, 7, 14, and 21 in the right and left eyes were counted to determine adenoviral-mediated gene expression in fibroblasts. RESULTS: In the adenoviral vector-treated eyes, the median number of cells per high-power field with both beta-galactosidase and vimentin expression on days 3, 7, 14, and 21 was 83,100, 1, and 0, respectively. No beta-galactosidase activity was noted in the saline-treated eyes. CONCLUSIONS: Adenoviral vectors can transfer genes to fibroblasts in filtering blebs after glaucoma surgery. The peak efficiency of gene transfer to fibroblasts occurred 7 days after glaucoma surgery. These studies show a potential for genetic manipulation of fibroblast activity in filtering blebs after glaucoma surgery.

Detection of T cell receptor delta gene rearrangements in childhood B and T lineage acute lymphoblastic leukaemia by southern blot and PCR: technical comparison of two methods of analysis.

Molecular analysis of antigen receptor genes (Ig and TCR) has been useful for clonal studies in acute lymphoblastic leukaemia (ALL) patients. Rearrangements of these genes can be used to track the persistence of the leukaemic clone during the therapy. The purpose of our study was to analyse the percentage and the pattern of the rearrangements at the TCR D locus in a series of ALL patients, comparing the results obtained by Southern blot and PCR. Genomic DNA was extracted from mononuclear BM cells of 40 paediatric ALL cases, digested with different restriction enzymes and hybridized to TCRDJ1 probe to study the TCR delta locus. Amplification of the rearranged TCR delta genes was performed by PCR to define the gene segments involved. The junctional region was deduced from the sequence to obtain patient-specific primers. Among the 31 B lineage ALL samples, one or two TCR delta alleles proved to be rearranged in 53% of cases. Two different types of rearrangements were chiefly detected: Vdelta2Ddelta3 and Ddelta2Ddelta3. In T-ALL patients, the predominant rearrangement involved the Vdelta1 and the Jdelta1 gene segments.

The expression of metalloproteinase-2, -9, and -14 and of tissue inhibitors-1 and -2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase-14.

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14. These, along with the tissue inhibitors TIMP-1 and -2, are developmentally regulated according to the maturation stage of osteoblasts. Their levels change in a similar association with osteoblast phenotypic maturation in different populations of ROB, which take different times to complete osteogenesis in vitro. MMP-14 expression coincides in both cell populations with the mature osteoblastic phenotype and is localized in the cells forming nodules. MMP-2 and -9 are expressed diffusely in the osteoblast population. Developmentally associated changes in the activation of MMP-2 are detected, associated in their timing with the expression of MMP-14 in both populations of ROB, and MMP-14 activates pro-MMP-2 in vitro. Expression of messenger RNAs (mRNAs) for the three MMPs increases up to the time of nodule formation. At this stage, TIMP-1 mRNA levels are lowest. TIMP-2 mRNA decreases throughout osteogenesis. In situ hybridization in 7-day-old rat tibias shows the strongest expression of MMP-14 among osteogenic cells, in lining osteoblasts on the newly formed trabeculae under the growth plate, and on the endosteal surface of cortical bone. Our data support the concept that the developmentally regulated expression of MMP-14 triggers localized proteolysis within the osteogenic population, concomitant in vitro to nodule formation.

Late Epstein-Barr virus infection of a hepatosplenic gamma delta T-cell lymphoma arising in a kidney transplant recipient.

BACKGROUND AND OBJECTIVE: gd T-cell lymphomas are only exceptionally observed in transplanted patients. Aim of this study was the detailed characterization of one such case. DESIGN AND METHODS: The patient developed spontaneous splenic rupture six years after kidney transplantation. The splenic red pulp was infiltrated by medium-sized and large lymphoid cells with two or more nucleoli. At autopsy, similar lymphoid cells infiltrated the hepatic sinusoids. Histologic, immunologic and molecular studies were carried out. RESULTS: By immunohistochemistry, the atypical lymphoid cells were found to express CD3, CD45 and CD43, indicating their T-lineage origin. Approximately 99% of spleen mononuclear cells (MNC) were CD3(+), gammadelta TcR+, CD4-, CD8-, alphabeta TcR-. A clonal gammadelta TcR rearrangement (Vgamma1-Jgamma1.3/2.3-Cgamma2; Vdelta1-Ddelta2-Jdelta1) was detected. The final diagnosis was peripheral T-cell lymphoma, hepato-splenic gammadelta-type. EBV infection of spleen MNC was documented by molecular studies. However, in situ hybridization for EBER-1 (EBV-RNA) showed that only a minority of malignant lymphoid cells (5-7%) were EBV-infected. INTERPRETATION AND CONCLUSIONS: It is concluded that EBV infection was as a late event involving an already transformed gd T-cell clone.

Exocrine pancreatic function and fat malabsorption in human immunodeficiency virus-infected patients.

BACKGROUND: Nutrients malabsorption frequently occurs in human immunodeficiency virus (HIV)-infected patients, but very few studies have investigated exocrine pancreatic digestive capacity in these patients. We therefore evaluated the frequency of exocrine pancreatic impairment and its eventual relation with fat malabsorption in HIV-infected patients. METHODS: Thirty-five HIV-infected patients (30 male, 5 female: mean age +/- standard deviation, 33.6 +/- 7.2 years) and 51 sex- and age-matched controls without gastroenterologic diseases were studied. In all subjects fecal elastase 1 (EL-1) was assayed, and fecal fat excretion was evaluated with the steatocrit test. RESULTS: Nineteen of 35 (54%) HIV-infected patients showed subnormal EL-1 values, whereas all the controls had normal values; furthermore, EL-1 values were significantly lower in patients than in controls: mean (95% confidence intervals), 207 ( 164-251 ) microg/g versus 312 (291-332) microg/g (P < 0.0001). Increased fecal fat excretion was observed in almost all (25 of 35) HIV-infected patients, and an inverse but not significant correlation was found between fecal EL-1 and steatocrit values. No association was found between reduced fecal EL-1 and the severity of HIV disease or nutritional and immunologic status. Opportunistic infections and drug administration had no influence on EL-1 concentrations in stools. CONCLUSIONS: Reduced exopancreatic function is frequent in HIV-infected patients but does not seem to be a major factor contributing to fat malabsorption.

Pancreatic dysfunction and its association with fat malabsorption in HIV infected children.

BACKGROUND: Nutrient malabsorption frequently occurs in HIV infected children, but very few studies have investigated exocrine pancreatic digestive capacity in these cases. AIMS: To investigate pancreatic function in HIV infected children and to determine whether faecal fat loss, a prominent feature of intestinal dysfunction, is associated with pancreatic dysfunction. PATIENTS: Forty seven children with HIV infection without apparent pancreatic disease and 45 sex and age matched healthy controls. METHODS: Pancreatic function was evaluated by measuring elastase 1 concentration and chymotrypsin activity in stools by ELISA and colorimetric methods, respectively. Intestinal function was evaluated by measuring fat and protein loss by the steatocrit method and by faecal alpha1 antitrypsin concentration. RESULTS: 14 (30%) had abnormal pancreatic function tests: seven had isolated elastase activity deficiency, three isolated chymotrypsin deficiency, and four pancreatic deficiencies in both enzymes. Patient enzyme values were significantly lower than those of controls. Low faecal pancreatic enzymes were not associated with symptoms. Twelve children had steatorrhoea and four had increased alpha1 antitrypsin. Steatorrhoea was significantly associated with reduced faecal pancreatic enzymes. There was a significant negative correlation between elastase 1 concentration and steatocrit. Children with pathological faecal elastase 1 or chymotrypsin values did not differ from the other HIV infected children with respect to nutritional and immunological status, stage of HIV disease, presence of opportunistic infections, or drug administration. CONCLUSIONS: Abnormal pancreatic function tests are a frequent feature of paediatric HIV infection; this condition is associated with steatorrhoea, which probably contributes to the disease.

Rapid detection of gammaT cell receptor gene rearrangements in acute lymphoblastic leukemia by electrophoresis and silver staining: implications for detection of minimal residual disease.

Minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) was studied using polymerase chain reaction (PCR). GammaT cell receptor (TCRG) genes are ideal targets for PCR-based detection of MRD due to their molecular characteristics. Polyacrylamide gel electrophoresis (PAGE) analysis of PCR products followed by silver staining was performed for 72 children with ALL at the onset of disease. Silver staining is an effective technique to detect gene rearrangements without the use of ethidium bromide. Moreover, this method may show heteroduplex bands of a clonal nature when both TCRG alleles are rearranged. PCR products subjected to a rapid staining protocol were recovered from the gel, reamplified by a second PCR and directly sequenced. After sequencing, we identified the junctional region and obtained patient-specific probes. In more than half of the patients we detected TCRG rearrangements that were used as molecular markers for residual disease.

Serum HCV-RNA and liver histologic findings in patients with long-term normal transaminases.

In this study we aimed to correlate liver histology and the presence of hepatitis C virus (HCV) viremia, genotype, and quantity of HCV genome in 19 positive and 11 RIBA II indeterminate patients presenting persistently normal ALT values over 24 months before biopsy. In addition, after biopsy serum ALT values were monitored monthly for a mean follow-up period of 24.8 months, after which patients were reevaluated for RIBA II and the presence of viremia. Sixteen patients (53%) were serum HCV-RNA-positive; 13 of them (68%) were confirmed positive and 3 (27%) indeterminate on RIBA II. Histology of the HCV-RNA-positive patients showed eight cases of CPH (one case of genotype 1a; four cases type 1b; three cases type 2), six cases of CAH (three cases type 1b, three cases type 2), one case of CLH (type not determined), and one case of normal liver (NL) (type 1b). Histology of the HCV-RNA-negative patients showed four cases of CPH, one case of CAH, two cases of CLH, and seven cases of NL. During the follow-up period nine patients (30%) presented slight increases in ALT values (< 2 x N), and in particular, flares of ALT were observed four times in the CAH and five times in the CPH patients, who were all viremic, but never in the NL subjects. These results indicate that subjects positive on RIBA II, but with persistently normal ALT values, had a high probability of being serum HCV-RNA-positive and that almost all these viremic subjects presented histologic signs of liver disease. In contrast, RIBA II indeterminate subjects had a moderate probability of being HCV-RNA-positive, but a number of these may present signs of liver disease. In both cases there was no association with genotype or HCV-RNA serum levels. The other nonviremic cases included subjects with hepatic changes going toward resolution or with normal liver in whom hepatic biopsy can be avoided. Only one case was a true carrier since he was viremic with normal liver and persistently normal ALT values.