Comparison of Two- and Three-dimensional Ultrasonography in the Evaluation of Lesion Level in Fetuses with Spina Bifida

Purpose To evaluate the precision of both two- and three-dimensional ultrasonography in determining vertebral lesion level (the first open vertebra) in patients with spina bifida. Methods This was a prospective longitudinal study comprising of fetuses with open spina bifida who were treated in the fetal medicine division of the department of obstetrics of Hospital das Clínicas of the Universidade de São Paulo between 2004 and 2013. Vertebral lesion level was established by using both two- and three-dimensional ultrasonography in 50 fetuses (two examiners in each method). The lesion level in the neonatal period was established by radiological assessment of the spine. All pregnancies were followed in our hospital prenatally, and delivery was scheduled to allow immediate postnatal surgical correction. Results Two-dimensional sonography precisely estimated the spina bifida level in 53% of the cases. The estimate error was within one vertebra in 80% of the cases, in up to two vertebrae in 89%, and in up to three vertebrae in 100%, showing a good interobserver agreement. Three-dimensional ultrasonography precisely estimated the lesion level in 50% of the cases. The estimate error was within one vertebra in 82% of the cases, in up to two vertebrae in 90%, and in up to three vertebrae in 100%, also showing good interobserver agreement. Whenever an estimate error was observed, both two- and three-dimensional ultrasonography scans tended to underestimate the true lesion level (55.3% and 62% of the cases, respectively). Conclusions No relevant difference in diagnostic performance was observed between the two- and three-dimensional ultrasonography. The use of three-dimensional ultrasonography showed no additional benefit in diagnosing the lesion level in the fetuses with spina bifida. Errors in both methods showed a tendency to underestimate lesion level.

A and B mode ultrasonography in preoperative evaluation of lens and posterior segment of dogs eyes with cataract

Ultrasonography of the lens and posterior segment is an indispensable step in the preoperative evaluation of dogs with cataracts, since ophthalmoscopy is not feasible when there is opacification of the lens. This study evaluated the echographic conditions of cataractous lens and fundus of the eye in dogs affected by cataracts. The study was conducted in 30 dogs (56 eyes), 10 males and 20 females, with different types of cataracts at different stages of development. Echography in A and B modes, simultaneously, was carried out for the examination of the lens and posterior segment. The examinations revealed anterior cortical, posterior cortical and nuclear cataract in 12 eyes (21.4%), anterior cortical, posterior cortical, nuclear and posterior capsular in 23 eyes (41%), anterior cortical, posterior cortical and posterior capsular cataract in one eye (1.7%), anterior cortical and nuclear cataract in one eye (1.7%), anterior cortical, nuclear and posterior capsular cataract in five eyes (8.9%), and anterior cortical cataract in seven eyes (12.5%). Abnormal ultrasonographic alterations were observed in the posterior segment in 26 eyes evaluated (46.4%). Vitreal degeneration was detected in 12 eyes (21.4%), images of vitreal exudate or hemorrhage in seven eyes (12.5%), persistence of hyaloid artery in four eyes (7.1%) and lens subluxation in three eyes (5.3%). The results obtained reiterate the importance of ultrasonography in canine patients presented for cataract surgery given that alterations of the posterior segment are difficult to identify in a clinical examination when the lens is opacified.

Interphase cytogenetics using fluorescence in situ hybridization: an overview of its application to diffuse and solid tissue

Interphase cytogenetics, utilizing fluorescence in situ hybridization (FISH) techniques, has been successfully applied to diffuse and solid tissue specimens. Most studies have been performed on isolated cells, such as blood or bone marrow cells; a few have been performed on cells from body fluids, such as amniotic fluid, urine, sperm, and sputum. Mechanically or chemically disaggregated cells from solid tissues have also been used as single cell suspensions for FISH. Additionally, intact organized tissue samples represented by touch preparations or thin tissue sections have been used, especially in cancer studies. Advantages and pitfalls of application of FISH methodology to each type of specimen and some significant biological findings achieved are illustrated in this overview.

Acute promyelocytic leukemia: the study of t(15;17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93% (mean: 56%). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80% of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100% of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40% of 5' breaks and 60% of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

In situ variation of cervical mucus pH during exposure to atmospheric air

The objective of the present study was to determine if exposure of cervical mucus to air during specular examination could modify mucus pH. Detection of changes is justified because of their possible interference with sperm-mucus interaction, since an acidic pH is unfavorable to sperm penetration and is associated with infertility due to the cervical factor. Twenty women with good quality mucus were evaluated. pH measurements of ecto- and endocervical mucus were made in situ using a glass electrode after 0-, 5- and 10-min exposure to air. There was a progressive alkalinization of mucus pH. Mean values of ectocervical mucus pH were 6.91, 7.16 and 7.27, while mean values of endocervical mucus pH were 7.09, 7.34 and 7.46 at 0, 5 and 10 min, respectively. Significant differences were found between the mean values obtained at 0 and 5 min, and at 0 and 10 min (P<0.05), whereas the differences in mean values at 5 and 10 min were not significant at either site. We conclude that 5 to 10 min of exposure to atmospheric air affects cervical mucus pH in a significant way. Since tests used to evaluate sperm-mucus interaction generally have not considered this possibility, we suggest that they should be performed immediately after mucus collection in order to avoid misinterpretation of the results.

Presence of ductal carcinoma in situ confers an improved prognosis for patients with T1N0M0 invasive breast carcinoma

We have retrospectively analyzed a series of 155 sequential cases of T1N0M0 ductal carcinomas of which 51 tumors had a ductal carcinoma in situ (DCIS) component for correlation between the presence of DCIS and clinicopathological variables, recurrence and patient survival. No correlations between the presence of DCIS and age, menopausal status, size, estrogen or progesterone receptors were found. High-grade infiltrative tumors tended not to present a DCIS component (P = 0.08). Patients with tumors associated with DCIS form a subgroup with few recurrences (P = 0.003) and good survival (P = 0.008). When tumors were classified by size, an association between large tumors (>1.0 cm) and increased recurrence and shortened overall survival was found. The presence of DCIS in this subgroup significantly reduced the relative risk of death.

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

In vitro evaluation of the cytotoxic and trypanocidal activities of Ampelozizyphus amazonicus (Rhamnaceae)

Ampelozizyphus amazonicus Ducke is a tree commonly found in the Amazon region and an extract of its stem bark is popularly used as an antimalarial and anti-inflammatory agent and as an antidote to snake venom. Ursolic acid; five lupane type triterpenes: betulin, betulinic acid, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid, and three phytosteroids: stigmasterol, sitosterol and campesterol, have been isolated from stem extracts of A. amazonicus Ducke. Their structures were characterized by spectral data including COSY and HMQC. In an in vitro biological screening of the isolated compounds, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid was cytotoxic against the SKBR-3 human adenocarcinoma cell line (1 to 10 mg/mL), while 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid exhibited cytotoxicity against both SKBR-3 human adenocarcinoma and C-8161 human melanoma tumor cell lines (>0.1 mg/mL). In the present study, different extracts and some fractions of this plant were also investigated for trypanocidal activity due to the presence of pentacyclic triterpenes. The triterpene classes are potent against Trypanosoma cruzi. The bioassays were carried out using blood collected from Swiss albino mice by cardiac puncture during the parasitemic peak (7th day) after infection with the Y strain of T. cruzi. The results obtained showed that A. amazonicus is a potential source of bioactive compounds since its extracts and fractions isolated from it exhibited in vitro parasite lysis against trypomastigote forms of T. cruzi at concentrations >100 µg/mL. Fractions containing mainly betulin, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid showed more activity than crude extracts.