Validation and reproducibility of computerised cell-viability analysis of tissue slices

Background

The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results.

Methods

To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes.

Results

Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77).

Conclusions

This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

     

Epidermal cyst of the breast: Detection in a screening programme

Two cases of epidermal cyst of the breast, a rare benign condition, were detected during a 3-year period in a mammographic screening programme, from 57 954 screening examinations. It is not uncommon for epidermal cysts to be initially misdiagnosed. The mammographic, ultrasound and histological features are presented. It is recommended that these lesions be resected because they possibly have malignant potential.     

TRACHEOSTOMY COMPLICATIONS AND THEIR MANAGEMENT

The word tracheostomy derived from two greek words meaning ‘I cut the trachea’ has been known for about 3500 yrs. The process has evolved over the years and has undergone revolutionary changes in the methodology, instrumentation and indications. Although tracheostomy is now commonly used the complication rate remains high. In our series it was 48% which is comparable with other series. The purpose of this paper is to discuss the complications of tracheostomy with special attention to their management and prevention.KEY WORDS: Complications, Tracheostomy     

Erythromycin treatment for gastrointestinal dysmotility in preterm infants

To report our clinical experience on the use of oral erythromycin for the treatment of severe gastrointestinal dysmotility in preterm infants.

Methodology:

A case series study of seven preterm infants (six were very low birthweight) with severe intestinal dysmotility in a tertiary neonatal centre.

Results:

All responded favourably without adverse effects and tolerated full enteral feeding within 1–2 weeks of the commencement of the drug.

Conclusions:

As prolonged total parenteral nutrition carries significant risk of complications, this therapy could be considered in selected preterm infants who fail to establish enteral feeding after an extended period, and in whom an anatomically obstructive lesion of the gastrointestinal tract has been excluded. Meanwhile, we would caution against the widespread implementation of this therapeutic approach until formal evaluation by randomized controlled trials have established the exact role of erythromycin, or its analogues, in the treatment of intestinal dysmotility in preterm infants.     

Structural genomic variation in childhood epilepsies with complex phenotypes

A genetic contribution to a broad range of epilepsies has been postulated, and particularly copy number variations (CNVs) have emerged as significant genetic risk factors. However, the role of CNVs in patients with epilepsies with complex phenotypes is not known. Therefore, we investigated the role of CNVs in patients with unclassified epilepsies and complex phenotypes. A total of 222 patients from three European countries, including patients with structural lesions on magnetic resonance imaging (MRI), dysmorphic features, and multiple congenital anomalies, were clinically evaluated and screened for CNVs. MRI findings including acquired or developmental lesions and patient characteristics were subdivided and analyzed in subgroups. MRI data were available for 88.3% of patients, of whom 41.6% had abnormal MRI findings. Eighty-eight rare CNVs were discovered in 71 out of 222 patients (31.9%). Segregation of all identified variants could be assessed in 42 patients, 11 of which were de novo. The frequency of all structural variants and de novo variants was not statistically different between patients with or without MRI abnormalities or MRI subcategories. Patients with dysmorphic features were more likely to carry a rare CNV. Genome-wide screening methods for rare CNVs may provide clues for the genetic etiology in patients with a broader range of epilepsies than previously anticipated, including in patients with various brain anomalies detectable by MRI. Performing genome-wide screens for rare CNVs can be a valuable contribution to the routine diagnostic workup in patients with a broad range of childhood epilepsies.     

Centrilobular emphysema: CT-pathologic correlation

Over a 5-year period, 25 patients who had undergone chest computed tomography (CT) died and were autopsied. Their lungs were fixed in the inflated state and were assessed for the presence and severity of centrilobular emphysema (CLE). Three radiologists independently evaluated the CT scans for nonperipheral low-attenuation areas, peripheral low-attenuation areas, pulmonary vascular pruning, pulmonary vascular distortion, and pulmonary density gradient. The CT criterion that best correlated with the presence and severity of CLE was the nonperipheral low-attenuation area. With this CT criterion, lung destruction was correctly identified in 13 of 15 cases. The absence of this criterion resulted in correct identification of eight of ten normal lungs. These preliminary data suggest that CLE can be reliably identified and quantified with current CT scanners.     

A comparison of excitatory amino acid antagonists acting at primary afferent C fibres and motoneurones of the isolated spinal cord of the rat.

The potency of 7 excitatory amino acid antagonists had been measured at kainate receptors on primary afferent C fibres using isolated dorsal roots from immature rats. Two of the compounds were tested as antagonists of excitant amino acids at motoneurones using isolated hemisected spinal cord preparations. Mean dose-ratios for antagonism of kainate at C fibres, produced by 1 mM antagonist in at least four preparations, ranged from 2.1 +/- 0.5 (mean +/- s.e.mean) with 2-amino-5-phosphonopentanoate (AP5) to 17.6 +/- 2.4 with 1-(p-bromobenzoyl)-piperazine-2, 3-dicarboxylate (BBpzD). The rank order of potency of antagonists at C fibres was similar to that obtained previously for antagonism of kainate at motoneurones. The potency of kynurenate as an antagonist of kainate at C fibres (apparent Kd 70 +/- 4.3 microM (mean +/- s.e.mean), n = 12) was significantly different (P less than 0.005, Wilcoxon rank sum) from its potency at motoneurones (apparent Kd 164 +/- 14 microM, n = 13). Kynurenate also was significantly (P less than 0.025 Wilcoxon rank sum) more potent at antagonizing kainate- than quisqualate (apparent Kd 258 +/- 28 microM, n = 12)-induced depolarization of motoneurones. Kynurenate and BBpzD (0.25, 1.0 and 4.0 mM) were compared as antagonists of N-methyl-D-aspartate (NMDA) at motoneurones and the slope of the Gaddum-Schild plot for kynurenate was markedly greater than 1 (2.01 +/- 0.22, 95% confidence limits). A greater than additive antagonism of NMDA-induced depolarizations was produced by combinations of kynurenate with, either AP5, or magnesium ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostatic evaluation by transrectal sonography with histopathologic correlation: the echopenic appearance of early carcinoma

Fifty-two patients with clinical stage A and B carcinomas of the prostate were imaged by ultrasound (US) transrectally with a 5-MHz linear array transducer and transabdominally with a 3-MHz sector scanner prior to radical prostatectomy. The fresh specimens of 44 prostate glands were scanned in a water bath with a 5-MHz linear array transducer in multiple planes. In all cases, histopathologic correlation was obtained. Prostatic carcinoma presented as an echopenic lesion in 54% of the specimens, as a slightly hypoechoic area in 22%, and could not be identified in 24% because of its isoechoic characteristics. In contrast to many previous reports, no instance of echogenic cancer was observed.