Optimization of immunocytochemistry in cytology: comparison of two protocols for fixation and preservation on cytospin and smear preparations

Objective: A new protocol for fixation and slide preservation was evaluated in order to improve the quality of immunocytochemical reactions on cytology slides. Methods: The quality of immunoreactions was evaluated retrospectively on 186 cytology slides (130 direct smears, 56 cytospins) prepared from different cytology samples. Ninety-three of the slides were air dried, stored at -20 °C and fixed in acetone for 10 minutes (Protocol 1), whereas the other 93 were immediately fixed in methanol at -20 °C for at least 30 minutes, subsequently protected with polyethylene glycol (PEG) and stored at room temperature (Protocol 2). Immunocytochemical staining, with eight primary antibodies, was performed on a Ventana BenchMark Ultra instrument using an UltraView Universal DAB Detection Kit. The following parameters were evaluated for each immunoreaction: morphology preservation, intensity of specific staining, background and counterstain. The slides were blinded and independently scored by four observers with marks from 0 to 20. Results: The quality of immunoreactions was better on methanol-fixed slides protected with PEG than on air-dried slides stored in the freezer: X¯ = 14.44 ± 3.58 versus X¯ = 11.02 ± 3.86, respectively (P < 0.001). Conclusion: Immediate fixation of cytology slides in cold methanol with subsequent application of PEG is an easy and straightforward procedure that improves the quality of immunocytochemical reactions and allows the storage of the slides at room temperature.

Children visual screening protocol: validation by a focus group panel of experts

Screening programs, particularly the inclusion of specific orthoptic tests to detect visual abnormalities, varies among countries. This study aims to: 1) describes expert perception of issues related with children visual screening; 2) identify specific orthoptic tests to detect visual abnormalities in children visual screening.

A novel flow cytometric protocol for assessment of yeast cell adhesion

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner.

Optimising protocol of acquisition and post-processing procedures in the assessment of coronary artery disease by computed tomography

Coronary artery disease (CAD) is currently one of the most prevalent diseases in the world population and calcium deposits in coronary arteries are one direct risk factor. These can be assessed by the calcium score (CS) application, available via a computed tomography (CT) scan, which gives an accurate indication of the development of the disease. However, the ionising radiation applied to patients is high. This study aimed to optimise the protocol acquisition in order to reduce the radiation dose and explain the flow of procedures to quantify CAD. The main differences in the clinical results, when automated or semiautomated post-processing is used, will be shown, and the epidemiology, imaging, risk factors and prognosis of the disease described. The software steps and the values that allow the risk of developingCADto be predicted will be presented. A64-row multidetector CT scan with dual source and two phantoms (pig hearts) were used to demonstrate the advantages and disadvantages of the Agatston method. The tube energy was balanced. Two measurements were obtained in each of the three experimental protocols (64, 128, 256 mAs). Considerable changes appeared between the values of CS relating to the protocol variation. The predefined standard protocol provided the lowest dose of radiation (0.43 mGy). This study found that the variation in the radiation dose between protocols, taking into consideration the dose control systems attached to the CT equipment and image quality, was not sufficient to justify changing the default protocol provided by the manufacturer.