Computerised image analysis of split-drop micropuncture data

A technique is described for rapid and reproducible analysis of split-drop micropuncture sequences recorded on film. Automatic identification of the droplet menisci in each cine frame, and subsequent evaluation is performed by a computerised T.V. image analysis system. In comparison with a more conventional technique, the improvement in reproducibility of the analysis is achieved without loss of accuracy and is accompanied by a three fold increase in speed.     

Computerised image analysis in conjunction with fluorescence microscopy for the study of blood-brain barrier permeability in vivo

The present paper describes a new method using computerised image analysis techniques for quantification of tracer extravasation over the blood-brain barrier as studied by intravital fluorescence microscopy. Cats were equipped with an open cranial window and continuously infused with fluorescein isothiocyanate-labelled dextran (FITC-dextran, mol. wt. 70 000) to maintain a steady plasma concentration. Several cortical fields were recorded in each experiment and the images stored on video tape for off-line analysis. This procedure, which largely eliminates the superficial pial vasculature and allows extraction of the extravasation areas, consists of the following steps: (1) averaging of images, (2) software shading correction based on the original images for compensation of optical non-uniformity, (3) correction of displacement artefacts, (4) intensity adjustment, (5) generation of subtraction images by subtracting the first image of a series from the subsequent ones, (6) median filtering and thresholding, (7) a length recognition algorithm, and (8) elimination of small areas. Compared to the previously described method, step (2) has been newly developed and steps (4) and (8) added to enhance sensitivity for detecting tracer extravasation. The degree of extravasation in a cortical field at a given time point [E(f) value] was calculated as the mean intensity of the remaining pixels. TheE(f) is a quantitative value computed by a fully automatised procedure which takes into account the number, as well as the size and intensity, of extravasation areas in a given cortical field. TheE(f) values obtained at different times in a series of experiments were averaged to give theE(I) value. TheE(I) value did not alter when hypercapnia was employed to induce pure vasodilatation. On the other hand it increased dramatically, indicating tracer extravasation, during topical application of high concentrations of adenosine (10^–5–10^–3 M). The new computerised image analysis procedure may therefore be suitable for measuring quantitatively tracer extravasation over the blood-brain barrier in vivo under different experimental conditions. It may also be applicable to study changes of vascular permeability in peripheral vascular beds.     

Safety management in pathology laboratory: from specimen handling to confirmation of reports

Medical errors in pathological diagnosis give a huge amount of physical and psychological damage to patients as well as medical staffs. We discussed here how to avoid medical errors in surgical pathology laboratory through our experience. Handling of surgical specimens and diagnosing process requires intensive labor and involves many steps. Each hospital reports many kinds of accidents or incidents, however, many laboratories share common problems and each process has its specific risk for the certain error. We analyzed the problems in each process and concentrated on avoiding misaccessioning, mislabeling, and misreporting. We have made several changes in our system, such as barcode labels, digital images of all specimens, putting specimens in embedding cassettes directly on the endoscopic biopsied specimens, and using a multitissue control block as controls in immunohistochemistry. Some problems are still left behind, but we have reduced the errors by decreasing the number of artificial operation as much as possible. A pathological system recognizing the status of read or unread the pathological reports by clinician are now underconstruction. We also discussed about quality assurance of diagnosis, cooperation with clinicians and other comedical staffs, and organization and method. In order to operate riskless work, it is important for all the medical staffs to have common awareness of the problems, keeping careful observations, and sharing all the information in common. Incorporation of an organizational management tool such as ISO 15189 and utilizing PDCA cycle is also helpful for safety management and quality improvement of the laboratory.     

Computerised data reduction

The use of personal computers in accountancy and business generally has been stimulated by the availability of flexible software packages. We describe the implementation of a commercial software package designed for interfacing with laboratory instruments and highlight the ease with which it can be implemented, without the need for specialist computer programming staff.     

An image processing software applied to oral pathology

This study presents the development and evaluation of an image processing software for computer-assisted cellular structure counting. The proposed software consists of a set of processing and analytical tools which allows its use in several applications of cell and cellular structure counting. A particular application in AgNOR quantitative analysis is presented. The knowledge obtained from experienced pathologists has been codified in a sequence of processing steps in order to allow automatic estimation of the mean number of AgNORs per cell in ameloblastomas. The performance of the presented software in such application was verified by comparing the data provided by visual analysis, by two observers previously calibrated and under supervision of two experienced specialists (Group 1) and by the computer program (Group 2). No statistical difference was observed (p < 0.05) between the two groups. The use of the proposed method in AgNOR applications permitted attainment of accurate and precise data without the difficulties frequently found in the traditional visual analysis method (time, training and subjectivity). The developed software is an interesting tool as an aid in the study (estimation of the number of cells and cellular structures) of malignant and benign neoplasms.     

A practical application of quantitative vascular image analysis in breast pathology

Aims

Quantitative image analysis of histopathology slides is becoming an important technology in diagnostic pathology. To this end, it is essential to combine a robust image analysis software with the most commonly used immunohistochemical staining methods. In this investigation, we describe a practical application of NIH ImageJ software for quantitative vascular image analysis for diaminobenzene chromogen-based CD34 immunostain in breast cancer. CD34 immunostain is in a unique position to identify lymphangiogenesis and angiogenesis simultaneously in a given tumor tissue. This investigation aims at establishing a practical quantitative vascular image analysis solution for diagnostic pathologists by using ImageJ, and CD34 immunostain.

Methods and results

Tissue microarray slides containing breast cancer tissue were immunostained for CD34 for simultaneous identification of lymphatic endothelial cells (LEC) and blood vessel endothelial cells (BEC). Digital images were analyzed using NIH ImageJ software. A CD34 score was quantified for each tissue core as a percentage (CD34-positive area/area of tissue core). The mean CD34 scores were 0.24%, 0.40%, 1.30%, 2.33%, 2.64%, and 3.44% for normal breast tissue, in stage IIA, IIB, IIIA, IIIB, and IIIC breast cancer tissue cores, respectively (p < 0.0001). The mean CD34 scores were 0.70% and 2.21% for lymph node-negative and lymph node-positive breast cancer patients, respectively (p < 0.0001).

Conclusions

ImageJ software seems to be an attractive quantitative image analysis tool for diagnostic pathology for immunohistochemistry-based applications because of its capabilities, availability, and ease of use with most image formats. Our results show the feasibility, versatility, and ease of use of ImageJ and CD34 immunohistochemistry for vascular image analysis in breast pathology. Given the prospects of novel lymphatic and vascular endothelium-targeting therapeutics in breast oncology, the practical analysis of combined LEC and BEC density described in this report could enable diagnostic pathologists to apply quantitative vascular image analysis easily in their pathology practice and translational research.     

Intrarenal calcium in phosphate handling.

The intrarenal role of plasma ionized calcium (Ca), on fractional phosphate excretion (FE PO4) was investigated in dogs with control of parathyroid hormone (PTH). In series 1, acute thyroparathyroidectomy was immediately followed by a constant infusion of bovine PTH (0.01 U/kg per min). Subsequent calcium chloride infusions increased Cai in plasma phosphate and decreased in the percentage of ultrafiltrable phosphate. A 20% increase in Cai significantly increased FE PO4 by +3.82 +/- 0.97% (P less than 0.01) when infused intravenously and by +2.62 +/- 1.06% (P less than 0.05) when infused in the renal artery. In contrast, a 75% increase in Cai did not significantly change FE PO4. In series 2, dogs were thyroparathyroidectomized 18 h before experiments, and no PTH infusion was initiated. A bolus of bovine PTH (30 U/kg) increased FE PO4 + 8.9 +/- 0.9% (P less than 0.001) in hypocalcemic dogs, +19.1 +/- 4.4% (P less than 0.001) in normolcalcemic dogs, and +15.5 +/- 1.5% (P less than 0.001) in hypercalcemic dogs. We conclude that increases in plasma calcium potentiate the phosphaturic effect of PTH. This potentiating effect is attenuated in marked hypercalcemia by superimposed hemodynamic and/or metabolic changes.     

Computerised monitoring of integrated cervical screening. Indicators of diagnostic performance

In a previous issue of this journal, we presented the background, rationale, general methods, and indicators of participation of a computerised system for the monitoring of integrated cervical screening, i.e. the integration of spontaneous Pap smear practice into organised screening. We also reported the results of the application of those indicators in the general database of the Pathology Department of Imola Health District in northern Italy. In the current paper, we present the rationale and definitions of indicators of diagnostic performance (total Pap smears and rate of unsatisfactory Pap smears, distribution by cytology class reported, rate of patients without timely follow-up, detection rate, positive predictive value, distribution of cytology classes reported by histology diagnosis, and distribution of cases of CIN and carcinoma registered by detection modality) as well as the results of their application in the same database as above.     

Antigen handling in ageing. III. Age-related changes in antigen handling by liver parenchymal and nonparenchymal cells

This is a continuation of our earlier studies on antigen handling and the ageing process which have shown the liver to be a major site of antigen retention in the body. This paper reports on the uptake and cellular distribution in relatively pure isolates of the principal cell types of the liver of [35S]sulfanilate-azo-bovine serum albumin (35S-BSA) injected into F-344 rats of ages 4, 12 and 28 months. In all three cell types [hepatocytes (H), Kupffer cells (K) and endothelial cells (E)] antigen retention peaks in the 12-month age class and then exhibits a significant decline in the 28-month age class; K and E cells retain more antigen per cell than do H cells. All three cell types metabolize antigen to nucleopeptides and this transformed antigen is immunologically active; K and E cells metabolize antigen to nucleopeptides more effectively than do H cells. A significant age-related difference is found when nucleopeptides from either H, K or E cells are used to stimulate an antigen-specific lymphoproliferative response in vitro: nucleopeptides from 4-month and 12-month rats stimulate the proliferation of lymphocytes to a significantly higher degree than do nucleopeptides from 28-month old rats. The experiments therefore identify in all three cell populations an age-related defect in antigen uptake and in metabolism of antigen to bioactive nucleopeptides, and they demonstrate that the nonparenchymal cells are more effective in antigen retention and in metabolism of antigen to nucleopeptides than are parenchymal cells. Further, the experiments support the thesis that the liver must be considered to be a principal part of the total immune system, its role much more fundamental than heretofore recognized.     

International society of urological pathology consensus conference on handling and staging of radical prostatectomy specimens

The 2009 International Society of Urological Pathology consensus conference on handling and staging of radical prostatectomy specimens issued recommendations for standardization of pathology reporting of radical prostatectomy specimens. The conference addressed specimen handling, T2 substaging, prostate cancer volume, extraprostatic extension, lymphovascular invasion, seminal vesicle invasion, lymph node metastases, and surgical margins. This review summarizes the conclusions and recommendations resulting from the consensus process.