A novel mitochondria-targeting method using special staining for the detection of apoptotic hepatocytes

ABSTRACT

Early detection of apoptotic cells on histological slides is of major importance for both diagnostic and research areas. In the current study, the aim was to propose a convenient method to stain the mitochondria and establish whether hepatocytes undergoing apoptosis can be identified in tissue sections using the proposed method. Liver tissue from five adult chinchillas was fixed with 10% neutral buffered formalin for Goldner’s trichrome (GT) and Groat’s iron hematoxylin and eosin (HE) stains and with Kolster’s fixative for the Heidenhain’s iron hematoxylin procedure. The HE and GT-stained sections showed the morphological features consistent with apoptosis i.e., homogenous intensely acidophilic cytoplasm, cell shrinkage with an irregular outline, nuclear shrinkage with cloudy karyoplasm, and karyopyknosis in the late stage. Sections stained with Heidenhain’s iron hematoxylin method was used to pinpoint mitochondria and revealed cells which were undergoing the first stages of the apoptosis process i.e., disappearance of mitochondria from the cell, chromatin condensation and margination, paracentral localization of nucleoli, and vacuolated nuclei. In more advanced stages of apoptosis, cells presented significant nuclear and cytoplasmic changes. It was concluded that this is the first report targeting the mitochondria, by performing inexpensive histological staining techniques, in order to assess dead cells in situ.     

Diagnostic utility of a p63/alpha-methyl-CoA-racemase (p504s) cocktail in atypical foci in the prostate.

Prostatic needle biopsy is the preferred method for diagnosing early prostate cancer, providing specific information. In cases of histological cancer mimics, a diagnosis of atypical small acinar proliferation suspected of but not diagnosed as malignancy can be made. In such cases, and in small focus carcinomas, pathologists use 34betaE12, cytokeratin (CK) 5/6 or p63 immunostaining to label basal cells, and alpha-methylacyl-CoA racemase (AMACR/p504s) immunostaining as a positive prostate cancer marker on two distinct slides. However, in cases of small foci, ambiguous lesions might disappear. The purpose of our study was to improve the sensitivity of a cocktail of two antibodies (p63/p504s) with a sample incubation on 260 prostatic specimens, in order to help make a decision in conjunction with standard histology and CK 5/6 immunostaining. We tested 101 small focus prostatic cancers, 104 atypical small acinar proliferation, 19 high-grade prostatic intraepithelial neoplasia, two atypical adenomatous hyperplasia and 34 benign mimics of cancer. After p63/p504s immunostaining, the final diagnoses retained were as follows: 154 prostatic cancers, 14 atypical small acinar proliferation, 30 high-grade prostatic intraepithelial neoplasia, three atypical adenomatous hyperplasia and 62 benign mimics of cancer. To differentiate malignant from benign lesions, we used the criteria of greater sensitivity to p504s/p63 (95%) than to CK 5/6 (57%) or p63 (86%), and higher specificity for p504s/p63 (95%) than for CK 5/6 (88%) or p63 (81%). With the p504s/p63 cocktail, 89% of the ambiguous lesions were classified vs 53% for CK 5/6. Combined use of the two antibodies, one (p504s) as a positive marker and the other (p63) as a negative marker, with a simple immunostaining procedure, may improve diagnostic performance, sensitivity and specificity, leading to a reduction in the risk of false negatives; this technique in cases of atypical small acinar proliferation should reduce the percentage of residual ambiguous lesions and the need for additional biopsies.     

Histological evidence concerning the osseointegration of titanium implants in the fractured rabbit femur

Stabilization of the broken bone is achieved using biocompatible materials. Since histology is still considered the gold standard technique for the assessment of bone formation around metallic implants, this report investigated the titanium implant integration in the accidentally broken bone in rabbits. The experimental protocol was reviewed and approved by the Ethical Committee of the Faculty of Medicine and Pharmacy Oradea, Romania. Holes were drilled in the diaphysis of the femur, and titanium implants were inserted in the created bone defect. In two subjects, fractures occurred on days two and three after the metallic alloy implantation. The other two rabbits presented no fractures following the surgical procedure. The rabbits were euthanized and the bones (with metallic implants) were harvested for histopathological investigation. Following decalcification, the bone samples were processed using the standard paraffin technique and stained by Goldner’s trichrome procedure. In subjects with a perfect immobilization of the titanium implants, the osseointegration occurred with minimal callus formation (i.e. primary cortical healing). In rabbits with bone fractures, the callus was more exuberant. A progressive replacement of the granulation tissue with hyaline cartilage and woven bone occurred soon after. The former aspects suggested an indirect metaplasia in the created callus. In all subjects, no inflammatory cells were identified in the created callus. The bone regeneration occurred either by primary cortical healing (in perfectly immobilized titanium implants) or by a process similar to the endochondral ossification (in poorly immobilized titanium implants following accidental post-implantation bones fracture).     

Risk factors associated with fatal influenza,Romania, October 2009 – May 2011

Background

Limited data are available from Central and Eastern Europe on risk factors for severe complications of influenza. Such data are essential to prioritize prevention and treatment resources and to adapt influenza vaccination recommendations.

Objectives

To use sentinel surveillance data to identify risk factors for fatal outcomes among hospitalized patients with severe acute respiratory infections (SARI) and among hospitalized patients with laboratory-confirmed influenza.

Methods

Retrospective analysis of case-based surveillance data collected from sentinel hospitals in Romania during the 2009/2010 and 2010/2011 winter influenza seasons was performed to evaluate risk factors for fatal outcomes using multivariate logistic regression.

Results

During 2009/2010 and 2010/2011, sentinel hospitals reported 661 SARI patients of which 230 (35%) tested positive for influenza. In the multivariate analyses, infection with influenza A(H1N1)pdm09 was the strongest risk factor for death among hospitalized SARI patients (OR: 6·6; 95% CI: 3·3–13·1). Among patients positive for influenza A(H1N1)pdm09 virus infection (n = 148), being pregnant (OR: 7·1; 95% CI: 1·6–31·2), clinically obese (OR: 2·9;95% CI: 1·6–31·2), and having an immunocompromising condition (OR: 3·7;95% CI: 1·1–13·4) were significantly associated with fatal outcomes.

Conclusion

These findings are consistent with several other investigations of risk factors associated with influenza A(H1N1)pdm09 virus infections. They also support the more recent 2012 recommendations by the WHO Strategic Advisory Group of Experts on Immunization (SAGE) that pregnant women are an important risk group for influenza vaccination. Ongoing sentinel surveillance can be useful tool to monitor risk factors for complications of influenza virus infections during each influenza season, and pandemics as well.     

Biomarkers

Epidemiologic, clinical, and basic research has identified several antecedent conditions that predispose individuals to heart failure and its predecessor, asymptomatic left ventricular remodeling and dysfunction (stage B heart failure). Many biochemical markers have been described that characterize the remodeling process and the development of cardiac dysfunction. Although natriuretic peptides and cardiac troponin are currently used in the context of diagnosis, risk stratification, and management of stage C and D heart failure, many other biomarkers provide insights into the underlying pathophysiology of left ventricular dysfunction, suggesting new directions for fundamental research or the development of new therapies.     

Diagnosis of renal metanephric adenoma: relevance of immunohistochemistry and biopsy

Most renal tumors of the adult are carcinomas. Their treatment is surgical, consisting of limited excision or nephrectomy. In some instances, biopsy of the tumor can be performed in order to adapt treatment. We report the case of a 45 year-old woman presenting with renal tumor. A biopsy of the mass showed a metanephric adenoma. No surgical excision was performed because of the benignity of this tumor. Here we develop the interest of immunohistochemistry for differential diagnosis of metanephric adenoma and other "basophilic small cell tumors" of the kidney. We also put the stress on the growing role of biopsy of renal tumor allowing optimal treatment.     

Surrogate end points in heart failure

Because of the increasing number of pharmacologic strategies available for treatment of heart failure (HF), the time has come to reassess the adequacy of end points used to evaluate therapeutic efficacy. Interest in the use of surrogate end points in clinical studies is increasing. A surrogate end point is defined as a measurement that can substitute for a true end point for the purpose of comparing specific interventions or treatments in a clinical trial. A true end point is one that is of clinical importance to the patient (e.g., mortality or quality of life), whereas a surrogate end point is one biologically closer to the disease process (e.g., ejection fraction or left ventricular volume in HF). The prime motivation for the use of a surrogate end point concerns the possible reduction in sample size or trial duration. Such reductions have important cost implications and in some cases may influence trial feasibility. Another, perhaps more important, aspect of measuring surrogate end points is that they increase our understanding of the mechanism of action of drugs and thus may help physicians to take a more enlightened approach in managing their patients. In this article we have analyzed the possible potentials of the surrogate end points in clinical studies of patients with chronic HF. Other uses of possible surrogates are discussed, and the limitations in finding true surrogates are mentioned. At this time we conclude there is no well established surrogate in HF.     

A prospective study on the safety and efficacy of angiographic embolization for pelvic and visceral injuries

BACKGROUND: Angiographic embolization (AE) is used with increasing frequency as an alternative to surgery for control of intraperitoneal and retroperitoneal bleeding. There are no prospective studies on its efficacy, safety, and indications. PATIENTS: From April 1999 to June 2001, patients with abdominal visceral organ injuries or major pelvic fractures sent for AE were prospectively studied. Patients were transported to the angiography suite either because they were hemodynamically unstable ("emergent" angiography) or hemodynamically stable but had injuries likely to bleed ("preemptive" angiography). The efficacy of AE was derived from its ability to control bleeding radiographically and clinically; the safety of AE was determined by the complications related to transport, vascular access, catheter insertion, contrast administration, and tissue necrosis after interruption of blood supply to organs. Predictors of bleeding were identified by comparing patients who showed contrast extravasation on angiography with those who did not by univariate and multivariate analysis. RESULTS: Of 100 consecutive patients evaluated by angiography for bleeding from major pelvic fractures (n = 65) or solid visceral organ injuries (n = 35), 57 were found to have active contrast extravasation and were embolized, 23 were found to have indirect signs of vascular injury or ongoing hemodynamic instability and were embolized, and 20 had no signs of bleeding and were not embolized. AE was effective and safe in 95% and 94%, respectively, of 80 patients who were embolized. Four patients had recurrent bleeding after AE and five developed AE-related complications. In three of the four patients, bleeding was controlled by repeat AE. In all five patients, the complications were managed with no further sequelae. Three independent factors were predictive of bleeding identified on angiography: age older than 55 years, absence of long-bone fractures, and emergent angiography. The presence of all three independent predictors was associated with a 95% probability of bleeding; however, the probability of bleeding was still 18% when all three independent predictors were absent. CONCLUSION: AE is highly effective in controlling bleeding caused by abdominal and pelvic injuries and difficult to manage by surgery. Older age, the absence of long-bone fractures, and emergent angiography increase the likelihood of finding active bleeding angiographically. However, there are no clinical characteristics to exclude reliably all patients who are not actively bleeding internally. Because of this and its reasonable safety profile, AE should be offered liberally in patients with selected injuries of the pelvis and abdominal visceral organs.