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.     

A comparison of standard PCNL and staged retrograde FURS in pelvis stones over 2 cm in diameter: a prospective randomized study

The aim of the study was to compare percutaneous nephrolithotomy (PCNL) and staged retrograde flexible ureteroscopy (FURS) methods used in the treatment of kidney stones of 2 cm or more in diameter. The study comprised a total of 60 patients with a diagnosis of kidney pelvic stones more than 2 cm in diameter, for whom surgery was planned between January 2013 and January 2014. The patients were randomly allocated to two groups as staged retrograde FURS (Group A) and PCNL (Group B). Comparison of the groups was made with respect to operating time, number of procedures, total treatment time, length of hospital stay, stone-free rates and complications according to the Clavien–Dindo classification. In Group A, the total operating time of multiple sessions was 114.46 min. In Group B, a single session of PCNL was applied to all patients and the mean operating time was 86.8 min (p = 0.014). Mean total treatment time was 2.01 weeks in Group A and 1 week in Group B (p < 0.01). The mean total hospitalization time was 3.66 days in Group A and 3.13 days in Group B (p = 0.037). At the end of the sessions, clinically insignificant residual fragments were observed in ten patients of Group A and one patient of Group B (p = 0.03). No statistically significant difference was determined between the groups in terms of stone-free rates or complications. Although current technology with FURS is effective on large kidney stones, it has no superiority to PCNL due to the need for multiple sessions and long treatment time.     

Early Contribution of Pericytes to Angiogenic Sprouting and Tube Formation

Immunostaining with endothelial and pericyte markers was used to evaluate the cellular composition of angiogenic sprouts in several types of tumors and in the developing retina. Confocal microscopy revealed that, in addition to conventional endothelial tubes heavily invested by pericytes, all tissues contained small populations of endothelium-free pericyte tubes in which nerve/glial antigen 2 (NG2) positive, platelet-derived growth factor beta (PDGF beta ) receptor-positive perivascular cells formed the lumen of the microvessel. Perfusion of tumor-bearing mice with FITC-dextran, followed by immunohistochemical staining of tumor vasculature, demonstrated direct apposition of pericytes to FITC-dextran in the lumen, confirming functional connection of the pericyte tube to the circulation. Transplantation of prostate and mammary tumor fragments into NG2-null mice led to the formation of tumor microvasculature that was invariably NG2-negative, demonstrating that pericytes associated with tumor microvessels are derived from the host rather than from the conversion of tumor cells to a pericyte phenotype. The existence of pericyte tubes reflects the early participation of pericytes in the process of angiogenic sprouting. The ability to study these precocious contributions of pericytes to neovascularization depends heavily on the use of NG2 and PDGF beta -receptor as reliable early markers for activated pericytes.     

Low eradication rate of Helicobacter pylori with triple 7-14 days and quadriple therapy in Turkey

AIM:The eradication rate of Helicobacter pylori (H pylon) shows variation among countries and regimens of treatment.We aimed to study the eradication rates of different regimens in our region and some factors affecting the rate of eradication.METHODS:One hundred and sixty-four Hpylori positivepatients (68 males, 96 females; mean age:48&#177;12 years)with duodenal or gastric ulcer without a smoking history were included in the study. The patients were divided into three groups according to the treatment regimens. Omeprazole 20mg, clarithromycin 500mg, amoxicillin 1g were given twice daily for 1 week (Group I) and 2 weeks (Group Ⅱ).Patients in Group Ⅲ received bismuth subsitrate 300mg,tetracyline 500 mg and metronidazole 500mg four times daily in addition to Omeprazole 20mg twice daily.Two biopsies each before and after treatment were obtained from antrum and corpus, and histopathologically evaluated.Eradication was assumed to be successful if no Hpylorus was detected from four biopsy specimens taken after treatment. The effects of factors like age, sex, Hpyloridensity on antrum and corpus before treatment, the total Hpylori density, and the inflammation scores on the rate of Hpylori eradication were evaluated.RESULTS:The overall eradication rate was 42%. The rates in groups Ⅱ and Ⅲ were statistically higher than that in group I (P&lt;0.05). The rates of eradication were 24.5%,40.7% and 61.5% in groups Ⅰ, Ⅱ and Ⅲ, respectively. The eradication rate was negatively related to either corpus Hpylori density or total Hpyloridensity (P&lt;0.05).The median age was older in the group in which the eradication failed in comparison to that with successful eradication (55yr vs 39yr, P&lt;0.001). No correlation between sex and Hpylori eradication was found.CONCLUSION: Our rates of eradication were significantly lower when compared to those reported in literature.We believe that advanced age and high Hpyloridensity are negative predictive factors for the rate of Hpylorieradication.     

Effects of 2-Hydroxypropyl-Beta-Cyclodextrin on Cardiovascular Signs of Amitriptyline Poisoning in a Rat Model

The aim of this study was to investigate the efficacy of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) as an antidotal treatment for the in vivo cardiovascular effects of amitriptyline poisoning. Experiments were carried out on 33 Wistar rats. To evaluate cardiovascular effects of HPBCD, rats were infused with dextrose or HPBCD. In the poisoning model, amitriptyline (0.94 mg/kg/min) was infused until the mean arterial blood pressure (MAP) dropped to 50 % of the baseline. Following amitriptyline infusion, dextrose, low-dose HPBCD (4.19 mg/kg/min), or high-dose HPBCD (16.76 mg/kg/min) was infused, and MAP, heart rate (HR), and electrocardiogram were recorded for 60 min. Hearts were examined for tissue damage and apoptosis. HPBCD infusion alone did not yield significant difference for MAP, HR, QRS duration, QT interval, and cardiac tissue damage when compared to dextrose (p > 0.05). In the poisoning model, MAP and HR decreased, while QRS duration and QT interval prolonged significantly following amitriptyline infusion (p < 0.0167). Dextrose, low-dose HPBCD, and high-dose HPBCD infusion similarly corrected MAP, HR, QRS duration, and QT interval values at the end-experiment time point (p > 0.05). Histological scores for tissue damage and apoptosis showed no significant difference between the groups (p > 0.05). Based on our results, HPBCD did not show cardiovascular toxicity, while it was not more effective than dextrose for the treatment of amitriptyline poisoning. Further antidotal studies of cyclodextrins with higher doses and/or binding affinities are needed for poisonings.     

Effects of glutamine on wound healing

Studies reporting the need for replacing amino acids such as glutamine (Gln), hydroxymethyl butyrate (HMB) and arginine (Arg) to accelerate wound healing are available in the literature. The primary objective of this study was to present the effects of Gln on tissue hydroxyproline (OHP) levels in wound healing. This study was conducted on 30 female Sprague Dawley rats with a mean weight of 230 ± 20 g. Secondary wounds were formed by excising 2 × 1 cm skin subcutaneous tissue on the back of the rats. The rats were divided into three equal groups. Group C (Control): the group received 1 ml/day isotonic solution by gastric gavage after secondary wound was formed. Group A (Abound): the group received 0·3 g/kg/day/ml Gln, 0·052 g/kg/day/ml HMB and 0·3 g/kg/day/ml Arg by gastric gavage after secondary wound was formed. Group R (Resource): the group received 0·3 g/kg/day/ml Gln by gastric gavage after secondary wound was formed. The OHP levels of the tissues obtained from the upper half region on the 8th day and the lower half region on the 21st day from the same rats in the groups were examined. Statistical analysis was performed using the statistics program SPSS version 17.0. No statistically significant differences were reported with regard to the OHP measurements on the 8th and 21st days (8th day: F = 0·068, P = 0·935 > 0·05; 21st day: F = 0·018, P = 0·983 > 0·05). The increase in mean OHP levels on the 8th and 21st days within each group was found to be statistically significant (F = 1146·34, P = 0·000 < 0·001). We conclude that in adults who eat healthy food, who do not have any factor that can affect wound healing negatively and who do not have large tissue loss at critical level, Gln, Arg and HMB support would not be required to accelerate secondary wound healing.     

Thrombus aspiration during primary percutaneous coronary intervention associated with reduced platelet activation

Objectives:

To determine the effect of thrombectomy on platelet function in patients undergoing primary percutaneous coronary intervention (PPCI) for ST segment elevation myocardial infarction (STEMI).

Methods:

This retrospective study included 413 consecutive STEMI patients who underwent PPCI between March 2012 and September 2013 at Kartal Kosuyolu High Specialty Education and Research Hospital, Istanbul, Turkey that were assigned to the thrombus aspiration (TA) group or the non-TA group. Platelet count and mean platelet volume (MPV) were obtained at baseline and 24 hours (h), 48 h, and 72 h post PPCI.

Results:

Baseline MPV was similar in both groups, whereas the baseline platelet count was higher in the TA group (p=0.42 and p=0.002). The platelet count was higher in the TA group 24 h post PPCI (p=0.02), but was similar in both groups 48 h and 72 h post PPCI (p=0.18 and p=0.07). The MPV 48 h and 72 h post PPCI was higher in the non-TA group than in the TA group (8.4 ± 1.3 fL versus 8.7 ± 1.6 fL [p=0.04] and 8.5 ± 1.1 fL versus 8.9 ± 1.5 fL [p=0.04]).

Conclusion:

Thrombectomy reduced platelet activity via removal of thrombi from the coronary arteries in patients undergoing PPCI for STEMI.In acute ST-segment elevation myocardial infarction (STEMI), the main initiating factor is atherosclerotic plaque rupture or erosion, then platelets become activated and aggregated leading to acute coronary artery occlusion.1 Primary percutaneous coronary intervention (PPCI) is the standard treatment in patients with STEMI.2 In recent years, implementation of adjunctive mechanical and pharmacological therapies during PPCI, including manual thrombus aspiration (TA), has improved myocardial reperfusion and clinical outcome in STEMI patients.3-5 In daily practice, thrombectomy devices are used to remove thrombi or to prevent embolization of thrombi and plaque during PPCI.6 Platelet volume is a marker of platelet activation and is measured via mean platelet volume (MPV).7 An increase in MPV can occur in cases of acute myocardial ischemia, acute myocardial infarction, coronary atherosclerosis,8,9 and early stent thrombosis (ST).10 The Percutaneous Coronary Intervention in Acute Myocardial Infarction (TAPAS) study5 showed that TA therapy during PPCI decreases mortality and reinfarction rate.5 We speculate that the good results obtained with TA therapy are not only related to improved tissue perfusion but also may be related to reduced platelet activation. Therefore, the present study aimed to determine the effect of thrombectomy on platelet function and ST in patients undergoing PPCI for STEMI.     

Cystatin C is better than albuminuria as a predictor of pulse wave velocity in hypertensive patients

Introduction: Arterial stiffness is important in the evaluation of the cardiovascular risk in both general population and hypertensive patients. In this study, we aimed to investigate the associations of both serum cystatin C levels and albuminuria with arterial stiffness in healthy controls and hypertensive patients.

Patients and methods: Seventy-six healthy controls (male/female?=?44/32) and 76 hypertensive patients (male/female?=?43/33) were enrolled. Arterial stiffness parameters such as augmentation index (AIx) and pulse wave velocity (PWV) were non-invasively measured with the Arteriograph (Tensiomed Ltd., Budapest, Hungary).

Results: AIx (31.92?±?14.31 vs. 27.95?±?11.03, p?=?0.03) and PWV (9.84?±?1.62 vs. 8.87?±?2.04, p?p?=?0.002) and higher serum cystatin C levels [0.76 (0.67–0.95) vs. 0.68 (0.62–0.78) mg/L, p?=?0.03]. In the hypertensive group, AIx was significantly correlated with PWV (r?=?0.519, p?r?=?–0.438, p?=?0.003), mean arterial pressure (MAP) (r?=?0.288, p?=?0.015) and urinary albumin–creatinine ratio (ACR) (r?=?0.386, p?=?0.004). PWV was associated with serum cystatin C (r?=?0.442, p?=?0.003) and MAP (r?=?0.377, p?=?0.001). In the linear regression analysis (model r?=?0.577, p?=?0.006) for the prediction of PWV in hypertensive patients, MAP, urinary ACR, age and serum cystatin C levels were included as independent variables. Cystatin C was found to be the significant determinant of PWV in hypertensive patients.

Conclusion: Multivariate analysis revealed that serum cystatin C but not albuminuria was significantly associated with PWV in hypertensive patients. Serum cystatin C may be better than albuminuria as a predictor of arterial stiffness in hypertensive patients.