Application and comparison of silver intensification methods for the diaminobenzidine and diaminobenzidine-nickel endproduct of the peroxidation reaction in immunohistochemistry and in situ hybridization.

Silver-intensification methods described in the literature for the diaminobenzidine (DAB) and diaminobenzidine-nickel (DAB/Ni) endproduct of the peroxidase reaction were compared in model systems after immunoperoxidase and in situ hybridization. First, these methods were compared in immunohistochemical model systems, using the demonstration of glial fibrillar acidic protein (GFAP) and prostate-specific antigen (PSA) in paraffin sections of human brain and prostate tissue, respectively. When DAB without Ni was used as substrate, tissue argyrophilia caused considerable background staining. Only when this tissue reactivity was quenched with, e.g., CuSO4 with H2O2 or thioglycolic acid, were the results acceptable. A considerable improvement in the signal-to-noise ratio could be obtained when nickel was included in the substrate mixture. The methods that proved to be best for demonstration of GFAP and PSA made use of acid developer solutions. Subsequently, these methods were compared with other sensitive immunostaining methods for demonstration of the gamma-delta T-cell receptor in frozen lymphoid tissue. In this model a considerable increase in the number of positive cells could be obtained using silver intensification. The different methods using DAB/Ni were also compared for use in DNA in situ hybridization (DISH). In this case two model systems were used: human papilloma virus type 11 (HPV-11) DNA in condyloma tissue (abundant target model) and Epstein-Barr virus (EBV) DNA in a mononucleosis lymph node (low target model). For demonstration of HPV-11, all methods gave more or less satisfactory results, which were best with the acid developer solutions. Moreover, for demonstration of EBV DNA, a signal could be obtained only with these developer solutions. Such a method also proved suitable in double immuno-hybrido stainings for the demonstration of EBV DNA in specific antigen-positive Reed-Sternberg cells in paraffin sections of Hodgkin lymph nodes.     

Enterotoxigenic Bacteria in Food and Water from an Ethiopian Community

Food and water samples from an Ethiopian community were screened for the presence of enterotoxin-producing bacteria. Using the Chinese hamster ovary cell assay, 40 of 213 isolates (18.8%) produced heat-labile (LT) enterotoxin. These LT-producing isolates comprised 33 of 177 (18.6%) strains from 24 of 68 food samples (35.3%) and 7 of 36 (19.4%) isolates of 4 of 17 water samples (23.5%). One LT-producing strain each of Salmonella emek and of Shigella dysenteriae was found. Three pseudomonads, all LT producers, produced heat-stable enterotoxin as gauged by the suckling mouse test. Two strains of LT-enterotoxigenic Escherichia coli O68 were found in water samples. No enterotoxigenic E. coli were isolated from food samples, but 13 of the LT-producing strains were Enterobacter, Klebsiella, Serratia, and Proteus species, and 7 food samples yielded more than one species of enterotoxigenic bacterium. Of the enterotoxigenic isolates from food, 15 were oxidase-positive strains of the genera Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Vibrio. LT-enterotoxigenic Enterobacter, Acinetobacter, Klebsiella, Proteus, Providencia, and Serratia species represented 20 of the food and water isolates. Culture supernatant fluids of representative strains of oxidase-positive and oxidase-negative species giving positive reactions in Chinese hamster ovary cell tests induced fluid accumulation in rabbit ileal loops. Eight of the food samples and two of the water samples contained more than one isolate or species of enterotoxigenic bacterium. The stability of the LT production by oxidase-positive bacteria and non-E. coli strains was assessed by the rabbit skin and adrenal cell tests after 9 months and 1 year of storage, respectively, in Trypticase soy broth with glycerol at −70°C. Only 33% of the oxidase-positive strains were still LT enterotoxigenic. Of the oxidase-negative strains, 50 and 33% were LT producing at 9 months and 1 year, respectively. None of the E. coli isolates, both enterotoxigenic and nonenterotoxigenic, possessed K88, K99, or colonization factor antigen. The survey demonstrates the presence in food and water of enterotoxigenic bacteria of the same species as those isolated from cases of infantile diarrhea in the same community, although a correlation between these sources and infantile diarrhea remains to be established.     

Evaluation of Mitotic Activity Index in Breast Cancer Using Whole Slide Digital Images

Introduction

Mitotic Activity Index (MAI) is an important independent prognostic factor and an integral part of the breast cancer grading system. Thus, correct estimation of this prognostically relevant feature is essential for guiding treatment decision and assessing patient prognosis.The aim of this study was to validate the use of high resolution Whole Slide Images (WSI) in estimating MAI in breast cancer specimens.

Methods

MAI was evaluated in 100 consecutive breast cancer specimens by three observers on two occasions, microscopically and on WSI with a wash out period of 4 months. MAI was also translated to mitotic scores as in grading. Inter- and intra-observer agreement between microscopic and digital MAI counts and scores was measured.

Results

Almost perfect inter-observer agreements were obtained from counting MAI using a conventional microscope (intra-class correlation coefficient (ICCC) 0.879) as well as on WSI (ICCC 0.924). K coefficients reflected good inter-observer agreements among observers'' microscopic mitotic scores (average kappa 0.642). Comparable results were also observed among digital mitotic scores (average kappa 0.635). There was strong to perfect intra-observer agreements between MAI counts and mitotic scores for the two diagnostic modalities (ICCC 0.716–0.863, kappa 0.506–0.617). There were no significant differences in mitotic scores using both diagnostic modalities.

Conclusion

Scoring mitoses using WSI in breast cancer seems to be just as reliable and reproducible as when using a microscope. Further development of software and image quality will definitely encourage the use of WSI in routine pathology practice.     

Urinary Protein Profiles in a Rat Model for Diabetic Complications

Diabetes mellitus is estimated to affect ∼24 million people in the United States and more than 150 million people worldwide. There are numerous end organ complications of diabetes, the onset of which can be delayed by early diagnosis and treatment. Although assays for diabetes are well founded, tests for its complications lack sufficient specificity and sensitivity to adequately guide these treatment options. In our study, we employed a streptozotocin-induced rat model of diabetes to determine changes in urinary protein profiles that occur during the initial response to the attendant hyperglycemia (e.g. the first two months) with the goal of developing a reliable and reproducible method of analyzing multiple urine samples as well as providing clues to early markers of disease progression. After filtration and buffer exchange, urinary proteins were digested with a specific protease, and the relative amounts of several thousand peptides were compared across rat urine samples representing various times after administration of drug or sham control. Extensive data analysis, including imputation of missing values and normalization of all data was followed by ANOVA analysis to discover peptides that were significantly changing as a function of time, treatment and interaction of the two variables. The data demonstrated significant differences in protein abundance in urine before observable pathophysiological changes occur in this animal model and as function of the measured variables. These included decreases in relative abundance of major urinary protein precursor and increases in pro-alpha collagen, the expression of which is known to be regulated by circulating levels of insulin and/or glucose. Peptides from these proteins represent potential biomarkers, which can be used to stage urogenital complications from diabetes. The expression changes of a pro-alpha 1 collagen peptide was also confirmed via selected reaction monitoring.Diabetic nephropathy (DNP)¹ accounts for ∼44% of new cases of end stage renal disease (ESRD) (1). This high morbidity is the result of the impact of a growing population and longer life expectancy. With an increase in the prevalence of DM and a corresponding reduction in the mortality associated with both type 1 and type 2 DM, patients are living longer and are therefore at higher risk to develop complications such as nephropathy (2). Moreover, Type 1 DM patients who progress to ESRD have a substantial risk of mortality with estimated annual health care costs in the United States to be approximately $1.9 billion (3, 4). Two key therapies for the prevention and management of ESRD are aggressive glycemic control and blood pressure regulation (5, 6). Early intervention is essential in reducing the severity and course of this complication (6), and changes in urine biomarkers have historically been used to diagnose and monitor disease progression. In addition, urine represents a desirable matrix in which to detect biomarkers of nephropathy as urinary protein excretion profiles are reflective of functional changes within the kidney, such as glomerular filtration rate. Clinical determinations of urinary total protein and urinary albumin excretion are commonly used measurements to monitor and/or determine the onset of diabetic nephropathy. Unfortunately, these measurements often lead to improper diagnoses for at risk DM patients (7, 8). Therefore, new prognostic indicators are required to accurately target these patients for therapeutic intervention earlier in the course of the disease as well as identify patients who are unlikely to progress, as therapy may be of little or no benefit to them.Utilizing experimental models to study the pathophysiological changes that occur as function of disease progression has provided an approach for biomarker discovery. In diabetes, animal models have been widely used in the investigation of the progression of diabetes complications such as nephropathy. Research conducted on the association between hyperglycemia and microvascular disease in diabetes as well as the study of the effect of extracellular matrix protein expression on changes in morphology in the diabetic kidney are two such examples (9). In addition, these models have assisted in developing appropriate clinical trials for the prevention and treatment of these complications. One such example is the use of anti-hypertensive treatment regimes in genetically hypertensive rats; these have examined whether early intervention may be renoprotective and therefore delay or prevent the onset of diabetic nephropathy (10–12).STZ-induced hyperglycemia in rodents is the most extensively studied model of diabetic nephropathy and associated complications (9, 13). Hyperglycemia occurs in this model because of the toxin''s destruction of pancreatic Beta-islet cells, which are essential to the production of insulin. STZ-induced hyperglycemia is associated with reliable and consistent structural and functional deficits in specific urogenital organ function (i.e. kidney and bladder). Increased glomerular filtration and hypertrophy, as well as increased urgency and morphology changes, are structural and functional abnormalities that have been observed in the kidney and bladder, respectively, in both in humans and the STZ rat model (14–19).Currently, there are two primary methods used to monitor disease progression in diabetes. The measurement of urinary albumin excretion rates and total protein concentration are routinely used to monitor disease progression as they reflect structural and functional changes in the kidney. Measurements of albumin by immunochemical assays and size exclusion high performance liquid chromatography are routinely employed (7). However, urine consists of a multitude of proteins, many of which are also reflective of pathophysiological changes because of DM urogenital complications (20–23). Proteomics provides a powerful approach for the detection of urinary protein changes as a result of disease, and multiple proteomic techniques are available in large scale protein profiling to discover new biomarkers (24–26). To date, proteomic strategies for biomarker discovery in urine have primarily included top-down approaches, for example two-dimensional gel electrophoresis coupled with mass spectrometry and/or surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) analyses (27–30). In addition a number of studies using capillary electrophoresis mass spectrometry, which have a number of advantages for analysis of urine, have been successfully carried out (31–33). While these approaches can easily detect and quantify a variety of proteinaceous species including isoforms, posttranslational modifications, or degradation products, other methods could be used to expand the number of proteins that are both quantified and identified providing an expanded set of biological targets to understand the complications of disease and its progression. Recent advances in both chromatography and mass spectrometry have enabled bottom-up approaches that identify and quantify at the peptide level (34–36). One advantage of bottom-up proteomics is increased overall proteome coverage. Moreover, most bottom-up methods provide both qualitative and quantitative data in a single run, and quantifying at the peptide level leads directly into a bottom-up confirmation/validation analysis thereby avoiding the peptide selection step in this procedure. Approaches to bottom-up proteomics include specific peptide labeling or label-free analysis. Specific labeling approaches such as isobaric tag for relative and absolute quantification (iTRAQ) and ¹⁸O employ differential stable isotope labeling strategies that create specific mass tags for different samples, which are mixed and then identified and quantified using mass spectrometry (37). The utility of these techniques is that they accommodate a wide range of pre-fractionation strategies thereby improving proteome coverage.The label-free approach capitalizes on the highly reproducible chromatography and high mass accuracy available in current LC/MS systems. This method observes all detectable peptides and if interrogated by MS/MS their corresponding fragment ions. This approach quantifies a peptide by its intensity and groups each peptide across individual samples based on its accurate mass and retention time (38, 39). These intensities associated with specific mass and retention time values are organized into peptide array tables that may be further processed using statistical techniques that accommodate high-dimensional data. As with other bottom-up approaches, this method is also amenable to pre-fractionation strategies, but unlike labeled approaches, the removal of chemical or metabolic labeling steps simplifies the overall approach.Here we use a comparative label-free LC/MS/MS approach to identify and rank candidate biomarkers of urogenital complications from an STZ rat model of diabetes. We describe further technical validation of our approach by confirming the changes observed with the putative biomarker, pro-alpha (2) with an alternative method: selected reaction monitoring (SRM).     

An improved immunocytochemical method for the detection of human cytomegalovirus antigens in peripheral blood leucocytes

A recently described immunoperoxidase method for the detection of nuclear human cytomegalovirus (HCMV) immediate early antigen (IEA) directly on peripheral blood leucocytes suffers from the drawback that the antigen is vulnerable to endogenous peroxidase inactivation procedures. To solve this problem a procedure is developed in which endogenous peroxidase is inactivated after binding and immobilization of the primary antibody with 4% formaldehyde. In combination with this procedure, three types of inactivation were investigated: glucose/glucose oxidase, hydrochloric acid and methanol/H2O2. Of these three, the first gives optimal results, especially in combination with methanol/acetic acid (20/1 v/v) as the primary fixative. This procedure results in preparations which allow for a more objective evaluation and enable automated examination using bright field microscopy. As a second improvement we developed a simple adherence method in order to diminish the risk of infection for the laboratory staff during processing of unknown blood samples. The protocol described shows great clinical potential for the diagnosis of HCMV infections.     

Design and analysis of quantitative differential proteomics investigations using LC-MS technology

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is becoming an increasingly important tool in characterizing the abundance of proteins in biological samples of various types and across conditions. Effects of disease or drug treatments on protein abundance are of particular interest for the characterization of biological processes and the identification of biomarkers. Although state-of-the-art instrumentation is available to make high-quality measurements and commercially available software is available to process the data, the complexity of the technology and data presents challenges for bioinformaticians and statisticians. Here, we describe a pipeline for the analysis of quantitative LC-MS data. Key components of this pipeline include experimental design (sample pooling, blocking, and randomization) as well as deconvolution and alignment of mass chromatograms to generate a matrix of molecular abundance profiles. An important challenge in LC-MS-based quantitation is to be able to accurately identify and assign abundance measurements to members of protein families. To address this issue, we implement a novel statistical method for inferring the relative abundance of related members of protein families from tryptic peptide intensities. This pipeline has been used to analyze quantitative LC-MS data from multiple biomarker discovery projects. We illustrate our pipeline here with examples from two of these studies, and show that the pipeline constitutes a complete workable framework for LC-MS-based differential quantitation. Supplementary material is available at http://iec01.mie.utoronto.ca/~thodoros/Bukhman/.     

Added Value of HER-2 Amplification Testing by Multiplex Ligation-Dependent Probe Amplification in Invasive Breast Cancer

Background

HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH).

Methods

As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases).

Results

The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001). Overall concordance between IHC and MLPA/CISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining.

Conclusions

MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.     

An improved immunocytochemical method for the detection of human cytomegalovirus antigens in peripheral blood leucocytes

Summary A recently described immunoperoxidase method for the detection of nuclear human cytomegalovirus (HCMV) immediate early antigen (IEA) directly on periphiral blood leucocytes suffers from the drawback that the antigen is vulnerable to endogenous peroxidase inactivation procedures. To solve this problem a procedure is developed in which endogenous peroxidase is inactivated after binding and immobilization of the primary antibody with 4% formaldehyde. In combination with this procedure, three types of inactivation were investigated: glucose/glucose oxidase, hydrochloric acid and methanol/H₂O₂. Of these three, the first gives optimal results, especially in combination with methanol/acetic acid (20/1 v/v) as the primary fixative. This produre results in preparations which allow for a more objective evaluation and enable automated examination using bright field microscopy.As a second improvement we developed a simple adherence method in order to diminish the risk of infection for the laboratory staff during processing of unknown blood samples. The protocol described shows great clinical potentual for the diagnosis of HCMV infections.Supported in part by the Nier Stichting Nederland (grants No's: C-83404 and C-86606) and Het Praeventiefonds, The Netherlands (grant No: 28-1346)