Utilizing image analysis by optical density to evaluate changes in hematoxylin and eosin staining quality after reagent overuse

With immunohistochemical (IHC) staining increasingly being used to guide clinical decisions, variability in staining quality and reproducibility are becoming essential factors in generating diagnoses using IHC tissue preparations. The current study tested a method to track and quantify the interrun, intrarun, and intersite variability of IHC staining intensity. Our hypothesis was that staining precision between laboratory sites, staining runs, and individual slides may be verified quantitatively, efficiently and effectively utilizing algorithm-based, automated image analysis. To investigate this premise, we tested the consistency of IHC staining in 40 routinely processed (formalin-fixed, paraffin-embedded) human tissues using 10 common antibiomarker antibodies on 2 Dako Omnis instruments at 2 locations (Carpinteria, CA: 30 m above sea level and Longmont, CO: 1500 m above sea level) programmed with identical, default settings and sample pretreatments. Digital images of IHC-labeled sections produced by a whole slide scanner were analyzed by a simple commercially available algorithm and compared with a board-certified veterinary pathologist's semiquantitative scoring of staining intensity. The image analysis output correlated well with pathology scores but had increased sensitivity for discriminating subtle variations and providing reproducible digital quantification across sites as well as within and among staining runs at the same site. Taken together, our data indicate that digital image analysis offers an objective and quantifiable means of verifying IHC staining parameters as a part of laboratory quality assurance systems.

Microscopic anatomy of the liver.

Template for Reporting Results of Biomarker Testing of Specimens From Patients With Carcinoma of the Endometrium.

Background: The potential problems related to the use of formalin in histopathology, such as health hazards, deterioration of nucleic acids are well-known. The aim of this study was to evaluate the utilization of a Carnoy's solution fixation in comparison with formalin on subsequent tissue sectioning and histochemical staining. Materials and Methods: Corresponding sections of 25 tissue biopsies of rabbit's different organs were fixed in Carnoy's solution and in 10% neutral buffered formalin. Samples were processed using the conventional method and then stained applying five histochemical methods. The degree of the quality of the staining was assessed for each method by scoring system (1-10) depending on comparison of the stained tissue sections with illustrated photomicrographs. Results: For the quality of cutting, the best quality was obtained by Formalin (mean = 4.76) then Carnoy's fixative (mean =3.84). The best quality of Haematoxylin and Eosin staining was obtained by formalin (mean =5.28) then Carnoy's (mean = 4.00). For Alcian blue and Perl's Prussian blue, the best staining qualities were obtained by Formalin (mean = 4.76 and 5.64 respectively) followed by Carnoy's (mean = 2.88 and 3.92 respectively). For periodic Acid Schiff's the best staining quality was obtained following Carnoy's fixation (mean = 4.52) then, the formalin (mean = 3.76). Conclusion: Although, Carnoy's fluid is a save fixative and can rapidly penetrate the tissues, but it can't be a substitute for formalin.

Alopecia is a psychologically devastating complication of chemotherapy for which there is currently no effective therapy. PTH-CBD is a collagen-targeted parathyroid hormone analog that has shown promise as a therapy for alopecia disorders. This study compared the efficacy of prophylactic versus therapeutic administration of PTH-CBD in chemotherapy-induced alopecia using a mouse model that mimics the cyclic chemotherapy dosing used clinically. C57BL/6J mice were treated with a single subcutaneous injection of PTH-CBD (320 mcg/kg) or vehicle control before or after hair loss developing from three courses of cyclophosphamide chemotherapy (50-150 mg/kg/week). Mice receiving chemotherapy alone developed hair loss and depigmentation over 6-12 months. Mice pretreated with PTH-CBD did not develop these changes and maintained a normal-appearing coat. Mice treated with PTH-CBD after development of hair loss showed a partial recovery. Observations of hair loss were confirmed quantitatively by gray scale analysis. Histological examination showed that in mice receiving chemotherapy alone, there were small, dystrophic hair follicles mostly in the catagen phase. Mice receiving PTH-CBD before chemotherapy showed a mix of normal-appearing telogen and anagen hair follicles with no evidence of dystrophy. Mice receiving PTH-CBD therapy after chemotherapy showed intermediate histological features. PTH-CBD was effective in both the prevention and the treatment of chemotherapy-induced alopecia in mice, but pretreatment appears to result in a better cosmetic outcome. PTH-CBD shows promise as an agent in the prevention of this complication of chemotherapy and improving the quality of life for cancer patients.

To the Editors: We read with interest the August 2012 case report by Yeh et al1 in which they presented a spindle cell melanoma with features of neurofibroma. Their case report was quite timely as we were facing the same dilemma with exact opposite outcome. We recently encountered a dermal-based spindle cell tumor that appeared to be a neurofibroma, except it had numerous lymphoid aggregates throughout, which resulted in spindle cell melanoma being added as a differential diagnosis. This diagnostic dilemma left us in a transposed position as the authors of the aforementioned case report. We received multiple fragments of an entirely dermal neoplasm removed from the jaw of 49-year-old woman with the clinical description of "atypical nevus." The tumor was nonencapsulated and composed of delicate spindle-shaped cells embedded in a pink, mildly fibrotic stroma with scattered mast cells throughout (Fig. 1). There were numerous lymphoid aggregates of varying size throughout the tumor, some with germinal center formation (Fig. 2). Only 1 of the 4 fragments received contained epidermis. Close review of the epidermis showed no significant increase in melanocytes (Fig. 3). The tumor cells were diffusely positive for S100 and negative for melan-A, tyrosinase, Mitf, and HMB-45 by immunohistochemical staining (Fig. 4).FIGURE 1: The excision demonstrates a nonencapsulated dermal nodule composed of delicate spindle-shaped cells embedded in a pink, mildly fibrotic stroma. It is accompanied by numerous mast cells and lymphoid aggregates of varying size with germinal center formation (hematoxylin and eosin stain, ×40).FIGURE 2: This higher power view of the specimen highlights lymphoid aggregates with reactive germinal center formation surrounded by bland spindle cells (hematoxylin and eosin stain, ×100).FIGURE 3: This lower power view of the specimen demonstrates histology more consistent with a classic neurofibroma. There is no epidermal involvement by the neoplasm (hematoxylin and eosin stain, ×40).FIGURE 4: The specimen demonstrates diffuse positive staining for S100 (hematoxylin and eosin stain, ×40).Although the clinical impression of melanocytic nevus is not uncommonly seen in cases of neurofibroma, it added to our diagnostic concern in light of the numerous lymphoid aggregates, with no explanation for the clinical pigmentation. The patient had no history of autoimmune disorders or other significant clinical disease. Review of the literature did not yield any reported cases of neurofibroma with lymphoid aggregates, although the occasional presence of lymphoid collections in neurofibromas is recognized.2 The finding of lymphoid aggregates, commonly used as a clue to the diagnosis of desmoplastic melanoma, is nonspecific and recently demonstrated to be associated with the diagnosis of desmoplastic melanocytic nevi, including Spitz melanocytic nevi.3 As suggested by the authors, we performed CD34 immunohistochemical staining, which was diffusely positive, supporting the diagnosis of neurofibroma with numerous lymphoid aggregates (Fig. 5).4 A comparative genomic hybridization profile was not completed, as we believe the aforementioned methods of analysis provide substantial information to support this diagnosis.FIGURE 5: The specimen is diffusely and strongly immunoreactive for CD34 (hematoxylin and eosin stain, ×40).

Metastasis is innate tendency of malignancy. Lymphatic spread plays a crucial role in the progression of Head and Neck Squamous Cell Carcinoma and significantly influences tumor staging. The presence of regional lymph node involvement reduces survival rates by nearly half. The presence of single micrometastasis affects the prognosis, recurrence and overall survival rate of the patient. Macrometastasis is the nodal deposit examined by surgeon on clinical examination or by radiologist in the imaging studies and can be routinely diagnosed in Hematoxylin and Eosin staining. But, micrometastasis cannot be determined easily by clinical and radiographic examination. Even in Hematoxylin and Eosin staining, micrometastasis is often obscure. The current research focuses on identifying the efficacy of special stain Papanicolaou to detect the micrometastasis in lymphnode, which was undetected in Hematoxylin and Eosin staining. The study included 432 lymph nodes obtained from 40 patients diagnosed with Oral Squamous Cell Carcinoma. Hematoxylin and Eosin staining was done and 100 lymph nodes were proven non-metastatic. Modified Papanicolaou stain was used for identification of micrometastatic deposit. Out of 100 Hematoxylin and Eosin stained non-metastatic lymph nodes, Papanicolaou stain was useful in detecting micrometastasis in 9 lymph nodes. This accounted to 9% of non-metastatic lymph node sections used in our study. Papanicolaou staining has shown greater effectiveness than routine Hematoxylin and Eosin in detecting micrometastasis, while also being cost-effective, widely accessible, and less technique-sensitive.

After analysing the qualitative and quantitative characteristics of organized crime that produces and sells forged banknotes and securities, its development, it may be concluded that effective counteraction to this negative phenomenon is possible with cooperation of all nations. It is caused by the fact that organized criminal groups counterfeit money, produce and sell hard currency (US dollars and euro), cooperate closely and broaden their criminal activity outside regions and countries. International Criminal Police Organization (Interpol) plays the main role in cooperation and investigation of money counterfeiting. It is the largest intergovernmental body consisting of 179 member-states. The USSR became a member-state of Interpol on September 27, 1990, at the 59th session of General Assembly of International Criminal Police Organization. Later the Russian Federation became its assignee. The Russian National Central Bureau is the Interpol representative in Russia. It has authority and recognition abroad. In the Ministry of Internal Affairs, in the Central Internal Affairs Directorate, and in the Directorate of Internal Affairs of 59 subjects of the Russian Federation, the branches of Interpol are established that make sufficient contribution in combating international crime. International Centre on combating money counterfeiters and securities forgers is the most important subdivision of Interpol. It directly realizes the objectives in the sphere of combating banknotes counterfeiting. International cooperation in the sphere of combating banknotes and securities counterfeiting is at the high level due to the effective and operative cooperation of law enforcement agencies. At the same time some provisions of the Geneva Convention of 1929 about combating banknotes counterfeiting is outdated, and its text requires revising. Firstly, the concept banknotes should be broadened: but for them, other securities (promissory notes, bills, letters of credit, signs of post payment) are forged.

Objective To explore the expression of surface glycoprotein protein hepatocyte 14 (CD14), vascular endothelial growth factor 34 (CD34) and transforming growth factor-β1 (TGF-β1) in liver biopsy of biliary atresia (BA) and examine the role of liver fibrosis. Methods From January 2013 to January 2014, liver biopsy specimens were collected intraoperatively from BA patients undergoing Kasai procedure (Kasai group), BA patients after Kasai procedure with liver transplantation (transplantation group) and congenital biliary dilation patients (CBD group as control) (n=15 each). The hematoxylin & eosin staining and immunohistochemistry were used to observe the degree of liver fibrosis and detect the expression of CD14, CD34 and TGF-β1 and determine their localizations. The image analysis software of Image-Pro-Plus was used to analyze the values of integrated optical density (IOD) for CD14 and TGF-β1 proteins in different groups and CD34 positive area. SPSS 17.0 statistical software was used for single factor ANOVA analysis (P<0.05). Pearson correlation analysis (P<0.01) showed that there was correlation between three groups about the expression of different proteins. Results CD14 was expressed in myeloid liver sinusoidal endothelial cells, fibroblasts and inflammatory cells. And TGF-β1 was expressed in fibroblasts, biliary epithelial cells and inflammatory cells while CD34 in portal area and proliferating vascular endothelial cells. Compared with CBD group, the expressions of CD34, CD14 and TGF-β1 proteins were up-regulated in experimental group. From the perspective of liver fibrosis grading (stage I-IV) in BA. The expression of CD14 and TGF-β1 proteins decreased gradually while the expression of CD34 increased. The difference was statistically significant between three groups about the expression of CD14, TGF-β1 and CD34 proteins (P<0.05), the expression of CD14 and TGF-β1 proteins were significantly correlated (P<0.05) whereas the expression of TGF-β1 and CD34 proteins significantly positively correlated. Conclusions The expression of CD14 and TGF-β1 in liver fibrosis increase gradually in BA. And liver cirrhosis may be accelerated. In advanced disease, all cells are damaged and become substituted by fibrosis. And the levels of three proteins gradually decline. Key words: Biliary atresia; Antigens, CD14; Antigens, CD34; Transforming growth factor beta1

Short tandem repeat (STR) genotyping provides parental origin information about aneuploidy pregnancy loss and has become the current gold standard for hydatidiform mole diagnosis. STR genotyping diagnostic support most commonly relies on formalin-fixed paraffin-embedded samples, but maternal contamination is one of the most common issues based on traditional unstained sections. To evaluate the influence of hematoxylin and eosin (H&E) staining on DNA quality and STR genotyping, DNA was isolated from unstained, deparaffinized hydrated, and H&E-stained tissue sections (i.e. 3 groups) from each of 6 formalin-fixed paraffin-embedded placentas. The macrodissected view field, DNA quality, and polymerase chain reaction amplification efficiency were compared among groups. STR genotyping analysis was performed in both the test cohort (n = 6) and the validation cohort (n = 149). H&E staining not only did not interfere with molecular DNA testing of formalin-fixed paraffin-embedded tissue but also had a clearer macrodissected field of vision. In the test cohort, H&E-stained sections were the only group that did not exhibit maternal miscellaneous peaks in STR genotyping results. In the validation cohort, 138 (92.62%) cases yielded satisfactory amplification results without maternal contamination. Thus, H&E staining helped to reduce maternal contamination in STR genotyping for hydatidiform mole diagnosis, suggesting that H&E-stained sections can be incorporated into the hydatidiform mole molecular diagnostic workflow.

Diagnosis of initial epithelial pathology maybe difficult in Squamous Cell Carcinoma (SCC), Carcinoma In Situ and other atypical epithelial malignancies, under routine Haematoxylin and Eosin (H and E) stain. The detection of minor basement membrane alterations in doubtful cases is both time consuming and confusing. To evaluate efficacy of Modified Cajal's Trichrome Stain (CTS) in relation to Haematoxylin and Eosin for study of epithelial dysplasia, carcinoma in situ, micro invasive SCC, frank SCC, and SCC in lymph nodes. Formalin-fixed, paraffin-embedded tissue blocks of mild epithelial dysplasia (n = 2), moderate epithelial dysplasia (n = 2), severe epithelial dysplasia (n = 4), carcinoma in situ (n = 1), micro-invasive SCC (n = 4), verrucous carcinoma (n = 1), and frank OSCC (n = 5) were stained with CTS and H&E. The sections were compared based on set histopathological criteria. In SCC cases stained with CTS, invasion into connective tissue and keratin pearls were strikingly evident. Depth of invasion could be more accurately determined. Tumour cells in lymph node were intensely contrasted and easily discernible. Thus, CTS is a good differential stain, clearly delineating the epithelial elements from the connective tissue elements visually. This helps in tracing the basement membrane very clearly. It is an economic, rapid and easy to use method which cannot replace Haematoxylin and Eosin stain in cancer diagnosis, but can definitely be used adjunctive to it. Prompt diagnosis is crucial to effective treatment, and this stain assists in early and rapid diagnosis of cancer.

Although peptide nucleic acid (PNA), fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) are the reference tools in the study of bacterial aggregates/biofilms, it may also be rather time-consuming. This study aimed to investigate the sensitivity and specificity between bacterial aggregates identified by haematoxylin and eosin (HE) staining vs bacterial aggregates in corresponding PNA-FISH samples. Axillary biopsies were obtained in 24 healthy controls. HE-stained and PNA-FISH samples were investigated using traditional light microscopy and CLSM, respectively. The data demonstrate that HE staining identifies large bacterial aggregates (>10μm) with a sensitivity of 0.43 and specificity of 1. The methods, however, are not equivalent as demonstrated by a McNemar's test (P=.04). Where bacterial aggregates >10μm in diameter, HE staining may offer a rapid and practical low-cost tool to evaluate bacterial aggregates.

Advancements in genomic, computing, and imaging technology have spurred new opportunities to use quantitative image analysis (QIA) for diagnostic testing. To develop evidence-based recommendations to improve accuracy, precision, and reproducibility in the interpretation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) for breast cancer where QIA is used. The College of American Pathologists (CAP) convened a panel of pathologists, histotechnologists, and computer scientists with expertise in image analysis, immunohistochemistry, quality management, and breast pathology to develop recommendations for QIA of HER2 IHC in breast cancer. A systematic review of the literature was conducted to address 5 key questions. Final recommendations were derived from strength of evidence, open comment feedback, expert panel consensus, and advisory panel review. Eleven recommendations were drafted: 7 based on CAP laboratory accreditation requirements and 4 based on expert consensus opinions. A 3-week open comment period received 180 comments from more than 150 participants. To improve accurate, precise, and reproducible interpretation of HER2 IHC results for breast cancer, QIA and procedures must be validated before implementation, followed by regular maintenance and ongoing evaluation of quality control and quality assurance. HER2 QIA performance, interpretation, and reporting should be supervised by pathologists with expertise in QIA.

The study aimed to find a more appropriate method to detect eosinophils in formalin- fixed nasal polyps, since there is no consensus on the standard counting method of eosinophils now. Four 5 μm serial sections were obtained from each 10% neutral formalin-fixed paraffin block and were stained with Chromotrope 2R, Congo red, MBPmAb immunohistochemistry, and conventional hematoxylin and eosin stain respectively. Each section was scanned by the Aperio digital section scanner. The same selected areas were procured for assessment in the serial sections. Chromotrope 2R and MBPmAb immunohistochemistry were specific in detecting eosinophils, which had the lower background staining compared with Congo red and conventional hematoxylin and eosin stain. There were significant differences among the four methods in terms of the eosinophil counting data (p < 0.05), while no significant difference between Chromotrope 2R and Congo red (P = 0.1413). The eosinophil counts in nasal polyps could be more accurately assessed by Chromotrope 2R and Congo red compared with MBPmAb immunohistochemistry and conventional hematoxylin and eosin stain. The popularization of Chromotrope 2R and Congo red may help to unify the eosinophil count in the definition of eosinophilic CRSwNP.

A 38-year-old woman presented with left lower chest pain. At the age of 3 years, a commissurotomy of the pulmonary valve and infundibulectomy was performed to correct severe infundibular pulmonary stenosis. The initial thoracic CT diagnosed bilateral pulmonary embolism. Echocardiography revealed a filiforme mass in the pulmonary artery 33 mm in length, floating in the right outflow tract, suggesting a pulmonary sarcoma. In addition, a moderate pulmonary valve insufficiency with mild stenosis was diagnosed. Cardiac magnetic resonance (CMR) confirmed the mass in the left pulmonary artery (PA) with floating parts and a small mass in the right PA (Figure 1) and provided complementary findings identifying …