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Paired box protein 8 (PAX8) is a transcription factor that is considered a relatively specific marker of carcinomas of the thyroid, kidney, and Müllerian/Wolffian duct derivatives. Unexpected PAX8 immunoreactivity has occasionally been reported in other tumors. The frequency of PAX8 expression in carcinomas of the biliary tract is not well studied. We evaluated the immunohistochemical expression of PAX8 in 73 cases of biliary tract carcinoma. We found that 28 of 73 (38%) biliary tract carcinomas had variable immunoreactivity for PAX8, assessed by a widely used polyclonal antibody (ProteinTech Group, Chicago, IL). This included 3 (4%) of cases with strong diffuse, and 14 (19%) of cases with strong focal staining. Strong PAX8 expression was more frequent in distal bile duct carcinomas than other biliary sites (p = 0.015), and showed a weak association with advanced T stage (T3-T4 versus T1-T2; p = 0.09). No correlation was observed between PAX8 positivity and age at diagnosis, gender, or lymph node metastasis. The 28 polyclonal PAX8-positive cases were largely negative for monoclonal PAX8 and PAX6 immunostains, with only rare tumor cells with weak immunoreactivity being present in a subset of cases. We show that a substantial fraction of biliary tract carcinomas exhibit immunoreactivity with a widely used polyclonal PAX8 antibody. Pathologists should be aware of this potential pitfall during the diagnostic workup of hepatobiliary lesions to avoid misdiagnosis as a metastasis from a PAX8-positive tumor., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Histological analysis to tissue samples is elemental for diagnosing the risk and severity of ovarian cancer. The commonly used Hematoxylin and Eosin (H&E) staining method involves complex steps and strict requirements, which would seriously impact the research of histological analysis of the ovarian cancer. Virtual histological staining by the Generative Adversarial Network (GAN) provides a feasible way for these problems, yet it is still a challenge of using deep learning technology since the amounts of data available are quite limited for training. Based on the idea of GAN, we propose a weakly supervised learning method to generate autofluorescence images of unstained ovarian tissue sections corresponding to H&E staining sections of ovarian tissue. Using the above method, we constructed the supervision conditions for the virtual staining process, which makes the image quality synthesized in the subsequent virtual staining stage more perfect. Through the doctors' evaluation of our results, the accuracy of ovarian cancer unstained fluorescence image generated by our method reached 93%. At the same time, we evaluated the image quality of the generated images, where the FID reached 175.969, the IS score reached 1.311, and the MS reached 0.717. Based on the image-to-image translation method, we use the data set constructed in the previous step to implement a virtual staining method that is accurate to tissue cells. The accuracy of staining through the doctor's assessment reached 97%. At the same time, the accuracy of visual evaluation based on deep learning reached 95%., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Xiangyu Meng et al.)

Background/aim: Programmed death ligand-1 (PD-L1) is a predictive marker for immunotherapeutic agents. However, heterogeneous staining of PD-L1 can cause false-negative results. The aim of this study is to evaluate the importance of histological patterns on PD-L1 staining heterogeneity in lung adenocarcinomas (LAC)., Materials and Methods: PD-L1 immunohistochemistry (IHC) stain was performed to two different tissue cores of 128 LAC cases, and cut-off values are given for grouping the cases according to the percentage of staining (1%-10%, 11%-49%, 50%-100%). Staining rates between cores were compared and analyzed by their histological patterns. Also, the relation of the PD-L1 expression with the clinicopathological characteristics of the cases was analyzed., Results: Overall, PD-L1 expression was observed in 53 of 128 cases (41.4%, 1% cut-off), 23.5% of them were positive at 10% cut-off and 14.1% at 50% cut-off. PD-L1 expression was significantly related to the high grade micropapillary and solid patterns of adenocarcinomas (p:0.01). Staining cut-offs were mostly similar between cores (43/50, 86%) (k:0.843). However, 14% of them were positive only in one core (7 of 50). This false negativity was mostly related to the histological patterns., Conclusion: Our data reveal the heterogeneous staining of PD-L1 expression, also micropapillary and solid patterns show higher rates of PDL expression. Therewithal, these findings also highlight the importance of taking into consideration of histological patterns, when choosing a paraffin block for the PDL1., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (This work is licensed under a Creative Commons Attribution 4.0 International License.)

Background: Classification of thyroid follicular neoplasms can be challenging for pathologists. Introduction of noninvasive follicular thyroid neoplasms with papillary-like nuclear features, the utilization of immunohistochemistry, and molecular analysis are all thought to be valuable diagnostic adjuncts. Our aim was to determine whether interobserver variability for follicular neoplasms has improved since the application of these adjuncts., Methods: One representative section from a cohort of follicular neoplasms previously proven difficult for pathologists were examined independently by 7 pathologists and assigned to 1 of 3 diagnostic categories (benign, neoplasms with papillary-like nuclear features, or malignant). This process was carried out separately 3 times: (1) after viewing hematoxylin and eosin stain slides, (2) hematoxylin and eosin stain in conjunction with immunohistochemistry, and (3) hematoxylin and eosin stain/immunohistochemistry in conjunction with molecular analysis. The interobserver variability and overall agreement were then calculated using the free-marginal kappa coefficient., Results: Agreement on hematoxylin and eosin stain was 57%, with a kappa coefficient of 0.36 (minimal agreement). The agreement improved slightly with the application of immunohistochemistry (kappa coefficient = 0.49 [weak agreement] and a percentage agreement 67%). The level of agreement decreased slightly after the addition of molecular analysis (kappa coefficient = 0.43 [weak agreement] and percentage agreement 62%)., Conclusion: Despite attempts to standardize the diagnostic criteria for neoplasms with papillary-like nuclear features and the utilization immunohistochemistry and molecular analysis, attaining pathologic consensus for difficult follicular neoplasms of the thyroid remains a challenge., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The visualization of glycogen deposits in cells and tissues is important for studying glycogen metabolism as well as diagnosis of glycogen storage diseases. Evidence suggests that the demonstration of glycogen can better be enhanced by factors such the choice of fixative and temperature during fixation. Here, we assessed efficacy of neutral buffered formalin (NBF), alcoholic formalin (AF) and paraformaldehyde (PFA) at 4 °C, 37 °C and 40 °C using Periodic Acid Schiff's staining method. Each liver specimen was fixed in NBF and AF while the brain tissues were fixed in NBF, AF and PFA. We found that there was a better PAS staining intensity with the liver tissues fixed in AF compared with NBF. Also, there was no difference in the quality of the staining for tissues fixed in AF at 37 °C, 4 °C and 40 °C, but fixation with NBF at 4 °C gave the best staining quality when compared with 40 °C and 37 °C. Furthermore, hippocampal tissues fixed in AF showed better quality of PAS staining compared with NBF and PFA. A significant increase in staining intensity was observed for PFA when compared with NBF. Superior staining intensity for PAS was observed at 4 °C for hippocampal tissues fixed with NBF, AF and PFA. Taken together our results show that AF at a temperature of 4 °C gave the best result. Hence, glycogen demonstration can better be enhanced by the choice of fixative and temperature during fixation., Competing Interests: Declaration of competing interest The authors stated that there are no conflicts of interest that can affect the decision to publish this manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of competing interest All authors were active participants of this study, and none has connection to any companies or products mentioned in this manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Background: Tumor budding is known predictors of lymph node metastasis from esophageal squamous cell carcinoma. However, it is not easy to detect such small cell clusters on hematoxylin-eosin (HE) staining. Therefore, we evaluated tumor budding using immunohistochemistry (IHC) for epithelial cell markers., Method: We analyzed tumor budding in 50 cases of superficial esophageal squamous cell carcinoma. We evaluated the impact of clinicopathological factors and tumor budding to predict lymph node metastasis. A total of 565 tumor sections were assessed using HE staining and IHC for cytokeratin 5/6., Results: Based on receiver operating characteristic curves, the cut-off values for high-grade tumor budding evaluated using HE staining or IHC were 2 and 11, respectively. High-grade tumor budding evaluated using HE staining (P = 0.007) and IHC (P ≤ 0.001) were significantly correlated with lymph node metastasis. For tumors with pT1a-MM to pT1b-SM1, high-grade tumor budding evaluated using IHC was correlated with lymph node metastasis (P = 0.050)., Conclusions: Tumor budding was significantly associated with lymph node metastasis. The optimal cut-off values of tumor budding on HE staining and tumor budding on IHC were 2 and 11, respectively. Even though both tumor budding on HE staining and tumor budding on IHC were significantly associated with lymph node metastasis, tumor budding on IHC tend to be more associated with lymph node metastasis.

Background/aim: The dramatic color change after iodine staining (from white-yellow to pink after 2-3 min), designated as the "pink-color sign" (PCS), is indicative of esophageal high-grade intraepithelial neoplasia (HGIN) or an invasive lesion. However, no study has yet examined the association between the time of PCS appearance and histopathology. We investigated the association between the time of PCS appearance and esophageal histopathology in 456 lesions of 438 patients who were examined for suspected esophageal cancer., Materials and Methods:: The records of 495 consecutive patients who had suspected esophageal cancer based on gastroscopy and who underwent Lugol's chromoendoscopy from January 2015 to March 2018 were retrospectively reviewed. The time of PCS appearance was recorded in all patients, and tissue specimens were examined., Results: We examined 456 lesions in 438 patients. Use of PCS positivity at 2 min for the diagnosis of HGIN/invasive cancer had a sensitivity of 84.1%, a specificity of 72.7%, and an accuracy of 80.4%. We classified the PCS-positive patients in whom the time of PCS appearance was recorded (168 lesions) into 4 groups: 0-30, 31-60, 61-90, and 91-120 s. Based on a 60-s time for appearance of the PCS, the area under the receiver operating characteristic curve was 0.897, indicating good validity. At the optimal cutoff value of 60 s, the sensitivity was 90.2% and the specificity was 82.3%. The appearance of the PCS within 60 s had a diagnostic accordance rate of 88.6%, significantly higher than appearance of the PCS within 2 min (79.7%, P < 0.05)., Conclusion: Appearance of the PCS within 1 min after iodine staining has a higher diagnostic accordance rate for esophageal HGIN/invasive cancer than appearance of the PCS at 2 min., Competing Interests: None

Stain colour estimation is a prominent factor of the analysis pipeline in most of histology image processing algorithms. Providing a reliable and efficient stain colour deconvolution approach is fundamental for robust algorithm. In this paper, we propose a novel method for stain colour deconvolution of histology images. This approach statistically analyses the multi-resolutional representation of the image to separate the independent observations out of the correlated ones. We then estimate the stain mixing matrix using filtered uncorrelated data. We conducted an extensive set of experiments to compare the proposed method to the recent state of the art methods and demonstrate the robustness of this approach using three different datasets of scanned slides, prepared in different labs using different scanners., Competing Interests: The authors have declared that no competing interests exist.

Background: Timely pathologic diagnosis relies on the communication of specific clinical information in the requisition form (RF). Clinical information may use nonspecific terms such as "rule out", the use of which may result in diagnostic delays and the unnecessary application of pathology stains and sections., Objective: This study was designed to evaluate the relationship between use of the term "rule out" and time to diagnosis, and the use of additional pathology stains and sections in integrated and non-integrated dermatopathology practices., Methods: A retrospective double-cohort study of 475 RFs from the integrated practice (of which 182 used the term "rule out" [RO] and 293 did not [NRO]) and 412 RFs from the non-integrated practice (RO, n = 126; NRO, n = 286) was performed., Results: No significant differences emerged between groups of patients with, respectively, RO and NRO RFs in the integrated practice with respect to time to diagnosis, and numbers of additional tissue sections or stains applied. By contrast, the use of RFs containing the term "rule out" was associated with significantly longer times to diagnosis and higher rates of use of pathology stains and sections in comparison with NRO RFs in the non-integrated practice. However, the study is limited by its status as a retrospective review of data sourced from a single institution., Conclusions: Use of the term "rule out" in RFs may not significantly impact key care delivery outcomes in an integrated practice. However, it may cause diagnostic delays and the use of unnecessary pathology services in a non-integrated practice., (© 2016 The International Society of Dermatology.)

Fluorescent in situ hybridization (FISH) is a powerful technique for the detection of RNA or DNA within cells and tissues, which provides a unique link between molecular and cell biology. This technique is broadly applicable across a range of biological systems. While FISH has been previously adapted to flow-based platforms, their use remains limited because of procedural challenges and costs associated with commercial kits. Herein we present a protocol that modifies existing techniques to sensitively and specifically detect and examine RNA expression patterns in primary cells and cell lines using flow cytometry (expression-FISH; X-FISH). As relevant examples, we show how this technique can be used to monitor changes in mRNA expression following activation, how it can be combined with antibody staining to study RNA and protein in the same sample, and how it can help distinguish among subsets in a mixed cell population. X-FISH can integrate multiple probes and can be performed in conjunction with other assays, allowing for informative multiparametric analyses and increased statistical robustness. For non-classical comparative animal models this procedure provides a time saving alternative to de novo production of antibody-based markers. Finally, X-FISH provides an economical solution that is applicable to conventional as well as multi-spectral imaging flow cytometry platforms., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Luminal-type breast cancer is divided into types A and B, depending on the Ki-67 labeling index (LI). However, the area at which Ki-67 is measured and the choice of specimen greatly affects the results. The aim of the present study was to evaluate the Ki-67 LI variability using different measurement methods and specimens. We also evaluated how the chemotherapy indication changed for luminal-type breast cancer using the different measurements., Materials and Methods: The Ki-67 levels in 87 patients with breast cancer were assessed, and the Ki-67 LI was calculated. Five measurement sites were randomly selected, including the most densely labeled areas (hot spots) in both core needle biopsy (CNB) and surgical specimens., Results: The intraclass correlation coefficient of the CNB and surgical specimens was 0.91 and 0.95, respectively. If the hot spot was used, the correlation coefficient (CC) between the CNB and surgical specimens was 0.635. If the average score was used, the CC was 0.730. If the average score was used, the CNB specimens indicated that 49 patients had a high Ki-67 LI, and 48 patients had a high Ki-67 LI using surgical specimens. If the hot spot was used, 60 patients using the CNB specimens and 58 patients using the surgical specimens had a high Ki-67 LI. If the average score was used, 17 patients were identified as being in different groups, and if the hot spot was used, 16 patients were identified as being in different groups, depending on the specimens that were used., Conclusion: The results differed according to the method and specimen type that was used., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Immunohistochemistry (IHC) is considered a valuable ancillary tool for dermatopathology diagnosis, but few studies have measured IHC utilization by dermatopathologists or assessed its diagnostic utility. In a regionalized, community-based dermatopathology practice, we measured IHC utilization (total requests, specific antibodies requested, and final diagnosis) over a 12-month period. Next, we assessed diagnostic utility by comparing a preliminary "pre-IHC" diagnosis based on routine histochemical staining with the final diagnosis rendered after consideration of IHC results. The dermatopathology IHC utilization rate was 1.2%, averaging 3.6 stains requested per case. Melanocytic, hematolymphoid, and fibrohistiocytic lesions made up 23%, 18%, and 16%, respectively, of the total cases requiring IHC. S100 and Melan A were the most frequently requested stains, ordered on 50% and 34% of IHC cases, respectively. The utility study revealed that IHC changed the diagnosis in 11%, confirmed a diagnosis, or excluded a differential diagnosis in 77%, and was noncontributory in 4% of cases. Where IHC results prompted a change in diagnosis, 14% were a change from a benign to malignant lesion, whereas 32% changed from one malignant entity to another. IHC is most commonly used in cutaneous melanocytic and hematolymphoid lesions. In 11% of dermatopathology cases in which IHC is used, information is provided that changes the H&E diagnosis. Such changes may have significant treatment implications. IHC is noncontributory in only a small percentage of cases.

This study compares the use of immunohistochemistry (IHC) for diagnosing carcinoma in private practice and commercial settings with use in a single academic center. H&E-stained slides and IHC stains, when present, of recently diagnosed carcinomas (n = 200) from patients referred to our institution for treatment were reviewed by a resident and mid-and senior-level pathologists. Diagnostic agreement between academic and referral pathologists was 98%; the former group used IHC stains in 11% and the latter in 26% of cases (P < .0001). Pathologists from commercial laboratories (12% of referrals) used IHC in 38% of cases, whereas private/hospital-based community laboratories (86% of referrals) used them in 24%. The average number of stains ordered per case was similar among all groups. We suggest that the use of IHC may reflect both the degree of experience of the pathologist and the pathology practice setting.

Carboxy-fluorescein diacetate succinimidyl ester (CFSE) labeling is an important experimental tool for measuring cell responses to extracellular signals in biomedical research. However, changes of the cell cycle (e.g., time to division) corresponding to different stimulations cannot be directly characterized from data collected in CFSE-labeling experiments. A number of independent studies have developed mathematical models as well as parameter estimation methods to better understand cell cycle kinetics based on CFSE data. However, when applying different models to the same data set, notable discrepancies in parameter estimates based on different models has become an issue of great concern. It is therefore important to compare existing models and make recommendations for practical use. For this purpose, we derived the analytic form of an age-dependent multitype branching process model. We then compared the performance of different models, namely branching process, cyton, Smith-Martin, and a linear birth-death ordinary differential equation (ODE) model via simulation studies. For fairness of model comparison, simulated data sets were generated using an agent-based simulation tool which is independent of the four models that are compared. The simulation study results suggest that the branching process model significantly outperforms the other three models over a wide range of parameter values. This model was then employed to understand the proliferation pattern of CD4+ and CD8+ T cells under polyclonal stimulation.

Purpose: To compare the proteomic profiles of formalin-fixed, paraffin-embedded (FFPE) liver biopsy material and matched frozen liver tissue from patients with nonalcoholic steatohepatitis (NASH)., Experimental Design: A label-free mass spectrometry-based approach was used to profile global protein expression in FFPE and frozen liver biopsy specimens from five patients with NASH., Results: Eight hundred and sixty proteins were identified with >75% confidence: 225 common proteins were identified in both the FFPE and frozen tissues, and an additional 142 and 493 proteins were identified in the FFPE and frozen tissues, respectively. Functional analyses revealed a general, nonspecific reduction in the number of proteins identified in FFPE tissue compared with frozen tissue. No bias toward proteins located in any specific subcellular compartments or implicated in any particular biological functions was observed. The relative abundance of several proteins with functions relating to the pathogenesis of NASH (peroxiredoxin-1, fatty acid binding protein 1, fatty acid synthase, vimentin, catalase, and glutathione S-transferase A1) was similar in FFPE and frozen liver tissues., Conclusions and Clinical Relevance: FFPE liver biopsy material from NASH patients can be used for global proteomic analysis and biomarker identification studies, although a universal reduction in the number of identified proteins compared with frozen tissue is likely., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Objectives: Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. In current clinical practice, diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign. This case-control study investigated the prognostic role of mere C4d staining for graft outcome during an ARE in a well defined cohort of similarly ARE-treated patients., Design, Setting, Participants, & Measurements: All kidney transplant recipients in the authors' center from January 1, 1995 to December 31, 2005 were reviewed. From these patients, 151 had a clinical ARE. Paraffin and/or frozen material was available for 128 patients showing a histologically proven ARE within the first 6 months after transplantation. All ARE patients were treated similarly with high-dose pulse steroids and in the case of steroid unresponsiveness with anti-thymocyte globulin. Biopsies were scored according to Banff criteria. Frozen and paraffin sections were stained by immunofluorescence (IF) and immunohistochemistry (IHC) for C4d, respectively, and scored for PTC positivity., Results: Diffuse C4d+ staining in PTCs was found in 12.5% and 4.2% sections stained by IF or by IHC, respectively. Four patients showed diffuse positive staining with both methods but showed no different risk profile from other patients. No relation between C4d staining and clinical parameters at baseline was found. C4d staining was not associated with steroid responsiveness, graft, or patient survival., Conclusions: This study shows that C4d staining is not related to clinical outcome in this cohort of histologically proven early AREs., (Copyright © 2011 by the American Society of Nephrology)

Purpose: To validate lysyl oxidase (LOX), a hypoxia-related protein, as a marker for metastasis in an independent head and neck cancer (HNC) patient group enrolled onto a prospective trial., Patients and Methods: We performed traditional immunohistochemical (IHC) staining and automated quantitative analysis (AQUA) for LOX expression in 66 HNC patients from one institution. We also performed AQUA staining for LOX in 306 of 1,113 patients treated on a phase III trial comparing four radiation fractionation schedules in locally advanced HNC (RTOG 90-03). Pretreatment characteristics and outcome were similar between patients with and without LOX assessment. We correlated AQUA LOX expression with time to metastasis (TTM), time to progression (TTP), and overall survival (OS)., Results: LOX expression from both staining methods predicted for TTM in the first 66 patients. Multivariate analysis, controlling for significant parameters including nodal stage and performance status, revealed tumor LOX expression, as a continuous variable, was an independent predictor for TTM (hazard ratio [HR], 1.21; 95% CI, 1.10 to 1.33; P = .0001), TTP (HR, 1.06; 95% CI, 1.02 to 1.10; P = .0069), and OS (HR, 1.04; 95% CI, 1.00 to 1.07; P = .0311) in RTOG 90-03 patients. This translates into a 259% increase in metastatic risk for a patient at the 75th percentile of LOX compared with one at the 25th percentile., Conclusion: AQUA LOX expression was strongly associated with increased metastasis, progression, and death in RTOG 90-03 patients. This study validates that LOX is a marker for metastasis and survival in HNC.

Objective: To determine the impact on patients' outcome of clinical effectiveness of empirical antimicrobial treatment of nosocomial bacteremia., Setting: 904-bed hospital, comprising acute care wards, rehabilitation and intermediate care wards and long-term care wards, that provides care primarily for aged (880 beds); design: prospective cohort analysis to access the evidence regarding the effectiveness of empirical antimicrobial treatment in patients with nosocomial bacteremia, when the Gram stain result was communicated to the physician; data collection: data collected concerned patients' characteristics at the early bacteremia, microbiology, antimicrobial treatment and patients' outcome within 30 days; analysis: patients with clinical signs of bacteremia after the Gram stain result were compared with those without symptoms, in univariate analysis., Results: Significant differences were not found for age, sex, underlying diseases, comorbidities, hospitalization wards, sources of bacteremia, microorganisms or patients' outcome. However, antimicrobial therapies were more often changed in symptomatic patients after the announcement of the Gram stain result (RR = 1.87; [1.03-3.37]; p = 0.04)., Conclusion: This study supports the notion that the outcomes for patients are similar whether patients have symptoms or not when the Gram stain result of the first positive blood culture is communicated to the clinician.

LC-MS/MS with certain labeling techniques such as isotope-coded affinity tag (ICAT) enables quantitative analysis of paired protein samples. However, current identification and quantification of differentially expressed peptides (and proteins) are not reliable for large proteomics screening of complex biological samples. The number of replicates is often limited because of the high cost of experiments and the limited supply of samples. Traditionally, a simple fold change cutoff is used, which results in a high rate of false positives. Standard statistical methods such as the two-sample t-test are unreliable and severely underpowered due to high variability in LC-MS/MS data, especially when only a small number of replicates are available. Using an advanced error pooling technique, we propose a novel statistical method that can reliably identify differentially expressed proteins while maintaining a high sensitivity, particularly with a small number of replicates. The proposed method was applied both to an extensive simulation study and a proteomics comparison between microparticles (MPs) generated from platelet (platelet MPs) and MPs isolated from plasma (plasma MPs). In these studies, we show a significant improvement of our statistical analysis in the identification of proteins that are differentially expressed but not detected by other statistical methods. In particular, several important proteins - two peptides for beta-globin and three peptides for von Willebrand Factor (vWF) - were identified with very small false discovery rates (FDRs) by our method, while none was significant when other conventional methods were used. These proteins have been reported with their important roles in microparticles in human blood cells: vWF is a platelet and endothelial cell product that binds to P-selectin, GP1b, and GP IIb/IIIa, and beta-globin is one of the peptides of hemoglobin involved in the transportation of oxygen by red blood cells.

Purpose: To compare immunostaining quantification obtained by a digital computer-assisted method with the well-established semiquantitative analysis., Methods: Cytoplasmic staining of galectin-3 was obtained by standard immunohistochemical reactions in 25 cases of well-differentiated thyroid carcinoma. The expression index that associates the conventional area fraction of labeled cells with the immunostaining intensity score based on visual qualitative observation was used as the semiquantitative analysis. A digital computer-assisted method is described based on the use of an image processing program (ImageLab). Three parameters were obtained: (1) percentage of labeled cells; (2) digital immunostaining intensity, and (3) digital expression index. The proposed method allows numerical analysis of the immunostaining intensity., Results: There was a strong correlation between the immunostaining intensity obtained by the two methods (Pearson correlation coefficient, r = 0.71, P = 0.0001). The same was observed between expression indexes (Pearson correlation coefficient, r = 0.66, P = 0.0001)., Conclusion: Results obtained with our proposed digital computer-assisted method for immunoexpression analysis were concordant with the semiquantitative analysis. In addition, digital values can also resolve disagreement among different observers about the quality of staining intensity because the digital method does not classify the results into groups, but rather provides a numerical value for each individual case; thus, it increases the diagnostic and, more importantly, the prognostic sensitivity of the immunohistochemical analysis.

A simple Gram stain evaluation for the diagnosis of bacterial vaginosis was assessed in comparison to Nugent's method. The sensitivity (90%), specificity (94%), positive and negative predictive values (90% and 94%), and good interobserver agreement obtained indicate that the test method can be performed reliably in the routine clinical microbiology laboratory.

The current situation of the malaria inspection in our laboratory was investigated. Malaria was detected by three different methods, May Giemsa staining(MG), acridine orange staining(AO), and antigen detecting method using NOW ICT Malaria P.f./P.v. kit(Ag). There were 207 requests a year(17.3 per month), and the holiday/night request occupied 12%. Fifteen patients were positive, 5 with plasmodium falciparum (p.f.) and 10 with plasmodium vivax(p.v.), including 3 relapsed cases. All the patients with p.f. were suffered in Africa, and 6 with p.v. were in Southeast Asia, and one with p.v. was in Central America. The rate of coincidence between MG/Ag and MG/AO were 94.4% and 96.9%, respectively. There were 7 samples that were MG negative and Ag positive, but all of these samples were obtained after the initiation of the treatment. There was no sample that showed MG positive and Ag negative. Our data suggested that no difference in detection sensitivity was found between microscopic observation and the antigen detection kit. Thus it would be a very useful and accurate strategy to use this antigen detection kit in a routine laboratory check up.

Context: Surface swab cultures have attracted attention as a potential alternative to biopsy histology or quantitative culture methods for microbiological burn wound monitoring. To our knowledge, the utility of adding a Gram-stained slide in this context has not been evaluated previously., Objective: To determine the degree of correlation of Gram stain with culture for the microbiological analysis of burn wound surfaces., Design: Prospective laboratory analysis., Setting: Urban health region/centralized diagnostic microbiology laboratory., Patients: Burn patients hospitalized in any Calgary Health Region burn center from November 2000 to September 2001., Intervention: Gram stain plus culture of burn wound surface swab specimens obtained during routine dressing changes or based on clinical signs of infection., Main Outcome Measures: Degree of correlation (complete, high, partial, none), including weighted kappa statistic (kappa(w)), of Gram stain with culture based on quantitative microscopy and degree of culture growth., Results: A total of 375 specimens from 50 burn patients were evaluated. Of these, 239 were negative by culture and Gram stain, 7 were positive by Gram stain only, 89 were positive by culture only, and 40 were positive by both methods. The degree of complete, high, partial, and no correlation of Gram stain with culture was 70.9% (266/375), 1.1% (4/375), 2.4% (9/375), and 25.6% (96/375), respectively. The degree of correlation for all 375 specimens, as expressed by the weighted kappa statistic, was found to be fair (kappa(w) = 0.32).Conclusion.-The Gram stain is not suitable for the microbiological analysis of burn wound surfaces.

Data from five different Institution of Pathological Anatomy Hospital Services are presented in order to show one (the benchmark) of the multiple existing ways to approach the budget problem and the macroeconomic management of our Services. The aim of this work is not to show the "best" way to work in terms of cost-efficacy but only a methods to compare our results with others. Nevertheless from this study is possible also to make some considerations about medical and technical workload in different services with different habits.

The developing human cerebrum displays age-specific changes in its patterns of lamination. Among these, the subplate zone is the most prominent transient compartment because growing major afferent systems temporarily reside in this zone, establish synapses and take part in cellular interactions that are crucial for subsequent cortical development. We explored the potential of magnetic resonance imaging (MRI) for tracing the developmental history of the most prominent cortical layer (the subplate zone) and other laminar compartments of the fetal cerebral wall between 15 and 36 weeks post-ovulation. We found that changes in the MRI lamination pattern of the human fetal cerebral wall are predominantly caused by changes in the subplate zone. Histochemical staining of the extracellular matrix (ECM) enables selective visualization of the subplate zone and correlation with an increase in MRI signal intensity in the subplate zone and ingrowth and accumulation of thalamocortical and corticocortical afferents and their subsequent relocation to the cortical plate. Thus, dynamic changes in the MRI appearance of the subplate zone and histochemical staining of its ECM can be used as indirect parameters for an assessment of normal versus disturbed unfolding of crucial histogenetic events that are involved in prenatal shaping of the human cerebral cortex.

The identification of CTL-defined tumor-associated Ags has allowed the development of new strategies for cancer immunotherapy. To potentiate the CTL responses, peptide-based vaccines require the coadministration of adjuvants. Because oligodeoxynucleotides (ODN) containing CpG motifs are strong immunostimulators, we analyzed the ability of CpG ODN to act as adjuvant of the CTL response against tumor-derived synthetic peptide in the absence or presence of IFA. Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG ODN alone or CpG ODN emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro. Overall, these results indicate that CpG ODN by itself is a good candidate adjuvant of CTL response and can also enhance the effect of classical adjuvant.

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.

From February 1995 through May 1997, we prospectively studied 533 patients with community-acquired pneumonia requiring hospitalization in order to assess the current usefulness of sputum Gram stain in guiding the etiologic diagnosis and initial antibiotic therapy when applied routinely. Sputum samples of good quality were obtained in 210 (39%) patients, 175 of whom showed a predominant morphotype. Sensitivity and specificity of Gram stain for the diagnosis of pneumococcal pneumonia were 57% and 97%, respectively; the corresponding values for Haemophilus influenzae pneumonia were 82% and 99%. Patients with a predominant morphotype were more frequently treated with monotherapy than were patients without a demonstrative sputum sample (89% vs. 75%; P<.001). Analysis of our data shows that a good-quality sputum sample can be obtained from a substantial number of patients with community-acquired pneumonia. Gram stain was highly specific for the diagnosis of pneumococcal and H. influenzae pneumonia and may be useful in guiding pathogen-oriented antimicrobial therapy.

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols., (Copyright 1999 Academic Press.)

Ewing's sarcoma (ES) and peripheral neuroectodermal tumor (PNET) are considered in the differential diagnosis of small round blue cell tumors of infancy and childhood which includes neuroblastoma, rhabdomyosarcoma and malignant lymphoma. Fine-needle aspiration diagnosis of these neoplasms can be particularly difficult when the neoplasms are composed of poorly differentiated cells or fail to produce a stroma. MIC-2 is a highly sensitive and specific marker for the PNET/ES group of neoplasms and has been studied extensively in surgical pathology. Other small blue cell neoplasms including rhabdomyosarcoma, blastemal Wilm's tumor, and lymphoblastic lymphoma have also shown positivity, but the staining reactions are usually weak and focal. The utility of this marker in the differential of small blue cell neoplasms in cytologic material has not been examined. Twenty cases of small blue cell neoplasms obtained by fine-needle aspiration (FNA) were studied. MIC-2 antibody was applied retrospectively to formalin-fixed cell block material and destained alcohol-fixed and air-dried cytologic preparations. These cases include primitive neuroectodermal tumor (five cases), Ewing's sarcoma (two cases), neuroblastoma (four cases), Wilms's tumor (four cases), lymphoblastic lymphoma (two cases), and small-cell carcinoma (three cases). The cases were judged positive when the majority of the cells showed cytoplasmic staining. Diffuse cytoplasmic staining was observed in all seven cases of PNET/ES. Staining could be seen on the destained air-dried smears (three cases), fixed smears (two cases), or the cell block material (two cases). None of the other 13 small blue cell neoplasms showed positive staining. We conclude that MIC-2 is a sensitive and specific marker for the PNET/ES group of neoplasms in specimens from formalin-fixed cell block, air-dried, and alcohol-fixed cytologic material and is useful in the differential diagnosis of small blue cell tumors.

A method for quantification of images of immunohistochemically stained cell nuclei by computing area proportions is presented. The image is transformed by a principal component transform. The resulting first component image is used to segment the objects from the background using dynamic thresholding of the P2/A-histogram, where P2/A is a global roundness measure. Then the image is transformed into principal component hue, defined as the angle around the first principal component. This image is used to segment positive and negative objects. The method is fully automatic and the principal component approach makes it robust with respect to illumination and focus settings. An independent test set consisting of images grabbed with different focus and illumination for each field of view was used to test the method, and the proposed method showed less variation than the intraoperator variation using supervised Maximum Likelihood classification.

Mass spectrometric techniques for the identification of proteins either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

This paper describes a new, sensitive (in the nanogram range), and rapid (two-step) technique for the negative staining of proteins in polyacrylamide gels in the presence or absence of sodium dodecyl sulfate. After separation, gels are incubated with 8% methyl trichloroacetate ester in 38% isopropanol and then washed in water to produce a negative image of colorless proteins against an opaque background. The technique allows unmodified proteins to be recovered for biological studies or transblot for amino acid sequence. Finally, owing to the reversibility of the process, gels can be restained after rapid visualization. For these reasons, negative staining with methyl trichloroacetate should become the method of choice for rapid and sensitive staining of proteins prior to further processing, including stable staining with silver ions.

Following a major reorganization of the National Health Service, competition for resources will depend to a great extent on the quality of the service offered, and audit will be of increasing importance. The present investigation deals with the quantitative aspects of the histopathology workload. A retrospective analysis of the biopsy handling of 1,000 consecutive cases was conducted in each histopathology lab of seven general hospitals. The data collected included information on specimen type, diagnosis, block/section/stain details for each specimen, as well as information about human resources, total workload and technical equipment. Among the seven labs, the histopathology workload ranged between 6,600 and 15,600 cases/year. The workload per person/year ranged between 1,400 and 2,600 (mean 1,900) for pathologists, between 800 and 3,000 (mean 1,500) for technicians, and between 2,200 and 8,300 (mean 4,400) for secretarial staff. The prevalence of biopsy fragments (endoscopic, endometrial curettage , etc.), "small" surgery (skin, appendix, gallbladder, etc.) and surgery was, respectively, between 42 and 50%, 34 and 47%, 9 and 21%. In all labs but one, 80% of cases were within 1-3 blocks; the seven labs had a mean of blocks/case ranging from 1.8 to 4.0 (total mean = 2.8). Differences in performing special stains were astonishing: one lab performed special stains in about 40%, and another in only 0.6% of cases (mean of the seven labs = 15%). Finally, the labs performed immunohistochemical stains within a range of 2.7-8% of cases (mean of the seven labs = 4.6%). The data we have collected provided baseline information on the laboratory running, and this is likely to be an integral part of laboratory administration in the near future.

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 microliters of stool to detect one microsporidian after viewing 50 fields at a final magnification of x1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence of microsporidia by transmission electron microscopy (TEM). All TEM-positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An additional seven TEM-negative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identification as specific antibodies become available.

To evaluate the usefulness of cytocentrifugation for detection of acid-fast bacilli (AFB) in sputum specimens, we compared this method to a traditional concentration method for the preparation of smears. A total of 844 sputum specimens (from 579 patients) of adequate volume that were submitted for detection of mycobacteria were evaluated. A portion of each specimen was used for cytocentrifugation; the remainder was processed by our decontamination-concentration protocol (2% sodium hydroxide-N-acetyl-L-cysteine; centrifugation at 3,600 x g for 15 min) for preparation of smears and culture. All smears were stained with auramine O. Ninety-four cultures from 46 patients gave positive results, and AFB were seen in one or both smears from 53 specimens; 3 of the latter specimens (positive by both smear methods) were culture negative. Of the 50 AFB smear-positive and culture-positive specimens, 46 were smear positive by traditional concentration, and 47 were positive by the cytocentrifugation smear (P, not significant). Cultures of all specimens that were smear positive by only one method grew nontuberculous mycobacteria. The routine use of cytocentrifugation for concentrating sputum specimens increases the cost of smear preparation but does not increase detection of AFB by auramine O staining; however, it would be useful in handling emergent requests for AFB smears.

Differential staining of spindle and chromatin was adapted to meiotic cells of male mice. Immunostaining with antitubulin antibodies was developed. Both methods were used to test acrylamide, diazepam and vinblastine for their potential to cause spindle disturbances in male mouse germ cells. The analyses were performed after in vivo treatment, comparing the classical safranin/brilliant blue spindle staining to an immunofluorescent assay. The two staining methods complemented each other. Differential staining seems to be more sensitive to the detection of misplaced chromatin whereas the immunostaining is superior when scoring for spindle structure aberrations. All three chemicals showed spindle activity. Acrylamide mainly caused multipolar spindles which possibly develop from a separation of mother and daughter centrioles during an unspecific meiotic block. Diazepam elevated the level of monopolar spindles and induced the loss of single chromosomes, suggesting an effect on motor proteins. Vinblastine was the strongest spindle poison by far. It produced high numbers of shortened and monopolar spindles as well as multiple chromosome loss.

Newly available assays offer alternatives to conventional microscopic examination for Cryptosporidium spp. We compared two enzyme immunoassays, ProSpect Cryptosporidium microtiter assay (Alexon, Inc., Mountain View, Calif.) and Color Vue Cryptosporidium assay (Serady, Indianapolis, Ind.), and a direct immunofluorescent assay, Merifluor Cryptosporidium kit (Meridian Diagnostics, Cincinnati, Ohio), with acid-fast Kinyoun-staining for the detection of Cryptosporidium spp. Examinations were performed on 129 stool specimens received from patients during a recent waterborne outbreak. A specimen was considered positive when organisms could be identified visually by acid-fast and immunofluorescent stains or if organisms could be visualized by either acid-fast or immunofluorescent stain and detected by both enzyme immunoassays. The final number of positive specimens was 55. No single procedure detected all 55 positive specimens. Of these, ProSpect and Color Vue detected 52 (sensitivity, 94.5%), and the Kinyoun stain and Merifluor detected 53 (sensitivity, 96.4%). The final number of negative specimens was 74. One false-positive result was seen with both the Kinyoun stain and the ProSpect assay. The Color Vue and ProSpect assays required the most hands-on technologist time. The ProSpect assay and Merifluor kit were easiest to perform. The acid-fast stain was difficult to interpret. The Merifluor kit was easiest to read and was adaptable to both batch and single testing. Overall, the Kinyoun stain and the Merifluor test were preferable to both of the enzyme immunoassays because of the high reagent cost and hands-on time required for the enzyme immunoassays. The difficult interpretation of the Kinyoun stain smears made the Merifluor a more desirable test despite its higher cost. We conclude that all methods tested were equally sensitive and specific for the detection of Cryptosporidium spp. Ease of use, adaptability to batch testing, and cost are important criteria in determining the method of choice.

Gram's stain (GS) and culture of sputum are used routinely for diagnosis of lung infection. We prospectively studied physicians' use of sputum GS and culture for management of lung infection, the correlation between GS and culture in 224 adequate sputum specimens from patients admitted to our hospital over a 15-month period, and its impact on the antibiotic used. GS correlated with sputum culture in one third of the cases in the most predictive group. Gram-negative rods including Haemophilus influenzae formed the majority of the positive cultures and were also the most poorly predicted by GS. Physicians used broad spectrum antibiotics empirically in most cases. These choices were not altered by the culture results in most instances. It may be reasonable to use broad spectrum antibiotics empirically for lung infection. Sputum GS and culture may be helpful in patients at risk for high morbidity and mortality.

We examined the staining characteristics and degranulation of mast cells in bronchial biopsy specimens taken by fiberoptic bronchoscopy from 13 stable asthmatic patients and eight normal nonsmoking subjects. Specimens were fixed in periodate-lysine-paraformaldehyde, embedded in glycol methacrylate, and stained with toluidine blue (2%) for 30 min (pH 2.7) and 7 days (pH 0.5). The number of mast cells in the epithelium and in the lamina propria was counted under light microscopy. In addition, the distribution of mast cells with different granule contents, arbitrarily defined as degranulated or partly degranulated and fully granulated, was estimated at the two levels. In asthmatic subjects, the number of mast cells in the epithelium after either staining method was significantly higher compared with that in control subjects. The number of mast cells in the lamina propria, but not in the epithelium, was significantly higher after 7 days compared with 30-min toluidine blue stain both in asthmatic (135.6/mm2 versus 74.8/mm2; p < 0.001) and control subjects (121.5/mm2 versus 71.5/mm2; p < 0.01). There was evidence of a progressive mast cell degranulation when moving toward the airway lumen in both groups. However, degranulation was more evident in asthmatic subjects. In both groups, granulated mast cells were absent in the epithelium, whereas in the lamina propria granulated mast cells were approximately one-third of total in asthmatic and two-thirds of total in normal subjects. These observations suggest that mast cells in human bronchial mucosa are heterogeneous with respect to histochemical characteristics. They provide evidence that degranulation of mast cells occurs in both asthmatic and normal subjects and that degranulation is greater in asthmatics.

The usefulness of five dying techniques to detect oocysts of Cryptosporidium spp in stools is evaluated in a healthy children preschool population, taking from a smear of the sediment obtained in the formalin-ether concentration technique (Ritchie's test). 10 positive stool samples were detected of a total of 115 samples (8.7%). Dying with Lugol the sediment of Ritchie's test only detected 40% of positive samples. Modified Ziehl-Neelsen dye, auramine dye and indirect immunofluorescence dye with monoclonal antibodies, yielded an excellent diagnostic correlation (each of them detected 90% of the positive samples). Indirect immunofluorescence dye with specific human antibodies detected one case more of parasites than the rest of dyes, is not expensive and easy to perform.