Confocal Raman spectroscopy coupled with in vitro permeation testing to study the effects of formalin fixation on the skin barrier function of reconstructed human epidermis.

Confocal Raman Spectroscopy is recognised as a potent tool for molecular characterisation of biological specimens. There is a growing demand for In Vitro Permeation Tests (IVPT) in the pharmaceutical and cosmetic areas, increasingly conducted using Reconstructed Human Epidermis (RHE) skin models. In this study, chemical fixation of RHE in 10 % Neutral Buffered Formalin for 24 h has been examined for storing RHE samples at 4 °C for up to 21 days. Confocal Raman Spectroscopy (CRS), combined with Principal Components Analysis, revealed the molecular-level effects of fixation, notably in protein and lipid conformation within the stratum corneum and viable epidermis. IVPT by means of high-performance liquid chromatography, using caffeine as a model compound, showed minimal impact of formalin fixation on the cumulative amount, flux, and permeability coefficient after 12 h. While the biochemical architecture is altered, the function of the model as a barrier to maintain rate-limiting diffusion of active molecules within skin layers remains intact. This study opens avenues for enhanced flexibility and utility in skin model research, promising insights into mitigating the limited shelf life of RHE models by preserving performance in fixed samples for up to 21 days.

Oil blotting paper for formalin fixation increases endoscopic ultrasound-guided tissue acquisition-collected sample volumes on glass slides.

OBJECTIVES: Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is used for pathological diagnosis and obtaining samples for molecular testing, facilitating the initiation of targeted therapies in patients with pancreatic cancer. However, samples obtained via EUS-TA are often insufficient, requiring more efforts to improve sampling adequacy for molecular testing. Therefore, this study investigated the use of oil blotting paper for formalin fixation of samples obtained via EUS-TA. METHODS: This prospective study enrolled 42 patients who underwent EUS-TA for pancreatic cancer between September 2020 and February 2022 at the Osaka International Cancer Institute. After a portion of each sample obtained via EUS-TA was separated for routine histological evaluation, the residual samples were divided into filter paper and oil blotting paper groups for analysis. Accordingly, filter paper and oil blotting paper were used for the formalin fixation process. The total tissue, nuclear, and cytoplasm areas of each sample were quantitatively evaluated using virtual slides, and the specimen volume and histological diagnosis of each sample were evaluated by an expert pathologist. RESULTS: All cases were cytologically diagnosed as adenocarcinoma. The area ratios of the total tissue, nuclear, and cytoplasmic portions were significantly larger in the oil blotting paper group than in the filter paper group. The frequency of cases with large amount of tumor cells was significantly higher in the oil blotting paper group (33.3%) than in the filter paper group (11.9%) (p = 0.035). CONCLUSIONS: Oil blotting paper can increase the sample volume obtained via EUS-TA on glass slides and improve sampling adequacy for molecular testing.

Impact of Formalin- and Cryofixation on Raman Spectra of Human Tissues and Strategies for Tumor Bank Inclusion.

Reliable training of Raman spectra-based tumor classifiers relies on a substantial sample pool. This study explores the impact of cryofixation (CF) and formalin fixation (FF) on Raman spectra using samples from surgery sites and a tumor bank. A robotic Raman spectrometer scans samples prior to the neuropathological analysis. CF samples showed no significant spectral deviations, appearance, or disappearance of peaks, but an intensity reduction during freezing and subsequent recovery during the thawing process. In contrast, FF induces sustained spectral alterations depending on molecular composition, albeit with good signal-to-noise ratio preservation. These observations are also reflected in the varying dual-class classifier performance, initially trained on native, unfixed samples: The Matthews correlation coefficient is 81.0% for CF and 58.6% for FF meningioma and dura mater. Training on spectral differences between original FF and pure formalin spectra substantially improves FF samples' classifier performance (74.2%). CF is suitable for training global multiclass classifiers due to its consistent spectrum shape despite intensity reduction. FF introduces changes in peak relationships while preserving the signal-to-noise ratio, making it more suitable for dual-class classification, such as distinguishing between healthy and malignant tissues. Pure formalin spectrum subtraction represents a possible method for mathematical elimination of the FF influence. These findings enable retrospective analysis of processed samples, enhancing pathological work and expanding machine learning techniques.

Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Effects of Extended Fixation on Advanced Gastric Cancer HER2 Status Assessment Using IHC and FISH.

BACKGROUND/AIM: In gastric cancer, accurate determination of human epidermal growth factor receptor type 2 (HER2) status is crucial for treatment decision-making. However, the optimal formalin fixation time of gastric cancer specimens for HER2 status determination remains unestablished. Here, we investigated real-world data on formalin overfixation and its effect on HER2 status determination in gastric cancer. PATIENTS AND METHODS: We comprehensively analyzed HER2 testing results in 228 gastric cancer specimens, including those subjected to formalin overfixation. Subsequently, we divided 52 resected specimens of advanced gastric cancer into three groups and studied the effects of short-term (6-72 h) and long-term (1 and 2 weeks) fixation on HER2 status determination using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). RESULTS: A total of 21.5% (49/228) of the specimens were HER2-positive, whereas 78.5% (179/228) were negative. Among the HER2-negative specimens, no biopsies were overfixed, whereas 12.5% (9/72) of the surgical resection specimens were overfixed. The HER2 status of the 6-72-h group was 82.7% and 76.9% identical to that of the 1- and 2-week groups, when determined using IHC, and 73.1% and 36.5%, when determined using FISH, respectively. However, HER2 determination was not feasible in 26.9% and 63.5% of the specimens in the 1- and 2-week groups, respectively. CONCLUSION: Formalin overfixation may hinder the determination of HER2 status and should be avoided in gastric cancer sample preparation.

P53 loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded leiomyosarcoma (LMS): a novel report.

BACKGROUND: The occurrence of p53 loss of heterozygosity (LOH) is a common genetic event in malignancy. LOH occurs when a heterozygous locus loses one of its two parental alleles, becoming homozygous at that locus, by either copy number loss (CNL-LOH) or by becoming copy number neutral (CNN-LOH). A role for CNL-LOH (cnLOH) has been postulated in cancer aetiology. Loss of heterozygosity (LOH) results in irreversible genetic loss. AIMS: LOH was determined in DNA extracted from formalin-fixed paraffin-embedded (FFPE) leiomyosarcoma (LMS) specimens in a retrospective study from 30 patients, to assess the prognostic significance of LOH. The findings were analysed and their validity assessed. LOH was an adverse prognostic factor in LMS. Prospective uniform standardisation of formalin-fixation techniques is required. METHODS: DNA was extracted from 169 formalin-fixed paraffin blocks of 30 patients with LMS, following extensive tissue microdissection. Genomic DNA was amplified using the polymerase chain reaction (PCR) technique. Fluorescence-based microsatellite PCR was used to detect and quantitate heterozygosity loss. RESULTS: LOH was detected at gene locus 17p13 in 16 LMS (Four grade 2 and 12 grade 3 LMS). LOH was not detected in 14 LMS cases (one grade 1, five grade 2 and eight grade 3 LMS). LOH was associated with shorter patient survival. CONCLUSIONS: The results reported herein endorse the value of utilizing FFPE DNA in identifying LOH as a prognostic factor in LMS. The results have implications for tumour biobanking and precision medicine in patients with sarcomas.

Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine.

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Impact of formalin fixation on mismatch repair protein evaluation by immunohistochemistry.

Mismatch repair/microsatellite instability (MMR/MSI) status in colorectal cancer (CRC) has become fundamental as a diagnostic, prognostic, and predictive factor. MMR immunohistochemistry (IHC) is considered a simple and reliable approach; however, its effectiveness depends on pre-analytic factors. Aim of this study was to investigate the impact of different fixation times/protocols on MMR protein IHC quality. Left over tissue from surgically resected CRC samples (cold ischemia time < 30 min) where fixed as follows: standard formalin fixation (24-48 h); hypo-fixation (<20 h); hyper-fixation (>90 h); cold (4°C) fixation (24-48 h); standard fixation for small sample size (0.5×0.5 cm). Samples for each group were collected from 30 resected CRC and the following parameters were evaluated on 600 immunohistochemical stains: intensity of expression; patchiness of staining; presence of central artefact. Forty-six immunoreactions were inadequate (score 0 intensity), the majority regarding MLH1 or PMS2 in the hypo-fixation group (47.8%), followed by the hyper-fixation group (28.1%); cold formalin fixation showed the least inadequate cases. Patchiness and central artefact were more frequent in hypo-fixation and standard fixation group compared to the others. MLH1 (closely followed by PMS2) performed worse with regard to immunostaining intensity (p=0.0002) in the standard and in the hypo-fixation group (p< 0.00001). Using a small sample size improved patchiness/central artefacts. This is the first study specifically created to evaluate the impact of fixation on MMR protein IHC, showing that both formalin hypo- and hyper-fixation can cause problems; 24-h formalin fixation as well as cold (4°C) formalin fixation are recommended for successful IHC MMR evaluation.

The risk of weekend biopsy: Impact of specimen source and fixation status on HER2 assessment in the treatment of advanced gastric cancer (The HER_WEEKEND study).

Accurate evaluation of human epidermal growth factor receptor type 2 (HER2) expression is crucial for determining chemotherapy regimens in gastric cancer. However, formalin fixation status has been identified as an important factor affecting HER2 assessment reliability. This retrospective cohort study aimed to investigate the correlation between sample collection day (weekday vs. weekend) and source (biopsy vs. surgical specimens) in assessing HER2 expression in patients with unresectable advanced/recurrent gastric cancer. Data were collected from gastric cancer patients who received chemotherapy at a single public hospital in Japan from 2008 to 2021. The analysis included 177 patients (109 men, 68 women) with a median age of 68.0 (21-88) years, and the primary outcome was the HER2 positivity rate. The overall HER2 positivity rate was 18.1%, with higher rates on weekdays (20.0%) compared to weekends (12.8%). Biopsies had higher positivity rates on weekdays (23.9%) but lower rates on weekends (11.1%) than surgical specimens. Significant differences were observed in formalin fixation times between weekdays and weekends for both biopsies and surgical samples. The study findings suggest that longer formalin fixation times on weekends may lead to underestimating HER2 expression, particularly in biopsies. Therefore, it is crucial to be cautious of excessive formalin fixation when collecting samples, especially during weekend biopsies.

Effects of formalin fixation on polarimetric properties of brain tissue: fresh or fixed?

Significance: Imaging Mueller polarimetry (IMP) appears as a promising technique for real-time delineation of healthy and neoplastic tissue during neurosurgery. The training of machine learning algorithms used for the image post-processing requires large data sets typically derived from the measurements of formalin-fixed brain sections. However, the success of the transfer of such algorithms from fixed to fresh brain tissue depends on the degree of alterations of polarimetric properties induced by formalin fixation (FF). Aim: Comprehensive studies were performed on the FF induced changes in fresh pig brain tissue polarimetric properties. Approach: Polarimetric properties of pig brain were assessed in 30 coronal thick sections before and after FF using a wide-field IMP system. The width of the uncertainty region between gray and white matter was also estimated. Results: The depolarization increased by 5% in gray matter and remained constant in white matter following FF, whereas the linear retardance decreased by 27% in gray matter and by 28% in white matter after FF. The visual contrast between gray and white matter and fiber tracking remained preserved after FF. Tissue shrinkage induced by FF did not have a significant effect on the uncertainty region width. Conclusions: Similar polarimetric properties were observed in both fresh and fixed brain tissues, indicating a high potential for transfer learning.

A New Medium (HistoCold) for Surgical Specimens Preserving to Improve the Preanalytic Issues in Histopathological Samples Handling: Morphologic and Antigenic Analysis.

Introduction: The onset of precision medicine has led to the integration of traditional morphologic tissues evaluation with biochemical and molecular data for a more appropriate pathological diagnosis. The preanalytic phase and, particularly, timing of cold ischemia are crucial to guarantee high-quality biorepositories of formalin-fixed paraffin-embedded (FFPE) tissues for patients' needs and scientific research. However, delayed fixation using the gold-standard and carcinogenic fixative neutral-buffered formalin (NBF) can be a significant limitation to diagnosis and biopathological characterization. HistoCold (patented; Bio-Optica Milano S.p.A., Milano, Italy) is a nontoxic, stable, and refrigerated preservative solution for tissue handling. This study examined HistoCold's potential role in improving the preanalytic phase of the pathological diagnostic process. Materials and Methods: Breast, lung, or colorectal cancers (20, 25, and 10 cases, respectively) that were to be surgically resected were recruited between 2019 and 2021. Once specimens were surgically removed, three residual samples for each patient were first promptly immersed into HistoCold for 24, 48, and 72 hours and then FFPE. These were compared with routine specimens regarding morphologic features (hematoxylin and eosin) and tissue antigenicity (immunohistochemical stains). Results: Good concordance regarding both the morphologic characteristics of the neoplasms and their proteins expression between the routine and HistoCold handled tissues were found. The tissue handling with the solution never affected the histopathological diagnosis. Conclusions: The use of HistoCold for samples transporting is easy, allows for improving the management of cold ischemia time, and monitoring the fixation times in NBF, resulting in good quality tissue blocks for biobanking. Moreover, it could be a candidate to eliminate formalin from operating theaters. HistoCold looks very promising for the preanalytic phase of human tissues handling in the era of precision medicine, to provide the best service to patients, and to scientific research.

Prediction Formula for Pathological Depth of Invasion From Clinical Depth of Invasion in Tongue Squamous Cell Carcinoma (SCC) Stage I/II Cases.

BACKGROUND:  The depth of invasion (DOI) of tongue squamous cell carcinoma (SCC) is an important prognostic factor. The definition is clear for pathological DOI (pDOI), but the treatment strategy is determined by the preoperative clinical DOI (cDOI). Few studies have investigated the difference between these DOIs. The purpose of this study was to obtain the correlation equation between cDOI and pDOI for Stage I/II tongue SCC and to consider the points to be noted in actual clinical practice. METHODS:  In this retrospective study, 58 patients with clinical stage I/II tongue SCC were included. Correlations between cDOI and pDOI were obtained for all 58 cases, as well as for 39 cases which excluded superficial and exophytic lesions. RESULTS:  The overall cDOI and pDOI median values were 8.0 and 5.5 mm, respectively; the 2.5 mm reduction was significant (p < 0.01). The correlation equation was pDOI = 0.81 × cDOI-0.23 (r = 0.73). Furthermore, re-analysis of the 39 cases revealed that pDOI = 0.84 × cDOI-0.37 (r = 0.62). Hence, a derived equation pDOI = 0.84 × (cDOI-0.44) was obtained to predict pDOI from cDOI. CONCLUSIONS:  This study indicated that it is necessary to consider contraction due to specimen fixation by subtracting the thickness of the mucosal epithelium. Clinical T1 cases with a cDOI of 5 mm or less had a pDOI of 4 mm or less, and it would be expected to have low positive rate of neck lymph node metastasis.

Effects of different preservation on the mechanical properties of cortical bone under quasi-static and dynamic compression.

Introduction: Mechanical properties of biological tissue are important for numerical simulations. Preservative treatments are necessary for disinfection and long-term storage when conducting biomechanical experimentation on materials. However, few studies have been focused on the effect of preservation on the mechanical properties of bone in a wide strain rate. The purpose of this study was to evaluate the influence of formalin and dehydration on the intrinsic mechanical properties of cortical bone from quasi-static to dynamic compression. Methods: Cube specimens were prepared from pig femur and divided into three groups (fresh, formalin, and dehydration). All samples underwent static and dynamic compression at a strain rate from 10-3 s-1 to 103 s-1. The ultimate stress, ultimate strain, elastic modulus, and strain-rate sensitivity exponent were calculated. A one-way ANOVA test was performed to determine if the preservation method showed significant differences in mechanical properties under at different strain rates. The morphology of the macroscopic and microscopic structure of bones was observed. Results: The results show that ultimate stress and ultimate strain increased as the strain rate increased, while the elastic modulus decreased. Formalin fixation and dehydration did not affect elastic modulus significantly whereas significantly increased the ultimate strain and ultimate stress. The strain-rate sensitivity exponent was the highest in the fresh group, followed by the formalin group and dehydration group. Different fracture mechanisms were observed on the fractured surface, with fresh and preserved bone tending to fracture along the oblique direction, and dried bone tending to fracture along the axial direction. Discussion: In conclusion, preservation with both formalin and dehydration showed an influence on mechanical properties. The influence of the preservation method on material properties should be fully considered in developing a numerical simulation model, especially for high strain rate simulation.

Developmental and regional dependence of macromolecular proton fraction and fractional anisotropy in fixed brain tissue.

An important advantage of imaging fixed tissue is a gain in signal-to-noise ratio and in resolution due to unlimited scan time. However, the fidelity of quantitative MRI parameters in fixed brain tissue, particularly in developmental settings, requires validation. Macromolecular proton fraction (MPF) and fractional anisotropy (FA) indices are quantitative markers of myelination and axonal integrity relevant to preclinical and clinical research. The goal of this study was to assert the correspondence of MR-derived markers of brain development MPF and FA between in vivo and fixed tissue measures. MPF and FA were compared in several white and gray matter structures of the normal mouse brain at 2, 4, and 12 weeks of age. At each developmental stage, in vivo imaging was performed, followed by paraformaldehyde fixation and a second imaging session. MPF maps were acquired from three source images (magnetization transfer weighted, proton density weighted, and T1 weighted), and FA was obtained from diffusion tensor imaging. The MPF and FA values, measured in the cortex, striatum, and major fiber tracts, were compared before and after fixation using Bland-Altman plots, regression analysis, and analysis of variance. MPF values of the fixed tissue were consistently greater than those from in vivo measurements. Importantly, this bias varied significantly with brain region and the developmental stage of the tissue. At the same time, FA values were preserved after fixation, across tissue types and developmental stages. The results of this study suggest that MPF and FA in fixed brain tissue can be used as a proxy for in vivo measurements, but additional considerations should be made to correct for the bias in MPF.

Transcriptional Profiling Identifies Prognostic Gene Signatures for Conjunctival Extranodal Marginal Zone Lymphoma.

This study characterizes the transcriptional profile and the cellular tumor microenvironment of conjunctival extranodal marginal zone lymphoma (EMZL) and identifies prognostically relevant biomarkers. Ten formalin-fixed and paraffin-embedded conjunctival EMZL and eight healthy conjunctival specimens were analyzed by Massive Analysis of cDNA Ends (MACE) RNA sequencing. The 3417 upregulated genes in conjunctival EMZL were involved in processes such as B cell proliferation and Rac protein signaling, whereas the 1188 downregulated genes contributed most significantly to oxidative phosphorylation and UV protection. The tumor microenvironment, as determined by deconvolution analysis, was mainly composed of multiple B cell subtypes which reflects the tumor's B cell lineage. However, several T cell types, including T helper 2 cells and regulatory T cells, as well as innate immune cell types, such as anti-inflammatory macrophages and plasmacytoid dendritic cells, were also strongly enriched in conjunctival EMZL. A 13-biomarker prognostic panel, including S100A8 and S100A9, classified ocular and extraocular tumor recurrence, exceeded prognostic accuracy of Ann Arbor and American Joint Committee on Cancer (AJCC) staging, and demonstrated prognostic value for patient survival in 21 different cancer types in a database of 12,332 tumor patients. These findings may lead to new options of targeted therapy and may improve prognostic prediction for conjunctival EMZL.

Micronucleus test using formalin-fixed rat glandular stomach and colon.

BACKGROUND: Genotoxicity in tissues other than hematopoietic tissues, such as the liver and gastrointestinal (GI) tract, is an important focus in the risk assessment of chemicals in humans. We previously developed a rat micronucleus test for the GI tract, which is the first contact tissue where chemicals are introduced into the body through oral exposure. Target cells were obtained from fresh tissue samples by ethylenediaminetetraacetic acid disodium salt (EDTA) treatment. As an improvement to this method, we have used formalin-fixed tissues instead of fresh tissues; this approach can be used for tissues that are sampled from other toxicological tests and that are archived for several years. This new method can be used for examining micronucleus induction retrospectively when needed. In the present study, we compared the performance of the EDTA method and the new method with formalin-fixed tissues (formalin-fixation method). RESULTS: Histological examination showed that both the EDTA and formalin-fixation methods could be used for collecting cells located in or above the proliferative zone of the GI tract tissues of rats. In addition, the collected cells were similar in shape. We conducted micronucleus tests with rat GI tract tissues by the two methods using model chemicals, which were used as positive control chemicals (a combination of diethylnitrosamine, 1,2-dimethylhydrazine dihydrochloride, and potassium bromate). The two methods showed similar results. We additionally evaluated the aging effect of tissues stored in formalin fixative. The results showed that 1 year of storage did not affect the frequency of micronucleated cells. CONCLUSION: The equivalence of the EDTA and formalin-fixation methods was confirmed, and micronucleus analysis was possible up to at least 1 year after formalin fixation of the GI tract, indicating that the formalin-fixation method is valuable for the rat GI tract micronucleus test.

Effect of 80% ethanol or 10% formalin fixation, freezing at -20 °C and staining on Myxobolus (Myxosporea) spores to be deposited in parasitological collections.

The preparation of myxosporeans for the description of myxospores and their preservation as type material in parasitological collections show great variations. Most frequently, formalin and ethanol are used for fixation and Giemsa solution for staining spores. In this work, authors studied the effect of 80% ethanol and 10% formalin fixation, freezing at -20 °C and staining on the size and transparency of two Myxobolus species of cyprinid fishes, M. bramae and M. bliccae spore, and recommended a new method for the deposition of type material to parasitological collections in museums. The studies have commended that fresh spores from mature plasmodia are the best material for measuring the size and studying the inner structures, the number of polar tubules in polar capsules and the morphological characters of the intercapsular appendix. The obtained quantitative data suggest that cryo- and chemical preservation do not have a notable negative effect on spores compared to fresh samples but they decrease the transparency of spores. Staining the spores with Ziehl-Neelsen has proved to be a useful method for studying the fine structure without size reduction, while Giemsa staining induced a shrinkage of spores so it seems to be not ideal for description of a new species. When treating spores of Myxobolus spp. with Lugol's solution, iodinophilous vacuoles in the sporoplasm were not recognised but visualisation of the coils of polar tubules was enhanced. As a type material for newly described species, authors suggest phototypes and spores fixed in 80% ethanol to be deposited into collections, as this preservation method is suitable for subsequent research, such as re-measurements and molecular analysis.

Quantification of Canine Apocrine Gland Anal Sac Adenocarcinoma (AGASACA) Tumor Specimen Shrinkage after Formalin Fixation.

The aim was to prospectively measure the shrinkage of primary apocrine gland anal sac adenocarcinoma (AGASACA) tumors after 24 and 48 h of formalin fixation. Dogs that were diagnosed with AGASACA pre-operatively by aspiration cytology were prospectively enrolled in the study. Tumor extirpation was performed in a closed technique. The tumor and associated tissues were examined on the back table away from the patient and the widest dimension of the tumor was measured using a sterile ruler (Medline®; Northfield, IL, USA). This measurement was recorded in mm (t0). The tissue was placed in 10% buffered formalin and stored at room temperature. Two further measurements were taken after 24 (t24) and 48 (t48) hours of formalin fixation. Once the 48 h measurement was taken, the tissue was submitted for histopathology. The percentage of shrinkage between time points was calculated by using the following equation: (1 - [time b/time a]) × 100. Overall, 23 dogs with 23 tumors were enrolled. The mean percentage of shrinkage after 24 and 48 h of formalin fixation was 4.8% and 7.2%, respectively. The median diameter of the tumors reduced by 1 mm over 48 h and was not significantly different at any time point. These data will aid clinicians in interpreting measurements of AGASACA tumors following formalin fixation and shows that minimal change in tumor size is expected following 48 h.

Immunocytochemistry of effusion fluids: Introduction to SCIP approach.

Due to the remarkably wide morphologic spectrum of reactive mesothelial cells, some of the effusion fluids may be difficult to interpret with objective certainty by cytomorphology alone. Cytomorphology of well to moderately differentiated adenocarcinomas (responsible for the bulk of malignant effusions) may overlap with floridly reactive mesothelial cells. Even mesotheliomas including diffuse malignant epithelioid mesothelioma, are usually cytomorphologically bland without unequivocal features of malignancy. The intensity of challenge depends on the interpreter's training or experience level, institutional demographics of patients (such as type of prevalent diseases, predominant sex and age group), technical support, and quality of cytopreparatory processing. In general immunocytochemistry is valuable adjunct to facilitate objective interpretation with or without other ancillary techniques as indicated. An increasing number of immunomarkers is further refining the contribution of immunohistochemistry to this field. However, application of immunohistochemistry to effusion fluids is relatively challenging because of many variables. Multiple factors such as delay after specimen collection, specimen processing related factors including fixation and storage; ambient conditions under which paraffin blocks are archived (for retrospective testing); antigen retrieval method; duration of antigen retrieval step; antibody clone and dilution; and antibody application time are identical to application of immunohistochemistry in other areas. The significant challenge related to the potential compromization of the immunoreactivity pattern due to exposure to non-formalin fixatives / reagents is also applicable to effusion fluid specimens. The immunoreactivity results would be compared and corelated with cumulative metadata based on the reported studies performed and validated on formalin-fixed paraffin-embedded tissue sections. Deviating from such protocols may lead to suboptimal results, which is not uncommon in clinical practice with potential compromization of patient care and related liability. Because of this, it is critical to perform immunocytochemistry on formalin-fixed cell-block sections only. In addition, unless the interpretation criteria for immunohistochemical evaluation of effusion fluids are not modified specifically, it may not be productive in resolving some challenging cases. However, this aspect is not well elaborated in the literature. A basic and critical challenge is finding and locating the cells of interest in cell-block sections of effusion fluids. A unique approach is to choose a fundamental immunopanel which highlight the mesothelial and inflammatory cells in reactive effusion fluids to create the basic map. This allows detection of a 'second-foreign' population which can be immunocharacterized further with the help of subtractive coordinate immunoreactivity pattern (SCIP) approach elaborated here.

Comparative study of two reprocessing methods for formalin fixed paraffin embedded tissue.

Formalin fixed paraffin embedded tissue occasionally requires reprocessing if the histologic quality of a section is inadequate for clinical diagnosis. The Pat Dry (PD) and the Serial Xylene (SX) methods are two techniques described in the literature to reprocess under-fixed and/or under-processed tissue samples. To date, no study has compared the effects of these methods on the histologic quality of tissue sections, cost, and turnaround times. In the present study, these two methods were evaluated on 129 tissue samples taken from 40 submitted clinical specimens, 3 blocks per sampled location. Before processing, sample Group 1 (Control) was cut at routine 3-5 mm thickness. Sample Groups 2 and 3 were cut at 10 mm to ensure the thicker tissues would be poorly processed. Histotechnicians performed a subjective evaluation of all the samples at the time of embedding and microtomy. Hematoxylin and eosin stained sections from all samples were scored for histologic quality by two pathology residents. Thicker samples (Groups 2 and 3) were then reprocessed using either PD or SX methods, re-sectioned, stained, and then re-scored by the pathology residents. The two reprocessing methods equally improved quality scores and reduced the fraction of slides that were rejected. The PD method average preparation time was 66 minutes as compared to 250 minutes for the SX method. The PD method was easier to perform than the SX method, required less reagent, and was less susceptible to reagent spillage than the SX method.