Cell block cellularity: A comparison of two fixatives and their impact on cellularity.

BACKGROUND: Ancillary testing including immunohistochemistry and molecular diagnostics has become an increasingly important component for the evaluation of cytologic specimens. Ancillary testing is important not only for diagnosis but also for predictive and prognostic evaluation. While a number of substrates are appropriate for ancillary testing, cell block specimens are commonly utilized and the success of ancillary testing depends on cell-block cellularity. METHODS: Forty-six pairs of cases each fixed in both formalin and CytoLyt were each analyzed by two evaluators for overall cellularity. Linear regression was used to assess inter-rater reliability of cell counts for each method. Cellularity scores for each case were obtained by averaging the scores for each rater and cellularity was compared between the methods. RESULTS: Inter-rater agreement was very good for both methods. The coefficient of determination was 1.0 and 0.99 for the CytoLyt and formalin methods respectively. Cell blocks using the CytoLyt method have lower levels of cellularity than cell blocks performed by the formalin method. CONCLUSIONS: Cell blocks prepared using a formalin fixative yield significantly greater cellularity than those produced by the CytoLyt method. Formalin fixation appears to optimize cellularity of cell blocks useful for ancillary testing.

Bee Honey as a Safer Alternative for Routine Formalin Fixation.

Background Formaldehyde (10% buffered formalin) is still in use as the gold standard fixative in the field of biology however, as reported by Occupational Safety and Health Administration (OSHA) the use of formalin causes health hazards due to its toxicity. Hence, we considered to substitute formalin with natural Bee-Honey to achieve a formalin free laboratory for preservation of the biological specimens. Objective To assess the efficacy of honey as a fixative agent for the preservation of the tissue specimens and to study their cellular and structural characteristics by using routine stains, special stains and Immunohistochemistry (IHC) and compare its effectiveness with the currently, universally accepted formalin fixation. Method Our study contained sample size of 10 tissue specimens. All samples were fixed in two different solutions one in honey and other in conventionally used formalin solution for 24 hrs in room temperature and then were routinely processed, sectioned and stained using routine, special stains and with immuno-histochemical markers. The slides were viewed by two independent examiners and the entire procedure was blind folded. Result We obtained good comparable results with bee honey for Hematoxylin and Eosin, special stains including immunohistochemistry when compared to formalin fixed tissues. Conclusion Based on the observations of this study, it can be suggested that natural bee honey could be a safer alternative to formalin as a fixative, considering the health hazards of formalin.

Fixation strategies for retinal immunohistochemistry.

Immunohistochemical and ex vivo anatomical studies have provided many glimpses of the variety, distribution, and signaling components of vertebrate retinal neurons. The beauty of numerous images published to date, and the qualitative and quantitative information they provide, indicate that these approaches are fundamentally useful. However, obtaining these images entailed tissue handling and exposure to chemical solutions that differ from normal extracellular fluid in composition, temperature, and osmolarity. Because the differences are large enough to alter intercellular and intracellular signaling in neurons, and because retinae are susceptible to crush, shear, and fray, it is natural to wonder if immunohistochemical and anatomical methods disturb or damage the cells they are designed to examine. Tissue fixation is typically incorporated to guard against this damage and is therefore critically important to the quality and significance of the harvested data. Here, we describe mechanisms of fixation; advantages and disadvantages of using formaldehyde and glutaraldehyde as fixatives during immunohistochemistry; and modifications of widely used protocols that have recently been found to improve cell shape preservation and immunostaining patterns, especially in proximal retinal neurons.

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. CONCLUSIONS: In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation.

Effects of sample site and size, skin tension lines, surgeon, and formalin fixation on shrinkage of skin samples excised from canine cadavers.

OBJECTIVE: To assess the effects of sample size and location, skin tension lines, surgeon, and formalin fixation on the extent of shrinkage that occurs in excised canine skin samples. ANIMALS: Cadavers of 4 adult purpose-bred mixed-breed hound dogs with grossly normal skin. PROCEDURES: 54 circular areas of skin (2-, 4-, and 6-cm-diameter samples from each of 9 body regions on each side) were excised by 1 of 2 surgeons from each cadaver. The diameter of each sample was measured in 4 orientations (parallel to previously reported tension lines, perpendicular to tension lines, in a dorsoventral orientation, and in a craniocaudal [or rostrocaudal] orientation) at 3 time points (before and immediately after excision and after 24 hours of formalin fixation). RESULTS: 216 samples were measured in all 4 orientations at all 3 time points. For all samples, mean ± SE decrease in diameter after fixation, compared with pre-excision findings, was 6.2 ± 0.7 mm. No significant correlations were found between percentage of skin shrinkage and surgeon, body side or region, or measurement orientation in relation to skin tension lines. The mean sample diameter immediately after excision differed significantly from that before excision (mean diameter decrease, 5.5 ± 0.7 mm). Overall, sample diameter immediately after excision and after formalin fixation did not differ. CONCLUSIONS AND CLINICAL RELEVANCE: The extent of shrinkage of skin samples from hound cadavers that occurred immediately after excision was notable. A better understanding of the effectors of excised skin sample shrinkage is needed, especially when histopathologic findings provide guidelines for surgical margins.

Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials.

High dimensional flow cytometry is best served by centralized facilities. However, the difficulties around sample processing, storage and shipment make large scale international studies impractical. We therefore sought to identify optimized fixation procedures which fully leverage the analytical capability of high dimensional flow cytometry without the need for complex cell processing or a sustained cold chain. Whole blood staining procedure was employed to investigate the applicability of fixatives including Cyto-Chex® Blood Collection tube (Streck), Transfix® (Cytomark), 1% and 4% paraformaldehyde to centralized analysis of field trial samples. Samples were subjected to environmental conditions which mimic field studies, without refrigerated shipment and analyzed across 10 days, based on cell count and marker expression. This study showed that Cyto-Chex® demonstrated the least variability in absolute cell count relative to samples analyzed directly from donors in the absence of fixation. Transfix® was better at preserving the marker expression among all fixatives. However, Transfix® caused marked increased cell membrane permeabilization and was detrimental to intracellular marker identification. Paraformaldehyde fixation, at either 1% or 4% concentrations, was unfavorable for cell preservation under the conditions tested and thus not recommended. Using these data, we have created an online interactive tool which enables researchers to evaluate the impact of different fixatives on their panel of interest. In this study, we have identified Cyto-Chex® as the optimal cellular preservative for high dimensional flow cytometry in large scale studies for shipped whole blood samples, even in the absence of a sustained cold chain.

Nucleic acid quantity and quality from paraffin blocks: defining optimal fixation, processing and DNA/RNA extraction techniques.

Although the extraction and analysis of nucleic acids from formalin-fixed paraffin-embedded tissues is a routine and growing part of pathology practice, no generally accepted recommendations exist to guide laboratories in their selection of tissue fixation, processing and DNA/RNA extraction techniques. The aim of this study was to determine how fixation method and length, paraffin embedding, processing conditions and nucleic acid extraction methods affect quality and quantity of DNA and RNA, and their performance in downstream applications. Nine tissue samples were subjected to freezing, fixation in formalin for <24 h and 7 days followed by conventional processing, and fixation in molecular fixative for <24 h and 7 days followed by rapid processing. DNA and RNA were isolated using in-house extraction and commercial kits, and assessed by PCR reactions for amplicons with varying sizes ranging from 268 to 1327 bp and one-step RT-PCR for 621 bp and 816 bp amplicons of housekeeping genes. Molecular fixative (MF) appeared to perform well under nearly all circumstances (extraction methods, fixation lengths and longer amplicons), often performing as well as frozen samples. Formalin fixation generally performed well only for shorter length amplicons and short fixation (<24 h). WaxFree kit showed consistently higher success rates for DNA and poorer rates for RNA. RecoverAll kit generally performed suboptimally in combination with prolonged formalin fixation. In conclusion, the Molecular Fixative regardless of fixation length, and the rapid tissue processing system were able to preserve large DNA and RNA fragments in paraffin blocks, making these techniques preferable for use in downstream molecular diagnostic assays.

Proposals for uniform collection of biospecimens from neoadjuvant breast cancer clinical trials: timing and specimen types.

In this Personal View, we outline proposals for uniform collection of biospecimens obtained in neoadjuvant breast cancer trials undertaken by the Breast International Group (BIG) and the National Cancer Institute-sponsored North American Breast Cancer Group (NABCG). These proposals aim to standardise collection of high-quality specimens, with respect to both type and timing, to enhance and allow integration of results obtained from neoadjuvant trials done by several groups. They should be considered in parallel with recommendations for tissue-specimen collection and handling previously developed by BIG and NABCG. We propose that tumour tissue (formalin-fixed, paraffin-embedded and samples dedicated for molecular studies) should be taken at baseline, 1-3 weeks after the start of treatment, and at definitive surgery, with clear prioritisation in the study protocol of number, order, and preservation of samples to be gathered. This step should be accompanied by blood collection (plasma, serum, and whole blood) whenever possible. We advocate strongly a move towards one diagnostic and research biopsy procedure in all women with breast cancers potentially suitable for neoadjuvant treatment. If possible, patients should be referred at the outset to specialised centres to give them the opportunity to participate in neoadjuvant clinical trials, thereby avoiding several biopsy procedures.

SpermBlue: a new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis.

Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue fixative and SpermBlue stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue fixative or by adding 1 ml SpermBlue fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue stain or adding four drops of SpermBlue stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue staining process. SpermBlue stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 x or 1000 x magnification for most of the species studied.

A system for quantitative evaluation of fixatives for light microscopy using paraffin sections of kidney and brain.

A numerical scoring system is presented for evaluating structural fixation of certain mammalian tissues for light microscopy. Small pieces of rat's kidney and brain, tissues for which artifacts of fixation are well documented, were fixed in various fluids. Random code numbers hid their identities, and paraffin sections were stained to show nuclear chromatin, cytoplasm and extracellular materials. Two sections of each specimen were examined and awarded scores according to described schemes, for microanatomical and cytological fixation. The assessment was confined to preservation of structure; chemical changes were not taken into account. When the code was broken, the scores for both sections of each specimen from each fixative were added. The scores obtained (24 best to eight worst) are generally comparable to the grades (I-V) given for traditional fixatives by JR Baker. The criteria for quality of fixation were defined more explicitly than those for Baker's grading method, which used mouse testis as the test object. Assessments are presented for several traditional fixatives and for four zinc-formaldehyde mixtures. The scoring system should be useful for evaluating newly developed fixatives for animal tissues for light microscopic examination of paraffin sections. In an evaluation of four traditional fixatives and four zinc-formaldehyde mixtures, variation among three different observers was only +/-1 point on either side of the median score for microanatomical or cytological preservation by any of the eight fixatives. This approach has certain limitations, notably that the criteria for cytological fixation do not include the preservation of chromosomes or specific cytoplasmic organelles.

[Fixation, staining and preparation of permanent mounts of helminths]. / Helmintlerde Tespit, Boyama ve Kalici Preparat Yapimi.

The techniques for the collection, relaxation, preservation and staining of helminths are very important for parasitologists. Parasites should be collected alive and fixed directly in the living condition. These procedures insure proper preservation of internal and external details of parasites. There are various methods for relaxing and preserving the normal morphology of helminths. These methods are absolutely essential for permanent preservation of the specimens. Staining and mounting techniques vary depending upon size of specimens, species, and stage of development of the organisms. In this review, the preparation of permanent mounts, relaxation, fixation and staining methods of helminths has been discussed.

Comparison of preparation methods for the evaluation of nerve axons.

We have conducted a study to compare various preparation methods, including a certain number of new methods, to find which ones are best suited to the morphological evaluation of nerve fibers in the pyramidal tract of the human medulla oblongata. Our main concern was to find fixation and staining methods that would minimize errors, especially regarding the tissue shrinkage ratio and the ease of staining. From the two fixation methods we examined, the most satisfactory was the secondary chromic acid fixation (which gave the best overall results when followed by nitrocellulose embedding), as it gives the lowest shrinkage ratio with the narrowest range (10 +/- 0%). Among the ten staining methods we tested, we found that the most suitable for morphological evaluation were the discriminative staining methods (Luxol fast blue-Periodic acid-Schiff-Hematoxylin stain, Masson-Goldner-Goto method and modified Hematoxylin-Eosin stain) and the silver impregnation methods (Luxol fast blue-Silver impregnation and Luxol fast blue-Silver impregnation-Periodic acid-Schiff-Hematoxylin).

Neuropathology of brain death in the modern transplant era.

BACKGROUND: Autopsy studies in patients who have been declared brain dead are rare. Total brain necrosis ("respirator brain") has been a common finding in the distant past. The time to brain fixation has been shortened as a result of timely organ transplant protocols, therefore the neuropathologic findings may be different than previously described. METHODS: We reviewed macroscopic and microscopic brain pathology for ischemic neuronal damage in 41 patients who fulfilled the clinical criteria of brain death. Hematoxylin and eosin stained brain tissue slides were retrieved and available wet tissue was additionally stained to complete a series of samples of the hemispheres, brainstem, and cerebellum for each patient. Neuronal ischemic change was semiquantitatively graded for severity (mild 0 to 5%, moderate >5 to 75%, and severe >75%). RESULTS: After the clinical diagnosis of brain death and terminal cardiac arrest, 12 brains were fixated in less than 12 hours and 29 brains were fixated between 12 and 36 hours. The frontal lobe, temporal lobe, parietal lobe, occipital lobe, and basal ganglia showed moderate to severe ischemic change in 53 to 68% of the cases. Moderate to severe neuronal ischemic change was found in the thalamus in 34%, midbrain in 37%, pons in 41%, medulla in 40%, and cerebellum in 52% of the cases. CONCLUSIONS: No distinctive neuropathologic features were apparent in our series of patients with brain death. Neuronal ischemic changes were frequently profound, but mild changes were present in a third of the examined hemispheres and in half of the brainstems. Respirator brain with extensive ischemic neuronal loss and tissue fragmentation was not observed. Neuropathologic examination is therefore not diagnostic of brain death.

Evaluation of the value of frozen tissue section used as "gold standard" for immunohistochemistry.

To examine the use of acetone- or ethanol-fixed frozen tissue sections as the "gold standard" for immunohistochemical analysis, we evaluated frozen sections with various conditions of fixation and antigen retrieval (AR). Fresh human tissues were frozen in OCT. An adjacent tissue block was fixed in 10% neutral buffered formalin (NBF) and paraffin embedded (FFPE). Frozen sections were fixed by 6 protocols: acetone, ethanol, NBF (2 durations), and NBF + calcium chloride (2 durations). AR was used for all NBF-fixed sections. More than half of the antibodies (16/26 [62%]) showed immunohistochemical results indistinguishable between acetone- and NBF-fixed sections; 8 (31%) showed better immunohistochemical signals following NBF and AR; 2 gave better immunohistochemical results for acetone-fixed sections. Most cytoplasmic proteins (10/13) showed comparable immunohistochemical signals between acetone- and NBF-fixed sections. For nuclear proteins, NBF-fixed sections gave better immunohistochemical signals than did acetone-fixed sections. In most cases, NBF yielded stronger signals with less background and better morphology. The data do not support the use of acetone-fixed frozen tissue sections as the gold standard for immunohistochemical analysis. In evaluating new antibodies, a combination of acetone- and NBF-fixed frozen sections should be used, although in practice, FFPE tissue sections may serve as the standard for most antigens for immunohistochemical analysis.

Effects of fixative and fixation protocols on assessment of Her-2/neu oncogene amplification status by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is used to determine amplification status of the Her-2/neu gene in specimens of newly diagnosed breast carcinoma. The Vysis kit for FISH analysis stipulates that the tissue be formalin-fixed and paraffin-embedded. Concerns regarding carcinogenicity of formalin and environmental effects of formalin waste have led to the development of formalin replacement products. An increasing number of breast biopsy specimens are being fixed in these substitutes. We tested 6 non-formalin-based fixatives to determine their impact on FISH testing for Her-2/neu gene amplification status by comparison with formalin-fixed control specimens from the same neoplasm. Specimens fixed in Pen-Fix, Prefer, Histochoice, UniFix, and GTF were associated with absent or technically compromised staining in at least one of the 3 neoplasms tested for each fixative when compared to the formalin-fixed control. O-Fix did not seem to compromise staining quality in 3 paired specimens tested.

Recommendations for improved standardization of immunohistochemistry.

Immunohistochemistry (IHC) continues to suffer from variable consistency, poor reproducibility, quality assurance disparities, and the lack of standardization resulting in poor concordance, validation, and verification. This document lists the recommendations made by the Ad-Hoc Committee on Immunohistochemistry Standardization to address these deficiencies. Contributing factors were established to be underfixation and irregular fixation, use of nonformalin fixatives and ancillary fixation procedures divested from a deep and full understanding of the IHC assay parameters, minimal or absent IHC assay optimization and validation procedures, and lack of a standard system of interpretation and reporting. Definitions and detailed guidelines pertaining to these areas are provided.

The fixation of living skin equivalents.

Histologic evaluations and immunohistochemical characterizations are important in studies of artificial organs such as skin equivalents. However, tissue compact organization is not easy to obtain when the artificial organ is constructed in vitro. Thus, appropriate fixation methods must be selected according to the compactness of the artificial organ and tissue engineering methodologies. The effects of several fixatives-Carnoy, Bouin's solution, formalin, paraformaldehyde, and paraformaldehyde-glutaraldehyde-were examined to select the best fixation method for preserving the structural and molecular markers of skin equivalents. Formalin-based fixatives ware found to be relatively free of the histologic problems (e.g., tissue shrinkage, poor structural preservation, weak stainability, and nonspecific immunolocalization) presented by the soft tissue fixatives (i.e., Carnoy or Bouin's solution). Unfortunately, the standard concentration of formalin induced detachment of epidermis from dermis, but this was prevented by reducing the concentration of the fixative. These findings suggest that fixation procedures should be selected for particular tissue and specific goals; in particular, they show that the paraformaldehyde-glutaraldehyde combination performed best in terms of preserving the histologic features of skin equivalents.

Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system.

A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.