Prolonged Formalin Fixation Research Articles

Factors impacting tumor displacement artifact and pseudo-lymphovascular invasion in testicular germ cell tumors.

Testicular germ cell tumors are susceptible to tumor displacement artifact (TDA), which produces pseudo-lymphovascular invasion (LVI) and confounds the identification of true LVI. Our study aimed to evaluate tumor displacement artifact and pseudo-LVI in testicular germ cell tumors and determine if prolonged fixation improves histological quality. A retrospective search identified 121 orchiectomies with slides that were reviewed to assess TDA and pseudo-LVI. Seminoma had more TDA (68% vs 45%, P= .01) and pseudo-LVI (53% vs 19%, P< .001) than mixed germ cell tumor. Seminoma and mixed germ cell tumor with TDA and pseudo-LVI were larger than those without. Mixed germ cell tumor with ≥50% composition of seminoma had a higher rate of TDA (89% vs 38%, P= .008) and pseudo-LVI (44% vs 15%, P= .06) than those with less. TDA was reduced in seminoma with >1 night fixation compared to no fixation (50% vs 82%, P= .046), with a similar trend in mixed germ cell tumor (31% vs 60%, P= .15). A trend in reduction of pseudo-LVI was seen with >1 night fixation compared to no fixation in seminoma (64% vs 39%, P= .12). Seminomas and larger germ cell tumors were more prone to TDA and pseudo-LVI. Prolonged formalin fixation improved histological quality intesticular germ cell tumors. Based on these data, we recommend fixation for at least 2 nights before sectioning orchiectomy specimens, particularly for larger tumors.

Retrospective Analyses of Porcine Circovirus Type 3 (PCV-3) in Switzerland.

Porcine circovirus 3 (PCV-3) has emerged as a significant pathogen affecting global swine populations, yet its epidemiology and clinical implications remain incompletely understood. This retrospective study aimed to investigate the prevalence and histopathological features of PCV-3 infection in pigs from Switzerland, focusing on archival cases of suckling and weaner piglets presenting with suggestive lesions. An in-house qPCR assay was developed for detecting PCV-3 in frozen and formalin-fixed paraffin-embedded tissues, enhancing the national diagnostic capabilities. Histopathological reassessment identified PCV-3 systemic disease (PCV-3-SD) compatible lesions in 19 (6%) of archival cases, with 47% testing positive by qPCR across various organs. Notably, vascular lesions predominated, particularly in mesenteric arteries, heart, and kidneys. The study confirms the presence of PCV-3 in Switzerland since at least 2020, marking the first documented cases within the Swiss swine population. Despite challenges in in situ hybridization validation due to prolonged formalin fixation, the findings indicate viral systemic dissemination. These results contribute to the understanding of PCV-3 epidemiology in Swiss pigs, emphasizing the need for continued surveillance and further research on its clinical implications and interaction with host factors. Our study underscores the utility and limitations of molecular techniques in confirming PCV-3 infections.

The impact of formalin fixation in the elemental content of tissues: Parametrization up to 48 h

Formalin fixation is a crucial step in the preparation of tissue samples for anatomic pathology studies, however, research showed that prolonged formalin fixation, can alter the elemental composition of tissues with implications for pathology and biomedical research. In this study we aim to assess and parametrize the influence of short-term formalin fixation in tissues.Six sets of human colon tissue samples were exposed to different formalin fixation times, up until 48 h. The elemental content throughout time was compared to the elemental content of the snap-frozen sample of the same tissue that was not exposed to formalin using Energy Dispersive X-ray Fluorescence.Results showed a clear decrease of Cl and K concentration in the tissues solution reaching a plateau between 1 h and 3 h of fixation. Also, there is an uptake of P in the tissue, likely due to the buffered formalin solution, within the first 15 min of fixation. This behaviour was concomitant with an increase of Na determined using Particle Induced gamma-ray emission analysis (PIGE) and Elastic Backscattering Spectroscopy (EBS). The dynamic changes determined in the studied elements demonstrate that a better understanding of the fixation-related mechanisms is in demand as well as optimization of the fixation protocols implemented in anatomic pathology laboratories.

Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

Quality assessment of histopathological stainings on prolonged formalin fixed thrombus tissues retrieved by mechanical thrombectomy.

Formalin-fixed retrieved clots from mechanical thrombectomy (MT) are now routinely studied using both conventional histopathologic techniques and immunohistochemistry (IHC). However, the effects of prolonged formalin fixation on the histological results of clot analysis remain unknown. The objective of this study was to investigate the effects of prolonged formalin fixation on quality of histopathologic stainings of thrombus tissues retrieved by MT. As part of the multicenter EXCELLENT registry, a total of 80 clots extracted by MT from acute ischemic stroke patients were randomly selected from the tissue database and assigned into four groups according to 10% neutral buffered formalin (NBF) fixation duration (1-30, 30-60, 60-90, and 90+ days, up to 2 years). Samples underwent processing and sectioning. Two serial sections for each case were stained with hematoxylin and eosin (H&E), Martius Scarlet Blue (MSB), and IHC for CD42b (platelet marker). An expert pathologist, who was blinded to tissue fixation duration and patient clinical data, assessed the quality of each stain including stainability, sensitivity, specificity, and consistency of stainings. No significant issues were encountered during tissue processing and sectioning. On H&E stain, 97.5% (78/80) of slides showed good-quality staining, demonstrating clear histological properties of the thrombus tissue as red blood cells (RBC) stained in red, fibrin/platelet stained in pink, and nuclei stained in blue with intranuclear detail. The same histological features were also successfully demonstrated on MSB for all 80 samples. One of the 80 samples (1.2%) showed that RBC lost stainability on H&E due to tissue autolysis. Clear positive signal of platelet staining was expressed in 98.8% of the samples (79/80) with minimal background staining on IHC. There was no significant difference in staining quality across different formalin fixation groups. A good quality of histopathological staining is achievable for the thrombus tissue fixed in 10% neutral buffered formalin for up to 2 years. The findings are limited to the thrombus tissue retrieved by MT and specific fixation and staining protocols used in the study. To apply these results to other tissue or experimental setups, further studies and validations would be necessary. This study was conducted as part of the EXCELLENT study: www.clinicaltrials.gov, unique identifier: NCT03685578.

CytogeneticsTissue Fixation Significantly Impacts MLH1 Promoter Methylation Analysis: A Quality Improvement Study

Abstract MLH1 promoter methylation (MPM) analysis is an invaluable tool for distinguishing sporadic from inherited colorectal and endometrial cancers. At our institutional clinical genomics lab, we observed an unusually high failure rate of MPM testing in endometrial (EM) cancers. This triggered a quality improvement (QI) project to identify the underlying cause and improve the success rate of our MPM assay. We retrospectively identified all MPM cases (EM and non-endometrial (NEM) cancers) between January 2019 and October 2022 through querying our departmental database. The failure rates of these two groups were juxtaposed with the following variables: DNA concentration, performing technologists, operating surgeons, and specimen collection day (Monday through Saturday). A subsequent fishbone analysis was performed to evaluate the impact of specimen handling, fixation, and embedding procedure/timeline in the gross room and histology labs. We identified one-hundred thirty-one EM cases and eighty-two NEM cases that underwent MPM testing. Thirty-one EM cases (24%) and seven NEM cases (9%) failed, re-demonstrating the higher failure rate (X2=7.9, P-value=0.005) of EM cases. There was no significant difference in DNA concentration, performing technologists, and operating surgeons when compared to passed cases, however, a highly significant difference was seen concerning day of specimen collection (X2=53, P-value&amp;lt;0.0001). Most notably, all failed EM (n=31) and NEM cases (n=7) were collected on either Thursday or Friday (failure rate of 26% and 59%, respectively, 44% jointly). Our fishbone analysis revealed that specimens accessioned Thursday afternoon and Friday were fixed throughout the weekend before embedding on Monday, resulting in prolonged fixation (one extra day). This is consistent with previous studies’ conclusions showing DNA quality (but not quantity) significantly worsened with longer formalin fixation. Upon presenting these data to lab personnel, workflow changes were implemented. Cancer tissue that would have originally fixed over the weekend was instead processed on Saturday starting December 2022. This QI project revealed that prolonged formalin fixation of specimens over the weekend dramatically reduces the success rate of MPM analysis of EM cancer cases. Subsequently, we expanded our investigation to include other molecular assays at our institution that have returned similar results. Since the implementation of the revised workflow in December 2022, a preliminary prospective analysis showed the MLH1 failure rate of EM cancer tissues collected on Thursday and Friday has decreased from 44% to 11% in the past three months (December 2022 – February 2023). The current findings have important implications for the use of FFPE specimens in genetic analysis and highlight the need for careful consideration of fixation workflows, especially over the weekend, in sample processing protocols.

The effect of prolonged formalin fixation on the expression of proteins in human brain tissues

Formalin-fixed, paraffin-embedded (FFPE) tissues have been widely used in researches. Proteins and nucleic acids in prolonged FFPE tissues display different degrees of degradation. We investigated the effect of prolonged formalin fixation on protein expression in human brain tissues. Twenty-eight middle prefrontal front cortex tissue blocks from human brains prefixed in formalin were obtained from a brain bank. The tissue blocks were divided into two groups, the control group and the prolonged fixation group. Quantitative immunocytochemistry was used to analyse the biological markers of Fox-3, Rbfox3 (NeuN), glial fibrillary acidic protein (GFAP), ionized calcium binding adapter molecule-1 (IBA-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nissl staining showed that positive signaling of Nissl body was significantly decreased by 16.6% in the prolonged fixation group. In addition, the staining intensity of Nissl body was negatively correlated with fixation time. The level of NeuN immunoreactivity (ir) was significantly reduced by 19.31% in the prolonged fixation group. Moreover, there was a significant negative correlation between NeuN-ir and fixation time. There were no significant changes in GFAP-ir, IBA-1-ir and GAPDH-ir between control group and the prolonged fixation group. Prolonged formalin-fixed tissues showed time- and molecule-dependent protein changes, which may be potential confounders in the clinic and researches. Our study suggested short formalin fixation time is recommended when using PPFE brain tissues.

The effect of prolonged formalin fixation on the staining characteristics of archival human brain tissue.

Neurodegenerative disorders include wide range of conditions, which affect millions of people worldwide. Unfortunately, they are incurable and irreversibly progressive. Immunohistochemical staining of paraffin-fixed tissues for both diagnostic and research purposes are widely used. However, large amount of brain tissues are fixed but little is known about whether they are suitable for retrospective studies. The study aimed at investigating the effects of prolonged formalin fixation time on immunohistochemical expression of some common neurodegenerative markers in archival brain specimens. Twenty brain specimens were obtained from hu- man cadavers in the Anatomy Department of King Abdulaziz University that were prefixed in 10% formalin. They were divided into two equal groups according to time of fixation: group 1 - less than 1 year, group 2 - up to 20 years. Histological examination of white and grey matter was done using haematoxylin and eosin, luxol fast blue (LFB) for myelin staining, Con- go red for amyloid plaques, CD 68 for microglial cells, tenascin-C (large ex- tracellular matrix glycoprotein) and caspase 3 antibody for apoptotic cells. For both groups, corpus callosum sections displayed myelination with LFB staining. The distribution of CD 68 positive microglial cells was evi- dent in frontal and temporal grey matter, but not in corpus callosum sections. Strongly positive masses were seen in Congo red-stained frontal and temporal sections. Anti-caspase 3 immunostaining revealed positively stained neurons. Histological and immunohistochemical techniques yielded repro- ducible staining results when applied to human brain tissue stored in formalin for long periods; so they can be used in well preserved biobank material which are the most targeting research areas in neuropathology.

Factors affecting KRAS mutation detection in colorectal cancer tissue

BackgroundWith the recent development of molecular tests for various biomarkers, it has become even more important to prepare adequate tissue samples. However, little is known about how the effect of cold ischemia time or formalin fixation time can affect KRAS mutation detection in colorectal cancer. MethodsThis study included the results of KRAS mutation tests for colorectal cancer in 401 specimens. We investigated clinicopathologic factors that may affect DNA quality of formalin-fixed, paraffin-embedded (FFPE) tissue including specimen type, cold ischemia time, and formalin fixation time and assessed the detection rate of the KRAS mutation in samples with varying DNA quality. ResultsSample DNA quality for KRAS mutation test was better in biopsy specimens, which showed markedly shorter cold ischemia time and shorter formalin fixation time compared to resection specimens. A cold ischemia time of one hour or less was associated with better sample DNA quality. But the formalin fixation time was not a significant factor when it fell within the range performed in routine pathology diagnosis. When prolonged formalin fixation was tested, we confirmed that the specimen DNA quality gradually got worse from one month to three months. ConclusionsThe biopsy specimens showed better sample DNA quality for KRAS mutation test compared to resection specimens. In a routine diagnostic pathology setting, the cold ischemia time was an important factor affecting DNA quality and the formalin fixation had a wide time range for optimal DNA quality.

The effects of prolonged formalin fixation on immunohistochemical staining of PCNA and Dcx proteins in the mouse hippocampus

We have performed a quantitative analysis of temporal pattern of immunohistochemical staining to PCNA and Dcx in brain tissue under the influence of 10% neutral buffered formalin (fixation for 3.5 h–7 weeks). After 7 days of fixation we found a decrease in Dcx and, to a lesser extent, PCNA immunoreactivity. Later, no further decline in immunoreactivity occurred. Our data may be useful for optimization of neurogenesis assessment protocols in mammalian and human brains.

Mitochondrial DNA from archived tissue samples kept in formalin for forensic odontology studies

BackgroundSamples used for DNA isolation to be used for forensic odontology studies are often limited. The possibility to use tissue samples stored in formalin for a prolonged period, which contains nucleic acids of questionable quality, opens exciting possibilities for genetic and molecular biology studies useful in speciality of forensic odontology. AimThe present study defines substantial modification of existing protocols for total genomic isolation including mitochondrial DNA and proves the utility of such obtained mitochondrial DNA in microsatellite analyses. Methods50 dental tissue samples which were kept in neutral buffered formalin liquid bottles were taken for DNA isolation and subsequent analysis. For the isolation of total genomic DNA from tissue samples, a new protocol with substantial modifications from routine ones was adopted by us. Total genomic DNA from matched blood samples were extracted using standard phenol-chloroform extraction method. ResultsPolymerase Chain Reaction and Sequencing of such extracted DNA samples for mitochondrial D loop region were successful and the results were comparable with DNA extracted from normal sources of samples. ConclusionThe present study reports for the first time that nucleic acids extracted from human dental tissue samples under prolonged formalin fixation times can be used for forensic odontology studies using the described methodology.

Effects of Fixation and Storage of Human Tissue Samples on Nucleic Acid Preservation

BackgroundBecause of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice.MethodsTo evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), β-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for β-actin (97 bp) were performed.ResultsAll formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and β-actin by PCR. hGH was successfully detected in all MFPE samples, but in only 46.7% of the FFPE samples. Prolonged formalin fixation resulted in fewer GAPDH and β-actin PCR products, and amplification of hGH was not successful. The PCR and reverse transcription-PCR results were significantly affected by the duration of decalcification. The yield, purity, and integrity of mRNA progressively decreased with increased storage periods of paraffin blocks.ConclusionsFixation and storage should therefore be standardized in order to improve the quality of molecular pathologic diagnosis.

Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral (Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents (Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.

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 <24h and 7days followed by conventional processing, and fixation in molecular fixative for <24h and 7days 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 1327bp and one-step RT-PCR for 621bp and 816bp 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 (<24h). 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.

Effect of prolonged formalin fixation on immunohistochemical staining for the proliferation marker Ki67

Effect of prolonged formalin fixation on immunohistochemical staining for the proliferation marker Ki67

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

A total of 29 brain tissue samples (BTS) were examined for rabies infection by different diagnostic techniques. None of the examined brain tissues were presented as a whole intact brain. Twenty-seven brain tissue samples from various animal species - dog (13 cases), cat (one case), fox (one case), pig (one case), cow (three cases), sheep (two cases), goat (one case), camel (one case), horse (one case) and donkey (three cases) - were provided by the Vaccine and Sera Department/Al-Bashir Central Hospital in Amman/Jordan from July 2009 up to May 2010. All these samples were frozen at -20°C, for a period of time and then fixed in 10% formalin after being tested for rabies virus by fluorescence antibody test (FAT). The results showed that 21 (77.77%) of 27 BTS were positive for rabies by FAT. Seventeen samples (58.62%) of 29 were positive by histopathology, 2 (6.90%) were positive by histopathology, immunohistochemistry (IHC) and of those which were fixed for 24h only, and 21 (72.42%) were positive using RT-PCR assay. Five of 29 BTS had no pathological lesions, 17 had Negri bodies and the remaining had non-suppurative encephalitis and necrosis. Thirteen BTS that were diagnosed positive by FAT were also positive by RT-PCR and histopathology, but negative by IHC. Four BTS that were positive by FAT were negative by histopathology, IHC and RT-PCR. Also, 3 BTS (cases 19, 22, and 25) that were negative by FAT were positive by RT-PCR and negative by IHC. One of these was negative, while two were positive by histopathology. Therefore, definitive diagnosis of rabies under these conditions in Jordan needs one or more other diagnostic tests in addition to FAT. Also, freezing and prolonged formalin fixation of BTS is not suitable for the detection of rabies virus antigen using IHC.

Lymph node harvest in colorectal adenocarcinoma specimens: the impact of improved fixation and examination procedures

A review of 1050 pathology reports from colorectal adenocarcinoma specimens examined at the Department of Pathology, Sørlandet sykehus HF, Kristiansand, Norway during the period 1995-2006 revealed a poor performance of most doctors concerning lymph node harvest. A mean of 8.1 nodes per specimen (range 12.3-2.1) and a mean proportion of 22.3% of specimens with ≥12 lymph nodes (range 47.1-0%) were found. A small pilot study was undertaken in 2007 to evaluate the effect of prolonged formalin fixation and the use of a special lymph node fixative [glacial acetic acid, ethanol, water and formaldehyde (GEWF) solution] with regard to the number of retrieved nodes. This showed that one extra day formalin fixation and the use of GEWF solution considerably enhanced the detection of lymph nodes, particularly those of smaller size. Based on these findings, our routines concerning handling of colorectal cancer specimens were changed during 2007. After this time all specimens have been fixed in a mixture of GEWF solution and formalin for at least 48 h and the doctors have been encouraged to find as many lymph nodes as possible. In cases revealing <12 nodes after microscopical examination, the specimens have been re-examined and searched for additional nodes. A review of lymph node retrieval in 423 cases of colorectal cancer during the period 2008-2010 showed that the mean number of nodes per specimen had increased to 16.8 (range 29.0-13.3) and the proportion of specimens with ≥12 nodes to 78.0% (range 96.8-63.6%). Thus, these changes of routines which were easy to implement without significant extra costs have considerably improved lymph node harvest at our department. The use of a special lymph node fixative (e.g. GEWF solution) is highly recommended not only for detection of nodes in colorectal specimens, but also for retrieval of lymph nodes embedded in fat tissue generally.

Effects of Prolonged Formalin Fixation on the Immunohistochemical Detection of Infectious Agents in Formalin-Fixed, Paraffin-Embedded Tissues

Immunohistochemistry is commonly used to detect and characterize infectious agents in diagnostic pathology. The principal advantage of immunohistochemistry over other antigen detection techniques is the ability to identify antigen within the context of histologic lesions. Although epitope masking attributed to formalin fixation, especially prolonged fixation, has been considered a limiting factor in diagnostic immunohistochemistry, only a few studies have evaluated the immunohistochemical detection of infectious agents following prolonged formalin fixation. Therefore, the goal of this study was to evaluate the effects of prolonged formalin fixation on the immunohistochemical detection of 21 infectious agents. Tissue slices about 5 mm thick were fixed in 10% neutral-buffered formalin, processed, and paraffin embedded at day 1 or 2 and then at approximately weekly intervals. Three pathologists graded immunoreactivity according to a four-tier grading system: negative, weak, moderate, strong. Canine parvoviral immunoreactivity was markedly decreased following 2, 7, and 10 weeks of fixation in myocardium, small intestine, and spleen, respectively. Bovine respiratory syncytial virus immunoreactivity was markedly decreased following 7 weeks of fixation. Bartonella henselae had an abrupt loss of immunoreactivity following 9 weeks of fixation. Despite variation among time points, immunoreactivity remained moderate to strong throughout the study period for the other 18 antigens. These results suggest that prolonged formalin fixation of up to 7 weeks generally does not limit immunohistochemical detection of infectious agents. However, the effects of prolonged fixation depend on the targeted antigen and the selected antibody. The results of this study further validate the utility and reliability of immunohistochemistry in diagnostic pathology.

Effects of Prolonged Formalin Fixation on Diagnostic Immunohistochemistry in Domestic Animals

Immunohistochemistry (IHC) is routinely used in diagnostic pathology to detect infectious agents, to immunophenotype neoplastic cells, and to prognosticate neoplastic diseases. Formalin fixation is considered a limiting factor for IHC because formalin can cross-link antigens and mask epitopes. Prolonged formalin fixation is presumed to result in decreased antigen detection; however, this effect has only been evaluated with a few antibodies. The goal of this study was to evaluate the effect of prolonged formalin fixation on the immunohistochemical detection of 61 different antigens. Approximately 5-mm-thick tissue slices were fixed in 10% neutral-buffered formalin. Tissue slices were removed from formalin, processed, and paraffin-embedded at 1-day, 3-day, and then at approximately 1-week intervals. IHC was performed on all sections in tandem after all tissues were processed. Immunoreactions were evaluated by three pathologists according to a four-tier grading system. Immunoreactivity of cytokeratin 7, high-molecular-weight cytokeratin, and laminin was diminished by prolonged formalin fixation. However, immunohistochemical reactivity remained moderate to strong with up to 7 weeks of fixation for all other antibodies. These results suggest that prolonged formalin fixation has minimal effects on antigen detection for most commonly used antibodies. These results further validate the use of IHC in diagnostic pathology.

Detection of viral RNA from paraffin-embedded tissues after prolonged formalin fixation

Background Isolating amplifiable RNA from formalin-fixed, paraffin-embedded (FFPE) tissues is more difficult than isolating DNA because of RNases, chemical modification of the RNA, and cross-linking of nucleic acids and proteins. Tissues containing infectious disease agents that require biosafety level (BSL)-3 and -4 necessitate fixation times of 21 and 30 days, respectively. Objective To improve procedures for extracting RNA from these FFPE tissues and detect the RNA with the more sensitive TaqMan ®-based reverse transcriptase (RT)-PCR. Study design Through a single modification of a commercially available kit, we were able to extract amplifiable RNA and detect West Nile virus (WNV), Marburg virus (MARV), and Ebola virus (EBOV)-infected tissues using TaqMan ® assays. Results Formalin fixation results in an approximately 2 log 10 reduction in detection limit when compared to fresh tissues. Increasing proteinase K digestion (24 h) improved extraction of amplifiable RNA from FFPE tissues. The TaqMan ® results were comparable to more traditional detection results such as virus isolation. Conclusion This improved extraction procedure for obtaining RNA combined with the TaqMan ® RT-PCR assays permit retrospective and prospective studies on FFPE tissues infected with BSL-3 and -4 pathogens.