Role of the frozen tissue bank in molecular pathology.

The new discipline of molecular pathology requires that high-quality, intact genomic DNA, mRNA, and proteins be available from frozen tissue samples. It is now necessary for pathology laboratories to establish consistent guidelines for the preparation and storage of frozen tissue samples in order to have properly preserved tissues available for diagnostic molecular techniques. Maintaining a frozen tissue bank requires a pathologist to oversee this program and to integrate it into the routine surgical pathology activities. A member of the laboratory technical staff can serve as a tissue bank coordinator and have responsibility for preparation of tissue samples, their systematic storage and retrieval, and routine maintenance of equipment and supplies. Tissue sampling must be done as soon as possible after excision of the specimen and is the responsibility of a qualified pathologist. The samples may be snap frozen without cryoprotection at -78 degrees C or colder for subsequent use in procedures requiring the extraction of genomic DNA, mRNA, or protein. To preserve tissue architecture and cytologic features for immunohistochemistry and in situ hybridization, the tissue should be frozen at -78 degrees C or colder with a cryoprotectant such as OCT. Long-term storage of the frozen tissue is recommended at -140 degrees C or colder in a locked liquid nitrogen freezer, and the record of sample inventory can easily be kept in a computerized database. Tissues sampled and stored under these conditions have been used successfully in a wide variety of molecular techniques. In addition to malignant tumor tissue, samples from benign lesions and normal tissues should be frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation of hybridomas

Summary A method for the long-term storage of hybridoma cells in liquid nitrogen is described. Hybridoma cells are harvested in logarithmic phase of growth and suspensed in freeze medium containing dimethylsulfoxide. Using a programmable freezer, the cells are frozen very slowly (1°C/min) and then transferred rapidly to liquid nitrogen refrigerators. Consequently, the hybridoma cells can be stored for long periods without substantial loss of viability and with minimal biochemical changes.     

Genotoxicity of doxorubicin in F344 rats by combining the comet assay,flow‐cytometric peripheral blood micronucleus test,and pathway‐focused gene expression profiling

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7‐week‐old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes‐modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non‐neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species. Environ. Mol. Mutagen. 55:24–34, 2014. © 2013 Wiley Periodicals, Inc.^?     

T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells

A criticism of current techniques for monitoring changes in T-cell subset numbers over extended periods in individuals with disease states in which such changes might provide insight is the fact that serial samples taken are usually analysed fresh and therefore not in the same assay. To try to overcome this problem we have stored peripheral blood mononuclear cells frozen in liquid nitrogen and thence examined their ability to form sheep red blood cell (E) rosettes and to label with OK monoclonal antibodies. Results obtained show that cell viabilities following freezing and T-cell subset analysis of E-rosette positive cells are no different when fresh or frozen and subsequently thawed peripheral blood mononuclear cells are used.     

Tissue freeze-drying with a closed drying tube stored in a freezer

A simple method of freeze-drying tissue is described that does not require a continuously running vacuum pump. After the frozen tissue is positioned a few centimeters from the dessicant in a drying tube, a vacuum pump is attached briefly to lower the pressure in the drying tube. The drying tube is closed at a pressure of 5 X 10(-3) mm Hg or less, and the tube is stored in a freezer set at the desired temperature. Frog kidneys, 300-400 mg, were dry in about 24 hr at -32 degrees C and 48 hr at -40 degrees C. Since the tissue is dried in individual drying tubes, many tissue samples can be dried at one time.     

A novel method for freezing and storing research tissue bank specimens

Preserving small pieces of frozen tissue for possible future ancillary studies ("tumor banking") usually involves either placing a small piece of tissue in a cryovial and snap freezing it in liquid nitrogen or embedding and freezing the tissue in a block of cryopreservation medium, such as optimal cutting temperature (OCT) compound. The cryovial storage method leaves an irregularly shaped piece of tissue frozen to the side of the plastic vial, where it is exposed to air, subject to desiccation ("freezer burn"), and difficult to remove, but the vials are easy to store. The OCT method results in good morphological preservation, but yields a large awkwardly stored block from which it may be difficult to locate and recover small specimens. We have proposed a novel method of storing tissue bank specimens, the "capsule-freeze" method, which combines the advantages of OCT specimen preservation with those of cryovial specimen storage. Using this method, a tissue specimen is "snap-frozen" in OCT within a size "00" VCap pharmaceutical capsule, then the capsule is stored within a 1.5-mL cryovial. The specimen is harvested by simply cutting a slice out of the capsule and sealing the cut ends with OCT before their return to the freezer. The slice is then embedded en face within an OCT block before frozen sectioning. Morphological preservation is excellent, and the capsules are very easy to store. Occasional cracking that we found with the use of gelatin capsules is greatly diminished with the use of VCaps cellulose-walled capsules. The OCT can be easily removed by rinsing in cold 70% ethanol solutions. This method of tissue storage is ideal for the small specimens that are now commonly archived in these days of tissue sparing surgery.     

Comet assay application in environmental monitoring: DNA damage in human leukocytes and plant cells in comparison with bacterial and yeast tests

Urban airborne particulate is a complex mixture of air pollutants, many of which have not been identified. However, short-term mutagenesis tests together with chemicophysical parameter analysis are able to better assess air quality and genotoxic load. The findings of continuous monitoring (January 1991-August 1998) of urban air genotoxicity of a Po Valley town (Italy) on Salmonella typhimurium and Saccharomyces cerevisiae are reported. During this period, various measures (catalytic devices, unleaded fuels, annual vehicle overhaul, etc.) to improve air-dispersed pollutant control were enforced. However, a continuous presence of genotoxic compounds is shown and more qualitative than quantitative changes are evident. We also demonstrate the ability of the Comet assay to detect DNA-damaging agents in airborne particulate samples. We applied the test to human leukocytes and, with major improvements, to plant cells (Allium cepa roots and epigean tissues of Impatiens balsamina). The first findings on human leukocytes confirm the sensitivity of this assay, its peculiarity and its applicability in assessing genotoxicity in environmental samples. The capability of plants to show the response of multicellular organisms to environmental pollutants largely counterbalances a probable lowering in sensitivity. Moreover, application of the Comet test to epigean tissues could be useful in estimating the bioavailability of and genotoxic damage by air pollutants, including volatile compounds (ozone, benzene, nitrogen oxides, etc.) to higher plants.     

Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations

The alkaline Comet assay was used to investigate the in vivo genotoxicity of 17 compounds. Altogether 21 studies were conducted with these compounds. The investigations were triggered for various reasons. The main reason for performing the studies was to evaluate the in vivo relevance of in vitro genotoxicity findings with 10 compounds. Eight of these compounds showed no effects in the in vivo Comet assay while two compounds induced altered DNA migration patterns in specific organs. The remaining seven compounds were tested to follow up on neoplastic/preneoplastic or chronic toxicity changes as detected in specific target organs identified in rodent studies, to investigate the possibility of site-of-contact genotoxicity and to test the liver as a target organ for a suspected reactive metabolite. For the studies, various organs of rodents were analyzed, depending on the suspected properties of the compounds, including liver, jejunum, leukocytes, stomach mucosa, duodenum, lung and kidney. All tissues were amenable to investigation by gel electrophoresis after simple disaggregation of organs by means of mincing or, in the case of epithelial cells from the gastrointestinal tract, scraping off cells from the epithelium. In conclusion, the Comet assay was found to be a reliable and robust test to investigate in vivo genotoxicity in a variety of rodent organs. Therefore, it is concluded that in vivo Comet assay data are useful for elucidating positive in vitro genotoxicity findings and to evaluate genotoxicity in target organs of toxicity.     

Reliability of the comet assay in cryopreserved human sperm

BACKGROUND: Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. METHODS: Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. RESULTS: The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. CONCLUSIONS: Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.     

Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus

Cell-mediated immunity appears to be critical for the prevention and control of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersome or lack sensitivity. We have developed a gamma interferon ELISPOT assay that provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (SFC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This assay has been validated by demonstrating the following: (i) the response detected is mediated almost entirely by CD4+ T cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those from freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a computer-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the use of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either during a herpes zoster disease outbreak or following vaccination.     

Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus

Cell-mediated immunity appears to be critical for the prevention and control of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersome or lack sensitivity. We have developed a gamma interferon ELISPOT assay that provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (SFC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This assay has been validated by demonstrating the following: (i) the response detected is mediated almost entirely by CD4⁺ T cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those from freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a computer-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the use of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either during a herpes zoster disease outbreak or following vaccination.     

A procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking in surgical pathology

Different methods for snap freezing surgical human tissue specimens exist. At pathology institutes with higher work loads, solid carbon dioxide, freezing sprays, and cryostat freezing are commonly used as coolants for diagnosing frozen tissue sections, whereas for tissue banking, liquid nitrogen or isopentane cooled with liquid nitrogen is preferred. Freezing tissues for diagnostic and research purposes are therefore often time consuming, laborious, even hazardous, and not user friendly. In tissue banks, frozen tissue samples are stored in cryovials, capsules, cryomolds, or cryocassettes. Tissues are additionally embedded using freezing media or wrapped in plastic bags or aluminum foils to prevent desiccation. The latter method aggravates enormously further tissue handling and processing. Here, we describe an isopentane-based workflow which concurrently facilitates tissue freezing and processing for both routine intra-operative frozen section and tissue banking and satisfies the qualitative demands of pathologists, cancer researchers, laboratory technicians, and tissue bankers.     

DNA damage in mice treated with sulfur dioxide by inhalation

Sulfur dioxide (SO2) is a ubiquitous air pollutant produced by the burning of fossil fuels. In this study, single-cell gel electrophoresis (the Comet assay) was used to evaluate the DNA damage produced by inhalation exposure of mice to SO2. Male and female mice were housed in exposure chambers and treated with 14.00 +/- 1.25, 28.00 +/- 1.98, 56.00 +/- 3.11, and 112.00 +/- 3.69 mg/m3 SO2 for 6 hr/day for 7 days, while control groups were exposed to filtered air. Comet assays were performed on blood lymphocytes and cells from the brain, lung, liver, spleen, kidney, intestine, and testicles of the animals. SO2 caused significant, dose-dependent increases in DNA damage, as measured by Olive tail moment, in all the cell types analyzed from both sexes of mice. The results indicate that inhalation exposure to SO2 damages the DNA of multiple organs in addition to the lung, and suggests that this damage could result in mutation, cancer, and other diseases related to DNA damage. Further work will be required to understand the ultimate toxicological significance of this damage. These data also suggest that detecting DNA damage in blood lymphocytes, using the Comet assay, may serve as a useful tool for evaluating the impact of pulmonary SO2 exposure in human biomonitoring studies.     

Analysis of macrophage interactions with cryopreserved amastigotes of Leishmania tropica.

Amastigotes of Leishmania tropica were harvested from infected footpads of BALB/cJ mice and cryopreserved, using a controlled-rate freezer. This technique produced inocula with reproducible viability and infectivity for peritoneal macrophages from C3H/HeN mice. Cryopreserved amastigotes, stored for up to 17 weeks in a liquid nitrogen freezer, were similar to freshly harvested parasites in susceptibility to lymphokine-induced intracellular killing. Interactions of cryopreserved parasites with macrophages were best evaluated at 72 h, since macrophages preferentially ingested viable amastigotes and rapidly cleared ingested nonviable parasites.     

DNA damage and acute toxicity caused by the urban air pollutant 3-nitrobenzanthrone in rats: characterization of DNA adducts in eight different tissues and organs with synthesized standards

3-Nitrobenzanthrone (3-NBA) is an urban air pollutant and rat lung carcinogen that is among the most potent mutagens yet tested in the Salmonella reversion assay. In the present study, 1 mg 3-NBA was administered orally to female F344 rats and DNA adduct formation was examined in liver, lung, kidney and five sections of the gastrointestinal (GI) tract at 6 hr, and 1, 2, 3, 5, and 10 days after administration. The DNA adduct patterns, analyzed by (32)P-postlabelling followed by HPLC separation, were similar in all tissues and organs. Five of the adduct peaks cochromatographed with synthesized DNA adduct standards. Three of these unequivocally determined standards, dGp-C8-N-ABA, dGp-N2-C2-ABA, and dAp-N6-C2-ABA, were of the nonacetylated type, suggesting that at least part of the pathway for activation of 3-NBA proceeds through O-acetylation of the hydroxylamine intermediate. The two other DNA adduct standards, dGp-C8-C2-N-Ac-ABA, and dGp-N2-C2-N-Ac-ABA, were of the acetylated type, but there was some ambiguity in the characterization of these DNA adducts, since they varied inconsistently between samples and they also aligned with peaks found in controls. At 6 hr after treatment, the level of DNA adducts was highest in glandular stomach (relative adduct labeling (RAL), approximately 70 adducts/10(8) normal nucleotides (NN)); adduct levels in this organ decreased at 24 hr, but increased afterwards. DNA adduct levels in the majority of organs were characterized by an early increase (from 6 hr to 3 days), which was followed by a decrease at 5 days and a maximum level 10 days after administration (RAL approximately 120 adducts/10(8) NN for the lung, kidney and glandular stomach, approximately 80 adducts/10(8) NN for the forestomach and ceacum, and approximately 40 adducts/10(8) NN for the liver, small intestine, and colon). This pattern was consistent with pathological observations during autopsy showing high levels of tissue damage in the GI tract; the tissue damage included hemorrhages, loss of villous surface structure in the small intestine, as well as intestine fragility and oedema of the adipose tissue around the GI-tract. Tissue damage decreased and DNA adduct levels increased at 10 days after administration. These observations suggest that 3-NBA not only exerts acute toxic effects, but that the bioavailability is affected by storage in tissues and later becomes available, resulting in the increased DNA adduct levels at the later time points of collection.     

The effects of freeze/thawing process on cryopreserved equine umbilical cord blood-derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs)-derived equine umbilical cord blood in cell grafts transplantation is advantageous; therefore, preservation of these cells is of utmost importance in repair therapies. To evaluate the viability ratio of the MSCs obtained from equine umbilical cord blood after cryopreservation, umbilical cord blood obtained from nine 46–48-week-old foals were purified for harvesting MCs. The purified cells were frozen from the first to tenth passages and stored in liquid nitrogen. After thawing, the cell viability was assessed through trypan blue staining procedure. The highest viability (>80%) ratio was observed with the cells derived from the first passage in 1 and 8?weeks after cryopreservation. However, the viability of cells was dependent on the passage used for cryopreservation. Results in this study demonstrated that equine umbilical cord blood stem cells could successfully be frozen and stored in liquid nitrogen for 8?weeks without significant change in the characterization of the cells cryopreserved as regards their viability, growth ability, and differentiation potential.     

Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality

Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin.     

Analysis of the viability of freezer stored serum samples for hepatitis C virus RNA analysis by the SUPERQUANT method: results of a 16 year retrospective study

Prior to the discovery of the hepatitis C virus (HCV), virological analysis of serum from patients with non-A non-B hepatitis was not possible. Since the finding that HCV is the causative agent of most non-A non-B hepatitis, several reliable methodologies have been developed that allow for quantification of HCV RNA. To determine the viability of stored serum samples for HCV RNA analysis. 256 samples were examined for HCV RNA using a multi-cycle RT-PCR assay. All samples were stored unopened in a -70 degree C freezer until the time of testing. Collection years ranged from 1981 to 1995. To examine the integrity of stored serum samples, the distribution of quantitative HCV RNA values for each year was compared: year-to-year; and, to the distribution of HCV RNA concentrations from 1510 chronic HCV patients determined by the same assay in 1996 and 1997. Pairwise year-to-year analysis revealed that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). Likewise, comparison of the stored samples to the 1510 fresh samples demonstrated that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). The results demonstrate a method for determination of the integrity of stored serum samples from chronic HCV patients. The mechanism of RNA degradation is unknown but it is most likely to be due to poor sample collection procedures in place prior to 1991.     

Nonmetabolizable glucose compounds impart cryotolerance to primary rat hepatocytes

We herein report a novel method for the cryopreservation of hepatocytes using a non-metabolizable glucose derivative in an attempt to mimic the natural cryoprotective adaptations observed in freeze-tolerant frogs. Primary rat hepatocytes were loaded with 3-O-methyl glucose (3OMG) through endogenous glucose transporters without evident toxicity. The 3OMG-loaded hepatocytes were then frozen in a controlled rate freezer down to -80 degrees C and stored in liquid nitrogen at -196 degrees C. Hepatocytes cryopreserved with a relatively small amount of intracellular 3OMG (<0.2 M) showed high post-thaw viability and maintained long-term hepatospecific functions, including synthesis, metabolism, and detoxification. Metabolite uptake and secretion rates were also largely preserved in the cryopreserved hepatocytes. This is the first study to demonstrate the use of the non-metabolizable glucose derivative 3OMG in hepatocyte cryopreservation.