The utility of vapor fixation for cell block preparation in fine needle aspiration of malignant lesions of lymph nodes

Cell blocks overcome certain limitations of fine needle aspiration cytology (FNAC), such as study of tissue architecture and application of immunohistochemistry (IHC). The main disadvantage of cell block by conventional method is cell loss. This study compares formaldehyde vapor-fixed cell blocks with conventional formalin-fixed blocks obtained from aspirates of malignant lesions of lymph nodes. Seventy-nine patients with suspected primary or metastatic malignancy in lymph nodes were studied. Cytologic smears were prepared in all the cases. Conventional formalin-fixed cell blocks were made in 34 cases, vapor-fixed cell blocks in 34 cases, and in the remaining 11 cases, both methods were employed. Cell block sections were compared for cellularity, fixation artifacts, morphologic details, and crispness of immunohistochemical staining using defined subjective criteria. Cellular yield was slightly less with vapor-fixed cell blocks in comparison with conventional formalin-fixed cell blocks. Staining artifacts were more frequent with vapor fixation (33.3%) as compared to conventional formalin fixation (20%). Also, vapor fixation required more time (6 h) as compared to conventional formalin fixation (1 h). There was no difference in the two techniques with respect to immunohistochemical staining. Cell blocks prepared by vapor fixation are comparable to conventional formalin-fixed cell blocks.     

Cell block cytology. Improved preparation and its efficacy in diagnostic cytology

Cell blocks prepared from residual tissue fluids and fine-needle aspirations can be useful adjuncts to smears for establishing a more definitive cytopathologic diagnosis. They can be particularly useful for categorization of tumors that otherwise may not be possible from smears themselves. A modified cell block technique using an improvised ethanol formalin fixative (Nathan alcohol formalin substitute) followed by a simple paraffin processing schedule is described. This improved preparation offers excellent cytomorphologic features corresponding closely to cells in Papanicolaou-stained smears and ensures optimal preservation of histochemical and immunocytochemical properties. The technique is simple and reproducible and uses routine safe laboratory chemicals. The efficacy of cell blocks also is discussed.     

Estrogen,progesterone, and HER‐2 receptor immunostaining in cytology: The effect of varied fixation on human breast cancer cells

The ASCO/CAP Expert Panel recommends that all invasive breast carcinomas and breast cancer recurrences be tested for ER, PR and HER‐2 expression. The guidelines for testing of surgical specimens by immunohistochemistry (IHC) are well defined, whereas they are lacking for cytological samples. We evaluated various fixation protocols for optimal receptor testing by immunohistochemistry and immunocytochemistry (ICC) of human breast cancer cell lines MCF‐7 (ER/PR positive) and SKBR‐3 (overexpressing HER‐2). The cells were fixed in 10% neutral buffered formalin or Saccomanno Fixative (SF) for various time points, and either embedded in paraffin as cell blocks or prepared as cytospins. ER and PR slides were assigned a proportion score (PS; 0–5), an intensity score (IS; 0–3) and a total score (TS = PS + IS). Standard DAKO scoring system ranging from 0 to 3+ was used for the evaluation of HER‐2 staining. Human breast cancer cells stained successfully for ER, PR and HER‐2 when fixed in formalin and prepared as cell blocks. The optimal fixation time for formalin‐fixed cells ranged from 2 to 96 hours. Cells fixed in SF from 2 to 96 hours also stained well for ER and PR. However, SF produced variable results for HER‐2 staining; particularly, SF fixation beyond 24 hours caused false negative results. The interpretation of HER‐2 staining on cytospins was not feasible irrespective of the fixative and fixation time. In summary, formalin fixation from 2 to 96 hours and preparation of cells as cell blocks produces optimal results for ER, PR, and HER‐2 testing in human breast cancer cells. Diagn. Cytopathol. 2013;41:864–870. © 2013 Wiley Periodicals, Inc.     

Using Kryofix as alternative for formalin results in more optimal and standardized immunostaining of paraffin sections.

Although used for over one century formalin has several disadvantages which Kryofix, an alternative fixative for paraffin blocks used in Leiden for 6 years, does not have. In this study the effects of Kryofix on tissue regarding immunohistochemistry are compared with those of buffered formalin. All markers studied showed enhanced staining in the Kryofix blocks after 4 hours of fixation, whilst in some cases the immunostaining of the formalin blocks was even negative. For all markers, the sera could be further diluted for the Kryofix sections, for some with as much as a factor 20, enhancing the cost-effectiveness of the method. We established that for formalin, the fixation time strongly influenced the results. For Kryofix there was no time factor: the immunostaining results of 1 hour fixation were identical to those after 3 months of fixation. This study shows that by this method of fixation, in which there is no cross-linking of proteins, immunostaining of paraffin sections is optimized and standardized.     

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples

Background  

Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material.     

High quality of DNA retrieved for Southern blot hybridization from microwave-fixed, paraffin-embedded liver tissues

To overcome the degradation problem encountered in DNA extracted from formalin-fixed, paraffin-embedded tissue blocks, several methods of tissue fixation were examined in order to improve the quality of the DNA recovered for use in nucleic acid analysis. The fixation methods included formalin fixation alone, alcohol fixation alone, and microwave fixation with tissues immersed in phosphate-buffered saline (PBS), alcohol, or formalin. Unfixed fresh frozen tissue served as the control. Using hepatitis B virus (HBV) DNA sequences and the type I human procollagen gene as markers and liver tissue as a target, microwave fixation, with formalin omitted, not only preserved the DNA very well, but also the labile viral antigen. Both high molecular weight-integrated and free-form HBV DNAs were well preserved, and suitable for polymerase chain reaction and Southern blot analysis. The restriction enzyme fragment pattern of DNA recovered from these paraffin blocks was identical to that of unfixed fresh frozen tissue. Microwave fixation also preserved the labile preS2 epitope of the hepatitis B surface antigen (HBsAg) considerably better than formalin. These results suggest that microwave fixation is superior to routine formalin fixation for the preservation of excellent quality of genomic and viral DNAs for nucleic acid hybridization analysis. Alcohol, often used for nucleic acid purification, was also a good fixative for preserving DNA and the antigenicity of the labile antigen, especially when carried out in combination with microwave fixation.     

Use in immunohistochemical studies of a method of restoration of antigenic specificity as affected by microwaves in tissue fixed with formalin and embedded in paraffin]

Recovery of specific antigenic characteristics using microwave treatment of paraffin sections of tissues fixed by formalin allows to extend spectrum of antibodies for immunohistochemical diagnosis. Microwaves enable the reaction on the material prepared according to the standard technique, and macropreparations long stored in formalin and archive blocks.     

Comparison of proliferation rates assessed using "multiblock" and conventional tissue blocks of lung carcinoma.

AIMS: To compare the proliferative rates, assessed immunohistochemically, of human lung tumours using conventional paraffin wax blocks and the multitumour tissue block (MTTB) technique. METHODS: A multiblock containing 20 lung tumours (eight adenocarcinomas, five squamous cell, five small cell and two carcinoid tumours) was constructed. Sections were also cut from the original blocks of formalin fixed, paraffin wax embedded tissue used to construct the multiblock. Sections were stained with the monoclonal antibody PC10, which recognises a proliferating cell nuclear antigen, using the three stage immunoperoxidase technique. RESULTS: The proliferation rates of the lung tumours obtained using both techniques were, overall, significantly different (p = 0.05), although most cases showed good correlation. Some tumours displayed a high degree of intratumoral variation in PC10 staining. The degree of PC10 staining was in keeping with the known proliferative state of particular histological subtypes--that is, carcinoid tumours showed little staining and small cell carcinomas showed extensive positivity. CONCLUSION: The MTTB technique is a less suitable means of assessing proliferation rate in lung carcinomas than conventional tissue blocks. It should be restricted to qualitative antibody studies or quantitative studies using tumours with little intratumoral variation.     

<Emphasis Type="Italic">In vitro</Emphasis> mutation artifacts after formalin fixation and error prone translesion synthesis during PCR

Background  

Clinical specimens are routinely fixed in 10% buffered formalin and paraffin embedded. Although DNA is commonly extracted from fixed tissues and amplified by PCR, the effects of formalin fixation are relatively unknown. Formalin fixation is known to impair PCR, presumably through damage that blocks polymerase elongation, but an insidious possibility is error prone translesion synthesis across sites of damage, producing in vitro artifactual mutations during PCR.     

Immunological equivalence between mouse brain-derived and Vero cell-derived Japanese encephalitis vaccines

The persistent spread via animal reservoirs urges expanding vaccination programs against pathogens like the Japanese encephalitis virus, JEV. The JEV is spreads to new areas by domestic as well as by wild animals. Although there is a safe and efficient vaccine on the market, this is derived from infected mouse brains, why today's situation requires overcoming the potential risk caused by using animal tissues. To meet this demand we have developed a Vero cell-derived JEV vaccine, using the same virus strain as in the established one. A phase III clinical study of the new vaccine has recently been completed with positive outcome. Like the established mouse brain-derived vaccine, the Vero cell-derived one is a formalin inactivated whole virus vaccine. We here demonstrate the very good agreement in immunological tests between the two antigens. The study includes analyses with two neutralizing monoclonal antibodies that blocks cell entry at a late stage in infection, assumedly interfering with fusion-related refolding in the virus fusion protein. It is obvious that the formalin inactivation treatment, with both virus preparations, retains these essential vaccine epitopes.     

Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma

To identify the minimum time necessary for consistent immunohistochemical estrogen receptor (ER) results in our laboratory, we evaluated results in timed fixation blocks and cases with disparate and similar needle core biopsy and partial mastectomy specimens. Tissue sections of 24 ER-positive, invasive breast carcinomas were fixed for 3, 6, 8, and 12 hours and 1, 2, and 7 days. ER values were quantified using the Q score (0-7). In timed fixation blocks, the mean Q score per block was 2.46 for blocks fixed for 3 hours, 5.75 for blocks fixed for 6 hours, and 6.70 for blocks fixed for 8 hours (P < .001). The difference between the case maximum and mean block Q scores was a plateau of almost 0 at 6 to 8 hours of formalin fixation. For needle core biopsy specimen fixation times, the means for specimens with ER-disparate and ER-similar results were 1.2 and 6.3 hours, respectively (P = .01). The minimum formalin fixation time for reliable immunohistochemical ER results is 6 to 8 hours in our laboratory, regardless of the type or size of specimen.     

BIOMED-2聚合酶链反应Ig基因重排对成熟非霍奇金B细胞淋巴瘤诊断的价值

目的 探讨BIOMED-2聚合酶链反应(PCR)在成熟非霍奇金B细胞淋巴瘤(B-NHL)诊断中的价值.方法 收集成熟B-NHL组织标本72例,其中弥漫性大B细胞淋巴瘤37例,黏膜相关淋巴组织结外边缘区淋巴瘤35例为研究对象,并以反应性增生病变25例作为对照.提取以上组织的DNA,并以PCR来检测其完整性和可扩增性,选取质量合格的DNA.85.6%(83/97)的样品DNA长度>300 bp,其中60例成熟B-NHL和23例反应性增生可用于BIOMED-2 PCR检测免疫球蛋白重链(IgH)和kappa轻链(IgK)基因重排的克隆性.结果 利用BIOMED-2 PCR检测的60例成熟B-NHL中,57例存在Ig基因的克隆性重排,其检测敏感性为95%,23例反应性增生病例中未出现Ig基因的克隆性重排,其检测特异性为100%.结论 BIOMED-2 PCR适用于石蜡包埋组织.该方法具有很高的敏感性和特异性,对成熟B-NHL诊断的辅助价值很高.     

Effective application of the methanol‐based PreservCyt™ fixative and the Cellient™ automated cell block processor to diagnostic cytopathology,immunocytochemistry, and molecular biology

We studied the feasibility of immunocytochemistry (ICC), in situ hybridization (ISH), and polymerase chain reaction (PCR) after Cellient^? automated cell block processing, and tested whether methanol‐based PreservCyt^? fixation could replace formalin fixation, in an attempt to eliminate toxic formaldehyde vapors. Immunostaining with 30 different antibodies was performed on cell blocks from 73 FNA specimens and 42 body cavity fluid specimens prepared by Cellient^? automated processing that uses the methanol‐based fixative (PreservCyt^?). For each antibody we evaluated ICC in at least three different cell block specimens and compared it with immunohistochemistry (IHC) in formalin‐fixed, paraffin‐embedded (FFPE) histological sections from the corresponding tumors. The quality of DNA and RNA in Cellient^? blocks was analyzed by ISH, applying a SYT gene break‐apart assay and EBER probes, respectively. Moreover, DNA quality was analyzed by PCR by using primer sets for DNA products of 100, 200, 300, 400, 500, and 600 base pairs, and evaluated by gel electrophoresis. When compared with IHC results in corresponding FFPE tumor tissue from the same patient, 24 out of 30 antibodies showed concordant ICC results. With FISH, distinctive hybridization signals were observed for SYT DNA sequences and EB virus RNA sequences. With PCR, DNA products, up to 600 base pairs in size, were readily observed after gel electrophoresis. The antibodies that showed concordant immunostaining in Cellient^? blocks could be applied to diagnostic algorithms that proved to be helpful in the discrimination of major tumor types (carcinoma, lymphoma, melanoma, and germ cell tumors), discrimination of carcinoma subtypes, and determination of primary tumor site in cases of metastatic carcinoma. In a separate study, we found that the application of ICC to this cell block technique provided additional diagnostic and clinically important information in 24% of 100 consecutive cases. The high quality of DNA and RNA in Cellient^? cell blocks allowed sensitive and specific molecular biologic analysis, in particular FISH and PCR. Diagn. Cytopathol. 2013;41:734–741. © 2013 Wiley Periodicals, Inc.     

Lymph node revealing solution: a rapid method for the fixation of cystectomy specimens

The objective of this study was to describe the use of the lymph node revealing solution (LNRS) for rapid fixation of total cystectomy specimens, and to compare it with formalin fixation. LNRS is a mixture of 95% ethanol, diethyl ether, glacial acetic acid and buffered formalin (65:20:5:10 v/v) prepared under a fume-hood. Sixteen consecutive cystectomy specimens were fixed for two hours either in LNRS or in buffered formalin. Representative sections were embedded in paraffin, sectioned and stained with H&E, periodic acid Schiff, alcian-blue, and immunostained for cytokeratins 20, high and low molecular weight cytokeratins, prostatic specific antigen, Factor VIII related antigen, s-100 protein, and protein kinase C isoenzymes. Results showed that the tissues were well fixed after 2 hours in LNRS, and were not fixed after 2 hours in formalin. Processing and sectioning of the paraffin blocks of the LNRS fixed tissue was excellent; it was impossible in the sections fixed for 2 hours in formalin. All the stains were excellent after LNRS fixation. We conclude that fixation of cystectomy specimens in LNRS requires only two hours and results in excellent stained slides. It is therefore recommended for cystectomy specimens.     

Effect of formalin fixation and epitope retrieval techniques on antibody 34betaE12 immunostaining of prostatic tissues.

Basal cell-specific, anti-high molecular weight cytokeratin (HMCK) antibody is often used to confirm the diagnosis of prostatic adenocarcinoma, particularly if limited amounts of tissue are available. HMCK is formalin sensitive and requires pretreatment by enzymes or heat if formalin-based fixatives are used. To date, the effect of prolonged formalin fixation on HMCK immunoreactivity has not been systematically studied; this is critical, because the diagnosis of malignancy is based on a negative immunoreaction. In this study, 5 tissue blocks obtained from each of 10 radical prostatectomy specimens were fixed in formalin from 6 hours to 1 month. HMCK immunostaining was performed with monoclonal antibody clone 34betaE12 after pretreatment of the sections by either enzymatic predigestion with pepsin, heat-induced epitope retrieval (HIER) with a microwave, or HIER with a hot plate. For scoring, the staining intensity at 6 hours of formalin fixation was considered as the baseline for that particular antigen retrieval technique. After pepsin predigestion or microwaving, there was progressive loss of HMCK immunoreactivity from 1 week or longer of formalin fixation. HIER with a hot plate yielded consistent results with no decrease in HMCK immunoreactivity with as long as 1 month of formalin fixation. The staining intensity was consistently stronger at all periods of formalin fixation when the hot plate method was used, compared with pepsin predigestion or microwaving. Generally weak HMCK positivity was observed in rare neoplastic cells of 3 of 10 specimens after hot plate HIER but not with pepsin predigestion or microwave antigen retrieval. This sporadic immunostaining of malignant cells was quantitatively and qualitatively distinct from the pattern seen in benign epithelium. We conclude that formalin fixation affects HMCK immunoreactivity over time and might impact its diagnostic usefulness. Efficacies of different antigen unmasking/epitope retrieval techniques vary and must be standardized for individual laboratories.     

Immunocytochemical detection of Candida albicans in formalin fixed, paraffin embedded material

AIM: To assess the ability of the commercially available monoclonal antibody 1B12 (BioGenex, San Ramon, USA) to identify C albicans in formalin fixed, paraffin wax embedded material (FFPE). METHODS: Broth cultures of 20 strains of seven Candida species were resuspended in 4% agarose blocks, fixed in formalin for 24 hours, and embedded in paraffin wax. In addition, 16 blocks of FFPE tissue known to contain periodic acid-Schiff positive fungal hyphae were examined. Antigen retrieval involved microwave treatment of specimens in citrate buffer (0.01 M; pH 6.5) before addition of 1B12 antibody for 24 hours. Bound antibody was subsequently detected using a biotinylated link antibody and a peroxidase conjugated streptavidin. RESULTS: Only C albicans strains were 1B12 positive in the agarose blocks. All FFPE tissue blocks were found to contain 1B12 positive hyphal structures, indicating the presence of C albicans. CONCLUSIONS: The ability to identify candida organisms penetrating the lesional tissue in cases of chronic hyperplastic candidosis will help to clarify the role of individual Candida spp in this important form of oral candidosis.     

Microwave fixation and rapid processing in a large throughput histopathology laboratory.

Technical details of a method of microwave fixation and rapid processing are described. Fresh biopsy specimens are sampled and trimmed into 2 mm thick blocks and irradiated to a temperature of 68 degrees C. They are then processed through cycles of absolute alcohol (75 min), xylene (50 min) and wax (50 min). It is possible, in this manner, to run several 'short' cycles of about 3 hrs each during the working day so that stained sections are available on the same day as receipt of the specimens. Endoscopic biopsies are processed through a shorter cycle comprising 30 min of absolute alcohol (4 changes), 20 min of xylene (3 changes) and 20 min of wax (2 changes). For convenience we also employ an overnight cycle whereby microwave-fixed blocks are processed through several changes of absolute alcohol, xylene and wax without the use of formalin. This method of processing not only removes the use of noxious and potentially toxic formalin but also allows rapid preparation of good quality diagnostic sections with superior antigen preservation compared to formaldehyde fixation.     

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.     

Demonstration of AA-protein in formalin-fixed, paraffin-embedded tissues.

AA-protein was identified by SDS-acrylamide electrophoresis in amyloid fibrils fixed in formalin after isolation from fresh-frozen tissues obtained from patients with familial Mediterranean fever (FMF) amyloidosis and idiopathic AA-amyloidosis and, following deparaffination, rehydration and homogenization of embedded formalin-fixed tissues of old autopsy cases of the hereditary amyloidosis of FMF and amyloidosis acquired in association with tuberculosis, bronchiectasis, and rheumatoid arthritis. That AA-protein is unaltered by formalin was firmly established by agar gel diffusion using specific rabbit anti-AA serum. By contrast, AL proteins could not be demonstrated either in formalin-fixed amyloid fibrils derived from fresh-frozen tissues of a patient with presumably AL-amyloidosis dominated by cardiomegaly and one with AL-kappa amyloidosis or in blocks of cases of familial neuropathic amyloidosis, multiple myeloma, and idiopathic amyloidosis with cardiopathy. AA-protein is not denatured by formalin and retains its typical electrophoretic, chromatographic, and immunologic characteristics even 30 years after fixation and paraffin-embedding.