Tissue shrinkage after fixation with formalin injection of prostatectomy specimens

Prostate cancer volume correlates with stage, grade, and progression after prostatectomy. When tumor volume is measured planimetrically, results are multiplied by a correction factor to compensate for tissue shrinkage caused by processing. Injection of formalin into prostatectomy specimens was suggested for improved fixation. Our aim was to investigate how this affects the prostate volume. We studied 142 radical prostatectomy specimens. All prostates were immersed in 10% formalin. In 84 prostates (59%) we also injected 20 ml of formalin before routine fixation. The prostates were weighed unfixed after injection and after final fixation. The specimens were sliced and totally embedded. The transverse diameters of the prostates were measured on unfixed specimens and microscopic sections. The average weight loss after final fixation was 5.8 and 8.6% for formalin-injected specimens and standard-fixed specimens, respectively (p<0.001). However, when total shrinkage was estimated from the transverse diameters, there was no difference related to fixation technique (p=0.59). The average linear shrinkage was 4.5%, corresponding to a volume correction factor of 1.15. We conclude that formalin injection for fixation of prostate tissue does not influence tumor volume calculation compared to conventional fixation.     

Comparison of formalin and Bouin's reagent for fixation of coagulase negative staphylococcal biofilm.

Methodological modifications, particularly the use of different fixatives, may account for discrepancies between studies of the relation between virulence and biofilm production in vitro by isolates of coagulase negative staphylococci. The efficacy of formalin and Bouin's reagent for fixing coagulase negative staphylococcal biofilms in a microtitre tray assay was compared. The optical density of stained adherent growth by three strains was reduced by an average of 20% following fixation with 10% formaldehyde compared with Bouin's reagent. This difference seemed to be mainly because of increased background staining and blackening of the biofilm when Bouin's reagent was used. Formalin fixation was also effective at identifying early and late biofilm production in adherence growth kinetic experiments with 10 coagulase negative staphylococcal clinical isolates.     

Comparison of formalin, buffered formalin, and Bouin's fixation on the detection of human papillomavirus deoxyribonucleic acid from genital lesions

Detection of nucleic acid sequences homologous to human papillomavirus (HPV) relies primarily on their extraction from unfixed tissue. We detected HPV sequences in DNA extracted from paraffin-embedded tissue fixed in formalin (buffered and unbuffered) and Bouin's solution by dot blot hybridization. A detectable hybridization signal was noted in 32% of these fixed tissues which were chosen from cases where HPV DNA was detected in the unfixed tissue. When using a homologous 32P-labeled probe and a high stringency wash, the hybridization signal was lost if DNA was extracted after Bouin's fixation and diminished after formalin fixation, more so with unbuffered formalin. Similar differences in the hybridization signals among the different fixatives after high stringency wash were noted with in situ hybridization. Southern blot analysis showed that DNA extracted from tissues fixed in Bouin's was degraded and ranged in size from 100 to 500 base pairs as compared with 100 to 900 base pairs for DNA extracted from tissue fixed with unbuffered formalin. In contrast, no degradation was noted after fixation with buffered formalin. These results demonstrate that HPV sequences can be identified in DNA extracted from paraffin-embedded, fixed tissue. However, use of some fixatives may preclude identification of HPV type, by either dot blot or in situ hybridization.     

Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues

Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.     

A molecular mechanism of formalin fixation and antigen retrieval

Despite the popularity of antigen-retrieval techniques, the precise molecular mechanism underlying the process remains enigmatic. We examined the molecular features underlying the loss of immunoreactivity following formalin fixation, with subsequent recovery by antigen retrieval. To do this, we first created a molecular model using short peptides that mimic the antibody-binding site of common clinical protein targets. The advantage of this model is that we know the amino acid sequence in and around the antibody-binding site. We observed that some, not all, of the peptides exhibited the formalin-fixation and antigen-retrieval phenomenon. Other peptides did not lose their ability to be recognized by antibody, even after prolonged incubation in formalin. A third, intermediate group exhibited the formalin-fixation and antigen-retrieval phenomenon only if another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicates that fixation and antigen retrieval are associated with a tyrosine in or near the antibody-binding site and with an arginine elsewhere, implicating the Mannich reaction as important in fixation and antigen retrieval.     

Influence of formalin fixation on tissue dimensions in palatal tonsils

Aim

To investigate the change of tissue dimensions after formalin fixation, and to determine the optimal time of fixation.

Hypothesis

Formalin fixation may lead to shrinkage in tissue dimensions and may thus alter tumor stages.

Background

It is often observed in tumor surgery that the dimensions in vivo seem larger than after resection, and tissue appears to shrink further after formalin fixation. This might alter dimensions and assessment of spread of the tumor and thus lead to a lesser tumor classification and stage. In cases where the decision for adjuvant chemoradiation is based upon the stage, it may thus be of relevance for the patient to evaluate the pathologic and not the in vivo dimensions of the tumor.

Material and methods

In order to obtain comparable tissues, we investigated 100 palatal tonsils after cold steel dissection tonsillectomy for chronic tonsillitis.There were four time points investigated: directly after excision in the operating room and after four, 24 and 72 h of fixation in formaldehyde (4% Formaldehyde in phosphate buffer pH 7.4). The tissue was measured in the following dimensions: volume (ml), weight (g) and length, broadness and width (mm).

Results

The tissue size did not change significantly in dimensions except for an increase in length. The time of fixation did not influence the size.

Discussion

Formalin fixation does not significantly influence the tissue dimensions of palatal tonsils in comparison to direct ex vivo measurements. A minimal time of fixation of 20 h is required in order to stop all degenerative processes; however, longer fixation does not change the dimensions of the specimen.

Conclusion

The null hypothesis has to be withdrawn that tissue dimensions are altered by formalin fixation. Thus, the histopathological measurements do not influence TNM staging.     

Bouin液和4%中性甲醛固定液对睾丸活检组织固定和HE染色差异的比较

在男性学发展过程中,睾丸活检曾经是惟一对无精子症或少精子症有诊断价值的检查.随着精液内一些附性腺的功能性指标检测技术的建立,生殖激素测定及与精子发生有关基因诊断技术的开展,睾丸活检已成为一种既可协助临床诊断又可指导临床治疗的方法.     

Rapid fixation and immunofluorescent staining of cultured cells using microwave irradiation

Abstract

Microwave irradiation during tissue fixation and immunostaining reduces the sample preparation time. Microwave irradiation also facilitates the penetration of fixatives and antibody solutions into the tissues, resulting in efficient fixation and reduction of non-specific antibody binding, respectively. Experimental procedures involving immunofluorescence microscopy are time-consuming as this method relies on antigenantibody reaction. Here, we utilized a technique involving exposure of cultured cells and tissues to intermittent microwave irradiation and immunostaining of fixed samples. Intermittent microwave irradiation during fixation reduces the required incubation time with blocking and antibody solutions, and results in good preservation of the immunoreactivity of fixed cells. Microwave irradiation also reduces the non-specific binding of fluorescein-labeled antibodies. These microwave-assisted rapid immunofluorescence techniques are useful for analysis of molecular compositions in cultured cell systems.     

Effects of formalin fixation on tissue optical polarization properties

Formalin fixation is a preparation method widely used in handling tissue specimens, such as biopsies, specifically in optical studies such as microscopy. In this note, we examine how formalin fixation affects the polarization properties of porcine myocardium and liver as assessed by optical polarimetry. Spatial maps of linear retardance and depolarization were derived from four myocardial and four liver samples before and after formalin fixation. Overall, linear retardance and depolarization increased after fixation for both myocardium (15% and 23% increase, respectively) and liver (38% and 51%, respectively). The relative increase in retardance was greater in liver compared to myocardium, although the absolute increase in retardance was comparable for both. The effect of fixation on bulk optical properties was also investigated for myocardium where the scattering coefficient increased from 92 to 132 cm(-1) and the absorption coefficient remained constant at 1.1 cm(-1).     

Histochemical and immunohistochemical evaluation of mouse skin histology: comparison of fixation with neutral buffered formalin and alcoholic formalin

Abstract

Histological analysis is a cornerstone of skin disease diagnosis, and the stratified nature of the skin presents many technical problems in the preparation of sections of acceptable quality. Furthermore, histological artifacts may be exacerbated in pathological states that compromise cutaneous integrity. Therefore, great care must be taken when selecting the conditions, particularly fixative type, to ensure that meaningful conclusions may be drawn. The goal of this work was to compare the morphology of mouse skin as revealed using a range of histological stains, and the antigenicity of key cutaneous proteins using immunohistochemistry (IHC) following fixation in either 10% neutral buffered formalin (NBF) or alcoholic formalin (AF). Although 10% NBF fixation is routinely used for histology, providing good retention of morphology and structure for special staining, AF fixation more effectively maintained antigenicity for the cutaneous markers investigated in this study. A comprehensive assessment of the two fixatives and histological techniques to optimize morphology, structure, and antigenicity in the analysis of mouse skin is presented.     

Biopsy findings in kidney transplants after treatment with cyclosporin A

51 renal transplant biopsies of 37 patients immunosuppressed with Cyclosporin A were histologically reevaluated. Lesions related as well as unrelated to Cyclosporin A treatment were found. Cyclosporin A related findings corresponding to the so-called Cyclosporin A nephropathy were divided into early postoperative (diffuse fibrosis), acute (toxic tubulopathy and others) and chronic changes (arteriolopathy, striped fibrosis with tubular atrophy). Since there is often a combination of Cyclosporin A related and unrelated lesions, it is necessary to define the main lesion in the individual case. Accordingly, the following diagnostic groups were present in the biopsies: Cyclosporin A nephropathy (19 cases), rejection nephropathy (18), rejection and Cyclosporin A nephropathy (9), acute renal failure (3) and other diagnoses (2 cases). For the further management of the patients, the exact diagnosis of Cyclosporin A related and unrelated pathology is important in renal biopsies.     

Pitfalls of formalin fixation for determination of antineutrophil cytoplasmic antibodies

Sera can produce nuclear or perinuclear immunofluorescence staining in neutrophils which may be caused by antibodies with differing antigenic specificities. These include perinuclear antineutrophil cytoplasmic antibodies (P-ANCA), granulocyte specific antinuclear antibody (GS-ANA), and antinuclear antibody (ANA). There is controversy over the value of formalin fixation of neutrophils in differentiating antibodies giving selective or preferential reaction with the nuclear or perinuclear area of neutrophils. In a comparative study of 77 sera, formalin fixation caused inconsistency, nonspecific effects, and false positivity owing to enhanced fluorescence. If formalin fixed neutrophils are used in the routine diagnostic laboratory, this will add confusion to the interpretation of the ANCA assay.     

Effect of heat-induced antigen retrieval following inconsistent formalin fixation.

The variable effects that inconsistent lengths of formalin fixation are known to have on the reactivity of tissue antigens were addressed by the application of a method for their heat-induced retrieval. Uniformly sized blocks of tonsil were fixed for increasing periods within 12 hours to 8 days before the heat retrieval and immunostaining of 30 tonsil antigens. The results were compared with their staining in the absence of heat retrieval, after prolongation of the standard retrieval time, and by use of a higher retrieval temperature. Consistent optimal staining of 26 of the 30 antigens was achieved despite the variable length of fixation. It is anticipated that the results will make a useful contribution to ongoing efforts toward the standardization of immunohistochemistry.