Influence Of Formalin Fixation Research Articles

Determination of phosphorylated proteins in tissue specimens requires high‐quality samples collected under stringent conditions

For selection of patients who will benefit from targeted therapies, identification of biomarkers predictive of treatment response is desirable. Activation of the targeted pathway becomes apparent by protein phosphorylation. Determination of this phenomenon is therefore considered a promising biomarker approach. To date, however, it is unclear whether routinely collected tissue specimens allow determination of in-vivo phosphorylation states. To investigate whether routinely collected tissue specimens retain the true phosphorylation states of a tumour's proteins, we compared protein phosphorylation states between matched tumour samples that were subjected to different ischaemic times by immunohistochemistry. The influence of formalin fixation and paraffin-embedding on phosphorylation states was investigated by comparison of matched fresh frozen and formalin-fixed paraffin-embedded surgical specimens as well as small biopsies. We show that ischaemia influences protein phosphorylation in a tumour-specific, unpredictable manner. Formalin fixation and paraffin-embedding lead to a decrease in detectable protein phosphorylation in larger surgical specimens, but not in small biopsies. Determination of protein phosphorylation using routinely collected surgical specimens results in artefacts which do not reflect a tumour's true states of pathway activation. Valid measurement of phosphorylated biomarkers requires that tissue collection procedures are tightly controlled, avoiding ischaemia and large-specimen fixation.

Influence of formalin fixation on tissue dimensions in palatal tonsils

AimTo investigate the change of tissue dimensions after formalin fixation, and to determine the optimal time of fixation. HypothesisFormalin fixation may lead to shrinkage in tissue dimensions and may thus alter tumor stages. BackgroundIt 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 methodsIn 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 72h 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). ResultsThe tissue size did not change significantly in dimensions except for an increase in length. The time of fixation did not influence the size. DiscussionFormalin fixation does not significantly influence the tissue dimensions of palatal tonsils in comparison to direct ex vivo measurements. A minimal time of fixation of 20h is required in order to stop all degenerative processes; however, longer fixation does not change the dimensions of the specimen. ConclusionThe null hypothesis has to be withdrawn that tissue dimensions are altered by formalin fixation. Thus, the histopathological measurements do not influence TNM staging.

Formalin Fixation Could Interfere with the Clinical Assessment of the Tumor-Free Margin in Tumor Surgery: Magnetic Resonance Imaging-Based Study

Objectives: The tumor-free margin in bone and soft-tissue cancer is a key factor for subsequent treatment. While flattening and shrinkage of specimens after formalin fixation have been described in breast cancer, there are no data for bone and soft tissue sarcoma. Fixation could interfere with the accuracy of the assessment of the tumor-free margin. Methods: The influence of formalin fixation was assessed on forelimb specimens in a preclinical porcine model. The specimens were subjected to magnetic resonance imaging before and after formalin fixation. Weight, width and height of the specimen were measured and different consecutive volumes (total, muscles, bones and fatty tissue) were obtained by segmentation. Results: After formalin fixation, the weight increased, total volume and muscle volume slightly increased while bone did not change and fatty tissue decreased. The width of the specimens increased while their height decreased. Conclusions: Formalin fixation caused slight muscle expansion, fatty tissue shrinkage and flattening of the specimen. These changes could interfere with the assessment of the tumor-free margin in clinical practice.

Measuring mouse sperm parameters using a particle counter and sperm quality analyzer: A simple and inexpensive method

This study examined a method for analyzing the count, motility, and morphology of mouse epididymal sperm, optimizing the diluent, incubation time, sample concentration, and temperature, using a particle counter (CDA-500) to count and size sperm and a sperm quality analyzer (SQA-IIC) to measure sperm motility, quantified as the sperm motility index (SMI). The optimal conditions consisted of a 30-min incubation in D-MEM (Dulbecco's modified Eagle's medium; considering cost and availability) at 37 °C, with 5 × 10 6 cells mL −1 in the original solution. Furthermore, the influence of formalin fixation, and the correlation between the automated counter and a manual method were investigated. The sample fixation had no marked effect on the sperm count or morphology assessment. A linear correlation was observed between the manual and automated methods ( y = 0.920 x + 0.276; r 2 = 0.571; p < 0.001; range: (3–6) × 10 6). The suitability of the proposed method was confirmed using spermatozoa prepared from mice treated with the reproductive toxin diethylstilbestrol (DES). Using sperm from the cauda epididymidis on one side per mouse, we confirmed that measurement of these sperm parameters using the two devices was simple, rapid, inexpensive, and reproducible.

Anisotropy of the slope of ultrasonic attenuation in formalin fixed human myocardium.

Clinical implementation of quantitative ultrasonic tissue characterization is likely to require imaging the heart with sound propagating at varying angles relative to the fibers of the heart. Under these circumstances, the variation of the ultrasonic properties of myocardium with the angle of propagation relative to the myofibers may represent a significant source of potential misinterpretation. In the present study, the systematic approach of assessing the impact of anisotropy on quantitative myocardial tissue characterization is extended by reporting results of a recent in vitro study to measure the anisotropy of the slope of ultrasonic attenuation in specimens of formalin fixed human myocardium. Data obtained from regions of remote infarct are presented and compared to data acquired from regions identified to be free of infarct. The slope of attenuation for both regions exhibit a sinusoid-like dependence on angle that is approximately doubled for propagation parallel to the fibers as compared to perpendicular. These results are, in turn, compared to an earlier study from the laboratory that examined the effects of myocardial infarction on ultrasonic attenuation and interstitial collagen content in freshly excised canine hearts. Discussion regarding the analysis and interpretation of measurements of slope of attenuation is presented as well as a discussion of the possible influence of formalin fixation on our results.

Influence of formalin fixation on the detection of cytomegalovirus by polymerase chain reaction in immunocompromised patients and correlation to in situ hybridization, immunohistochemistry, and serological data.

The possibility of detecting cytomegalovirus (CMV) in formalin-fixed tissues by polymerase chain reaction (PCR) was evaluated in necroposies from lung tissues in a total of 24 patients who either had received organ transplants or were immunocompromised. PCR using two different pairs of primers for amplification of the major immediate early antigen of CMV was performed on fresh tissues and tissues fixed for 24, 48, and 72 h in neutral buffered formalin and compared to immunohistochemistry (IHC) and in situ hybridization (ISH). The fresh tissues of nine patients with serological evidence for acute CMV infection were all positive for CMV by PCR. After formalin fixation, the majority of the patients failed to show distinct signals with one or both pairs of primers as measured by densitometry. In contrast to this, fresh tissues of 15 patients without signs of an acute CMV infection were found either negative or weakly positive by PCR. Using IHC or ISH, positive results were observed only in five of nine and four of nine patients with acute CMV infection, respectively. These data demonstrate that, if only formalin-fixed tissue is available, PCR for CMV detection should be performed using two pairs of primers and should be supported by IHC.

The use of tetranitro-blue tetrazolium for the cytochemical localization of succinic dehydrogenase. Cytochemical and cytological studies of sarcoma 37 ascites tumor cells.

Succinic dehydrogenase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor were evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazan- or iron hematoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone pretreatment, on mitochondrial morphology and enzymatic staining. Data from these studies appear to indicate that TNBT is more suitable than NBT for use as a cytochemical reagent in oxidative and/or dehydrogenase enzyme histochemistry and cytochemistry.