An impeccable sample fixation is a prerequisite for a correct histological diagnosis. Tissue samples must be immersed in an optimally chosen fixative immediately after sampling, because a timely fixation will prevent autolysis, putrefaction and other unwanted cellular changes. Although there are hundreds of histological fixatives and at least tens of formaldehyde-based fixatives, neutral buffered formaldehyde solutions with a concentration range from 4% to 10% are the most commonly used fixatives, primarily because of their simple and universal application.
ImmunoForm is a fixative produced by melting paraformaldehyde in buffer solution, without added methyl alcohol. The final formaldehyde concentration is 4% (10% formalin solution), and advantage in using paraformaldehyde is higher oxidation stability and creates 10 times lesser amount of formic acid, chemical responsible for formalin pigment.
Tissue fixation using a buffered formaldehyde solution results in forming cross-links, i. e. it forms methylene bridges between proteins, that is, it results in keeping tissue components in their in vivo relation. If fixated properly, the tissue sample can withstand additional histological tissue processing and staining. It is suitable for fixating bioptic materials, smaller tissue samples and long-term fixated samples storing. Optimal molarity phosphate buffer (0.05 M) is used for securing permanent pH range between 6.90 and 7.1 at 25C. It is suitable for usage in all automated devices for tissue processing, as well as for manual histological techniques. It is conveniently packaged in 1 liter bottles, 5 and 10 liter canisters.
IMMUNOFORM - 10% neutral buffered and stabilized formalin solution produced by melting paraformaldehyde, without added methyl alcohol. Suitable for fixating histology samples, recommended for immunohistochemistry methods.
If the tissue was not properly stored or stabilized during the fixation process, or if an unsuitable fixative was used, all the subsequent procedures in tissue processing and diagnostics will be of mediocre quality or useless. If the fixative is of inferior quality, pH value over its physiological bounds, or if the volume ratios between tissue and active substance in the fixative are not suitable, improper fixation can occur as well as tissue degradation and misdiagnosis. For that reason the fixative must be produced in accordance with the in vitro diagnostic devices norms and must bear the CE marking of conformity, and the processes of fixating, processing and staining must be carried out by a qualified person (histotechnician). In order to avoid mistakes during the procedure, a suitable fixative must be applied in accordance with the standard norms of histotechnology. If there is uncertainty regarding the choice of the fixative and possibility that the tissue would not be stored in a satisfactory manner, consultation with an experienced histotechnician is required.
Always wear protective gloves while handling formaldehyde and fixated tissue samples. The rooms in which the buffered formaldehyde is being used should be well aerated by using an exhaust fan or a digester in order to remove toxic evaporation. Additional security information can be found in the Material Safety Data Sheet of this product.
Before the process a fixative should be chosen in accordance with the subsequent histological, histochemical or immunohistochemical diagnostic methods. If ImmunoForm was chosen as an optimal fixative, the tissue sample should be immediately immersed in the solution container.
The sample should be fixated as soon as possible in order to prevent autolysis, putrefaction, and other changes. If it is not possible to put the sample in the fixative immediately, it is advised to maintain it moist and keep it in a cold place. The sample should not be bent or folded in the fixation container. The samples must not be thicker than 1 cm for adequate fixation. All the samples should be clearly marked.
During the fixation the sample should be immersed in an adequate amount of fixative. Tissue and fixative ratio must be 1:50.
The fixative can also be poured into hollow organs, and before immersing into the fixative container they can be filled with gauze soaked with the fixative. Certain organs, such as the colon, can be opened and pinned on a board before immersing in the fixative. Encapsulated tissue should be processed by an expert in order for the fixation to be successful.
Fixation time can vary from a few hours to a few weeks. That depends on the type of tissue and sample thickness, fixation temperature, tissue and fixative volume ratio, as well as the concentration of formaldehyde in the fixative.
Selection of concentration of formaldehyde and fixation time must be determined in accordance with the norms of histotechnology and professional experience. Samples up to 5 mm thick should be fixed for 5 hours at room temperature, while thicker samples should be fixed for 1-2 days. The process can be shortened by fixating the sample in an incubator or a microwave oven.
If the tissue has not been dimensioned for processing prior to fixation, after the fixation it should be processed down to thickness of 3-5 mm. The first solution to come into contact with the tissue should be 70% alcohol solution in order to avoid the subsidence of phosphate salts used to neutrally buffer the formaldehyde solution. Fixing can be conducted using formalin fixatives containing buffers soluble in alcohol.