Neutral buffered formalin, usually simply shortened to NBF, has become the standard fixative for use in a diagnostic setting. It is more effective than the simple formalin mixtures as the phosphate salts present make it unlikely that erythrocytes will be damaged, and the neutral pH inhibits the formation of formalin pigment. The phosphates will adjust the pH to about 7.0 as the “neutral” infers, but there is no need to be ultra precise if it is slightly different.

Although the formation of formalin pigment is inhibited it is not stopped altogether, and it may slowly form in very bloody tissues, or in tissues stored for a long time in NBF without it being changed.

NBF is useful as a fixative for museum and photography specimens as it permits restoration of natural color to the specimen.

Time

This fixative should be applied overnight as a minimum, but fixation is not complete until applied for a few days, and a week or two is not too long. For thorough fixation the proteins in the tissue need to be crosslinked, but it is well known that simple formalin mixtures discolor tissues well before they crosslink the proteins. For that reason visual observation does not give any indication as to the degree of fixation, and discoloration should never be used as an indicator that it is complete.

It is essential that the time in fixative be noted, and sufficient time be allowed for the chemical reactions to occur. Time measured in a few hours is not adequate, and the lack of crosslinking in tissues treated for such a short time will not give adequate protection to the tissue from the fixation effects of dehydrating ethanols. Smaller pieces of tissue do not fix appreciably faster than larger pieces. Fine needle biopsies require the same length of time as 3 mm thick slices of solid organs.

On a practical basis in a diagnostic laboratory the slowness of simple formalin fixation is a distinct drawback. If the constraints of making a diagnosis are a major problem, then consideration should be given to increasing the temperature of the fixative solution. It must be emphasized that, although increasing the temperature of the fixative may increase the speed of fixation, it will cause a reduction in the quality of the final stained section and there may also be effects on some staining or immunohistochemical reactions. The temperature increase should be kept to a minimum consistent with accomplishing the goal of faster fixation, since heat itself is a means of fixation. If increased temperatures are used then formaldehyde fumes will be generated, more so as the temperature is increased, so fixation should be done in a fume chamber to protect personnel.

Complete organs are often placed in containers filled with NBF. Although formalin penetrates fairly rapidly, it will not reach the center of a large mass for some time and the delay may permit some autolysis in the middle of the tissue. Large organs should be described as soon as possible, then sliced into 1 cm or so slices to allow the fixative to gain access. Similarly, intestines should be opened and cleaned of any contents, then returned to a container large enough to permit the fixative to contact all the tissue surfaces, or pinned out on a board with rust free pins and placed into the fixative.

Tissues may be stored in this solution for long periods, but the fixative should be changed at least every six months.

Aftertreatment

NBF contains salts which have limited solubility in high concentrations of ethanol. For that reason the tissues should be transferred to a dehydrant containing 60% ethanol, or less, for a short time in order to give the salts an opportunity to be removed. If transferred directly to 95% or absolute ethanol the phosphates will most likely precipitate on and in the tissue, causing difficulties in sectioning, such as tearing and scoring. Processing machines should be flushed periodically with water to remove accumulated salts.

Secondary fixation

Other fixatives may be applied after formalin fixation, and some of their characteristics will be obtained. It must be realized that secondary fixation in any fixative will not give the same results as would have been obtained if the secondary fixative had been applied to fresh tissue.