Compound fixatives

Pathologists use formaldehyde-based fixatives to ensure reproducible histomorphometric patterns. Other agents may be added to formaldehyde to produce specific effects that are not possible with formaldehyde alone. The dehydrant ethanol, for example, can be added to formaldehyde to produce alcoholic formalin. This combination preserves molecules such as glycogen and results in less shrinkage and hardening than pure dehydrants.

Compound fixatives are useful for specific tissues, e.g. alcoholic formalin for fixation of some fatty tissues, such as breast, in which preservation of the lipid is not important. In addition, fixation of gross specimens in alcoholic formalin may aid in identifying lymph nodes embedded in fat. Some combined fixatives including alcoholic formalin are good at preserving antigen immunorecognition, but non-specific staining or background staining in immunohistochemical procedures can be increased. Unreacted aldehyde groups in glutaraldehydeformaldehyde fixation for example may increase background staining, and alcoholic formalin may cause non-specific staining of myelinated nerves (Grizzle et al. 1995, 1997, 1998a, 1998b; Arnold et al. 1996; Grizzle 1996b).

Factors affecting the quality of fixation Buffers and pH The effect of pH on fixation with formaldehyde may be profound depending upon the applications to which the tissues will be exposed. In a strongly acidic environment, the primary amine target groups (–NH2) attract hydrogen ions (–NH+3) and become unreactive to the hydrated formaldehyde(methylene hydrate or methylene glycol), and carboxyl groups (–COO−) lose their charges (–COOH). This may affect the structure of proteins. Similarly the hydroxyl groups of alcohols (–OH) including serine and threonine may become less reactive in a strongly acidic environment. The extent of formation of reactive hydroxymethyl groups and cross-linking is reduced in unbuffered 4% formaldehyde (Means &Feeney 1995), which is slightly acidic (French & Edsall 1945), because the major methylene crosslinks are between lysine and the free amino group on side chains. The decrease in the effectiveness of formaldehyde fixation and hence cross-linking in such a slightly acid environment has led some

authors to suggest that unbuffered formalin is a better fixative than NBF with respect to immunorecognition of many antigens (Arnold et al. 1996; Eltoum et al. 2001b). This no doubt aided the detection of antigens before the early 1990s, prior to the advent of heat- induced epitope retrieval methods in immunocytochemistry. However, minimal delay in effectively fixing very labile antigens, such as the estrogen receptor, is vital in the immunohistochemical testing for a range of clinically important prognostic and predictive biomarkers. Whilst formaldehyde fixation remains the recommended method for optimal preservation of morphological features, proteins and nucleic acids in a clinical environment, the most reliable way of achieving optimal formalin fixation is through its buffering at pH 7.2–7.4 (i.e. neutral buffered formalin). At the acidic pH of unbuffered formaldehyde, hemoglobin metabolic products are chemically modified to form a brown-black, insoluble, crystalline, birefringent pigment. The pigment forms at a pH of less than 5.7, and the extent of its formation increases in the pH range of 3.0 to 5.0. Formalin pigment is recognized easily and should not affect diagnoses except in patients with large amounts of hemoglobin breakdown products secondary to hematopoietic diseases.

The pigment is removed easily with an alcoholic solution of picric acid. To avoid the formation of formalin pigment, neutral buffered formalin is used as the preferred formaldehyde-based fixative.

Acetic acids and other acids work mainly through lowering pH and disrupting the tertiary structure of proteins. Buffers are used to maintain optimum pH. The choice of specific buffer depends on the type of fixative and analyte. Commonly used buffers are phosphate, cacodylate, bicarbonate, Tris, and acetate. It is necessary to use low salt-buffered formalin in the new complex tissue processors in order to keep the machine ‘clean’, and reduce problems in its operation.

Duration of fixation and size of specimens The factors that govern diffusion of a fixative into tissue were investigated by Medawar (1941). He found that the depth (d) reached by a fixative is directly proportional to the square root of duration of fixation (t) and expressed this relation as: d = k ⌡ t