Formaldehyde Substitute Fixatives Appraised for Histology

In histology and pathology laboratories, neutral buffered formalin (NBF) has been the gold standard fixative for decades, but formaldehyde is known to be toxic.

Other less toxic alcohol-based cross-linking or non–cross-linking fixatives have advantages that include faster fixation; elimination of carcinogenic vapors; better preservation of glycogen, DNA, and RNA; greater staining avidity; and no need for enzyme predigestion for immunohistochemical analysis.

Scientists at the University Medical Center Utrecht (the Netherlands) tested three cross-linking and non–cross-linking alcohol-based fixatives for routine staining in comparison with NBF, an aqueous solution of 4% buffered formaldehyde. The aim was to observe the fixation on tissue morphologic features and the quality of special histological stains and immunohistochemical stains in comparison with NBF.

Fresh tissue samples from many different organs were divided into four equal pieces and fixed in all fixatives for varying times. After paraffin embedding, staining with hematoxylin and eosin (H&E), seven common histochemical stains, and nine common immunohistochemical stains were performed. Fixation with the non-linking RCL2 (Alphelys; Plaisir, France) resulted in soft and slippery tissue, causing sectioning difficulties. The cross-linking the aldehyde-containing F-Solv (Adamas; Rhenen, The Netherlands) and the non–cross-linking FineFIX (Milestone; Bergamo, Italy) led to partial tissue disintegration during fixation.

F-Solv performed morphologically similar to NBF but needed considerable protocol adjustments before being applicable in daily histology and immunohistochemical practice. FineFIX did not necessitate major protocol changes but caused shrinkage artifacts, degranulation, and lysis of red blood cells (RBC). RCL2 generated morphologically overall good results without major protocol changes, but caused pigment deposition, degranulation, and erythrocytic lysis. The alcohol-based fixatives had positive and negative attributes and environmental drawbacks, and none was overall comparable to NBF with regard to macroscopy, morphologic evaluation, and immunohistochemical studies.

The authors concluded that before replacing formalin with another fixative in a surgical pathology laboratory, all levels of pathology must be examined thoroughly. At the histochemical and the immunohistochemical levels using the usual NBF-based protocols, NBF performance was superior, followed by FineFIX/RCL2 and F-Solv. The study was published online in for the October 2011 edition of the Journal of Clinical Pathology.

Related Links:
University Medical Center Utrecht
Alphelys
Adamas