Comparing the Quality of Fixation for Gel-based Formalin (Formagel) versus Traditional Liquid-Based Formalin for Immunohistochemistry Brian H. Le, M.D., Reading Hospital Reviewed by Michael R. LaFrinere, HT (ASCP) Introduction As the first stage of a multistep process to prepare surgical specimens for microscopic analysis, tissue fixation must demonstrate the ability to preserve the architectural and morphologic integrity of cells and their supporting matrix. Additional criteria for an effective fixative medium includes the ability to withstand the treatments involved in all steps associated with tissue preparation, including specimen transport, and the processing steps that encompass tissue dehydration, embedding, microtomy and staining. For specialized techniques, such as immunohistochemistry, an effective fixative must also demonstrate the ability to retain tissue antigenicity such that the subsequent steps of antigen retrieval can be optimally executed. Currently, 10% neutral buffered formalin serves as the gold standard for fixation of tissue in preparation for routine histologic, histochemical, and immunohistochemical assessments. However, as a non-viscous liquid preparation, it is prone to leakage during transport of pre-filled specimen containers, thus posing potential hazards for laboratory couriers, as well as for commercial ground and air transportation carriers. Additionally, in a liquid form, volatility of the substance is high, and this may further increase the health hazards to laboratory personnel. To address these concerns, a 10% formalin gel preparation (Formagel) was formulated by Azer Scientific, Inc. (Morgantown, PA) as a substitute to the traditional liquid based, 10% neutral buffered formalin. This formalin preparation is more viscous, less prone to leakage in
prefilled specimen containers, and has less volatility as a mucosal irritant. Validation testing performed by two independent laboratories (LaFriniere and Lu, unpublished data) has demonstrated equal efficacy between Formagel and 10% neutral buffered formalin with respect to hematoxylin uptake, eosin uptake, cellular preservation and detail, staining as well as background cleanliness. This study attempts to additionally characterize the efficacy of Formagel as a fixative for the purpose of immunohistochemistry, an important and commonly employed technique in modern anatomic pathology. Materials and Methods A prospective, side-by-side study was undertaken to compare the quality and acceptability of tissue fixation with 10% neutral buffered formalin versus Formagel for the purpose of immunohistochemistry. Twenty cases were randomly selected for study, sampling tissues commonly encountered in surgical pathology. For each specimen that arrived in the laboratory without fixative, a block of tissue was obtained from a representative area, demonstrating either grossly normal organs, or part of a large neoplasm. The block of tissue, measuring approximately 1 cm cubed, was divided in half; one half was placed in a jar prefilled with approximately 10 ml of Formagel, while the other half was placed in a jar with approximately 10 ml of 10% neutral buffered formalin. Tissue was allowed to fix, with fixation times (varying from 5 hours to 26 hours) matched for the Formagel and formalin samples. Subsequent to fixation, Formagel and formalin treated samples were treated identically and placed in histologic cassettes for processing using the Sakura Tissue-
Tek VIP processor. No special techniques were required in the handling of the Formageltreatment samples. Following routine histologic processing, an H&E stained section of each sample (20 formalin fixed, 20 Formagel fixed) was examined to ensure acceptable morphology. Subsequently, based on the origin of the tissue, commonly utilized antibodies were selected for comparative assessment. The immunoreactivity for each tissue section under study (20 Formagel-fixed, 20 formalin-fixed) was examined and scored for intensity on a tier-system (0, 1, 2, 3) by a pathologist who was blinded to the fixation medium. Results A summary of the immunoreactivity (intensity) of tissues fixed with Formagel versus traditional neutral buffered formalin is presented in Table 1. In terms of the quality of histology, formalin and Formagel treated samples yielded tissue sections on slides demonstrating clean backgrounds. No excess stains or chromogenic substrates were noted on slides with tissue treated by either methods, neither around the tissue edges or in blank areas of the slides. With respect to the various intermediate filaments associated with epithelial differentiation (pan-cytokeratin, AE1/AE3, CK 5/6, CK7, CK20, K903), there was no difference in immunoreactivity for Formagel-fixed versus formalin-fixed samples. In addition, no
significant difference in immunoreactivity for Formagel-fixed and formalin-fixed samples was observed with mesenchymal markers (vimentin, desmin, smooth muscle actin). Additionally, the immunoreactivity for membrane-associated antigens (epithelial membrane antigen, placental alkaline phosphatase), nuclear antigens (p63, estrogen receptor, progesterone receptor), and selected clustered differentiation hematolymphoid markers (CD34, CD45) was identical for Formagel-treated samples versus formalin-treated samples. Image Results Image 1: Breast Tissue, ER Stain Image 2: Breast Tissue, PR Stain
Image 3: Fallopian Tube Tissue, Pan-Cytokeratin Stain Image 4: Kidney Tissue, CD34 Stain Image 5: Tonsil Tissue, LCA Stain
Discussion This is a prospective, side-by-side study comparing the quality and acceptability of 10% gel-based formalin preparation (Formagel) versus the traditional, liquid-based 10% neutral buffered formalin as it pertains to immunohistochemical assessments. For each sample, unfixed tissue arriving from the operating room was procured and divided into two small, similarly sized fragments, each of which was allowed to fix in either of the formalin preparation for an identical period of time prior to further processing; tissue origin, sample size, fixative amount and fixation times were thus variables controlled across the two fixatives. Based on site of origin, antibodies were selected to accentuate key cellular constituents. Tissue immunoreactivity was assessed with regards to intensity on a tiered grading system. The global results demonstrate that Formagel is just as effective as the traditional liquidbased, 10% neutral buffered formalin in terms of intensity of immunoreactivity across all major classes of antibodies commonly employed in diagnostic surgical pathology. This includes antibodies directed against intermediate filaments (cytokeratins, vimentin), and other mesenchymal markers (smooth muscle actin, desmin), hematolymphoid markers (LCA, CD34). Immunoreactivity was also similar among liquid formalin treated specimens versus gel-based formalin fixed specimen with respect to antigen localization within a cellular structure, such as membrane-bound (epithelial membrane antigen, placental alkaline phosphatase), intracytoplasmic (keratins), and nucleus-associated (ER, PR, Ki-67). As a formalin product with identical concentration to its traditional liquid counterpart, Formagel is expected to facilitate retention of antigenicity and immunoreactivity. It is possible that as a gel-based preparation with less volatility, Formagel may be better able to prevent
tissue disintegration and antigenic dissipation; as a result, this physical property may in fact even enhance morphologic and antigenic preservation for the purpose of routine microscopic examination, and for specialized techniques such as immunohistochemistry. Conclusion Formagel is an effective and practical alternate fixative to traditional, neutral buffered formalin for the purpose of antigenicity retention and preservation. The immunoreactivity of tissue fixed in Formagel is at least equivalent to that of tissue fixed in neutral buffered formalin across a wide spectrum of antibody/antigen types and classes.