COMPARATIVE STUDY BETWEEN OPTIMAL AND REDUCED FORMALIN-BASED FIXATIVE FOR NORMAL AND PATHOLOGICAL TISSUES

Objectives: This study aims to use an alternative fixative compound with reduced concentration of formalin to minimize the exposure and related side effects. Methods: Human normal and pathological tissue samples of different organs were immediately immersed in a modified fixative containing formalin, glycerin, ethanol, and hypotonic saline with pH around 7. Two time schedules were used at 8 and 12 h. Tissue samples were undergone routine processing procedure used in histopathological laboratories, scoring of samples was done, tissue sections of new compound fixative were examined for cytoplasmic, nuclear, and architectural properties and compared with 10% neutral buffered formalin (NBF). Results: Tissue samples treated with modified fixative showed good and acceptable preservation of structural, cellular details with less harmful effects when compared with those preserved with routine 10% (NBF). Conclusion: The suitability and effectiveness of reduced formalin-based fixative in histological and histopathological routine examination with minimal harmful effects for laboratory worker were clearly indicated in this study.


INTRODUCTION
Fixation of tissues is necessary for effective dissection, processing, and microscopical inspection of histopathological specimens. Phosphatebuffered formalin (10%) is used long time ago as fixative of choice. Formalin has many characteristic properties such as easy to use, low cost, and good morphological conservation, it infiltrates tissue rapidly and tolerates specific stains [1,2]. Formalin toxicity remains a major threatening factor for laboratory workers due to chronic and long use through daily practice [3].
Formaldehyde is known to have acute and chronic damaging effects on the health of exposed groups such as anatomy students, anatomists, embalmers, cadaver handlers, and pathologists [4][5][6]. Formalin is absorbed efficiently from the mucous membranes of eyes and respiratory tract causing local irritation, a sudden contact with high doses may cause pulmonary edema while repeated or prolonged contact can lead to allergic manifestations [7][8][9][10]. Many studies linked formalin to the high incidence of hematopoietic and lymphoid malignancies among anatomists and embalmers in the United States [11][12][13]. It may alter gene expression and affect the signaling associated with cancer, inflammatory response, and endocrine system [4,14,15]. Multiple human and animal studies are available in literatures which support the mutagenic, carcinogenic, and teratogenic potential of formaldehyde [16,17].
A number of reports from the International Agency for Research on Cancer and others correlate leukemia to formalin exposure; therefore, strict limits for exposure were monitored worldwide [18][19][20][21]. This study aims to evaluate the suitability of reduced formalin compound fixative and to minimize the exposure with reasonable validity and less harmful effects.

METHODS
A compound fixative contains different minimal concentrations of 10% formalin 7 ml in addition to glycerin 5 ml, absolute alcohol 20 ml, and hypotonic saline, the pH is adjusted near 7. Alcohol used for dehydration can cause shrinkage of cells while adding of hypotonic saline is beneficial to reduce the dehydration in addition, glycerin will minimize evaporation, the fixative prepared has a light blue color by adding methylene blue 0.05 g. Buffers such as sodium dihydrogen phosphate monohydrate 4 g and anhydrous disodium hydrogen phosphate 6 g were added. The pH is maintained near 7 and then the solution is completed to 100 ml by adding of hypotonic saline.
Different human histological samples from various sites were arranged into two categories, normal and pathological; fixation was done by two methods, the first using 10% neutral buffered formalin (NBF) and the second by the new fixative, in which samples were directly fixed at two different time schedules; 8 and 12 h then completed by classical conventional tissue processing procedure. Four-micron thickness sections were obtained, stained with hematoxylin and eosin, examined, and scored for nuclear, cytoplasmic, and architectural assessments; the time table for each procedure was linked to both modified and conventional 10% NBF. The nuclear assessment was evaluated by examining the size, membrane maintenance, chromatin pattern, and mitotic entity, while cytoplasmic features were shown for the color, abundance, granules, and mucin, to assess the architectural criteria, the staining properties, integrity of membranes, pigment, and artifacts deposition were observed.
In general, if nuclear, cytoplasmic, or structural features are ill defined, it is considered as poor with score 1, less defined features were given score 2, and score 3 was given for good or well-preserved Alharoon et al.
tissues (Table 1). For 10% NBF fixative, the nuclear, cytoplasmic, and architectural properties were considered as an absolute fixation (optimal) and given score 3. The results were tabulated and analyzed. p<0.05 is regarded as statistically significant.
The architectural, cellular, and nuclear details of normal and pathological tissues fixed in the modified compound were compared with optimal fixative, for the consistency of tissues both illustrate the same features, but for cytoplasmic changes, the comparison showed that only two specimens had faint color and less noticeable cytoplasmic granules at 8 h, Fig. 1 (score 2). Commonly, a good preservative feature was obtained by the new fixative for both normal and pathological specimens at 12 h (score 3). No statistically significant differences were found when it is compared with 10% NBF (Table 2 and Figs. 1-3).
For nuclear changes, the majority of specimens (46) fixed in the new fixative obtained the same nuclear criteria (no nucleoli changes and constant mitotic figures) at 8 and 12 h. No significant difference was detected when compared with 10% NBF (Table 3 and Figs. 4-6).

DISCUSSION
An optimal fixative should be non-toxic with detailed morphology, highquality histochemical and immunohistochemical staining properties, respectable preservation of nuclear details, and a reasonable price [22].
The toxicity of formaldehyde is evolving as the main issue for its diminishing as a general fixative used in large amounts in histopathology. Laboratory workers are frequently in contact with different formalin concentration and vapor in addition to the role of formalin as chemical carcinogen which should be given attentions. Recently, chromosomal variations have been noticed in laboratory workers of pathological fields [16,23,24].
Many attempts have been tried to replace formalin with other fixatives being harmless and frequently used. A non-cross-linking fixative such as FinFIX (Milestone, Bergama, Italy) [25] and RCL2 (Alphelys, Plaisir, France) [26] has been proposed as NBF alternatives. Advantages of this type of fixation include quick fixation, dismissal of carcinogenic vapor.
DNA and RNA glycogen defined preservation, the disadvantages are tissues that have faint color, variability of tissue staining, hardening, artifact pigment deposition in bloody specimens, partial or complete lysis of erythrocytes, and increased flammability compared with 10% NBF. Other alternatives include PAGA, ZBF, Z7, and cell block which show faint color and tissues are soft with slippery consistency and difficult for processing [26].
The current study showed that the new fixative had light blue color with good consistency, had significantly less irritant odor; besides, it is easily processed and suitable for light microscopical examination. The two fixatives showed no significant differences at 8 and 12 h and both are acceptable for nuclear and cytoplasmic preservation, by reducing the formalin concentration from 10 to 7, the exposure will be reduced, furthermore, alcohol acts by eliminating water molecule from tissues which lead to cell contraction [27], to tolerate this, the hypotonic saline is added, good and acceptable fixative will be obtained.

CONCLUSION
The present study demonstrates that reduced formalin fixative can easily be prepared and replaced the 10% NBF for routine histopathological laboratory work with easy and less toxic side effects. Further studies involving this new fixative in histochemical and immunohistochemical tissue procedures will be recommended.

ACKNOWLEDGMENT
The authors are especially grateful to the laboratory assistants who have helped to complete this research.

AUTHORS' CONTRIBUTIONS
Dr. Nawal and Dr. Sawsan contributed to the collection of the tissue samples and preparing the slides for examination; Dr. Nawal