Tissue fixation is a fundamental step in histopathology, ensuring the preservation of cellular and tissue architecture for diagnostic accuracy. The ideal fixative should maintain morphological integrity while preserving antigenicity for downstream applications such as immunohistochemistry (IHC). Among various fixatives, 10% neutral buffered formalin (NBF) has long been regarded as the gold standard due to its ability to provide excellent structural preservation through protein cross-linking (1). However, formalin fixation has limitations, including prolonged fixation time, potential health hazards due to formaldehyde exposure, and reduced immunoreactivity of some antigens resulting from epitope masking (2,3).

In recent years, alcohol-based fixatives have gained attention as alternatives to formalin. These fixatives, typically composed of ethanol or methanol in combination with acetic acid, act by protein precipitation rather than cross-linking, thereby minimizing epitope alteration and improving antibody accessibility during IHC (4). Several studies have demonstrated that alcohol-based fixatives can preserve tissue morphology adequately while enhancing immunostaining results for specific markers such as cytokeratin and CD antigens (5,6).

Despite these findings, the adoption of alcohol-based fixatives in routine diagnostics remains limited, primarily due to concerns over tissue shrinkage, brittleness, and variability in staining characteristics. Therefore, comparative studies evaluating both morphological and immunohistochemical performance are essential to assess the feasibility of replacing or supplementing formalin fixation in routine histopathology (7). This study aims to evaluate and compare the effects of formalin and alcohol-based fixatives on tissue morphology and immunostaining outcomes using commonly employed markers, thereby providing insights into optimizing fixation protocols for improved diagnostic accuracy.

A total of 60 freshly obtained human tissue samples were included, comprising 30 liver biopsies and 30 lymph node specimens. The tissues were collected immediately after surgical excision and were divided into two equal portions. One half of each specimen was fixed in 10% neutral buffered formalin (NBF), while the other half was fixed in an alcohol-based fixative composed of 70% ethanol, 5% acetic acid, and 25% methanol.

After fixation for 24 hours at room temperature, all tissue samples were processed routinely using a standard paraffin-embedding protocol. Sections of 4–5 µm thickness were prepared from each paraffin block and mounted on glass slides. Hematoxylin and eosin (H&E) staining was performed for morphological evaluation. The parameters assessed included nuclear detail, cytoplasmic clarity, tissue shrinkage, and overall architectural preservation, graded on a semi-quantitative scale from 0 (poor) to 3 (excellent).

For immunohistochemical (IHC) analysis, two markers were used: cytokeratin (for epithelial cells) and CD3 (for T lymphocytes). IHC staining was performed using the standard avidin-biotin peroxidase complex method. Antigen retrieval was done using citrate buffer (pH 6.0) in a microwave oven for 15 minutes. Diaminobenzidine (DAB) was used as the chromogen, followed by counterstaining with hematoxylin.

The intensity of immunostaining was evaluated under a light microscope and graded as 0 (no staining), 1+ (weak), 2+ (moderate), or 3+ (strong). Background staining and tissue integrity were also noted. Statistical analysis was performed using SPSS software (version 25.0). Differences in morphological and immunohistochemical scores between the two groups were assessed using the paired t-test and chi-square test, with p < 0.05 considered statistically significant.

A total of 60 tissue samples were evaluated, including 30 liver and 30 lymph node specimens. All tissues were assessed for morphological preservation using H&E staining and immunohistochemical staining for cytokeratin and CD3.

Morphological Evaluation

Formalin-fixed tissues displayed superior nuclear detail and cytoplasmic clarity in comparison to alcohol-fixed specimens. The average nuclear detail score for formalin-fixed tissues was 2.7 ± 0.3, while that for alcohol-fixed tissues was 2.3 ± 0.4. Similarly, the architectural integrity score was higher in formalin-fixed tissues (2.6 ± 0.2) than in alcohol-fixed samples (2.1 ± 0.3). However, alcohol-fixed tissues exhibited more noticeable shrinkage artifacts.

Table 1: Comparison of Morphological Features Between Formalin and Alcohol-Fixed Tissues (n=60)

Table 1 shows that formalin-fixed samples scored significantly better for nuclear and cytoplasmic preservation, while alcohol-fixed tissues showed increased shrinkage.

Immunohistochemical Evaluation

Immunostaining intensity was higher in alcohol-fixed tissues for both cytokeratin and CD3 markers. Strong (3+) cytokeratin staining was observed in 86.6% of alcohol-fixed samples versus 63.3% in formalin-fixed ones. Similarly, CD3 showed 3+ staining in 83.3% of alcohol-fixed versus 66.6% of formalin-fixed tissues. Background staining was more prominent in formalin-fixed samples.

Table 2: Immunohistochemistry Staining Intensity Scores (n=60)

As illustrated in Table 2, alcohol-fixed tissues showed stronger IHC staining with less background noise compared to formalin-fixed samples.

These results suggest that while formalin fixation provides better morphological preservation, alcohol-based fixatives enhance antigen retrieval and staining intensity in IHC (Tables 1 and 2).

The present study aimed to evaluate and compare the effects of formalin and alcohol-based fixatives on tissue morphology and immunohistochemical (IHC) staining outcomes in routine histopathology. Our findings reaffirm the conventional understanding that formalin fixation provides superior morphological preservation, while alcohol-based fixatives enhance antigenicity, making them more favorable for IHC applications.

Formalin, particularly 10% neutral buffered formalin (NBF), remains the most widely used fixative due to its strong cross-linking ability, which preserves cellular detail and prevents autolysis (1). It has been the standard fixative in diagnostic histopathology for decades because of its consistency in preserving nuclear and cytoplasmic structures (2). In our study, formalin-fixed tissues showed better nuclear detail and architectural integrity compared to alcohol-fixed samples, consistent with findings from previous studies (3,4).

However, the formalin fixation process can mask antigenic sites due to the formation of methylene bridges between proteins, resulting in reduced antibody binding during IHC procedures (5). This limitation often necessitates rigorous antigen retrieval techniques, which may not always yield optimal results for all markers (6). In contrast, alcohol-based fixatives act through protein coagulation and dehydration, which preserve antigenic sites more effectively, resulting in stronger immunoreactivity (7).

Our results showed enhanced staining intensity for both cytokeratin and CD3 markers in tissues fixed with alcohol-based fixatives, corroborating previous reports that demonstrated similar improvements in IHC staining, especially for epithelial and lymphoid markers (8,9). Studies have also reported that alcohol fixation reduces the need for harsh antigen retrieval methods, thereby preserving tissue integrity and reducing processing time (10).

Despite these advantages, alcohol-based fixatives are not without drawbacks. They can cause significant tissue shrinkage and brittleness, which may complicate microtomy and morphological evaluation (11). In our study, these issues were evident in the increased shrinkage scores and occasional fragmentation of alcohol-fixed samples. These artifacts may pose challenges in assessing subtle histopathological changes, especially in small biopsy specimens (12).

Moreover, background staining was observed more frequently in formalin-fixed tissues, a finding that aligns with previous observations suggesting that formalin may increase non-specific binding during IHC (13). Alcohol-fixed tissues exhibited cleaner staining profiles, enhancing visualization and interpretation of antigen-specific reactions (14).

Given these findings, the choice of fixative should be determined based on the diagnostic requirements. While formalin is more suited for routine morphological assessment, alcohol-based fixatives offer a valuable alternative when IHC is the primary investigative modality. Hybrid or sequential fixation strategies may be worth exploring to optimize both morphological and immunological outcomes (15).

Formalin provides superior morphological preservation, making it ideal for routine histopathology. However, alcohol-based fixatives offer better immunostaining quality with enhanced antigen preservation. A combined or case-specific fixative approach may optimize diagnostic accuracy in surgical pathology.

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