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Volume 13 Issue 5 (May) 2024

Original Articles

Comparison of Rubeanic acid and Rhodanine staining for detection of copper in liver with copper quantitation by Inductively Coupled Plasma Mass Spectrometry (ICPMS) Method
Asha Raby N, Dr. Ajas SN

Background: Copper is an essential trace element that plays vital roles in various physiological processes. However, excessive accumulation of copper in tissues, particularly in the liver, can lead to severe health complications, including liver damage and neurological disorders. Wilson's disease, characterized by abnormal copper metabolism, underscores the importance of accurate methods for detecting copper deposits in liver tissues. Several staining techniques have been developed to visualize copper deposits in histological sections. Among these, Rubeanic Acid and Rhodanine staining methods have gained prominence for their ability to selectively bind to copper ions, producing distinctive colour changes. While both methods have been used for detecting copper in liver tissues, their comparative efficacy remain unclear. Aim and Objective: To compare two different histochemical stains ie, Rubeanic acid and Rhodanine for detection of copper in liver with copper quantitation by Inductively Coupled Plasma Mass Spectrometry (ICPMS) Method. Materials and Methods: Between 2016 and 2021, a total of 33 patients diagnosed with Wilson's disease or suspected cases were included in the study. Cell blocks from these patients were gathered and subjected to standardized Rhodanine staining. Subsequently, both Rubeanic acid and Rhodanine staining procedures were conducted on all collected samples. The comparison of the histochemical stains with copper quantitation using ICPMS method was then carried out for each stain individually. Results: The research involved 33 cases, consisting of 26 males and 7 females, aged between 4 and 93 years, with mean age of 46.70 and a standard deviation of 32.95.Out of these cases, after employing the copper quantitation test and reviewing records from clinical and other biochemical tests, 22 cases were definitively diagnosed as Wilson's disease. The remaining 11 cases included diagnoses such as Alcoholic Liver Disease (ALD), Non-Alcoholic Fatty Liver Disease (NAFLD), Autoimmune Hepatitis (AIH), and Drug-Induced Liver Injury (DILI). Rubeanic acid staining revealed that among the 22 cases diagnosed with Wilson's disease, 10 exhibited positive staining: 5 cases at 1+, 4 at 2+, and 1 at 3+. Rhodanine staining, on the other hand, showed positive results for 7 cases: 3 at 1+, 3 at 2+, and 1 at 3+. The remaining cases in both groups were graded as 0. While Rubeanic acid staining identified 10 cases compared to Rhodanine, no statistically significant association was found between the two methods (p = <0.07). Additionally, a moderate positive correlation was observed between Rubeanic acid staining and copper quantitation (r = 0.365, p < 0.001), suggesting that Rubeanic acid may be a more reliable indicator of copper levels than Rhodanine staining. Conclusion: Rubeanic acid outperformed Rhodanine in detecting liver copper accumulation, but larger tissue sections showed staining variability. Negative staining in small biopsies does not rule out Wilson's disease. Some cases with elevated copper levels showed negative staining, indicating early-stage accumulation. So, to conclude, Histochemical stains can support diagnosis alongside clinical factors, but copper quantitation remains the gold standard.

 
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