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HERDIN Record #: R07-CIM-18061316503469 Submitted: 14 June 2018 Modified: 07 November 2018

Acid alcohol extract of ripe Alugbati (Basella rubra) fruit: a possible alternative to hematoxylin stain?.

Kimberly Anne T. Aznar,
Angelie P. Castro,
Efren M. Empanado,
Allyson Therese Y. Larcena,
Mary Grace  S. Nepomuceno,
Lorenz Robert Y. Ong,
Nadjel L. Opamen,
Janessa Kim C. Padin,
Mary Frances U. Penalosa,
Mark Benett M. Reroma,
Christian Micheal  J. Saga,
Patricia Llana P. Suson,
Cristoper Jay P. Tagalog,
Nika Q. Tansingco

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Context/ Background: The market for hematoxylin, the most widely used nuclear histologic stain, is a volatile one. There have been efforts to find more sustainable, readily available and cost-effective alternatives in order to curb the fluctuations in hematoxylin prices. Of these alternatives - the anthocyanins, have shown great promise as a hematoxylin substitute and are found in many plants including Alugbati (Basella rubra). Alugbati is a plant that is commonly found in the Philippine setting and whose anthocyanin-rich fruits may be extracted and used as a hematoxylin substitute. Objective: To determine the efficacy of acid alcohol extracts of ripe Alugbati (Basella rubra) fruit as a hematoxylin substitute for nuclear staining on formalin fixed tissue. Study Design: An analytical experimental study design was utilized. Study Setting: Samples were collected from a plantation in Barangay Adloan, Cebu City. Tests were conducted at the Cebu Velez General Hospital Clinical Laboratory- a tertiary hospital-based laboratory. Study Population: Pig livers were utilized for tissue staining in this study. Maneuver: Ripe Alugbati fruits were obtained from a local plantation, washed and dried. Dried fruits were then blended with ethanol and added to either (1) ethanol HCI (Solution A) or (2) ethanol HAc (Solution B). Solutions were left to stand for 32 hours, filtered and stored. Formalin-fixed pig liver was processed and sectioned and stained by either: (1) Solution A with 10% FeCl3 (Stain A), (2) Solution B with ammonium alum (Stain B), (3) hematoxylin (positive control). Slides were counterstained with eosin and mounted. Negative control slides were stained with eosin only. All slides were sent to participating pathologists along with a scoring sheet based on the Leica Microsystems Scoring System. Scoring sheets were collected and analyzed. Results: Scores from all score sheets were tallied and their means obtained. One Way Analysis of Variance was used. A significant difference was determined. Tukey's test was used and showed that Stained A and B performed similarly to the Negative Control. Conclusion Acid alcohol stains of Basella rubra are not suitable substitutes of hematoxylin in terms of nuclear staining. Recommendations: (1) Better sample size calculations be used (2) Better dye extraction be used (3) Plants be grown in similar conditions (4) Better attention be given to mordants used (5) Other tissue types be used (6) More funding and resources be allocated for further studies (7) Other alternatives to hematoxylin as nuclear stain be sought.      


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