Acid Alcohol Extract of Ripe Alugbati (Basella Rubra) Fruit - A Possible Alternative to Hematoxylin Stain

 Acid Alcohol Extract of of Ripe Alugbati Ripe  Alugbati (Basella rubra) Fruit rubra)  Fruit:: A Possible Alternative to Hematoxylin Hematoxylin Stain?

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

ICC-1 (PBL-2) Group 8 Cebu Institute of Medicine  AY 2016 – 2017

 ABSTRACT  AB STRACT Fruit:  A Possible Alternative to Title:  Acid Alcohol Extract of Ripe  Alugbati (Basella rubra)  rubra)   Fruit: Hematoxylin Stain?  Auth  Au th or s:  Ong, L.R., Aznar, K.A., Castro, A., Empanado, E., Larcena, A.T., Nepomuceno, M. G., Opamen, N. , Padin, J.K.,  Penalosa, M. F., Reroma, M. B., Saga, C.M. Suson, P.L., Sy, D.C., Tagalog, C.J. Tansingco, N. Context/ Backgroun Backgroun d: The market for hematoxylin, the most widely used n uclear 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  – the anthocyanins, have shown great promise as a hematoxylin substitute and are found in many rubra) .  Alugbati   Alugbati  is a plant that is commonly found in the plants including alugbati (Basella rubra). Philippine setting and whose anthocyanin rich fruits may be extracted and used as a hematoxylin substitute. To determine the efficacy of acid alcohol extracts of ripe Alugbati ripe  Alugbati (Basella Objective: (Basella rubra) fruit as a hematoxylin substitute for nuclear staining on formalin fixed tissue. study design was utilized Study Design: Design:  A analytical experimental study Study Setting: Samples were collected from a plantation in Barangay Adlaon, Cebu City. Tests were conducted at the Cebu Velez General Hospital Clinical Laboratory – Laboratory – a  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 HCl (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 a mmonium 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 o n 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 Stain A and B performed perf ormed similarly to the Negative Control.

Conclusion:  Acid alcohol stains of Basella rubra are 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. i

TABLE OF CONTENTS

CONTENT

PAGE

 Abstract Table of Contents

i ii

INTRODUCTION Background of the Study Significance of the Study Research Question General Objective Specific Objectives Scope and Limitations

1 1 2 3 3 3 4

REVIEW OF RELATED LITERATURE

5

CONCEPTUAL FRAMEWORK

13

METHODOLOGY Study Design Operational Definitions Study Setting Study Population Methodological Flowchart Maneuvers Data Analysis

14 14 14 14 15 16 17 19

RESULTS AND DISCUSSION

20

CONCLUSION AND RECOMMENDATIONS

27

References  Appendix A (Data Collection Collection Parameters) Parameters)  Appendix B (Data Collection Collection Sheet)  Appendix C (Order of Stained Slides)  Appendix D (Gantt Chart) Chart)  Appendix E (CIM-CVGH (CIM-CVGH IRB Review) Review)  Appendix F (Transmittal Letter to CVGH)  Appendix G (CVGH (CVGH Histopathology Laboratory H&E Staining Process)  Appendix H (Manual Tissue Processing Processing Schedule)  Appendix I (Transmittal Letter to Pathologists) Pathologists)  Appendix J (Budget)

28 31 32 34 35 36 37 38 39 40 43

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INTRODUCTION Background of t he Study Study The use of Hematoxylin as a dye dates back to the time of the Aztecs when extracts were obtained from the logwood tree (Haematoxylon campechianum). campechianum) . Hematoxylin was then introduced to Europe where it became a means for trading between countries. In the 1850s, the hematoxylin pigment became a source for dyeing cloth. As the field of science expanded, Hematoxylin became valuable to pathologists especially with its ability to bind to mordants such as metal salts which produced a high range of colors. 1 Hematoxylin is one of the few f ew dyes derived from natural resources that are still being used in the modern histology laboratory. 2 In 19733 and 20084, a worldwide shortage of hematoxylin took place. This shortage is probably due to factors such as a high demand for Hematoxylin, the limited availability of commercially usable Hematoxylin, and the complicated process of purifying Hematoxylin powder from the presence of tannins.4 The need for Hematoxylin stain alternatives prompted the search for other synthetic or natural resources. One of the alternatives for Hematoxylin staining would be anthocyanins which have been used as histological stains since the late 19th century. Anthocyanins can be found in plants such as red cabbage, elderberry, dahlia, blackberry5, and alugbati6,7. Recent studies have been conducted with regard to the potential use of Alugbati ((Basella Basella rubra) as a stain for peripheral blood smears and are easily grown in countries such as the Philippines and in the Southeast of Brazil8. In this study, the researchers will be using paraffin-fixed liver tissues since its hepatic portal vein is histologically similar to human hepatic portal vein.9, 10 In addition, one study suggests that urea synthesis is similar in human a nd pig liver and the h high igh metabolic activity of cultured pig liver cells makes it a potential substitute for human cells in bioartificial liver devices.11

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Significance of t he Study Study The hematoxylin and eosin (H&E) staining method has been profoundly used in histologypathology laboratories worldwide for many years. Hematoxylin has proven to serve many uses in biological staining and not a single synthetic dye exists that can substitute it for all its applications.4 Cooksey (2010), stated that hematoxylin is a compound obtained from the heartwood of a logwood tree.12 Up to date, there remains only one true supplier of logwood chips that meets international demand: Mexicana De Extractos, Extractos , in Campeche, Mexico. However, there have been repeated shortages in the supply of hematoxylin due to interruptions in its extraction and the greater profit that can be achieved in production of chemicals for purposes other than staining. The decreasing supply cannot meet the increasing demand, which then lead to an increase in the cost of the compound and consequently, affecting the cost of diagnostic histopathologic examinations.1 Hence, many synthetic dyes have been tried and tested as an alternative to hematoxylin.1, 4, 5, 14, 15, 16, 17

Since many synthetic dyes pose a hazard to the health of technicians and those who prepare them, this study also aims to make use of a natural alternative to hematoxylin. Roots, fruits and leaves from dye-producing plants can hypothetically provide an effective stain.18 An abundant number of potential plants with staining properties sprout throughout the Philippine setting, and one of them is Basella rubra (alugbati). rubra (alugbati).14, 6  The Basellaceae family Basellaceae  family in general has anthocyanins, which are water-soluble, vacuolar pigments capable of staining cellular structures.19,

20

  Therefore, Basella rubra  rubra  presents a promising alternative as a non-toxic,

accessible and cost-efficient organic chemical stain.14

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Research Question Is there a comparable difference between Hematoxylin staining and staining with acid alcohol extracts of ripe alugbati (Basella rubra) fruits?

General objective To determine the efficacy of acid alcohol extracts of  Alugbati (Basella rubra) fruit as a hematoxylin substitute for nuclear staining on formalin fixed tissue.

Specifi Specifi c Objective Objective 1. To compare the mean staining scores of pig liver tissues stained with: (1) ripe alugbati fruit alugbati  fruit extracted with ethanol-hydrochloric acid and (2) ripe alugbati fruit alugbati  fruit extracted with ethanol-acetic acid and (3) hematoxylin based on the following parameters: a.

Nuclear staining

b.

Nuclear Detail

c.

Cytoplasmic staining

d.

Uniformity of Stain

e.

Absence of Precipitates

3

Scope and and Limi tations of the Study

 Acid alcohol extracts of ripe alugbati fruit alugbati  fruit together with eosin stain were used as substitutes of hematoxylin stain. In addition, the specimen used was pig liver tissue since it can be easily obtained and prepared. Pig liver tissue was chosen by the researchers instead of actual human specimens as obtaining them would involve a more complicated process in terms of bioethics and acquiring patient’s permission. permission . The researchers were not able to use the most ideal extraction method which involves ether/ chloroform because it is a health hazard. 21 Ethanol was also used in extraction instead of methanol as methanol fumes are highly toxic.22 Alugbati fruits were also obtained from various sources and therefore growth conditions – conditions – such as: soil and water water quality, age of ripe alugbati fruit, and other factors that contribute to alugbati growth – growth – were  were not taken into account.

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REVIEW OF RELATED LITERATURE

The hematoxylin and eosin (H&E) staining method is the standard method for the visualization and examination of human and animal tissue. Most cellular organelles and extracellular matrix are eosinophilic, while the nucleus, rough endoplasmic reticulum, and ribosomes are basophilic.23, 24

  The H&E method aids in the visualization of changes in nuclear and cytoplasmic staining

characteristics which are indicative of various disease states and thus is an indispensable tool in surgical pathology for the diagnosis of disease ranging from viral infections to cancer.25

Hematoxylin, the nuclear stain component of H&E, is a natural product extracted from the Haematoxylin campechianum campechianum). heartwood of the logwood tree ((Haematoxylin ).2 Hot water or stream is used to obtain the crude product from the milled heartwood; it is purified through ether extraction and then dried. With this, hematoxylin powders have an orange-brown color due to the tannins that are removed from the extraction. It can be oxidized to haematein naturally and artificially. 26  In the natural process, prepared hematoxylin solution is exposed to air and sunlight for 6 to 8 weeks before it is used. Oxidation in this process is slow and gradual which makes the solution last longer. In the artificial process of oxidation, oxidizing agents such as sodium iodate, hydrogen peroxide, mercuric oxide and potassium permanganate are added to the solution of hematoxylin.27 Oxidation in this process is fast but the solution has a shorter lifespan compared to the natural process because the solution is quickly over oxidized. Hematoxylin cannot stain a tissue unless a mordant is incorporated into the dye such as aluminum alum and potassium alum.28 Haematin has indicator-like properties.12 It appears blue and less soluble in aqueous alkaline conditions and red and more soluble in alcoholic acidic conditions.13 It binds to lysine re sidues of nuclear histones by linkage via a me tallic ion mordant such as aluminum in acidic conditions. The

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stain is applied longer than necessary, to ensure saturation of chemical binding sites resulting in the over staining of the tissues with with much non-specific background coloration. coloration. This undesirable coloration is selectively removed by controlled filtering in an alcoholic acidic solution, (acid alcohol), the process being being termed "differentiation". Differentiation is arrested arrested by returning to an alkaline environment, whereupon the haematin takes on a blue hue, the process of "blueingup". The haematin demonstrates cell nuclei.27 Hemalums are used to provide blue stain th at largely restricted to nuclei of cells. Hemalum staining solutions are made using hematoxylin because they also contain hematein, which is the oxidized form of hematoxylin, an aluminum salt, and other ingredients such as alcohols and organic acids. In most histopathologic laboratories, hemalum is followed by eosin in H&E method. This method provides a range of colors between orange and purple to cytoplasmic and extracellular materials.15 Mechanized H&E staining should provide blue to purple nuclei and the rest should be pink. Aside from aluminum, hematoxylin can also be used in combination with other metallic salts such as iron. There are several iron hematoxylin stains for nuclei, nuclei, myelinated nerve fibers, cytoplasmic organelles, and protozoa.15 Luna (1991) believes that an H&E-stained slide should contain the following tectorial qualities: (1) Nuclear chromatin should be blue to bluish purple and very noticeable. (2) Nucleoli should stain reddish purple. Too much hematoxylin will remain if there is overstaining by hematoxylin. (3) Collagen and muscle should stain well and a fibrillar pattern should be seen (4) distinct eosinophilic granules should appear orange. Overstaining by either hematoxylin or eosin will prevent this.29

Due to its versatility as a nuclear stain, increasing demand for hematoxylin has led to shortages in the past. One of the main causes of shortage of hematoxylin is due to limited sources of logwood trees from where it is extracted. Because of this, supply may decrease as a result of climate political or economic forces and thus fail to meet demand.2 Increased shortages not only

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happened during both World Wars, but also in the late 1920’ 1920’s and early 1970’ 1970’s. 3, 17 Unfortunately, there remains only one true supplier of logwood chips in the Mexicana De Extractos Company in Campeche, Mexico. Moreover, the lack of suppliers cannot provide enough hematoxylin dye for the ever-increasing demand for hematoxylin.1  It also became apparent that formulators and vendors of the staining solution found it difficult to secure supplies of hematoxylin of high purity at affordable prices and inconveniences were experienced lasting for weeks or a few months. Lastly, some studies have suggested that hematoxylin shortage is caused by using logwood trees on botanicals used for cosmetic and skincare industries instead of extracting the hematoxylin from it. 2

In addition to the volatility of the hematoxylin market, the extraction and preparation of the dye is detrimental to human health. Hematoxylin causes acute and chronic health hazards such as irritation to skin, eyes, nose, mouth, throat and stomach pains. Prolonged or repeated skin contact may lead to dermatitis and eventually lead to irreversible damage to health.21

Due to the uncertainty of the future of hematoxylin supplies, there have been studies which aimed to find an alternative to hematoxylin. Among these substitutes was the class of pigments called anthocyanin. Anthocyanins are natural pigments that are responsible for revealing the colors in fruits, flowers, stem and leaves. The anthocyanin extract is non-toxic and can be used as food colorants that impart a reddish color.30 Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2- phenyl- benzopyrylium salts that belong to a widespread class of flavonoids. A free malonyl group attached to the glucose at C3 position of anthocyanin mainly preserves the color of anthocyanin pigments.18 Anthocyanins are most stable and most highly colored at low pH value; its optimum pH range is 2.5-4.0.5 At pH of 3 or lower, the anthocyanin is orange or red in color due to the presence of flavylium cation. It gradually loses color as the pH

7

increases, becoming almost colorless between pH 4.0 and 5.0. At a pH of 6 to 7, anthocyanin has a purplish color due to the formation of resonance stabilized quinonoid anions.31 Pigment color loss is due to anthocyanin hydrolysis. Heat causes anthocyanins to undergo hydrolysis at glycoside linkages to produce chalcone and, later, later, alpha-diketones. Color loss is a reversible process; the red color returns when acidified.8  Additionally, unacylated anthocyanins are only stable at low pH values where flavylium flavylium cation is present. At pH 5-6, unacylated anthocyanins anthocyanins are unstable and decolorize quickly.18 According to Amon et al. al. 2012, the anthocyanin and DPPH (di(phenyl) - (2,4,6-trinitrophenyl) iminoazanium) radical scavenging activity are present in the extract that marks its its potential as a food colorant. Anthocyanin pigments appear to have have many therapeutic benefits such as vasoprotective, anti-inflammatory, anti-cancer, chemo-protective, anti-neoplastic properties, moreover, its ability for reversing age related deficits, controlling oxidative stress during pregnancies, stabilizing DNA triple helical complexes, and protecting chloroplast against high light intensities.30 In addition, owing to dye shortage especially hematoxylin, anthocyanin has been considered to be as an effective alternative for hematoxylin staining because of its availability, easy preparation and usage, and resistance to fading.5 Since anthocyanin molecule is usually found in plants, extraction of this molecule involves boiling and acidification procedures which causes the removal of the sugar moieties from the anthocyanin.4  One of the unique characteristic of this molecule is that it does not require a r ipening process, which makes it different from hematoxylin.  Also, these substitutes resist extraction by solvents that are used for dehydration, clearing, and coverslipping, just like hematoxylin.5

Basella sp., Basella  sp., also known as Alugbati, Malabar spinach, Indian spinach, Ceylon spinach and vine spinach, belongs to the family Basellaceae. Basellaceae . It is commonly found in tropical regions like

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Southeast Asia and grows abundantly in the Philippines. It is also a fast-growing perennial climber that can grow up to 9 m in length. Its stem is green or purplish with a thickness of abo ut 2 to 3 cm and a quadrangular shape. The leaves are dark green, fleshy, and ovate or heart shaped, with sizes varying from 3 to 9 cm in length and 4 to 8 cm in width. The stem and leaves have no odor and taste bland. Its flowers are inconspicuous, bisexual, white, and borne on axillary spikes or branching peduncles. Its fruits are fleshy and have no stalks, 5 to 6 mm with an ovoid or spherical shape; it turns purple when the fruit becomes mature. The useful parts of the plant include its leaves, young stem, mature fruit, and ro ots.32 The fruit contains a major red pigment, gomphrenin-I, a betalain pigment, and other substances including betanidin dihexose, betalins and isobetanidin dihexose. Betalains are bioactive compounds in pigments that need further exploration; hence some studies have demonstrated their potential use as antioxidant pigments. Betalains are water-soluble nitrogencontaining pigments that are synthesized from the tyrosine into two structural groups namely the red-violet betacyanins and the yellow-orange betaxanthins. They are antioxidants, sources of food colorants with nutraceutical benefits, and have anti-inflammatory properties that are being studied for protection against cancer.33,34  A study by Mundo Mundo et al. 1995 reported that Basella sp. Basella  sp. fruit pulp produces a stain that can be used as a substitute for crystal violet or safranin in the Gram staining; its extract stained the bacteria red-orange. Their study also showed that Basella  Basella   sp. extract was evaluated as a favorable stain for plant nuclei and its organelles which produced a red stain on plant nuclei and light pink stain on the cytoplasm.35 Alugbati anthocyanin pigments are also water soluble; hence, they are a potential source of natural dye.8 At a pH of 3 to 7, the Basella sp. fruit pulp pigment is known to have a good stability.30 Light and high temperatures are factors that result to acceleration of anthocyanin degradation. Light accelerates anthocyanin degradation.18 Basella rubra  rubra   has anthocyanin production when exposed to UV light. Research done by Pumchaosuan and 9

Wongroung 2009 shows that exposure of the callus of Basella rubra  rubra   to UV light for 20 to 30 minutes led to the production pro duction of anthocyanin.7

 A research study by Cruz 2007, used Basella sp. fruits and Dioscorea alata (ubi) alata (ubi) roots as potential histologic stains for human tissue. The extract was obtained by cutting and grinding Basella sp. fruits and Dioscorea alata roots. alata  roots. The properties such as pH, specific gravity color, odor, taste, turbidity and viscosity, presence of reducing sugar and hemolytic property were all observed. Phytochemical profile such as alkaloids, saponins, flavonoids, cardenolides and tannins were also determined. Basella  (alugbati)) fruit gave a positive result in saponins. Basella   sp. (alugbati Dioscorea alata (ubi) alata  (ubi) roots contain protein, saponins and flavonoids. The extract of both samples contained carbohydrates and both did not show hemolytic properties. A blood smear was stained using the extract and both extracts have the ability to stain cells. It was also noted that the addition of mordants such as Potassium Alum and Potassium iodate improved the staining property of both extract, which is comparable with the Hematoxylin staining.14

Dye extraction from the plant matter is a necessary procedure in the process of manufacturing a stain. There are various methods in extracting anthocyanins from plant sources. According to  Al Tikriti (1977), anthocyanin from blackberry can be extracted extracted by filtering or or centrifuging the crude  juice from the shredded fruit to make a clear juice.5 Another study by Metivier et. al. shows that anthocyanins are best extracted by using methanol with 10% HCl. They also noted th at extraction using ethanol with 10% HCl, ethanol with citric acid and ethanol with acetic acid are also possible but do not yield as much dye as methanol.36 De Leon (et. al.) used methanol alone to extract anthocyanins from Basella rubra rubra..37  Other methods also include ether extraction but pose too much of a health hazard to be considered by the research group. 9, 26, 38, 40

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Various staining procedures were previously developed f or tissue staining using anthocyanin as a stain, but one particular procedure, developed by Al Tikriti (1977) used anthocyanin for histological purposes. Two formulas were developed by Al Tikriti (1977); the first formula used 100ml of clear extract from blackberries added with 1.0 g aluminium chloride and 1.2ml ferric chloride solution. The second formula was the combination of 100ml clear blackberry extract, 5.0g sodium chloride 1.2ml 10% ferric chloride solution and 3ml glacial acetic acid. Both formulas were used the same way: paraffin embedded tissues were brought to water, and were then stained with either one of the formulas for 10-12 minutes. The stained slides were then washed with running water for at least two minutes, stained with eosin, dehydrated, cleared then mounted. The result was that nuclei were specifically stained a dark violet blue color distinguishable from hematoxylin staining only by the faintest of green casts. The stains were also effective on tissues which were fixed with formalin, Zenker’s and Carnoy’s fixatives. 5  A similar study was done by Co et al. (2001), where, where, instead of using using blackberries, blackberries, ripe Basella rubra ( Alugbati)  Alugbati) and Biva arellana (atsuete atsuete)) seeds were used to stain albino mouse liver and kidney. Results of their study noted that without mordants, nuclear stain uptake was close to nil.41  Another method worth mentioning mentioning is the work of De Leon (et. al.) on using the methanolic fruit extract of Basella rubra as rubra  as a hematologic stain. Few drops of the Basella rubra methanol rubra methanol extract was added on a blood smear. The extract was allowed to dry and was not washed. Upon drying, two drops of diluted Methylene blue were added to contrast the color. The results showed that methanolic fruit extract of Basella rubra was an effective stain to red blood cells.4 In order to assess the quality of a specific stain, certain control slides are utilized. Ideally, tissue specimens from humans are used to assess stains. However, because of the difficulty in obtaining human tissue specimens, pig tissues – tissues – which  which resemble human tissue, can be used as a substitute. According to the Tissue Quality Assessment of Leica Microsystems, pig liver tissue can be excellent for stain quality control as hepatocytes have characteristic morphological 11

features in association with delicate sinusoidal vessels. Also present is connective tissue containing vessels and ducts.39 The structure of cell layers of hepatocytes and liver sinusoids in hematoxylin and eosin is better b etter understood using H&E-stained liver specimens; moreover, this is significant for differential diagnosis of liver diseases. Hepatic cells are arranged in a radial pattern originating from a central vein. Any irregularities observed in the stained specimen are used for histological classification and diagnosis.10, 42

 According to Barczak (2005), most synthetic dyes, especially substances derived from coal tar, are highly toxic and might actually pose health hazards. For this reason, finding a safe, economical, and widely available organic chemical stain remains as a challenge in the biochemical field. field.18 Aside from its issues in availability, its preparation in the lab also poses as a health hazard especially to the medical technologists who work in close proximity to the solution because of the toxic gases that the working hematoxylin solution emits.21

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CONCEPTUAL FRAMEWORK Tissue Type

Dehydrating Agent

Embedding Medium

Tissue

Duration of Fixation

Processing Fixative

Mounting Medium

Stain Used*

Duration of Staining*

Counterstain Used

Staining pH of Stain Solution *

Temperature

Procedure

Mordant Used*

Dye Extraction*

Nuclear Detail*

Uniformity of Stain*

Staining Quality

Nuclear Staining*

Precipitates*

Cytoplasmic Staining*

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METHODOLOGY Study Design  An analytical experimental study design was utilized. Study Setting Ripe alugbati (Basella rubra) fruits were obtained from a plantation in Barangay Adlawon, Cebu City while the pig liver specimen was obtained from a local slaughterhouse. Preparation of the dye extract and processing of tissues for staining was conducted at the Cebu Velez General Hospital Clinical Laboratory – Laboratory – a  a tertiary, hospital based laboratory. Stained slides, along with the scoring sheets were then submitted to participating pathologists to be read at their respective laboratories. Operational Operational Definitions: 1. Nuclear Detail - clarity and sharpness of chromatin and visibility of the nuclear membrane when stained.43 2. Nuclear staining – staining – the  the intensity, sharpness and contrast of the nucleus when stained.43 3. Cytoplasmic staining  –   –  the intensity, sharpness and contrast of the cytoplasm and contents when stained.43 4. Uniformity of Stain – Stain – the  the homogeneity of stained areas and the absence of uneven stained areas.

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Study Population Inclusion Criteria Samples used in this study were freshly dissected pig liver obtained from a local slaughterhouse. Exclusion Criteria Not Applicable Sampling Procedure Purposive convenience sampling was utilized. Sample Size Calculation Not Applicable

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Methodologi Methodologi cal Flow Chart Stain A Slides

1Stain B Slides

Positive Control Slides

Negative Control Slides

Stain Extraction and Preparation Obtain ripe alugbati  fruits  fruits Wash, dry Add a small amount of 95% ethanol Blend To 500 g of blended alugbati :

Add 500 ml Ethanol-Acetic acid solution

Add 500 ml Ethanol-HCl solution

Mix, stand for 32 hours, and filter Filtered Solution A To 300 ml: Add: - 3g AlCl3 - 3.6 ml 10% FeCl3

Stain Solution A

Filtered Solution B To 300 ml: Add: - 20ml 50% (w/v) aq. NH4 alum solution

Stain Solution B

Tissue Processing Obtain Pig Liver

Fix, dehydrate, clear, paraffinize, section, deparaffinize and rehydrate tissue

Stain with Stain Soln’ A

10-12 10-12 minutes minutes

Stain with Stain Soln’ B 10-12 10-12 minute minutess

Stain with Harris Hematoxylin (5 minutes) Differentiate (1% Acid Alcohol); Blue (NH3 water)

Wash in tap water (10 dips)

Counterstain with eosin (2 minutes) Wash, Dehydrate, Clear and Mount Slides

Label and submit slides for scoring; collect slides afterward for data analysis

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Maneuvers Data Collection Tool Scoring of stained slides was performed “blind” blind” with the participating pathologists unaware of the tissue processing and staining procedure. Stained slides were scored with a fourpoint scale whose parameters were based on the Leica Microsystems Scoring System. The parameters included were: nuclear staining, nucleolar detail, cytoplasmic staining, uniformity of stain and absence of precipitates. A score sheet will be submitted with each slide. (See  Appendices A and B) Data Collection Procedure Ripe alugbati fruits alugbati  fruits were obtained from a plantation in Barangay Adlaon, Cebu City. Collected fruits were washed washed and dried to remove remove water and other impurities. impurities. The cleaned alugbati fruits were blended with a small amount of 95% ethanol in a (insert brand and model here) blender. Two solutions: (1) an ethanol-hydrochloric acid  solution (Servadio), a 15% (v/v) solution of 0.1N hydrochloric acid in 95% ethanol (2) an ethanol-acetic acid  solution (Metivier), a 3% (v/v) solution of glacial acetic acid in 95% ethanol were prepared for dye extractions. Five hundred grams of blended alugbati were mixed with 500 ml of both the ethanol-hydrochloric acid and the ethanol-acetic acid solution, mixed, sealed with Parafilm and allowed to stand for 32 hours with agitation at room temperature. After which both dye dye extracts were clarified clarified by crude filtration to a filtration cloth and fine filtration with Whatman filter number 1. Dye extracts will be placed in dark colored glass bottles for storage. Working solutions were made from both dye extracts. From the ethanol-hydrochloric acid dye extract , (1) 300 ml of extract, 3 grams of aluminum chloride and 3.6 ml of 10% ferric chloride (Al-Tikriti) was added and designated as Stain A. A . From the ethanol-acetic acid dye extract: extract : to 300 ml of extract, 20 ml of 50% (w/v) aqueous ammonium alum was added and designated as Stain B. B. 17

Pig liver obtained from a recently slaughtered pig was obtained from a local slaughterhouse and immediately fixed in 10% Buffered Neutral Formalin for 12 hours. Fixed tissues were dehydrated, cleared, embedded in paraffin, sectioned with a microtome and then sections are placed on slides. A total of forty slides were made for this study. Unstained slides were divided into four groups of 10 each: the positive control slides, negative control slides, slides stained with Stain A and slides stained with Stain B. Unstained slides were deparaffinized with xylene, rehydrated with decreasing concentrations of ethanol and rinsed with tap water. The positive control group were stained with Harris Hematoxylin for 1 minute and the test groups were stained with either stain A or stain B for 10-12 minutes. All slides were then rinsed in tap water to remove excess stain. Positive control slides were differentiated in 1% acid alcohol and blued in ammonia water. All slides were stained with eosin as the counterstain, dehydrated with increasing concentrations of alcohol, cleared in xylene and then mounted. Negative control slides were stained with eosin only. Slides were randomly given numerical designations in order to prevent interpretation bias by the participating pathologists.

Slides were submitted to three participating pathologists, all of whom have more than 5 years’ experience, along experience,  along with a score sheet, based on the Leica Microsystems Scoring System, containing parameters such as: nuclear detail, nuclear staining, cytoplasmic staining, uniformity of stain and absence of precipitates. Questionnaires precipitates. Questionnaires were then collected and analyzed.

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Data Analysis

Scores from all score sheets were tallied. The mean scores from each test group, positive p ositive control and negative control were obtained. One Way Analysis of Variance was used to analyze differences among the mean scores. A significant difference was determined using a post hoc analysis which is Tukey’s test using SPSS using  SPSS and the level of significance was set at 0.05. To interpret the results, the following hypothetical mean ranges and their corresponding interpretations are proposed: 2.25-3.00

Chromatin clear and sharp; nuclear border visible Nuclei stain intensely; sharpness and contrast of nucleus excellent Cytoplasm stains intensely; sharpness and contrast of cytoplasm excellent Stained areas homogeneous; unevenly stained areas non-existent Precipitates absent Chromatin somewhat clear and somewhat defined; nuclear border somewhat delineated

1.50-2.24 Nuclei stain satisfactorily; sharpness and contrast of nucleus satisfactory Cytoplasm stains satisfactorily; sharpness and contrast of cytoplasm satisfactory Stained areas show some heterogeneousity; unevenly stained areas noted (<25% of slide) Few precipitates seen 0.75-1.99

Chromatin unclear and poorly defined; nuclear border ill defined Nuclei stain poorly; sharpness and contrast of nucleus non-existent Cytoplasm stains poorly; sharpness and contrast of cytoplasm non-e xistent  Absence of homogeneousity; unevenly stained areas widespread (<50%of slide) Precipitates widespread

0.00-0.74

Chromatin and nuclear border not visible Nuclei not stained Cytoplasm not stained  All of the slide is stained stained unevenly Precipitates over majority of the slide

19

RESULTS AND DISCUSSION

Tables 1 through 4 are presented for the purpose of giving descriptive results of the groups used in this study. Interpretations are based o n the hypothetical mean ranges of each parameter and their interpretations are presented in the previous section.

Table 1. Descriptive Results of Positive Control Slides Nuclear Detail

Nuclear Staining

Cytoplasmic Staining

Stain Uniformity

Precipitates

Pathologist 1

2.80

2.90

2.90

2.90

1.50

Pathologist 2

2.80

2.80

2.90

2.90

2.00

Pathologist 3

3.00

3.00

3.00

3.00

3.00

Factor  Average

2.87

2.90

2.93

2.93

2.17

Interpretation

Chromatin clear and sharp; nuclear border visible

Nuclei stain intensely; sharpness and contrast of nucleus excellent

Cytoplasm stains intensely; sharpness and contrast of cytoplasm excellent

Stained areas homogeneous; unevenly stained areas non-existent

Few precipitates seen

Table 1 shows the descriptive results of the positive slides used in this study. Being the positive control, all parameters ought to have better scores as shown above. Nuclear detail showed clear and sharp chromatin and a visible nuclear border. The nuclear staining was also intense. Sharpness and contrast of the nuclei were excellent. There was intense cytoplasmic staining and contrast and sharpness were excellent. There was also excellent stain uniformity. As with all slides in this study, few precipitates were seen by the pathologists. This may be due to the age of the counterstain.

20

Table 2. Descriptive Results of Negative Control Slides Nuclear Detail

Nuclear Staining

Cytoplasmic Staining

Stain Uniformity

Precipitates

Pathologist 1

1.00

1.10

1.10

1.30

1.40

Pathologist 2

1.80

1.00

2.00

2.00

1.90

Pathologist 3

1.00

1.00

1.00

1.30

1.30

Factor  Average

1.27

1.03

1.37

1.53

1.53

Chromatin unclear and poorly defined; nuclear border illdefined

Nuclei stain poorly; sharpness and contrast of nucleus non-existent

Cytoplasm stains poorly; sharpness and contrast of cytoplasm non-existent

Stained areas show some heterogeneousity ; unevenly stained areas noted

Interpretation

Few precipitates seen

Table 2 shows the descriptive results of the negative control slides, i.e. slides stained with eosin only. Nuclear detail showed unclear chromatin and ill-defined nuclear borders. Nuclear staining was poor and there was no sharpness of contrast of nuclei. There was also poor cytoplasmic staining and sharpness and contrast of cytoplasm were non-existent. There were also unevenly stained areas noted with regards to stain uniformity. Few precipitates were also observed.

21

Table 3. Descriptive Results of Slides Stained with Stain A Nuclear Detail

Nuclear Staining

Cytoplasmic Staining

Pathologist 1

0.90

1.00

1.10

1.20

1.60

Pathologist 2

1.60

1.00

2.00

1.90

2.00

Pathologist 3

1.00

1.00

1.00

0.90

1.10

Factor Average

1.17

1.00

1.37

1.33

1.57

Chromatin unclear and poorly defined; nuclear border ill-defined

Nuclei stain poorly; sharpness and contrast of nucleus nonexistent

Interpretation

Stain Uniformity Precipitates

Cytoplasm  Absence of Few stains poorly; homogeneousity precipitates sharpness and ; unevenly seen contrast of stained areas cytoplasm non- widespread existent

Table 4. Descriptive Results of Slides Stained Stained with Stain Stain B Nuclear Detail

Nuclear Staining

Cytoplasmic Staining

Stain Uniformity

Precipitates

Pathologist 1

0.70

1.00

1.00

1.20

1.40

Pathologist 2

1.50

1.00

1.90

1.90

2.00

Pathologist 3

1.00

1.00

1.00

1.20

1.90

Factor  Average

1.07

1.00

1.30

1.43

1.77

Interpretation

Chromatin unclear and poorly defined; nuclear border illdefined

Nuclei stain poorly; sharpness and contrast of nucleus non-existent

Cytoplasm  Absence of stains poorly; homogeneousi sharpness ty; unevenly and contrast stained areas of cytoplasm widespread non-existent

Few precipitates seen

22

Tables 3 and 4 shows the descriptive results of slides stained with Stain A and Stain B, respectively. Parameters for both sample populations have similar interpretations. Nuclear detail showed poorly defined and unclear chromatin and nuclear borders were ill-defined. Nuclei stained poorly and did not show sharpness nor contrast. Cytoplasm also stained poorly and sharpness and contrast were non-existent. Stain uniformity was poor as it was not homogeneous and unevenly stained areas were widespread. As with both positive and negative controls, few precipitates were observed.

Figure 1. Comparison of Factor Averages between Groups

3.00

2.93

2.90

2.87

2.93

2.50

2.17

2.00 1.50

1.77 1.27

1.17

1.37 1.37 1.30 1.07

1.53 1.33

1.43

1.53 1.57

1.03 1.00 1.00

1.00 0.50 0.00 Nuclear Detail

Nuclear Staining Positive Control

Cytoplasmic Staining Stain Uniformity Negative Control Stain A Stain B

Precipitates

Figure 1 compares the factor averages given for each group of samples. Positive control slides have the highest factor averages for all parameters compared to the other groups. The negative control slides, slides stained with Stain A and Stain B have similar factor averages for all parameters.

23

Table 5. One Way ANOVA for the Comparison of Mean Ratings Sum of Squares Between Groups Within Groups Total

df

Mean Square

23.499

3

7.833

9.568

56

.171

33.067

59

F

Sig.

45.846

.000

The ANOVA table above shows that there is a significant difference of the average ratings of the pathologists across parameters. This is evidenced by the p-value = 0.00 which is lesser than the level of significance of 0.05. Indeed, the different groups performed significantly different among the others. The test is carried using the different hypotheses: Null hypothesis: There is no significant difference among the average ratings of Hematoxylin stained slides and Acid Alcohol extracts of Alugbati stained slides.  Alternative hypothesis: There is a significant difference among the average ratings of Hematoxylin stained slides and Acid Alcohol extracts of Alugbati stained slides. To show which group combinations necessarily n ecessarily have significant difference, the following post-hoc post-hoc Turkey’s test result are derived.

24

Table 6. Post hoc Analysis for Piecewise Comparison of Mean Ratings Dependent Variable: Average Ratings Tukey HSD (I) Factor

(J) Factor

Mean Difference (I-

Std. Error

Sig.

95% Confidence Interval

J)

1

2

3

4

Lower Bound

Upper Bound

2

1.41333*

.15093

.000

1.0137

1.8130

3

1.47333*

.15093

.000

1.0737

1.8730

4

1.44667*

.15093

.000

1.0470

1.8463

1

-1.41333*

.15093

.000

-1.8130

-1.0137

3

.06000

.15093

.979

-.3397

.4597

4

.03333

.15093

.996

-.3663

.4330

1

-1.47333*

.15093

.000

-1.8730

-1.0737

2

-.06000

.15093

.979

-.4597

.3397 .3397

4

-.02667

.15093

.998

-.4263

.3730 .3730

1

-1.44667*

.15093

.000

-1.8463

-1.0470

2

-.03333

.15093

.996

-.4330

.3663

3

.02667

.15093

.998

-.3730

.4263

*. The mean difference is significant at the 0.05 level. Legend: 1  – Positive Contro l (H&E) (H&E)

3  – Stain A (ethanol-hydrochloric acid dye extract)

2  – Negative Contro l (Eosin)

4  – Stain B (ethanol-acetic acid dye extract)

It is evident from the table above that the performance of Groups 2, 3 and 4 vary significantly from that of 1. However, it is to be noted that groups 2, 3 and 4 perform similarly and showed no statistical difference as reflected by p -values >0.05.

25

A

B

C

D

Figure 2: Microscopic findings of pig liver stained with: A (Stain A), B (Stain B), C (H&E), D (Eosin). X400

Figures 2A – 2A  – 2D  2D demonstrate the microscopic appearance of pig liver using the different stains used in this study. Nuclear staining and nuclear detail were best demonstrated on Figure 1C which was stained with Hematoxylin and Eosin whereas Figures 1A, 1B and 1D, which were stained with Stain A, Stain B and Eosin re spectively, all demonstrated poor to nil nuclear staining and nuclear detail. Results of this study have similar findings to that of Co et al. (2001) which also  Alugbati have demonstrated that extracts of of Alugbati  have poor nuclear staining and nuclear detail. It is to be noted, however, that mordants were not added to the Alugbati the Alugbati stains  stains by Co et al.

26

CONCLUSION AND RECOMMENDATIONS RECOMMENDATIONS

Conclusion Based on the results and microscopic findings presented above, it is demonstrated that acid alcohol stains of ripe Basella rubra fruit rubra fruit are not suitable substitutes of hematoxylin in terms of nuclear staining.

Recommendations Due to financial, time and technical limitations, the research group would wish to recommend the following: (1) A better statistical method be employed to calculate for the sample size. (2) A more efficient dye extraction procedure be used in order to obtain more soluble dye pigments suitable for tissue staining. (3) Plants to be studied as potential sources of stains be grown in similar conditions as variations in fluid, carbohydrate and p igment content play a role in staining tissues. (4) Better attention be given to mordants used, their concentrations and proportions with regards to staining solutions. (5) More tissue types be used in order to ascertain efficacy over all tissue types. (6) More funding and resources be given to future studies in order to secure better equipment, reagents and specimens. (7) Other more renewable, affordable and less hazardous nuclear stain alternatives to hematoxylin be sought in order to provide economic benefits not only to stain manufacturing companies and histopathology laboratories but also to the patients and the communities involved.

27

References: 1. Groover A, Geddis C, Finney Finney A, Charleston SC. Recent Hematoxylin Shortage Shortage and Evaluation of Commercially Available Substitutes. 2. Titford M. The long history of hematoxylin. Biotechnic Biotechnic & histochemistry. histochemistry. 2005 Jan 1;80(2):73-8. 3. Lillie RD. The hematoxylin shortage and the availability of synthetic substitutes. substitutes. The  American journal of medical technology. technology. 1974 Nov;40(11):455. 4. Dapson R, Horobin RW, Kiernan J. Hematoxylin shortages: their causes and duration, and other dyes that can replace hemalum in routine hematoxylin and eosin staining. Biotechnic & Histochemistry. 2010 Jan 1;85(1):55-63. 5. Al-Tikritti SA, SA, Walker F. F. Anthocyanin BB: BB: a nuclear stain substitute for haematoxylin. Journal of clinical pathology. 1978 Feb;31(2):194. 6. "Alugbati / Basella Rubra L./ Malabar Nightshade / Spinach Vine: Philippine Medicinal Herbs / Philippine Alternative Medicine". Stuartxchange.com. Web. 18 Aug. 2016. 7. Pumchausuan T, Wongroung S. In vitro propagation of Ceylon Ceylon spinach Basella rubra L.  As. J. Food Ag-Ind. Ag-Ind. 2009 Aug:31-S36. 8. Ferreira Ozela E, E, Stringheta PC, PC, Cano Chauca M. Stability of anthocyanin in spinach spinach vine (Basella rubra) fruits. Ciencia e investigación agraria. 2007 Aug;34(2):115-20. 9. Zhang MR, Zhang H, Zhu HD, Wang RR, Wei Y. The study of Basella rubra red’s extraction, separation, analysis and stability. China Food Additives. 2010;1:17. 10. Yifei Z, Tiezhu H, Peijun W, Wenchun L, Minghua Y. Comparison of morphology and microstructural components of hepatic portal vein between human and pig. Journal of Huazhong University of Science and Technology [Medical Sciences]. 2005 Aug 1;25(4):419-22. 11. Vilei MT, Granato A, Ferraresso C, Neri D, Carraro P, Gerunda G, Muraca M. Comparison of pig, human and rat r at hepatocytes as a source of liver specific metabolic functions in culture systems--implications for use in bioartificial liver devices. T he International journal of artificial organs. 2001 Jun;24(6):392-6. 12. Cooksey C. Hematoxylin and related compounds –an  –an annotated bibliography concerning their origin, properties, chemistry, and certain applications. Biotechnic & Histochemistry. 2010 Jan 1;85(1):65-82. 13. Myers R. The Basic Chemistry of Hematoxylin. Leica Biosystems. Luettu. 2011. 14. Cruz, Rogelio. "Basella Rubra (Alugbati) And Dioscorea Alata Linn. (Ubi): Potential Histologic Stains For Human Tissue". Herdin.ph. N.p., 2007. Web. 10 Sept. 2016. 15. Carleton, H. M, and E. A Wallington. Carleton's Histological Technique Histological Technique. 4th ed. New York: Oxford University Press, 1967. Print. 16. Lillie RD, Pizzolato P, Donaldson PT. Hematoxylin substitutes: a survey of mordant dyes tested and consideration of the relation re lation of their structure to performance as nuclear stains. Stain technology. 1976 Jan 1;51(1):25-41. 17. Phoescheh F, Arkush AS. Metallic lakes of the oxazines (gallamin blue, gallocyanin and coelestin blue) as nuclear stain substitutes for h ematoxylin. Stain Technology. 1928 Jan 1;3(1):28-38. 18. Bkowska-Barczak Bkowska-Barczak A. Acylated anthocyanins as stable, natural food colorants –a  –a review.

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19. Amon, MFL, and LP Pladio. "Potential Food Colorant From The Extracts Of Alugbati (Basella Rubra L.)". Eurasia 12 Conference on Chemical science , Corfu, Greece (2012): 16-21. Print. 20. Deshmukh SA, Gaikwad DK. A review of o f the taxonomy, ethnobotany, phytochemistry and pharmacology of Basella alba (Basellaceae). ( Basellaceae). 21. "Hematoxylin Safety Data Sheet". Leica Biosystems. N.p., 2015. Web. 23 Oct. 2016. 22. Kavet R, Nauss KM. The toxicity of inhaled methanol vapors. Critical re views in toxicology. 1990 Jan 1;21(1):21-50. 23. El-Shishtawy, El-Shishtawy, Reda M. "Functional dyes, and some hi-tech applications." International Journal of Photoenergy 2009 (2009). 24. Bancroft JD, Layton C. The Hematoxylin and eosin. Theory & Practice of histologica l techniques. 7th ed., Churchill Livingstone of El Sevier, Philadelphia. 2013:173 -214. 25. Reifschneider O, Wehe CA, Diebold K, Becker C, Sperling M, Karst U. Elemental bioimaging of haematoxylin and eosin-stained tissues b y laser ablation ICP-MS. Journal of Analytical Atomic Spectrometry. 2013;28(7):989-93. 26. Dapson RW. Dye Quality and Implications for Biomedical Staining. Education Guide Special Stains and H & E Second Edition. 2010:44. 27. Lillie RD. Histopathologic technic and practical histochemistry. Histopathologic technic and practical histochemistry.. 1954. 28. Awvioro OG. Histochemistry and Tissue Pathology Principles and Techniques. ISBN. 2002;978356279:154162. 29. Luna LG. The quality control dilemma in Histotechnology: A possible answer. Histologic. 1991;21(3):245-50. 30. Adhikari R, Naveen Kumar HN, Shruthi SD. A review on medicinal importance of Basella alba L. International Journal of Pharmaceutical Sciences and Drug Research. 2012;4(2):110-4. 31. Fossen T, Cabrita L, Andersen OM. Colour and stability of pure anthocyanins influenced by pH including the alkaline region. Food Chemistry. 1998 Dec 31;63(4):435-40. 32. Kumar S, Prasad AK, Iyer SV, Vaidya SK. Systematic pharmacognostical, phytochemical and pharmacological review on an ethno medicinal plant, Basella alba L. Journal of Pharmacognosy and Phytotherapy. 2013 Apr 3 0;5(4):53-8. 33. Reshmi SK, Aravindhan KM, Suganyadavi P. The effect of light, temperature, pH on stability of betacyanin pigments in basella alba fruit. Asian J. Pharm. Clin. Res. 2012;4(3):107-10. 34. Oloyede FM, Oloyede FA, Obuotor EM. Comparative studies of chemical compositions of two species of Basella. Journal of applied science reports3. 2013;2:121-4. 35. Mundo L, Gorospe KJ, Lorilla L, Serquina AK, Torres E. The feasibility of Basella rubra L. as a biological stain. Bato Balani Sophomore. 1995;96(14):16-8. 36. Metivier RP, Francis FJ, Clydesdale FM. Solvent extraction of anthocyanins from wine pomace. Journal of Food Science. 1980 Jul 1;45(4):1099-100. 37. De Leon M, Latoza AG, Nues A, Pilac MR, Sistoza C. METHANOLIC FRUIT EXTRACT OF Basella rubra: ORGANIC STAIN FOR HEMATOLOGIC BLOOD SMEAR. SMEAR. 38. Fuleki T, Francis FJ. Quantitative Methods for Anthocyanins. Journal of food science. 1968 May 1;33(3):266-74.

29

39. Revilla E, Ryan JM, Martín-Ortega G. Comparison of several procedures used for the extraction of anthocyanins from red grapes. Journal of Agricultural and Food Chemistry. 1998 Nov 16;46(11):4592-7. 40. Rodriguez‐Saona LE, Wrolstad RE. Extraction, isolation, and purification of anthocyanins. Current protocols in food analytical chemistry. 2001. 41. Co, NL et al. "The Potential Use Of Ripe Basella Rubra (Alugbati) And Biva Arellana (Atsuete) Seeds Extract As A Stain For Paraffin-Embedded Animal Tissues". Herdin.ph. N.p., 2013. Web. 16 Sept. 2016. 42. Ishikawa M, Ahi ST, Kimura F, Yamaguchi M, Nagahashi H, Hashiguchi A, Sakamoto M. Segmentation of sinusoids in hematoxylin and eosin stained liver specimens using an orientation-selective filter. Open Journal of Medical Imaging. 2013 Dec 5;3(04):144. 43. Rolls G, Farmer N, Tarbet F. Assessing the Quality of Tissue Processing and the Performance of Peloris using the Leica Microsystems Scoring System. Leica Microsystems. 2008.

30

 Appen  Ap pendi di x A Data Collection Parameters Parameters: Nuclear Detail: (clarity and sharpness of chromatin; visibility of the nuclear border)

Nuclear Staining: (intensity, sharpness and contrast of the nucleus

Cytoplasmic Staining (intensity, sharpness and contrast of the cytoplasm and contents)

Uniformity of Stain: (homogeneity of stained areas and the absence of uneven stained areas)  Ab sen ce o f Precipitates

RATING 3

2

1

0

Chromatin clear and sharp; nuclear border visible

Chromatin somewhat clear and somewhat defined; nuclear border somewhat delineated

Chromatin unclear and poorly defined; nuclear border ill defined

Chromatin and nuclear border not visible

3

2

1

0

Nuclei stain intensely; sharpness and contrast of nucleus excellent

Nuclei stain satisfactorily; sharpness and contrast of nucleus satisfactory

Nuclei stain poorly; sharpness and contrast of nucleus non existent

Nuclei not stained

3

2

1

0

Cytoplasm stains intensely; sharpness and contrast of cytoplasm excellent

Cytoplasm stains satisfactorily; sharpness and contrast of cytoplasm satisfactory

Cytoplasm stains poorly; sharpness and contrast of cytoplasm non existent

Cytoplasm not stained

3 Stained areas homogeneous; unevenly stained areas non existent

2 Stained areas show some heterogeneousity; unevenly stained areas noted (<25% of slide)

1  Absense of homogeneousity; unevenly stained areas widespread (<50%of slide)

0  All of the slide is stained unevenly

3

2

1

0

Precipitates absent

Few precipitates seen

Precipitates widespread

Precipitates over majority of the slide

31

   t    e    e     h    B    S    x    n    i    o    i     d    t    n    c    e    e     l    p    l    p    o    A    C    a    t    a    D

32

33

 Appen  Ap pendi di x C Order Stained Slides

Slide Number

Stain

Slide Number

Stain

1

Positive

21

Stain A

2

Negative

22

Stain B

3

Positive

23

Stain A

4

Stain B

24

Positive

5

Positive

25

Stain B

6

Negative

26

Positive

7

Negative

27

Negative

8

Negative

28

Stain A

9

Stain A

29

Negative

10

Stain A

30

Positive

11

Stain A

31

Negative

12

Positive

32

Stain B

13

Positive

33

Stain B

14

Stain B

34

Stain A

15

Negative

35

Stain B

16

Stain A

36

Negative

17

Stain B

37

Stain B

18

Negative

38

Positive

19

Positive

39

Stain A

20

Stain A

40

Stain B

34

    h    7    c    r    1    a    0    M   2    y    r    a    7    u    r    1     b    0    e    2    F    y    r    a    7    u    1    n    0    a    2    J    r    e     b    6    m   1    e    0    c    2    e    D    r    e     b    6    m   1    e    0    v    2    o    N    r    e     b    6    1    o    t    0    c    2    O

   t    D   r   a   x   h    i    d   C   n   t   e   t   p   n   p   a    A   G

   r    e     b    6    m   1    e    t    0    p    2    e    S    t    s    u    6    g    1    u    0    2    A

   y    6     l    1    u    0    J    2

   e    6    n    1    u    0    J    2

   6    1    0    2    y    a    M      5    1    0    2    t    s    u    g    u    A

   y    t     i    v     i    t    c    A

   n    o    i    t    a    r    a    p    e    r    P     l    o    c    o    t    o    r    P

   n    o    i    s    s    i    m     b    u    S     l    o    c    o    t    o    r    P

   g    n    i    t    s    e    T    t    o     l    i    P

   n    o    i    t    c    e     l     l    o    C    a    t    a    D

   s    i    s    y     l    a    n    A    a    t    a    D

   t    p    i    r    c    s    u    n    a    M     d    e    t    e     l    p    m    o    C     f    o    p    u      e    t    i    r    W

35

 Appen  Ap pendi dix xE CIM-CVGH IRB Review

36

 Appen  Ap pendi di x F Transmittal Letter to CVGH

37

 Appen  Ap pendi di x G CVGH CVGH Histop Histop athology Laboratory H&E Staining Staining Process

38

 Appen  Ap pendi di x H Manual Manual Tissue Processing Schedule Schedule

39

 Appen  Ap pendi dix xI Transmi Transmi ttal Letters to Pathologis Pathologis ts January 17, 2017 Dr. Judito Caballero Faculty, PBL-2 Cebu Institute of Medicine F. Ramos Street, Cebu City

Dear Dr. Caballero, We are second year medical students of the Cebu Institute Institute of Medicine. As part of our research curriculum, we (Research Group 8) are conducting a study entitled "Acid Alcohol Extract of  Alugbati  Anthocyanin: A Possible Alternative to Hematoxylin Stain?" The general objective of the study is to determine the efficacy of acid alcohol extracts of Alugbati (Basella rubra) fruit as a hematoxylin substitute for nuclear staining. The extracts are prepared by the members of the group and the tissue preparation, staining and slide preparations are performed under the guidance of the section head of the department of histopathology of the Cebu Velez General Hospital. Survey forms will then be given out to you, the participating pathologist, who will be comparing the efficacy of the experimental histochemical stain against a positive and negative control. We are therefore inviting you to share with us your knowledge and technical expertise on the qualities of histopathologic sections. Tokens of appreciation will be given to to you for your participation. No evaluator identifier information information will be included in the collection of data. Your refusal to participate in this this study will not affect your your relationship with our school. You may opt to discontinue your participation at any time without need for explanation. We will be providing each study participant with a copy of our protocol should you request for it. Attached to this letter is the score sheet and the criteria for scoring. Samples are arranged randomly to prevent bias. This protocol was approved by the Ethics Review Committee of the Cebu Institute of Medicine (ERC-CIM). This study was duly approved by the Cebu Velez General Hospital - Laboratory Department through the hospital director, Dr. Josefina Poblete. You may contact me through this number (09255057362) if you have any inquiries regarding our study.

Hoping for your kind support.

Humbly yours,

Lorenz Robert Y. Ong Leader, Research Group 8 PBL-2 Cebu Institute of Medicine

40

January 17, 2017 Dr. Ruby Rusia-Uy Faculty, PBL-2 Cebu Institute of Medicine F. Ramos Street, Cebu City

Dear Dr. Uy, We are second year medical students of the Cebu Institute Institute of Medicine. As part of our research curriculum, we (Research Group 8) are conducting a study entitled "Acid Alcohol Extract of  Alugbati  Anthocyanin: A Possible Alternative to Hematoxylin Stain?" The general objective of the study is to determine the efficacy of acid alcohol extracts of Alugbati (Basella rubra) fruit as a hematoxylin substitute for nuclear staining. The extracts are prepared by the members of the group and the tissue preparation, staining and slide preparations are performed under the guidance of the section head of the department of histopathology of the Cebu Velez General Hospital. Survey forms will then be given out to you, the participating pathologist, who will be comparing the efficacy of the experimental histochemical stain against a positive and negative control. We are therefore inviting you to share with us your knowledge and technical expertise on the qualities of histopathologic sections. Tokens of appreciation will be given to to you for your participation. No evaluator identifier information information will be included in the collection of data. Your refusal to participate in this this study will not affect your your relationship with our school. You may opt to discontinue your participation at any time without need for explanation. We will be providing each study participant with a copy of our protocol should you request for it. Attached to this letter is the score sheet and the criteria for scoring. Samples are arranged randomly to prevent bias. This protocol was approved by the Ethics Review Committee of the Cebu Institute of Medicine (ERC-CIM). This study was duly approved by the Cebu Velez General Hospital - Laboratory Department through the hospital director, Dr. Josefina Poblete. You may contact me through this number (09255057362) if you have any inquiries regarding our study.

Hoping for your kind support.

Humbly yours,

Lorenz Robert Y. Ong Leader, Research Group 8 PBL-2 Cebu Institute of Medicine

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January 17, 2017 Dr. Cecila L. Capatoy Dean, College of Medical Technology Velez College F. Ramos Street, Cebu City

Dear Dr. Capatoy, We are second year medical students of the Cebu Institute Institute of Medicine. As part of our research curriculum, we (Research Group 8) are conducting a study entitled "Acid Alcohol Extract of  Alugbati  Anthocyanin: A Possible Alternative to Hematoxylin Stain?" The general objective of the study is to determine the efficacy of acid alcohol extracts of Alugbati (Basella rubra) fruit as a hematoxylin substitute for nuclear staining. The extracts are prepared by the members of the group and the tissue preparation, staining and slide preparations are performed under the guidance of the section head of the department of histopathology of the Cebu Velez General Hospital. Survey forms will then be given out to you, the participating pathologist, who will be comparing the efficacy of the experimental histochemical stain against a positive and negative control. We are therefore inviting you to share with us your knowledge and technical expertise on the qualities of histopathologic sections. Tokens of appreciation will be given to you for your participation. No evaluator identifier information information will be included in the collection of data. Your refusal to participate in this this study will not affect your your relationship with our school. You may opt to discontinue your participation at any time without need for explanation. We will be providing each study participant with a copy of our protocol should you request for it. Attached to this letter is the score sheet and the criteria for scoring. Samples are arranged randomly to prevent bias. This protocol was approved by the Ethics Review Committee of the Cebu Institute of Medicine (ERC-CIM). This study was duly approved by the Cebu Velez General Hospital - Laboratory Department through the hospital director, Dr. Josefina Poblete. You may contact me through this number (09255057362) if you have any inquiries regarding our study.

Hoping for your kind support.

Humbly yours,

Lorenz Robert Y. Ong Leader, Research Group 8 PBL-2 Cebu Institute of Medicine

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 Appen  Ap pendi di x J Budget

Item Apparatus: Beaker 250ml Stirring Rod Reagent Bottle 1000ml Reagent Bottle 500ml Mortar and Pestle Plastic Funnel Blender (Hanabishi HJB-115) Slide box (50's)

Reagents: Potassium Alum (Technical) Ferric Chloride Laboratory Processing Alugbati fruit Honorarium Gasoline Printing costs

TOTAL

Unit Price

Quantity

Amount

₱65.00

1 1 2 2 2 2 1 1

₱65.00

₱25.00 ₱180.00 ₱120.00 ₱130.00 ₱7.80 ₱719.00 ₱120.00

₱30.00/kg ₱50.00/L

1kg 1L

₱25.00 ₱360.00 ₱240.00 ₱260.00 ₱15.60 ₱719.00 ₱120.00

₱30.00 ₱50.00 ₱1,855.00

₱500.00 /kg

3kg

₱1,500.00 ₱2,380.00 ₱540.00

₱30.00

3

₱90.00

₱8,249.60

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