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

Hematoxylin Stain?

Ong, Lorenz Robert Y.

Aznar, 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

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

Authors: 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/ 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: A analytical experimental study design was utilized

Study Setting: Samples were collected from a plantation in Barangay Adlaon, 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 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 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. Tukeys test was used and
showed that Stain 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.

i
 TABLE OF CONTENTS

CONTENT PAGE

Abstract i
Table of Contents ii

INTRODUCTION 1
Background of the Study 1
Significance of the Study 2
Research Question 3
General Objective 3
Specific Objectives 3
Scope and Limitations 4

REVIEW OF RELATED LITERATURE 5

CONCEPTUAL FRAMEWORK 13

METHODOLOGY 14
Study Design 14
Operational Definitions 14
Study Setting 14
Study Population 15
Methodological Flowchart 16
Maneuvers 17
Data Analysis 19

RESULTS AND DISCUSSION 20

CONCLUSION AND RECOMMENDATIONS 27

References 28
Appendix A (Data Collection Parameters) 31
Appendix B (Data Collection Sheet) 32
Appendix C (Order of Stained Slides) 34
Appendix D (Gantt Chart) 35
Appendix E (CIM-CVGH IRB Review) 36
Appendix F (Transmittal Letter to CVGH) 37
Appendix G (CVGH Histopathology Laboratory H&E Staining Process) 38
Appendix H (Manual Tissue Processing Schedule) 39
Appendix I (Transmittal Letter to Pathologists) 40
Appendix J (Budget) 43

ii
 INTRODUCTION

Background of the 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). 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 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

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 and pig liver and the high metabolic activity of cultured pig liver

cells makes it a potential substitute for human cells in bioartificial liver devices.11

1
Significance of the Study

The hematoxylin and eosin (H&E) staining method has been profoundly used in histology-

pathology 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, 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).14, 6 The Basellaceae family in general has

anthocyanins, which are water-soluble, vacuolar pigments capable of staining cellular

structures.19, 20
Therefore, Basella rubra presents a promising alternative as a non-toxic,

accessible and cost-efficient organic chemical stain.14

2
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.

Specific Objective

1. To compare the mean staining scores of pig liver tissues stained with: (1) ripe alugbati fruit

extracted with ethanol-hydrochloric acid and (2) ripe 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 Limitations of the Study

Acid alcohol extracts of ripe 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 patients 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 such as: soil and water quality,

age of ripe alugbati fruit, and other factors that contribute to alugbati growth were not taken into

account.

4
 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

heartwood of the logwood tree (Haematoxylin campechianum).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 residues of

nuclear histones by linkage via a metallic ion mordant such as aluminum in acidic conditions. The

5
stain is applied longer than necessary, to ensure saturation of chemical binding sites resulting in

the over staining of the tissues with much non-specific background coloration. This undesirable

coloration is selectively removed by controlled filtering in an alcoholic acidic solution, (acid

alcohol), the process being termed "differentiation". Differentiation is arrested by returning to an

alkaline environment, whereupon the haematin takes on a blue hue, the process of "blueing-

up". The haematin demonstrates cell nuclei.27

Hemalums are used to provide blue stain that 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, 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

6
happened during both World Wars, but also in the late 1920s and early 1970s. 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, 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 cation is present. At pH 5-6, unacylated anthocyanins

are unstable and decolorize quickly.18 According to Amon et al. 2012, the anthocyanin and DPPH

(di(phenyl) - (2,4,6-trinitrophenyl) iminoazanium) radical scavenging activity are present in the

extract that marks its potential as a food colorant. Anthocyanin pigments appear to 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 ripening 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., also known as Alugbati, Malabar spinach, Indian spinach, Ceylon spinach and

vine spinach, belongs to the family Basellaceae. It is commonly found in tropical regions like

8
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 about 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 roots.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 nitrogen-

containing 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 et al. 1995 reported that 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 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 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 to UV light for 20 to 30

minutes led to the production of anthocyanin.7

A research study by Cruz 2007, used Basella sp. fruits and Dioscorea 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. 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 sp. (alugbati) fruit gave a positive result in saponins.

Dioscorea 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 by filtering 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 that 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.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

10
 Various staining procedures were previously developed for 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, Zenkers and Carnoys fixatives.5

A similar study was done by Co et al. (2001), where, instead of using blackberries, ripe Basella

rubra (Alugbati) and Biva arellana (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 is the work of De Leon (et. al.) on using the methanolic fruit

extract of Basella rubra as a hematologic stain. Few drops of the Basella 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 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 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.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

12
CONCEPTUAL FRAMEWORK

Tissue Type Embedding Medium

Dehydrating Agent Tissue Duration of Fixation

Processing

Fixative Mounting Medium

Stain Used* Duration of Staining*

Counterstain Used
Staining Temperature
pH of Stain Solution* Procedure

Mordant Used* Dye Extraction*

Nuclear Detail* Uniformity of Stain*

Staining Quality

Nuclear Staining* Precipitates*

Cytoplasmic Staining*

13
 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 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 Definitions:

1. Nuclear Detail - clarity and sharpness of chromatin and visibility of the nuclear membrane

when stained.43

2. Nuclear staining 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 the homogeneity of stained areas and the absence of uneven stained

areas.

14
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

15
Methodological Flow Chart
Stain A Slides 1Stain B Slides Positive Control Slides Negative Control Slides
Stain Extraction and Preparation

Obtain ripe alugbati fruits

Wash, dry

Add a small amount of 95% ethanol

Blend

To 500 g of blended alugbati:

Add 500 ml Add 500 ml

Ethanol-HCl Ethanol-Acetic acid
solution solution
Mix, stand for 32 hours,
and filter
Filtered Solution A Filtered Solution B

To 300 ml: To 300 ml:

Add: Add:
- 3g AlCl3 - 20ml 50% (w/v) aq. NH4
- 3.6 ml 10% FeCl3 alum solution

Stain Solution A Stain Solution B

Tissue Processing
Obtain Pig Liver

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

Stain with Stain Soln A Stain with Stain Soln B Stain with Harris
(10-12 minutes) (10-12 minutes) 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
16
Maneuvers

Data Collection Tool

Scoring of stained slides was performed blind with the participating pathologists

unaware of the tissue processing and staining procedure. Stained slides were scored with a four-

point 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 were obtained from a plantation in Barangay Adlaon, Cebu City.

Collected fruits were washed and dried to remove water and other 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 extracts were 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. From the ethanol-acetic acid dye

extract: to 300 ml of extract, 20 ml of 50% (w/v) aqueous ammonium alum was added and

designated as Stain 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 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 were then collected and analyzed.

18
Data Analysis

Scores from all score sheets were tallied. The mean scores from each test group, positive

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 Tukeys test 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-existent
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 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 on 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 Nuclear Cytoplasmic Stain
Precipitates
Detail Staining Staining Uniformity
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 2.87 2.90 2.93 2.93 2.17

Average

Interpretation Chromatin Nuclei stain Cytoplasm Stained areas Few

clear and intensely; stains homogeneous; precipitates
sharp; sharpness intensely; unevenly seen
nuclear and contrast sharpness stained areas
border visible of nucleus and contrast non-existent
excellent of cytoplasm
excellent

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 Nuclear Cytoplasmic

Stain Uniformity Precipitates
Detail Staining Staining

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

Interpretation Chromatin Nuclei stain Cytoplasm Stained areas Few

unclear poorly; stains show some precipitates
and poorly sharpness poorly; heterogeneousity seen
defined; and contrast sharpness ; unevenly
nuclear of nucleus and contrast stained areas
border ill- non-existent of cytoplasm noted
defined non-existent

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 Cytoplasmic
Nuclear Detail Stain Uniformity Precipitates
Staining 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

Interpretation Chromatin Nuclei stain Cytoplasm Absence of Few

unclear and poorly; stains poorly; homogeneousity precipitates
poorly defined; sharpness and sharpness and ; unevenly seen
nuclear border contrast of contrast of stained areas
ill-defined nucleus non- cytoplasm non- widespread
existent existent

Table 4. Descriptive Results of Slides Stained with Stain B

Nuclear Nuclear Cytoplasmic Stain

Detail Staining Staining 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 1.07 1.00 1.30 1.43 1.77

Average

Interpretation Chromatin Nuclei stain Cytoplasm Absence of Few

unclear and poorly; stains poorly; homogeneousi precipitates
poorly sharpness sharpness ty; unevenly seen
defined; and contrast and contrast stained areas
nuclear of nucleus of cytoplasm widespread
border ill- non-existent non-existent
defined

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

2.87 2.90 2.93 2.93

3.00

2.50
2.17
2.00 1.77
1.53 1.53 1.57
1.50 1.27 1.17 1.37 1.37 1.30 1.33 1.43
1.07 1.03 1.00 1.00
1.00

0.50

0.00
Nuclear Detail Nuclear Staining Cytoplasmic Staining Stain Uniformity Precipitates
Positive Control Negative Control Stain A Stain B

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 df Mean Square F Sig.

Between Groups 23.499 3 7.833 45.846 .000

Within Groups 9.568 56 .171
Total 33.067 59

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 have significant difference, the following

post-hoc Turkeys 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) Lower Bound Upper Bound

2 1.41333* .15093 .000 1.0137 1.8130

1 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

2 3 .06000 .15093 .979 -.3397 .4597
4 .03333 .15093 .996 -.3663 .4330
1 -1.47333* .15093 .000 -1.8730 -1.0737
3 2 -.06000 .15093 .979 -.4597 .3397
4 -.02667 .15093 .998 -.4263 .3730
1 -1.44667* .15093 .000 -1.8463 -1.0470

4 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 Control (H&E) 3 Stain A (ethanol-hydrochloric acid dye extract)
2 Negative Control (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 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 respectively, 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

demonstrated that extracts of Alugbati have poor nuclear staining and nuclear detail. It is to be

noted, however, that mordants were not added to the Alugbati stains by Co et al.

26
 CONCLUSION AND RECOMMENDATIONS

Conclusion

Based on the results and microscopic findings presented above, it is demonstrated that

acid alcohol stains of ripe Basella 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 pigment 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
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tested and consideration of the relation of their structure to performance as nuclear
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18. Bkowska-Barczak A. Acylated anthocyanins as stable, natural food colorantsa review.

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19. Amon, MFL, and LP Pladio. "Potential Food Colorant From The Extracts Of Alugbati
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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
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1968 May 1;33(3):266-74.

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39. Revilla E, Ryan JM, Martn-Ortega G. Comparison of several procedures used for the
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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.
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Segmentation of sinusoids in hematoxylin and eosin stained liver specimens using an
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30
 Appendix A
Data Collection Parameters

Parameters: RATING

Nuclear Detail: 3 2 1 0
(clarity and
sharpness of Chromatin clear Chromatin somewhat Chromatin unclear Chromatin and
chromatin; and sharp; clear and somewhat and poorly defined; nuclear border
visibility of the nuclear border defined; nuclear nuclear border ill not visible
nuclear border) visible border somewhat defined
delineated

Nuclear 3 2 1 0
Staining:
(intensity, Nuclei stain Nuclei stain Nuclei stain poorly; Nuclei not
sharpness and intensely; satisfactorily; sharpness and stained
contrast of the sharpness and sharpness and contrast of nucleus
nucleus contrast of contrast of nucleus non existent
nucleus satisfactory
excellent

Cytoplasmic 3 2 1 0
Staining
(intensity, Cytoplasm stains Cytoplasm stains Cytoplasm stains Cytoplasm not
sharpness and intensely; satisfactorily; poorly; sharpness stained
contrast of the sharpness and sharpness and and contrast of
cytoplasm and contrast of contrast of cytoplasm cytoplasm non
contents) cytoplasm satisfactory existent
excellent

Uniformity of 3 2 1 0
Stain: Stained areas Stained areas show Absense of All of the slide is
(homogeneity of homogeneous; some homogeneousity; stained unevenly
stained areas unevenly stained heterogeneousity; unevenly stained
and the areas non unevenly stained areas widespread
absence of existent areas noted (<50%of slide)
uneven stained (<25% of slide)
areas)

Absence of 3 2 1 0
Precipitates
Precipitates Few precipitates Precipitates Precipitates over
absent seen widespread majority of the
slide

31
 Appendix B
Data Collection Sheet

32
33
 Appendix 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
 Appendix D
Gantt Chart

June July August September October November December January February March
Activity August 2015 - May 2016
2016 2016 2016 2016 2016 2016 2016 2017 2017 2017
Protocol Preparation

Protocol Submission

Pilot Testing

Data Collection

Data Analysis

Write-up of Completed Manuscript

35
 Appendix E
CIM-CVGH IRB Review

36
 Appendix F

Transmittal Letter to CVGH

37
 Appendix G
CVGH Histopathology Laboratory H&E Staining Process

38
 Appendix H
Manual Tissue Processing Schedule

39
 Appendix I
Transmittal Letters to Pathologists
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 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 will be included in the collection of data. Your refusal to participate in this
study will not affect 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 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 will be included in the collection of data. Your refusal to participate in this
study will not affect 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

41
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 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 will be included in the collection of data. Your refusal to participate in this
study will not affect 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

42
 Appendix J
Budget

Item Unit Price Quantity Amount

Apparatus:
Beaker 250ml 65.00 1 65.00
Stirring Rod 25.00 1 25.00
Reagent Bottle 1000ml 180.00 2 360.00
Reagent Bottle 500ml 120.00 2 240.00
Mortar and Pestle 130.00 2 260.00
Plastic Funnel 7.80 2 15.60
Blender (Hanabishi HJB-115) 719.00 1 719.00
Slide box (50's) 120.00 1 120.00

Reagents:
Potassium Alum (Technical) 30.00/kg 1kg 30.00
Ferric Chloride 50.00/L 1L 50.00

Laboratory Processing 1,855.00

Alugbati fruit 500.00/kg 3kg 1,500.00
Honorarium 2,380.00
Gasoline 540.00
Printing costs 30.00 3 90.00

TOTAL 8,249.60

43