Chapter I

THE PROBLEM AND ITS BACKGROUND

INTRODUCTION
Blood which is derived from the greek word haima, together with phlegm,
yellow bile and black bile are the four humors that Hippocrates proposed
contriuting in the cuse of disese chller, Gerber,& Kaempfer, 2008).
Blood is one of the most important components in our body that carries oxygen
and nutrients to the tissues and remove carbon dioxide. And it is used as a
specimen in order to detect abnormality within our body. It contains the three
important cells which are erythrocytes, leukocytes and thrombocytes that may
reflect our body physiological state.
Although advance technology is now used nowadays, still, the manual
method, peripheral blood smear is used in many laboratories especially in the
Philippines. It is essential to know, especially for the medical technologist the
proper making of peripheral blood smear since it is used to manually check and
assess blood cells morphology that automated machine cannot identify.
The peripherial blood smear must be well-made in order to have a reliable
identification, quantification and evaluations of peripheral blood cells. This is
made on the glass slides by smearing the blood properly then staining it with the
special stains like the Wright-Giemsa stain which is the most commonly used
stain in routine blood smears. (Valenciano, owell, & Rizzi, 2013). The purpose
of staining the blood smears is to identify and differentiate blood cells through the
use of microscope. A well-stained smear is important to have an accurate
interpretation (Rodak & Carr, 2012)
The Philippines is blessed with many plants that can be used as an
alternative in many ways especially when some products are not readily available
at use. Examples are the plants Alugbati (Basella rubra) and Gumamela
(Hibiscus rosa sinensis) leaves which can be used as an alternative stain.
Basella rubra and Hibiscus rosa sinensis is said to have anthocyanin property
which contributes to its ability to stain cells. Anthocyanins are vacuolar pigments
most commonly responsible for red or purple coloration.
With this background information, the researchers would like to determine
Basella rubra (Alugbati) as an alternative stain and Hibiscus rosa sinensis
(Gumamela) as counterstain to be used in routine blood cells differentiation. The
dyes extracted from Alugbati will stain the nuclear part of the cells while dyes
from Gumamela will stain the cytoplasm. Furthermore, this study is conducted to
provide additional option in staining routine peripheral blood smears. The
researchers also intend to provide cheaper and effective alternatives to
commercially prepared stain using dyes extracted from natural sources.

CONCEPTUAL FRAMEWORK

Figure 1. Schematic Diagram of Gumamela as Counterstain for Alugbati in
Staining Routine Peripheral Blood Smear.


STATEMENT OF THE PROBLEM
This study will focus in the use of gumamela as an counterstain of alugbati
used for peripheral blood smears. Specifically, the researchers will seek to
answer the following questions:
1. Can gumamela extract be used as an counterstain of alugbati in routine
hematological blood smear?
INPUT
Anticoagulated
blood
methanolic
extract of
alugbati as basic
stain
PROCESS
methanolic
extract of
gumamela as
counterstain for
alugbati
stbilization of
the organic stain
staining
peripheral blood
smear
OUTPUT
demosntration
of nucleus and
cytoplasm of
peripheral blood
cells
stained non-
pathogenic
blood smear
stained
peripheral blood
smears
containing
immature blood
cells
2. Does it stain all blood cells?
a) Erythrocytes
b) Leukocytes
c) Thrombocytes?
3. Does it provide differential staining of white blood cells?
a) Neutrophils
b) Lymphocytes
c) Monocytes
d) Eosinophils
e) Basophils
4. What properties of alugbati and gumamela contribute to the staining
procedures?
5. Does it stain immature blood cells?


OBJECTIVES
The general objective of the study is to use Gumamela extract as an
counterstain in routine peripheral blood smear.
Specific objectives aim:
1. To improvise an counterststain to alugbati
2. To provide a counterstain made from a gumamela extract that is common
in the Philippines
3. To produce a less expensive stain for blood smears


STATEMENT OF HYPOTHESIS
To answer the research questions in this study, the following hypotheses
will be tested:
H
o
: Gumamela extract is not a good counterstain to alugbati for routine
hematological blood smears.

H
1
: Gumamela extracts gives good staining quality as counterstain of
alugbati for routine hematological blood smears.


ASSUMPTIONS OF THE STUDY
The research is based on the following assumptions:
1. That Gumamela extract can be used as a counterstain of Alugbati in
peripheral blood smear.
2. That Gumamela and Alugbati extract can be used in differentiating
peripheral blood cells.
SIGNIFICANCE OF THE STUDY
This study would be most beneficial to the following:
1. Medical Technologist, who is most responsible behind the diagnosis,
prognosis and prevention of disease that can used the alugbati and
gumamela extract as their alternative stain in analyzing peripheral blood
smears obtained from patient.

2. Future Researchers and students, which can serve as a study that
provide evidences on the effectiveness of alugbati and gumamela extract
as an alternative hematological stain.

3. Clinical Laboratories, which performs hematology testing. Using this
stain is very economical and cost-effective that can be used anytime in
case of emergency.

4. Community, for them to appreciate the importance of alugbati and
gumamela as useful plants.

SCOPE AND DELIMITATION
This study will focus on the effectiveness of gumamela extract as a
counterstain of alugbati in staining peripheral blood smear. It will be used as a
routine stain in blood smears.
The researchers will use 20 random blood samples in different individuals
around University of Perpetual Help System Binan Campus. Anticoagulant to be
used will be EDTA. The study will only focus for the examination of non-
pathologic blood smears, non-febrile and non-anemic patients.
There are few limitations inherent in the study that is beyond the control of
the researchers. First,


DEFINITION OF TERMS
The following terms that will be used in this study are operationally and
conceptually defined as follows:
Extract. To draw or pull out a substance, using force or effort. This also refers to
substance obtain from herb or plants by chemical or mechanical action, as by
pressure, distillation or evaporation then mixed with a solvent like alcohol, water
or other solvent.
Alternative. This refers to the choice that comes from two or more possible
choices.
Counterstain. This refers to the second stain used to contrast the other cell
elements that not made visible by the primary stain.
Peripheral Blood Smear. This refers to the blood prepared on a slide that will be
stained in order to analyze blood cells.
















Chapter II

REVIEW OF RELATED LITERATURE AND RELATED STUDIES

RELATED STUDIES
Extraction of Anthocyanin
An aqueous two-phase system composed of hydrophilic solvent and an
inorganic salt, especially ethanol and ammonium sulfate, is suitable for the
extraction of anthocyanins from V. uliginosum residue. The effect of ethanol and
ammonium sulfate on the partition of anthocyanins was investigated to obtain the
optimum condition for the extraction. The ATPS composed of 30% (w/w)
ethanol/19% (w/w) ammonium sulfate was found to yield the best result.
Meanwhile, the AB-8 resin fit for the purification of anthocyanins. Compared with
conventional extraction based on acidified ethanol, an integrated process of
ATPE combined with column chromatography has some advantages, such as
short treatment time, lower ethanol consumption and no heating requirement to
yield anthocyanins from the fruit residue of V. uliginosum. This new technology
might be a suitable extraction method for other natural pigments and bioactive
natural products on industrial scale.
(Extraction and Purification of Anthocyanins from the Fruit Residues of
Vaccinium uliginosum LinnZhang Hua, Dong Yuesheng, Xu Ge, Li Menglu, Du
Liya, An LiJia and XiuZhilong)

Anthocyanin of Basella rubra as microbiological stain
This study was undertaken to extract anthocyanin from Basella rubra berries and
utilize the extract as microbiological stain. It is an inexpensive, indigeneous and
abundant raw material. The alugbati berries were macerated in a blender and
extracted with 1% HCl in 95% methanol. The extract obtained was filtered and
then concentrated. Thin layer and column chromatography methods were used
to isolate and purify the anthocyamin. The samples were analyzed using infrared
spectra and ultraviolet spectra. FT-IR revealed the presence of a hydroxyl group
which is prominent in the structure of anthocyanin pigment at 3385cm'1. The
C=O stretching for aromatic ring were indicated by a peak at 1635.20 cm'l,
1513.38 em-I for C=O bending, 1439.91 cm'I for O-H bending, 1207.42 cm,l for
c-o stretching, 1151.92cm" for alkane and 1100.77 em-I for C-C stretching. The
structure of anthocycanin was further established by the e maximum of the
ultraviolet spectrum at 510.0 nm. For the application, the crude extract was used
as a stain for Staphyloccus aureus, a gram positive bacteria and Escherichia coli,
a gram negative bacteria. The staining process for the microorganism used
mordants like potassium alum, calcium oxide and copper sulfate for fixing the
color. Only copper sulfate and lime responded positively as a mordant that gave
favorable outcome in fixing the color of alugbati. The samples were screened
based on the criteria of color retention and evenness. The structure of the
microorganisms with respect to shape and size and certain cellular components
were identified using a microscope and photomicrographs. The alugbati extract
produced stain that was comparable with synthetic stains like crystal violet and
safranin and can, therefore, be used as an alternative stain.
Utilization of an indigenous dyestuff from Basella rubra (alugbati) as
microbiological stain