An Alternative anticoagulant for Hematologic Analysis,
Leaf Extract of Jatropha gossypifolia (Tuba-Tuba).

A Research Proposal
Presented to the Faculty of the
College of Medical Technology/Medical Laboratory Science

In Partial Fulfillment of the Requirements for the Degree
Bachelor of Science in Medical Technology/Medical Laboratory Science

By:

Cagandahan, John Paul M.
Carizon, Jay Niño P.
Estillore, Michael-Andrew B.
Resonable, Bryan H.
Sasaki, Sen M.
Veloso, Jerome A.

July 26, 2013

Chapter 1

The Problem and its Background

Introduction

The majority of the products for medical purposes are made from chemicals that harm the environment. Many medical researchers are modifying medical products that are natural in origin and one of them is anticoagulants.

In the laboratory the most widely used anticoagulant is the EDTA, ammonium-potassium oxalate (Heller and Paul double oxalate) and Heparin. Most anticoagulants can alter cell structure as well as coagulation and hereby affects the result of the laboratory analysis. There are certain plants that have anticoagulant properties and one of these plants is Jatropha gossypifolia or Tuba-Tuba, this plant is found widely in the Philippines and was shown in some previous studies its leaf extract has an anticoagulant property and has been used as an anticoagulant, because of this property this plant is a potential alternative for the commercially prepared anticoagulant used in the laboratory, but there is no known published article or existing literature that determines its effectiveness as an anticoagulant for blood specimens used in hematological analysis. So that is why in this study, the researcher decided to determine the anticoagulant effectiveness of the leaf extract of the plant, Tuba-Tuba, for potential cheap alternative for commercially prepared anticoagulant.

Statement of the Problem
1. Does the leaf extract of Tuba-Tuba have an anticoagulant property.
2. How effective is the Tuba-Tuba as an anticoagulant with regards: 1. With preservation of the morphology of: 1. White blood cell/WBC 2. Red blood cell/RBC 3. Platelet Significance of the Study The findings of this research will be benificial to the following: patients, for them to have lower costs as compared with commercially prepared anticoagulant and have a more affordable laboratory tests. Medical technology students, for them to be less exposed to hazardous materials in the laboratory, and may lessen the costs in laboratory supples. Producers of laboratory materials may lessen the use of hazardous chemicals in making anticagulants compared to the amount of chemicals they will use in making synthetic anticoagulant.

Conceptual Framework [pic] Conceptual Framework The plant of interest was selected based on the gathered related researches and literatures about the chemical properties of anticoagulants and those plants that may exhibit high content of these chemicals. Jathropa gossypifolia(Tuba-Tuba) is determined as one of the candidates for the study because of its fruits high oxalate content. After the selection, healthy leaves of the plant will be collected. After completing the input, the researchers will proceeded to the throughput in which the method of extraction will be determined and will quickly proceeded to the extraction of the sample that will be tested to the blood. The pH will be adjusted to different levels to avoid the hemolysis of the cells that could be seen in the plasma and the different concentration of the extract will be tested and different blood extract with the chosen concentration had been performed thrice to validate the anticoagulant property of Jathropa gossypifolia (Tuba-Tuba).
As an output, the produced anticoagulant had been determined to be ideal for white blood cell demonstration because the results of the extracts has been recorded and evaluated against EDTA which is considered as the standard.

Scope and Llimitation The focus of the study will determine if the plant has an anticoagulant property that can be used in hematologic test. The researcher will determine the concentration of the plant extract to be used with the blood. The reseacher will determine the volume ratio of both blood and plant extract. The researcher will not further elaborate the effectiveness of the anticoagulant property of the said plant. The researcher will not give dose of the anticoagulant assess for physical and chemical needed for hematologic in anticoagulant in oral in vivo.

Hypothesis There is or there is no presence of anticoagulant in terms of using the leaf extract from Jathropa gossypifolia (Tuba-Tuba).

Definition of Terms 1.) Anticoagulant – are used to prevent the clotting of the blood specimen and the reagent employed should not bring about alteration of blood components. (Methods of Hematology, 2004) 2.) EDTA (K2EDTA) – a crystalline acid that acts as a strong chelating agent and that forms a sodium salt used as an antidote for metal poisoning and as an anticoagulant. (The Free Dictionary by Farlex, 2013) 3.) Hematology – includes the study of blood cells and coagulation. It encompasses analyses of the concentration, structure, and function of the cells in blood. (McPherson, 2009) 4.) Red Blood Cells – any of the haemoglobin-containing cells that carry oxygen to the tissues and are responsible for the red color of vertebrate blood. It is also called as erythrocyte, red blood corpuscle, red cell, red corpuscle (Merriam Webster, 2012) 5.) Wright stain - is a methyl alcoholic solution of eosin and a complex mixture of thiazines, including methylene blue (usually 50-75%), azure B (10-25%), and other derivatives. (Henry’s 2009) 6.) Extraction - are a way to separate a desired substance when it is mixed with others. The mixture is brought into contact with a solvent in which the substance of interest is soluble, but the other substances present are insoluble. (Chemicool, 2013)

CHAPTER III RESEARCH METHODS This chapter contains the study design, locale of the study, sampling procedures, statistical treatment, screening of blood sample, the process of extraction and the tests done on the extract.

STUDY DESIGN The study design of this research is of experimental type wherein the control that is being used is the commercially prepared EDTA anticoagulant compared to the extract from Jatropha gossypifolia (“Tuba-Tuba”). Two variables were used namely: the independent variable and the dependent variable. The EDTA or the commercially prepared anticoagulant served as the independent variable (IV) because its value and performance was constant, whereas the extract from the leaf of interest was the dependent variable (DV) because its value and performance was still unknown or it may change due to several contributing factors. A series of experimental test was done to determine the anticoagulant property of the extract and also its capability to demonstrate the morphology of the white blood cell. The researchers carefully conducted the experimental procedure and followed a time table for each tests to confirm the validity of the results.

LOCALE OF THE STUDY The study was conducted at the Arellano University – Manila. All the laboratory equipments were also provided by the university. The leaf of Jatropha gossypifolia came from one of the researchers family friend residing in Antipolo, Rizal City. The patient who participated in the study are all Medical Technology students, third year level, currently studying at the Arellano University - Manila, and ages 18 – 21 years old.

SAMPLING PROCEDURES The chosen populations for this study were the third year medical technology students from Arellano University – Manila because a larger number of populations would be inconvenient and expensive. The twenty percent of the sample from the qualified third year population is obtained through random sampling by the process of random computer generated numbers. Random sampling is done to avoid biases in the results and to provide more convincing results.

STATISTICAL TREATMENT The morphological assessment of the white blood cells done in this research was qualitative in nature and to be able to assess statistically a grading system was adopted. The obtained data had been subjected for the computation of the weighted mean that had provided the average degree of satisfaction of the morphology of the white blood cells that are garnered in the results of different experiments and trials. The weighted mean is obtained with the following formula:

The computed results had been used as one of the criterion for the selection of dilutions utilized for the progression of research.

PLANT MATERIAL Healthy plant of Jatropha gossypifolia (“Tuba-Tuba”) freshly picked from Antipolo, Rizal City.

SCREENING OF BLOOD SAMPLE Blood samples are collected from healthy volunteer donors from the Medical Technologist students, third year lever of both genders, ages 18-21 years old. A survey form was prepared for the determination if the volunteers are healthy.

BLOOD COLLECTION Blood samples were drawn via venipuncture at the antecubital region of the arm.

DECOCTION PROCESS The 100 g of fresh leaves of J. gossypifolia was crushed in a blender for 10 min and the raw juice collected by straining the crushed leaves. The researchers will centrifuge the raw juice at 3,500 rpm for 10 minutes and after that, the researchers will also collect the clear supernatant that will be produce. The minimum volume of the extract that achieved anticoagulation was determined by the method of Lee and White (1968). Freshly collected whole blood was added to different Khan tubes containing 0.5, 1.0, 0.5, 1.0, 0.5, 1.0 ml of leaf extract. The time taken for clot formation in each tube will be recorded. The anticoagulant effect of the extract was achieved at 0.1 ml of the leaf extract, which was then pipetted into plain specimen bottles and allowed to dry for 72 h at room temperature.

EXTRACT AGAINST BLOOD The extract will be poured into different test tubes with labels 1ml blood:0.5 ml extract, 1 ml blood:1 ml extract, 3 ml blood:0.5 ml extract, 3 ml blood:1 ml extract, 5 ml blood:0.5 ml extract, 5 ml blood:1 ml extract for every concentration of extract. The test tubes were then carefully inverted for 8 times to properly distribute the extract.

BLOOD SMEAR PREPARATION Right after mixing the tubes, a drop of blood was placed in a slide and smeared with the use of another slide. The test tubes were then set aside for another 30 minutes then smeared again. Another set of smears were made after 2 hours. The time difference was used to check whether the white blood cells can sustain their morphology after being mixed with an anticoagulant.