METHANOLIC FRUIT EXTRACT OF Basella rubra: ORGANIC STAIN FOR HEMATOLOGIC BLOOD SMEAR

De Leon, Manuel ; Latoza, Ani Gold ; Nues, Angelique ; Pilac, Maria Rae-Ghine ; Sistoza, Christina Joy ; Soliman, Donna Grace ; Zuiga, Maria Christina ;Mortel, Ferdinand

ABSTRACT:
The emergent need and campaign of the DENR and other international agencies in promoting a greener and cleaner environment should urge different agencies including clinical laboratories in using a safe and environment friendly chemicals. This urgency leads to the desire of the researchers to develop a safe, non-toxic and organic chemical stain. The researchers thought of extracting dye from fruit of Basella rubra that produces a rich purplish juice when it ripens. This vegetable grows abundantly in the Philippines and when developed could be a source of inexpensive and yet safer blood stain compared to the expensive commercially and toxic stains available in the market. The researchers utilized an experimental study by doing a miss and hit method. The Alugbati fruits were extracted using 95% Methyl Alcohol and the obtained extract was filtered and centrifuged at 3000 rpm for 5 minutes. The isolated colored extract was then used to stain the prepared normal blood smears. After several trials the need for contrast stain was identified on which so far methylene blue was found to be effective. Methylene blue gave favorable outcome in enhancing the color of White Blood Cell. Then cells were screened based on criteria of color retention and blood cell morphological structure visibility using a compound light microscope. At present study the blood cells are visibly observed but the researchers further recommends that several factors must be studied well by the future researchers to come up with a better reliability of results. GENERAL OBJECTIVE: To produce an organic, safe and cost effective dye for blood smears. SPECIFIC OBJECTIVES: 1. To determine if the extracted dye from the fruit of Basella rubra can stain the blood cells. 2. To determine the staining capability of extract from the fruit of Basella rubra on blood smear even in the presence of varying factors: contrast chemicals, anticoagulants and ratio of blood to extracted dye. INTRODUCTION: A well-made, well-stained, and carefully examined peripheral blood smear can provide valuable information regarding a patients health. Through the years many blood film dyes have been developed. Most of these dyes are synthetic chemical compounds made from substances found in coal tar which are highly toxic and may cause adverse effects to peoples health according to Anderson (1998). This fact led the researchers to investigate a certain species of plant in Philippine biosphere of raw material for the production of natural dyes with suitable properties for use in staining. A great source of natural dyes can be found in roots, nuts and flowers. One of the sources of natural dye is Basella rubra (alugbati). Basella rubra is from the family Basellaceae and genus Basella Linnaeus. The potential of the fruits of Basella rubra as a blood smear dye has not yet been established, but with the growing harmful effects of synthetic dyes, the need of biologically prepared stains from dye-producing plants like Basella rubra would probably prove to be a solution.

METHODOLOGY The researchers utilized an experimental design of study. With the consent of volunteer patients their blood were extracted and examined if free of pathological findings prior to use in the procedure. The fruits of Basella rubra used in this study were obtained particularly from a farmer growing these vegetables at Brgy. Helera Jaen, Nueva Ecija and was certified by Ms. Nieves Capili a botanist from Manila Central University. The extraction of dye from the fruit of Basella rubra was conducted at the PGT Laboratory. The ripe fruit were carefully chosen and properly washed to remove dirt and some debris. Using a top load, 5 grams of fruits were weighed added with 5ml of Methyl Alcohol as extracting agent and macerated. After maceration, it was filtered using a funnel with 0.1 filter paper and erlenmeyer flask. The filtrate was then centrifuged at 3000 rpm for 3-5 minutes and ready to be used as stain for blood smears.

The staining of blood cells was done by adding few drops of Basella rubra extract on a previously prepared blood smear. Allow to dry, do not wash. Upon drying add two drops of diluted Methylene blue to contrast the color and make white blood cells more visible. Allow it to dry completely and observe under Oil immersion objective. The picture below shows the reaction of blood cells using the Basella rubra dye (picture on left) and the standard wrights stain (picture on right). The picture at the center is the ripe fruit of Basella rubra.

Alugbati stain

Alugbati (Basella rubra) Fruit

Wrights stain

RESULTS AND DISCUSSIONS The results appeared that the temperature and the anticogulant used did not greatly affect the reaction of blood cells in terms of morphology and staining reaction using the extracted dye of Basella rubra. After several trials and testing of the correct and proper dilutions, the researchers finally identified that there is an ideal ratio of blood and the amounts of Basella rubra extract that must be combined in order to get a satisfactory results. Table I Effects of increasing ratio of Methanol-extracted Alugbati stain to blood smear
Blood to stain Ratio 1 is to 1 1 is to 10 1 is to 15 1 is to 20 =3 1 2 3 4 5

The table above shows that blood smears stained with an increasing amount of dye extracted from Basella rubra produces a more defined visibly stained blood cells. 1 is to 1 ratio shows no observable morphology of the cell, on the contrary a 1:15 and 1:20 ratio exhibited more visible stained blood cells. Red Blood Cells (RBC) and platelets are visible with no morphological alteration.

Effectiveness of Stains on blood film with Contrast Reagents

6 5 4 3 2 1 0

stains

The figure above shows the effectiveness rating of the stain on different blood cells when added with certain reagents. When the methanolic extract alone was tested it stained all blood cells but cannot be differentiated. But when the extract was added with certain chemicals such as potassium alum, copper sulfate, methylene blue and iodine it produces contrast. Thereby, the blood cells are differentiated from one another. The nucleus of white blood cells became clearly visible and platelets are identifiable. Among the tested chemicals methylene blue gave a very satisfactory result.

Table II. Rate of Stain Effectiveness with Verbal Interpretation

NS et S ha m n ol et w/ han P. o l al w/ w u M /C m et hy uSO le ne 4 b w/ lue io di ne

Figure 1

Observations very good visibly stained rbc platelets, and wbc with no morphological alterations good - visibly stained rbc, platelets, with no morphological alterations satisfactory/average- visible stained rbc, platelets with some morphological alterations poor- stained rbc only, morphological alterations present very poor- no observable morphology of cells

Equivalent rate

5 4 3 2 1

The table above shows the verbal interpretation on how the blood films are graded. It shows that the grade of the dye depends on the observable blood cells seen under the microscope.

CONCLUSIONS: Based on the study conducted by the researchers the following conclusions were drawn: 1. They dye produced from Basella rubra can be extracted using methyl alcohol. 2. The blood cells can be stained by the extracted dye coming from fruit of Basella rubra 3. The ratio of blood to dye that yield a visible blood cells on blood smear is 1:15 and 1:20. 4. Demonstration of WBC and platelets can be achieved by adding methylene blue to the extracted dye. SUMMARY In summary after the untiring hardwork and perseverseverance, the researchers were able to isolate an organic dye from the fruit of Basella rubra and found to be an effective stain for normal blood cells. The use of methyl alcohol as extracting agent produces a better dye yield, and the correct ratio of blood to the dye was also identified. The dye was able to stain blood cells particularly red cells and even enhance with the use of contrast reagents making white cells and platelets more visible.

RECOMMENDATIONS: 1. Since the researchers delimit the study on testing the dye on normal blood smears, the future researchers can use the extracted dye in pathological blood samples, other body fluids or even microbiological specimens. A more sophisticated approach of extracting the dye from Basella rubra may also be done by the future researchers. The future researchers can also explore on possibilities of using other organic dye to contrast the color of other cells and make the dye completely organic, safe, environment friendly and nontoxic. Other gaps of research such as testing the extracts pH, its shelf-life, and its effect on the presence of other anticoagulants.

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