organic stain for hematologic blood smear

8/13/2019 Organic Stain for Hematologic Blood Smear

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METHANOLIC FRUIT EXTRACT OF Basella rub ra: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

rubrathat 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 wasthen 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 presentstudy 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 ofBasellarubracan stain the blood cells.

2. To determine the staining capability of extractfrom 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 ledthe 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 befound 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 Basellarubraas 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 ofBasella rubraused 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. Theripe 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. Aftermaceration, 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.


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The staining of blood cells was done by adding few drops of Basella rubra extract on a previouslyprepared 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 rubradye

(picture on left) and the standard wrights stain (picture on right). The picture at the center is the ripe fruitofBasella rubra.

RESULTS AND DISCUSSIONSThe 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 rubraextract 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

The table above shows that blood smears stained with an increasing amount of dye extracted fromBasella rubraproduces 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.

Blood tostain

1 2 3 4 5Ratio

1 is to 1

1 is to 10

1 is to 15

1 is to 20

= 3

Alugbati stain Alugbati (Basell a rubra) Fruit Wrights stain


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Effectiveness of Stains on blood film with Contrast Reagents

0

1

2

3

4

5

6

NSS

etha

nol

metha

nol

w/P.alum

w/CuS

O4

w

/Methyle

neblue

w/iodin

e

stains

Figure 1

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 thetested chemicals methylene blue gave a very satisfactory result.

Table II. Rate of Stain Effectiveness with Verbal Interpretation

ObservationsEquivalent

rate

very goodvisibly stained rbcplatelets, and wbc with nomorphological alterations 5

good - visibly stained rbc, platelets, with no

morphological alterations 4

satisfactory/average- visible stained rbc, platelets with

some morphological alterations 3

poor- stained rbc only, morphological alterations

present 2

very poor- no observable morphology of cells 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.


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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 rubra3. 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 extracteddye.

SUMMARY

In summary after the untiring hardwork and perseverseverance, the researchers were able to isolate an

organic dye from the fruit of Basella rubraand 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 futureresearchers can use the extracted dye in pathological blood samples, other body fluids or even

microbiological specimens.

2. A more sophisticated approach of extracting the dye from Basella rubramay also be done by thefuture researchers.

3. The future researchers can also explore on possibilities of using other organic dye to contrast thecolor of other cells and make the dye completely organic, safe, environment friendly and non-

toxic.

4. Other gaps of research such as testing the extracts pH, its shelf-life, and its effect on the presenceof other anticoagulants.

REFERENCES:

Kiernan JA (2008) Histological and Histochemical Methods. Theory and Practice. Bloxham, UK:Scion.

Rodak, B.F; Fritsma, G.A; Doig, K; (2009) Hematology: Clinical Principles and Applications, 3rd

ed. Elsevier, INC.

Palada, M.C.; Chang, L.C (2003) Suggested cultural practices for Basella. AVDRC InternationalCooperators Guide. AVDRC Pub # 03-553. 4p

Service, United States Department of Agriculture. Last Modified: 10/05/2007

Bruce-Gregorios. J.H (2006) Histopathological Techniques. 2nded: Goodwill Trading Co.,INC

Siemonsma, J.S.; Piluek, K. (1994) Plant Resources of South-East Asia, Number 8. , Bogor, Indonesia:

PROSEA.Sukprakam, S.; Juntakool, S.; Huang, R.; Kalb, T. (2005) Saving your own vegetable seeds: A guide for

farmers. AVDRC Publication Number 05-647. Shanhua, Taiwan: AVDRC-The World Vegetable

Center. 25p

Vadhwa, O.P.; Reddy, C. R.; Spiers, J.M.; Marshall, D.A.(2003) Different trellis system for MalabarSpinach (Basella alba L.) production. Association of Research Directors. Agricultural Research

Rodak, B.F; Fritsma, G.A; Doig, K; (2009) Hematology: Clinical Principles and Applications, 3rd

ed. Elsevier, INC.

organic stain for hematologic blood smear

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