Journal of Indonesian Medical Laboratory and Science pISSN 2775-0108 An eISSN 2774-2504 Parijoto (Medinilla specios.
extract as a natural dye alternative for peripheral blood smears: A comparative study with giemsa stain Meida Trisna1*.
Indanah2.
Yunita Rusidah1.
Arief Adi Saputro1.
Anisa Sholikhati1.
Sherly Nur Ekawati1 Department of Medical Laboratory Technology.
Faculty of Health Sciences.
Universitas Muhammadiyah Kudus.
Kudus.
Indonesia.
Department of Nursing.
Faculty of Health Sciences.
Universitas Muhammadiyah Kudus.
Kudus.
Indonesia.
Correspondence Meida Trisna Jepara.
Central Jawa Ae 59466.
Indonesia Email: 32022150002@std.
Received: 2026-01-21 Revised: 2026-04-21 Accepted: 2026-04-25 Available online: 2026-04-27 DOI: https://doi.
org/10.
53699/joimedlabs.
Citation Trisma M.
Indanah I.
Rusidah Y.
Saputro AA.
Sholikhati A.
Ekawati SN.
Parijoto (Medinilla specios.
extract as a natural dye alternative for peripheral blood smears: A comparative study with giemsa stain.
Journal of Indonesian Medical Laboratory and Science, 7.
, 1-11.
Abstract Background: Peripheral blood smear staining is an essential hematological procedure used to evaluate blood cell morphology.
The commonly used standard stain is Giemsa stain.
however, its chemical components may pose potential risks to health and the Therefore, safer natural alternatives are needed.
Previous studies have reported that anthocyanin-containing plant extracts, such as Hibiscus sabdariffa.
Tectona grandis, and Garcinia mangostana, can effectively stain blood cells.
Medinilla speciosa .
also contains anthocyanins and has potential as a natural Objective: This study aimed to evaluate the staining quality of peripheral blood smears using Medinilla speciosa extract at different concentrations and to compare the results with a 10% Giemsa stain Methods: This laboratory-based experimental study used a post-test only control group design.
A total of 30 blood smears were stained using Medinilla speciosa extract at concentrations of 25%, 50%, and 70%, along with a 10% Giemsa stain control.
The extract was prepared using maceration with pH adjustment to optimize anthocyanin stability.
Staining quality was assessed based on color intensity, background clarity, and erythrocyte morphology using a semi-quantitative scoring system.
Data were analyzed using the KruskalAeWallis test and MannAeWhitney U test.
Results: Increasing extract concentration was generally associated with improved staining quality.
A significant difference among groups was observed .
= 0.
Pairwise analysis showed that the 70% extract was not significantly different from the 10% Giemsa stain control .
= 0.
while lower concentrations differed significantly.
Conclusion: The 70% Medinilla speciosa extract showed the best performance among extract groups and demonstrated comparable staining quality in certain parameters.
However.
Giemsa stain remains the more consistent standard.
Further optimization is required before routine Keywords Erythrocyte morphology.
Giemsa.
Natural dyes.
Parijoto.
Peripheral blood smear Copyright: A 2026 by the authors.
Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY SA) license.
ttps://creativecommons.
org/licenses/by-sa/4.
0/).
Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
Introduction Blood cell morphology examination is an important parameter in hematology testing.
Blood disorders such as anemia are generally detected through blood morphology using the peripheral blood smear method (Khasanah et al.
, 2.
Hematology examination using the peripheral blood smear method is carried out by making a thin layer of blood on a glass object, then fixing it and coloring it with a certain substance, then viewing it using a microscope (Alawiyah, 2.
The addition of dye to the peripheral blood smear preparation aims to clarify and provide contrasting color to the blood cell structure, making it easier to observe using a microscope (Varghese et al.
, 2.
Giemsa is a commonly used to stain for staining blood smears (Alawiyah, 2.
Although it produces sharp and high-contrast results, this type of stain has negative impacts, particularly on the environment.
This is due to its main component, methanol, which is toxic and therefore hazardous to aquatic organisms and human health.
Additionally, synthetic stains such as eosin and methylene blue are persistent, difficult to degrade, and have the potential to cause toxicity in aquatic microorganisms.
If Giemsa waste is disposed of directly without treatment, the accumulation of these chemical compounds can disrupt ecosystem balance, degrade water quality, and contribute to environmental pollution (N.
Putri et al.
, 2.
Growing awareness of the environmental impact of synthetic dyes has spurred the exploration of natural and eco-friendly materials as potential alternatives (Agustin et al.
Previous research using related natural materials has shown that natural dyes derived from plant extracts have the potential to be used as stains for peripheral blood In this study, rosella flower extract (Hibiscus sabdariff.
, with its anthocyanin content, can impart a reddish-purple color to erythrocytes.
A study using teak leaf extract (Tectona grandi.
on blood cells produced a reddish-purple color.
In another study, mangosteen peel (Garcinia mangostan.
, with its anthocyanin content, also provided good staining on blood cells (Ghofur et al.
, 2.
Based on this research, the materials used contain natural pigments that can bind to blood cell components through electrostatic bonds and chemical interactions without damaging the cell structure (Nuraini & Tianto.
The parijoto plant (Medinilla specios.
is an endemic species that grows in highland areas such as mountain slopes and forests in Indonesia.
According to research conducted by Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
Sholikhati et al, .
, parijoto fruit has been shown to possess pharmacological activities such as antidiabetic, antioxidant, antibacterial, and anticancer properties.
Its various chemical constituents include saponins, flavonoid glycosides, and tannins.
Flavonoid compounds are divided into several groups: chalcones, anthocyanins, anthocyanidins, isoflavones, flavanones, flavonols, and flavones.
These pigment compounds produce the colors found in plants, such as anthocyanins, which produce blue, purple, and red colors (Ifadah et al.
, 2.
The anthocyanin pigments in parijoto are deeply colored and water-soluble.
These pigments are found in the leaves, flowers, and fruits of higher plants.
Structurally, the hydroxyl and methoxyl groups in anthocyanins influence the intensity of the color produced by these compounds (Garcia et al.
, 2.
An increase in hydroxyl groups can produce a strong blue color, while an increase in methoxyl groups produces a reddish The parijoto plant (Medinilla specios.
is generally used only as a food ingredient, such as in jams and syrups.
The use of parijoto (Medinilla specios.
as a natural dye has not yet been explored.
Furthermore, to date, no studies have examined the use of natural parijoto extract (Medinilla specios.
as a natural dye for peripheral blood smears.
This study aimes to determine the staining ability of parijoto extract (Medinilla specios.
in peripheral blood smears compared to the standard Giemsa stain.
In addition to identifying a safe and environmentally friendly alternative stain, this study may also contribute to scientific advancement, innovation in medical laboratory technology, and the sustainable use of local natural resources.
Materials and Methods Study Design This study employed a laboratory-based experimental design using a control group approach without a pretest.
The treatment group consisted of peripheral blood smears stained with Medinilla speciosa extract at various concentrations, while the control group was stained using Giemsa stain as the standard reference.
The primary variable observed was the quality of blood cell staining produced by each extract concentration when compared to the control group.
Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
Materials and Equipment The materials used include parijoto fruit, 70% ethanol, distilled water, venous blood human samples, absolute methanol, 3% citric acid, 10% Giemsa, 25%, 50%, and 70% parijoto extract, immersion oil.
The instruments and equipment used include an oven (Memmert.
German.
Erlenmeyer flask (Iwaki.
Tokyo.
Japa.
Whatman filter paper no.
1, digital analytical balance (Ohaus PA214C.
Parsippany.
NJ.
USA), stirring rod (Iwaki.
Tokyo.
Japa.
, aluminum foil, volumetric flask (PyrexA.
Corning.
NY.
USA), measuring cylinder (PyrexA.
Corning.
NY.
USA), glass object (Citotest.
Jiangsu.
Chin.
, glass beaker (Iwaki.
Tokyo.
Japa.
, staining rack, dropper pipette, binocular microscope (Olympus CX23.
Olympus Corporation.
Tokyo.
Japa.
, gloves (Sensi.
Medisafe.
Indonesi.
, surgical masks (One Car.
Preparation of Making Parijoto Extract Fresh parijoto fruits were cleaned to remove impurities, washed under running water, and oven-dried at 50AC for four hours.
The dried material was ground into powder and macerated using acidified 70% ethanol .
H 2Ae.
at a ratio of 1:10 for 24Ae72 hours under light-protected conditions (Wijaya et al.
, 2.
The maceration process is carried out at room temperature for 24-72 hours in an airtight container that is tightly closed and protected from light (Garcia et al.
, 2.
The maceration solution is homogenized periodically 2-3 times.
The resulting maceration solution is filtered using filter paper until 100% extract filtrate is obtained.
To achieve the desired concentration, mix:
Tabel 1.
Concentration of Parijoto Extract Concentration 100% Concentrated Extract .
L) Aquadest .
L) Extract 25% Extract 50% Extract 70% Preparation and Examination of Peripheral Blood Smear One drop of venous blood sample obtained in a vacuum tube was placed onto a glass slide.
Each sample on the slide was assigned a code for each respondent.
Another glass slide was Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
placed on the drop of blood on the slide at a 30-45A angle until the blood spread and pushed forward quickly.
The resulting blood smear was air-dried and fixed with absolute methanol for 3 minutes (Ghofur et al.
, 2.
The fixed blood smears were immersed in a staining solution .
% Giemsa, 25%, 50%, and 70% parijoto extrac.
for 10Ae15 minutes, adjusted for each treatment.
The staining solution was discarded, and the smears were rinsed with running water.
After the smears were dry, one drop of immersion oil was added for microscopic observation.
Observations were made starting at 40x to 100x magnification to observe the morphology of red blood cells .
Data Analysis This study was conducted using a quantitative comparative approach involving univariate and bivariate analysis.
Staining quality scores are treated as ordinal data.
Staining quality is evaluated using a semi-quantitative scoring system based on color intensity, background clarity, and erythrocyte morphology, with scores ranging from 1 .
, 2 .
airly clea.
, 3 .
, to 4 .
ery clea.
Univariate analysis was performed using frequency distributions, percentages, medians, and interquartile ranges to describe the data in each group, including the groups treated with 25%, 50%, and 70% Medinilla speciosa extracts, as well as the control group stained with Giemsa stain.
Bivariate analysis was performed using non-parametric tests.
Differences among the four groups were analyzed using the KruskalAeWallis test, followed by pairwise comparisons using the MannAeWhitney U test.
All preparations were evaluated by trained laboratory personnel using standard evaluation criteria to ensure consistency and minimize subjectivity.
Results and Discussion Result Overall, the study results indicate that an increase in the concentration of Medinilla speciosa extract is associated with an improvement in color quality.
These findings are consistent with the role of anthocyanins as natural pigments that contribute to coloring ability, although the effect is not uniform across all parameters.
Table 2 shows that a 25% extract showed relatively low staining quality with moderate variation among samples, as reflected by the distribution of scores.
As shown in Table 3, an increase in concentration of up to 50% was associated with improved staining quality and a more consistent distribution of scores compared to a 25% extract.
Among the extract Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
group, a concentration of 70% showed the highest overall coloring quality, as presented in Table 4 The distribution of scores shows a tendency towards higher values, although some variability among the samples is still observed.
These findings suggest that increased extract concentrations are generally associated with improved coloring quality.
the effect is not consistent across all parameters evaluated.
The control group stained with Giemsa staining showed consistently high staining quality with a stable distribution of scores, as shown in Table 5 Although the coloring quality of Medinilla speciosa extract does not fully correspond to Giemsa coloring in all parameters, the 70% extract concentration shows comparable performance in certain aspects, which shows its potential as an alternative to natural coloring.
Table 2.
Univariate Analysis Results of 25% Extract Parameter Color Intensity Background Clarity Erythrocyte Morphology Clarity Mean Median Mode Table 3.
Univariate Analysis Results of 50% Extract Parameter Color Intensity Background Clarity Erythrocyte Morphology Clarity Mean Median Mode Table 4.
Univariate Analysis Results of 70% Extract Parameter Color Intensity Background Clarity Mean Median Mode Erythrocyte Clarity Morphology Table 5.
10% Giemsa Univariate Analysis Results Parameter Color Intensity Background Clarity Erythrocyte Morphology Clarity Mean Median Mode Table 6.
Results of the Shapiro-Wilk Normality Test Concentration Extract 25% Extract 50% Extract 70% Extract 10% Sig Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
Table 7.
Results of the Kruskal-Wallis Intergroup Difference Test Result Sig Table 8.
Results of the - Mann Whitney Advanced Test for Differences Between Treatment Groups Group Comparison Sig Extract 25% vs Giemsa 10% Extract 50% vs Giemsa 10% Extract 70% vs Giemsa 10% .
Figure 1.
Microscopic Observation 100x of Red Blood Cells with Parijoto Extract Staining at Concentrations of 25% .
, 50% .
, 70% .
and 10% Giemsa as Control .
Discussion A comparison between the treatment and control groups of 10% Giemsa staining using the Mann-Whitney U test, as shown in Table 8, showed that the concentrations of the 25% and 50% extracts differed significantly from those of the control group .
< 0.
In contrast, the 70% extract concentration showed no statistically significant difference compared to the control group .
> 0.
These findings suggest that a 70% Medinilla speciosa extract showed comparable staining performance to controls.
however, these results should be interpreted with caution, as insignificant differences do not imply equivalence.
The flavonoid and tannin compounds in parijoto extract can bind to erythrocyte membrane proteins, producing a stable and even color at certain concentrations.
This mechanism is similar to the principle of synthetic dyes, although the colors are obtained from natural Staining can identify the condition of erythrocytes, including the degree of hypochromia or hyperchromia, as well as abnormalities in shape and size (Olyvia Manihiya et al.
, 2.
The difference in the intensity of the color of erythrocytes is associated with the concentration of the Medinilla speciosa extract used.
Statistical analysis using the Kruskal7 Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
Wallis test .
showed significant differences in the treatment group .
= 0.
further pairwise comparison using the Mann-Whitney U test .
showed that the concentrations of the 25% and 50% extracts differed significantly from the Giemsa 10% staining control .
< 0.
In contrast, the 70% extract concentration showed no statistically significant difference compared to the control .
> 0.
These findings suggest that increased concentrations of Medinilla speciosa extract are generally associated with increased erythrocyte color intensity.
This effect can be attributed to the presence of anthocyanins, which are known to produce reddish purple pigments.
However, this mechanism was not directly evaluated in this study and should be interpreted The high protein content in erythrocytes allows anthocyanin pigments to have an affinity for red blood cell components.
Meanwhile, antioxidants can maintain morphological stability during the staining process.
Therefore, as the extract concentration increases, the anthocyanin flavonoid pigments bind well to erythrocyte proteins, producing a clear and even color (Pertiwi et al.
, 2.
The concentration of parijoto extract also affected the clarity of the blood smear background.
Statistical tests showed that, compared to other concentrations, the 70% concentration was not significantly different from the 10% Giemsa control.
It can be concluded that the 70% extract can produce a clear and clean The chemical properties of anthocyanins significantly influence the staining results, which are related to pH stability, temperature, and environmental conditions.
Under acidic conditions, anthocyanin pigments are more stable, producing a purplish-red color.
However, at alkaline pH, anthocyanins degrade, producing a pale, unstable color.
The amount of anthocyanin pigment is not the only factor contributing to the improved staining quality at a concentration of 70%.
Chemical stability can also influence the staining results, allowing the anthocyanin pigment to be active and effective in coloring erythrocytes (Raihani & Wahab, 2.
The staining results of peripheral blood smears are influenced not only by concentration and chemical stability but also by staining time, preparation method, and fixation Furthermore, the presence of artifacts or residual pigment in the background can also interfere with the contrast of red blood cells.
Other factors that can affect the quality of staining with natural extracts include the extraction method, rinsing process, preparation technique, and observation.
To maintain anthocyanin stability, extraction is Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
carried out using a maceration method using 70% anthocyanin acidified with citric acid .
H 2-.
for 72 hours (S.
Putri et al.
, 2.
The findings of this study are consistent with previous reports of anthocyanin-based natural dyes, which have shown improved coloring quality at higher pigment Compared with Roselle and mangosteen peel extracts reported in previous studies.
Medinilla speciosa extract at 70% concentration showed comparable erythrocyte staining clarity with relatively minimal background artifacts.
The study has several limitations, including a relatively small sample size and reliance on visual assessment performed by a single observer, which can give rise to subjectivity and potential bias.
Efforts such as the use of standardized evaluation criteria are implemented to improve consistency.
however, further refinement of the methodology is still needed.
In addition, the morphology of leukocytes and platelets was not evaluated in the study.
Future research should include more objective methods of image analysis and assessing staining performance in different types of blood cells to provide a more comprehensive Conclusions Overall, the findings of this study indicate that Medinilla speciosa extract has potential as a natural dye for staining peripheral blood smears.
Among the concentrations evaluated, the 70% extract showed a coloring quality most comparable to the Giemsa 10% coloring Statistical analysis showed that there was no significant difference between the 70% extract and the control group, although this does not imply equivalence.
Nevertheless.
Giemsa tinting remains the standard tinting with more consistent and superior tinting performance.
Therefore, further optimization of Medinilla speciosa extract, including improvements in extraction methods, staining protocols, and color stability, is required before it can be considered as an alternative for routine laboratory In addition, while Medinilla speciosa extract may offer potential advantages in terms of natural origin and environmental considerations, its broader application, including use in resource-limited settings, requires further investigation.
This study provides a basis for future research focusing on standardization of concentration, staining reproducibility, and evaluation across a broader range of hematologic parameters.
Acknowledgments: The author would like to express his gratitude to the Institute for Research and Community Meida Trisna, et al J Indones Med Lab Sci.
1:1-11
Service (LPPM) of Muhammadiyah University of Kudus for granting permission for this research.
The author also thanks the staff of the Clinical Pathology Laboratory of Muhammadiyah University of Kudus for their technical support.
He would also like to express his gratitude to his parents for their support and to his friends who helped him throughout the research.
Funding: This research did not receive funding from any specific grants from any institution, whether from the public, commercial or non-profit sectors.
Conflicts of Interest: The author declares that there is no conflict of interest related to this research.
Author Contributions: MT: Conceptualization.
Methodology.
Research Implementation.
Data Analysis.
Preparation of the Original Draft of the Manuscript.
I: Research Supervision.
Data Analysis Direction.
Manuscript Review.
YR: Validation of Methodology and Research Results.
Manuscript Review.
AA: Manuscript Review.
AS: Manuscript Review.
Data Analysis Review.
SN: Provider of Input in Manuscript Improvement.
References