Efficacy of Anticoagulant Extract out of Averrhoa bilimbi (Kamias) in Treating Blood Samples

Keywords: Anticoagulants, Averrhoa bilimbi, blood samples, CBC, EDTA, kamias 

Statement of the Problem

The objective of this research was to modify A. bilimbi extract by introducing additive solutions to the extract to potentially create a more effective alternative anticoagulant. 

1. At which volume of additive solution added to 1 mL of A. bilimbi extract to will the sample remain uncoagulated the longest:

  1.1 250 µL;

  1.2 500 µL;

  1.3 750 µL; and

  1.4 1000 µL (1 mL)?

2. How different are the effects of the modified A. bilimbi extracts, the unmodified A. bilimbi extract, and EDTA to the CBC parameters of the samples, namely:

    2.1 Red Blood Cell (RBC);

    2.2 Hemoglobin (Hb);

    2.3 Hematocrit (Hct);

    2.4 White Blood Cell (WBC);

    2.5 Neutrophil;

        2.6 Eosinophil;

    2.7 Lymphocyte;

    2.8 Monocyte;

    2.9 Platelet count;

    2.10 Mean Corpuscular Volume (MCV);

    2.11 Mean Corpuscular Hemoglobin (MCH); and

    2.12 Mean Corpuscular Hemoglobin Concentration (MCHC)?

3. How different are the formations observed in the samples treated with the extracts and EDTA in terms of:

3.1 Hemolysis; and

3.2 Rouleaux formation?

Methods

Research Design

The researchers will use a quantitative method and an experimental design to collect the data. Quantitative research is typically used to explain and examine a phenomenon. This technique allows the researchers to observe the connections, correlations, and casual connections between variables. Furthermore, the experimental quantitative process relies on the scientific approach. It creates procedures that enable the researchers to test a hypothesis and study the causal connections between variables in a systematic and scientific manner. (Grand Canyon University, 2021). 

The A. bilimbi extract from the previous study showed almost similar results with EDTA, with differences only between its white blood cell and platelet count. Given this, the researchers then aimed to modify the extract to make it a more suitable alternative to the standard anticoagulant the researchers used for CBC testing. To achieve this, the researchers extracted A. bilimbi and made it undergo several tests and experiments to ensure its suitability and investigate its properties, thus making the research an experimental type of research. 

Research Instrument

The researchers used the fruit of A. bilimbi for the extraction of the fruit. A blender was used, alongside a rudimentary substitute to water bath, and a centrifuge. For the preparations of extracts, the fresh fruits were first washed with distilled water, then peeled to remove overripe or bruised parts. Then, they were blended using a blender, filtered using gauze to remove solid particles, and were then transferred to test tubes. In lieu of a water bath, the test tubes were placed inside a flask filled with hot water, where the tubes were submerged for at least 30 minutes. A temperature of around 70ºC was maintained by putting in hot water from an electric kettle and removing cooled water using a syringe. These were then centrifuged for 10 mins, and filtered using Whatman no. 1 filter paper. To store it until CBC could be performed, it was frozen.

Testing of CBC and neutralizing the acidity of the fruit extract is the critical part of this research in order to identify if the additives made a difference to the blood’s CBC parameters. The previous study shows that slight hemolysis was observed after the extract was centrifuged, caused by its acidity (Dizon et al., 2024); as such, the researchers aim to neutralize the extract to prevent hemolysis. The researchers used sodium hydroxide and potassium hydroxide with sodium fluoride as neutralizing agents to prevent these effects. The additive extract was applied to 1 mL of A. bilimbi extract in the ranges of 0.25, 0.5, 0.75, and 1 mL additive. This resulting modified extract was then applied to 1 mL of blood, which was done to investigate differences in anticoagulative ability based on the amount of extract applied.

Data Gathering Procedure

First, the researchers wrote a formal permission letter to use the laboratory. Following this, the researchers purchased one kilogram of A. bilimbi. Second, the researchers used the procedure of their prior study (Dizon et al., 2024), which was adapted from Sales et al. (2018). Third, the researchers took a few test runs by adding the mentioned solutions to the A. bilimbi extract and observing it through the microscope. This was done to monitor for improvements in the anticoagulative ability of the extract, and in preventing formation of hemolysis and rouleaux formation. Fourth, the researchers, having prior established a relationship with chosen diagnostic clinic ACE Diagnostics Valenzuela, corresponded and established a schedule for experimentation of the study. Fifth, the clinic aided the researchers for the experimentation and testing of the study. The researchers performed titration on the extract until it achieved the desired neutral pH level of the extract. The researchers then had their blood drawn to be used for testing the extract. The data from these tests was then collected. After the observation, the researchers summarized and interpreted the data.

Conclusion

The chemicals that were used in an attempt to neutralize the acidity of A. bilimbi, namely sodium hydroxide, potassium hydroxide, and sodium fluoride, may have interfered with the mechanism of A. bilimbi that causes anticoagulation, making them unfit as additives. This is because blood samples treated with modified A. bilimbi extract clotted within 2-4 minutes, making them unsuitable for CBC testing, unlike samples treated with unmodified A. bilimbi extract. 

None of the CBC parameters were preserved by the modified A. bilimbi extract, as all samples treated with it clotted. Unmodified A. bilimbi extract remains more in line with EDTA, except for in platelet count, akin to findings of similar previous studies (Lao, 2015; Sales et al., 2018). WBC count is better preserved by unmodified A. bilimbi extract in this trial than the researcher’s previous trial (Dizon et al., 2024).

Even with very alkaline solutions reaching up to pH level 14, the introduction of additives to A. bilimbi extract could not neutralize the acidity of the oxalic acid. Modified A. bilimbi extract still has a pH level of 1-2, similar to unmodified A. bilimbi extract, regardless of the alkalinity of the applied additive. Thus, the appearance of these formations could not be stopped by the modified extract.