Research Proposal 2

1
Z ST. ALEXIUS COLLEGE

Gen. San. Drive, City of Koronadal, South Cotabato, Philippines 09506, Tel.: (083) 228-2019, Fax: (083) 228-4015, Email: [email protected]
COMPARISON OF MANUAL PLATELET COUNT BETWEEN

ETHYLENEDIAMINETETRAACETIC ACID AND CITRATED BLOOD AMONG 3 RD
YEAR STUDENTS OF ST. ALEXIUS COLLEGE
______________________________________________________________________
A Research Proposal Submitted in Partial Fulfilment
of the Requirements in Introduction to Medical Laboratory Science Research (MTR-A)
______________________________________________________________________
By:
JANINE ALYZZA V. CRUZ
IAN GERALD BAJADE
AMEER HAZAROUQ M. CORO
MAUREEN GEM M. NAVAJA
RICHELLE ANN L. QUE
Second Semester
School Year 2022-2023
JANINE TEZA S. VILLENA, RMT, MSMT
Research Adviser
April 2022
2

CHAPTER I
THE PROBLEM AND ITS BACKGROUND
This chapter presents the background of the study, statement of the problem,
purpose of the research and significance of the study.
Introduction
Platelets are tiny, anucleated fragments of cells. They are regarded as the
second most prevalent in circulation of blood. They are generated from the cytoplasm of
megakaryocytes, which are giant cells that exist in the bone marrow and lungs. Its
primary function is to prevent a person's blood loss. The bloodstream is what carries
them around for a period of seven to ten days. After they develop into senile, they are
eliminated in the liver or spleen. In the locations where endothelial damage has
occurred, platelets become activated when they come into contact to protein molecules
from the extracellular matrix. The number of platelets that are moving through the
bloodstream is known as the platelet count. Normally, there are between 140,000 and
440,000 platelets per microliter. The menstrual cycle may have an impact on it. When
inflammation occurs, it might rise and then drop as the pregnancy progresses. Both of
these illnesses are not severe, and the majority of affected individuals experience no
symptoms at all. In line with this, our goal as educators is to create insight comparing
manual platelet counting with the use of various anticoagulants such as EDTA and
Citrate. (Shevchuk et al., 2021)
The platelet count is a significant test for handling patients and a crucial
diagnostic tool for hemorrhagic conditions. As of now, accessible hematology analyzers
count platelets utilizing a variety of innovations, including impedance, optical
procedures, and immunofluorescence methods. Using a hemocytometer, a microscope,
and a commercial dilution system, one can manually count platelets. Due to the difficulty
in separating platelets from debris, these counts are less reliable than automated
3

counts. The use of various anticoagulants may be implemented to give manual platelet
counting an entirely new perspective. Anticoagulants work to prevent blood clotting or
thickening. They work by inhibiting the production or activity of certain coagulation
factors that are naturally present in blood in order to generate their desired effects.
There are several types of anticoagulants. Each of the types operates on the blood
coagulation pathway at a different level. Heparin, citrate, and EDTA
(Ethylenediaminetetraacetic acid) are the three anticoagulants that are usually utilized.
Furthermore, a platelet count that is abnormal or uncertain needs to be thoroughly
examined with the aid of trusted decision trees. To obtain the proper outcome, it is
important to be aware of the analytical difficulties posed by the various hematology
analyzers' technology. (Baccini et al., 2020)
Hematology analyzers are known to be imprecise and inconsistent when
counting platelets in patients with severe thrombocytopenia, despite the fact that their
linearity constraints allow them to produce valid full blood counts. In high-risk patients
who require platelet transfusions, overestimation of thrombocytopenic platelet counts
can result in a major under transfusion of platelets, posing a serious danger of under
transfusion. Because of this, manual platelet estimation utilizing a variety of techniques
has been widely employed in situations with low platelet counts to precisely confirm the
auto analyzer numbers. Platelet size, shape, granulation, and platelet aggregation or
satellitism can all be assessed further by manual blood film evaluation. Any test that is
part of the standard of care must also be consistent. When a consistent method is
applied, it has been shown that manual platelet count estimation is reproducible in
skilled, trained hands. In addition, some investigations have recommended
standardizing manual platelet count techniques with respect to the microscopes used
for the counts, the area of the smears viewed on the slides, and the laboratory workers
involved. (Muhammed, 2020)

4

Calcium is removed by EDTA and sodium citrate, which is necessary for
coagulation. It is either bonded in a non-ionized state or precipitates as insoluble
oxalate, which crystals may be visible in oxalated blood. Ethylenediaminetetraacetic
acid, also known as EDTA, is an aminopolycarboxylic acid. It is a common binding
agent for both iron and calcium ions and appears as a white, water-soluble solid. It is
utilized for the majority of hematology operations, including determining the complete
blood count, making EDTA plasma, collecting whole blood, and obtaining bone marrow
samples. In aqueous solutions, it can be used as a sequestering agent to remove metal
ions. Sodium citrate is an inorganic compound with a changeable ratio of citrate anions
to sodium cations. It has been employed as an anticoagulant to regulate blood. Citrate
prevents the coagulation cascade by chelating calcium in the filter, which reduces the
concentration of ionized calcium. Citrate is quickly processed in the systemic circulation
after being extracted from the calcium citrate complexes in part by the filter. In general,
EDTA and citrate are essential agents. These anticoagulants have the ability to bind
calcium ions in free plasma, which stops blood clotting. (Madhu, 2022)
Statement of the Problem
This study aims to investigate the comparison of EDTA and Citrate
anticoagulants in Manual Platelet Count.
Specifically, this study seeks to answer following:
1. What are the differences of platelet count in EDTA anticoagulated blood and
citrate anticoagulated blood?
2. Which of the two anticoagulants performs manual platelet counts more
accurately?
3. What are the effects of using EDTA and citrate as anticoagulant to the manual
platelet count?
5

Hypothesis
1. H0: There is no significant relationship between the differences of platelet count
and the EDTA anticoagulated blood and Citrate anticoagulated blood.
H1: There is significant relationship between the differences of platelet count and
the EDTA anticoagulated blood and Citrate anticoagulated blood.
2. H0: There is no significant relationship between the accuracy of EDTA
anticoagulant and Citrate anticoagulant and manual platelet counting.
H1: There is significant relationship between the accuracy of EDTA anticoagulant
and Citrate anticoagulant and manual platelet counting.
3. H0: There is no significant relationship between the effects of EDTA anticoagulant
and Citrate anticoagulant to the manual platelet count.
H1: There is significant relationship between the effects of EDTA anticoagulant
and Citrate anticoagulant to the manual platelet count.
Scope and Delimitation
The scope of this study is to compare manual platelet counts in EDTA and
citrated blood in 18 students from St Alexius College. The study will be conducted on
healthy students without any history of hematological disorders. The manual platelet
count will be performed using a hemocytometer for both anticoagulants. The study will
aim to determine if there is a statistically significant difference in manual platelet counts
between EDTA and citrated blood. The study will also aim to identify any factors that
may affect the accuracy of manual platelet counts in both anticoagulants. The results of
this study will provide insights into the use of EDTA and citrated blood for manual
platelet counting and may help in the selection of the appropriate anticoagulant for
platelet counting in clinical practice.

6

Only 18 healthy students from St. Alexius College will participate in the study, thus
patients who have hematological diseases may not be able to benefit from the results
and with a smaller sample size, the study’s findings might only be applicable to the
specific population and may not be representative of a larger population. The study will
not compare automated platelet counts or other anticoagulants; instead, it will compare
manual platelet counts in EDTA and citrated blood. Other manual counting techniques
will not be investigated; the study's manual platelet counting will only use a
hemocytometer. The impact of platelet aggregation or blood storage duration on manual
platelet counts will not be examined in this study.
Significance of the Study
This study on the comparison of manual platelet count in EDTA and citrated
blood will be beneficial to the following:
HEALTHCARE PROFESSIONALS
The results of this study will provide insights into the use of EDTA and citrated
blood for manual platelet counting, and healthcare professionals who rely on platelet
counts in their practice may use this information to choose the appropriate anticoagulant
for blood collection.
MEDICAL TECHNOLOGISTS AND LABORATORY TECHNICIANS
This study may help laboratory technicians to improve the accuracy of manual
platelet counts by selecting the appropriate anticoagulant for blood collection.
FUTURE RESEARCHERS
The results of this study may provide a basis for further research on the effects of
different anticoagulants on platelet counts, which may lead to improvements in
laboratory techniques and clinical practices.

7

MEDICAL STUDENTS AND EDUCATORS
Medical students and educators may use the study's findings to enhance their
understanding of the use of anticoagulants in laboratory procedures.

8

CHAPTER II
REVIEW OF RELATED LITERATURE
This chapter summarizes relevant books, theses, and online articles authored by
both international and domestic writers that are crucial to the subject.
Review of Related Literature
PLATELETS
Platelets or thrombocytes are blood cells that serve as the first line of defense
when the body is facing problems concerning with loss of blood. It is known to play a
vital role in hemostasis- maintaining the body to not cause excess bleeding or clotting.
Platelets are produced from the cytoplasm of the bone marrow megakaryocytes. In
blood circulation, the platelet is biconvex when it is in its resting phase. This cell is
small, usually 2–5 µm in diameter, making its internal structure difficult to examine using
the light microscope. To examine its structure in vitro, a wedge-prepared blood film
stained with Wright stain is used making the appearance of a platelet from its circular
form to an irregular one. Normally, the platelet count is 150 to 400x109/L. This count will
change depending on different factors and conditions such as aging, injury, and
disorders. (Walenga et al., 2019)
Platelets also can induce an immune response in the presence of pathogens
present intravascularly. It possesses immunoreceptors that allow the detection of
foreign microorganisms. But, this may cause inflammation and certain thrombotic
events. According to reports from the World Health Organization, infectious diseases
contribute to high rates of death and morbidity annually. These build observations and
conclusions that platelets contributed greatly to defending individuals against
pathogens. On the contrary, overwhelming infections caused by foreign

9

microorganisms cause the activation of platelets that can damage blood vessels. In
addition, inflammation caused by non-infectious conditions can also be the origin of
thromboinflammation. Thromboinflammation is a state that correlates inflammation to
thrombosis. Other conditions linked to thromboinflammation are low platelet count or
Thrombocytopenia, and increased permeability of blood vessel walls. (Campbell &
Portier, 2020)
THROMBOCYTOPENIA
Low platelet count can be caused by different factors such as decreased platelet
production, high platelet consumption, high sequestration, and destruction of platelets.
Decreased platelet production can be due to infections caused by viruses, aplastic
anemia, excessive intake of alcohol, nutritional deficiencies, hematologic malignancies
such as lymphoma and leukemia, radiation, and chemotherapy. High platelet
consumption can be through excessive blood loss, trauma, sepsis, disseminated
intravascular coagulation, and renal dialysis. In Intensive Care Unit, the usual causes of
thrombocytopenia are major bleeding, microangiopathic hemolytic anemias, drug-
induced thrombocytopenia, hemolytic uremic syndrome, and intake of glycoprotein
IIb/IIIa inhibitor. GPIIb-IIIa inhibitors can also cause Pseudothrombocytopenia- a type of
low platelet count that can happen when using an automated particle counter. This can
occur due to the clumping of platelets that is caused by antibodies IgG or IgM that are
dependent on ethylenediaminetetraacetic acid. To confirm the presence of
pseudothrombocytopenia, the platelet count is repeated but with the use of different
anticoagulants such as heparin, oxalate, or citrate. The presence of giant platelets and
platelet satellitism can also cause thrombocytopenia when analyzed on automated
counting machines. Thrombocytopenia that is observed using automated counting
machines should be confirmed by examining a peripheral blood smear manually.
(Santoshi et al., 2022) Thrombocytopenia has a platelet count of less than 150x109 per
10

liter (<150x109/L). Patients exhibiting platelet count of >50x109/L are usually
asymptomatic and do not show bleeding. Spontaneous bleeding is common in patients
having a platelet count of less than 20x109/L. During pregnancy, it is expected for a
pregnant woman to have a low platelet count. This is due to platelet activation and
haemodilution which usually happens in the different trimesters of pregnancy. In the first
and second trimesters of pregnancy, pregnant women usually experience Primary
immune thrombocytopenia (PIT). PIT is a type of isolated thrombocytopenia that can be
asymptomatic, exhibit minimal bruising or the presence of petechial rash, or could
cause bleeding. This type of thrombocytopenia is not limited to pregnant women but
also children and adults. In the third trimester of pregnancy, Gestational
Thrombocytopenia is usually present but this condition is mild and may disappear within
1-2 months after giving birth. (Ashworth et al., 2022)
As stated by Moore (2019), thrombocytopenia was first thought to be caused by
only one reason and that is the active sequestration of the spleen that is caused by
congestive splenomegaly. Several proposals state that liver cirrhosis can also be a
cause of thrombocytopenia. The liver is known to produce thrombopoietin which is the
principal hormone that regulates the production of thrombocytes or platelets. The more
the liver is damaged, the more the thrombopoietin levels decreased causing the low
production of platelets leading to thrombocytopenia.
As the COVID-19 Pandemic spread throughout the whole world, medical
practitioners observed that it has the potential to make hematological changes to the
infected patients. According to the study of Chen et al. (2020), patients infected with
COVID-19 have a normal white blood cell count and decreased lymphocyte and platelet
count. 1,099 patients from different provinces and municipalities in China recorded that
82.1% of the patients experienced lymphopenia, 33.7% of patients had leukopenia,
while thrombocytopenia was experienced by 36.2% out of the total observed patients.
11

In addition, a study conducted in 3 hospitals in Beijing, the capital of China stated that
out of 13 patients, 72.5% developed thrombocytopenia. In COVID-19 patients, it has
been studied that this infection can result to decrease platelet production because
coronaviruses can invade bone marrow cells which lead to abnormal hematopoiesis and
low platelet count. (Chang et al., 2020) Furthermore, it can lead to increase platelet
consumption due to damaged pulmonary endothelial cells and lung tissues that result in
the activation of platelets that results in the formation of microthrombi, and increase
platelet destruction. COVID-19 infection can increase the levels of immune complexes
and autoantibodies that lead to the destruction of platelets due to an active immune
system. (Xu et al., 2020)
THROMBOCYTOSIS
Thrombocytosis or also known as Thrombocythemia is a condition wherein the
platelet count exceeds its normal value. Patients diagnosed with thrombocytosis have a
platelet count of greater the 450x10 9/L. Thrombocytosis have two types: Primary
Thrombocytosis and the Reactive (Secondary) Thrombocytosis. Primary thrombocytosis
is caused by abnormal productions of platelets by the bone marrow progenitor cells and
is associated with myeloproliferative neoplasm. This type of thrombocytosis has an
increased risk of bleeding and thrombosis than secondary thrombocytosis. Reactive
(Secondary) thrombocytosis is due to several factors such as the use of certain
medications, diseases, and other conditions. This can be resolved when the underlying
cause of the condition of the patient is resolved. (Rokkam & Kotagiri, 2022)
Essential thrombocytosis is a type of myeloproliferative disorder that is described
as a thrombocytosis associated with the existence of megakaryocytic hyperplasia in the
bone marrow. As indicated by the World Health Organization, essential thrombocytosis
occurs when a patient has a platelet count of greater than 450 x109/L accompanied by
Janus kinase 2, Calreticulin, and mutations from myeloproliferative leukemia virus

12

oncogene. The presence of these genes plays a role in the development of
myeloproliferative neoplasm. Janus Kinase 2 (JAK2) is a type of non-receptor tyrosine
kinase that originates in the cytoplasm. The mutation of this gene will lead to the
activation of signaling pathways of hematopoietic cytokines such as erythropoietin,
thrombopoietin, and granulocyte-stimulating factors. Calreticulin (CALR) gene is
involved in cellular proliferation, differentiation of cells, and apoptosis. It is reported that
myeloproliferative leukemia (MPL) virus oncogene is found in patients experiencing
essential thrombocytosis. (Ashorobi, 2022)
Reactive thrombocytosis is characterized by an elevated platelet count due to
extrinsic factors that can stimulate megakaryocytopoiesis such as acute and chronic
inflammation. It is a phenomenon where there is a dysregulation of thrombopoiesis.
Secondary or reactive thrombocytosis can be caused by different conditions such as
viral or bacterial infections, deficiency in iron, tissue damage, autoimmune diseases,
malignancies, hemolytic anemia, and the effects of certain medications or drugs.
(Stockklausner et al., 2021)
PLATELET COUNT
Platelet count is an important step to assess the risk of bleeding and thrombosis.
It is used to assess certain pathologies and conditions associated with thrombocytes. It
is crucial for the diagnosis of certain hemorrhagic diseases and conditions such as
dengue fever, malaria, leukemia, pregnancy-induced sepsis, and hypertension and the
management of patients undergoing chemotherapy. (Bhasker et. al, 2022)The common
methods used to assess platelet count are using a counting chamber or hemocytometer
to count manually, utilization and evaluation of peripheral smear, and the use of
automated hematology analyzers. (Mahajan et al., 2019)
Manual platelet count using a counting chamber utilizing a hemocytometer such
as the Neubauer chamber. This method requires 1% ammonium oxalate that acts as a
13

diluting fluid to lyse red blood cells that are present and preserve white blood cells and
platelets. (Jain, 2020) This method is observed under the microscope preferably a
phase contrast microscope as proposed by Brecher in 1953. Evaluating platelets using
a peripheral blood smear is an alternative procedure for manual platelet count. It serves
a double purpose by providing a simple procedure of counting platelet and a
confirmatory method when errors are encountered while using automated hematology
analyzers. However, performing manual methods can be prone to imprecision and poor
reliability. In addition, this requires a longer time to be performed and is considered as
labor-intensive. (Baccini et.al, 2020)
As technology emerged, automated hematology analyzers were discovered and
invented. Several automated cell counting methods are discovered such as
Immunoplatelet counting, Impedance Platelet Counting, Optical Platelet counting, and
Fluorescence Platelet Counting. This produces fast, accurate, and precise counts of
platelet but some interferences can affect and compromise the results generated by the
automated hematology analyzers such as the unusual size of platelets, small
erythrocytes, or cell debris and fragments. (Roshal & Gil, 2019)
According to the report of Bhasker et.al (2022), they concluded that manual
method of platelet count provides higher count compared to automated hematology
analyzers. In this study, blood samples are utilized from 600 patients composed of 375
females and 225 males. The blood samples are placed in a tube containing
Ethylenediaminetetraacetic acid as an anticoagulant. In the manual method, blood
smears are prepared and stained it using Leishman stain. The blood smear was
observed using an Olympus cx21i microscope under the oil immersion lens. In the
automated counting, Mindray BC-6200 auto-hematology analyzer 5 is used.
Similar to the study of Jain (2020), the researcher stated that the platelet count
through the manual method is higher than that of the automated counting procedure.
14

Though the automated counting procedure produces rapid results, it cannot analyze
low-level and abnormal platelets. This should be taken into consideration because of
some conditions that result in low platelet count such as thrombocytopenia. Manual
platelet count plays a vital role when these kinds of problems are encountered. Even
though it is labor intensive, it provides support to automated hematologic analyzers. In
addition, this procedure can be utilized in small laboratories where patients are less and
there is no automated hematologic analyzer.
ETHYLENEDIAMINETETRAACETIC ACID
Ethylenediaminetetraacetic acid (EDTA) is a known anticoagulant that is used to
preserve the morphology and cellular components of blood cells. This anticoagulant
contains four carboxylic acid groups, two amine groups, and some other metal ions. It
also contains lone pair of electrons that play a vital role in chelating calcium. Removal of
calcium in the blood will prevent the blood from clotting. In hematological testing, EDTA
is always the anticoagulant of choice. (Wright, 2021)
This anticoagulant can stimulate thrombocytopenia termed EDTA-induced
thrombocytopenia. EDTA can chelate cation that leads to a conformational change in
the GPIIb-IIIa a complex of the platelet membrane. This change will expose platelet
antigen which can attract anti-platelet autoantibodies leading to in-vitro clumping and
aggregation of platelets that results to low-platelet count. An automated hematologic
analyzer evaluates platelet clumping and aggregation as giant platelets or as
lymphocytes in the white blood cell lineage. When this occurs, this may result in a
slightly increased count of white blood cells. This occurrence is suspected using a
platelet histogram and is reviewed using a peripheral blood smear to confirm the
presence of platelet aggregates. To differentiate EDTA-induced thrombocytopenia from
real thrombocytopenia, other anticoagulants are used for blood sample such as heparin,
sodium citrate, and oxalate. Thrombocytopenia can still be observed in the said
15

anticoagulants but the probability of forming thrombocytopenia is lower compared to
EDTA-anticoagulated blood samples. (Pitkin, 2018)
The occurrence of EDTA-induced thrombocytopenia can also be termed as
Pseudothrombocytopenia stated by Saenz-Flor et.al (2020) Ethylenediaminetetraacetic
acid can stimulate agglutination of platelets with visible platelet agglutination in
peripheral blood smear. This condition is prevalent in hospitals and if not identified
properly and resolved, this may lead to inappropriate clinical decision-making and
diagnosis. When an automated hematologic analyzer generates a low platelet count, a
manual method using a peripheral blood smear should be examined. Furthermore, it is
also recommended to incubate blood for 15 minutes at 37°C for autoantibodies to react
and new readings on the automated hematologic analyzers should be conducted.
CITRATE
Citrate is an anticoagulant that neutralizes the clotting effect of blood samples.
This anticoagulant came from the salt of citric acid that can bind with free calcium
(Ca2+) and removes it from the coagulation cascade. The mechanism of citrate as an
anticoagulant was discovered by Pekelharing in the year 1891 when he observed that
the clotting of blood can be neutralized with the addition of sodium citrate. As
investigated by Perrotta and colleagues, citrate is used as an alternative to
Ethylenediaminetetraacetic acid to improve hematological investigation. However, as
studied by Thompson and colleagues, the use of sodium citrate can decrease mean
platelet volume (MPV) after 8 hours compared to EDTA which the MPV result is
increased after 2 hours and can remain stable up to 8 hours. (Mannuß, 2020)
EDTA is known to cause pseudothrombocytopenia - a rare phenomenon when
automated hematologic analyzers detect low platelet count due to the presence of
clumping or aggregation of platelet, while artifactual platelet count may occur when
using sodium citrate as an anticoagulant. Artifactual platelet count is associated with

16

satellitism and platelet clumping in blood samples with the presence of sodium citrate.
Platelet clumping can be observed in artifactual platelet count due to the presence of
agglutinating antibodies that links to the surface of platelet glycoproteins. Platelet
satellitism occurs simultaneously with platelet clumping because of the presence of
antiplatelet antibodies that is stimulated by the presence of sodium citrate. This induces
alterations in the surface proteins of white blood cells and platelets leading to the
binding of the two blood cells. (Dima et.al, 2020)
Platelets are blood cells that are responsible for maintaining hemostasis to
ensure that there is no excess bleeding or clotting. It also plays a role during infection.
Platelets can trigger immune responses through the presence of pathogens and foreign
materials. A platelet count lower than the normal values which are 150x109/L-
450x109/L results into condition termed thrombocytopenia. This may be due to different
factors such decrease platelet count, high platelet consumption, high sequestration, and
destruction of platelets. Different conditions such as COVID-19 infection, liver cirrhosis,
primary immune thrombocytopenia, and gestational thrombocytopenia are just a few of
the conditions that are associated with thrombocytopenia. High platelet count or
thrombocytosis is a condition wherein platelet count exceeds the normal reference
range. It is classified into 2 types which are Primary thrombocytosis – which is
associated with myeloproliferative disorders and Secondary (Reactive) thrombocytosis.
Reactive thrombocytosis is related to extrinsic factors that can trigger
megakaryocytopoiesis which may lead to increased platelet production.
To assess platelet count, two methods are considered: manual platelet count and
automated platelet count. Manual platelet count utilizes a counting chamber and
examination of peripheral blood smear to evaluate platelet count. Automated platelet
count uses automated hematologic analyzers that apply different principles and
mechanisms to assess platelet count. However, both methods have their advantages
17

and disadvantages. Automated hematologic analyzer produces rapid and accurate
platelet count but it cannot detect low levels of platelet. It is also prone to error due to
the clumping and aggregation of platelets. When this error is encountered, a manual
platelet count should be performed to confirm the presence of clumped, aggregated, or
giant platelets. While manual platelet count uses cheaper materials compared to
automated platelet count, this method requires a longer time to perform and is labor-
intensive.
Anticoagulants such as Ethylenediaminetetraacetic acid (EDTA) and Citrate are
used to preserve platelet integrity in vitro. However, these anticoagulants possess
different conditions that may affect the platelet count when presented to automated
hematologic analyzers. EDTA can cause pseudothrombocytopenia that triggers the
clumping and aggregation of platelets which may lead to low platelet count. This is
because clumped and aggregated platelets are recognized by automated hematologic
analyzers as lymphocytes. Citrate on the other hand also can cause artifactual platelet
count that is associated with the clumping of platelets and platelet satellitism
Definition of Terms
 Accurate (Conceptual Definition)
-It is defined as a degree of being true, correct, or exact without any mistakes.
(Cambridge Dictionary, N.D)
 Accurate (Operational Definition)
-It is the extent how true or reliable is the count of platelet using the manual
platelet count.
 Anticoagulant (Conceptual Definition)
-It is defined as a substance that prevents blood from clotting. (National
Cancer Institute, N.D)

18

 Anticoagulant (Operational Definition)
-Anticoagulants are used in the study to preserve the integrity of platelets
before testing.
 Blood (Conceptual Definition)
-It is defined as a reddish fluid that is pumped by the blood and supplies
oxygen and nutrients all throughout the body and removes waste. It consist cells
such as red blood cells, white blood cells, and platelets. (Collins Dictionary, N.D)
 Blood (Operational Definition)
-It is the variable that the researchers will use and manipulate in the study to
observe about the platelet count between citrate- anticoagulated blood and EDTA-
anticoagulated blood.
 Citrate (Conceptual Definition)
-Citrate is defined as an anticoagulant that is capable of binding free calcium
in the blood. (Drugs.com, 2022)
 Citrate (Operational Definition)
-It is one of the anticoagulants that will be used to examine the platelets under
manual method of platelet count.
 Ethylenediaminetetraacetic Acid (Conceptual Definition)
-Ethylenediaminetetraacetic acid is defined as a chemical that is used as an
anticoagulant that contains metal ions that inhibits clotting of blood. It can remove
calcium and lead from the body. (National Cancer Institute, N.D)
 Ethylenediaminetetraacetic Acid (Operational Definition)

19

-It is an anticoagulant that will be utilized to assess platelet using manual
platelet count.
 Platelet Count (Conceptual Definition)
-It is defined as a procedure that evaluates the number of platelets on a
certain individual. (University of California, N.D)
 Platelet Count (Operational Definition)
-It is a variable in the study that is dependent to the anticoagulant used to
treat the blood sample.
 Platelet (Conceptual Definition)
-Platelet is defined as a small and disc-shaped cell that responsible of forming
blood clot to stop wounds from bleeding. (National Cancer Institute, N.D)
 Platelet (Operational Definition)
-It is a variable that will be observed by the researchers to determine the
difference between the anticoagulants that will be used in the study.
Conceptual/ Theoretical Framework
INDEPENDENT VARIABLE DEPENDENT VARIABLE
TYPE OF ANTICOAGULANT
USED FOR BLOOD MANUAL PLATELET COUNT
COLLECTION:
EDTA AND CITRATE
INTERVENING VARIABLE
TIME INTERVAL BETWEEN
BLOOD COLLECTION AND
MANUAL PLATELET
COUNTING
20

CHAPTER III
METHODOLOGY
This chapter presents the methods that will be used by the researchers to gather
needed data and how the data will be gathered.
Research Design
This study will utilize quantitative research, specifically an experimental research
design. Experimental research is commonly used when the study involved a laboratory
test that manipulates variables and applies an experimental and controlled treatment to
gather data. The sample will be collected from the participants involved in the study.
The blood will be stored in tubes containing an anticoagulant additive, which is the
EDTA and Sodium Citrate. After that, a manual platelet count will be conducted. The
researchers will use certain laboratory procedures and guidelines to distinguish
noticeable changes in the variables involved in the study. The process of the
experiment will be recorded throughout the procedures. Interpretation and comparison
of results will be observed. Given the nature of this research, it is appropriate to use
experimental research as a research design. This is essential in gathering data and
proving the hypothesis.
Selection of Respondents
The participant of the study will be the 3 rd year, Section A, Regular Bachelor of
Science in Medical Technology students of St. Alexius College. The researchers
estimate that there will be 20 3 rd year, Section A, Regular BSMT students enrolled from
the school. Through this, the researchers utilized the Slovin’s formula with 5% margin of
error and a confidence level of 95% to determine the sample size. This will help the
researchers to discover how many participants are needed for the study.
21

Slovin’s formula is presented below:
Where:
n= Sample size
N= Population size (19)
e= Margin of error (5%)
19
n=
1+¿ ¿
n= 18.138 or 18 participants
According to Sloven's formula, the needed participant in this research is 18. Where the
9 participants' blood samples will be stored in an EDTA tube and the remaining 9 will
use a Sodium citrate tube.
Research Instruments
Whole blood sample
EDTA Tube
Sodium Citrate Tube
Stains (such as Wright-Giemsa Stain)
Diluting Fluid
Platelet counting chamber (such as a hemocytometer)
Hemocytometer chamber holder
Microscope slides
22

Coverslips
Pipettes (for transferring the diluting fluid and blood sample)
Data Gathering Procedure
All materials needed for the laboratory testing are gathered including test tubes
with respective anticoagulant additives used to store the blood sample. The researchers
will seek permission first from specific individuals if they are willing to take part in our
experiment. This will be done through a waiver which states that they are voluntarily
participating in our study. Then whole blood samples will be taken from the participants,
following the proper procedures for collecting blood samples. The blood will be stored in
EDTA and Sodium Citrate Tubes with proper identification information.
After the sample collection, the researchers will prepare a blood smear by
placing a drop of blood from an EDTA tube using a pipette on a microscope slide and
spreading evenly with the use of the spreader creating a thin layer of blood. After
preparing a blood smear, stain the smear with the use of Wright-Giemsa stain which will
enhance the distinguishable feature of the platelets. Then put the blood smear with the
stain on the microscope slide holder. The researcher will use a magnification of 100x or
higher, then view the smear under the microscope. When viewing, they will locate an
appropriate field of view, count the number of platelets, calculation, and record the
result (Brahim, 2020).
The researchers can also utilize an alternative method of manually counting the
platelets. This involves a counting chamber, which is the hemocytometer, and a
microscope for counting platelets using phase contrast light microscopy. It also a red
cell lysing reagent, such as ammonium oxalate, to treat the diluted whole blood. Then,
platelets are counted microscopically using an improved Neubauer counting chamber
(Jain, 2020).
23

After the manual counting of platelet in EDTA, the researchers will repeat the
laboratory procedures but instead of using the blood samples with EDTA, they will
utilize the blood samples with sodium citrate. The result of manual platelet count in
EDTA and Citrated Blood will be compared to know the difference between using the 2
anticoagulant additives.
Data Analysis
The platelet counts obtained from the manual counting procedure will be
recorded on a data sheet. The data sheet will include the average platelet count for
each EDTA and citrated blood sample. To ensure accuracy, the platelet counts will be
recorded by two independent observers, and the average of their counts will be used for
analysis.
The mean and standard deviation of the platelet counts for each sample will be
calculated to determine the central tendency and variability of the data. These
calculations will be done using appropriate statistical software.
To test the hypothesis that there is a significant difference between the platelet
counts in EDTA and citrated blood samples, appropriate statistical methods such as the
t-test or ANOVA will be used. The choice of statistical method will depend on the nature
and distribution of the data.
If the null hypothesis of no significant difference between the platelet counts in
EDTA and citrated blood samples is rejected, further analysis will be done to determine
the source of the difference. This may involve comparing the effects of different
anticoagulants on platelet counts and analyzing the possible causes of measurement
error.
The results of the data analysis will be presented in tables, graphs, and charts to
aid in the interpretation of the findings. The statistical significance level will be set at
24

0.05. The findings will be discussed in the context of previous studies on manual
platelet counting, and recommendations will be made on the most accurate and reliable
method for manual platelet counting.
Ethical Considerations
To ensure the ethical standards of the "Comparison of Manual Platelet Count in
EDTA and Citrated Blood" study, various considerations should be taken into account.
These include acquiring informed consent from patients prior to utilizing their blood
samples for research and ensuring compliance with all relevant ethical regulations and
guidelines. This may require obtaining approval from an ethics committee or institutional
review board and ensuring that the study adheres to the principles of the Declaration of
Helsinki.
Furthermore, the safety and well-being of patients must be given the highest
priority, and the collection and analysis of blood samples must not cause any harm.
Patient privacy and confidentiality should also be maintained throughout the study, with
all data collected and analyzed anonymously to protect their identities.
It is crucial to bear in mind that ethical considerations are an essential part of
research and must be given due attention throughout all stages of the study. By
following ethical guidelines, researchers can ensure that their study is conducted in a
responsible and ethical manner, while also producing meaningful and valuable results.
25

BIBLIOGRAPHY
Ashorobi, D. (2022, June 21). Essential Thrombocytosis. StatPearls. Retrieved from
https://www.statpearls.com/ArticleLibrary/viewarticle/30099#ref_28529308
Ashworth, I., Thielemans, L., & Chevassut, T. (2022, May). Thrombocytopenia: the
good, the bad and the ugly. Clinical Medicine, 22(3), 214–217.
https://doi.org/10.7861/clinmed.2022-0146
Baccini, V., Geneviève, F., Jacqmin, H., Chatelain, B., Girard, S., Wuilleme, S.,
Vedrenne, A., Guiheneuf, E., Toussaint-Hacquard, M., Everaere, F., Soulard, M.,
Lesesve, J. F., & Bardet, V. (2020, March 16). Platelet Counting: Ugly Traps and
Good Advice. Proposals from the French-Speaking Cellular Hematology Group
(GFHC). Journal of Clinical Medicine, 9(3), 808.
https://doi.org/10.3390/jcm9030808
Bhasker, N.,Parashar,B., & Aghinotri,M. (2022). Comparison Between Manual Platelet
Count and Automated platelet count inThrombocytopenia patients. European
Journal of Molecular & Clinical Medicine, 9(4),2515-826. Retrieved from
https://ejmcm.com/article_19251_7b3f7e418c4f0ed7d1760ea10138e4a6.pdf
Brahim, M., et.al. (2020, September). The estimation of platelet count from a blood
smear on the basis of the red cell: platelet ratio. Journalagent. Retrieved April 26,
2023, from https://jag.journalagent.com/tjh/pdfs/TJH_26_1_21_24.pdf
Cambridge University Press & Assessment. (N.D). Accurate. Cambridge Dictionary.
Retrieved from https://dictionary.cambridge.org/us/dictionary/english/accurate
Chang, D., Lin, M., Wei, L., Xie, L., Zhu, G., Dela Cruz, C. S., & Sharma, L. (2020,
March 17). Epidemiologic and Clinical Characteristics of Novel Coronavirus
Infections Involving 13 Patients Outside Wuhan, China. JAMA, 323(11), 1092.
https://doi.org/10.1001/jama.2020.1623
26

Chen, N., Zhou, M., Dong, X., Qu, J., Gong, F., Han, Y., Qiu, Y., Wang, J., Liu, Y., Wei,
Y., Xia, J., Yu, T., Zhang, X., & Zhang, L. (2020, February). Epidemiological and
clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in
Wuhan, China: a descriptive study. The Lancet, 395(10223), 507–513.
https://doi.org/10.1016/s0140-6736(20)30211-7
Collins. (N.D). Blood. Collins Dictionary. Retrieved from
https://www.collinsdictionary.com/dictionary/english/blood
Dima, F., Salvagno, G. L., Danese, E., Veneri, D., & Lippi, G. (2020, September 16). An
unusual case of sodium citrate-dependent artifactual platelet count. Interventional
Medicine and Applied Science, 11(3), 193–196.
https://doi.org/10.1556/1646.11.2019.24
Drugs.com. (N.D). Sodium Citrate Anticoagulant Solution Prescribing Information.
Retrieved from https://www.drugs.com/pro/sodium-citrate-anticoagulant-
solution.html
eClinpath. (2019, February 13). Platelet count | eClinpath.
https://eclinpath.com/hematology/tests/platelet-count/#:~:text=Platelet%20counts
%20can%20be%20done%20manually%20with%20a%20commercial
%20diluting,decrease%20the%20hemocytometer%20platelet%20count
Jain, D. K. (2020, September 24). Comparison of platelet count by manual and
automated method. International Journal of Research in Medical Sciences, 8(10),
3523. https://doi.org/10.18203/2320-6012.ijrms20204011
Madhu. (2022, May 17). What is the Difference Between EDTA and Sodium Citrate.
Compare the Difference Between Similar Terms.
https://www.differencebetween.com/what-is-the-difference-between-edta-and-
sodium-citrate/#Summary
27

Mahajan, D., Aashna, K. Koul, K., & Jandial, A. (2019, August 15). Platelet count
correlation: automated versus manual on peripheral smear. Indian Journal of
Pathology and Oncology, 6(3), 381–387. https://doi.org/10.18231/j.ijpo.2019.074
Mannuß, S. (2020, September 17). Influence of different methods and anticoagulants on
platelet parameter measurement. Journal of Laboratory Medicine, 44(5), 255–
272. https://doi.org/10.1515/labmed-2020-0037
Moore, A. H. (2019, November). Thrombocytopenia in Cirrhosis: A Review of
Pathophysiology and Management Options. Clinical Liver Disease, 14(5), 183–
186. https://doi.org/10.1002/cld.860
Muhammed. (2020, April 4). Comparison Of Platelet Count By Automated And Manual
Methods, A Study And Review Of Literature In A Medical College Hospital In
Kashmir. BJMHS450083.pdf.
http://www.jmhsci.org/wp-content/uploads/2020/05/BJMHS450083.pdf
National Institutes of Health. (N.D). EDTA. National Cancer Institute. Retrieved from
https://www.cancer.gov/publications/dictionaries/cancer-terms/def/edta
National Institutes of Health. (N.D). Platelet. National Cancer Institute. Retrieved from
https://www.cancer.gov/publications/dictionaries/cancer-terms/def/platelet
Pitkin, F. (2018). Ethylenediaminetetraacetic Acid (EDTA)-Induced Thrombocytopenia:
A Case Report. Journal of Traditional Medicine & Clinical Naturopathy, 2573-
4555. Retrieved from
https://www.omicsonline.org/open-access/ethylenediaminetetraacetic-acid-
edtainduced-thrombocytopenia-acase-report.php?aid=85757
Portier, I., & Campbell, R. A. (2020, October 29). Role of Platelets in Detection and
Regulation of Infection. Arteriosclerosis, Thrombosis, and Vascular Biology,
https://doi.org/10.1161/atvbaha.120.314645
28

Roshal, M., & Reyes Gil, M. (2019). Measurement of Platelet Count, Mean Platelet
Volume, and Reticulated Platelets. Transfusion Medicine and Hemostasis, 811–
815. https://doi.org/10.1016/b978-0-12-813726-0.00137-9
Sáenz-Flor KV, Velepucha Fernandez PS, Muñoz Crespo FA, Ochoa Toasa EN (2020)
Pseudothombocytopenia in Total Blood with Ethylenediaminetetraacetic Acid.
Res Trends Lab Med Pathol: RTLMP-103. DOI: 10.29011/RTLMP-103.1000033
Santoshi, R. K., Patel, R., Patel, N. S., Bansro, V., & Chhabra, G. (2022, August 5). A
Comprehensive Review of Thrombocytopenia With a Spotlight on Intensive Care
Patients. Cureus. https://doi.org/10.7759/cureus.27718
Stockklausner, C., Duffert, C. M., Cario, H., Knöfler, R., Streif, W., & Kulozik, A. E.
(2021, March 12). Thrombocytosis in children and adolescents—classification,
diagnostic approach, and clinical management. Annals of Hematology, 100(7),
1647–1665. https://doi.org/10.1007/s00277-021-04485-0
The Regents of The University of California. (N.D). Platelet count. UCSF Health.
Retrieved from https://www.ucsfhealth.org/medical-tests/platelet-count
Walenga, J. M., Keohane, E. M., & Otto, C. N. (2019, March 11). Rodak’s Hematology:
Clinical Principles and Applications. Saunders.
World Medical Association. (2013). World Medical Association Declaration of Helsinki:
ethical principles for medical research involving human subjects. JAMA, 310(20),
2191-2194. doi:10.1001/jama.2013.281053
Wright, A. (2021). Characterizing the Presence of EDTA in Blood Samples. VCU
Scholars Compass. https://doi.org/10.25772/d18m-7b40
Xu, P., Zhou, Q., & Xu, J. (2020, April 15). Mechanism of thrombocytopenia in COVID-
19 patients. Annals of Hematology, 99(6), 1205–1208.
https://doi.org/10.1007/s00277-020-04019-0
29


Research Proposal 2

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Research Proposal 2

Uploaded by

Copyright:

Available Formats

1

Z ST. ALEXIUS COLLEGE

COMPARISON OF MANUAL PLATELET COUNT BETWEEN

A Research Proposal Submitted in Partial Fulfilment

of the Requirements in Introduction to Medical Laboratory Science Research (MTR-A)

JANINE ALYZZA V. CRUZ

IAN GERALD BAJADE

AMEER HAZAROUQ M. CORO

MAUREEN GEM M. NAVAJA

RICHELLE ANN L. QUE

School Year 2022-2023

JANINE TEZA S. VILLENA, RMT, MSMT

Z ST. ALEXIUS COLLEGE

THE PROBLEM AND ITS BACKGROUND

purpose of the research and significance of the study.

Citrate. (Shevchuk et al., 2021)

diagnostic tool for hemorrhagic conditions. As of now, accessible hematology analyzers

count platelets utilizing a variety of innovations, including impedance, optical

procedures, and immunofluorescence methods. Using a hemocytometer, a microscope,

Z ST. ALEXIUS COLLEGE

counting an entirely new perspective. Anticoagulants work to prevent blood clotting or

thickening. They work by inhibiting the production or activity of certain coagulation

coagulation pathway at a different level. Heparin, citrate, and EDTA

Furthermore, a platelet count that is abnormal or uncertain needs to be thoroughly

important to be aware of the analytical difficulties posed by the various hematology

analyzers' technology. (Baccini et al., 2020)

Hematology analyzers are known to be imprecise and inconsistent when

who require platelet transfusions, overestimation of thrombocytopenic platelet counts

transfusion. Because of this, manual platelet estimation utilizing a variety of techniques

skilled, trained hands. In addition, some investigations have recommended

involved. (Muhammed, 2020)

Z ST. ALEXIUS COLLEGE

Calcium is removed by EDTA and sodium citrate, which is necessary for

coagulation. It is either bonded in a non-ionized state or precipitates as insoluble

oxalate, which crystals may be visible in oxalated blood. Ethylenediaminetetraacetic

acid, also known as EDTA, is an aminopolycarboxylic acid. It is a common binding

samples. In aqueous solutions, it can be used as a sequestering agent to remove metal

to sodium cations. It has been employed as an anticoagulant to regulate blood. Citrate

concentration of ionized calcium. Citrate is quickly processed in the systemic circulation

Statement of the Problem

This study aims to investigate the comparison of EDTA and Citrate

anticoagulants in Manual Platelet Count.

Specifically, this study seeks to answer following:

citrate anticoagulated blood?

2. Which of the two anticoagulants performs manual platelet counts more

Z ST. ALEXIUS COLLEGE

1. H0: There is no significant relationship between the differences of platelet count

and the EDTA anticoagulated blood and Citrate anticoagulated blood.

the EDTA anticoagulated blood and Citrate anticoagulated blood.

2. H0: There is no significant relationship between the accuracy of EDTA

anticoagulant and Citrate anticoagulant and manual platelet counting.

H1: There is significant relationship between the accuracy of EDTA anticoagulant

and Citrate anticoagulant and manual platelet counting.

3. H0: There is no significant relationship between the effects of EDTA anticoagulant

and Citrate anticoagulant to the manual platelet count.

H1: There is significant relationship between the effects of EDTA anticoagulant

and Citrate anticoagulant to the manual platelet count.

Scope and Delimitation

aim to determine if there is a statistically significant difference in manual platelet counts

platelet counting in clinical practice.

Z ST. ALEXIUS COLLEGE

hemocytometer. The impact of platelet aggregation or blood storage duration on manual