1.
INTRODUCTION
COVID-19,
caused by the Severe Acute Respiratory
Syndrome Coronavirus 2 (SARS-CoV-2), rapidly became a pandemic after emerging
in Wuhan, China, in December 2019 (Zhou et al, 2020). SARS-CoV-2
infections can lead to a wide range of clinical symptoms, with some individuals
remaining completely asymptomatic (Xie et al, 2020; Lippi &
Favaloro, 2020). The disease can cause severe complications, including acute
respiratory distress syndrome, coagulation dysfunction, and death. By November
2020, there had been 55 million infections globally, resulting in about 1.34 million
fatalities (Sohrabi et al, 2020). Clinical signs can range from mild
acute upper respiratory symptoms (fever, cough, and body aches) to multiorgan
failure and catastrophic disease. Of these, almost 80% experienced mild
symptoms, 13% had severe illness, and just 5% had critical symptoms that required
to be admitted to the intensive care unit (ICU) (Tang et al, 2020).
The human immune system is unfamiliar with this unique coronavirus, and
no natural immune system had evolved to combat it. As a result, this could
explain why SARS-CoV-2 spread so quickly throughout the world. Disease
resistance and infection prevention rely heavily on the immune system (Calder,
2020).
The
results of COVID-19 can be predicted by a number of various laboratory markers,
such as high-sensitivity cardiac troponin, LDH, D-dimer levels, and CRP (Flower et al, 2021.
Globally, as of 17 June 2022, the confirmed cases of COVID-19 were 535,863,950,
with 6,314,972 estimated deaths, and the number of vaccine doses administered had
reached 11,902,271,619 according to WHO. In Iraq, between 3 January 2020 and
22 June 2022, there were 2,333,443 confirmed cases of COVID-19, with 25,229
deaths reported to WHO. As of 20 June 2022, a total of 18,589 vaccination
doses had been administered (WHO, 2022). The COVID-19 pandemic affected Iraqi
Kurdistan in three waves: the first occurred from March to December 2020, the
second from January to June 2021, and the third from July to December 2021
(Merza et al, 2021). Phylogenetic analysis revealed
similarities between the viral genome in Kurdistan and isolates from USA,
UK, Germany, Austria, Canada, Australia, Denmark, Poland, and Colorado,
indicating multiple virus introductions. Further research could clarify its
infectivity and virulence (Taher et al, 2023).
The increasing trends of COVID-19 in the two neighboring countries, Iran
and Turkey, had alarming effects on Iraqi Kurdistan. The first cases were
reported in Iraq and Iraqi Kurdistan on February 24, 2020 and March 1, 2020
respectively (Merza et al, 2020; Merza, et al, 2021). Saudi
Arabia is one of the countries that battled the pandemic in its early phases
(Khan et al, 2021). The first case of COVID-19
in the world was discovered in March 2020, and once the first wave of COVID-19
ended, 557,082 individuals had been infected with SARS-CoV-2, and 8878 deaths
were reported at the end of 2021 (Sharma et al., 2020). The second
COVID-19 wave is in its early stages in Saudi Arabia, while the third wave had already
begun in some countries. This rapid progression has been driven by emerging
variants of the virus, which have required new preventive and corrective
measures to combat the emergence of each variant (Otto et al, 2021).
Coagulation
indicators and D-dimer identification early on can help manage and control
COVID-19 (Huang et al, 2021). Thus, in the context of a prothrombotic state,
elevated D-dimer levels in COVID-19 patients may help in the early
identification of individuals with major medical issues, pulmonary
difficulties, and a high risk of venous thromboembolism. This would support
early therapeutic strategy deployment and risk classification, both of which
could lower COVID-19 morbidity and mortality (Khan et al, 2021). C-reactive
protein (CRP),
a plasma protein produced by hepatocytes, can be elevated by various
inflammatory mediators, including IL-6. CRP is an acute-phase protein that
serves as an early warning indication of infection or inflammation (Elbe &
Buckland‐Merrett, 2017). When clinical features are paired with
biological markers like CRP, they become more understandable. As a result, CRP levels may
be necessary for early detection and treatment of COVID-19-related diseases
(Upadhyay et al, 2020). All patients receive a Complete Blood Count (CBC), a routine test that
provides vital information that may aid in clinical care. This information includes the stage of
inflammatory disease, etiology, anemia, and therapeutic response, presence of
infection or inflammation, and diagnosis. Individuals who are more likely to
have COVID-19 could be identified with the help of basic blood testing and
clinical observations (Li et al, 2020).
The purpose of
this study is to compare the levels of specific biomarkers through estimated
D-dimer, CRP, and Complete Blood Count (CBC) levels between COVID-19 patients
and healthy individuals to assess disease severity. Elevated D-dimer and CRP
levels are associated with inflammation and coagulation abnormalities.
Identifying CBC patterns can help understand the impact on blood parameters,
potentially improving patient care and outcomes.
2.
MATERIALS AND METHODS
Clients
and Study Designs
This study took place from April to August 2022 at the
private medical Jin Complex laboratory. Patients who visited the laboratory
provided samples. Fifty COVID-19 patients and fifty healthy controls, ranging
in age from 20 to 65, participated in the study. The positive samples
were confirmed as COVID-19 using molecular diagnostic tools (PowerChek
2019-nCoV Real-time PCR Kit) according to the manufacturer's procedure. A
medical expert identified the people who tested positive for the virus and had
elevated CRP as COVID-19 patients.
Collection
of samples
A nasopharyngeal swab was collected from 100
samples (50 COVID-19 patients and 50 non-COVID-19 samples). Ten milliliters of blood were collected from each subject using
disposable syringes and vein punctures. Five milliliters were placed in an
Ethylene diamine tetraacetic acid EDTA tube, three milliliters in buffered
sodium citrate, and two milliliters in gel tubes.
To perform CBC and D-dimer assays, whole blood and plasma samples were
refrigerated at 2 to 4 °C post-collection. The gel tube samples were
centrifuged at 3000 g for 10 minutes for CRP detection, and the resulting serum
was stored at -20 °C until analysis.
Sample Analysis.
Using the ELFA technique (Enzyme Linked Fluorescent Assay) and a commercially
available protocol, the D-dimer was evaluated using VIDAS D-DIMER Exclusion II
(60 Tests) equipment for the immunoenzymatic assessment of fibrin degradation
products (FbDP) in human plasma (sodium citrate) (Chen et al, 2020). CRP was quantified using Eurolyser
Diagnostics GmbH Bayemstrabe II a 5020 in Austria. The concentration was
determined kinetically by photometric detection of the antigen-antibody
interaction between the sample's CRP and antibodies to human CRP attached to
polystyrene particles at 546 nm or 700 nm. For the CBC profile, the Swelab
AlfaDiluent and RFID Swelab AlfaLyse system (Spnga, Sweden) were used.
Statistical Analysis.
Statistical tests were considered
significant at p < 0.05 and highly significant at p < 0.01, using a 95%
confidence range. The independent samples students’ t-test and descriptive
statistics were tools used to analyze all the data in the Statistical Package
for Social Sciences (SPSS) version 25 and RStudio version 4.3.3. The data were analyzed
using descriptive statistics and the Independent Samples Students’ t-test. To
identify the most significant predictors, stepwise regression analysis was
employed. This method was chosen to optimize model selection by including only
those variables that significantly contributed to the model’s predictive power.
The results of the stepwise regression are presented in Table 3 of the Results
section, where the mean and standard deviation of the results are also
displayed. For each regression model, an Analysis of Variance (ANOVA) table was
generated to assess the overall significance of the predictors. The ANOVA table
includes the following key components.
4.
DISCUSSION
D-dimer is a fibrin decomposition product
that consists of several cross-linked D domains and/or E domains from the
original fibrinogen molecule, and its formation is only theoretically
conceivable when both the hemostasis and fibrinolysis pathways are engaged. In
the early stages of COVID-19 illness, our study found from the data in Table 2
that D-dimer in patients was increased compared to the control, which means it
is statistically significant (p = 0.001). This data was in agreement with that
of (Rostami & Mansouritorghabeh, 2020). Regarding the specific relationship
between D-dimer levels and COVID-19, severe COVID-19 can lead to a
hypercoagulable state, which increases the risk of blood clot formation. Recent
findings show that D-dimer elevations are common in patients with
SARS-CoV-2infection (especially in those with thrombosis), that its value
predicts the clinical severity (up to death) of COVID-19, and that it remains
more frequently elevated in COVID-19 patients with post-discharge clinical
sequelae (Lippi et al, 2023). CRP levels were statistically
significantly elevated (p = 0.001), in patients compared to the control group,
so by comparing our findings with those of other researchers, we found that
high levels of CRP in the blood (Ali, 2020). Other studies have shown that
higher CRP levels are associated with a more severe course of COVID-19.
In the context of COVID-19, CRP levels
can be elevated, particularly in severe cases or individuals with complications
such as pneumonia (Abdelhadi & Kassem, 2021), and this may be an
increase in CRP produced by the liver in response to inflammation, infection,
and tissue damage. CRP elevations in the blood can be utilized as a biomarker
of inflammation since they signify an acute-phase response (xu et al, 2020). This is most likely
because the immune system responds to viral infections more forcefully by
producing a variety of immunological chemicals. In COVID-19 patients, excessive
CRP production above normal limits may result in organ failure. CBC results can help providers assess overall
health, detect infections or abnormalities, and monitor response to treatment.
CBC results in individuals infected with COVID-19 may show specific changes
that indicate the presence of the infection or its impact on the body. These
changes may include alterations in WBC counts, among the different subtypes of
WBCs, such as lymphocytes, neutrophils, and monocytes. COVID-19 can cause
Lymphopenia (decreased lymphocyte count) and Neutrophilia (increased neutrophil
count) in some cases as happened with our patients' results, although
individual variations exist (Petrone et al, 2021). However, many studies
have investigated the trends of lymphocyte count during the progression of the
disease in COVID-19 patients. It was found that lymphocytes declined over time
in non-survivors and increased in survivors in the early hospitalization stage.
Within 10 days after admission, the difference in lymphocyte counts between the
two groups increased by 0.0731 × 109/L per day (Chen et al,
2023).
The granulocyte count, particularly
neutrophils, is often elevated in COVID-19, reflecting an intense inflammatory
and immune response. This study and our findings confirm significantly higher
granulocyte levels in patients compared to controls, aligning with other
research indicating that elevated granulocytes are a hallmark of severe
COVID-19 and are associated with poor (Li et al, 2023). Thromboembolic
problems and systemic inflammation are associated with higher COVID-19 death
rates. Neutrophilia is involved in the necrotic inflammatory response and is a
sign of venous thrombosis. Although protective, neutrophil extracellular traps
can have adverse effects when an infection is present. Cytokine storms, which
result in acute respiratory distress syndrome (ARDS) and severe inflammatory
response syndrome (SIRS), can be caused by improper phagocytosis and excessive
extracellular trap formation. The immune system's homeostasis and inflammatory
reactions depend heavily on lymphocytes. Research indicates that individuals
with elevated WBC levels and reduced levels of lymphocytes, monocytes,
basophils, and eosinophils may be prone to inflammation. In COVID-19 patients,
eosinophils are helpful and reduce neutrophil-induced inflammation (Qin et al, 2020).
Hemoglobin and hematocrit levels reflect
the oxygen-carrying capacity of the blood. In COVID-19, these levels may be
influenced by inflammatory processes and comorbidities. The lack of significant
differences between patients and controls is similar to our results from the
data in Table 2, which suggests that they might not vary as markedly as other
hematologic parameters in response to COVID-19 (Li et al., 2020). Anemia
is not a common finding in patients suffering from COVID-19. Other different
results indicate lower hemoglobin-related parameters and a reduced RBC volume
in the COVID groups compared to control subjects, thus supporting recent data
(Bros et al, 2023). RBC counts can be affected by COVID-19 through
mechanisms such as inflammation-induced anemia or hemoconcentration due to
dehydration. Although RBC counts were lower in patients compared to controls,
the lack of statistical significance (p-value = 0.174) from our findings
suggest that RBC count alone may not be a strong indicator of COVID-19 severity
(Henry et al, 2020).
This variability could be due to the
complex interplay of factors such as the body’s inflammatory response and
pre-existing conditions, while RBC deformability has been reported to be
reduced after SARS-CoV-2 infection in critically ill patients, but also in
patients showing a relatively mild disease course (Bros et al, 2023).
This study showed that low MCV and MCH were both with (p = 0.001) significantly
associated with the severity of the illness. So, a comprehensive analysis
of the RBC parameters would be helpful for early identification and better
management of severe COVID-19 disease. A significantly higher level of MCHC
(p-value = 0.022) was observed with our data results shown n table 2, where
similarly lower levels of MCHC are reported to be associated with disease
severity and mortality in COVID-19 of this study (Mao et al, 2021). However,
in a previous study, significantly higher MCHC levels were found in COVID-19
survivors regardless of the post-COVID-19 symptoms compared to healthy subjects
(Gameil et al 2021). While other different results of COVID-19 cases
were found to have significantly lower levels of Hb concentration MCH, MCHC,
and MCV in comparison with negative patients, this result is quite similar to
that of previously reported studies (Elkhalifa et al, 2022). Platelet
counts in COVID-19 can vary, with some studies reporting thrombocytopenia and
others showing normal or even elevated counts. In this study, the mean platelet
count was lower in patients compared to controls, which is consistent with
findings that thrombocytopenia is common in severe COVID-19 cases (Lippi et
al, 2020). However, the lack of significant difference (p-value = 0.147)
suggests that platelet count alone might not be a reliable marker for disease
severity in all cases. A detailed understanding of immune responses following
SARS-CoV2 infection will enable better treatment and diagnostic procedures, as
well as the development of successful vaccines that will help to control the
global COVID-19 pandemic. In this regard, it is essential to better understand
the presence of neutralizing anti-SARS-CoV-2 serum antibodies in the
population, as they potentially prevent (re)infection and might be a treatment
option (Abdullah et al, 2023). Our research has significant limitations.
First, COVID-19 patients' BMI and treatments were not taken into account.
Second, the research exclusively covered patients from the Duhok City region.
CONCLUSION
The study highlights the significant
elevation of D-dimer and CRP levels in COVID-19 patients compared to controls,
indicating a hypercoagulable state and inflammatory response that correlate
with disease severity. Elevated D-dimer is associated with worse outcomes and
thrombosis, while CRP serves as a biomarker of inflammation, particularly in
severe cases. Hematological findings reveal changes in white blood cell counts,
including lymphopenia and neutrophilia, which reflect the immune response to
the virus. Although red blood cell parameters showed variability, low MCV and
MCH were linked to illness severity. Thrombocytopenia was noted, but without
significant differences in platelet counts. Overall, understanding these
hematological and inflammatory markers can aid in the management of COVID-19.
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