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\begin{document}

\title{Diagnostic Value of Monocyte, Eosinophil, Platelet-Lymphocyte Ratio And
Monocyte Lymphocyte Ratio In Previously Healthy Covid-19 Patients}



\author[1]{Ibrahim Koc}%
\author[2]{Mesiha Babalik}%
\author[1]{Ozlem Yildiz}%
\author[1]{Rahime Ozgur Isıgıbol}%
\author[1]{Sevda Unalli Ozmen}%
\author[1]{Duygu Zeytinoglu}%
\author[1]{Nazan Nalci Savas}%
\author[1]{Ilhami Yapici}%
\author[1]{Halil Celikten}%
\author[1]{Sedat Demirsoy}%
\author[2]{Olgun Deniz}%
\affil[1]{Affiliation not available}%
\affil[2]{Bursa City Hospital}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Aim: In this study we aimed to investigate diagnostic usefullness and
optimal cut-off values of monocyte, mean platelet volume (MPV),
eosinophil, neutrophil lymphocyte ratio (NLR), platelet lymphocyte ratio
(PLR), monocyte lymphocye ratio (MLR), red blood cell distribution width
(RDW) in laboratory confirmed Covid-19 cases with and without pneumonia
who were totally healthy before being infected with Covid-19. Materials
and Methods: Data of a total 10000 patients who were admitted to
hospital with complaints apropriate with Covid-19 in six months
(March--Agust 2020) interval and underwent SARS-CoV-2 Polymerase Chain
Reaction (PCR) investigated. Out of 10000 patients we included only
previosly healthy 174 patients with Covid-19 pneumonia, 144 SARS-CoV-2
positive but no pneumonia and SARS-CoV-2 PCR negative 156 healthy
controls. The laboratory results and demographic findings were collected
from patient files retrospectively before undergiong treatment for
Covid-19. Results: We found wbc levels lower in Covid-19 patients with
pneumonia (p \textless{}0.001 vs healthy), as well as neutrophil (p
\textless{}0.001 vs healthy), Monocyte (p \textless{}0.001 vs healthy
and Covid-19 without pneumonia), Eosinophil (p \textless{}0.001 vs
healthy and Covid-19 without pneumonia) and Platelet count (p
\textless{}0.001 vs healthy). MLR remained higher in patients without
pneumonia (p \textless{}0.001 vs Covid-19 with pneumonia and healthy
controls) as well as PLR (p \textless{}0.001 versus Healthy).
Conclusion: In the light of the obtained results, decrease in wbc,
neutrophil, monocyte and eosinophil levels support the diagnosis of
Covid-19 pneumonia on the other hand increase in MLR and PLR may
indicate Covid-19 without pneumonia. Keywords: Covid-19 pneumonia,
esoinophil, monocyte%
\end{abstract}%



\sloppy


\textbf{Introduction}

Coronavirus disease-2019 (COVID-19), caused by the severe acute
respiratory syndrome-coronavirus 2 (SARS-CoV-2) is cause of illness and
death of millions since December 2019. It is a highly contagious disease
that spreads via contact and aerosol transmission. There are much more
cases than reported because many are asymptomatic but infectious. Gold
standart diagnostic tool is testing nasopharyngeal swab samples by
real-time reverse-transcription polymerase chain reaction (PCR). But PCR
is not available in every health-care institution due to its cost and
working technique, elsemore getting results takes time. Computed
tomography (CT) of the chest is indicated in those patients suspicious
of pneumonia with a prior chest x-ray that is undiagnostic. CT is
expensive, not available in every healthcare institution and patients
are exposed to radiation. There is a need for fast diagnostic parameters
to assist doctors in the diagnostic phase and to help differentiating
patients with Covid-19 pneumonia than those without. Comlpete blood
parameters are studied in many patients at admittion in Covid-19
clinics. Biomarkers of inflammation derived from the peripheral blood,
hemogram parameters such as white blood cell (WBC) count, neutrophil
lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR) have been
investigated as independent predictors for prognosis of systematic
inflammatory diseases \textsuperscript{1}. Monocyte-macrophage
activation markers are increased and correlate with other inflammatory
markers in SARS-CoV-2 infection, in association to hospital admission.
The red blood cell distribution width (RDW) is a measure of the
variation of red blood cell volume. There are recent studies focusing on
RDW \textsuperscript{2}. Mean platelet volume (MPV) is one of the
platelet function indices which reflects the platelet production rate
and stimulation \textsuperscript{3}. Platelet distribution width (PDW)
is a~direct measure of platelet size variation and a~ marker of platelet
activation. High levels of PDW have been associated with Covid-19
mortality \textsuperscript{4}. All above mentioned parameters are
studied by routine complete blood count test that might be overlooked by
clinicians. The major difficulty for physicians is to translate the
recommended guidelines into clinical practice. In this study we aimed to
determine wheather monocyte levels, eosinophil levels, MPV, PDW, RDW,
NRL, MLR and PLR can be used as biomarkers in the early diagnosis of
laboratory-confirmed COVID-19 patients as well as to explore the most
useful diagnostic biomarkers and optimal cut-off values. Else more to
investigate if these parameters can be used in differentiation of
Covid-19 patients with pneumonia than those without and healthy
controls. To the best of our knowledge this is the first study
investigating above mentioned parameters in previously totally healthy
Covid-19 patients.

\textbf{Materials and methods}

We investigated files of those patients admitted to Bursa City
Hospital/Bursa/Turkey between March-August 2020 with complaints
compatible with COVID-19. In a total 10000 patients retrospectively
evaluated. Out of 10000 patients those who were found to be positive for
Covid-19 and were completely healthy before the disease were included in
the study. In a total 318 Covid-19 patients who met the specified
criteria and 156 healthy controls those tested negatively and had no
chronic diseases included. We divided Covid-19 patients into two groups,
the main group consisted of those patients who had penumonia on computed
tomography of the lungs, the second gruop consisted of those patients
tested positively with PCR but had no penumonia on computed tomography
of the chest. And the third group consisted of healthy subjects. The
demographic and clinical data of all patients is shown on table 1. The
three patient groups were compared by examining the hemogram parameters
(WBC, neutrophil, lymphocyte, monocyte, eosinophil, platelets, PDW, MPV
and RDW) obtained at the time of hospital admission before using any
kind of medication. NLR, PLR and MLR values were calculated by dividing
neutrophil, platelet and monocyte levels to lymphocyte count. Patients
with chronic diseases, those using a medication and pregnants were
exluded. After obtaining scientific research approval for the study from
the Ministry of Health General Directorate of Health Services, the
ethics committee approval was obtained from Bursa City Hospital Clinical
Research Ethics Committee (Ethics Committee Approval No:2021-2/2). All
the statistical analyses were carried out using SPSS 25.0 software. A
Kolmogorov-Smirnov test was performed for the normality of the sample
data, and the continuous variables were defined by the mean \selectlanguage{ngerman}± standard
deviation, median (interquartile range \%25--\%75), while the
categorical variables were expressed as frequency and percent. In order
to compare the independent groups, One-way ANOVA Bonferroni for the
parametric test assumptions, and a MannWhitney U test for non-parametric
assumptions were used. The Roc analysis was performed for optimal
cut-off values to predict SARS-CoV-2 positivity. We exploited Youden
Index values to identify the optimal cut-off values. In addition, p
value less than 0.05 was set as the statistical significance level.

\textbf{Results}

The median age turned out to be 42 (19-82) in the Covid\selectlanguage{english}-19 pneumonia, 33
(19-81) in Covid-19 patients without pneumonia and 34 (19-87) in the
healthy group (Table 1). The median age was significantly higher in the
pneumonia group than those without pneumonia (p \textless{}0.001) and
healthy controls (p \textless{}0.001). Although the gender distribution
in the Covid\selectlanguage{english}-19 pneumonia group was 43\% (75) women and 57\% (99) men,
it was 45.8\% (66) women and 54.2\% (78) men in those without pneumonia.
WBC, neutrophil, platelet and lymphocyte levels remained significantly
low (Table 2) in patients with and without pneumonia compared to healthy
subjects (both p \textless{}0.001). When we compared WBC, neutrophil,
lymphocyte and platelet levels of Covid-19 patients with pneumonia to
those without we found no difference (p=0.482, 0.923, 0.164 and 0.447
respectively). Covid-19 patients with pneumonia had the lowest monocyte
levels compared to those without pneumonia and healthy controls (Both p
\textless{}0.001) but we found no difference between Covid-19 patients
without pneumonia and healthy controls (p=0.261). Eosinophil levels were
lower in patients with Covid-19 pneumonia compared to those without and
healthy controls as well as in Covid-19 patients without pneumnoia
compared to healthy controls (p \textless{}0.001 for three comparisons).
There was no difference between MPV, RDW, PDW and NLR levels of all
groups. MLR was higher in Covid-19 patients without pneumonia compared
to those with pneumonia and healthy controls (both p \textless{}0.001)
yet no difference between patients with pneumonia versus healthy
controls (p=0.045). PLR was higher in Covid-19 patients without
pneumonia compared to control group (p \textless{}0.001) but no
difference when compared to those with pneumonia (p=0.025) and patients
with pneumoia compared to healthy controls (p=0.013). We found no
difference between PLR of Covid-19 patients with and without pneumonia
(p=0.025). We analyzed the optimal cut-off values calculated by the ROC
analysis, and the ROC curves are presented in Figure 1, Figure 2. When
we compared Covid-19 patients with pneumonia to healthy controls the
areas under the curve (AUC) of WBC, Neutrophil, Lymphocyte, Monocyte,
Eosinophil and Platelet were found as 0.828 (p \textless{} 0.0001),
0.769 (p \textless{}0.001), 0.727(p \textless{}0.001), 0.657 (p
\textless{} 0.0001), 0.815 (p \textless{}0.0001) and 0.688 (p
\textless{}0.001) respectively (Table 3). For MLR and PLR 0.574 (p
=0.21), 0.591 (p =0.05) respectively (Table 3). When we compared
Covid-19 patients without pneumonia to healthy controls AUC of WBC,
Neutrophil, Lymphocyte, Eosinophil, Platelets, Monocyte, MLR and PLR
were found as 0.826 (p \textless{} 0.0001), 0.775 (p \textless{}0.001),
0.756 (p \textless{}0.001), 0.724 (p \textless{} 0.0001), 0.684 (p
\textless{} 0.0001), 0.535 (p = 0.304), 0.714 (p \textless{}0.001) and
0.657 (p \textless{}0.001) respectively (Table 4). Significant levels
for comparison between Covid-19 patients with and without pneumonia were
observed between monocyte levels (AUC 0.66 P \textless{}0.001) and
Eosinophil levels (AUC 0.612 P \textless{}0.001).

\textbf{Discussion}

In suspicious cases of Covid-19 PCR is used in the diagnosis and is the
gold standard, in some patients suspicious of having pneumonia may have
to undergo a CT scanning. Yet PCR and CT both are not availbale in every
healtcare institution. Therefore, there is a need for simpler and more
practical methods to detect Covid-19 cases and else more Covid-19
patients with pneumonia which may have to be internated or carefully
followed.

Our study demonstrated that neutrophil levels decrease in Covid-19
patienets with and without pneumonia compared to healthy subjects (p
\textless{}0.001 for both comparisons). The data on neutrophil levels in
Covid-19 are incomplete and have not been widely studied. The available
data suggest that neutrophilia is an expression of the cytokine storm
and hyperinflammatory state which have an important pathogenetic role in
COVID-19 and related infections. In previous studies neutrophilia is
reported in patients treated in intensive care unit \textsuperscript{5}.
Yet neutrophil levels may be effected by many secondary diseases. In our
study we included previously totally healthy patients at admission
before using any kind of medicine. In a study Gumus et al. demonstrated
low neutrophil levels in pediatric Covid-19 patients\textsuperscript{6}.
Similar to our study Xie et al. reported low levels of neutrophils in
Covid-19 patients \textsuperscript{7} yet their study contained patients
who had chnronic diseases like asthma, chronic obstructive pulmonry
disease, coronary heart disease etc.

Lymphocytes play an important role in the maintenance of immune system
function. After a viral infection, changes in total lymphocyte numbers
varies with different virus types. Wang et al. reported decreased
lymphocyte levels in patients with Covid-19 \textsuperscript{8}. Similar
to their study we found low lymphocyte levels in patients with Covid-19
pneumonia. Eventhough low lymphocyte levels have been demontrated
previously our study demonstrated that lymphocyte levels decrease in
preiously healthy Covid-19 patients but no statistically difference
between those with and without pneumonia.

Monocytes and macrophages are the most enriched immune cell types in the
lungs of Covid-19 patients and appear to have a central role in the
pathogenicity of the disease \textsuperscript{9}. Current data suggest a
preponderant role for monocyte-macrophage activation in the development
of immunopathology of Covid-19 patients \textsuperscript{10}.In our
study we found lowe monocyte levels in Covid-19 patients with pneumonia
compared to healthy controls (p \textless{}0.001) and patients without
pneumonia (p \textless{}0.001). Zhang et al observed that during
SARS-CoV-2 infection, there are morphological and inflammation-related
phenotypic changes in peripheral blood monocytes that correlate with the
patient's outcome \textsuperscript{11}. Zhang et al. did not detect
significant differences in the number of monocytes between patients with
Covid-19 and normal healthy individuals. In a study Kos et al. reported
reduced rate of activated monocytes mainly observed in patients with
severe Covid-19\textsuperscript{12}. Eventhough our study demonstrated
that monocyte levels decrease in previously healthy Covid-19 pneumonia
and in those without these results reflect begining of the disease
before treatment. Levels may differ after progression of the disease and
may be effected by drugs used in treatment.

Eosinophils are circulating and tissue-resident leukocytes that have
potential of proinflammatory effects in some diseases. Eosinophils also
have been shown to have various other functions like immunoregulation
and antiviral activity. Yet their role in Covid-19 is not well known.
Previosly it has been reported that Covid-19 patients had a decrease in
circulating eosinophil counts, which was significantly more frequent
than other types of pneumonia patients \textsuperscript{7}. Zhang et al.
has previously reported eosinopenia in patients with acute respiratory
deterioration during infection with SARS-CoV-2\textsuperscript{13}.
Zhanh et al. included 140 Covid-19 patients with comorbidities which may
effect eosinophil levels yet similar to their study we also found low
levels of circulating eosinophils in patients with Covid-19. Our study
demonstrated that circulating eosinophil counts decrease in preivously
totally healthy patients with Covid-19 and much more in Covid-19
patients with pneumonia. These results may indicate involvement of the
lungs is associated with decrease in eosinophil levels. So a treatment
strategy which increases eosinophil levels or targeting reasons of
eosinopenia may help in dealing with Covid-19.

Covid-19 causes a spectrum of disease; some patients develop a severe
proinflammatory state which can be associated with a unique coagulopathy
and procoagulant endothelial phenotype. We found Covid-19 patients with
pneumonia had the lowest platelet counts. Trombocytopenia is a big
problem during Covid-19 because many patients need anticoagulant
prophylaxy which implies that thrombocytopenia needs to be attached more
importance in the follow-up of Covid-19 patients. The difficulty is to
choose the apropriate anticoagulant while on the other side there is
trombocytopneia.

Prveiosly it has been reported that increased MPV levels was associated
with mortality in Covid-19 patients \textsuperscript{14}. In another
study Ozcelik et al. compared Covid-19 patients with influenza pneumonia
and reported low MPV levels in patients with
Covid-19\textsuperscript{15}. MPV levels also found significantly high
in asymptomatic children infected with Covid-19 \textsuperscript{6}. In
this study we found no difference in MPV levels between groups. MPV can
be effected from many diseases and conditions and the teqnique used
which may effect the results. In this study similar to MPV levels we
didnt find statistically differences between PDW and RDW levels. In a
study Henry et al. found a progressive increase of RDW with advancing
Covid-19 severity \textsuperscript{16}. The discrepency between two
studies might be due to patients grops, Henry et al. included patients
with comorbidities yet we aimed to see if RDW can be usefull in the
early diagnosis of previously healthy Covid-19 patients.

In recent years, some ratios have been in use by researchers in the
diagnosis and prognosis of many inflammatory conditions. Some of these
are neutrophil/lymphocyte, platelet/lymphocyte, and monocyte/lymphocyte
ratio. In a study Seyit et al. Reported high NLR in patients tested
positive for SARS-CoV-2 compared to controls \textsuperscript{17}. In
our study we found no differences between NLR of three groups. In a
study from Wuhan/China Yang et al. reported elevated NLR significantly
associated with illness severity \textsuperscript{18}. Difference
between our study and the study conducted by Seyit et al. migt be due to
patient characteristics or to the heterogenisity of Covid-19. Seyit et
al found high levels of PLR in patients with Covid-19. In acoordance
with their study we also found high levels of PLR in patients with
Covid-19 pneumonia compared to healthy subjects (p \textless{}0.013) and
Covid-19 patients without pneumonia compared to healthy controls (p
\textless{}0.005). Our study demonstrated that PLR is high in Covid-19
patients but it might not be associated with disease severity at least
at the begining stage of the disease. Yang et al. investigated 93
Covid-19 patients and found out that lyphocyte to monocyte ratio (LMR)
levels of severe patients were significantly higher than those of
non-severe patients.\textsuperscript{18}. In our study we aimed to
observe if MLR could be used in the diagnosis of Covid-19 patients at
the admmision before undergoing treatment. We found highest MLR in
patients without Covid-19 pneumonia versus patients with pneumonia and
healthy controls (both p \textless{}0.001). More studies are needed to
obtain more detailed information.

\textbf{Limitations}

Since we only included previously healthy individuals in our study, the
results of the study do not provide information about those with
additional diseases which stands as a limitation. We aimed to evaluate
the above mentioned parameters the first hospital admission, thus
results do not give an idea about the progressive stages of the disease
and the prognosis.

\textbf{Conclusion}

In the light of the obtained results, decrease in wbc, neutrophil,
monocyte and eosinophil levels support the diagnosis of Covid-19
pneumonia on the other hand increase in MLR and PLR may indicate
Covid-19 without pneumonia. Deep fall in eosinophil and monocyte levels
may be indicating a possible Covid-19 pneumonia.

References

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2. Gisondi P, Geat D, Lippi G, Montagnana M, Girolomoni G. Increased red
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6. Gumus H, Demir A, Yukkaldiran A. Is mean platelet volume a predictive
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7. Xie G, Ding F, Han L, Yin D, Lu H, Zhang M. The role of peripheral
blood eosinophil counts in COVID-19 patients.
\emph{Allergy.}2021;76(2):471-482.

8. Wang F, Nie J, Wang H, et al. Characteristics of Peripheral
Lymphocyte Subset Alteration in COVID-19 Pneumonia. \emph{J Infect
Dis.}2020;221(11):1762-1769.

9. Bost P, Giladi A, Liu Y, et al. Host-Viral Infection Maps Reveal
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\emph{Cell.}2020;181(7):1475-1488 e1412.

10. Gomez-Rial J, Curras-Tuala MJ, Rivero-Calle I, et al. Increased
Serum Levels of sCD14 and sCD163 Indicate a Preponderant Role for
Monocytes in COVID-19 Immunopathology. \emph{Front
Immunol.}2020;11:560381.

11. Zhang D, Guo R, Lei L, et al. COVID-19 infection induces readily
detectable morphological and inflammation-related phenotypic changes in
peripheral blood monocytes, the severity of which correlate with patient
outcome. 2020:2020.2003.2024.20042655.

12. Kos I, Balensiefer B, Lesan V, et al. Increased B-cell activity with
consumption of activated monocytes in severe COVID-19 patients.\emph{Eur
J Immunol.} 2021;51(6):1449-1460.

13. Zhang JJ, Dong X, Cao YY, et al. Clinical characteristics of 140
patients infected with SARS-CoV-2 in Wuhan, China.
\emph{Allergy.}2020;75(7):1730-1741.

14. Guclu E, Kocayigit H, Okan HD, et al. Effect of COVID-19 on platelet
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15. Ozcelik N, Ozyurt S, Yilmaz Kara B, Gumus A, Sahin U. The value of
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16. Henry BM, Benoit JL, Benoit S, et al. Red Blood Cell Distribution
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17. Seyit M, Avci E, Nar R, et al. Neutrophil to lymphocyte ratio,
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Immunopharmacol.}2020;84:106504.

\textbf{Figure 1:} The ROC curve of White Blood Cell Count, Neutrophil,
Lymphocyte, Monocyte, Eosinophil, Platelets (Plt) (Figure 1-A), MLR and
PLR (Figure 1-B). for discriminating between Covid-19 patients with
pneumonia and healthy groups.

\textbf{Figure 2:} The ROC curve of White Blood Cell Count (WBC)
Neutrophil, Lymphocyte, Eosinophil and Platelets for discriminating
between Covid-19 patients without pneumonia and healthy group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`4.Tables.docx`  available at \url{https://authorea.com/users/732502/articles/710827-diagnostic-value-of-monocyte-eosinophil-platelet-lymphocyte-ratio-and-monocyte-lymphocyte-ratio-in-previously-healthy-covid-19-patients}

\selectlanguage{english}
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