Delta Neutrophil Index: A Potential Diagnostic Marker of Multisystem Inammatory Syndrome in Children (MIS-C)

Background Multisystem inammatory syndrome in children (MIS-C) is a life threatening hiperinamation syndrome emerging after COVID-19. The serum delta neutrophil index (DNI), reects the fraction of circulating immature granulocytes and elevated in infection and inammation. The aim of this study is to evaluate the usefulness of DNI as a diagnostic marker in patients with MIS-C and to assess its role in determining the severity of MIS-C. This retrospective, observational study included 83 patients of MIS-C and 113 patients of COVID-19 and control group. C-reactive protein, absolute neutrophil count, absolute lymphocyte count, DNI and platelet count were recorded.


Introduction
Coronavirus disease 2019 (COVID-19) spread all over the world in a short time and became a global pandemic. In April 2020, there has been apparent cluster of children presenting with Kawasaki disease (KD), toxic shock syndrome, and secondary hemophagocytic lymphohistiocytosis associated with COVID-19 in Italy, United Kingdom and United States [1][2][3]. This disease was later named multisystem in ammatory syndrome in children (MIS-C), which is characterized by persistent fever, increased in ammatory laboratory tests, and multi-organ system involvement. Researches are still ongoing on who will develop or progress to MIS-C after COVID-19.
Given the severity of MIS-C, its early diagnosis is essential. A tiered diagnostic approach is recommended in MIS-C patients without life-threatening manifestations. Baseline laboratory workup should include complete blood count, renal function tests, liver functions tests, an assay of in ammatory markers and testing for SARS-CoV-2 by PCR or serology. An advanced diagnostic work-up should be done in children whose cause of fever cannot be determined with a baseline laboratory tests [4][5][6].
Recently, immature granulocytes in peripheral blood have been started to be counted automatically by cytochemical myeloperoxidase reaction and light beam re ection methods during the routine complete blood count. This parameter, called delta neutrophil index (DNI), shows the ratio of immature granulocytes in peripheral blood. This rate has been studied in various diseases such as sepsis, disseminated intravascular coagulation, urinary tract infection and KD in the literature, and its use is recommended as a parameter that provides information about the clinical course, prognosis, evaluation of treatment success and mortality of these diseases [7][8].
The aim of this study is to determine the value of DNI, which is an easily accessible and inexpensive marker in the diagnosis of MIS-C, and its usefulness in determining the severity of MISC. In addition, it is to investigate whether it can be used as an early parameter to predict the development of MIS-C.

Study design and participants:
We conducted this retrospective cross-sectional study in patients diagnosed with COVID-19 and MIS-C in our center between March 2020 and January 2021. Local ethics committee approval was obtained prior to the study.
The study group consisted of 113 patients with COVID-19 and 83 patients with MIS-C. During the study period, 113 patients aged 0-18 years who presented with symptoms such as fever, cough, respiratory distress and weakness and were diagnosed with COVID-19 were randomly selected to be included in this study. The COVID-19 diagnosis was con rmed by detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in oro-nasopharyngeal swab samples.
Eighty-three patients between the ages of 0-18 diagnosed with MIS-C according to the The Centers for Disease Control and Prevention (CDC) diagnostic criteria were included in the study. CDC case de nition for MIS-C is used to de ne a con rmed case of MIS-C, which is as follows: an individual aged < 21 years presenting with fever, laboratory evidence of in ammation, and evidence of clinically severe illness requiring hospitalization, with multisystem (≥ 2) organ involvement; and evidence of SARS-CoV-2 infection or exposure, and exclusion of other potential causes.
Control group was consisted of healthy children admitted to pediatric outpatient clinic for noninfectious conditions such as vaccination, routine control, checkup etc. Control group was matched with MIS-C group in demographic characteristics.
Patients with clinically important additional diseases (chronic lung disease, congenital heart failure, malignancy etc.) and those whose medical records could not be obtained were excluded from the study.

Data collection:
Clinical and laboratory data were retrieved from electronic records. Clinical data included demographics, family history for SARS-CoV-2, presence of comorbidities, symptoms at presentation and follow-up. Creactive protein (CRP), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), DNI, and platelet count were recorded from the laboratory data at the time of diagnosis.

Delta-Neutrophil Index
Delta-Neutrophil Index levels were routinely studied in complete blood count with the Siemens-ADVIA 2120 model automatic test device. This hematologic analyzer uses two independent leukocyte count methods: myeloperoxidase channel (MPO) and the lobularity/nuclear density channel. For DNI; the value obtained by the formula (leukocyte subfraction assayed using the MPO channel by cytochemical reaction)-(leukocyte subfraction assayed using the nuclear lobularity channel based on re ected light beam measurements) was determined as the ratio of immature granulocytes (myelocytes, metamyelocytes and promyelocytes) in peripheral blood.

Statistical analysis
All of data were analyzed by SPSS-2020 statistical package, Categorical variables between groups were analyzed using the χ2 test. Comparison of means between two groups was examined by using a t-test and, Mann-Whitney U test. Categorical variables are shown as number (n), percentage (%) and continuous variables are shown as mean (±) standard deviation (SD). For comparison of more than two groups, ANOVA was used for normal distributions and the Kruskal-Wallis test for nonnormal distributions. Receiver operating characteristic (ROC) curve analysis was used to determine the power of variables to differentiate groups, and the area under the curve (AUC) was calculated; signi cant cut-off levels and sensivity, speci ty, positive predictive and negative predictive values were calculated using by Youden index. p < 0.05 was deemed to indicate statistical signi cance.

Results
Eighty-three patients with MIS-C and 113 patients with COVID-19 and control group were included in the study. While there was male dominance in both groups, there was no signi cant difference between the groups in terms of gender. The mean age was 9.40 ± 4.52 in the MIS-C group, it was 10.35 ± 5.64 in the COVID-19 group. The COVID-19 group had a signi cantly shorter length of hospital stay compared with the MIS-C group (8.81 ± 3.88 days versus 16.58 ± 8.34 days, respectively; p < 0.001). The demographic data of the patients are given in Table 1. The DNI levels at the time of diagnosis were 4.60 ± 5.70% in the MIS-C group and 0.30 ± 0.99% in the COVID-19 group and 0.20 ± 0.56% in the control group (p < 0.001). According to the severity of MIS-C, the DNI levels were found 1,22% (0.1 ± 2.6) in mild MIS-C, 4,3% (2.4 ± 10.7) in moderate MIS-C and 5,7% (2.8 ± 12.2) in severe MIS-C. There was a statistically signi cant correlation between the DNI levels and the severity of MIS-C. (p < 0.001). Similarly, CRP, ANC, ALC and platelet levels were higher in the MIS-C group and this difference was found to be signi cant between the groups. Laboratory data of the patients are given in Table 2. The diagnostic performance of DNI in patients with MIS-C was evaluated and the cut-off value of DNI was found to be 0.45% (AUC 0.893) (Fig. 1). In the correlation analysis of DNI with other parameters (CRP, ANC, ALC and platelet count) sensitivity, speci city, positive predictive value and negative predictive value were found to be 79.5%, 97.1%, 95.7%, and 85.3%, respectively (Table 3). Also, a scoring system was created with these 5 parameters, which are among the baseline laboratory tests in the diagnosis of MIS-C, compared with DNI. According to this model, 1 point was given to each parameter from increased DNI, increased ANC, increased CRP, decreased ALC and decreased platelet count. With this scoring system, which has the highest score of 5, MIS-C diagnostic power was found to be 83% for those with a score of 3 and above, while it was 100% for those who scored 4 and above.

Discussion
To the best of our knowledge this is the rst study in the literature on diagnostic value of DNI in MIS-C. Early diagnosis and treatment of MIS-C is very important in reducing mortality and morbidity. A speci c in ammatory marker that can be used in the diagnosis of MIS-C has not been reported yet. In this current study, the importance of DNI in the early diagnosis of MIS-C was evaluated and it was shown that DNI is an effective marker in the diagnosis and determination of the severity of MIS-C.
The diagnostic value of in ammatory markers that can be used in the diagnosis of MIS-C has been previously evaluated in many studies. Increased ANC, decreased ALC, decreased platelet count, hyponatremia and hypoalbuminemia that may accompany with increased CRP and, erythrocyte sedimentation rate were determined as the rst step tests in the diagnosis of MIS-C by the American College of Rheumatology [10]. These tests are easily accessible and affordable tests that pediatricians often prefer in their daily practice. Zhao et al. showed that ALC, ANC, platelet count and CRP changes in 787 MIS-C patients compared to active COVID-19 patients, as a result of increased in ammatory response, excessive release of in ammatory mediators and cytokine storm [11]. Similarly, in our study, increased CRP, decreased ALC, increased ANC and decreased platelet count were found to be signi cant in the MIS-C group compared to the COVID-19 and control groups. In addition, the DNI level was 4.60 ± 5.70 in the MIS-C group, 0.30 ± 0.99 in the COVID-19 group and 0.20 ± 0.56 in the control group. Early release of immature neutrophils from bone marrow into peripheral blood in correlation with in ammation has been reported, especially in many infectious diseases. Similarly, in COVID-19 patients, it has been reported that immature neutrophils increase in the circulation and are associated with increased in ammation and disease severity [12].
DNI is a simple test that can be quantitatively measured in routine complete blood counts. It has a diagnostic value in diseases such as pediatric sepsis, meningitis, appendicitis, urinary tract infection, and KD; it has even been also reported to be an important marker in determining prognosis [13][14][15][16]. Park et al. emphasized that DNI is a potentially useful diagnostic tool in infected patients and should be used more widely in clinical practice to predict mortality [7]. However, studies of DNI in COVID-19 patients are limited in the literature. Birben et al. showed that DNI is an effective predictor of the intensive care unit mortality in 388 COVID-19 adult patients [17]. In our study, prognosis of MIS-C was not evaluated, but DNI levels were found to be correlated with disease severity. DNI cut-off levels were 0.45% for MIS-C. It is important to pay attention to both this cut-off value and to be aware that the increase in DNI level is related to the MIS-C severity. Prediction of the MIS-C severity by the DNI level at the time of diagnosis may affect the management of the patient and the choice of treatment.
MIS-C is a new phenomenon that causes multiorgan involvement with cardiac involvement and hemodynamic instability. Monitoring of in ammatory markers in patients diagnosed with MIS-C is important in determining early diagnosis and treatment of patients who may require critical care [18]. The majority of children affected are RT-PCR negative for SARS-CoV-2 virus, but are antibody positive, indicating a post-infectious in ammatory response following SARS-CoV-2 infection. However, in some of the patients, both RT-PCR and serology tests are positive. Although it is known that there are clinical and laboratory ndings speci c to MIS-C, if RT-PCR is found positive for SARS-CoV-2 in MIS-C patients, differentiating COVID-19 from MIS-C may be challenging for the clinician. The optimal in ammatory markers to demonstrate the difference in these diseases are still unclear. Some laboratory ndings of active COVID-19 and MIS-C are similar (eg lymphopenia, increased CRP). In our study, it was shown that the DNI at the time of diagnosis was signi cantly higher in MIS-C compared to COVID-19. Moreover, the simple scoring system we created in our study, which can only be calculated with basal laboratory tests and DNI, may contribute to the diagnosis of MIS-C.
Our study has several limitations, rst of all, it was designed as a retrospective study. In addition, a limited number of in ammatory markers such as ANC, ALC, PLT and CRP have been evaluated in our study. Finally, the high DNI levels in the cases were not con rmed by ow cytometry or peripheral smear. However, our study is the rst study in the literature evaluating DNI in MIS-C patients and contributed to the literature with its signi cant results.
In conclusion, early diagnosis and management are important for optimal outcomes in children affected with MIS-C. DNI is an easily accessible, inexpensive and dynamic marker that can be used in the diagnosis of MIS-C such as ANC, ALC, platelet count and CRP. Diagnostic tests have not been clearly established in MIS-C, DNI levels in simple hemogram analysis will guide pediatricians in determining the diagnosis and severity. However, randomized, long-follow-up studies with larger patient groups are required to demonstrate the role of DNI.

Declarations
Funding: There is no funding to disclose Con icts of interest/Competing interests: All authors declare that they have no con ict of interest.
Availability of data and material: The data sets analyzed during the current study are available from the corresponding author on reasonable request. Data can be provided after the article is published.