Using HScore for Evaluation of Hemophagocytosis in Multisystem Inflammatory Syndrome Associated with COVID-19 in Children

Hemophagocytic syndrome is a key point in the pathogenesis of severe forms of multisystem inflammatory syndrome associated with COVID-19 in children (MIS-C). The factors associated with hemophagocytosis in patients with MIS-C were assessed in the present study of 94 boys and 64 girls ranging in age from 4 months to 17 years, each of whose HScore was calculated. In accordance with a previous analysis, patients with HScore ≤ 91 (n = 79) and HScore > 91 (n = 79) were compared. Patients with HScore > 91 had a higher frequency of symptoms such as cervical lymphadenopathy, dry cracked lips, bright mucous, erythema/swelling of hands and feet, peeling of fingers, edematous syndrome, hepatomegaly, splenomegaly, and hypotension/shock. They also had a higher erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and D-dimer levels, and a tendency to anemia, thrombocytopenia, and hypofibrinogenemia. They more often needed acetylsalicylic acid and biological treatment and were admitted to ICU in 70.9% of cases. Conclusion: The following signs of severe MIS-C were associated with HScore > 91: myocardial involvement, pericarditis, hypotension/shock, and ICU admission.

The pathophysiological mechanism of this condition is still not clear.It could be due to immune dysregulation leading to anomalous immune response to the virus, itself leading to massive production of cytokines such as interleukin-1β (IL)-1β, IL-6, IL-8, IL-10, and IL-18 [9,10].
The diagnosis and differential diagnosis of MIS-C is often complex because of its numerous clinical manifestations and considerable similarity with Kawasaki disease (KD).
In some cases, MIS-C patients closely resemble patients with KD due to similar clinical symptoms and even meet the criteria of the American Heart Association (AHA) for KD, but these diseases are different [4,5,7,8,11,12].
Hemophagocytic syndrome was proven to be one of the most important factors determining the severity of the course of MIS-C [13,20,21].It can also be considered a complication of Kawasaki disease [22].The signs of secondary hemophagocytic lymphohistiocytosis (HLH) are significantly more frequent in MIS-C than in KD (18.4% vs. 1.2%, respectively), according the literature [23].
The purpose of this study was to determine the features of hemophagocytosis in patients with multisystem inflammatory syndrome associated with COVID-19 in children.

Patients
The data from case histories (n = 166) of children with MIS-C associated with COVID-19 diagnosed between 2020 and 2022 were included in the present retrospective multi-central study.The diagnosis of MIS-C was made according to the criteria of the CDC and the WHO [24,25].
We excluded eight patients with missing data that did not allow confirmation or exclusion of hemophagocytosis and cases with an obvious microbial cause of multisystem inflammation (e.g., bacterial sepsis and staphylococcal/streptococcal toxic shock syndrome).

Study Design
We included all the related clinical data and the most significant laboratory test abnormalities obtained close to the time of hospital admission.
According to data from the previous analysis, patients with HScore ≤ 91 (n = 79) and HScore > 91 (n = 79) were compared because this cut-off is associated with the severe life-threatening course of MIS-C [20].

Statistics
The STATISTICA software package, version 10.0 (StatSoft Inc., Tulsa, OK, USA), was used for the data analysis.The median value (25th and 75th percentiles) was used to assess quantitative indicators.The Pearson criterion χ2 was used to compare qualitative indicators.Quantitative indicators were compared based on the Mann-Whitney criterion.Sensitivity and specificity analysis was used to evaluate the possibility of patient grouping based on binary characteristics.For quantitative variables, cut-off values were calculated using AUC-ROC analysis (AUC) with a 95% confidence interval (CI).The odds ratio (OR) calculation, independent of the time of event development, was performed using 2 × 2 tables.Sensitivity (Se) and specificity (Sp) were assessed for each parameter.Binary logistic regression was used to specify independent predictors, including quantitative and qualitative indicators associated with the dependent variable in the analysis.Differences or relatedness were considered statistically significant at p < 0.05.

General Characteristics of MIS-C Patients
One hundred and fifty eight (158) patients (94 boys and 64 girls) were included in the analysis.Boys suffered more frequently (59.6%) than girls.The patients were of different ages (from 4 months to 17 years; the median age was 8 years and 2 months).
Standard treatment in MIS-C patients was administered as follows: glucocorticosteroids (81.5%), acetylsalicylic acid (57.1%), intravenous immunoglobulin (44.7%, due to restricted access), and tocilizumab (4.9%).Supportive care was also administered via albumin, plasma, erythrocyte mass, and infusion and inotropic therapy.Patients stayed in the hospital for 18 days on average.No fatal cases were observed.All patients returned to full health.
Among laboratory parameters, differences in the levels of inflammatory markers were found, so ESR (47 mm/h vs. 34 mm/h, p = 0.0001) and CRP (175.5 mg/L vs. 125.8mg/L, p = 0.01) were more significantly elevated in patients with Hscore > 91.
Patients with HScore > 91 more often required ICU admission, the use of intravenous immunoglobulins, and biologic drugs.The clinical and laboratory signs associated with HScore > 91 are outlined in Table 3.

Discussion
In our study, we calculated the threshold value of the HScore index associated with more severe forms of MIS-C.Criteria of severity of MIS-C such as cardiac involvement, including myocardial and pericardial damage and hypotension/shock, were associated with intense hemophagocytosis (HScore > 91).Patients with intensive hemophagocytosis more often required the use of intravenous immunoglobulins and biological drugs.
The incidence of hemophagocytic syndrome in MIS-C is 18-76%, according to various studies [5,13,21,23,30,31].In our study, signs of hemophagocytic syndrome were recorded in 35.4% of patients and predominantly in patients with a severe course of disease which required ICU admission (48.8%).A similar pattern was described in other studies [30,31].The frequency of cytokine storm syndrome (CSS) in MIS-C and active COVID-19 depended on the scoring systems applied to identify macrophage activation syndrome (MAS) and hemophagocytic lymphohistiocytosis (HLH).Its frequency in patients with MIS-C was as high as 76% in an American study [30].We also used some of these scoring systems.For example, in our study, 5.7% of patients met the HLH-2004 criteria, and 11.5% fulfilled the criteria for MAS (Ravelli, 2016).In the study mentioned above, we can see quite comparable statistics: 2% of patients met the HLH-2004 criteria and 24% the 2016 sJIA/MAS criteria.
A similar pattern was observed in the research of Buda et al.: MIS-C patients fulfilling the MAS criteria were treated more often with IVIG (94.8 vs. 87.6%)and steroids (87.3 vs. 62.7%) than children without MAS, and they also needed ICU admission two times as often.The median hospitalization time was also significantly longer in patients with hemophagocytosis in our study (23 days vs. 14 days, p < 0.0000001) and also according to the data from Buda et al. (20 days vs. 12 days) [31].
The dissimilarities in treatment are related to different national recommendations.Our patients received glucocorticosteroids as the first-line treatment [32]; meanwhile, ACR guidelines [33] and British national consensus [34] recommend commencing treatment with IVIG, as in the treatment of Kawasaki disease.
The hemophagocytic syndrome associated with MIS-C can be attributed to MAS rather than to primary HLH.Attention is drawn to the more pronounced systemic inflammation in patients with MIS-C compared with patients with primary HLH in whom the increase in CRP is usually not as significant [35,36].This is partly explained by the different cytokine profiles.Interferon-gamma is the leading cytokine in primary HLH, whereas IL-1, 6, and 17 lead in MIS-C [9,10,37,38].MIS-C occupies an intermediate position between the secondary, virus-associated HLH to which it formally belongs and MAS in sJIA, which it more closely resembles.Options for the treatment of MAS in sJIA have shown the greatest effectiveness in MIS-C.
The phenomenon of hemophagocytosis/macrophage activation syndrome is a fairly frequent and very serious manifestation of MIS-C, which has been confirmed by several studies [23,30,31].
Unfortunately, at the moment there are no specific criteria for evaluating hemophagocytosis in MIS-C.Many authors use previously developed criteria for other diseases, but it is not entirely known how accurate this is [27][28][29].
It is necessary to develop and validate an individual set of criteria for assessing hemophagocytosis in patients with MIS-C [30,31].Future assessments are being planned to find the discriminative or diagnostic criteria of MAS validated in patients with MIS-C.
However, we can say with confidence that HScore works quite well in the assessment of hemophagocytosis in patients with MIS-C.

Study Limitations
The limitations of our study are related to the retrospective study design, missing data, relatively small sample size, different study time intervals, method of selection of clinical and laboratory parameters and different SARS-CoV-2 variants affecting patients during the pandemic, and the impossibility of having laboratory data from the same time point.We fully understand that these limitations can affect the study results.

Conclusions
Hemophagocytic syndrome was diagnosed in 35.4% of the children studied, and in 48.8% of patients with a severe course.HScore > 91 was associated with myocardial damage, pericarditis, hypotension/shock, and ICU admission.HScore is a simple tool that can also be used to assess the severity of MIS-C and for dynamic monitoring.Using this concept, we can suggest the following management scheme for patients with MIS-C (Figure 1).Nevertheless, it is necessary to develop and validate specific criteria for the assessment of hemophagocytosis in the course of MIS-C.
mophagocytosis in patients with MIS-C [30,31].Future assessments are being planned to find the discriminative or diagnostic criteria of MAS validated in patients with MIS-C.
However, we can say with confidence that HScore works quite well in the assessment of hemophagocytosis in patients with MIS-C.

Study Limitations
The limitations of our study are related to the retrospective study design, missing data, relatively small sample size, different study time intervals, method of selection of clinical and laboratory parameters and different SARS-CoV-2 variants affecting patients during the pandemic, and the impossibility of having laboratory data from the same time point.We fully understand that these limitations can affect the study results.

Conclusions
Hemophagocytic syndrome was diagnosed in 35.4% of the children studied, and in 48.8% of patients with a severe course.HScore > 91 was associated with myocardia damage, pericarditis, hypotension/shock, and ICU admission.HScore is a simple too that can also be used to assess the severity of MIS-C and for dynamic monitoring.Using this concept, we can suggest the following management scheme for patients with MIS-C (Figure 1).Nevertheless, it is necessary to develop and validate specific criteria for the assessment of hemophagocytosis in the course of MIS-C.

Figure 1 .
Figure 1.The algorithm for the management of patients with MIS-C, depending on the presence of signs of hemophagocytosis and severity of the disease.

Table 2 .
Comparison of characteristics of MIS-C patients with HScore > 91 and patients with HScore ≤ 91.