Owing to its complexity, heterogeneity, severity, and rapid evolution, severe systemic inflammation poses both biologic and clinical challenges. This complexity is reflected by the terms used to describe it, including SIRS, sepsis, HLH, MAS, cytokine storm, etc. Like most complex disorders, it results from the interplay of genetic susceptibility, background diseases, and inciting triggers, and these features all affect presentation and severity. Timely identification, proper diagnosis, and prompt context-specific management are integral to preventing immunopathology and clinical deterioration [8]. Ferritin is consistently cited as a critical screening biomarker for HLH and related disorders [5,6,7], potentially improved by dividing by ESR [18]. To aid in identifying and monitoring such patients, we implemented a novel hyperferritinemia alert system and ultimately screened 931 alerts from 180 patients over a two-year period. Overall, the process was feasible with minimal staff, clinically useful in identifying and tracking patients at risk for systemic immunopathology, and it significantly aided efforts to collect early research specimens. These features suggest feasibility and generalizability to other care settings. While this study used ferritin values over 1000 ng/ml as a tradeoff between sensitivity and specificity, future studies may consider lowering the threshold to 500-700 ng/ml to reflect different MAS/HLH criteria.

Clinical ferritin testing served as the basis for this study, and we did not advertise the study or attempt to influence ferritin ordering practices. Non-inflammatory hyperferritinemia, largely from hemoglobinopathies like sickle cell anemia and post-transplant patients, made up the majority of both total alerts and individual patients. Inflammation may contribute to hyperferritinemia in these patients, including HLH, but we excluded these groups from further analysis given their frequent ferritin testing as part of iron panels and the major contribution of iron overload to their hyperferritinemia. In general, the distribution of diagnoses associated with IHF and its correlation with HLH vary substantially by cut-off value and institutional testing practices [19,20,21]. In our cohort, patients with Stills Disease generated the second most alerts. Ferritin is used routinely for screening and monitoring in Stills, and these diagnoses are far more common than primary HLH. We did not identify ferritin values > 1000 in other rheumatic disease patients during this period, suggesting IHF is far more common in Stills Disease relative to other pediatric rheumatologic diseases, but also likely ferritin ordering practices and local prevalence/severity of other rheumatic diseases (particularly systemic lupus erythematosus).

Sepsis constituted a high proportion of alerts from the main study in Pittsburgh, whereas sepsis was the cause of IHF in only one patient from the CCHMC study. This likely represents local ordering practices, as there is long-standing institutional interest in hyperferritinemic sepsis in Pittsburgh [10, 22], and ferritin is only ordered when requested or as part of a clinical pathway at CCHMC. By contrast, the CCHMC screen identified IHF in a high proportion of MIS-C patients, where ferritin was part of an order set. Though the Pittsburgh and CCHMC screen occurred at different time points, ferritin ordering practice bias remains a large contributor to the patients and subsequently diseases identified in the screens. Ferritin > 1000 was observed in ~ 10% of pediatric sepsis patients at early timepoints in the PHENOtyping sepsis‐induced Multiple organ failure Study (PHENOMS) study [23], and we suspect institutional ferritin ordering practices explain the different rates of IHF in sepsis between institutions. Thus, institutional ferritin ordering practices will influence the patients identified and ultimately any samples collected.

Overall, our findings reinforce the reality that the readily-available components of the HLH/MAS classification criteria [5,6,7] lack specificity for underlying/driving diagnoses. Rheumatologic (Stills) patients had slightly higher mean ferritin values and trends toward less coagulopathy (higher platelet and fibrinogen levels) than the other two subgroups, consistent with prior literature [24, 25], but these findings appeared too non-specific to be of significant clinical utility in distinguishing causes of IHF and suffer from type 2 error given differences in ordering. Patients with “immune dysregulation” had lower maximum CRP levels, possibly reflecting differences in underlying inflammatory processes. As such, we measured total IL-18, IL-18BP, and CXCL9, grouped together because their degree of elevation in these diseases requires a high dilution [11]. Consistent with prior retrospective data [11, 26], and growing clinical experience, significantly elevated total IL-18 and a high IL-18/CXCL9 ratio distinguished hyperferritinemic Stills patients from the other two IHF subgroups [27]. As CXCL9 did not significantly differ between groups, the utility of this ratio is largely driven by IL-18 levels. We observed slightly lower IL-18BP levels in Stills versus “immune dysregulation”. IL-18BP and CXCL9 are both biomarkers of Interferon (IFN, particularly IFN-γ) and, this finding corroborates previous findings of relatively lower CXCL9 and sIL-2Ra in Stills/MAS patients when compared to primary HLH patients [28].

The ferritin alert system also facilitated longitudinal assessment of patients with chronic hyperferritinemia, enabling an anecdotal assessment of the utility of biomarker trends. We found that intermittent assessment of IL-18 over a patient’s clinical course was a useful residual disease activity marker, with elevation showing active or imminent flares and improvement/normalization predicting durable response (or lack thereof) to treatment (Figure S1). Thus, IL-18 may be useful both diagnostically and in assessing minimal residual disease activity in Stills disease.

In this study, IHF detection did not trigger any specific clinical intervention and members of the IHF screen review team did intervene with screened patients (other than to seek informed consent) based on alerts. Preliminary evidence suggests earlier involvement of HLH/cytokine storm specialists may improve patient outcomes [29]. This aligns with our anecdotal clinical experience and recent international consensus guidance efforts [8] in highlighting the likely utility of ferritin in sepsis order sets and institutional HLH/MAS response teams/pathways.

Multi-analyte panels typically use the same sample dilution for all analytes measured, rendering both antibody- and aptamer-based platforms susceptible to hidden hook effects, particularly at analyte levels above what assay developers might encounter in healthy and common disease controls. In a 96-plex “immuno-oncology” assay of 80 curated serum samples, our “positive control” analyte (IL-18) failed to distinguish Stills patients. Given prior experience [11], we suspected and subsequently identified a strong hook effect at high total IL-18 concentrations. We found similar hook effects for very high total IL-18 and CXCL9 levels in separate cohorts containing Stills and HLH patients. Though we did not observe a “hook effect” for CXCL9 or IL-18BP in an aptamer-based assay, the scarcity of very high CXCL9 or IL-18BP levels in that cohort precludes making any generalizations. These observations suggest that “proteomic” multi-analyte panels carry significant Type II error, and investigators should be cautious making any “failure to detect” conclusions from such data without proper controls.

Nevertheless, we identified a few differences of potential importance to our understanding of IHF immunopathology. First, biomarkers of T-cell activation and IFNγ-activity (e.g. GZMB, PDL1, LAG3, CXCL9) were present broadly in IHF, including hyperferritinemic sepsis, and were not unique to one diagnosis (Fig. 5B, PC1). By contrast, Lin et al. showed that IFN-induced chemokines (CXCL9, CXCL10, CXCL11) differentiated HLH from sepsis and SIRS patients, although ferritin may have been equally effective in this all-comers sepsis/SIRS cohort [30]. Biomarkers of T-cell activation appear to correlate with features of HLH (including ferritin) in patients with hyperferritinemic sepsis [31,32,33,34]. In HLH, T-cell activation is the primary cause of immunopathology, but it is unclear the extent to which T-cell activation drives immunopathology in individual hyperferritinemic sepsis patients.

By contrast, we identified an unexpected separation of hyperferritinemic sepsis samples from healthy controls and other IHF groups. This separation was driven by lower abundance of proteins like ANGPT1, EGF, and VEGFR2, and increased IL-15 (PC2, Fig. 5B). Our dataset is unable to determine whether these findings are related to hypotension (or use of vasopressor medications) and not directly to sepsis. Angpt-1 has been previously shown to be decreased in children with septic shock compared to critically ill children with either systemic inflammatory response syndrome (SIRS) or sepsis [35]. The utility of Angiopoietin family members as biomarkers useful in subcategorizing sepsis has been studied without definitive conclusions, but never in comparable hyperinflammatory cohorts [36,37,38,39]. A recent Olink study also found that EGF levels were lower and IL-15 levels higher in children with more severe Multiple Organ Dysfunction (MOD), whether due to sepsis or not [40]. Similar to our data, VEGFR2 was depressed and IL-15 elevated in a small Olink analysis of critically ill adults with sepsis (ferritin status unknown), but ANGPT1 and PDGF-subunit B levels were higher (not lower) [35] (Supplemental Fig. 7). If validated, these analytes could play a role in raising or lowering suspicion for sepsis in patients whose IHF etiology is unclear. We cannot rule out potential hook effects. Overall, the Olink screen suggests that biomarkers of T cell activation may not be clinically useful in distinguishing subtypes of IHF, but proteins like ANGPT1, VEGFR2, and IL-15 could play a role. Future studies should validate these findings and evaluate their clinical utility.

Ultimately, we implemented a hyperferritinemic screening system and found it to be feasible and (both clinically and academically) useful. The data generated from this study confirmed patterns distinct to certain underlying etiologies, especially those associated of IL-18 with Stills. It also highlighted a potentially major short-coming, “hook effects” at high levels of specific analytes in highly multiplexed biomarker discovery platforms. Finally, our data also identified some potential areas of further exploration, such as depression of vascular growth factors in sepsis. Thus, leveraging the sensitivity of hyperferritinemia for HLH and related disorders via an alert system may be a window to improved recognition, management, and differentiation of this nebulous and life-threatening group of disorders.