Hemophagocytic Lymphohistiocytosis (HLH) is a rare clinical condition characterized by sustained but ineffective immune system activation, leading to severe and systemic hyperinflammation. It may develop in the context of a familial disorder (FHL) or as a sporadic condition, in association with diverse triggers, such as infections, autoinflammation, acquired immune deficiencies, or malignancies. The identification of familial cases is crucial to allow a definitive cure by hematopoietic stem cell transplantation (HSCT). Clinical manifestations may include persistent fever, hepatosplenomegaly, multi lineage cytopenia, coagulopathy, hyperferritinemia, and central nervous system involvement. These signs and symptoms are the consequence of an exaggerated cytokine release and organ infiltration by activated lymphocytes and macrophages. Patients may rapidly deteriorate and develop multi organ failure and death. Thus, prompt recognition and treatment are essential for patients’ survival.

Historically, FHL has been associated to impaired cytotoxicity due to biallelic mutations in FHL-related genes. Nowadays, the advent of Next Generation Sequencing (NGS) and improvement of functional studies led to re-define HLH as a syndrome with etiopathogenetic heterogeneity, blowing the distinction between familial and sporadic cases. Starting from an historical perspective up to the current understanding of HLH, in this review we critically revise this complex condition and the available diagnostic tools, with the final aim to describe a comprehensive clinical and laboratory approach for the management of patients with suspected HLH.

The first description of HLH dates 1939, when Scott and Robb-Smith reported 4 patients with unremitting fever, lymphadenopathy, and hepatosplenomegaly (1) ( Figure 1 ). A few years later, Farquhar and Claireaux reported two siblings with a similar clinical syndrome. This was the first description of the familial form of the disease (2). In the next decades, similar features were observed both in familial clusters and as sporadic forms. In 1979, Risdall et al. described 19 patients with evidence of HLH and viral infection after transplantation, later defined ‘Virus associated hemophagocytic syndrome’ to denote any case of HLH without a genetic cause. Since other infective agents were shown to trigger HLH, these conditions were redefined as ‘infection associated hemophagocytic syndrome’ (IAHS) (3). The term ‘Macrophage activation syndrome’ (MAS) was firstly used in 1992 by Albert et al. to refer to a condition characterized by cytopenias, organ dysfunction, coagulopathy, and inappropriate activation of macrophages in a proinflammatory milieu (4).

The initial identification of a biological marker refers to the impairment of natural killer (NK) cell cytotoxic activity, which was observed in affected patients, compared with healthy relatives, in the 1980s (5–8). The first gene related to FHL, PRF1, was identified in December 1999 and PRF1-mutated FHL was then named FHL type 2 (FHL-2) (9). FHL-1 had been previously associated with an unknown gene mapping in position 9q21.3-2 (10). At the same time, mutations in SH2D1A affecting the SLAM associated protein (SAP) signaling have been recognized as the cause of a clinical syndrome phenotypically described in 1977 by Purtilo et al. (X-linked lymphoproliferative syndrome type 1, XLP-1) (2). Since then, the study of familial cases allowed the recognition of genetic alterations directly affecting proteins involved in cytotoxic activity, such as Munc13-4 (FHL-3), Syntaxin 11 (FHL-4), Munc18-2 (FHL-5), RAB27A (Griscelli Syndrome type 2, GS2), LYST (Chediak-Higashi Syndrome, CHS) (11–15). Lately, other variants were linked to dysregulated or persistent inflammasome activation involving XIAP, NLRC4 and CDC42 proteins (16). In recent years, NGS analysis led to the identification of an increasing number of genes predisposing to HLH.

Diagnostic guidelines were defined for the first time in 1991 and included fever and splenomegaly, hemophagocytosis in bone marrow, spleen, or lymph nodes, cytopenias, hypertriglyceridemia and/or hypofibrinogenemia (17). At that time, the first report of the international Registry of HLH on 122 patients reported a 5-year survival of only 21% (18). In January 1995, the HLH Study Group of the Histiocyte Society opened its first international treatment study, HLH-94, consisting of corticosteroids and the antineoplastic etoposide, followed by hematopoietic stem cell transplantation (HSCT) for patients with FHL (19). This trial led to a dramatic improvement of outcome up to a 5-year overall survival of 54%. Some years later, the HLH-2004 protocol showed that intensification with cyclosporine A upfront and intrathecal corticosteroids did not significantly improve the outcome, resulting in an OS of 62% (19, 20). With the HLH-2004 protocol the diagnostic criteria were updated with the inclusion of low or absent NK-cell activity, hyperferritinemia and high levels of soluble IL-2 receptor α chain (sIL-2r, also named sCD25) (21). In 2007, an alternative immunotherapeutic approach with antithymocyte globulin (ATG) and methylprednisolone followed by consolidation and HSCT was proposed, based on a single-center experience. It resulted in a higher and faster response rate at 8 weeks compared to HLH-94 at the price of a higher reactivation rate, ultimately not affecting OS (22). A combination between the HLH-94 and the ATG protocol (EURO-HIT-HLH, EudraCT 2011-002052-14) was then investigated, not resulting in significant improvement of outcomes (Jordan M., Sieni E., in preparation). Alternative or salvage treatment approaches have been proposed and are currently under investigation. Among them, monoclonal antibodies anti-CD52 (alemtuzumab), anti-interferon-γ (emapalumab) and anti-Janus Kinases (ruxolitinib) showed preliminary efficacy and are currently under investigation (23–25). Finally, preclinical studies on T cell-mediated gene therapy, by both lentiviral or gamma retroviral vectors, are promising for a definitive cure of FHL-2, FHL-3 and XLP-1 (26–28).

Traditionally, HLH is classified into primary and secondary. Primary HLH, or FHL, is caused by biallelic mutations in genes involved in the secretory lysosome-dependent exocytosis pathway and usually manifests in early infancy. The following FHL subtypes have been described: FHL-2 due to mutations in PRF1, FHL-3 (UNC13D), FHL-4 (STX11), FHL-5 (STXBP2), Griscelli Syndrome type 2 (RAB27A) and Chediack-Higashi syndrome (LYST) (9–15). Secondary HLH, also referred to as sporadic, which is more frequent in older children and adults, may develop in the context of infections, malignancies, rheumatic diseases, chemotherapy, or transplantation (29). In the setting of a rheumatologic condition, secondary HLH is referred to as MAS, a potentially life-threatening complication of systemic inflammatory disorders dominated by an overwhelming inflammation. It occurs mostly in in systemic juvenile idiopathic arthritis (JIA), but it is also described in other autoimmune or autoinflammatory conditions, including Systemic Lupus Erythematosus (SLE), Adult onset Still disease (AOSD), Kawasaki disease and periodic fever syndromes (30).

More recently, an increasing number of adults have been diagnosed with primary HLH (31) and monoallelic mutations of FHL-related genes have been identified in patients with HLH and MAS (32, 33). This blurred the distinction between primary and secondary HLH and prompted the hypothesis of HLH as a continuum where different weights of genetic and environmental factors contribute to the development of HLH. Indeed, biallelic disruptive mutations in FHL-related genes lead to severe cytotoxic defects and early onset FHL, even in the absence of environmental triggers, while hypomorphic or monoallelic mutations in the same genes are associated to a less complete cytotoxic defect and a later onset of the disease, as they require a more powerful trigger to elicit the HLH phenotype (34). In the last decade, the advent of NGS, from target resequencing to whole exome sequencing (WES), exponentially enhanced the ability to identify a growing number of variants, by expanding the spectrum of genetic predisposition to HLH ( Table 1 ). Biallelic RHOG loss-of-function variants were recently reported in a patient with early-onset HLH and defective lymphocyte exocytosis but reports of additional patients are needed to gain insights into penetrance (35). Besides variants in genes required for lymphocyte cytotoxicity, mutations leading to inflammasome activation (i.e. autosomal dominant mutations in NLRC4 or hemizygous mutations in XIAP) and mutations involved in immune response to viral infections (i.e. hemizygous mutations in SAP) have been related to HLH (36, 37). Recently, heterozygous mutations in CD48 have also been associated to HLH (38) as well as biallelic ZNFX1 loss-of-function variants that have been reported in three families with HLH or HLH-like disease (39).

On these bases, HLH can be now considered as a dynamic continuum of inflammatory conditions, where the risk of developing the syndrome is the result of the subtle balance between a predisposed genotype and environmental factors (33, 40).

Trying to divide patients with HLH into primary and secondary forms is likely to trap clinicians in a conceptual labyrinth that could lead to unrecognition or misclassification of blurred cases. Integrating the concept of HLH as a continuum with a deep genetic understanding will improve the management of patients with HLH.

HLH diagnosis is challenging due to clinical features overlapping with other conditions, including sepsis and severe inflammation (41). Although regularly adopted in clinical settings, the HLH-2004 criteria were developed in the context of a pediatric clinical trial. For this reason, despite their undeniable utility, they show some limitations. Firstly, they include non-specific markers of a common inflammatory phenotype, with particular emphasis on features that are likely related to HLH but are also found in other conditions. For example, the cutoff defined for ferritin levels (>500 ng/ml) may indicate a multiplicity of diseases characterized by inflammation, whether a threshold over 2000 ng/mL resulted in 70% sensitivity and 68% specificity in a cohort of 123 patients with HLH (42). Another pitfall is the inclusion of NK cell cytotoxicity assay, which was demonstrated to have poor sensitivity and specificity (60% and 72%, respectively), and it is now overcome by flow cytometry screenings (43). Finally, bone marrow aspirate before treatment initiation is mandatory to rule out hematological malignancies, whereas the absence of hemophagocytosis should not exclude HLH since it is detected in only 40% of patients at disease onset (HLH Italian Registry data 2014-2022, unpublished data). Despite these limitations, occurring when criteria are taken individually, their combinatory evaluation is still useful for a first clinical suspect and to monitor disease activity. In the attempt to simplify disease recognition, some years ago we proposed the combination of fever, splenomegaly, thrombocytopenia and hyperferritinemia as a minimal set of clinical criteria able to address the clinician to HLH suspicion, by observing a specificity of 84% in a cohort of FHL3 patients (44). Though not specific, high ferritin levels strongly support the HLH suspicion, while normal ferritin values point to other diagnosis.

HLH-2004 criteria are mainly useful to identify paediatric FHL, but show poor sensitivity for secondary HLH, MAS or other inflammatory conditions (45). To fill this gap, 2016 classification criteria for MAS complicating sJIA were validated (30). These criteria were developed by expert consensus and real-life data, by premising that clinical symptoms of suspected MAS are scarcely specific and may be deferred over time. On these bases, the only clinical symptom included was fever, leaving room to laboratory tests (ferritin, platelet count, AST, fibrinogen, and triglycerides). The validation of 2016 classification criteria resulted in a discrimination of MAS from other conditions with a sensitivity of 0.73, a specificity of 0.99, a positive predictive value of 97.4% and a negative predictive value of 85.9% (30). These criteria show two major limitations: on the one hand, they are reliable only on patients with sJIA/MAS, on the other, they were developed as classification criteria and cannot be considered diagnostic. To further simplify the distinction of MAS in the setting of sJIA from sJIA flares, a cut point interval for the ratio of ferritin to the erythrocyte sedimentation rate (ESR) was also proposed (46). Additionally, HScore was developed for adult cohorts on the basis of weighted clinical, biochemical, and cytological variables that were comparable to HLH-2004 criteria. The authors indicated a median score of 230 (IQR 203-257) in HLH positive cases and 125 (IQR 91–150) in negative cases (47). This scoring showed a 90% sensitivity and 79% specificity in adults (48). It is suitable to identify secondary HLH in general, but it is not validated in a pediatric cohort and was developed in the setting of patients with primarily malignancy related HLH.

In summary, the scores reported above and charted in Table 2 are a powerful tool for clinicians to rapidly identify patients with HLH allowing a prompt treatment start.

Once HLH is suspected, first line examinations are requested to confirm it ( Figure 2 ). Specialized laboratory tests are indeed supportive for the diagnosis of the syndrome and the monitoring of disease activity, but their integration with clinical data is decisive. Here, we report the available laboratory tests, some of which are increasingly used in clinical practice, while others are available for research only ( Table 3 ).

Despite the correlation between phenotype and gene variants has been reported in HLH (44, 87), the clinical distinction among different HLH subtypes is not easy. For this reason, genetic testing remains the gold standard for the diagnosis of inherited forms (88). From 1999, the year of the first identification of an HLH-related gene with Sanger sequencing, great progress has been made.

FHL is confirmed by the presence of biallelic pathogenic variants in the genes PRF1, UNC13D, STX11, STXBP2, RAB27A and LYST leading to impaired cytotoxicity. Other genes involved in control of infection and dysregulated inflammasome activity have been associated to HLH as a predominant manifestation. Among these, X-linked lymphoprolipherative diseases type 1 and 2 due to hemizygous pathogenic variants in SH2D1A and BIRC4 respectively, are the most known syndromes ( Table 1 ). In addition to them, several inherited errors of immunity or metabolic disorders may rarely cause the HLH phenotype. Thus, extending genetic investigation over the classical FHL-related genes is recommended in the suspicion of inherited conditions (29, 88). Heterozygous pathogenic variants in FHL-related genes may be associated with severe infection and MAS in rheumatologic conditions (89, 90). Namely, heterozygous mutation predisposing to sJIA/MAS were found in PRF1 and UNC13D (91–95). Even if the finding of heterozygous mutations in these patients rarely impacts the clinical management, it is worth proceeding with sequencing in these patients as well at least for research purposes.

Nowadays, several techniques are available for genetic analyses. Sanger sequencing is a targeted sequencing technique that uses dideoxyoligonucleotide to analyze specific DNA regions, and it can be considered a “first generation” sequencing method. This testing strategy counts several decades of intense application, since it is a robust technique to detect point mutations, small deletions, or duplications. High gene specificity is detrimental to the number of genes eligible for the analysis, which may be costly if compared to multiplexing testing systems. The method is fast, since it requires from a few days to a week, and less time-consuming, especially in the context of familial forms where the DNA region to be investigated is already known. Although in the last years NGS has been taking the lead, Sanger sequencing still serves as a complementary method to confirm the doubtful variants emerged and may be used to fill the gap of regions poorly covered by NGS due to GC-rich sequences or technical issues (96).

Despite the aforementioned limitations, NGS technology is a powerful method to investigate a targeted gene panel, optimized on the basis of the laboratory need in a cost-efficient manner. It may require from 2 to 6 weeks, but it can include up to 20 patients per experiment. One of the numerous examples of these platforms is the HLH sequencing panel of the Cincinnati Children’s Hospital Medical Center (CCHMC), which combines 15 HLH-associated genes and was launched in September 2013. Gadoury-Levesque et al. used this approach to perform a definite genetic diagnosis in 1892 patients with suspected HLH. The group obtained a diagnostic rate of 10.4%. This low value can be explained, by one hand, by lax inclusion criteria of patients, and on the other by the exclusion of newly reported HLH-associated genes, such as NLRC4 and CDC42 ( 97). Tesi et al. established a 12 HLH-associated comprehensive high-throughput platform to explain cases in which the demonstration of reduced NK cell function was not followed by a genetic diagnosis. The group reached an overall diagnostic rate of 38%, which was increased to 44% in the pediatric cohort. The mutation detection sensitivity was 97.3%, the average coverage per gene 98.0%, and the coverage of sites previously reported as mutated was over 98.6% (98).

To sum up, the NGS-based approach has high accuracy in detecting point mutations, small deletions, and insertions, which are the most frequent alterations in HLH. Notwithstanding, the method is not reliable to identify large deletions, duplications, or insertions. Considering that sequencing costs and timing are progressively decreasing, the detection of genetic causes of HLH may be more accurately provided by Whole Exome Sequencing (WES), which gives the opportunity to perform more comprehensive genetic analyses and to identify novel genes predisposing to HLH or HLH-mimicking phenotypes. WES is a method designed to perform a target resequencing of all protein-coding nuclear genes in the genome. Since non-coding sequences are generally characterized by variants with a mild or no effect on phenotype, the advantage of WES is to be directed on the small percentage of coding genome (1%) that is more likely to carry pathogenic variants (99). On the other hand, one of WES limitations is that it cannot identify structural and gross copy number variations and intronic variants affecting promoter or enhancer elements, which were reported in UNC13D, BIRC4, and RAB27A (36, 40, 100–102). This emphasizes the need for implementation of WES with specific probes, widely adopted in laboratory routine or other methods such as Whole Genome Sequencing (WGS) and RNA sequencing, which however are poorly available nowadays. Chinn et al. reported a promising use of WES in patients with HLH, in which the potential causing genes associated with HLH were identified in 58% of cases thanks to a precise patient selection by HLH-2004 criteria evaluation and a flow cytometry-based immunological screening (36).

Overall, genetic studies remain crucial for HLH diagnosis and allow familial counseling and prenatal diagnosis. The combination of immunological screenings with novel technologies, such as WES and WGS, will allow us to understand the significance of genetic variants, filling the gap between pathophysiological mechanisms and targeted treatments of HLH.

HLH is a rare syndrome with multiple clinical manifestations. Without prompt recognition, it is invariably fatal. Despite recent improvements in survival, around 40% of patients with HLH still die from refractory disease or toxicity. Thus, a rapid and effective diagnostic approach is crucial for early treatment start and HSCT decision.

In recent years, many novel approaches have been proposed to investigate patients with clinical and laboratory findings consistent with HLH. Their rationale and reliability has been widely analyzed in this review, and some of them might gain a pivotal role in HLH management in the future. However, there are extensively validated investigations that remain now essential in the diagnostic approach. In the view of the clinical and genetic heterogeneity of the syndrome, it may still be challenging for the clinician to make quick handling decisions when facing a patient with suspected HLH. For this reason, we recommend a comprehensive diagnostic approach to HLH that integrates clinical, functional, and genetic criteria, possibly in the setting, or with the advice, of a highly specialized team ( Figure 2 ). When HLH is suspected, diagnostic evaluations should immediately start to confirm the diagnosis. Since the available diagnostic criteria are non-specific for HLH, therapeutic decisions should be carefully discussed with expert physicians and individualized on each patient need. Disease severity, age at onset, underlying diseases and comorbidities need to be considered to avoid overtreatment or undertreatment. Patients with fever, splenomegaly and cytopenia should be investigated for HLH but atypical clinical presentations including newborn with acute liver failure and isolated central nervous system involvement should also be kept in mind for FHL. Hyperferritinemia is a hallmark of uncontrolled inflammation and supports the suspicion of HLH, though it is non-specific. Instead, normal ferritin levels should guide towards alternative diagnoses. Also, sCD25 and CXCL9 are increasingly used as parameters for a first HLH recognition but are still scarcely available. Patients with renal or liver failure, as well as hypofibrinogenemia can rapidly progress toward multi-organ failure indicating poor prognosis. Although not included in the HLH-2004 criteria, hyperbilirubinemia has been recently confirmed as predictor of early death (103). Among first level evaluations, bone marrow aspirate to rule out leukemia is imperative. It is also recommended to search for Leishmania, while looking for hemophagocytosis is not paramount for HLH diagnosis. First level imaging should also be performed, primarily to exclude neoplasia. In the meanwhile, etiological investigations should be initiated starting from screening for infections and rheumatological disease. Once HLH is confirmed, blood samples should be centralized to reference laboratories for functional and genetic evaluation. Indeed, flow cytometric screening of perforin expression and CD107a externalization allows a rapid (24-48h) identification of patients with FHL and guide genetic analysis (43, 51). A defective perforin expression prompt immediate Sanger investigation on PRF1 gene, whereas a defective degranulation assay is suggestive of mutations in UNC13D, STXBP2, STX11, and RAB27A. Other diagnostic tests, including cytokines dosage, provide supporting information (i.e. high IL-18 levels for inflammasome related diseases), but are not always timely accessible for clinical decisions. Under these conditions, cytofluorimetric tests have a high diagnostic value. Cardiac evaluation with echocardiography should be performed as soon as possible both to evaluate the cardiac function and to rule out coronary signs of Kawasaki disease. Additional investigations should address rarer conditions associated to HLH including other immunodeficiencies and LPI (Lysinuric Protein Intolerance), that is the only metabolic disease really mimicking FHL.

Nevertheless, a strong clinical suspicion of genetic HLH, even without functional defects nor mutations of FHL-related genes, should direct toward NGS investigation to identify rarer subtypes of genetic HLH beyond cytotoxicity defects, such as those involving impaired control of infections or dysregulated inflammasome activation ( Table 1 ). Indeed, it has been reported that only 8% of patients with suspected HLH do not carry mutations on FHL-related genes (34). Thus, in selected cases, we favor the use of WES instead of targeted resequencing panels to collect more information and, at the same time, direct the analysis towards a specific set of genes selected on a clinical basis. Furthermore, WES can also allow the identification of genetic predisposition in different conditions, including rheumatological and autoinflammatory diseases.

Thus, besides optimizing costs and timing, WES is also useful for expanding the knowledge of genetic variants associated with HLH. It is worth noting that the massive amount of information deriving from WES output should be interpreted with caution. The variants of uncertain clinical significance should be considered on a case-by-case basis, depending on the clinical phenotype, and should be validated by functional studies confirming the defect in vitro to clarify their biological impact. Despite WES is being increasingly adopted and easily accessible worldwide, we strongly recommend centralization of samples in reference laboratories to allow a reasoned approach starting from clinical evaluation towards functional and genetic analysis. Personnel expertise is indeed crucial to guide the diagnostic algorithm and to correctly interpret the results, with the final aim to support clinical and therapeutic decisions. Indeed, genetic findings do not always impact on patient management, including HSCT. In the last decades, great progress has been made, but novel technologies and therapeutic strategies are developing and will hopefully improve patient’s outcome in the next years.

In conclusion, as the Italian reference center for HLH, we suggest the integration between clinical evaluation, functional and genetic analysis to optimize diagnosis timing and accuracy, by following a reasoned clinical-based approach open towards novel diagnostic tools. To effectively realize this integration, we must keep in mind the knowledge collected so far by looking beyond at the same time, with the final aim to unravel the tangle of HLH spectrum, by allowing an increasingly personalized approach to each patient.

ES designed the study. AC and ES wrote the manuscript. AC, LB, FP, IT, MC, and ES reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.