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Clinical features and diagnosis of hemophagocytic lymphohistiocytosis Authors: Kenneth L McClain, MD, PhD, Olive Eckstein, MD Section Editor: Peter Newburger, MD Deputy Editor: Alan G Rosmarin, MD All topics are updated as new evidence becomes available and our peer review process is complete. Literature review current through: Aug 2019. | This topic last updated: Jul 11, 2019.

INTRODUCTION Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome of excessive immune activation. It most frequently affects infants from birth to 18 months of age, but the disease is also observed in children and adults of all ages. HLH can occur as a familial or sporadic disorder, and it can be triggered by a variety of events that disrupt immune homeostasis. Infection is a common trigger both in those with a genetic predisposition and in sporadic cases. Prompt treatment is critical, but the greatest barrier to a successful outcome is often a delay in diagnosis due to the rarity of this syndrome, variable clinical presentation, and lack of specificity of the clinical and laboratory findings. The clinical features and diagnosis of HLH and a related disorder, the macrophage activation syndrome (MAS), will be discussed here. The management of patients with these disorders is discussed separately. (See "Treatment and prognosis of hemophagocytic lymphohistiocytosis".)

TERMINOLOGY Terms used to describe HLH and related syndromes have evolved since the original patient was described as having "familial hemophagocytic reticulosis" in 1952 [1]. Other terms that were formerly used for HLH include virus-associated hemophagocytic syndrome, hemophagic histiocytosis, familial erythrophagocytic lymphohistiocytosis (FEL), and viral-associated

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hemophagocytic syndrome (VAHS) [2-7]. Use of the term "primary HLH" to denote the presence of an underlying genetic disorder and "secondary HLH" to denote presence of the HLH phenomenon occurring secondary to another condition has caused a great deal of confusion among clinicians. Both primary and secondary HLH can be triggered by infections or other immune activating events, and gene mutations can be found in individuals of any age and with any family history. In practice, a distinction between primary and secondary HLH is not essential for the initial diagnosis and management. However, identification of a gene mutation may be useful for subsequent management. (See 'Evaluation and diagnostic testing' below.) The following terms have been found in the literature: ●

Primary HLH, also called familial hemophagocytic lymphohistiocytosis (FHL), refers to HLH caused by a gene mutation, either at one of the FHL loci or in a gene responsible for one of several immunodeficiency syndromes. FHL loci include [7]:

• FHL1 (OMIM 267700) • FHL2 (OMIM 603553) • FHL3 (OMIM 608898) • FHL4 (OMIM 603552) • FHL5 (OMIM 613101) • GS2 (RAB27A) (OMIM 603868) • HPS2 (OMIM 608233) • XLP1 (OMIM 308240) • XLP2 (OMIM 300635) • BLOC1S6 (OMIM 604310) • CD27 (OMIM186711) • ITK (OMIM186973) • LYST (OMIM606897) • MAGT1 (XMEN) (OMIM300853) • SLC7A7 (OMIM 603593) • XIAP (BIRC4) (OMIM 300079) Whole exome sequencing of HLH patients who do not have any of the above mutations has identified a large number of genes associated with immune deficiencies and HLH [8], as discussed below. (See 'Genetics' below.) ●

Secondary (sporadic, acquired) HLH is generally used to describe patients without a known familial mutation and who typically have a clear trigger for developing acute HLH (eg, viral illness, autoimmune disease, lymphoma). However, this term can create confusion because many patients with "secondary HLH" have an underlying genetic defect

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associated with the syndrome (eg, heterozygous defect, mutation resulting in partial protein expression); conversely, many patients with primary HLH can develop an acute HLH flare in response to one of these triggers. (See 'Triggers' below.) ●

Macrophage activation syndrome – Macrophage activation syndrome (MAS) is a form of HLH that occurs primarily in patients with juvenile idiopathic arthritis or other rheumatologic diseases. Some authors call this "reactive hemophagocytic syndrome." (See 'Rheumatologic disorders/MAS' below.)

PATHOPHYSIOLOGY Immunologic abnormalities — HLH is a syndrome of excessive inflammation and tissue destruction due to abnormal immune activation. The hyperinflammatory/dysregulated immune state is thought to be caused by the absence of normal downregulation by activated macrophages and lymphocytes [9]. The cell types involved in the pathogenesis of HLH include the following: ●

Macrophages – Macrophages are professional antigen presenting cells derived from circulating monocytes; they present foreign antigens to lymphocytes. In HLH, macrophages become activated and secrete excessive amounts of cytokines, ultimately causing severe tissue damage that can lead to organ failure. (See "An overview of the innate immune system", section on 'Monocytes and macrophages'.)

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Natural killer cells and cytotoxic lymphocytes – Natural killer (NK) cells constitute 10 to 15 percent of lymphocytes. NK cells eliminate damaged, stressed, or infected host cells such as macrophages, typically in response to viral infection or malignancy, in an MHCunrestricted manner. (See "An overview of the innate immune system", section on 'Natural killer cells'.) Cytotoxic lymphocytes (CTLs) are activated T lymphocytes that lyse autologous cells such as macrophages bearing foreign antigen in association with class I histocompatibility proteins. Most CTLs express CD8. (See "The adaptive cellular immune response: T cells and cytokines", section on 'CD8+ T cell activation'.) In HLH, NK cells and/or CTLs fail to eliminate activated macrophages. This lack of normal feedback regulation results in excessive macrophage activity and highly elevated levels of interferon gamma and other cytokines. Other lymphocyte abnormalities include altered numbers of CD4 and CD8 lymphocyte subsets [10]. In a series of adult patients, those with increased CD8 numbers and decreased CD4/CD8 ratios had the best survival. Decreased total CD3 numbers portended

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a bad outcome. (See "Treatment and prognosis of hemophagocytic lymphohistiocytosis", section on 'Prognosis'.) Consistent with this mechanism, most patients with HLH have impaired cytotoxic function of NK cells and CTLs, coupled with excessive activation of macrophages [11-16]. Excessive cytokine production by macrophages, NK cells, and CTLs is thought to be a primary mediator of tissue damage [9]. (See 'Immunologic profile' below and 'Cytokine storm' below.) The normal elimination of activated macrophages by NK cells and CTLs occurs through perforin-dependent cytotoxicity. NK cells and CTLs lyse target cells in a series of steps that include formation of an immunologic synapse; creation of a pore in the macrophage membrane; and delivery of cytolytic granules into the macrophage. The granules contain a variety of proteases such as granzyme B that can initiate cell death, often through apoptosis. Most of the genetic defects in patients with familial HLH encode proteins involved in this process. (See "The adaptive cellular immune response: T cells and cytokines" and "NK cell deficiency syndromes: Clinical manifestations and diagnosis", section on 'Mechanisms of killing' and 'Genetics' below.) Toll-like receptor (TLR) activation of the immune system can be another cause of HLH [17]. TLRs are non-antigen-specific receptors on the surface of NK cells that are activated by components of bacteria, fungi, viruses, or mycoplasma. Normal mice with repeated TLR9 stimulation develop an illness similar to macrophage activation syndrome (MAS) [18]. Genes associated with TLR/interleukin 1 receptor (IL-1R) signaling are upregulated in patients with juvenile idiopathic arthritis and MAS [19]. Hemophagocytosis — In addition to antigen presentation and cytokine production, macrophages can also phagocytize host cells. Hemophagocytosis refers to the engulfment (literally "eating") of host blood cells by macrophages. Hemophagocytosis is characterized by the presence of red blood cells, platelets, or white blood cells (or fragments of these cells) within the cytoplasm of macrophages (picture 1 and picture 2). Hemophagocytosis can be observed in biopsies of immune tissues (lymph nodes, spleen, liver) or bone marrow aspirates/biopsies. Although it can be a marker of excessive macrophage activation and supports the diagnosis of HLH, hemophagocytosis alone is neither pathognomonic of, nor required for, the diagnosis of HLH. (See 'Bone marrow evaluation' below and 'Diagnosis' below.) Cytokine storm — The persistent activation of macrophages, NK cells, and CTLs in patients with HLH leads to excessive cytokine production (cytokine storm) by all of these cell types, and is thought to be responsible for multiorgan failure and the high mortality of this syndrome [9,20,21]. Cytokines found at extremely high levels in the plasma of patients with HLH include interferon

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gamma (IFN gamma); tumor necrosis factor alpha (TNF alpha); interleukins (IL) such as IL-6, IL-10, and IL-12; and the soluble IL-2 receptor (CD25) [22-24]. Elevated IL-16 levels may be important for a TH1-type response that recruits macrophages and other cells implicated in HLH [25]. In a study of adults with secondary HLH, markedly elevated levels of IL-18 and its binding protein were found [26]. Some of these cytokines can be measured in serum and are useful in distinguishing HLH from other conditions. A study of IFN gamma, IL-6, and IL-18 in patients with systemic JIA (sJIA) versus HLH showed higher levels of IFN gamma and IFN gammainduced proteins in HLH compared with sJIA, but the ratio of IL-18/IFN gamma was higher in sJIA [27]. (See 'Specialized testing' below.) An extensive study on the role of IL-18 in MAS and other rheumatologic conditions has shed light on the differences in pathophysiology of HLH and MAS [28]. Unbound (free) IL-18 levels >24,000 pg/mL could distinguish MAS from familial HLH with an 83 percent sensitivity and 94 percent specificity. Many patients with MAS had IL-18 levels >100,000 pg/mL, which helped distinguish MAS from other autoinflammatory conditions. A mouse model of MAS revealed that IL-18 was primarily produced by intestinal epithelium, which provides an intriguing biologic model for a syndrome of infantile enterocolitis and MAS caused by NLRC4 inflammasome hyperactivity [29]. Triggers — Patients with HLH can have a single episode of the disease or relapsing episodes, with relapses occurring most often in patients with familial HLH. The instigating trigger for an acute episode is often an infection or an alteration in immune homeostasis. The two broad categories of triggers include those that cause immune activation and those that lead to immune deficiency. Immune activation from an infection is a common trigger both in patients with a genetic predisposition and in sporadic cases with no underlying genetic cause identified. The most common infectious trigger is a viral infection, especially Epstein-Barr virus (EBV) [9]. Primary EBV infection can trigger HLH in individuals with a defect in perforin-dependent cytotoxicity, as well as in those without a known predisposition; patients with X-linked lymphoproliferative disease (XLP) are at particularly high risk [30]. Many other infectious organisms are also implicated. Kawasaki disease, a common vasculitis of childhood, can also trigger HLH and can often be misdiagnosed initially. The immune checkpoint inhibitors, nivolumab and ipilimumab, may be linked to development of HLH, but the incidence has not been defined [31]. (See 'Immunodeficiency syndromes' below and 'Infections' below and "Kawasaki disease: Clinical features and diagnosis".) Excessive cytokine release in patients with chronic granulomatous disease (CGD) may also lead to HLH. In one institution, 3 of 17 patients with CGD developed HLH [32]. Common causes of immunodeficiency triggers include inherited syndromes, malignancy, rheumatologic disorders, or HIV infection. (See 'Genetics' below and 'Malignancy' below and 'Rheumatologic

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disorders/MAS' below and 'Immunodeficiency' below.) The coexistence of immune dysregulation with unchecked inflammation distinguishes HLH from other syndromes of immune activation, immunodeficiencies, and inflammatory states [30].

GENETICS Genetic defects play a major role in childhood HLH and are increasingly found in adult cases [16,33-37]. Genetic information can be helpful in determining the likelihood of recurrence, the need for hematopoietic cell transplant, and the risk of HLH in family members. (See 'Diagnosis' below and "Treatment and prognosis of hemophagocytic lymphohistiocytosis".) Most of the implicated genes encode components of the machinery for perforin-dependent cytotoxicity (figure 1) [38] (see 'Pathophysiology' above). These genes act in an autosomal recessive fashion (ie, inheritance of a mutation at both alleles of a gene is required to manifest the disease) and many cases are related to consanguinity; however, heterozygosity for an HLH mutation is occasionally found in an individual (typically an adult) with HLH associated with another condition [39]. (See 'Associated illnesses' below.) In addition to homozygous mutation in a single HLH gene, individuals with HLH may be compound heterozygotes (ie, they may have a different mutation in each allele of the same gene) or they may show digenic inheritance (ie, they may have separate mutations in two different genes). A review of 2701 patients referred for genetic testing revealed that 225 (8 percent) were homozygous or compound heterozygous for mutations, and 28 (1 percent) showed digenic inheritance [37]. Another study reported similar findings, with monoallelic mutations of known familial HLH genes found in 43 of 281 patients classified as having "sporadic" disease, suggesting that this disorder is not a simple recessive one [40]. In a study that used whole exome sequencing, heterozygous variants in LYST, MUNC13-4, and STXBP2 were discovered in 5 of 14 patients with juvenile idiopathic arthritis (JIA) who had macrophage activation syndrome (MAS), but in only 4 of 29 patients with JIA who did not have MAS [41]. Several other recessive pairs and compound heterozygotes were found. The likelihood of identifying a gene mutation is highest in the youngest patients. In a review of 476 North American children, a gene mutation was identified in 45 percent of those less than one month of age [30]. In those aged between two months to one year, one to two years, and greater than two years, the frequencies of identifying a gene mutation were 39, 20, and 6 percent, respectively. In another study of 175 adults (age range, 18 to 75 years), 14 percent had gene mutations; these tended to cause partial defects in protein function rather than complete loss of the protein; this partial loss of function may explain the later age of HLH onset in some adults [42]. (See 'Features in adults' below.)

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In a study of 101 patients who met the HLH-2004 criteria for diagnosis of HLH, only 19 percent had biallelic mutations in the six primary genes associated with HLH [8]. Heterozygous variants in patients with potentially two HLH-associated gene mutations were not statistically different from the general population, suggesting these "digenic" cases were not disease causing. Of 47 patients with none of the expected HLH-associated gene mutations, 28 (58 percent) had potential disease-causing genetic defects. These defects were in genes associated with primary immunodeficiency disease or dysregulated immune activation or proliferation associated genes such as NLRC4 and NLRP12 as well as biallelic variants in NLRP4, NLRC3, and NLRP13. Mutations at FHL loci — Several HLH gene mutations map to loci that code for elements of the cytotoxic granule formation and release pathway, and have been labeled familial hemophagocytic lymphohistiocytosis (FHL) loci. (See 'Terminology' above.) ●

PRF1/Perforin – FHL2 results from mutations of PRF1, which encodes perforin. Perforin is delivered in cytolytic granules and forms pores in the membrane of target cells. Mutations in other genes that affect perforin expression have also been reported [34,43-45].

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UNC13D/Munc13-4 – FHL3 results from mutations of UNC13D, which encodes Munc13-4 [35,46]. Proteins of the Unc (uncoordinated) family regulate cytolytic granule maturation.

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STX11/Syntaxin 11 – FHL4 results from mutations of STX11, which encodes syntaxin 11. Syntaxins control granule exocytosis. Several syntaxin mutations were reported in a group of Kurdish families with HLH [36,47].

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STXBP2/Munc18-2 – FHL5 results from mutations of STXBP2, which encodes Munc18-2 (also called syntaxin binding protein 2) [39,48]. This protein binds to syntaxin 11 and promotes the release of cytotoxic granules.

The gene defect responsible for FHL1 remains uncharacterized. Immunodeficiency syndromes — Several mutations that cause congenital immunodeficiency syndromes are also associated with an increased incidence of HLH. These include the following: ●

Griscelli syndrome (GS) – GS type 2 is caused by mutations of RAB27A, which encodes a GTP-binding protein [49]. GS2 is characterized by hypopigmentation, immune deficiency, thrombocytopenia, and/or neurologic defects. (See "Syndromic immunodeficiencies", section on 'Griscelli syndrome'.)

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Chediak-Higashi syndrome (CHS) – CHS is caused by mutations of CHS1/LYST, which encodes a lysosomal trafficking regulatory protein [50]. CHS is characterized by partial oculocutaneous albinism, neutrophil defects, neutropenia, and neurologic abnormalities.

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(See "Chediak-Higashi syndrome".) ●

X-linked lymphoproliferative disease – X-linked lymphoproliferative disease type 1 (XLP1) is caused by mutations in SH2 domain protein 1A (SH2D1A), also called signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which encodes an activator of NK and T cells [51]. XLP2 is caused by mutations in X-linked inhibitor of apoptosis (XIAP), also called baculoviral IAP-repeat-containing protein 4 (BIRC4); the encoded protein protects cells from apoptosis [52]. XLP (also called Duncan disease) is characterized by an abnormal response to Epstein-Barr virus (EBV) infection. (See "Xlinked lymphoproliferative disease", section on 'Genetics'.)

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XMEN disease – X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease is another immunodeficiency syndrome with EBV-associated malignancies and rarely HLH [53]. A loss-of-function mutation in a gene encoding magnesium transporter 1 (MAGT1) leads to CD4 lymphopenia, chronic, high-level EBV infection, normal levels of NK-T cells, and dysregulated immune responses to EBV. (See "Malignancy in primary immunodeficiency", section on 'XMEN disease'.)

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Interleukin-2-inducible T cell kinase (ITK) deficiency – Patients with ITK deficiency, like those with XLP and XMEN deficiencies, are unable to control EBV infections. They have a variety of lymphoproliferative diseases, lymphomatoid granulomatosis, HLH, and dysgammaglobulinemia.

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CD27 (TNFRSF7) deficiency – Missense mutations that reduce expression of CD27 have been associated with a syndrome of severe EBV infections associated with HLH, Hodgkin lymphoma, uveitis, and recurrent infections [54].

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Hermanski-Pudlak syndrome (HPS) – HPS is a rare disorder characterized by oculocutaneous albinism and platelet storage pool deficiency. Several responsible gene mutations have been identified: HPS1, AP3B1 (HPS2), HPS3, HPS4, HPS5, HPS6, DTNBP1 (HPS7), BLOC1S3 (HPS8), and BLOC1S6 (PLDN). Patients with HPS type 2 have a lower risk of developing HLH than those with type 1 because of a milder defect in cytotoxicity [55]. (See "Congenital and acquired disorders of platelet function", section on 'Storage pool disorders'.)

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Lysinuric protein intolerance – Lysinuric protein intolerance (LPI; MIM 222700) is a recessive aminoaciduria caused by defective cationic amino acid transport in epithelial cells of the intestine and kidney. SLC7A7 (also called y+LAT1), the gene mutated in LPI, encodes the light subunit of a cationic amino acid transporter. Patients with LPI frequently display severe complications such as pulmonary disease, hematologic abnormalities, and disorders of the immune response [56].

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Chronic granulomatous disease (CGD) – CGD is a genetically heterogeneous condition associated with recurrent, life-threatening bacterial and fungal infections. (See "Chronic granulomatous disease: Pathogenesis, clinical manifestations, and diagnosis".)

Genotype-phenotype correlations — Patients with HLH gene mutations tend to present at a younger age than those without mutations. The affected gene and specific type and site of mutation may affect the age of presentation and clinical features, but there is controversy regarding the contribution of hypomorphic mutations to development of HLH [8,42]. Informative studies that evaluated genotype-phenotype correlations with HLH include: ●

Patients with PRF1 null mutations typically present within the first year of life, whereas those with missense mutations and variable degrees of perforin expression have a more variable age of presentation, even into adulthood [5,57-63].

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In a series of 76 patients with HLH, those with PRF1 mutations had a significantly higher risk of early disease onset (ie, 10,000 ng/mL was 90 percent sensitive and 96 percent specific for HLH, with very minimal overlap with sepsis, infections, and liver failure [82]. When the control group was re-analyzed with a comparison cohort of 120 patients with HLH, a ferritin level ≥2000 mcg/L had a 70 percent sensitivity and 68 percent specificity for diagnosing HLH [83]. There was no difference when primary and secondary HLH cases were analyzed separately. In adults and neonates, other potential causes of extremely high ferritin levels should also be evaluated. As an example, ferritin levels over 10,000 ng/mL can be seen in neonatal hemochromatosis or fulminant liver failure; however, the presence of cytopenias and fevers, as well as elevated soluble IL-2 receptor alpha (sIL-2R) and sCD163 in patients with HLH may help to exclude these other possible diagnoses [84]. (See 'Other diagnostic considerations' below and 'Differential diagnosis' below.) While a very high ferritin level is helpful in suggesting the possibility of HLH, a low ferritin (eg, ferritin 500 mcg/L over a two-year period [82]. In this cohort, a ferritin level >500 mcg/L was 100 percent sensitive for HLH, but less specific. A ferritin level >10,000 mcg/L in children was 90 percent sensitive and 96 percent specific for HLH, with very minimal overlap with sepsis, infections, and liver failure. (See 'Serum ferritin levels' above.)

• In adults, we rely less heavily on an isolated serum ferritin elevation, because serum ferritin is less specific for HLH in adults. (See 'Features in adults' above.) A scoring system has been developed to generate a diagnostic score referred to as an "Hscore" that estimates the probability of HLH [174]; this incorporates points for immunosuppression; fever; organomegaly; levels of triglycerides, ferritin, alanine aminotransferase, and fibrinogen; degree of cytopenias; and presence of hemophagocytosis on the bone marrow aspirate. An Hscore ≥250 confers a 99 percent probability of HLH, whereas a score of ≤90 confers a