Value of hemophagocytosis in the diagnosis of hemophagocytic lymphohistiocytosis in critically ill patients.

BACKGROUND: Ferritin is an established biomarker in the diagnosis of secondary hemophagocytic lymphohistiocytosis (HLH), which is diagnosed by the HLH-2004 criteria. Among these criteria, detection of hemophagocytosis through invasive procedures may delay early life saving treatment. Our aim was to investigate the value of hemophagocytosis in diagnosing HLH in critically ill patients. METHODS: In this secondary analysis of a retrospective observational study, we included all patients aged ≥18 years and admitted to any adult ICU at Charité-Universitätsmedizin Berlin between January 2006 and August 2018, who had hyperferritinemia (≥500 µg/L) and underwent bone marrow biopsy during their ICU course. RESULTS: Two hundred fifty-two patients were included, of whom 31 (12.3%) showed hemophagocytosis. In multivariable logistic regression analysis, maximum ferritin was independently associated with hemophagocytosis. By removing hemophagocytosis from HLH-2004 criteria and HScore, prediction accuracy for HLH diagnosis was only marginally decreased compared to the original scores. CONCLUSIONS: Our results strengthen the diagnostic value of ferritin and underline the importance of considering HLH diagnosis in patients with high ferritin but only four fulfilled HLH-2004 criteria, when hemophagocytosis was not assessed or not detectable. Proof of hemophagocytosis is not required for a reliable HLH diagnosis.

Consensus-Based Guidelines for the Recognition, Diagnosis, and Management of Hemophagocytic Lymphohistiocytosis in Critically Ill Children and Adults.

OBJECTIVE: Hemophagocytic lymphohistiocytosis is a hyperinflammatory syndrome that often requires critical care support and remains difficult to diagnose. These guidelines are meant to aid in the early recognition, diagnosis, supportive care, and treatment of patients with hemophagocytic lymphohistiocytosis in ICUs. DATA SOURCES: The literature searches were performed with PubMed (MEDLINE). STUDY SELECTION: Keywords and medical subject headings terms for literature search included "macrophage activation syndrome," hemophagocytic lymphohistiocytosis," and "hemophagocytic syndrome." DATA EXTRACTION: The Histiocyte Society developed these consensus recommendations on the basis of published reports and expert opinions with level of evidence provided for each recommendation. They were endorsed by the Society of Critical Care Medicine. DATA SYNTHESIS: Testing for hemophagocytic lymphohistiocytosis should be initiated promptly in all patients admitted to ICUs with an unexplained or disproportionate inflammatory response, especially those with rapid clinical deterioration. Meeting five or more of eight hemophagocytic lymphohistiocytosis 2004 diagnostic criteria serves as a valuable diagnostic tool for hemophagocytic lymphohistiocytosis. Early aggressive critical care interventions are often required to manage the multisystem organ failure associated with hemophagocytic lymphohistiocytosis. Thorough investigation of the underlying triggers of hemophagocytic lymphohistiocytosis, including infections, malignancies, and autoimmune/autoinflammatory diseases, is essential. Early steroid treatment is indicated for patients with familial hemophagocytic lymphohistiocytosis and is often valuable in patients with acquired hemophagocytic lymphohistiocytosis (i.e., secondary hemophagocytic lymphohistiocytosis) without previous therapy, including macrophage activation syndrome (hemophagocytic lymphohistiocytosis secondary to autoimmune/autoinflammatory disease) without persistent or relapsing disease. Steroid treatment should not be delayed, particularly if organ dysfunction is present. In patients with macrophage activation syndrome, whose disease does not sufficiently respond, interleukin-1 inhibition and/or cyclosporine A is recommended. In familial hemophagocytic lymphohistiocytosis and severe, persistent, or relapsing secondary macrophage activation syndrome, the addition of prompt individualized, age-adjusted etoposide treatment is recommended. CONCLUSIONS: Further studies are needed to determine optimal treatment for patients with hemophagocytic lymphohistiocytosis in ICUs, including the use of novel and adjunct therapies.

Recommendations for the management of hemophagocytic lymphohistiocytosis in adults.

Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome induced by aberrantly activated macrophages and cytotoxic T cells. The primary (genetic) form, caused by mutations affecting lymphocyte cytotoxicity and immune regulation, is most common in children, whereas the secondary (acquired) form is most frequent in adults. Secondary HLH is commonly triggered by infections or malignancies but may also be induced by autoinflammatory/autoimmune disorders, in which case it is called macrophage activation syndrome (MAS; or MAS-HLH). Most information on the diagnosis and treatment of HLH comes from the pediatric literature. Although helpful in some adult cases, this raises several challenges. For example, the HLH-2004 diagnostic criteria developed for children are commonly applied but are not validated for adults. Another challenge in HLH diagnosis is that patients may present with a phenotype indistinguishable from sepsis or multiple organ dysfunction syndrome. Treatment algorithms targeting hyperinflammation are frequently based on pediatric protocols, such as HLH-94 and HLH-2004, which may result in overtreatment and unnecessary toxicity in adults. Therefore, dose reductions, individualized tailoring of treatment duration, and an age-dependent modified diagnostic approach are to be considered. Here, we present expert opinions derived from an interdisciplinary working group on adult HLH, sponsored by the Histiocyte Society, to facilitate knowledge transfer between physicians caring for pediatric and adult patients with HLH, with the aim to improve the outcome for adult patients affected by HLH.

Clinical features, diagnosis and therapy of familial haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is an inherited immune deficiency with defective cytotoxicity of natural killer cells and cytotoxic T lymphocytes. A highly stimulated, but ineffective immune response leads to severe hyperinflammation. Clinical and laboratory features are characteristic, but unspecific; thus awareness of FHL is important for early diagnosis. FHL is rapidly fatal without treatment. Standard-of-care therapy is etoposide and corticosteroids, followed by haematopoietic stem cell transplantation (HSCT). CONCLUSION: FHL has become a curable disease with present treatment. Additional cytokine-directed therapy still has to prove its value. Earlier HSCT and less toxic conditioning regimens will lead to improved cure rates.

Treatment and Mortality of Hemophagocytic Lymphohistiocytosis in Adult Critically Ill Patients: A Systematic Review With Pooled Analysis.

OBJECTIVES: Hemophagocytic lymphohistiocytosis is a cytokine release syndrome caused by uncontrolled immune activation resulting in multiple organ failure and death. In this systematic review, we aimed to analyze triggers, various treatment modalities, and mortality in critically ill adult hemophagocytic lymphohistiocytosis patients. DATA SOURCES: MEDLINE database (PubMed) at October 20, 2019. STUDY SELECTION: Studies and case series of patients greater than or equal to 18 years old, of whom at least one had to be diagnosed with hemophagocytic lymphohistiocytosis and admitted to an ICU. DATA EXTRACTION: Source data of studies and case series were summarized and analyzed on an individual basis. Multivariable logistic regression analysis was performed adjusting for age, sex, and trigger groups. Each single treatment agent was entered as a dichotomous variable to determine treatments associated with survival, regardless if given alone or in combination. DATA SYNTHESIS: In total, 661 patients from 65 studies and case series were included. Overall mortality was 57.8%. Infections were the most frequent trigger (49.9%), followed by malignancies (28.0%), autoimmune diseases (12.1%), unknown triggers (9.4%), and drugs (0.6%). Treatment with IV immunoglobulins was associated with improved survival (odds ratio, 0.548; 95% CI, 0.337-0.891; p = 0.015), while treatment with cyclosporine was associated with increased risk of death (odds ratio, 7.571; 95% CI, 3.702-15.483; p < 0.001). Considering different trigger groups separately, same results occurred only for infection-triggered hemophagocytic lymphohistiocytosis. No information was available on disease severity and other confounding factors. CONCLUSIONS: Mortality of hemophagocytic lymphohistiocytosis in the ICU is high. Most common triggers were infections. Results of survival analyses may be biased by treatment indication and disease severity. Future studies prospectively investigating treatment tailored to critically ill hemophagocytic lymphohistiocytosis patients are highly warranted.

Hyperferritinemia in Critically Ill Patients.

OBJECTIVE: Hyperferritinemia is frequently seen in critically ill patients. A rather rare though life-threatening condition related to severely elevated ferritin is hemophagocytic lymphohistiocytosis. We analyze ferritin levels to differentiate hemophagocytic lymphohistiocytosis from other causes of hyperferritinemia in a mixed cohort of critically ill patients. DESIGN: Retrospective observational study. SETTING: Adult surgical, anesthesiologic, and medical ICUs of a university hospital. PATIENTS: Critical care patients (≥ 18 yr old) admitted to any of the adult ICUs at Charité - Universitätsmedizin Berlin between January 2006 and August 2018 with at least one ferritin value and hyperferritinemia (≥ 500 µg/L). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients were categorized into hemophagocytic lymphohistiocytosis, sepsis, septic shock, and other diagnoses. These were further categorized into 17 subgroups. Hemophagocytic lymphohistiocytosis diagnosis was based on Hemophagocytic Lymphohistiocytosis-2004 criteria and the HScore. Of 2,623 patients with hyperferritinemia, 40 were considered to have hemophagocytic lymphohistiocytosis (1.52%). Maximum ferritin levels were highest in hemophagocytic lymphohistiocytosis patients compared with all other disease groups (each p < 0.001). Sepsis and septic shock patients had higher maximum ferritin levels than patients with other diagnoses (each p < 0.001). A maximum ferritin value of 9,083 µg/L was at 92.5% sensitivity and 91.9% specificity for hemophagocytic lymphohistiocytosis (area under the curve, 0.963; 95% CI, 0.949-0.978). Of all subgroups with other diagnoses, maximum ferritin levels were highest in patients with varicella-zoster virus, hepatitis, or malaria (median, 1,935, 1,928, and 1,587 µg/L, respectively). Maximum ferritin levels were associated with increased in-hospital mortality (odds ratio, 1.518 per log µg/L [95% CI, 1.384-1.665 per log µg/L]; p < 0.001). CONCLUSIONS: This is the largest study of patients with ferritin available in a mixed ICU cohort. Ferritin levels in patients with hemophagocytic lymphohistiocytosis, sepsis, septic shock, and other conditions were distinctly different, with the highest ferritin levels observed in hemophagocytic lymphohistiocytosis patients. Maximum ferritin of 9,083 µg/L showed high sensitivity and specificity and, therefore, may contribute to improved diagnosis of hemophagocytic lymphohistiocytosis in ICU. The inclusion of ferritin into the sepsis laboratory panel is warranted.

Risk factors for mixed chimerism in children with hemophagocytic lymphohistiocytosis after reduced toxicity conditioning.

BACKGROUND: Reduced toxicity conditioning for hematopoietic stem cell transplantation of patients with hemophagocyticlymphohistiocytosis (HLH) results in favorable survival, however at the expense of relevant rates of mixed chimerism. Factors predisposing to mixed chimerism remain to be determined. PROCEDURE: Patients with primary HLH transplanted 2009-2016 after treosulfan- or melphalan-based conditioning regimens were analyzed in a retrospective multicenter study for survival, engraftment, chimerism, and adverse events. Mixed chimerism was considered substantial if < 25% donor chimerism occurred and/or if secondary cell therapy was administered. Donor type, graft source, type of alkylating agent, type of serotherapy, and remission status were analyzed as potential risk factors in a multivariable logistic regression model. RESULTS: Among 60 patients, engraftment was achieved in 95%, and the five-year estimated overall survival rate was 75%. Prevalence of any recipient chimerism was 48%. Substantial recipient chimerism was recorded in 32% of patients. Secondary post-HSCT cell therapy was administered in 30% of patients. A human leukocyte antigen (HLA)-mismatched donor (< 10/10) was the only significant risk factor for the occurrence of substantial recipient chimerism (P = 0.01; odds ratio, 5.8; CI 95%, 1.5-26.3). CONCLUSION: The use of an HLA-matched donor is the most important factor to avoid substantial recipient chimerism following treosulfan -or melphalan-based conditioning in primary HLH.

Hemophagocytic lymphohistiocytosis in critically ill patients: diagnostic reliability of HLH-2004 criteria and HScore.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare though often fatal hyperinflammatory syndrome mimicking sepsis in the critically ill. Diagnosis relies on the HLH-2004 criteria and HScore, both of which have been developed in pediatric or adult non-critically ill patients, respectively. Therefore, we aimed to determine the sensitivity and specificity of HLH-2004 criteria and HScore in a cohort of adult critically ill patients. METHODS: In this further analysis of a retrospective observational study, patients ≥ 18 years admitted to at least one adult ICU at Charité - Universitätsmedizin Berlin between January 2006 and August 2018 with hyperferritinemia of ≥ 500 µg/L were included. Patients' charts were reviewed for clinically diagnosed or suspected HLH. Receiver operating characteristics (ROC) analysis was performed to determine prediction accuracy. RESULTS: In total, 2623 patients with hyperferritinemia were included, of whom 40 patients had HLH. We found the best prediction accuracy of HLH diagnosis for a cutoff of 4 fulfilled HLH-2004 criteria (95.0% sensitivity and 93.6% specificity) and HScore cutoff of 168 (100% sensitivity and 94.1% specificity). By adjusting HLH-2004 criteria cutoffs of both hyperferritinemia to 3000 µg/L and fever to 38.2 °C, sensitivity and specificity increased to 97.5% and 96.1%, respectively. Both a higher number of fulfilled HLH-2004 criteria [OR 1.513 (95% CI 1.372-1.667); p <  0.001] and a higher HScore [OR 1.011 (95% CI 1.009-1.013); p <  0.001] were significantly associated with in-hospital mortality. CONCLUSIONS: An HScore cutoff of 168 revealed a sensitivity of 100% and a specificity of 94.1%, thereby providing slightly superior diagnostic accuracy compared to HLH-2004 criteria. Both HLH-2004 criteria and HScore proved to be of good diagnostic accuracy and consequently might be used for HLH diagnosis in critically ill patients. CLINICAL TRIAL REGISTRATION: The study was registered with www.ClinicalTrials.gov (NCT02854943) on August 1, 2016.

Confirmed efficacy of etoposide and dexamethasone in HLH treatment: long-term results of the cooperative HLH-2004 study.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome comprising familial/genetic HLH (FHL) and secondary HLH. In the HLH-94 study, with an estimated 5-year probability of survival (pSu) of 54% (95% confidence interval, 48%-60%), systemic therapy included etoposide, dexamethasone, and, from week 9, cyclosporine A (CSA). Hematopoietic stem cell transplantation (HSCT) was indicated in patients with familial/genetic, relapsing, or severe/persistent disease. In HLH-2004, CSA was instead administered upfront, aiming to reduce pre-HSCT mortality and morbidity. From 2004 to 2011, 369 children aged <18 years fulfilled HLH-2004 inclusion criteria (5 of 8 diagnostic criteria, affected siblings, and/or molecular diagnosis in FHL-causative genes). At median follow-up of 5.2 years, 230 of 369 patients (62%) were alive (5-year pSu, 61%; 56%-67%). Five-year pSu in children with (n = 168) and without (n = 201) family history/genetically verified FHL was 59% (52%-67%) and 64% (57%-71%), respectively (familial occurrence [n = 47], 58% [45%-75%]). Comparing with historical data (HLH-94), using HLH-94 inclusion criteria, pre-HSCT mortality was nonsignificantly reduced from 27% to 19% (P = .064 adjusted for age and sex). Time from start of therapy to HSCT was shorter compared with HLH-94 (P =020 adjusted for age and sex) and reported neurological alterations at HSCT were 22% in HLH-94 and 17% in HLH-2004 (using HLH-94 inclusion criteria). Five-year pSu post-HSCT overall was 66% (verified FHL, 70% [63%-78%]). Additional analyses provided specific suggestions on potential pre-HSCT treatment improvements. HLH-2004 confirms that a majority of patients may be rescued by the etoposide/dexamethasone combination but intensification with CSA upfront, adding corticosteroids to intrathecal therapy, and reduced time to HSCT did not improve outcome significantly.

Primary and secondary hemophagocytic lymphohistiocytosis have different patterns of T-cell activation, differentiation and repertoire.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening inflammatory syndrome characterized by hyperactivation of lymphocytes and histiocytes. T cells play a key role in HLH pathogenesis, but their differentiation pattern is not well characterized in patients with active HLH. We compared T-cell activation patterns between patients with familial HLH (1°HLH), 2°HLH without apparent infectious trigger (2°HLH) and 2°HLH induced by a viral infection (2°V-HLH). Polyclonal CD8+ T cells are highly activated in 1°HLH and 2°V-HLH, but less in 2°HLH as assessed by HLA-DR expression and marker combination with CD45RA, CCR7, CD127, PD-1 and CD57. Absence of increased HLA-DR expression on T cells excluded active 1° HLH with high sensitivity and specificity. A high proportion of polyclonal CD127- CD4+ T cells expressing HLA-DR, CD57, and perforin is a signature of infants with 1°HLH, much less prominent in virus-associated 2°HLH. The similar pattern and extent of CD8+ T-cell activation compared to 2° V-HLH is compatible with a viral trigger of 1°HLH. However, in most 1°HLH patients no triggering infection was documented and the unique activation of cytotoxic CD4+ T cells indicates that the overall T-cell response in 1°HLH is different. This may reflect different pathways of pathogenesis of these two HLH variants.

The minimum required level of donor chimerism in hereditary hemophagocytic lymphohistiocytosis.

Reduced-intensity conditioning has improved survival after hematopoietic stem cell transplantation (HSCT) for hemophagocytic lymphohistiocytosis (HLH) at the cost of more frequent mixed chimerism. The minimum level of donor chimerism (DC) required to prevent HLH reactivation in humans remains to be determined. In a multicenter retrospective study, 103 patients transplanted for hereditary HLH (2000-2013) and DC permanently or transiently <75% (overall, CD3(+), CD56(+)) were analyzed regarding DC, specific immunologic function, occurrence of systemic reactivations (≥5/8 HLH criteria), partial systemic flares (<5 criteria and HLH-directed treatment), isolated central nervous system reactivations, and management. Recurrence was reported in 18 patients (systemic reactivation n = 11, partial flare n = 3, isolated central nervous system reactivation n = 4). Ten events occurred during profound immune suppression before day 180 (median DC, 10%; range, 1-100%; CD3(+) if available, otherwise overall DC), which renders a differentiation between secondary post-HSCT HLH and HLH related to the genetic defect difficult. Eight events occurred between 0.5 and 6.7 years post-HSCT (median DC, 13%; range, 0-30%). In 5 patients, overall and lineage-specific DC were ≤10% for >6 months (median, 5.1; range, 1.1-10 years) without reactivation. A second HSCT was performed in 18 patients (median, DC 4%; range, 0-19%). Death from reactivation occurred in 4 patients (22% of recurrences). Six patients died of transplant complications following a second HSCT (33% of second HSCT). We conclude that a DC >20%-30% is protective against late reactivation. Lower levels do not, however, inescapably result in recurrences. The decision for or against second HSCT must be based on a thorough risk assessment.

Is an infectious trigger always required for primary hemophagocytic lymphohistiocytosis? Lessons from in utero and neonatal disease.

In this report, we evaluate the hypothesis that hemophagocytic lymphohistiocytosis in patients with defects of lymphocyte cytotoxicity is usually triggered by infections. We show that in the majority of patients, extensive virus PCR panels performed in addition to routine microbiological investigations remain negative and summarize 25 patients with onset of hemophagocytic lymphohistiocytosis in utero or within the first 10 days of life, in none of which an associated bacterial or viral infection was reported. These observations, even though preliminary, invite to consider a key role of lymphocyte cytotoxicity in controlling T-cell homeostasis also in the absence of apparent infectious stimuli.

Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis.

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.

Lack of bone lesions at diagnosis is associated with inferior outcome in multisystem langerhans cell histiocytosis of childhood.

Skeletal involvement is generally, but not universally, characteristic of Langerhans cell histiocytosis (LCH). We investigated whether the presence of bone lesions at diagnosis is a prognostic factor for survival in LCH. Nine hundred and thirty-eight children with multisystem (MS) LCH, both high (386 RO+) and low (RO-) risk, were evaluated for bone lesions at diagnosis. Risk organ (RO+) involvement was defined as: haematopoietic system (haemoglobin <100 g/l, and/or white blood cell count <4·0 × 10(9) /l and/or platelet count <100 × 10(9) /l), spleen (>2 cm below the costal margin), liver (>3 cm and/or hypoproteinaemia, hypoalbuminaemia, hyperbilirubinaemia, and/or increased aspartate transaminase/alanine transaminase). Given the general view that prognosis in LCH worsens with increasing extent of disease, the surprising finding was that in MS+RO+ LCH the probability of survival with bone involvement 74 ± 3% (n = 230, 56 events) was reduced to 62 ± 4% (n = 156, 55 events) if this was absent (P = 0·007). An even greater difference was seen in the subgroup of patients with both liver and either haematopoiesis or spleen involvement: 61 ± 5% survival (n = 105; 52 events) if patients had bony lesions, versus 47 ± 5% (n = 111; 39 events) if they did not (P = 0·014). This difference was retained in multivariate analysis (P = 0·048). Although as yet unexplained, we conclude that bone involvement at diagnosis is a previously unrecognized favourable prognostic factor in MS+RO+ LCH.

Malignancy-associated haemophagocytic lymphohistiocytosis in children and adolescents.

Haemophagocytic lymphohistiocytosis (HLH) in the context of malignancy is mainly considered a challenge of adult haematology. While this association is also observed in children, little is known regarding inciting factors, appropriate treatment and prognosis. We retrospectively analysed 29 paediatric and adolescent patients for presenting features, type of neoplasm or preceding chemotherapy, treatment and outcome. Haemophagocytic lymphohistiocytosis was considered triggered by the malignancy (M-HLH) in 21 patients, most of whom had T- (n = 12) or B-cell neoplasms (n = 7), with Epstein-Barr virus as a co-trigger in five patients. In eight patients, HLH occurred during chemotherapy (Ch-HLH) for malignancy, mainly acute leukaemias (n = 7); an infectious trigger was found in seven. In M- and Ch-HLH, median overall survival was 1·2 and 0·9 years, and the 6 month survival rates were 67% and 63%, respectively. Seven of 11 deceased M-HLH patients exhibited active malignancy and HLH at the time of death, while only two out of five deceased Ch-HLH patients had evidence of active HLH. To overcome HLH, malignancy- and HLH-directed treatments were administered in the M-HLH cohort; however, it was not possible to determine superiority of one approach over the other. For Ch-HLH, treatment ranged from postponement of chemotherapy to the use of etoposide-containing regimens.

Hemophagocytic lymphohistiocytosis in syntaxin-11-deficient mice: T-cell exhaustion limits fatal disease.

Syntaxin-11 (Stx11), an atypical member of the SNARE protein family, is part of the cytolytic machinery of T and NK cells and involved in the fusion of lytic granules with the plasmamembrane. Functional loss of syntaxin-11 in humans causes defective degranulation and impaired cytolytic activity of T and NK cells. Furthermore, patients with STX11 deficiency develop familial hemophagocytic lymphohistiocytosis type 4 (FHL4), a life-threatening disease of severe hyperinflammation. We established Stx11-deficient mice as an animal model for FHL4. Stx11-deficient mice exhibited severely reduced degranulation and cytolytic activity of CTL and NK cells and developed all clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) after infection with lymphocytic choriomeningitis virus (LCMV). The HLH phenotype was further characterized by hyperactive CD8 T cells and continuous IFN-γ production. However, in contrast to perforin-deficient mice, which represent a model for FHL2, progression of HLH was not fatal. Survival of Stx11-deficient mice was determined by exhaustion of antigen-specific T cells, characterized by expression of inhibitory receptors, sequential loss of effector functions, and finally T-cell deletion. Blockade of inhibitory receptors on T cells in Stx11-deficient mice converted nonfatal disease course into fatal HLH, identifying T-cell exhaustion as an important factor for determination of disease severity in HLH.

The risk of hemophagocytic lymphohistiocytosis in Hermansky-Pudlak syndrome type 2.

Genetic disorders of lymphocyte cytotoxicity predispose patients to hemophagocytic lymphohistiocytosis (HLH). Reduced lymphocyte cytotoxicity has been demonstrated in Hermansky-Pudlak syndrome type 2 (HPS2), but only a single patient was reported who developed HLH. Because that patient also carried a potentially contributing heterozygous RAB27A mutation, the risk for HLH in HPS2 remains unclear. We analyzed susceptibility to HLH in the pearl mouse model of HPS2. After infection with lymphocytic choriomeningitis virus, pearl mice developed all key features of HLH, linked to impaired virus control caused by a moderate defect in CTL cytotoxicity in vivo. However, in contrast to perforin-deficient mice, the disease was transient, and all mice fully recovered and controlled the infection. An additional heterozygous Rab27a mutation did not aggravate the cytotoxicity defect or disease parameters. In the largest survey of 22 HPS2 patients covering 234 patient years, we identified only 1 additional patient with HLH and 2 with incomplete transient HLH-like episodes, although cytotoxicity or degranulation was impaired in all 16 patients tested. HPS2 confers a risk for HLH that is lower than in Griscelli or Chediak-Higashi syndrome, probably because of a milder defect in cytotoxicity. Preemptive hematopoietic stem cell transplantation does not appear justified in HPS2.

Hemophagocytic lymphohistiocytosis in imported pediatric visceral leishmaniasis in a nonendemic area.

OBJECTIVES: To describe characteristics of visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (HLH) with focus on diagnostic clues and pitfalls, including the frequency of central nervous system (CNS) involvement, and to determine the efficacy of liposomal amphotericin B (L-AmB). STUDY DESIGN: We retrospectively analyzed clinical and laboratory features, diagnostic procedures, and treatment of 13 patients with HLH with imported visceral leishmaniasis, reported to the German HLH reference center (1999-2012). RESULTS: The spectrum of presentations was indistinguishable from patients with hereditary HLH or with acquired HLH because of infections with other pathogens. In 8 patients, disease onset occurred before the age of 2 years, coinciding with the typical age of manifestation of primary HLH. Two patients had mild nonspecific CNS findings. Misleading antiviral IgM (n = 6) and autoantibodies (n = 2) led to inaccurate interpretation of the etiology of HLH, sometimes with inappropriate therapeutic consequences. False negative results for Leishmania were obtained by initial bone marrow microscopy in 6/13, serology in 1/12, bone marrow culture in 2/5, and polymerase chain reaction of peripheral blood in 1/3 patients, and all bone marrow samples tested were Leishmania-positive by polymerase chain reaction (n = 7). L-AmB was administered to 12 patients, 5 of whom had no prior HLH-directed immunosuppressive therapy; sodium stibogluconate was administered to 1 patient. Persistent remission was achieved in 11 cases. Two patients required repeated or prolonged L-AmB therapy. CONCLUSIONS: Awareness of diagnostic pitfalls may save patients from unnecessary toxic treatment. CNS involvement is rare. L-AmB shows efficacy in visceral leishmaniasis-associated HLH.

A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes.

Familial hemophagocytic lymphohistiocytosis (FHL) is a life-threatening disorder of immune regulation caused by defects in lymphocyte cytotoxicity. Rapid differentiation of primary, genetic forms from secondary forms of hemophagocytic lymphohistiocytosis (HLH) is crucial for treatment decisions. We prospectively evaluated the performance of degranulation assays based on surface up-regulation of CD107a on natural killer (NK) cells and cytotoxic T lymphocytes in a cohort of 494 patients referred for evaluation for suspected HLH. Seventy-five of 77 patients (97%) with FHL3-5 and 11 of 13 patients (85%) with Griscelli syndrome type 2 or Chediak-Higashi syndrome had abnormal resting NK-cell degranulation. In contrast, NK-cell degranulation was normal in 14 of 16 patients (88%) with X-linked lymphoproliferative disease and in 8 of 14 patients (57%) with FHL2, who were identified by diminished intracellular SLAM-associated protein (SAP), X-linked inhibitor of apoptosis protein (XIAP), and perforin expression, respectively. Among 66 patients with a clinical diagnosis of secondary HLH, 13 of 59 (22%) had abnormal resting NK-cell degranulation, whereas 0 of 43 had abnormal degranulation using IL-2-activated NK cells. Active disease or immunosuppressive therapy did not impair the assay performance. Overall, resting NK-cell degranulation below 5% provided a 96% sensitivity for a genetic degranulation disorder and a specificity of 88%. Therefore, degranulation assays allow a rapid and reliable classification of patients, benefiting treatment decisions.