Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis

Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK-cell activity in 63 healthy Korean individuals using a flow-cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate-labeled K562 cells as target cells. NK-cell activity was calculated using the following equation: NK-cell activity (%) = (test lysis − spontaneous lysis) × 100/(maximum lysis − spontaneous lysis). NK-cell activity was analyzed in 13 known HLH patients and 16 suspected non-HLH patients using a flow-cytometric assay. The mean (±SD) cytotoxicity of PBMCs from healthy individuals was 20.9 ± 5.3% and the reference interval was 11.8–31.9%. The mean NK-cell activity of HLH patients (8.3 ± 8.9%) was significantly lower (P = 0.001) than that of non-HLH patients (20.1 ± 7.8%). The sequential changes in NK-cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK-cell activity test for use in the clinical laboratory and obtained a reference interval of NK-cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.     

Co-Occurrence of Familial Hemophagocytic Lymphohistiocytosis Type 2 and Chronic Active Epstein-Barr Virus in Adulthood

We report, to the best of our best knowledge, the oldest individual to ever be diagnosed with Familial Hemophagocytic Lymphohistiocytosis (FHL) Type 2 from homozygous c.1349C>T (p.T450M) missense variants in the PRF1 gene. This rare case advanced in complexity with a simultaneous diagnosis of Chronic Active Epstein-Barr Virus (CAEBV) – a distinct clinical entity from acute EBV infections and a well-described trigger of Hemophagocytic Lymphohistiocytosis (HLH). This is, to the best of our knowledge, the only individual to ever be diagnosed with CAEBV in the setting of this specific variant and the oldest to be diagnosed with a coexisting perforin variant. This case provides understanding of EBV, human genetics, and lymphoproliferative disorders while adding a unique differential diagnosis to adults who present with fever of unknown origin and diffuse lymphadenopathy without evidence of malignancy. This report explores the diagnosis and treatment of both HLH and CAEBV, encouraging discussion regarding current clinical management and future research needs.     

Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis

OBJECTIVES: Uncontrolled macrophage activation with hemophagocytosis is a distinctive feature of hemophagocytic syndromes (HPS). We examined whether lympho-histiocytic infiltration of the bone marrow and liver, as well as hemo-/erythrophagocytosis also occurs during sepsis and whether this process could account for the increased production of anti-inflammatory heme-oxygenase (HO-1) products observed during sepsis. METHODS: Hemophagocytosis and expression of CD163, HO-1, ferritin as well as CD8 and granzyme-B were examined in post-mortem bone marrow samples from 28 patients with sepsis and from eight control patients. RESULTS: Comparison of samples from non-septic patients with samples from patients with fatal sepsis revealed that the latter group displayed dense lympho-histiocytic bone marrow infiltration with CD163(+)/HO-1(+)/ferritin(+) macrophages as well as with CD8(+) and granzyme-B(+) T-cells. Hemophagocytosis with prominent phagocytosis of erythroid cells was readily apparent in septic patients, implying that this process is a likely stimulus for the up-regulation of macrophage HO-1 expression. CONCLUSIONS: Lympho-histiocytic activation with hemophagocytosis is a shared pathophysiologic mechanism in HPS and sepsis. Furthermore, the association of hemophagocytosis with an increase in HO-1 expression may indicate a novel role for this apparently futile process as a negative regulator of inflammation.     

A Case Report of Successful Treatment With Plasma Exchange for Hemophagocytic Syndrome Associated With Severe Systemic Juvenile Idiopathic Arthritis in an Infant Girl

Abstract: An infantile case of hemophagocytic syndrome (HPS) with systemic juvenile idiopathic arthritis (s-JIA), refractory to methylprednisolone pulse therapy and cyclosporine A administration, was successfully treated by plasma exchange. The patient was a one-year-old Japanese girl who had developed recurrent steroid-dependent signs, including fever, skin eruption, and hepatopathy, while in France, where she had been diagnosed as having s-JIA at eight months of age. As a high fever and rheumatoid rash were evident on arrival at our hospital, she was admitted and given intravenous methylprednisolone pulse therapy and cyclosporine A. She developed pancytopenia with a generalized clonic seizure, high fever, and liver dysfunction after her cytomegalovirus (CMV) titer became positive during the course of treatment; therefore, she was treated with ganciclovir. She was subsequently diagnosed as having HPS complicating s-JIA from the findings of a bone marrow aspirate. At this time, her blood examination data including a high level of C-reactive protein and hyperferritinemia, suggested that her s-JIA was very active, and the pancytopenia continued after her CMV titer became negative. Therefore, CMV infection against a background of active s-JIA could have triggered the HPS in this case. Because the HPS was resistant to an immunosuppressive regime of methylprednisolone pulse therapy and cyclosporine A, plasma exchange therapy was started. After three sessions of this therapy, the patient's symptoms and laboratory data were markedly improved. Our experience suggests that plasma exchange should be considered as a therapeutic tool for HPS refractory to conventional therapy.     

Familial hemophagocytic lymphohistiocytosis associated with disseminated T-cell lymphoma: A report of two siblings

Summary Two siblings with evidence of disseminated T-cell lymphoma at the time of diagnosis of familial hemophagocytic lymphohistiocytosis (FHL) are reported, an association which has not been described previously. The first child with typical clinical and laboratory features of FHL died shortly after admission, before diagnosis could be established. Retrospective analysis of autoptic tissue revealed marked hemophagocytosis as well as morphological and immunohistochemical features suggestive of disseminated T-cell lymphoma. In the second child, FHL was diagnosed in time. Subsequent histologic investigation of bone marrow biopsies displayed a focal infiltration by T-cell lymphoma. DNA hybridization studies provided evidence of a monoclonal T-cell receptor beta chain gene rearrangement. Following conventional chemotherapeutic induction for FHL, the patient received an allogeneic bone marrow transplant (BMT) from a related healthy donor. Currently, 17 months after BMT, the boy is in unmaintained remission from FHL and T-cell lymphoma. The current pathogenetic concepts for FHL and a possible relationship between T-cell lymphoma and FHL are discussed.     

A Recurrent Case of Adult-onset Still's Disease with Concurrent Acalculous Cholecystitis and Macrophage Activation Syndrome/Hemophagocytic Lymphohistiocytosis Successfully Treated with Combination Immunosuppressive Therapy

We herein report the case of 21-year-old female diagnosed with adult-onset Still''s disease (AOSD) three years earlier who presented with fever and right upper abdominal pain. She was diagnosed with acute acalculous cholecystitis (AAC) based on hepatic dysfunction, elevated C-reactive protein, and gallbladder wall thickening on abdominal ultrasound. Based on the presence of pancytopenia, hyperferritinemia, and hemophagocytosis by a bone marrow examination, she was diagnosed with macrophage activation syndrome (MAS)/hemophagocytic lymphohistiocytosis (HLH) which was refractory to glucocorticoid pulse therapy. The combination of intravenous cyclosporine A with glucocorticoids was able to successfully control the disease activity of AOSD-related AAC and MAS/HLH.     

Hemophagocytic lymphohistiocytosis in COVID-19: Case reports of a stepwise approach

Rationale:The immunologic syndrome induced by severe acute coronavirus disease 2019 (COVID-19) is yet not fully understood. Typical patterns of clinical and laboratory features match secondary hemophagocytic lymphohistiocytosis (sHLH). However, the optimal approach to COVID-19 patients testing positive for sHLH is still unclear.Patient concerns:Three patients with COVID-19 are reviewed. All showed hyperinflammation and cytokine storm, necessitating intensive care treatment including mechanical ventilation.Diagnosis:Secondary hemophagocytic lymphohistiocytosis due to severe COVID-19; diagnosed via HScore.Interventions:A treatment regimen of methylprednisolone, pentaglobin, and anakinra was developed and administered.Outcomes:One patient survived the ICU stay. Two other patients, in whom sHLH was diagnosed too late, deceased.Lessons:A routine screening of COVID-19 patients for secondary HLH by using the HScore is feasible; especially those patients deteriorating clinically with no sufficient response to shock management might be at particular high risk. A stepwise therapeutic approach comprising corticosteroids, immunoglobulins and anakinra, accompanied by immunoadsorption, may dampen cytokine storm effects, and potentially reduce mortality.     

A Novel Germline Mutation in Exon 10 of the SMAD4 Gene in a Familial Juvenile Polyposis

Familial juvenile polyposis (FJP) is a rare autosomal dominant hereditary disorder that is characterized by the development of multiple distinct juvenile polyps in the gastrointestinal tract and an increased risk of cancer. Recently, germline mutations, including mutations in the SMAD4, BMPR1A, PTEN and, possibly, ENG genes, have been found in patients with juvenile polyps. We herein report a family with juvenile polyposis syndrome (JPS) with a novel germline mutation in the SMAD4 gene. A 21-year-old man presented with rectal bleeding and was found to have multiple polyps in his stomach, small bowel, and colon. His mother had a history of gastrectomy for multiple gastric polyps with anemia and a history of colectomy for colon cancer. A review of the histology of the polyps revealed juvenile polyps in both patients. Subsequently, mutation screening in DNA samples from the patients revealed a germline mutation in the SMAD4 gene. The pair had a novel mutation in exon 10 (stop codon at tyrosine 413). To our knowledge, this mutation has not been previously described. Careful family history collection and genetic screening in JPS patients are needed to identify FJP, and regular surveillance is recommended.     

Fatal Thrombotic Microangiopathy and Posterior Reversible Encephalopathy Syndrome in a Patient with Anti-melanoma Differentiation-associated Gene 5 Antibody-positive Dermatomyositis

A 56-year-old woman presented with dermatomyositis positive for anti-melanoma differentiation-associated gene 5 antibody. No interstitial lung disease was detected. Despite treatment with methylprednisolone pulse therapy and cyclosporine, dysphagia developed. Furthermore, the presence of thrombocytopenia, elevated lactate dehydrogenase levels, and an undetectable haptoglobin level suggested the possibility of thrombotic microangiopathy (TMA). Disturbed consciousness developed shortly after TMA onset, and brain magnetic resonance imaging revealed hyperintensity lesions in the bilateral basal ganglia, thalami, and brainstem. The patient was diagnosed with atypical posterior leukoencephalopathy syndrome before dying of heart failure later that day. In conclusion, early TMA recognition and prompt intensive treatment are critical in such cases.     

异基因造血干细胞移植治疗原发性噬血细胞综合征合并中枢神经系统病变一例报告及文献复习

目的 探讨原发性噬血细胞综合征（HLH）合并中枢神经系统病变诊断要点以及异基因造血干细胞移植（Allo-HSCT）治疗情况。 方法：对1例根据HLH-2004诊断标准确诊的原发性HLH合并中枢神经系统病变的病例临床特点进行分析,完善基因测序、免疫学指标检测和家系调查,进行Allo-HSCT。 结果：确诊11岁男性病例1例,表现为反复发热、全血细胞减少,脾大、骨髓中可见噬血现象,NK细胞活性下降（10.39%）。基因检测和家系调查显示患者携带分别来自父系和母系的PRF1基因的复杂杂合改变,两位胞姐各自携带不同突变位点;全家成员穿孔素蛋白表达量均有不同程度下降。病程中出现癫痫,头颅核磁共振提 示多发病变。确诊原发性HLH合并中枢神经系统病变。给予HLH-2004方案治疗后,接受胞姐HLA 5/10相合Allo-HSCT。目前移植后14个月,一般情况良好。 结论 对于合并中枢神经系统病变的原发性HLH,尽早进行Allo-HSCT是获得长期生存及治愈的唯一方法。     

A Novel Mutation W252X in the WAS Gene in a Korean Patient with Wiskott-Aldrich Syndrome

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder characterized by recurrent infection, eczema, and microthrombocytopenia. WAS is inherited in an X-linked recessive pattern, and various mutations in the WAS gene on the X chromosome are the genetic basis of WAS. A 7-month-old Korean boy presented with recurrent bloody diarrhea, eczema, and persistent thrombocytopenia with small platelets. Direct sequence analysis of the entire coding region of the WAS gene showed a novel nonsense mutation with a G-to-A substitution at the nucleotide position 756 on exon 8, leading to a premature termination at codon 252 (c.756G>A; p.W252X). Family study revealed that neither of the parents had the mutation, indicating the de novo occurrence of the mutation.     

A Case of Familial Male-limited Precocious Puberty with a Novel Mutation

Familial male-limited precocious puberty (FMPP), also known as testotoxicosis, is a rare cause of precocious puberty in males. It is caused by a mutation in the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) gene, resulting in the receptor being constitutively activated. This causes excessive production of testosterone, leading to precocious puberty in males. Generally, boys present with signs of puberty, such as pubic hair growth, acne, and increased height velocity around the age of 2-4 years old. Like any other cause of precocious puberty, the goal of treatment is to prevent virilization and also delay closure of the epiphyseal plates to maintain adult height potential. Treatment, therefore, is aimed at decreasing the effects of testosterone, as well as stopping the conversion of testosterone to estrogen. Little is known about the long-term effects of treatment because the disorder is so rare. However, studies using bicalutamide and anastrozole have been promising. In this report, we present a boy with FMPP with a novel mutation in the LHCGR gene, who has been responding well to therapy using both drugs.     

Familial Mediterranean Fever and E148Q Pyrin Gene Mutation in Greece

Familial Mediterranean fever (FMF) is an inherited disease characterized by recurrent inflammatory polyserositis. Although FMF is classically expected only in Middle East populations, it is becoming evident that the disease affects more groups than initially thought. The disease is associated with a number of mutations of the MEFV gene, which codes for a protein named pyrin. The role of E148Q pyrin gene mutation in the development of FMF remains inconclusive. Some authors believe it causes the disease, whereas others favor the concept of a noncausative role. To understand better the role of this mutation, gathering data from different populations may be of value. We studied 60 Greek cases fulfilling the criteria for FMF diagnosis, 30 cases being a definite FMF diagnosis and 30 a probable diagnosis. Twenty-one of the patients, carried mutation E148Q. One was a homozygote (E148Q/E148Q), and 20 carried mutation E148Q in combination with other mutations (compound heterozygotes). In 6 of the 60 cases studied, no mutations were found. Compared with the results for healthy controls, E148Q mutation is significantly frequent. Because different populations may exhibit different patterns of pyrin mutations, association of the E148Q mutation with FMF should be considered in connection with origin data.     

A Novel XPA Gene Mutation and its Functional Analysis in a Japanese Patient with Xeroderma Pigmentosum Group A

Most Japanese patients with xeroderma pigmentosum group A (XPA) have the homozygous intron 3 splicing mutations ( Alw NI mutation). Here, we report a Japanese XPA patient, XP79KO, a compound heterozygote with a newly identified T to G transversion at splice donor site in intron 1 in one allele, and with the Alw NI mutation in another allele in the XPA gene. The mutation in intron 1 creates two new abnormal splice sites that resulted in two types of aberrant mRNA. These abnormal splicings cause frameshifts that make stop codons downstream. No XPA protein was detected in XP79KO fibroblasts.     

Successful use of subcutaneous anakinra in hemophagocytic lymphohistiocytosis precipitated by candidiasis in a patient with systemic lupus erythematosus: A case report and description of a novel therapeutic regimen

Hemophagocytic lymphohistiocytosis (HLH) is a rare and often fatal condition characterized by inappropriate immune system activation leading to a “cytokine storm”, and ultimately resulting in end-organ damage. Causes include primary defects in genes involved in immune-mediated cytolytic pathways, or secondary triggers such as infection or malignancy. We describe a case of HLH precipitated by fungal infection which occurred as a consequence of immunosuppression for management of systemic lupus erythematosus (SLE) and necrotizing myopathy. The patient presented with immune-mediated disease of the muscles and lung which was treated with high-dose corticosteroids and aggressive immunosuppression. HLH emerged in the context of confirmed candidiasis and features of severe sepsis. The patient responded rapidly to antifungal therapy and high-dose anakinra, which was administered subcutaneously and progressively weaned over 4 weeks. She completed HLH treatment as an outpatient and remains well at 12 months with controlled SLE and no recurrence of HLH.     

心脏型肌钙蛋白Ⅰ基因4693 C/T突变导致家族性心尖部肥厚型心肌病

目的 研究中国人肥厚型心肌病(HCM)致病基因,分析基因型与临床表型的关系.方法 在一HCM家系中进行心脏型肌钙蛋白Ⅰ基因(TNNI3)、心脏型肌钙蛋白T基因(TNNT2)、心脏型肌球蛋白结合蛋白C基因(MYBPC3)和β-肌球蛋白重链基因(MYH7)突变筛查,利用聚合酶链反应(PCR)扩增其功能区的外显子片段,双脱氧末段终止法测序.家系调查资料包括临床表现、体格检查、心脏超声和心电图.结果 在该家系接受家系调查的8例有亲缘关系的对象中5例携带TNNI3 4693C/T(R145W)突变,全部发病,外显率100%.正常对照组同一位置未见异常,该突变位点使TNNI3基因第7号外显子143位的精氨酸变为色氨酸,5例患者中4例表现为心尖部肥厚为主,1例表现为室间隔基底段肥厚为主,临床症状表现为轻微的胸闷.MYH7、MYBPC3及TNNT2基因未发现突变.结论 TNNI3基因4693C/T突变是该HCM家系的致病突变,其外显率为100%.其携带者主要表现为心尖部肥厚为主,临床表型较好.对于心尖部肥厚为主的HCM家系有必要进行TNNI3的突变筛查.     

A Novel PRPS1 Mutation in a Japanese Patient with CMTX5

The PRPS1 gene encodes phosphoribosyl pyrophosphate synthetase 1 (PRS-1). The phenotypes associated with PRPS1 mutations include DFN2 (mild PRS-1 deficiency), X-linked Charcot-Marie-Tooth disease type 5 (CMTX5) (moderate PRS-1 deficiency), Arts syndrome (severe PRS-1 deficiency), and PRS-1 superactivity1. CMTX5 is a very rare hereditary neuropathy characterized by deafness, optic atrophy, and polyneuropathy. We herein report a Japanese patient with CMTX5 who had a novel hemizygous mutation c.82 G>C in PRPS1. Despite showing a typical clinical picture, the decrease in enzyme activity measured in the patient''s erythrocytes was milder than in previously reported cases.     

Successful father-to-son stem cell transplantation in a child with hemophagocytic lymphohistiocytosis using a reduced-intensity conditioning regimen

OBJECTIVE: Hemophagocytic lymphohistiocytosis (HLH) is an uncommon disorder, usually lethal without allogeneic stem cell transplantation (SCT). MATERIALS AND METHODS: We report a 9-month-old boy, the first child of consanguineous parents, diagnosed with HLH and neurological involvement demonstrated by magnetic resonance imaging (MRI), who received an allogeneic SCT from his HLA genetically matched father. Transplant was performed after a reduced-intensity conditioning (RIC) regimen consisting of cyclophosphamide, fludarabine, and melphalan. Graft vs. host disease (GVHD) prophylaxis included cyclosporine a and methotrexate. RESULTS: An absolute neutrophil count of 0.5 x 10(9)/L was documented on day +20 and a platelet count >20 x 10(9)/L was shown by day 33. Full donor chimerism was showed on day +175. A follow-up brain MRI was reported normal. Twenty months after SCT, the child shows no evidence of HLH or GVHD activity, and has a normal psychomotor development. CONCLUSION: Given the reduced toxicity of SCT with RIC, it could represent an attractive transplant method for children with HLH, in whom myeloablation plays no role in disease eradication, and in whom mixed chimerism may be enough to cure the disease.