Clinical And Etiological Studies Of Hemophagocytic Syndrome In Children

PART1Clinical study of90children with hemophagocyticsyndromeObjectiveBy analyzing and summarizing the clinical features, the characteristics of laboratory findings,treatment, curative effect and outcome of children with hemophagocytic lymphohistiocytosis,our aim is to provide clinical data for revising HLH diagnosis and treatment protocol.ContentsThrough retrospective analysis of clinical data of children patients who were hospitalized inour hospital and fulfilled the "HLH-2004" diagnostic criteria from December2008to April2014, we summarized the clinical features, the characteristics of laboratory findings,treatment, curative effect and outcome of these patients, while at the same time, we observedthe clinical characteristics and their relationship with the causes of the disease, diseasecondition and prognosis.Methods1．Clinical observations Including the followings:①general information: age, gender;②family history;③symptoms: if there is a fever, fever peaks and duration, if thereaccompanied by the presence of other clinical manifestations;④physical examination: thesize of liver and spleen, the size of superficial lymph node, skin mucosa situation (if there isjaundice or skin rash);⑤laboratory tests: blood routine, coagulation function, liver function,serum ferritin, triglyceride, bone marrow smear, absolute count and percentage of NK cells. 2．Treatment protocol We took Histiocyte Society hemophagocytic syndrome2004protocol(hereinafter referred to as "HLH-2004") for reference and used chemotherapy ofcyclosporine A (CSA), dexamethasone (DEX) in combination with etoposide (VP-16). It isdivided into two phases: the initial treatment and the maintenance treatment, total of24weeks. For children patients with multiple organ dysfunction syndrome (MODS) in critically ill orextremely critical ill, we conducted plasma exchange and gave chemotherapy thereafter.3．Assessment methods According to the efficacy evaluation criteria of "HLH-2004"protocol, it is divided into four conditions: clinical response(CR), non-active disease(NAD),active disease(AD) and reactivation of disease(RD).4．Statistical analysis We used SPSS17.0statistical software for statistical analysis. Whenthe sample is larger than50, we used the Kolmogorov-Smirnov test for normality analysis, pvalues>0.1indicates a normal distribution. We used Levene’s test for analysis of variance, pvalues>0.1indicates homogeneity of variance. We selected t test when normal distributionand homogeneity of variance, while we selected Mann-Whitney U test when not normallydistributed and heterogeneity of variance.Results1．Among the90children patients with HLH,50cases (55.56%) were boys, while40cases(44.44%) were girls. The diagnosed age ranged from0.22to13.10years, while the medianage was1.89years.78.89%of the cases concentrated in the1~5age group.2．90children patients when they were definitely diagnosed with HLH, the main clinicalmanifestations were fever (100.00%), hepatomegaly (98.89%), splenomegaly (84.44%).Theremaining clinical manifestations were superficial lymph node enlargement (62.22%),respiratory symptoms (41.11%), jaundice (35.56%), skin rash (23.33%), gastrointestinalsymptoms (13.33%) and central nervous system symptoms (5.56%).3．90children patients when they were definitely diagnosed with HLH, the main laboratoryresults were serum ferritin≥500μg/L(100.00%), decreasing of two or more cell lineage in theperipheral blood (96.67%),finding of phagocytosis in the bone marrow (87.78%),fastingtriglyceride≥3.0mmol/L (76.67%), fibrinogen≤1.5g/L(54.44%).In addition, decreasing inabsolute count and percentage of NK cells were83.08%(54/65), only decreasing in absolutecount of NK cells were7.69%(5/65) and only decreasing in percentage of NK cells were 7.69%(5/65).4．The treatment of90children patients with HLH were as follows:48cases (53.33%)received chemotherapy. Among which, one patient only used dexamethasone because ofeconomic reasons, two cases used chemotherapy with dexamethasone and cyclosporin Abecause of family reasons,45cases used "HLH-2004" regimen.24cases (26.67%) whosecondition required for plasma exchange.6cases (6.67%) only gave anti-infection, andsymptomatic and supportive treatment.12cases (13.33%) gave up treatment due to economicreasons.5．Outcome Among the90children patients in our group, there were56cases at NAD state,2cases at AD state,5cases once at RD state,13cases died,12cases gave up treatment and19cases lost to follow-up. The overall NAD rate of90patients was62.22%. Usingchemotherapy immediately after definitely diagnosed with HLH, the NAD rate was93.33%.Using chemotherapy after plasma exchange, the NAD rate was66.67%. Not usingchemotherapy after plasma exchange, the NAD rate was22.22%.It is worth to notice that2cases only used chemotherapy with dexamethasone and cyclosporin A for8weeks, at thattime point they were assess as in NAD state. Currently, the2cases were in continuous NADstate for20week and29weeks, respectively.Conclusions1．In our group of90children patients with HLH, the top three clinical manifestations arefever, hepatosplenomegaly, superficial lymph node enlargement and respiratory symptoms,while the top three laboratory examinations are increasing of serum ferritin, decreasing of twoor more cell lineage of peripheral blood and finding of hemphagosis in the bone marrow.2．When making a diagnosis of HLH, the detection of cytotoxic activity of NK cells cannot bereplaced by the detection of absolute count and percentage of NK cells.3．Total treatment duration of24weeks’ chemotherapy can make NAD rate reaching93.33%,it is worth for further study.4．Using chemotherapy after plasma exchange, the NAD rate is66.67%, thus reminder us thatplasma exchange has encouraging therapeutic value for severe HLH. PART2Etiological study of90children with hemophagocyticsyndromeObjectiveThrough systematically, comprehensively summarize and analyze the etiology of90childrenpatients with HLH, combined with the clinical features, therapeutic response and prognosis ofthese patients, we preliminarily explore the clinical significance of genetic testing ofpHLH-related gene and summarize the distribution characteristics of the pathogen in childrenpatients with HLH. Our final goal is to provide some guidance for clinician about estimatingthe therapeutic response and judging the prognosis.ContentsIncluding analyzing the sequencing results of PRF1、UNC13D、STX11、SH2D1A、XIAP、STXBP2、LYST、RAB27A、AP3B1gene; analyzing etiological distribution characteristics;analyzing autoimmune disease-related indicators; analyzing malignancy-related indicators;analyzing metabolic disease-related indicators.Methods1．Detection of pHLH-related genes PCR amplification of PRF1、UNC13D、STX11、STXBP2、SH2D1A、XIAP、LYST、RAB27A、AP3B1gene. Sequencing the PCR products andanalyzing whether there exists mutations, deletions or insertions.2．Pathogen detection We use enzyme-linked immunosorbent assay (ELISA) to detect EBVvirus antibodies (EBVCA-IgM, EBVCA-IgG, EBVEA-IgG, EBVNA-IgG), and IgMantibodies of rubella virus, cytomegalovirus, herpes simplex virus, adenovirus, respiratorysyncytial virus, influenza A virus, influenza B virus and parainfluenza virus, legionellapneumophila type1and toxoplasma. We use quantitative PCR to detect plasma EBV-DNAload. All patients were routinely tested for TB IgG antibodies and PPD test. We use blood,bone marrow, sputum and other samples for bacterial culture and drug susceptibility test. Weuse indirect immunofluorescence to detect IgM antibodies of mycoplasma pneumoniae, Qfever rickettsia and chlamydia pneumoniae.3．Malignancy-related tests including (chest, abdomen, bone and/or skull) imaging to exclude lesions; tumor markers β2-microglobulin in blood and urine, carcinoembryonicantigen, alpha-fetoprotein, cancer antigen-125and cancer antigen-199; bone marrow cellmorphology was observed to eliminate infiltration with malignant cells.4．Autoimmune diseases-related tests We use ELISA to detect the following items:immunoglobulins IgG, IgE, IgM, IgA; autoantibodies (antinuclear antibodies, anti-nRNPantibody, anti-Smith antibodies, Sjogren’s syndrome-A antibodies, Sjogren’s syndrome-Bantibodies, anti Scl-70, anti-Jo-1, anti-centromere antibody, anti-double-stranded DNAantibodies, anti-nucleosome antibodies, anti-histone antibodies, anti-ribosomal P proteinantibodies); vascular inflammatory antibodies (perinuclear ANCA, cytoplasmic ANCA,myeloperoxidase antibody, proteinase3antibodies); rheumatoid factor IgG.5．Metabolic disease-related tests We use gas chromatography-mass spectrometry(GS-MS)method to detect plasma amino acid profile and urine organic acids, and use tandem massspectrometry method to detect blood acylcarnitine and use immune turbidimetric to detectceruloplasmin.Results1．In our study,20patients perform pHLH-related gene detection. The total detection rate is100%. The order of the detection rate of these genes are: PRF1and STXBP2(90%), UNC13D(60%), AP3B1(55%), XIAP (40%), SH2D1A (20%), RAB27A (15%), STX11and LYST (10%).2．In our study, we totally find out85mutations, and there are6mutations that have not beenreported by the literature, include: missense mutation c.1831G>A of UNC13D gene, missensemutation c.593C>T and c.613G>A of STXBP2gene, missense mutation c.5290G>A of LYSTgene, missense mutation c.362C>T of AP3B1gene, missense mutation c.35T>C of RAB27Agene.3．In our patients group, pathogens that is ordered by its detection rate are as follows: positiverate of EBV-IgM antibody is64.44%(58/90), positive rate of bacterial culture is20.51%(16/78), positive rate of CMV-IgM antibody is19.77%(17/86), positive rate ofmycoplasma pneumonia-IgM antibody is16.47%(14/85), positive rate of herpes simplexvirus-IgM antibody is9.30%(8/86).Conclusions1．Children patients who have the mutation c.1232G>A, c.368A>G, the condition is severe and the disease rapidly develop, while the mortality rate is high. Children patients who havethe mutation c.1033C>T, c.613G>A, the early treatment response is poor.2．EBV infection is the most common triggering factor of children patients with HLH. Theclinical condition of EBV-HLH is relatively severe, the mortality rate is high and theprognosis is poor, in addition, the prognosis associates with the load of EBV DNA.3．Among the severe cases or the dead cases, there are2cases have pHLH-related genemutation and coinfection with EBV, their primary and secondary relationship or causalrelationship in the genesis and development of the disease need further study.