| Hemophagocytic syndrome (HPS) also named hemophagocytic lymphohistiocytosis (HLH), is a rare disease with a high mortality, the patients usually undergo a very severe and critical process requiring an immediate clinical attention. It is a life-threatening disease characterized by fever, massive hepatosplenomegaly, hepatic dysfuncion, pancytopenia, coagulation disorder, hypertriglyceridemia, cytokinemia, elevated level of ferritin, and may be associated with severe neurological symptoms. Histopathological findings include a widespread accumulation of phagocytic cells which are mononuclear macrophages phagocytize erythrocytes, granulocytes, thrombocytes and lymphocytes, affecting the bone marrow, the liver and lymph nodes. There are two different catogories of HPS that may be difficult to distinguish from one another: one isprimary or familial form and another one is secondary form. The primary form, familial hemophagocytic lymphohistiocytosis (FHL), is a fatal disease with a median survival time of less than 2 months if no effective therapy such as intensive immunosuppressive therapy and/or hematopoietic stem cell transplantation is performed on time. Secondary HPS may develop when certain infections or malignancies are encountered with an up to 20%～70% of mortality rate. The earlier the disease occurs, the higher the mortality is.The most commonly used diagnostic criteria are the ones presented by the Histiocyte Society of Japan. Theses criteria were based on the common clinical symptoms, the laboratory and the histopathological findings. With the accumulation of the knowledge on its etiology and pathogenesis, the diagnostic criteria were amended in 2004 in which three additional parameters had been introduced. However, most of these diagnostic criteria are nonspecific clinical manifestations, and the histopathological findings of bone marrow or other organs involved with a few of phagocytized cells are not unique to this disease. When a patient meet all of the diagnostic criteria, he or she may already have been under a very serious condition, and lost the best time for patient to receive an effective therapy which may result in an increased mortality. Thus, early diagnosis of HPS followed by early intervention to the patients becomes an important issue in clinical practice.Until now, it is considered that HPS has close relationship with the abnormal immunological regulation. Most of clinical conditions are considered to result from hypercytokinemia. Histopathological findings including the widespread accumulation of phagocytic cells phagocytizing blood cells in the bone marrow, the liver, the spleen and the lymph nodes, resulting in the organ damage, are due to the cytotoxicity dysfunction of CTL and NK cells. Immunological cells especially macrophages and T lymphocytes produce a great deal of cytokines because of continuous stimulation of antigens. A Thl-Th2 imbalance in favor of Th1, releasing large quantity of cytokines such as IFN- γ, IL-6, GM-CSF known as "cytokine storm", causes an inappropriate activation and proliferation of macrophages, which are responsible for the clinical manifestations of HPS. Although stringent criteria for the diagnosis of HPS have been introduced, difficulties for the diagnosis are often encountered, especially during its early stage. Cytokine storm in this disease has been proposed for many years however, it only remains in the laboratory rather than for clinical diagnostic purpose. Can the detection of some cytokines provide an early diagnosis to HPS? If yes, then how can we choose an appropriate combination of cytokines to improve the accuracy of the diagnosis?In this study, a quick quantification of 6 cytokines with flow cytometry was performed in 16 cases of children with HPS to observe the dynamic changes of the cytokines in the course of the disease, therelationship of cytokines with the etiology, the severity and the prognosis of HPS to seek for an early diagnostic method for quick and correct diagnosis of the disease.Patients and Methods1 Patients16 cases of children with HPS in our hospital from the June of 2004 to the December of 2005 were enrolled into this study. All 16 patients were diagnosed as HPS according to the HLH Guideline 2004 criteria. 21 cases of healthy children, 17 cases of children with acute lymphoid leukemia (ALL) under remission, 17 cases of children with fever and positive blood culture were control groups.2 Collection of test samples2ml of aseptic peripheral blood without anticoagulant were obtained at the time of the disease onset, after the induction chemotherapy and/or when relapsed. The sera were collected for the determination of the 6 cytokines after clotting.3 Method of cytokines test6 cytokines in the serum including IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α were determined using cytometric bead assay (CBA) by flow cytometry. The procedures were following the instructions of the human Thl/Th2 cytokine kit II provided by the company.4 Statistical analysisData were expressed in mean±SD and analyzed with SPSS13.0 statistical kit. Paired T test was used to compare the cytokine levels before and after therapy. Spearman analysis was used to analyze the correlation between the cytokines and the clinical manifestations. P ≤ 0.05 was defined as statistical significance. Kaplan-Meier analysis was used to determine the correlation of the cytokines and the prognosis of the disease.Results1 The levels of cytokines in patients with HPS at the time of diagnosisIL-2(3.54±5.71pg/ml vs 1.85±1.11pg/ml, P>0.05) and TNF-α (3.76±6.5 1pg/ml vs 1.37±1.15pg/ml, P>0.05)didn't rise obviously, IL-4 (13.12±30.86pg/ml vs 2.79±1.90pg/ml, P>0.05)was slightly elevated while IL-6(61.03±75.61pg/ml vs 4.13±2.09pg/ml, P＜0.05),IL-10(5812G±967.15pg/ml vs 4.01±3.00pg/ml, P<0.05) and IFN-γ(2993.62±2237.80pg/ml vs 52.42±17.86pg/ml, P<0.05)increased significantly as compared to those from normal controls.2 Levels of cytokines between HPS patients and septicemiaIn order to demonstrate the differences of cytokine levels among different diseases, the levels of IFN-γ, IL-10 and IL-6 from patients with HPS were compared with those from patients with septicemia or ALL under remission. The level of IFN- γ in HPS patients was significantlyhigher than normal control(2993.62±2237.80pg/ml vs 52.42±17.86pg/ml, P<0.05), ALL patients under remission(2993.62±2237.80pg/ml vs 58.70 ±28.61pg/ml, P＜0.05) and septicemia patients (2993.62±2237.80pg/ml vs 255.66±302.55pg/ml, P<0.05). The level of IL-10 increased obviously higher than normal control(581.20±967.15pg/ml vs 4.01 ±3.00pg/ml, PO.05) and ALL patients (581.20±967.15pg/ml vs 4.59±1.85pg/ml, P<0.05). While the level of IL-6(61.03 ± 75.61 pg/ml vs 718.68 ± 678.63pg/ml, P<0.05) was significantly lower than that from patients with septicemia. The levels of IL-2, IL-4 and TNF-α were not significantly increased when compared to those from either patients with septicemia or ALL(P>0.05).3 Dynamic changes of cytokine levels before and after therapyAll 6 cytokines were analyzed for patients with HPS before and after therapy. The results showed that the levels of IL-2(3.66 ±5.02 vs 5.02 ± 8.54pg/ml) and IL-4(2.74±2.40 vs 5.78 ± 7.24pg/ml) were decreased after therapy, but there's no statistical significance (P>0.05). While those of IL-6 (11.22+18.44 vs. 52.76± 68.96pg/ml, P＜0.05), IL-10 (49.94±113.03 vs 374.13± 526.20pg/ml, P＜0.05) and IFN-γ(410.34±1131.52 vs 2528.52 + 2237.51pg/ml, P＜0.01) were dramatically decreased after therapy, indicating a good response of the patients to the HLH 94 and 2004 protocols. However, some patients did not show a good response to theprotocol with slightly changed or no change of the cytokines.4 Cytokines and prognosisThere were significant correlation between cytokines levels of IL-6, IL-10, IFN-γ and patients' prognosis. Patients with IL-6<30pg/ml, IL-10<300pg/ml and IFN-γ<300 pg/ml were better than those of IL-6>30pg/ml, IL-10>300pg/ml and IFN-γ>300 pg/ml (P＜0.05),5 Correlation between cytokines and clinical manifestationsIL-6 and IFN-γ showed a significant positive correlation, as well as IL-10 and IFN-γ, with Spearman's rank correlation coefficient of 0.688 and 0.870 (P=0.000). There was no statistical correlation between IL-2 and clinical manifestations as well as IL-4 and IFN- γ. IL-6 has a negative correlation with the level of platelets. IL-10 showed a significant positive correlation with LDH and the TNF- α with bilirubin (P＜0.05) .ConclusionsThe levels of IL-6, IL-10 and IFN-γ rose obviously in the HPS patients. The patients with notable high levels of these cytokines have poor prognosis. Most patients can achieve remission after chemotherapy, and with the levels of cytokines falling to normal. Cytokines like IFN-γ and IL-10 etc play an important role in the onset and progress of HPS. TheAbnormal cytokines pattern of the significant increase of IFN- γ and IL-10 with slight increase of IL-6 can become a reliable parameter for the diagnosis of HPS. The determination of the abnormal cytokine pattern IFN-γ/IL-10/IL-6 determined by cytometric bead assay (CBA) can provide a useful approach for the early quick diagnosis and the monitoring of HPS during therapy and follow-up. |
PDF Full Text Request