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At least one patient (No. 8) who developed HLH was suspected of primary HLH, but genetic testing was unrevealing. This patient was given the diagnosis of secondary HLH with no known etiology. Blood samples were collected by venipuncture in 5-mL vacu-tainer tubes without anticoagulant and allowed to clot for 10�C15 min. Samples were then centrifuged at 1000g for 10 min in a refrigerated centrifuge. http://www.selleck.cn/products/mi-773-sar405838.html The clot was then removed and 0.5 mL aliquots of serum were transferred to polypropylene tubes and stored at ?80��C. Serial or single measurements were obtained (depending on sample availability). The S-SMase activity assay was performed as previously described [23]. Briefly, porcine brain sphingomyelin (0.2 mM) mixed with 14C-sphingomyelin (radiolabeled in the choline moiety, specific radioactivity?=?1.5 �� 105 cpm/?L) were incubated with serum samples (1:10 dilution: 10 ?L serum?+?90 ?L of 0.2% Triton X-100, 50 mM Tris-HCl, pH 7.4, to a final volume of 100 ?L) in Triton X-100 micelles in a buffer containing 250 mM sodium acetate (pH 5.00) and 0.2 mM ZnCl2 (S-SMase) to a final concentration of 0.1 mM SM with either 0.1 mM ZnCl2 (S-SMase). The reaction was run for 3 hr at 37��C and terminated with the addition http://www.selleckchem.com/products/VX-770.html of 1.5 mL CHCl3: MeOH (2:1, v/v) followed by 0.4 mL water. Samples were vortexed, centrifuged (5 min, 3,000 rpm) and 0.8 mL of the aqueous/methanolic phase was removed for scintillation counting. The assay was linear with respect to time and volume of serum, and cleavage of substrate was http://www.selleckchem.com/products/Imatinib-Mesylate.html a multiple reaction-monitoring positive ionization mode, as described [30]. All sphingolipid analytes contain a d18:1 sphingoid backbone. Notation of SPL species therefore imply a d18:1 sphingoid base backone (e.g., C16-ceramide represents d18:1/16:0 ceramide) [31]. Data are represented as mean?��?standard error of the mean (S.E.M.), unless otherwise indicated. Non-Gaussian distribution was assumed and data were analyzed by non-parametric two-tailed, unpaired Mann�CWhitney U test or Kruskal�CWallis test with Dunn's post-test statistical analyses. Statistical analyses were performed using Prism/GraphPad software. After collecting sera from the 16 patients with documented HLH and 25 healthy volunteer controls, the activity of S-SMase was assessed, as described above in the ��Methods�� section. Serum S-SMase activity was elevated 2�C3 fold in patients with HLH (control: 4.28?��?0.49 nmol/mL/hr versus HLH: 12.30?��?0.86 nmol/mL/hr; P?