To optimize the ethanol extraction process for Shenlian formula. On the basis of the pharmacodynamics index for different extraction process routes, the contents of salvianolic acid B, tanshinone ⅡA and berberine, as well as the extraction ratio in different experimental schemes were used as the ethanol extraction examining indexes, and multi-criterion synthesizing grading method was used for data analysis to optimize and verify the ethanol-extraction process conditions in orthogonal experiment. The optimum ethanol extraction process was as follows: adding 60% ethanol, 10 times amount, extracting for 2.0 h each time for a total of 2 times. This extraction process showed good stability and availability.

OBJECTIVETo explore the coexistence of ASXL1 and CALR gene mutations in patients with essential thrombocytheima (ET) and with primary myelofibrosis(PMF), and to compare the differences of clinical characteristics between ET and PMF patients carrying ASXL1 and CALR mutations, and ET and PMF patients carrying solitary gene mutation, and ET and PMF patients without any mutations.

METHODSThe mutations of ASXL1 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 263 essential ET patients and 29 PMF patients were detected by PCR amplification followed by direct sequencing of genomic DNA. The JAK2V617F mutations were used by allele specific PCR detection.

RESULTS72.6%(212/292)of patients harbored at least one mutation. The incidences of ASXL1 and CALR mutations were 5.8% and 30.5%, respectively. The frequencies of JAK2V617F and MPL mutations were 39.0% and 2.4%, respectively. 5.1%(15/292) of patients had double mutations, including ASXL1 and CALR(n=11), ASXL1 and JAK2V617F(n=2), MPL and CALR(n=1) and ASXL1 and MPL(n=1). The frequency of concurrent ASXL1 and CALR mutations was found to be high. Significant difference was found on hemoglobin levels and platelet counts between CALR and ASXL1 mutations and single mutation (P<0.05),however, the difference on leukocyte counts and median age was not found. Compared with negative patients, the presence of ASXL1 and CALR mutations was found to be significantly correlative with lower hemoglobin level (P=0.045), lower leukocyte count (P=0.002) and with higher platelet counts(P=0.001), but the difference of median age was not found.

CONCLUSIONThe frequency of concurrent ASXL1 and CALR mutations is higher in ET patients. The coexistence of ASXL1 and CALR gene mutations significantly associated with lower hemoglobin level and higher platelet count.

This current cross-sectional study investigates the relationship between thyroid hormones and peripheral artery disease (PAD) among euthyroid Chinese population aged 40 years and above. Serum free triiodothyronine (FT3), free thyroxin (FT4), thyroid-stimulating hormone (TSH), and thyroid antibodies were measured. PAD was defined as ankle-brachial index (ABI) < 0.9. There were 91 (2.9%) PAD cases among the 3,148 euthyroid study participants. Participants in the highest quartile of FT3 and free-triiodothyronine-to-free-thyroxin (FT3/FT4 ratio) had a decreased risk of prevalent PAD (multivariate-adjusted odds ratio, 95% confidence interval: 0.32, 0.15-0.62, P for trend = 0.01 and 0.31, 0.13-0.66, P for trend = 0.004, respectively) compared to those in the lowest quartile. To conclude, FT3 levels and the FT3/FT4 ratio was inversely associated with prevalent PAD in euthyroid Chinese population aged 40 years and above.
Aged ; Aged, 80 and over ; Cross-Sectional Studies ; Female ; Humans ; Male ; Middle Aged ; Odds Ratio ; Peripheral Arterial Disease ; blood ; Risk Factors ; Thyroxine ; blood ; Triiodothyronine ; blood

BACKGROUNDThe established clinical staging systems (Rai/Binet) of chronic lymphocytic leukemia (CLL) cannot accurately predict the appropriate treatment of patients in the earlier stages. In the past two decades, several prognostic factors have been identified to predict the outcome of patients with CLL, but only a few studies investigated more markers together. To predict the time to first treatment (TTFT) in patients of early stages, we evaluated the prognostic role of conventional markers as well as cytogenetic abnormalities and combined them together in a new prognostic scoring system, the CLL prognostic index (CLL-PI).

METHODSTaking advantage of a population of 406 untreated Chinese patients with CLL at early and advanced stage of disease, we identified the strongest prognostic markers of TTFT and, subsequently, in a cohort of 173 patients who had complete data for all 3 variables, we integrated the data of traditional staging system, cytogenetic aberrations, and mutational status of immunoglobulin heavy chain variable region (IGHV) in CLL-PI. The median follow-up time was 45 months and the end point was TTFT.

RESULTSThe median TTFT was 38 months and the 5-year overall survival was 80%. According to univariate analysis, patients of advanced Rai stages (P < 0.001) or with 11q- (P = 0.002), 17p- (P < 0.001), unmutated IGHV (P < 0.001), negative 13q- (P = 0.007) and elevated lactate dehydrogenase levels (P = 0.001) tended to have a significantly shorter TTFT. And subsequently, based on multivariate Cox regression analysis, three independent factors for TTFT were identified: advanced clinical stage (P = 0.002), 17p- (P = 0.050) and unmutated IGHV (P = 0.049). Applying weighted grading of these independent factors, a CLL-PI was constructed based on regression parameters, which could categorize four different risk groups (low risk [score 0], intermediate low [score 1], intermediate high [score 2] and high risk [score 3-6]) with significantly different TTFT (median TTFT of not reached (NR), 65.0 months, 36.0 months and 19.0 months, respectively, P < 0.001).

CONCLUSIONSThis study developed a weighted, integrated CLL-PI prognostic system of CLL patients which combines the critical genetic prognostic markers with traditional clinical stage. This novel modified PI system could be used to discriminate among groups and may help predict the TTFT and prognosis of patients with CLL.

Adult ; Aged ; Aged, 80 and over ; China ; Chromosome Aberrations ; Chromosomes, Human, Pair 17 ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; metabolism ; In Situ Hybridization, Fluorescence ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Prognosis

Objective To evaluate the efficacy of rituximab in treating chronic lymphocytic leukemia (CLL). Methods The clinical data of CLL patients receiving fludarabine,cyclophosphamide±rituximab (with or without rituximab) regimen or cyclophosphamide,vincristine,and prednisone±doxorubicin±rituximab regimen in our hospital from March 2000 to February 2015 were analyzed retrospectively. Therapeutic efficacies and survivals of patients treated with different regimens were evaluated and compared. Results The complete response (CR) rate and the overall response rate (ORR) in 72 patients (43.6%) treated with rituximab were significantly higher than those treated without rituximab (38.9% vs. 21.5%,P=0.015;83.3% vs. 60.2%,P=0.001). The median PFS and OS for patients treated with rituximab were 53.0 (27.0-79.0) months and 112.0 (81.1-142.9) months,and the median PFS and OS for patients treated without rituximab were 28.0 (18.3-37.7) months and 89.0(72.0-106.0),but the results were not statistically significant (P=0.094,P=0.109). According to the cytogenetic features,patients were further divided into high-risk subgroup (with chromosome 17p deletion or 11q deletion) and non-high-risk subgroup. And in the high-risk subgroup,the ORR of patients treated with rituximab was 86.4%,which was significantly higher than that in patients treated without rituximab (53.3%)(P=0.012);in the non-high-risk subgroup,the PFS was marginally prolonged in patients treated with rituximab,but the difference was not statistically significant(P=0.050). Conclusions Compared with traditional chemotherapy,the chemoimmunotherapies with rituximab result in higher CR rate and ORR in CLL patients. In patients without 17p deletion or 11q deletion,the use of rituximab can marginally prolong PFS.

OBJECTIVETo compare the accuracy of serological and molecular approaches to identification of RhD-negative patients waiting for kidney transplantation.

METHODSA total of 103 RhD-negative blood samples by serological test were collected from patients waiting for kidney transplantation between January, 2006 and January, 2016. Quantitative PCR and sequencing were used to verify the results of RHD genotyping, and the false negative rates of the serological and molecular methods for RhD genotyping were compared.

RESULTSAmong the 103 blood samples, true RhD negativity (with all the 10 exons missing) was found in 56 samples (54.5%), and false RhD negativity (RhD positivity with loss, repetition, or missense mutation in the 10 exons) in 47 samples (45.6%). In the 47 false RhD-negative cases, weak D was detected in 1 case (2.1%), partial D in 13 cases (27.7%), and D-elution in 33 cases (70.2%). The detection rates of RhD negativity differed significantly between the serological and molecular methods (P<0.05).

CONCLUSIONSerological test is associated with a high false negative rate in detecting RhD blood group, and the use of the molecular approach has important clinical significance in accurate RhD genotyping for patients waiting for renal transplantation.

OBJECTIVETo determine the disease-causing mutation in a newborn with hereditary spherocytosis.

METHODSGenomic DNA was extracted from peripheral blood samples of the patient and her parents. Next-generation sequencing was used to analyze the related genes. Suspected pathogenic mutation was verified with polymerase chain reaction and Sanger sequencing.

RESULTSAn insertional mutation g.834_833insC was identified in the coding region of ankyrin-1 (ANK1) gene, which has caused a frame shift, resulting premature termination of protein translation.

CONCLUSIONThe hereditary spherocytosis in the neonate was probably due to the g.834_833insC mutation of the ANK1 gene.

Amino Acid Sequence ; Ankyrins ; genetics ; Base Sequence ; Female ; Humans ; Infant, Newborn ; Infant, Newborn, Diseases ; diagnosis ; genetics ; Molecular Sequence Data ; Mutation ; Spherocytosis, Hereditary ; diagnosis ; genetics

OBJECTIVETo investigate the optimal starvation conditions of human umbilical vein endothelial cells (HUVECs) and establish a highly efficient and stable method for separating HUVECs.

METHODSHUVECs harvested from human umbilical cords by digestion with 0.1% collagenase II for 15 min were cultured in endothelial culture medium (ECM) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth factor (ECGS) and 1% penicillin/streptomycin solution(P/S) at 37 degrees celsius; in 5% CO2. The cells were observed for cell morphology under an inverted microscope and identified with immunofluorescence assay. The purity of HUVECs was detected using flow cytometry (FCM). The cell cycles of HUVECs cultured in the presence of 0, 0.1%, 0.5%, and 1% FBS for 0, 6, 12, 18, and 24 h were analyzed with flow cytometry.

RESULTSs The purity of HUVECs harvested by digestion with 0.1% collagenase II reached 99.67%. The primary HUVECs showed a cobblestone or volute appearance in vitro. Immunocytochemistry showed that HUVECs highly expressed VIII-related antigen. Cell culture in the presence of different concentrations of FBS for 6 h resulted in 70% G0/G1 phase cells, which increased to 80%-90% at 12 h of cell culture, and further to around 95% at 18 and 24 h.

CONCLUSIONDigestion with 0.1% collagenase II can obtain high-purity primary HUVECs. Culturing HUVECs in serum-free medium for 12 h can result in a high purity (over 80%) of G0/G1 phase cells.

Cell Culture Techniques ; Cell Cycle ; Cells, Cultured ; Culture Media ; chemistry ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Matrix Metalloproteinase 8 ; chemistry ; Serum

OBJECTIVETo investigate the value of evaluating 5 platelet parameters in predicting delayed graft function (DGF) in patients following kidney transplantation.

METHODSWe retrospectively analyzed the pre- and postoperative (within 2 months) data of 330 renal transplant recipients. The cases with DGF and those without were analyzed to assess the association between relationship between DGF following transplantation and the variations of blood platelet parameters including platelet count (PLT), large platelet ratio (P-LCR), mean platelet volume (MPV), platelet volume distribution width (PDW) and platelet hematocrit (PCT).

RESULTSThe DGF and non-DGF cases were comparable for the platelet parameters before the operation. On postoperative day 7 when the diagnosis of DGF was made, PLT (P<0.05) and PCT (P<0.02) were significantly lower while MPV (P<0.01), PDW (P=0.036) and P-LCR (P=0.01) significantly higher in DGF group than in non-DGF group. The AUCs of P-LCR (0.611±0.047), PDW (0.603±0.048) and MPV (0.762±0.037) were significantly higher than the reference area (P<0.05) with cut-off values of 34.80%, 12.95fl and 11.55fl, respectively. MPV showed a high sensitivity, specificity and Youden index for predicting DFG; PDW and P-LCR had a high sensitivity but a low specificity for predicting DFG with a modest diagnostic value. PLT and PCT, with AUCs of were 0.37 and 0.38, respectively, did not have a predictive value for DGF.

CONCLUSIONSSignificant variations in platelet parameters occur in the event of DGF in renal transplant recipients, and monitoring the postoperative changes in MPV, PDW, and P-LCR can help in early diagnosis and treatment of DGF. MPV has a moderate value (0.7-0.9) in predicting DGF, and a MPV>11.55 fl suggests the risk of DGF.

Area Under Curve ; Blood Platelets ; Delayed Graft Function ; Humans ; Kidney ; physiology ; Kidney Function Tests ; Kidney Transplantation ; Mean Platelet Volume ; Platelet Count ; Postoperative Period ; ROC Curve ; Retrospective Studies ; Sensitivity and Specificity

OBJECTIVETo evaluate the effects of arthroscopic debridement for acute gouty arthritis of the ankle.

METHODSForty-one patients with acute gouty arthritis of the ankle were treated under arthroscopy from January 2010 to June 2012. All the patients were male, age in ranging from 28 to 69 years with an average of 43 years. Eighteen patients were in the left ankles and 23 in the right ankles; 12 cases were firstly attack and 29 cases were recurrent attack. Course of disease was from 2 weeks to 30 months. The American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Scale score was used to evaluate the clinical effects. Number of acute attacks of gouty arthritis were observed.

RESULTSAll the patients were followed up at least 12 months. The mean AOFAS Ankle-Hindfoot Scale score increased from 58.44 +/- 9.45 preoperatively to 86.15 +/- 7.36, 83.41 +/- 9.22, 84.10 +/- 8.22 postoperatively at 6, 12, months and the last follow-up respectively. Swelling of the ankle were improved significantly, pain was relieved and the mean number of acute attacks of gouty arthritis decreased significantly.

CONCLUSIONArthroscopy is helpful for the diagnosis of acute gouty arthritis of the ankle and improvement of clinical symptoms and ankle function.

Adult ; Aged ; Ankle Joint ; physiopathology ; surgery ; Arthritis, Gouty ; physiopathology ; surgery ; Arthroscopy ; Debridement ; Female ; Humans ; Male ; Middle Aged ; Treatment Outcome