BACKGROUND:As the sequencing technology has been widely used and high-resolution confirmation of organ transplant matching has been gradualy developed, new human leukocyte antigen (HLA) aleles are emerging. However, the gene frequency of some genes cannot be calculated accurately, and there are rare reports. These genes are often ignored, and it is easy to misjudge their genotypes only according to gene frequency. OBJECTIVE:To test and analyze a rare alele, HLA-C*08:99, from a volunteer donor of hematopoietic stem cel transplantation. METHODS: Genomic DNA was extracted automaticaly from the blood sample by using quick DNA purified kit and amplified by HLA-C locus commercial sequence-based typing kit. The purified PCR product was utilized as the DNA template in the sequencing reaction, and six direct sequencing reactions of PCR product covering exons 2, 3 and 4 in both directions were performed using commercial kit. Four direct sequencing reactions of PCR product covering exon 5 in both directions, exon 6 in forward direction and exon 7 in reverse direction were performed using in-house BigDye terminator cycle sequencing reaction kit. Sequencing reaction products purified by ethanol/sodium acetate/ ethylenediaminetetraacetic acid method were sequenced by ABI PrismTM3730 DNA Sequencer. RESULTS AND CONCLUSION:The alele assignment was analyzed with Assign-SBT 3.6+ software, and the sample HLA-C typing result was C*07:04, 08:99. Increasing the sequencing analysis at exons 5, 6 and 7 of HLA-C locus wil help to make clear the ambiguous SBT result and improve the accuracy of HLA-C typing when it is necessary, which shows important significance in clinical tissue matching. Cite this article:Wang DM, Zou HY, Nie DM, Gao SQ, Wang F.Sequencing analysis of a rare human leukocyte antigen, C*08:99, from a volunteer donor of hematopoietic stem cel transplantation. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):102-106.

Objective To investigate the variation of lymphocyte subgroup,lymphocyte and C-reaction protein in patients with acute and chronic Brucellosis,for auxiliary reference in differential diagnosis of acute and chronic Brucellosis.Methods All the blood samples were come from clinical laboratory of Heilongjiang Agricultural Reclamation Bureau General Hospital,which including 35 acute patients(acute group),62 chronic patients(chronic group) and 30 healthy people(control group).Flow cytometric method was used to determine the T lymphocyte subgroup(CD3+,CD19+,CD3+/CD4+,CD3+/CD8+) and the lymphocyte.Application of rate scattering turbidimetric method was used for determination of C-reactive protein.Results Percentages of CD3+ cells in acute and chronic groups were increased compared with that of control group(％:74.71 ± 6.79,70.61 ± 8.99 vs.65.00 ±7.50; F =12.40,P ＜ 0.05),and the acute group was higher than the chronic group(P＜ 0.05).Percentages of CD19+,CD3+/CD4+ cells in acute and chronic groups were decreased compared with that of control group(CD19+,％:7.79 ± 3.93,9.94 ± 4.53 vs.11.50 ± 3.25; CD3+/CD4+,％:30.56 ± 6.38,35.56 ± 8.22 vs.39.00 ± 4.00; F=18.62,13.03,all P＜ 0.05),and the acute group was lower than the chronic group (P＜ 0.05).Percentage of CD3+/CD8+ cells in the acute group was higher than those of the chronic and control groups(％:4.06 ± 8.35,29.50 ± 7.25 vs.28.72 ± 8.30; F =12.06,P ＜ 0.05).The lymphocyte counts in acute and chronic groups were higher than that of the control group(％:34.29 ± 10.91,39.12 ± 12.08 vs.30.84 ± 6.45; F=13.78,P ＜ 0.05),but the acute group was lower than the chronic group(P ＜ 0.05).C-reactive protein levels in acute and chronic groups were increased compared with that of the control group(mg/L:27.43,15.87 vs.0.60; H =19.42,P ＜ 0.05),and the acute group was higher than the chronic group(P ＜ 0.05).C-reactive protein positive rates in acute and chronic groups were increased compared with that of the control group(65.7％,61.3％ vs.3.3％; x2=27.54,23.16,all P ＜ 0.01).The difference between groups was statistically significant(x2 =9.94,P ＜ 0.01).Conclusion Lymphocyte subgroup,lymphocyte and C-reactive protein are changed differently in patients with acute and chronic Brucellosis which have a certain reference value in differential diagnosis and treatment of acute and chronic Brucellosis.

ObjectiveTo evaluate analytical performance of NT-proBNP on Electro-Chemiluminescence Immunoassay system.MethodsThe precision,accuracy,limit of blank ( LoB ),limit of detection (LoD),functional sensitivity (FS),analytical measure range (AMR),maximal dilution rate,clinical reportable range(CRR) and the analytical anti-interference ability of NT-proBNP were evaluated according to EP documents issued by CLSI and related references.The analytical performance data were compared to quality standards declared by the manusfacturers.According to CLSI C28-A2,80 healthy volunteers,aged from 18 to 74, were chosen and divided into 4 groups on average for biological reference intervals verification.Results The within-run CV and total CV were 1.1％ -2.2％and 1.5％ -2.9％ respectively.The deviations from controls distributed by National Center for Clinical Laboratory and affiliated calibrators were 2.7％ -5.9％ and 2.7％ -7.5％,respectively.The results of LoB,LoD and FS were 2.5,7.8 and 8.8 pg/ml,respectively.AMR was 8 -35 126 pg/ml,and the most suitable dilution rate was 1∶ 2,so the CRR was 9 -70 252 pg/ml.428 μmol/L bilirubin,2 g/L haematoglobin and 2 200 FIU chyle didn't interfere with the NT-proBNP assay.Moreover,almost all the data from different age groups were in the range of biological reference intervals declared by the manusfacturers, except one test data (167 pg/ml).Conclusions The analyticalperformance of NT-proBNP analyzed on Roche Cobas E601electrochemiluminescence immunoassay systemisconsistentwiththestandarlswhichmanufacturershas proclaimed.The establishment of LoD,FS,maximal dilution and CRR for NT-proBNP assay could provide the quality assurance for clinical use and the biological reference intervals declared by manusfacturers could meet the clinical needs.

Objective To explore the relationship between the prothrombotic state and blood coagulation-fibrinolysis system changes with deep venous thrombosis(DVT)in aged patients after total joint arthroplasty,and to propose preventive measures.Methods 400 patients who underwent total hip or knee replacement from January 2003 to June 2011 were classified into suspected DVT(n=200 cases)and non-suspected DVT(n=200 cases)according to Well's clinical scoring system.The patients were divided into 4 subgroups based on the measures to prevent DVT:low molecular weigh heparin group,intermittent pneumatic bag compression group,combined above measures group,control group who refused any preventive measures.Plasma thrombin-antithrombin complex(TAT),plasmin-α2 antiplasmin complex(PAP)were determined preoperatively.Venous Doppler ultrasound was performed before surgery,4 d and 10 d after surgery to detect the presence of DVT.Results Totally 71 cases(17.8％)were diagnosed as DVT and 5 cases(1.3％)as pulmonary thromboembolism(PTE).The incidence of DVT in suspected DVT group(28.5％,57 cases)was lower than in non-suspected DVT group(7.0 ％,14 cases)(x2 =31.66,P＜ 0.01).Among patients with suspected DVT,the DVT prevalence in combined measures group(2.0 ％,1 cases)was decreased than in low molecular weigh heparin group(14.0％,7 cases),intermittent pneumatic bag compression (34.0％,17 cases)and control groups(64.0％,32 cases)(x2=4.89,17.34,18.01,all P＜0.05).In the patients with non-suspected DVT,the DVT prevalence in combined measures group(0.0％)was lower than in control group(24.0 ％,12 cases)(x2 =13.64,P＜0.01).The average preoperative level of TAT[(9.63±3.06)μg/L]in patients with DVT was higher than without DVT[(2.59±0.87)μg/L](t=35.70,P＜0.01),while PAP level in patients with DVT[(38.52± 21.13)μg/L]was reduced than without DVT[69.75±30.26)μg/L](t=8.27,P＜0.01).Conclusions The levels of TAT and PAP before total hip or knee replacement are predictive for lower extremity DVT.The clinical effectiveness of Well's scoring on DVT prevention is dependent on the different TAT and PAP state.

Objective To explore the value of virtual touch tissue quantification(VTQ) on the early diagnosis of gouty nephropathy.Methods 180 healthy controls and 109 gout patients including 57 cases with normal renal function,and 52 cases with gouty nephropathy were measured by VTQ in this study.The shear wave velocity (SWV) of renal cortex,renal medulla and renal sinus were measured using virtual touch tissue quantification(VTQ).The recorded SWV value was compared.Results In all groups,the SWV of renal cortex was the highest with significant difference (P ＜0.01).The SWV of renal cortex in gouty nephropathy group was higher than those in control group (t =0.342,P =0.026).The SWV of renal medulla and sinus in gout with normal renal function group and gouty nephropathy group were higher than those in control group,respectively (gout with normal renal function group:t =-0.311,-0.628; P =0.012,0.000;gouty nephropathy group:t =-0.369,-0.701 ; P =0.000,0.000).The SWV of renal cortex,medulla and sinus had no significant difference between gout patients with normal renal function and gouty nephropathy patients(t =0.117,-0.059,-0.073; P =0.232,0.575,0.523).Conclusions The VTQ technology could quantitatively evaluate tissue elasticity of the gout patients and provide evidence for the early diagnosis of gouty nephropathy.

Objective To evaluate the value of chorionic villus cells karyotype analysis in prenatal diagnosis during the first tri-mester of pregnancy.Methods Pregnant women with prenatal diagnosis indications were punctured by guiding abdominal B-mode ultrasound to get villi tissue which was then to develop cell culture,chromosome preparation and karyotype analysis.Results A to-tal of 1 140 cases were successfully cultured,and the successful cultivating rate was 98.2% (1 140/1 160).Among them,chromo-somes of 62 cases were detected to be non-polymorphic structural abnormalities,including 32 abnormal chromosome number,5 chro-mosome balanced translocation,3 chromosome deletion,and 22 chimeras.What′s more,20 cases were detected to be chromosomal inversion,19 cases of chromosome 9 were inversion,and one with chromosome Y was inversion.Conclusion Karyotype analysis of villus cell could help to detect fetal chromosomal abnormalities during early pregnancy and get early intervention.It was significant to reduce the child′s birth with chromosome abnormalities.

Objective:To explore associations of subclinical hypothyroidism (SCH) with endocrine metabolic characteristics in women with polycystic ovary syndrome (PCOS).Methods:A total of 321 women who were newly diagnosed as PCOS were recruited from two endocrine outpatient clinics.The diagnosis of PCOS was established according to the 2003 Rotterdam consensus criteria.Thyroid function was examined by chemiluminescent immunoassay.Patients who had normal free thyroxine (FT4) were divided into different SCH subgroups according to two thyroid stimulating hormone (TSH) cutoffpoints (4.2 and 2.5 mU/L).Endocrine metabolic characteristics in different subgroups were compared and analyzed.Results:In PCOS women with normal FT4,the patients with TSH ≥ 4.2 mU/L had higher prolactin (PRL),luteinizing hormone-to-follicle stimulating hormone ratio,and visceral adipose index (all P<0.05).There were trends toward an increase in triglyceride (P=0.085) and a decrease in high-density lipoprotein cholesterol (HDL-C) (P=0.060) in the patients with TSH ≥ 4.2 mU/L compared with that in the patients with TSH<4.2 mU/L.Also in PCOS women with normal FT4,the patients with TSH ≥ 2.5 mU/L had higher body mass index,PRL,triglyceride,visceral adipose index and lower HDL-C in comparison of that in the patients with TSH<2.5 mU/L (all P<0.05).Conclusion:SCH is associated with more severe endocrine abnormality,dyslipidemia,and visceral obesity in PCOS women.PCOS women with normal FT4 and endocrine metabolic characteristics are more prone to be different between the SCH group and the euthyroid group when setting 2.5 mU/L as a TSH cutoff for SCH,indicating that 2.5 mU/L is a good TSH cutoff for SCH in PCOS women.

Objective: To explore the inlfuence of diastolic iflling pattern on cardiac resynchronization therapy (CRT) in patients with ischemic cardiomyopathy.
Methods: A total of 61 patients with ischemic cardiomyopathy received CRT in our hospital from 2012-03 to 2014-03 were studied. According to pre-CRT diastolic iflling pattern, the patients were divided into 2 groups:Non-restrictive iflling (NRF) group, n=36 and RF group, n=25. All patients were followed-up for 12 months, based on NYHA classiifcation, CRT efifcacy was assessed by echocardiography;the endpoints included re-hospitalization for heart failure or cardiac death. Kaplan-Meier survival curve was used to assess the prognosis.
Results: ①NRF group had CRT response rate at 66.7%(24/36) which was higher than RF group 28.0%(7/25), (χ2=8.826, P=0.003);the post-operative NYHA classiifcation, LVEF, FS, LVEDV and LVESV were signiifcantly improved, all P<0.01.② RF group showed the improved post-operative NYHA classification, P<0.01, while no obvious changes of LVEF, FS, LVEDV at 6 months after operation, and LVESV increased than it was before, P<0.05. Signiifcant differences were observed between 2 groups at 6 months after operation, P<0.01. Logistic regression analysis indicated that diastolic iflling pattern was the independent impact factor for CRT response. There were 2 patients died during 12 months of follow-up period;the endpoints in RF group was 76.0%(19/25) which was higher than NRF group 44.4%(16/36), (χ2=5.213, P=0.022).
Conclusion: Diastolic iflling pattern affected CRT efifcacy in patients with ischemic cardiomyopathy;NRF patients were more beneifciary for CRT, while RF patients had lower response to CRT which associated to poor prognosis.

Objective:To investigate the role of Th1/Th2 shift in unexplained recurrent spontaneous abortion (URSA) and to study the effect and molecular mechanism of Bushen Guchong decoction on URSA treatment by regulating Th 1/Th2 paradigm.Methods:30 cases of URSA patients were given Bushen Guchong decoction administration ; meanwhile,30 cases of normal pregnancy women were taken as control group.Th1/Th2 paradigm in peripheral blood monocytes of URSA patients before and after Bushen Guchong decoction administration was detected by flow cytometry.Moreover,the mRNA expression of the specific transcription factors for Th1 and Th2 subsets -T-bet and GATA-3 were detected by RT-PCR assay.Furthermore,the relationship between the Th1/Th2 paradigm and the uterine bleeding time after Bushen Guchong decoction administration was analyzed by Pearson correlation and re -gression tests.Results: Compared with normal pregnancy women , the proportion of Th1 subset of URSA patients was increased significantly (P<0.05),while the proportion of Th2 subset was decreased markedly (P<0.05).Additionally,the ration of Th1/Th2 subsets was increased in URSA women (P<0.05).After Bushen Guchong decoction treatment ,the proportion of Th1 subsets in URSA patients and T-bet mRNA expression were up-regulated significantly ( P<0.05 ) , whereas the Th2 proportion and GATA-3 mRNA expression were down-regulated obviously (P<0.05).Moreover,the ratio of Th1/Th2 was decreased significantly (P<0.05).Correlation analysis showed that the uterine bleeding time was positively related to both the proportion of Th 1 and the ratio of Th1/Th2, while negatively related to the proportion of Th 2 subsets.Conclusion:Th1/Th2 shift was involved in URSA.Bushen Guchong decoction up-regulated the transcription level of GATA-3 and down-regulated the transcription level of T-bet.Thereby, Bushen Guchong decoction exerted the curative effect on URSA by revising Th 1/Th2 shift.

High-affinity α-glucosidase inhibitors were screened from Cichorium glundulosum Boiss.et Hout seed (CGS) extract by ultra-filtration affinity-liquid chromatography-mass spectrometry (UF-LC-MS) and molecular docking.By taking 4-nitrobenzene-α-D-glucopyranoside (PNPG) as substrate and acarbose as positive control to evaluate the inhibitory activity of CGS extract, IC50 of acarbose and CGS extract were 0.003 mg/mL and 0.447 mg/mL, respectively.Meanwhile, 4 compounds from CGS extract by UF-LC-MS were screened and identified.Then by using autodock software, the compounds that combined with α-glucosidase were well screened out, including chlorogenic acid and isochlorogenic acid A.The inhibitory activity of chlorogenic acid and chlorogenic acid A against α-glucosidase was verified in vitro.The results showed that the inhibitory activity of the compounds toward α-glucosidase presented the sequence of acarbose>isochlorogenic acid A>chlorogenic acid.The inhibition rate of isochlorogenic acid A was close to acarbose.The experimental results illustrated that UF-LC-MS and molecular docking could be used to screen high affinity enzyme inhibitors from CGS.