Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization （WHO） manual-based semen parameter＇s concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay （SCSA）, a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection （ICSI）.

牛膝中齐墩果酸含量测定方法的研究

本文报道怀牛膝中齐墩果酸的薄层扫描测定法。用氯仿－甲醇－甲酸（２０：１：０．２）为展开剂，在硅胶Ｇ－ＣＭＣ－Ｎａ薄层板上分离齐墩果酸，１０％硫酸乙醇溶液显色，用ＣＡＭＡＧ Ⅱ型薄层扫描仪进行定量测定。测定参数：反射法单波长（λ＝５３０ｍｍ）锯齿扫描，背景补偿，线性参数ＳＸ＝３，狭缝０．６ｍｍ×０.６ｍｍ。同时测定了怀牛膝生品中齐墩果酸的含量为１．８４％。

仓储小麦中脱氧雪腐镰刀菌烯醇检测技术的优化

脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)是小麦仓储过程中的重要危害因子,其污染水平是仓储公司定期检测的重要指标。为提高DON检测的平行性和准确度,文章对仓储小麦DON检测过程中样品制备和酶联免疫吸附法(ELISA)检测关键技术进行优化。结果表明:样品制备过程中,将扦样后的样品先粉碎再分样的处理方法(方法 2)明显优于先分样再粉碎的方法(方法 1),测定样品的相对标准偏差由25.28%降至7.42%。另外,将ELISA检测过程中两种不同的移液枪使用方法(前进移液法和反向移液法)进行比较,发现前进移液法在10次测定中结果相对标准偏差较小,平行性较好;且加样过程中,采用不贴壁加样方式会使检测值更加准确,平行性也较好。优化后的仓储小麦中DON毒素ELISA快速检测技术具有较好的平行性和准确度,可用于实际仓储小麦中DON毒素的准确、快速检测。

Trends in feed evaluation for poultry with emphasis on in vitro techniques

Accurate knowledge of the actual nutritional value of individual feed ingredients and complete diets is critical for efficient and sustainable animal production.For this reason,feed evaluation has always been in the forefront of nutritional research.Feed evaluation for poultry involves several approaches that include chemical analysis,table values,prediction equations,near-infrared reflectance spectroscopy,in vivo data and in vitro digestion techniques.Among these,the use of animals(in vivo)is the most valuable to gain information on nutrient utilization and is more predictive of bird performance.However,in vivo methods are expensive,laborious and time-consuming.It is therefore important to establish in vitro methods that are reliable,rapid and practical to assess the nutritional quality of feed ingredients or complete diets.Accuracy of the technique is crucial,as poor prediction will have a negative impact on bird performance and,increase feed cost and environmental issues.In this review,the relevance and importance of feed evaluation in poultry nutrition will be highlighted and the various approaches to evaluate the feed value of feed ingredients or complete diets will be discussed.Trends in and practical limitations encountered in feed evaluation science,with emphasis on in vitro digestion techniques,will be discussed.

Analysis of immune responses against H pylori in rabbits

AIM： To investigate the immunogenicity of H pylori proteins, to evaluate the production rate of anti H pylori IgG antibodies in relation to time and to demonstrate the fidelity of newly optimized in-house enzymelinked immunosorbent assay （ELISA） technique as an alternative for H pylori infection assay. METHODS： In the present study, 100 μg of formalinfixed H pylori whole cell antigens was injected into an experimental animal （New Zealand white female rabbit） intramuscularly on d 0, 16, 27 and 35. The first two doses were injected with adjuvants. On d 0, a serum sample was collected from the rabbit before immunization and this pre-immunized serum was used as a negative control for the whole study. To evaluate the immunogenic responses of the injected antigen, serum samples were collected from the rabbit at regular intervals up to d 42. The sera were analyzed using inhouse ELISA and Western blot techniques.RESULTS： The production of anti Hpylor/IgG antibodies in the rabbit in response to the injected antigen increased almost exponentially up to d 14 and after that it was maintained at the same level until the last day （d 42）. By analyzing the immune profiles of immunized sera, 11 proteins were identified to be immunogenic, among them 2 （approximately 100 kDa and 85 kDa） were most prominent. CONCLUSION： Analysis of the immune responses against pathogenic microorganisms like H py/ori is necessary for the development of various diagnostic and preventive approaches. The results of this experiment reveal that the formalin-fixed H pylori whole cell antigens injected into the rabbit are highly immunogenic. These prominent proteins （approximately 100 kDa and 85 kDa） might have higher immunogenic effects among humans infected with H pylori and some of these immunogenic proteins can be included in diagnostic approaches based on serology and also for vaccine formulation. The in- house ELISA is a promising alternative compared to invasive techniques.

Field Evaluation of Alternative Testing Strategies for the Detection of HIV Infection in Beijing

To identify a cost-efficient altemative antibody testing strategy for screening and confirmation of HIV infection by rapid simple tests （RSTs） and enzyme-linked immunosorbent assays （ELISAs）. Methods Four RSTs （RST1, RST2, RST3, and RST4 ） and five ELISAs （ELISA1, ELISA2, ELISA3, ELISA4, and ELISA5） were evaluated in two phases by using banked and serum specimens prospectively collected at regional hospitals and voluntary counseling and testing （VCT） centers in Beijing. A total of 200 banked serum specimens were included in the first phase, including 62 HIV-positive, 127 HIV-negative and 11 indeterminate specimens. All specimens were tested by four RSTs and five ELISAs respectively. The second phase involved prospective testing of 389 routine specimens, including 92 HIV-positive, 287 HIV-negative, and 10 indeterminate specimens. All the specimens were tested by two RSTs （RST2 and RST4） and three ELISAs （ELISA1, ELISA3, and ELISA4）, which were selected for their respective excellent sensitivity and/or specificity. Westem blot （WB） was used as a gold standard for confirming the reactivity of all the specimens. Results Sensitivity, specificity, and efficacy were calculated for each assay in two phases. In the first phase, four assays （ELISA4, RST2, RST3, and RST4） had a specificity of 100%. For the determination of efficacy, ELISA4, RST2, and RST4 were selected in the second phase. ELISA1 and ELISA3 which have a sensitivity of 95.9% and 93.2% respectively also entered this phase. In the second phase, all the five assays （ELISA1, ELISA3, ELISA4, RST2, and RST4） had a sensitivity and specifity of over 90%. ELISA1 had a sensitivity of 99% and ELISA4 a specificity of 99%. Conclusion The sensitivity ELISA1 and the specificit of ELISA4 are comparable to ELISA/WB standard strategy. Application of this alternative testing strategy provides a cost-effective method for determining HIV prevalence in Beijing.

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years-implications for clinical transplantation and stability programs

BACKGROUND Peripheral blood stem cells(PBSC)are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant.Long term cryopreservation is commonly defined as five years or longer,and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft.Clinical programs,stem cell banks,and regulatory and accrediting agencies interested in product stability would benefit from such data.Thus,we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2 Rγnull(NSG)mice.AIM To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units.METHODS PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health.These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice,and the pre-freeze and post-thaw characteristics of the units were compared.Progenitor function was assessed using standard colony-forming assays.CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function.RESULTS Ten PBSC units with mean of 17 years in cryopreservation(range 13.6-18.3 years)demonstrated a mean total cell recovery of 88%±12%(range 68%-110%)and post-thaw viability of 69%±17%(range 34%-86%).BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw.Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units.All mice demonstrated long-term engraftment at 12 wk with mean34%±24%human CD45+cells,and differentiation with presence of human CD19+,CD3+and CD33+cells.Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies.CONCLUSION We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice,signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Evaluation of purified recombinant spike fragments forassessment of the presence of serum neutralizing antibodiesagainst a variant strain of porcine epidemic diarrhea virus

Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization(SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay(ELISA) results(involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics(ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient(r) and the area under curve(AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains(including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.

Accuracy of a new rapid diagnostic test for urinary antigen detection and assessment of drug treatment in opisthorchiasis

Background Screening for opisthorchiasis,a parasitic worm infection affecting many millions of people in Southeast Asia,has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique(FECT)and Kato-Katz method.Although the urinary enzyme-linked immunosorbent assay(ELISA)has been used more recently,we developed a urinary antigen-based rapid diagnostic test(RDT)to simplify diagnosis and as a point-of-care testing(POCT)and field applications for surveillance and control of opisthorchiasis.Methods A urinaryOpisthorchis viverrini(OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV.The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA(n=493).Cross-reactivities of urinary OV-RDT with other helminthiases coexisted withO.viverrini were determined(n=96).A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis(n=1629).The McNemar chi-square,Kruskal-Wallis and Cohen’s kappa coefficient(κ-value)tests were used for statistical analyses.Results Urinary OV-RDT had sensitivity of 94.2%and specificity of 93.2%,compared to faecal FECT.Urinary OV-RDT had high diagnostic agreement(Kappa=0.842-0.874,P<0.001)and quantitative correlation with urinary antigen ELISA(Kruskal-Wallis tests=316.2,P<0.0001)and faecal FECT(Kruskal-Wallis tests=362.3,P<0.0001).The positive rates by OV-RDT,ELISA and FECT were 48.9%,52.5%and 49.3%,respectively.Cross-reactions of urinary OV-RDT with other helminthiases were few(2%).Field trials of urinary OV-RDT yielded comparable prevalence ofO.viverrini between urinary OV-RDT(53.2%)and urinary antigen ELISA(54.0%).OV screening showed high diagnostic agreement(kappa>0.8,P<0.0001)between urinary OV-RDT and urinary antigen ELISA.The cure rates of opisthorchiasis at 1 month post-praziquantel treatment determined by urinary OV-RDT(86.6%)and urinary antigen ELISA(80.5%)were similar(P>0.05).Conclusions The urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis.The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening,control and elimination of opisthorchiasis,thereby contributing to a reduction in the disease burden in Southeast Asia.