Indirect Mixed Antiglobulin Reaction (MAR) as a Screening Procedure for Antisperm Antibodies. II. Clinical Studies

ABSTRACT: In this study the question was raised whether the indirect mixed antiglobulin reaction (MAR) using donor spermatozoa could substitute the direct MAR requiring motile spermatozoa in the patient's ejaculate in the screening for antisperm antibodies. Serum and seminal plasma from 48 men selected on the basis of the results in the direct MAR were tested by the indirect MAR. Both tests were carried out for IgG and IgA antisperm antibodies. A statistically significant correlation was found both between the sperm-bound IgG and the free IgG antisperm antibodies in serum as well as in seminal plasma and between sperm-bound IgA and the free IgA antisperm antibodies in seminal plasma whereas no correlation was found between the sperm-bound IgA and the free IgA antisperm antibodies in serum. This finding strongly supports the concept that the major part of the IgA antisperm antibodies in the ejaculate are locally produced.     

Indirect Mixed Antiglobulin Reaction (MAR) as a Screening Procedure for Antisperm Antibodies. I. Methodological Studies

ABSTRACT: Direct mixed antiglobulin reaction (MAR) cannot always be carried out because it requires motile spermatozoa in the patient's ejaculate. Therefore, the indirect MAR for IgG and IgA using donor spermatozoa sensitized with serum or seminal plasma from patients was investigated with the aim of developing a practical screening procedure for antisperm antibodies involving a characterization of the Ig-class of the antibodies. Samples from 23 men, 13 men with and 10 men without agglutinating antisperm antibodies, were used for the methodological experiments with the indirect MAR. Among three procedures to diminish the amount of nonbound immunoglobulin after sensitizing the donor spermatozoa with serial dilutions of serum or seminal plasma, a swim-up method was found to be optimal. The indirect MAR was carried out with glutaraldehyde-fixed sheep erythrocytes coated with IgG or IgA and various dilutions of anti-IgG and anti-IgA, respectively. These experiments led to a standardization of the indirect MAR, making it sensitive, easy to read, and allowing the reactions for IgG and IgA to be compared on an approximately equimolecular basis.     

Multivariant Analysis of Men from Infertile Couples With and Without Antisperm Antibodies

PROBLEM: Research studies in animal and human systems have demonstrated conclusively that antisperm antibodies can interfere with fertilization. In the male, autoantibodies to sperm can be detected both in the sera and seminal plasma. METHOD OF STUDY: Ninety-seven men who were tested for antisperm antibodies as a part of an infertility evaluation were identified. Complete medical history was obtained, including information related to events suspected of being associated with antisperm antibodies. History of surgery (varicocele repair, hernia repair, and vas reversal) and infection (epididymitis, sexually transmitted disease, and orchitis) were compared with semen parameters (motility less than 60%, concentration less than 20 times 10⁶, and volume less than 2 cc). These were compared to antisperm antibody results of mixed agglutination reaction (MAR) and direct immunobead binding test (IBT) for immunoglobulin G (IgG). Statistical analysis was performed using Fishers exact two-tailed test. RESULTS: As expected, prior vas reversal was significantly associated with the presence of antisperm antibodies (P = 0.0002) by MAR or IBT with a fivefold increased relative risk (95% confidence interval, 1.97-12.38). Other surgeries manipulating the cord structures independent of vas reversal were not associated with antisperm antibodies (P = 0.09). Prior infections, independent of vas reversal, were significantly associated with antisperm antibodies by MAR (P = 0.04) with a 3.8-fold increased relative risk (95% confidence interval, 1.06–13.87) but not by IBT. Sperm concentration less than 20 times 10⁶, motility less than 60%, and a volume less than 2 cc were not associated with antisperm antibodies by MAR or IBT. CONCLUSION: These findings suggest that manipulation of the cord structures excluding the vas were not associated with antisperm antibodies; however, vas reversal and prior infection are significant risk factors for the development of antisperm antibodies.     

Comparison of the Immunobead Binding Test (IBT) and Immunospheres (IS) Assay for Detecting Serum Antisperm Antibodies

PROBLEM: The IBT is considered the gold standard of sperm antibody assays. This test uses polyacrylamide beads labeled with antiglobulins (anti-IgG, anti-IgA, and anti-IgM), which bind to the corresponding antibody on the sperm surface. The IS uses color-coded latex beads of uniform 3.0 μm size coated with the antiglobulins which can be viewed with brightfield light microscopy. The purpose of the present study was to compare the IBT and IS in an indirect test using human serum. METHOD: Serum specimens (n=42) were tested for the presence of antibody isotypes IgG, IgA, and IgM to sperm using the standard protocol for IBT and IS. Donor sperm was washed in BWW with 5% BSA and diluted to a final concentration of 50 times 10⁶ motile sperm/ml. The sperm were incubated with a 1:10 dilution of test serum for 30 min to 1 h at 37°C and then washed by three cycles of centrifugation. The sperm and beads (IBT, IS) were mixed on a glass slide, covered with a coverslip, and observed within 5 min. At least 100 motile sperm were counted and scored for bead binding. A specimen was considered positive if 20% or more of the sperm were coated with one or more beads. The data were analyzed using calculation of the non-parametric kappa statistic with correction for chance expected agreement, and by calculating the proportion of specific agreement between the two methods. RESULTS: The results are summarized in the following table: The IS was able to detect 94% of IgG antibodies, 91% of IgA antibodies, and 100% of Ig M antibodies. One serum specimen was IgG negative by IS (14% binding), but positive by IBT (20%). A second serum specimen was IgA negative by IS (16%) yet positive by IBT (29%). There were no false positives with the IS assay. Of the IgM positives (five of six) occurred alone and not with IgG or IgA, suggesting the necessity for testing all specimens also for IgM. CONCLUSION: Antisperm antibody test results obtained by the IS assay are in agreement with the results obtained with the IBT test. The Immunospheres are monodispersed, color coded, and can be visualized with brightfield microscopy.     

The Value of the Mixed Antiglobulin Reaction (MAR Test) as an Addition to Routine Seminal Analysis in the Evaluation of the Subfertile Couple

ABSTRACT: A mixed erythrocyte-spermatozoa antiglobulin reaction (MAR test) for IgG antibodies has been done on semen specimens supplied by the male partners in 720 subfertile couples during a two-year period. The test was possible in all except 69 patients (9.5%). Antisperm antibodies were detected in 48 (10%) of 484 men with normal sperm counts, 18 (23%) of 78 with low sperm motility, and 19 (15%) of 128 with low counts. In 204 patients, antisperm antibodies were also measured by serum sperm-agglutination tests: The results showed a highly significant correlation with the results of MAR testing. It is concluded that the MAR test should be a routine part of seminal analysis, since the presence of IgG antisperm antibodies can be established in about 10% of men who might otherwise be passed as normal, and such antibodies can be positively excluded in a further 78% of the male partners of infertile marriages.     

An Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection and Quantitation of Antisperm Antibodies

ABSTRACT: Several recent comparative investigations using various assays to detect and quantitate levels of antibody to human spermatozoa have produced widely varying results. In an attempt to reduce test variability, an indirect enzyme-linked immunosorbent assay (ELISA) was devised to measure antisperm antibodies. A standardized protocol was adapted employing sperm adsorption to polystyrene microtiter plates, at a density of 10⁵ sperm per well, serum and enzyme-conjugate incubation conditions at 37°C for 60 min, and three ten-minute washes between incubations using phosphate-buffered saline containing Tween-20. Using antihuman sperm antisera generated in rabbits, the ELISA was shown to yield significantly detectable antibody at dilutions of 1/16,384. The ELISA demonstrated approximately 89% reproducibility (ie, 100% minus the coefficient of variation) for an “intraassay” study wherein 300 determinations were performed on the same day on sperm from ten donors. However, when sperm from one donor were used in 30 determinations during ten assays over a six-month period, “interassay” reproducibility was approximately 51%. The ELISA was compared with macroagglutination, microagglutination, and immobilization tests, using rabbit antisperm serum and human sera from vasectomized males. Results of this study indicated that the ELISA was more sensitive, less subjective, and easier to perform than these other commonly used techniques.     

Diagnostic Relevance and Clinical Significance of the New Enhanced Performance M2 (MIT3) ELISA for the Detection of IgA and IgG Antimitochondrial Antibodies in Primary Biliary Cirrhosis

Background/aims: Antimitochondrial antibodies (AMAs) are the serological hallmark of primary biliary cirrhosis (PBC). We evaluated the sensitivity and specificity of a new M2 enhanced performance enzyme-linked immunosorbent assay (ELISA) (MIT3) for the detection of IgG- and IgA-specific isotypes of AMA in PBC patients including a number of PBC patients negative for AMA by indirect immunofluorescence (IIF) as well as in patients with diverse, non-PBC disorders. We also investigated the clinical significance of IgG and IgA AMA in PBC. Methods: One hundred and three Greek PBC patients including 27 with AMA IIF-negative at the time of the investigation, 29 with autoimmune hepatitis-1 (AIH-1), 12 with primary sclerosing cholangitis (PSC), 26 with hepatitis C virus (HCV), 15 with hepatitis B virus (HBV), and 29 healthy were investigated for AMA (IgG and IgA) using the MIT3-based ELISAs (INOVA Diagnostics, San Diego, CA). The samples were also tested by conventional anti-M2 ELISA (INOVA Diagnostics, Inc.). Results: The IgG MIT3-based ELISA significantly increased AMA detection in the cohort of PBC patients, over 26% of whom were AMA IIF-negative, from 63.1% by the conventional anti-M2, and 73.7% by IIF to 79.6% by MIT3-based ELISA (p<0.001). IgA AMAs were detected in 47.6% patients. Overall, IgG/IgA AMAs were detected in 84/103 (81.6%). IgG MIT3-based ELISA detected 12/27 IIF AMA-negative samples (44.4%), while IgG/IgA MIT3-based ELISAs detected 13/27 IIF AMA-negative patients (48.1%). The specificities of MIT3-based ELISAs (IgG and IgA) were 82.8% and 89.7%, respectively, in AIH-1, 100% and 93.3%, respectively, in HBV, 100% in PSC, and 96% and 93.3%, respectively, in HCV. Patients positive for IgG AMA had significantly more severe disease as shown by worse histology and elevated biochemical markers; IgG and IgA AMA titers were associated positively with the Mayo risk score but none of the isotypes were able to predict disease outcome. Conclusions: The new IgG and IgA MIT3-based ELISAs seem to have higher specificity and sensitivity for AMA detection than IIF and the conventional anti-M2. Interestingly, these assays were able to unmask AMA presence in almost half of the AMA-negative samples by IIF. These findings may suggest the use of MIT3-based ELISAs as first-line investigation for AMA detection, particularly, when the laboratories are unfamiliar with the use and interpretation of the IIF patterns of AMA. The presence of IgG AMA seems to characterize PBC patients with more severe disease, but both IgG and IgA isotypes of AMAs were not predictive markers of disease outcome.     

Effects of the Reaction Conditions on the Syndiospecific Polymerization of Propene Promoted by Bis(phenoxyimine) Titanium Catalysts

Summary: The propene polymerization behavior of two typical bis(phenoxyimine) titanium catalysts has been investigated by varying reaction conditions, such as the monomer concentration, the solvent, and the cocatalyst. The experimental results indicate that the stereoregularity and regioregularity of the obtained poly(propylene)s are significantly affected by the reaction conditions. Fractionation of some poly(propylene) samples indicates the formation of macromolecules of different stereoregularity in the same run, suggesting that different active complexes can be generated in situ from these bis(phenoxyimine) titanium precatalysts.

     

ELISA法和RT-PCR法在麻疹病毒检测中的应用比较分析

目的 探讨酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和实时荧光定量PCR(quantitative real-time PCR,RT-PCR)对疑似麻疹病例的麻疹病毒的检测,为疾病的尽早确诊及治疗提供实验依据.方法 以上海市宝山区2014年4月至2015年12月的250例疑似麻疹病例为研究对象,采集其血清标本和咽拭子标本,分别应用ELISA法检测患者血清中的麻疹特异性的免疫球蛋白M(immunoglobulin M,IgM)抗体和RT-PCR法检测咽拭子中麻疹病毒的核酸.结果 250例疑似麻疹病例,排除风疹病毒阳性的34例病例,其中麻疹病毒(measles virus,MV) IgM抗体呈阳性的有80例,阳性率为37.04％;MV RT-PCR呈阳性的有128例,阳性率为59.26％.RT-PCR的检测阳性率显著高于IgM检测(x2=41.14,P＜0.001).结论 RT-PCR法可快速诊断麻疹病毒的病原学,且对出疹初期麻疹IgM呈阴性的病例可进～步确诊,是一种有效可靠的方法.     

慢性结膜炎患者泪液中腺病毒与单纯疱疹病毒的检测分析

目的 探讨腺病毒和单纯疱疹病毒在慢性结膜炎患者泪液中的分泌情况,为临床诊断提供参考.方法 收集慢性结膜炎患者、急性病毒性结膜炎患者及健康体检者的泪液标本,采用聚合酶链反应(PCR)法检测三组患者泪液标本腺病毒和单纯疱疹病毒核酸,并进行对比分析.观察腺病毒或单纯疱疹病毒阳性患者的临床症状.结果 慢性结膜炎组标本中腺病毒与单纯疱疹病毒核酸阳性率分别为48.5％(33/68)和17.6％(12/68).慢性结膜炎组有明确既往急性结膜炎病史的患者中腺病毒核酸阳性率为62.5％ (15/24),高于无既往病史组(20.0％),差异有统计学意义(P＜0.01);有既往病史的慢性结膜炎患者的单纯疱疹病毒感染阳性(29.2％)率亦高于无既往病史者(15.0％)(P＜0.05).急性结膜炎组患者的泪液中腺病毒和单纯疱疹病毒的核酸阳性率分别为100.0％(15/15)和86.7％(13/15),健康对照组标本中两种病毒核酸的检测结果全部为阴性.眼红、眼痒、下睑滤泡是感染以上两种病毒感染的主要临床表现.结论 腺病毒和单纯疱疹病毒在慢性结膜炎患者中的阳性率差异不明显.但有既往感染史患者上述两种病毒的阳性率显著高于无既往感染史患者,下睑滤泡症状对评估是否存在病毒感染有一定的参考价值.     

Modulating the Activities of Human Mesenchymal Stem Cells (hMSCs) and C3A/HepG2 Hepatoma Cells by Modifying the Surface Characteristics of Poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx)

The new biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx) has a potential application in tissue engineering. The aim of this study was to present a deeper picture of the relationship between the cellular behavior and the surface characteristics of PHBHHx films. The pristine PHBHHx film was prepared by adopting the compression-molding method, and then the acrylic acid molecules were grafted on PHBHHx membrane surface by UV irradiation. The hydrophilic nature and surface roughness of various PHBHHx films were controlled by adjusting the acrylic acid concentration and the UV irradiation time. Although the surface characteristics of various PHBHHx films could not affect the metabolic activity of hMSCs, the performance of morphology of hMSCs was deeply affected by the hydrophilic nature and the orientation of surface scars. The hydrophilic nature would effectively improve the spread of hMSCs, and the orientation of surface scars would guide the growth direction of cytoskeleton (actin) inside hMSCs. In contrast, the behaviors of C3A/HepG2 hepatoma cells presented an opposite outcomes. Those surface characteristics were obviously associated with the performance of metabolic activity of C3A cells, but not with the morphology of C3A cells. Both hMSCs and C3A cells have unique cellular characteristics; therefore, their responses to environmental stimulations are significantly different.     

Effect of low-density lipoprotein buoyant density and cholesterol content on the formation of lipoprotein(a) particles

Lipoprotein(a) [Lp(a)] is a unique lipoprotein which resembles low-density lipoprotein (LDL) both in lipid composition and the presence of apolipoprotein B-100 (apo B-100). Lp(a) is, however, distinguishable from LDL by the presence of an additional glycoprotein apolipoprotein(a) [apo(a)], which is covalently attached to apo B-100 by a single disulfide bond. It is now generally accepted that Lp(a) assembly is a two-step process in which the initial non covalent interaction between apo(a) and apo B-100 is mediated by the weak lysine binding sites present in kringle IV types 6, 7 and 8 of apo(a). In the present study, we have investigated the effect of LDL heterogeneity on Lp(a) assembly in a group of 111 individuals. The three parameters of LDL composition assessed in this study were the cholesterol content, the apo B content, and the relative flotation rate (a measure of LDL buoyancy and thus size). We found no correlation between the size of LDL particles and the extent of Lp(a) formation; a weak negative correlation was observed between cholesterol content of LDL and Lp(a) formation (P=0.042). This may suggest a role for free (i. e., surface-associated) cholesterol in the ability of LDL to form Lp(a) particles. Received: 1 June 2001 / Accepted: 2 July 2001     

241例孕妇血清中人微小病毒B_(19)-IgM与其他TORCH抗体检测结果的分析与比较

本文报告241例孕妇血清中人微小病毒B19－IgM及其他TORCH抗体（包括风疹病毒－IgM、巨细胞病毒－IgM、单纯疱疹病毒Ⅱ－IgM及弓形体抗体）的检测结果．其中B19－IgM阳性率为7．05％，与其余四项抗体阳性率比较，均呈显著性差异，表明我地区孕妇中人微小病毒B19。较其余四项TORCH因子有着较高的新近感染率，从而提示有必要将B19－IgM列为孕妇TORCH监测项目之一。     

12(S)-HETE对大鼠系膜细胞和肾小球内p27~(kip1)表达的影响

目的:探讨12-脂氧化酶的活性代谢产物12(S)-HETE对系膜细胞和肾小球内p27~(kip1)表达的影响.方法:12(S)-HETE刺激的系膜细胞和皮下包埋的微型渗透泵长期持续恒速注入12(S)-HETE刺激的大鼠提取的肾小球来观察p27~(kip1)的表达.采用总蛋白量/细胞数的比值作为细胞肥大的评价指标,利用RT-PCR和Western blot方法检测p27~(kip1) mRNA和蛋白的表达.结果:12(S)-HETE刺激能够直接诱导系膜细胞发生肥大.12(S)-HETE刺激的系膜细胞内p27~(kip1)mRNA水平没有变化,而p27~(kip1)蛋白的表达明显增加.然而,在微型渗透泵持续恒速注入12(S)-HETE刺激的大鼠提取的肾小球内p27kip1mRNA水平和蛋白的表达均增加.结论:12(S)-HETE能够通过促进p27~(kip1)的高表达而参与细胞周期的调控,是引起肾小球系膜细胞和肾小球肥大、衰老的重要原因之一.     

Investigation of the Physical Properties of Poly(ortho‐ and para‐trifluoromethoxy styrenes)

Ortho and para ‐OCF₃ substituted styrenes are prepared by the Grignard cross‐coupling reaction between ‐OCF₃ substituted bromobenzene and vinyl bromide and further polymerized in bulk using benzoyl peroxide. The glass‐transition temperatures (T_g) are 80 °C and 73 °C for the ortho and para –OCF₃ substituted polystyrenes, respectively, which are much lower compared with polystyrene (100 °C). The size of the ‐OCF₃ group is large and the ether bond is flexible; thus, the ‐OCF₃ substituted polystyrenes have larger free volumes, which has more effect on the T_g value compared with that of the steric hindrance effect. The refractive indices are 1.4908 and 1.4809 at 650 nm for ortho and para –OCF₃ substituted polystyrenes, respectively, which are much lower than that of polystyrene (1.5861) due to the presence of fluorine atoms in the polymer unit.

     

唾液SIgA、溶菌酶含量与慢性支气管炎关系的探讨

目的：探讨了慢性支气管炎患者唾液SIgA和溶菌酶含量的变化及临床意义。方法：应用放射免疫分析检测38例慢性支气管炎患者唾液SIgA含量,免疫扩散法测定溶菌酶含量,并与35名正常人作比较。结果：慢性支气管炎患者唾液SIgA和溶菌酶含量非常显著地高于正常人组（P〈0．01）,经2周治疗后仍有显著性差异（P〈0．05）。结论：检测慢性支气管炎患者唾液SIgA和溶菌酶含量的变化对临床观察预后有重要的临床价值。     

脉波指示剂连续心排血量(PiCCO)中的胸内血容量(ITBV)测定

在新近推向临床的脉波指示剂连续心排血量(PiCCO)监测中,用单一温度稀释法所测定的胸内血容量(IT-BV),已被许多学者证明是一项可重复、又敏感,而且比肺动脉阻塞压(PAOP)、右心室舒张末期压(RVEDP)、中心静脉压(CVP)更能准确反映心脏前负荷变化的实用指标.本文从心脏生理学、病理生理学及临床应用等方面,系统阐述了前负荷的定义及其影响因素,列举了上述压力监测数据不能准确反映心脏前负荷的机理及临床报道.详细讲述了单一温度稀释法测定ITBV的原理、计算方法、修正公式、测定中注意事项、正常范围值.列举了ITBV作为心脏前负荷的具有代表性的实验与临床统计分析资料.有鉴于左心功能减退伴有中度容量不足病人.恒速补液过程中显示充盈压比ITBV更敏感的报道,笔者建议:临床工作者在应用PiCCO新技术、确认ITBV作为前负荷指标的同时,仍然不应该忽略相关心血管腔的传统压力监测.     

Bioartificial materials based on collagen: 2. Mixtures of soluble collagen and poly(vinylalcohol) cross-linked with gaseous glutaraldehyde

Thin films of both pure soluble collagen (CLG) and poly(vinylalcohol) (PVA) and mixtures of the two, ranging from 20-80% PVA composition were studied to test the effects of PVA content and of glutaraldehyde vapour cross-linking. Both the thermal and mechanical behavior and, in addition, proteolytic stability were clearly influenced by the ratio of CLG/PVA. The experimental results indicate that no thermodynamic compatibility occurs between the two homopolymers. However, there is evidence that strong interactions, probably due to hydrogen bond formation, occur between the biological and synthetic polymers. The interactions appear stronger in those films with a lower PVA content and which were not cross-linked. Both the thermal and biological stability are increased and there is an improvement of the mechanical properties. The mutual intermolecular influence appears to allow the attainment of a good mechanical compatibility between CLG and PVA.     

妊高征患者外周血红细胞免疫功能的变化和血浆TXB2水平的相关性分析

目的:探讨妊高征患者外周血红细胞免疫功能的变化和血浆TXB2水平的相关性.方法:应用放射免疫分析和免疫法对33例妊高征患者进行红细胞免疫功能和血浆TXB2检测,并与35名正常人作比较.结果:妊高征患者RBC-C3bRR水平明显降低(P<0.01),而血浆TXB2水平显著升高(P<0.01),RBC-C3bRR与TXB2...