In vitro bioassays for androgens and their diagnostic applications

Androgen levels are measured in today's clinical practice almost exclusively by immunoassays. The androgen that is most frequently determined is testosterone (T), but sometimes also the levels of other testicular, ovarian and adrenal androgens such as 5alpha-dihydrotestosterone, androstenedione, dehydroepiandrosterone and its sulphate may be determined. In many instances, especially when androgen levels are low (as in women and children), the quality of the immunomeasurements is insufficient and the correlation between hormone levels and clinical symptoms is poor. One alternative to improve the clinical relevance of androgen measurements is provided by the recently developed in vitro bioassays of total androgen bioactivity in serum. These assays are not yet ready for routine laboratory diagnostics, but they provide a useful tool for clinical research in disturbances of androgen production. Another application of these assays is the screening for androgenic and antiandrogenic activity in chemical compounds, environmental samples and when suspecting androgen abuse. The purpose of this article is to introduce the current problems of androgen measurement by immunoassays, to describe the novel in vitro bioassay techniques and to review the current information on their application in clinical research.     

Laboratory diagnosis of heparin‐induced thrombocytopenia

Heparin‐induced thrombocytopenia (HIT) is a clinical‐pathological disorder; thus, laboratory testing for the pathogenic platelet‐activating antiplatelet factor 4 (PF4)/heparin antibodies is central for diagnosis. The “iceberg” model summarizes the inter‐relationship between platelet activation assays and PF4‐dependent immunoassays, with platelet‐activating antibodies comprising a subset of anti‐PF4/heparin antibodies. The platelet serotonin‐release assay (SRA), performed by reference laboratories, has high sensitivity and specificity for HIT (~95% each), and is especially suited for detecting highly pathogenic HIT sera containing both heparin‐dependent and heparin‐independent platelet‐activating antibodies; this latter subgroup of antibodies explains “autoimmune HIT” disorders (delayed‐onset, persisting, spontaneous, heparin “flush,” fondaparinux‐associated). Recently, SRA‐negative HIT has become recognized, in which serum from some HIT patients contains subthreshold levels of platelet‐activating antibodies (by SRA) that become detectable using a PF4‐enhanced platelet activation assay. Unusual immunologic features of HIT include early antibody detectability (at onset of platelet count fall) and antibody transience (seroreversion). Widely available PF4‐dependent enzyme immunoassays (EIAs) have high sensitivity but poor specificity for HIT, although specificity is enhanced with IgG‐specific EIAs and strong positive results; unfortunately, EIA results are usually not available in real time. Automated rapid immunoassays, such as the chemiluminescence immunoassay (CLIA) and latex immunoturbidimetric assay (LIA), facilitate real‐time laboratory diagnosis. Recently available likelihood ratio (LR) data for positive (LR+) and negative (LR?) test results allow clinicians to adjust their pretest probabilities for HIT, using Bayesian analysis, into real‐time posttest probabilities that are dramatically increased (test positive) or decreased (test negative). Moreover, (semi‐)quantitative CLIA‐ and LIA‐positive results (weak, moderate, strong positive) can further refine the posttest probability of HIT.     

Detectability of fentanyl and designer fentanyls in urine by 3 commercial fentanyl immunoassays

In recent times, structural variants of fentanyl (designer fentanyls) have appeared on the recreational drug market for new psychoactive substances (NPS). These potent opioids have caused harmful intoxications and increased opioid‐related mortality in many countries. This work evaluated 3 commercial immunoassays for fentanyl screening in urine and investigated whether they are useful also for screening of designer fentanyls. The assays examined were the Thermo DRI® Fentanyl Enzyme Immunoassay, the ARK? Fentanyl Assay homogeneous enzyme immunoassay, and the Immunalysis® Fentanyl Urine SEFRIA? Drug Screening Kit. A liquid chromatography–high‐resolution mass spectrometry method was used as reference. The DRI fentanyl immunoassay generated somewhat higher assay imprecision values (%CV) compared with the ARK? and SEFRIA? assays, but all assays showed %CV values acceptable for routine use. The 3 assays showed overall good detectability (33%–95% cross‐reactivity) for blank urine samples spiked with acetylfentanyl, acrylfentanyl, butyrfentanyl, 4‐chloroisobutyrfentanyl, 4‐fluorobutyrfentanyl, 4‐fluorofentanyl, 4‐fluoroisobutyrfentnyl, isobutyrfentanyl, methoxyacetylfentanyl, or tetrahydrofuranfentanyl, whereas 4‐methoxybutyrfentanyl (all assays) and 2‐fluorofentanyl (DRI assay) showed low cross‐reactivity. A good detectability of designer fentanyls was confirmed in urine samples from authentic acute intoxications. In conclusion, the present results demonstrate that the urinary fentanyl immunoassays are generally useful also for preliminary screening of fentanyl analogs sold as NPS. When the SEFRIA? assay was applied for testing of 980 urine samples from patients treated for drug dependence in Sweden, only 1 sample was confirmed positive for fentanyl.     

生化分析仪分析免疫五项检测的性能评价

目的:生化分析仪开展免疫五项检测的性能评价分析.方法:随机选取2019年2月~2019年8月于本院进行健康体检的40例健康体检人员作为观察对象,均无重大疾病史,身体健康.采集40例研究对象空腹状态下6mL静脉血,予以血清分离处理,把每位受检者的血清标本分别装在两个试管内,备用;采用生化分析仪(日立7600)检测血清五项免疫指标,按照检测结果对仪器的参考范围进行验证,选择传统方法对仪器开展免疫五项检验的精准度进行检验,根据国家卫生部室间质评结果评定免疫五项检测结果的正确度.结果:生化分析仪对IgA、IgG、IgM、补体C3以及补体C4进行检验,结果发现检验精准度都达到标准水平;检验正确度都达到标准;参考范围与厂家提供参考范围相符合.结论:生化分析仪(日立7600)开展免疫五项检测具有较好的精准度、正确度,性能较好.     

A simple and rapid assay to evaluate purity of foot-and-mouth disease vaccine before animal experimentation

Currently, foot-and-mouth disease (FMD) vaccine purity is tested in cattle to detect antibodies against the non-structural protein (NSP) after repeated immunization with the final vaccine product. In case of vaccine failure, the manufacturing company would suffer significant economic loss. To prevent such unfortunate losses with the final vaccine product, in vitro testing is required to quantitate an NSP antigen during the manufacturing process prior to animal experiments. A novel lateral-flow assay device was developed using a monoclonal antibody (MAb) against the 3B NSP. To determine the minimal amount of NSP required to elicit antibodies in livestock, goats were immunized several times with various concentrations of either the recombinant 3AB (rec.3AB) protein or FMD virus culture supernatant. Antibodies against 3AB were elicited after a second immunization with 10.6 ng to 42.5 ng of rec.3AB and a third immunization with a 10-fold diluted FMD virus culture supernatant in goats. The lateral-flow assay device detected the minimal amount of rec.3AB and native NSP in FMD virus culture supernatant required to induce NSP antibodies in goats. The in vitro assay device is simple and economical, provides rapid results, and should be useful for FMD vaccine-manufacturing companies prior to conducting animal experiments to test the vaccine purity.     

Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies

Antiphospholipid ELISAs are part of the Antiphospholipid Antibodies Syndrome classification criteria, having the same diagnostic value as lupus anticoagulant. However, sometimes their results appear scarcely meaningful especially when wide metanalyses studies are performed, probably because of their well-known inter-laboratory variability. The application of a common protocol was shown to improve the test reproducibility, but this observation did not have any influence on the routine performances.

After discussion among experts at the European level, we identified four conditions named “minimal requirements” considered useful to decrease the inter-laboratory variability:

(1) to run the samples in duplicate;

(2) to determine the cut off level in each laboratory analysing at least 50 samples from normal subjects, possibly age- and sex-matched with the patient population usually attending the Centre;

(3) to calculate the cut-off level in percentiles;

(4) to use stable external controls in the test.

A collaborative study involving 36 European centres proved that the use of monoclonal anti-beta2 glycoprotein I antibodies, HCAL (IgG) and EY2C9 (IgM) as standards, can help to reduce the inter-laboratory coefficient of variation both in anticardiolipin (aCL) and anti-β2GPI (anti-beta2 glycoprotein I) ELISA. Therefore, we propose HCAL and EY2C9 as external controls, but other monoclonal or polyclonal preparations may be considered.

During an interactive workshop held last May in Italy, 16 companies producing these tests agreed to consider the introduction of the “requirements” in their products. We suggest to adopt these “requirements” particularly in clinical studies, in order to compare more easily the literature data.     

Photoluminescence and chemiluminescence methods of drug analysis

The analysis of drugs in biological fluids requires sensitive, selective and convenient methods. Luminescence techniques provide extreme sensitivity and, alone or in conjunction with other methods, excellent selectivity. This paper assesses several recent developments in luminescence analysis, including the use of derivative and synchronous spectroscopy, luminescence immunoassays, room temperature phosphorimetry, and fluorescence excited by chemiluminescence energy transfer.     

雅培i4000快速测定血浆丙戊酸浓度及在儿科中的应用效果

目的通过评估丙戊酸钠（VPA）血浆浓度与疗效的关系,探讨用雅培i4000为儿科患儿提供VPA快速检测服务的可能性。方法收集122例患儿血浆,在i4000和AX—SYM上分别检测VPA浓度,比较VPA浓度与疗效的关系,然后按年龄和VPA浓度分组比较药物浓度分布规律,最后对两台仪器检测结果进行回归分析。结果VPA浓度50～100mg／L组和〈50mg／L组,总有效率分别为89％,66％,2组比较有显著性差异;不同年龄段患儿血浆VPA浓度分布不一致,5～10岁、〉10～15岁患儿在有效VPA浓度组内的例数显著高于0～5岁组;i4000和AXSYM结果回归方程为：y=2．3701＋0．949x,相关系数r=0．961。结论在雅培i4000上检测VPA血浆浓度操作简便、快捷,结果准确可靠,非常适合儿科患儿血药浓度的快速检测。     

空心莲子草有效部分的分离及抗病毒作用

采用铅盐沉淀法和两相溶剂萃取法提取空心莲子草的若干部分,进行抗病毒研究,结果表明石油醚,乙醚和醋酸乙酯提取物有抗EHFV作用。抗病毒有效成分为香豆精类化合物。     

Overcoming the challenge of diagnosis of early HIV infection: a stepping stone to optimal patient management

Prompt identification of individuals during the highly infectious acute or early stage of HIV infection has implications for both patient management and public health interventions. The studies on natural history of HIV infection over the last three decades have uncovered several clinical features and virological markers to diagnose early infection. However, the brevity of the acute symptomatic phase combined with the difficulty in identifying non-specific signs and symptoms poses diagnosis of early HIV infection as a remaining challenge. Furthermore, underestimation of risky behavior in the absence of detailed patient history and possible concurrent sexually transmitted infections render the diagnosis of recent infection difficult. Herein, we focus on the multifaceted clinical manifestations and the best usage of technological advancements to detect early HIV infection. Early diagnosis of HIV infection contributes to further improving patient outcomes and preventing transmission.