Preanalytical delay reduces sensitivity of QuantiFERON-TB gold in-tube assay for detection of latent tuberculosis infection

The effects of incubation delays on the accuracy of the QuantiFERON-TB gold in-tube assay (QFT-GIT) were measured. Compared to immediate incubation, 6- and 12-hour delays resulted in positive-to-negative reversion rates of 19% (5/26) and 22% (5/23), respectively. These findings underscore the need for standardizing QFT-GIT preanalytical practices.     

Impact of Blood Volume,Tube Shaking,and Incubation Time on Reproducibility of QuantiFERON-TB Gold In-Tube Assay

Gamma interferon (IFN-γ) release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-γ TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml; P = 0.002) or 1.2 ml (0.49 IU/ml; P < 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger proportions of false-negative results with 1.0 ml (29.4%; P = 0.2) and 1.2 ml (41.2%; P = 0.05) of blood for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-γ response in nil (0.04 versus 0.06 IU/ml; P < 0.001) and TB Ag (0.12 versus 0.24 IU/ml; P = 0.004) tubes and increased TB responses (TB Ag_vigorous minus nil_gentle) (0.02 versus 0.08 IU/ml; P = 0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.     

Reproducibility problems with the Abbott laboratories LCx assay for Chlamydia trachomatis and Neisseria gonorrhoeae

This study demonstrates that significant reproducibility problems can occur during routine use of the Abbott Laboratories LCx assay for Chlamydia trachomatis and Neisseria gonorrhoeae. These problems can go undetected by the quality control procedures outlined in the manufacturer's package insert. We outline here procedures for detecting and preventing contamination and reproducibility problems.     

Comparison of tuberculin skin test and QuantiFERON-TB gold in tube test in patients with chronic inflammatory diseases living in a tuberculosis endemic population

We aimed to compare tuberculin skin test (TST) and QuantiFERON-TB gold in tube (QFT-IT) in patients with chronic inflammatory diseases living in a population where tuberculosis is endemic and BCG vaccination is routine. Twenty-five patients with rheumatoid arthritis (RA), 16 patients with spondilarthropathy (SpA) and 7 healthy volunteers were recruited in the study. Blood samples were collected for QFT-IT test followed by TST. TST was considered to be positive with an enduration of 5 mm and over in patients with RA and SpA. The cutoff point for TST for the healthy control (HC) was 10 mm. Median TST result of patients who were using immunosuppressive drugs was significantly lower than that of patients who were not [5 (14.5), 13.5 (9.5), respectively, P = 0.04]. Poor agreement between TST and QFT-IT was seen in patients with RA (κ = 0.141) and in patients with SpA (κ = 0.190). We found poor concordance between QFT-IT and TST in patients with chronic inflammatory diseases, and TST response was suppressed in patients treated with immunosuppressive drugs.     

Reproducibility in quality control of protein (Western) immunoblot assay for antibodies to human immunodeficiency virus

The protein (Western) immunoblot assay (IB) for antibodies to human immunodeficiency virus, like other laboratory procedures, sometimes gives variable results. In the Transfusion Safety Study, indistinguishable aliquots of four quality control (QC) samples have been routinely submitted for IB with each group of patient specimens. A false negative IB result was obtained for 1.7% of 179 assays of two known positive (QC) standards and a false positive result for 2.0% of 101 assays of two known negative (QC) standards. In addition, a test panel of 24 samples was sent on a single occasion to three widely used laboratories. A false positive result was reported by one laboratory and a false negative by a second. Although generally reliable, IB results may occasionally be in error. There is much more technical variability in the relative frequencies of antigen-antibody bands than has been recognized. These interlaboratory and intralaboratory comparisons show quality control checks are essential for all laboratories, and more than one specimen should be tested if its applicability to a specific patient is questionable. Specific bands are sufficiently inconstant for the same specimen to make appearance or disappearance on successive specimens prognostically unreliable.     

点免疫金渗滤法筛选胶体金标记抗人IgG 的研究

目的:应用点免疫金渗滤法筛选适宜胶体金标记的抗人IgG(以下简称二抗),并运用多个评价指标进行比较和分析。方法:选择三个不同公司的二抗用胶体金标记,根据棋盘滴定法确定最佳标记条件;运用点免疫金渗滤法对三种二抗的胶体金标记后检测效能进行比较,采用包虫病人和健康对照血清,用包虫病特异性抗原检测包虫病患者血清中的特异性抗体,评价最优的二抗,并与酶联免疫吸附测定法进行比较。结果:A、B、C 三种二抗标记的最佳标记条件为:pH 均为8.5,加入量分别为38.4、24、19.2 g/ ml,综合评价二抗B 检测效能最优。将其与酶联免疫吸附测定法检测效能进行比较,两种方法检测结果的Kappa=0.895(P<0.05),吻合度较强。结论:应用点免疫金渗滤法,以及本文提出的评价指标,能够筛选出最佳二抗,对检测试剂盒中二抗的选择具有重要的参考价值。     

Development of a highly sensitive gold nanoparticle probe-based assay for bluetongue virus detection

A simple gold nanoparticle (GNP) probe based assay (GNPA) that was modified from a bio-barcode assay (BCA) technique, was developed for ultra-sensitive, rapid detection of the bluetongue virus (BTV) VP7 outer-core protein. This assay captures the VP7 target antigen using the GNP probe coated with anti-VP7 polyclonal antibodies and single-stranded signal DNA. Magnetic microparticle (MMP) probes coated with anti-VP7 monoclonal antibodies were then added to form a sandwich immuno-complex. The single-stranded signal DNA coated onto the GNP probe present in the immuno-complex could then be detected by PCR and real-time fluorescence PCR using a TaqMan probe. The assay has a purified VP7 detection limit of 10(-2)fg/ml which is 8 orders of magnitude greater than that of conventional antigen capture ELISAs and 1 order of magnitude more sensitive than that of a conventional BCA. These results indicate that the GNPA is a highly sensitive method for easy detection of BTV proteins and that it can be modified as needed to measure the presence of other proteins.     

Immediate Incubation Reduces Indeterminate Results for QuantiFERON-TB Gold In-Tube Assay

In vitro gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent Mycobacterium tuberculosis infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37°C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) (P = 0.10) and 17.1% (7/41) (P = 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h (P = 0.004) and 12 h (P < 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.Mycobacterium tuberculosis is among the leading infectious causes of death worldwide (12). Prophylactic treatment of individuals with latent infection is an effective strategy to prevent progression to active disease and further spread (9). After more than a century of using the tuberculin skin test (TST) for diagnosis of latent tuberculosis infection (LTBI), the development of T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) represents a significant advancement in the field of tuberculosis (TB) diagnostics (1, 14). These assays rely on the measurement of increased IFN-γ secretion by effector T cells which have previously been exposed to M. tuberculosis antigens (Ags) from an infection when the cells are stimulated in vitro with purified M. tuberculosis-specific antigens, such as the early secretory antigen 6 (ESAT-6) and culture filtrate protein 10 complex (CFP-10) (2). The advantages of IGRAs over TST include in vitro testing, which eliminates the boosting effect of TST; direct measurement of cell-mediated immune responses; the absence of cross-reactivity with M. bovis BCG vaccination; and the need for only one patient visit (10). The disadvantages of IGRAs include the requirement for laboratory infrastructure and, similar to TST, a lack of distinction between active and latent infection.A widely used commercial IGRA-based assay, the QuantiFERON-TB Gold assay (QFT-G), was approved for use by the FDA in December 2006. An in-tube version (QFT-IT), approved in October 2007, permits blood to be drawn directly into tubes precoated with antigens and incubated in the same tubes prior to IFN-γ measurement, thus minimizing the preanalytic processing of blood samples. Each QFT-IT consists of three tubes: (i) the TB antigen tube, containing the specific M. tuberculosis antigens, ESAT-6, CFP-10, and TB7.7; (ii) the mitogen control tube, containing nonspecific T-cell-stimulating antigens and serving as a positive control; and (iii) a nil control tube, containing no antigens and serving as a negative control. Each tube is inoculated with 1 ml of whole blood, and after vigorous shaking of the tubes, the tube set is incubated at 37°C for 16 to 24 h. According to the manufacturer''s instructions for use, initiation of the incubation can be delayed for up to 16 h. A quantitative enzyme-linked immunosorbent assay (ELISA) for IFN-γ is then performed and the results are interpreted as positive, negative, or indeterminate.Unambiguous positive and negative results are obviously desired, while indeterminate results, defined as an abnormally high level of IFN-γ in the nil tube (>8 international units [IU] per milliliter) or a low response in the mitogen tube (mitogen and nil tubes, <0.5 IU/ml), pose diagnostic challenges for clinicians and frustration for the individuals undergoing testing (7). The reported frequency of indeterminate results for QFT-IT ranges from 5% to 40% in all participants and 6% in health care workers (4, 8, 11). The manufacturer attributes these indeterminate results to technical error or the testing of patients with immunocompromised or hyperinflammatory states (3). The most common approach to resolving indeterminate results includes repeat testing, which requires a new visit and blood draw, which add to health care costs.Prior to large-scale implementation of QFT-IT as part of an employee screening program at our academic hospital (Stanford Hospital and Clinics), an in-house evaluation of the assay was performed. Prior studies using enzyme-linked immunospot (ELISpot) and whole-blood IGRAs showed that delays in processing and incubation of blood samples before stimulation with M. tuberculosis antigens result in a lower number of detectable IFN-γ-producing T cells (5, 6, 13). Thus, the focus of this study was to investigate the impact of incubation delay on QFT-IT results.     

Investigation of False-Positive Results Given by the QuantiFERON-TB Gold In-Tube Assay

We investigated a sudden increase in the rate of positive QuantiFERON-TB Gold In-Tube results from 10% to 31% at a U.S. academic institution. Direct comparison of the TB antigen tubes with tubes from a different lot number identified that a potential problem with the TB antigen vials in a certain tube lot was the likely cause of the elevated positive rate. The underlying defect remains unknown. This finding warrants refinement of quality control programs by the manufacturer and users.     

Reproducibility of skin tests

Fifty patients allergic to mite were tested for D. pteronyssinus using two prick-test techniques; the classical method by Pepys and the Morrow-Brown needle. Each patient received six tests with each method on the two forearms. The evaluation of the weal areas was conducted in two ways: by weighing the shapes of the tests transferred on to paper, and by measuring the two greatest diameter lengths. No significant difference was observed between the two methods. No significant difference was observed between the measurements of the same tests carried out at 2-monthly intervals by the same examiner. With the Pepys method the weal area sizes are significantly larger than with the Morrow-Brown needle, and the variance is much greater with the Pepys technique. However, the coefficients of variation are similar and in both cases the techniques are equally reproducible. No significant difference was found in the weal sizes with regard to the injection sites on the forearm with either of the two methods. The correlation between the weal area sizes and the log of the IgE levels is closer with the Morrow-Brown needle.     

A sensitive assay for cellular hypersensitivity based on the uptake of radioactive colloidal gold

The pinocytosis of radioactive colloidal gold by peritoneal exudate cells from guinea-pigs sensitized against tuberculin or ovalbumin was enhanced by specific antigen. The effect was mediated by soluble material (gold uptake stimulator (GUS) produced by lymphocytes and acting upon macrophages. GUS production began within the first 18 hours of culture and required 24 hours contact to maximally stimulate normal peritoneal exudate cells.

Comparison of the gold uptake and migration inhibition assays on identical populations of sensitive exudate cells showed in terms of minimum effective antigen dose that the gold uptake assay was considerably more sensitive. Similarly, when sensitive lymph node cells were incubated with antigen, activity could be detected in the culture supernatants at about sixty-four-fold greater dilution by the uptake of gold.

At low concentrations of GUS a log-linear dose response relationship held. At high concentrations, gold uptake levelled off or even fell, and this was true for concentrations of antigen greater than optimal when added to sensitive exudate cells.

Chromium uptake varied little at the concentrations of antigen and supernatant factors tested. Hence the effects of gold uptake are unlikely to be indirect effects on cell proliferation or viability.

     

柯萨奇病毒IgM抗体胶体金免疫层析快速诊断试纸条的研制

目的建立一种快速检测柯萨奇病毒IgM抗体的胶体金免疫层析试纸条，并优化制备中各关键步骤的实验条件。方法以柠檬酸三钠还原法制备20nm的胶体金溶液，标记羊抗人IgM，制备免疫胶体金复合物，组装胶体金免疫层析快速诊断试纸条。结果用柠檬酸还原法制备的20nm胶体金溶液呈亮红色。胶体金标记羊抗人IgM最低稳定量是1μg/ml，最适稳定量为1．5μg/ml。最适pH为8．2。血清标本检测结果与进口ELISA试剂盒比较差异无统计学意义。结论胶体金免疫层析试纸条制备质量不但与抗原抗体的质量、层析材料的选择、胶体金的制备与标记等因素密切相关，而且缓冲系统、辅助添加剂的选择与优化也非常重要。     

Matrix effect of swine urine on time-resolved fluorescent nanobeads and colloidal gold immunochromatographic assay

Immunochromatographic assay (ICA) is an efficient analytical technique and rapid, convenient, easy-to-use, low-cost, and on-site detection method that has been widely used to evaluate food safety. However, an important issue to be addressed for this method is its matrix effect. In this work, time-resolved fluorescent nanobead ICA (TRFN-ICA) and colloidal gold ICA (CG-ICA) were developed to detect clenbuterol in swine urine. Under optimized working conditions, the limits of detection of TRFN-ICA and CG-ICA were 16 and 68?pg/mL, respectively. The matrix effect on TRFN-ICA and CG-ICA was assessed in 20 swine urine samples. Results indicated that the sensitivity of TRFN-ICA was better than that of CG-ICA, and the detection time of the former was shorter than that of the latter. The matrix effect on TRFN-ICA was more serious than that on CG-ICA.     

玉米赤霉烯酮单克隆抗体的制备及胶体金免疫层析法的建立

目的 制备并鉴定玉米赤霉烯酮(ZEN)单克隆抗体(mAb),并初步建立ZEN的胶体金免疫层析检测方法.方法 采用活泼酯法制备人工抗原ZEN-OVA和ZEN-BSA,以ZEN-OVA作为免疫原制备ZEN mAb.ELISA法鉴定抗体效价、亚类、特异性等.ZEN mAb-胶体金复合物包被于胶体金结合垫上,检测原ZEN-BSA和山羊抗小鼠lgG分别结合于硝酸纤维膜上作为检测线(T线)和质控线(C线),建立ZEN的胶体金免疫层析检测法.结果 经紫外分光光度法和SDS-PAGE鉴定,ZEN人工抗原合成成功.经3次克隆化后,筛选出1株能稳定分泌ZEN mAb的杂交瘤细胞株1G4,腹水效价为1∶1.6×105;抗体亚类为IgG2b;对ZEN的IC50为10.2 ng/mL,检测限为0.58 ng/mL.mAb对ZEN具有高度的特异性,除与β-玉米赤霉烯醇的交叉反应率为12.8％外,与其他ZEN代谢物α-玉米赤霉醇、β-玉米赤霉醇、玉米赤霉酮及其他相似毒素呕吐毒素、伏马毒素、赭曲霉素A等几乎无交叉反应.制备的胶体金免疫层析试纸条在5 min内即可肉眼观察到结果,对ZEN的最低检测限为100 ng/mL.结论 成功制备出1株ZEN mAb,并初步建立了其胶体金免疫层析检测方法,最低检测限为100 ng/mL.     

Reproducibility of serological titers.

Serologists are making increasing use of the term "reproducibility" when referring to the reliability or repeatability of serological tests. The practical use of the concept, however, has been limited by the absence of an appropriate numerical scale on which differing reproducibilities can be quantified and objectively compared. This limitation can be overcome by adopting the proposed quantitative measure of reproducibility. The recommended measure is a natural extension of the common practice of considering a serological test to be acceptably reporducible so long as replicate titers remain within a twofold range. The measure can be readily used in the field of serology, and examples are given of how this can be done.