Comparison of QIAsymphony automated and QIAamp manual DNA extraction systems for measuring Epstein-Barr virus DNA load in whole blood using real-time PCR

Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAamp systems were similar (270 and 560 copies/mL, respectively). For samples estimated as having ≥10,000 copies/mL, the intrarun and interrun variations were significantly lower using QIAsymphony (10.0% and 6.8%, respectively), compared with QIAamp (18.6% and 15.2%, respectively); for samples having ≤1000 copies/mL, the two variations ranged from 27.9% to 43.9% and were not significantly different between the two systems. Among 68 paired clinical samples, 48 pairs yielded viral loads ≥1000 copies/mL under both extraction systems. Although the logarithmic linear correlation from these positive samples was high (r(2) = 0.957), the values obtained using QIAsymphony were on average 0.2 log copies/mL higher than those obtained using QIAamp. Thus, the QIAsymphony and QIAamp systems provide similar EBV DNA load values in whole blood.     

基于PCR的基因定量方法及方法学评价

基因定量检测已经成为研究遗传性疾病、感染性疾病以及某些易感基因引起的疾病的重要手段.在过去的几年中已相继出现了一些高效自动的技术方法.目前可用的基因定量方法大致可以分成3类:DNA印迹技术(Southern杂交),细胞遗传学方法和以PCR扩增为基础的基因定量方法.本文对基于PCR的基因定量方法作一综述,并进行方法学评价...     

Comparison of blood component preparation methods from whole blood bags based on buffy coat extraction

We compared the data of our quality control laboratory of the blood components according to the blood component preparation method that we used. We prepared blood components from top and top whole blood (WB) bags and manual pooling of buffy coats (BCs) (method I) or from top and bottom WB bags and automated pooling of BCs with OrbiSac (method II). Pooled platelet concentrates (PC) obtained with method II had higher platelet content when compared with pooled PCs obtained with method I (3.5 ± 0.7 × 10¹¹ vs. 2.6 ± 0.8 × 10¹¹; p < 0.001). The hemoglobin content in the RBCs obtained with method I was higher when compared with method II (55 ± 7 g vs. 52.5 ± 6.6 g; p < 0.001).     

Comparison of cyclosporin A measurement in whole blood by six different methods

In a multicentre trial, we analyzed and compared the cyclosporin concentrations in whole blood specimens (trough values) from renal and bone marrow transplant patients using six different methods: high-performance liquid chromatography (n-butyl reversed-phase column) as reference, and 5 immunoassay procedures using monoclonal specific or non-specific antibodies, or polyclonal antibodies, for measuring cyclosporin with and without its cross-reacting metabolites. Results obtained by the 2 specific RIAs show good correlations (r-values of 0.945 and 0.921) versus the HPLC method, with average assay ratios of: 1.52 for 3H-RIA/HPLC and 1.26 for 125I-RIA/HPLC. In contrast, ratios for non-specific immunoassay/HPLC show multiple-fold overestimations of cyclosporin with very wide variations: 4.58 for 3H-RIA/HPLC, 3.97 for 125I-RIA/HPLC and 3.87 for fluorescence polarization immunoassay (FPIA)/HPLC. These findings indicate 1) that the 'specific' 3H- and 125I-RIAs can be used for cyclosporin measurements in whole blood using normal therapeutic ranges established by HPLC, 2) that the important disparity and variable overestimations by 'non-specific' RIA or fluorescence polarization immunoassay, compared with HPLC, require adjustments in therapeutic ranges, and 3) that the available 'specific' and 'non-specific' immunoassays should enable establishment of within-house reference 'therapeutic/toxic' ranges for cyclosporin in individual centres, based on these 'specific' versus 'non-specific' assays.     

Evaluation of a rapid whole blood ELISA for quantification of troponin I in patients with acute chest pain.

BACKGROUND: Troponin I (cTnI) provides important prognostic information in patients with chest pain. We wished to evaluate a rapid, whole-blood analyzer for quantitative point-of-care testing. METHODS: A quantitative point-of-care test system (Stratus CS((R)); Dade-Behring) for cTnI with an incorporated centrifuge was evaluated in 412 patients with chest pain less than 12 h. RESULTS: Results were available within 15 min. CVs were 4.5% at 0.1 microgram/L, 4.2% at 0.25 microgram/L, and 6.5% at 0.82 microgram/L. The detection limit was 0. 01 microgram/L. The 97.5% percentile in a healthy population was 0.08 microgram/L. Based on ROC curve analysis, a threshold of 0.15 microgram/L was calculated for the detection of acute myocardial infarction (AMI). With it, sensitivity for the detection of patients with AMI (n = 62) was 63% at arrival and 98% after 4 h (Stratus II((R)), 48% and 85%, respectively; P <0.01). In 42% of patients with unstable angina (n = 121), cTnI was >/=0.08 microgram/L (Stratus II, 28%; P <0. 01). During 30 days, death or AMI occurred in 25.5% of these cTnI-positive vs 2.9% of cTnI-negative patients (Stratus II, 29.4% vs 5.8%). CONCLUSION: The Stratus CS provided better analytical performance and comparable or better prognostic information than the Stratus II.     

两种从陈旧血中抽提基因组DNA方法的比较

目的 比较盐析法和磁珠法从陈旧血中抽提基因组DNA的效果和特点,以便常规用于骨髓库HLA基因分型.方法 使用TECAN全自动工作站为平台,分别用Gentra盐析法DNA抽提试剂盒和Promega磁珠法DNA抽提试剂,从48份陈旧全血样本中抽提基因组DNA,提取的基因组DNA均溶于100 μlTE中;然后用紫外分光光度计测定其产量和纯度,并用琼脂糖凝胶电泳法测定DNA样本的完整性.结果 从400 μl陈旧全血中提取基因组DNA,用盐析法获得的DNA含量为28.6±8.7 ng/μl,A260/A280值平均为1.63±0.209;用磁珠法获得的DNA含量为94.2±10.3 ng/μl,A260-A320/A280-A320值平均为1.821±0.102;琼脂糖电泳法检测表明两种方法提取的DNA的分子量均大于15 kb.结论 使用磁珠法从陈旧血中所获得的DNA的产量和纯度明显高于盐析法,适合于骨髓资料库陈旧血样本的常规HLA基因分型实验以及下游的分子生物学实验.     

两种从陈旧血中抽提基因组DNA方法的比较

目的比较盐析法和磁珠法从陈旧血中抽提基因组DNA的效果和特点,以便常规用于骨髓库HLA基因分型。方法使用TECAN全自动工作站为平台,分别用Gentra盐析法DNA抽提试剂盒和Promega磁珠法DNA抽提试剂,从48份陈旧全血样本中抽提基因组DNA,提取的基因组DNA均溶于100μlTE中;然后用紫外分光光度计测定其产量和纯度,并用琼脂糖凝胶电泳法测定DNA样本的完整性。结果从400μl陈旧全血中提取基因组DNA,用盐析法获得的DNA含量为28.6±8.7ng/μl,A260/A280值平均为1.63±0.209;用磁珠法获得的DNA含量为94.2±10.3ng/μl,A260-A320/A280-A320值平均为1.821±0.102;琼脂糖电泳法检测表明两种方法提取的DNA的分子量均大于15kb。结论使用磁珠法从陈旧血中所获得的DNA的产量和纯度明显高于盐析法,适合于骨髓资料库陈旧血样本的常规HLA基因分型实验以及下游的分子生物学实验。     

全血糖化血红蛋白用常规方法测定的稳定性研究

目的 探讨全血糖化血红蛋白HbA1c在不同储存条件下分别用Tosoh G7、罗氏/日立7170A和NycoCard READERⅡ测定的稳定性.方法 收集3个糖化血红蛋白水平的乙二胺四乙酸抗凝及1个水平的肝素抗凝全血样本,冻存管分装,分别储存在-80℃、-20℃、4℃、室温(15～25 ℃)和37℃.分别在第1、2、5、7、9、14、21、28、35、42、49、56、63和70 d用3种方法进行测定.结果在-80℃储存的全血样本在研究期间无论是用东曹G7方法还是用罗氏/日立7170A方法测定结果稳定,东曹G7的变异系数(CV)为0.54%～1.22%;罗氏/日立7170A的CV为0.86%～1.82%;样本用东曹G7测定的稳定性为:-20℃可稳定14 d,4 ℃ 63 d,室温5 d,37 ℃少于1 d;罗氏/日立7170A方法 为:-20℃21 d,4 ℃ 42 d,室温7 d,37℃少于1 d;NycoCard READER Ⅱ:4℃9 d,室温少于1 d.结论 不同方法测定全血样本的稳定性是特异的,不同温度下允许保存时间不同.     

全血糖化血红蛋白用常规方法测定的稳定性研究

Objective To investigate the stability of glycated hemoglobin HbA1c in whole blood sample measured by Tosoh G7, Roche/Hitachi 7170A and NycoCard READER Ⅱ under different storage conditions. Methods Three whole blood samples (EDTA anticoagulated) with different glycated hemoglobin levels and one whole blood sample (heparin anticoagulated) were collected and stored at -80 ℃, -20 ℃, 4 ℃,room temperature(15 -25 ℃), and 37 ℃ HbA1c was analyzed by each method on days 1, 2, 5, 7, 9, 14, 21,28, 35, 42, 49, 56, 63 and 70 respectively. Results The results of sample stored at -80 ℃ appear to be stable for Tesoh G7 and Roche/Hitachi 7170A method. The coefficients of variation (CV) for Tosoh G7 was 0.54%-1.22%. The CV for Roche/Hitachi 7170A was 0.86% -1.82%. When samples was detected with Tosoh G7 method, the results was consistent when the sample was stored at -20 ℃ for 14 days, 4 ℃ for 63 days, room temperature for 5 days, and 37 ℃ for less than 1 day. When samples was detected with Roche/Hitachi 7170A method, the results was consistent when the samples was stored at -20 ℃ for 21 days, 4 ℃ for 42 days, room temperature for 7 days, and 37 ℃ for less than 1 day. The NycoCard READER Ⅱ showed stability at 4 ℃ for 9 days, and room temperature for less than 1 days. Conclusions The stability of whole blood samples is dependent on different methods. Storage time under different temperatures is different.     

固相法血型定型试剂在极端气候条件下使用的可行性评估

目的评估固相法血型定型试剂在极端条件下使用的可行性。方法模拟我国存在的4种极端气候条件(高温高湿、高温干燥、严寒干燥和低温高湿),选择全血标本120份,分析、比较固相法血型定型试剂在不同温度、湿度条件下测定ABO/RhD血型的准确性和反应时间。结果各组血型鉴定准确性均为100%。在高温条件下(高温干燥和高温高湿组)的反应时间与对照组相比无统计学差异,但低温高湿和严寒干燥2组与对照组相比,差异有统计学意义(P<0.05)。结论固相法血型定型试剂有良好准确性,由于其具备常温保存、结果易于判读等优点,可满足突发事件情况下血液应急保障的需求。     

Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.     

Evaluation of determination of uric acid in serum and whole blood with the Reflotron

The performance of the Reflotron system (Boehringer Mannheim) for the determination of urate in whole blood and serum was evaluated. Within-run and day-to-day imprecision of the system were comparable with those for a solution-chemistry enzymatic method (overall CVs in the range 2.2-2.5%). Results for 100 individual specimens with urate concentrations ranging from 16 to 134 mg/L agreed well with the comparison method, both for serum and whole blood. We saw no significant interference from lipemia or hemoglobin. Bilirubin interfered at concentrations greater than 100 mg/L. Hematocrit variation between 25% and 55% did not affect results for whole blood; variation of the applied sample volume from 28 microL to 35 microL (stated sample volume requirement: 30 microL) did not significantly influence the measured value. We consider results produced by the system to be of the same analytical quality as those obtained by the more conventional solution-chemistry methods that are currently available.     

血液净化技术在ICU急性肾功能衰竭的应用

目的：探讨ICU复杂性急性肾功能衰竭（ARF）血液净化治疗模式的选择及疗效和并发症。方法：回顾性分析1999年1月-2001年12月在我院ICU收治的20例ARF患的血液净化治疗情况。比较不同治疗模式的疗效的并发症。结果：5例接受普通间歇性血液透析（IHD）治疗。3例治愈，2例死亡。血尿素氮（BUN）、肌酐（Cr）和血钾透析后均明显下降，但透析间期波动大。补充的液体量受限制。2例透析后出现严重的并发症并死亡。13例行连续性肾脏替代治疗（CRRT），血BUN、Cr和血钾缓慢下降，波动小，并能持续于较低水平。允许补充大量液体和静脉营养。CRRT还能改善血流动力学状态，不加重脑水肿患的意识障碍。2例急性重症胰腺炎合并高乳糜微粒血症的ARF患接受非选择性血浆置换（PE）加CRRT治疗，胰腺炎得以较快控制，肾功能逐渐恢复。结论：对于ICU的重症ARF患，选择IHD应慎重。CRRT更适合复杂性ARF的肾替代治疗。血浆置换联合CRRT可能是治疗急性重症腺合并高脂血症的ARF患的有效方法。     

Evaluation of quantification methods for left arial late gadolinium enhancement based on different references in patients with atrial fibrillation

By using late gadolinium enhancement cardiac magnetic resonance (LGE-CMR) imaging, we compared left atrial late gadolinium enhancement (LA-LGE) quantification methods based on different references to characterize the left atrial wall in patients with atrial fibrillation (AF). Thirty-eight patients who underwent three-dimensional LGE-CMR imaging before catheter ablation for AF were classified into three groups depending on their clinical AF type: (1) paroxysmal AF (PAF; n = 12); (2) persistent AF (PeAF; n = 16); and (3) recurrent AF after catheter ablation (RAF; n = 10). To quantify LA-LGE on LGE-CMR imaging, we used the thresholds of 2 standard deviations (2-SD), 3-SD, 4-SD, 5-SD, or 6-SD above the mean signal from the unenhanced left ventricular myocardium, and we used the full width at half maximum (FWHM) technique, which was based on the maximum signal from the mitral valve with high signal intensity. The 6-SD threshold and FWHM techniques were statistically reproducible with an intraclass correlation coefficient >0.7. On applying the FWHM technique, the normalized LA-LGE volume by LA wall area showed a significant difference between the RAF, PeAF, and PAF groups (0.22 ± 0.04, 0.16 ± 0.06, and 0.09 ± 0.03 mL/cm², respectively) (P < 0.05). Furthermore, most of the fibrotic scarring and low-voltage tissue on the electroanatomic map corresponded well with the extent of LA-LGE. The FWHM technique based on the mitral valve can provide a reproducible quantification of LA-LGE related to AF in the thin LA wall.     

三种HBV DNA提取方法对荧光定量PCR检测结果影响的比较

目的 研究3种核酸提取方法对黄疸、溶血和脂血标本HBV DNA荧光定量结果的影响,为进一步提高临床检验质最和改进检测技术提供理论依据.方法 采用目前临床常用的多聚糖病毒沉淀浓缩法(PEG法)、碱液直接裂解和微量核酸释放剂(Micro-Nucleic-Releaser,MNR)等3种核酸提取方法,对含有HBV的黄疸、溶血和脂血标本进行荧光定量PCR检测,研究不同标本对荧光PCR定量结果和扩增效率的影响.选择酚一氯仿提纯法作为核酸提取对照方法.结果 黄疸、溶血和脂血标本,对3种不同核酸提取方法的荧光PCR定量结果和扩增效率都产生不同程度的影响,其中完全溶血的标本对PCR扩增效率的影响最大,如定量值为4×103拷贝/ml的低拷贝完全溶血标本,MNR法测定结果为2.21×103拷贝/ml,PEG法为1.02×103拷贝/ml,碱性直接裂解法的定量值为阴性,临床自然溶血较重的标本对荧光定量检测影响较小,脂血和黄疸标本对荧光定量PCR扩增效率有明显抑制作用,但对实际定最值无明显影响;其中,MNR法在不同标本的定量重复性和荧光PCR扩增效率最好,碱液直接裂解法较差.酚.氯仿提纯法虽然获得较高的扩增效率,但存在大约有1个数量级的核酸丢失.结论 基于MNR核酸提取方法的荧光定量PCR检测,对不同的临床标本均可获得较好的扩增效率、敏感性和重复性,操作简便、快速且无核酸丢失和污染,适合临床检验应用.     

血液净化技术在急性肾功能衰竭治疗中的应用评估

目的 评估血液净化疗法在急性肾功能衰竭(ARF)治疗中的应用及疗效。方法 总结我院1995年2月至2000年12月用间歇性血液透析(IHD)、持续性静-静脉血液滤过(CVVH)、持续性静-静脉血液滤过透析(CVVHDF)、血液透析 血液灌流(HD HP)技术治疗98例ARF患者的临床资料,分析病因、临床特点、治疗情况和预后。结果 IHD对血尿素氮(BUN)、肌酐(Cr)、血钾清除效果好:CVVH用于重症ARF患者血流动力学稳定,但BUN,Cr、血钾清除效果欠佳;CVVHDF介于二者之间,更适合于重症患者合并高分解代谢。结论 (1)ARF应根据病因、病情选择不同净化方式。(2)重症ARF预后和原发病的严重程度、病因及治疗方法有关。     

三种丙肝抗体检测方法用于献血员人群筛查的评价

目的探讨三种丙型肝炎病毒抗体检测方法的应用及可靠性。方法对150240份献血者用国产试剂检测阳性的516例标本再用ORTHO试剂及ABBOTT试剂检测,并做HCV RNA分析。结果516例国产试剂检测阳性的标本中ORTHO试剂检测阳性为226例,ABBOTT试剂检测阳性为245例,HCV RNA阳性为186例;ABBOTT试剂与ORTHO试剂检测符合率为90.7%,HCV RNA与ORTHO试剂检测符合率为90.3%。结论ABBOTT试剂与ORTHO试剂在检测抗HCV上有很好的一致性,而国产试剂则存在较大的差异,其判断标准需加以改进。