Comparison of five point-of-care D-dimer assays with the standard laboratory method

Introduction: D‐dimer (DD) assays are effective for the exclusion of deep‐vein thrombosis (DVT), but point‐of‐care (POC) DD assays have not been fully evaluated. Methods: We have compared five POC DD assays (Pathfast, Cardiac, Vidas, Stratus and NycoCard) with our routine DD method (Tinaquant), testing 60 samples from patients with suspected DVT. Results: Using 0.5 μg/mL as a cut‐off value, four samples tested negative with Tinaquant were positive with Pathfast. There were no Tinaquant‐positive samples tested negative with Pathfast, while the overall agreement (k = 0.81) was very good. Four samples were discrepant between Tinaquant and Cardiac (cut‐off, 0.4 μg/mL), while k = 0.72. One of two Tinaquant‐negative samples was shown to be positive for either Vidas (cut‐off, 0.5 μg/mL) or Stratus (cut‐off, 0.4 μg/mL), respectively. The agreement with Tinaquant was excellent for both Vidas (k = 0.92) and Stratus (k = 0.94). Total CV was <10% for all four assays. Eight samples (of 27) were negative with NycoCard although they were positive with Tinaquant, while CV was 41%. Conclusion: Vidas cannot be considered a POC assay because the sample has to be centrifuged before testing. Our findings have also shown that the use of NycoCard is inappropriate. Stratus and Pathfast have a similar analytical profile in comparison with the Tinaquant method. Cardiac is potentially less sensitive but may still be acceptable for use. It seems that the employment of these three assays for rapid bed‐side analysis offers a possibility to adequately rule out DVT in outpatients within minutes after admission.     

Determinants of ELISA D-dimer sensitivity for unstable angina pectoris as defined by coronary catheterization

Unstable angina pectoris is associated with elevated D-dimer levels. However, the operating characteristics (sensitivity, specificity, positive and negative predictive value) of the D-dimer assay for the diagnosis of coronary artery disease (CAD) are unknown. Using a prospective, observational design, we collected blood from 54 patients with unstable angina pectoris at admission and assayed for ELISA D-dimer levels. The sensitivity, specificity, and negative and positive prediction values for angiographically determined coronary artery disease were calculated at multiple discriminate levels. All patients underwent coronary catheterization. A statistically significant correlation was noted between ELISA D-dimer levels and age, male sex, hypertension, use of beta-blocker, fibrinogen levels and catheterization findings. No correlation was noted between ELISA D-dimer levels and degree of the coronary artery disease. Best results were provided at a discriminate level of 270 ng/ml, with sensitivity 70%, negative predictive value 72%, and overall accuracy 67%. All discriminate levels, however, provided values too low for diagnosis. In conclusion, ELISA D-dimer assay is a non-sensitive, non-specific test for coronary artery disease as defined by coronary catheterization. However, the assay adds information regarding the severity of disease in patients presenting with acute coronary syndrome.     

Usefulness of Ictal and Interictal 99mTC Ethyl Cysteinate Dimer Single Photon Emission Computed Tomography in Patients with Refractory Partial Epilepsy

Summary: Purpose: Ictal perfusion single photon emission computed tomography (SPECT), using HMPAO, has been shown to localize epileptic foci in ～90% of studies. Unfortunately, HMPAO decomposes rapidly, precluding the performance of ictal studies. Ethyl cysteinate dimer (ECD) is a SPECT perfusion agent recently approved by the Food and Drug Administration. After preparation, this compound is stable for ～6 h. facilitating the performance of ictal studies. Methods: In a prospective, open-label, uncontrolled, non randomized study, we evaluated the potential benefits of the use of ^99mTc-ECD SPECT for lateralization of the epileptic focus. Ten consecutive adult epilepsy surgery candidates were studied with ictal and interictal ^99mTc-ECD SPECT. Results: The mean delay between seizure onset and ictal SPECT injection was 23.2 s. The mean seizure duration was 84.1 s. Ictal studies agreement between the epilepsy focus and area of hyperperfusion was evident in 8 of 10 cases. In one case, SPECT was lateralized in a patient with bilateral temporal lobe epilepsy (TLE); however, hyperperfusion was observed on the same side of that particular seizure. In another case, there was location disagreement. Interictal SPECT showed focal hypoperfusion in three cases. Conclusions: ^99mTc-ECD proved to be an optimal tracer for ictal studies. Although this is a small series, the results of ictal and interictal findings using 99mTc-ECD are similar to those reported with ^99mTc-HMPA0. Because ^99mTc-ECD has a longer decomposition time, true ictal studies are easier to obtain. This new tracer will probably allow the use of ictal SPECT to become widely accepted in most epilepsy centers.     

从噬菌体抗体库中筛选D二聚体特异的Fab抗体

目的:从人源性噬菌体抗体库中筛选抗人D二聚体单克隆抗体并进行可溶性表达。方法:以人D二聚体标准品为抗原对所构建的噬菌体抗体库进行两轮“吸附-洗脱-扩增”筛选,从中筛选出抗人D二聚体的噬菌体Fab抗体,并在大肠杆菌细胞中进行可溶性表达。结果:经4次电转化构建了库容为2. 8×108cfu的抗体库,滴度为4. 1×1014PFU/mL,Fab基因重组率为46%。经过淘筛后携带抗人D二聚体抗体的特异性噬菌体得到了明显的富集。用试剂盒以ELISA法检测对人D二聚体的结合活性,得到抗人D二聚体单克隆抗体。筛选出的阳性克隆成功在大肠杆菌细胞中可溶性表达筛选出的阳性克隆在大肠杆菌细胞中可溶性表达。结论:人D二聚体标准品对抗体库的淘筛,富集了表面呈现抗人D二聚体的单克隆抗体的噬菌体,成功构建了人源性抗D二聚体噬菌体抗体,并成功在大肠杆菌细胞中可溶性表达。     

An immunochemical method for quantitative determination of latent antithrombin, the reactive center loop-inserted uncleaved form of antithrombin

Antithrombin (AT) is a serine protease inhibitor that has thrombin, factors IXa and Xa as target proteases. In addition to active native AT, two other forms have been identified in plasma: the reactive center loop inserted cleaved and latent, uncleaved forms. Both have been shown to be present in normal human blood. Latent AT forms a dimer with native AT in vitro, thus inactivating the native form. Here we describe a mouse monoclonal antibody, 8C8, that is specific for latent AT. The affinity of 8C8 was found to be 500-fold higher for latent than for native AT and 5000-fold higher for latent than for cleaved AT. A sandwich assay was developed to measure the concentration of latent AT in plasma, which was found to be approximately 4.8 mg L(-1) in healthy individuals. The K(D) of the interaction between native and latent AT was found to be 51 mum, i.e. far above the plasma concentration of both native and latent AT, indicating a negligible complex formation in blood.     

D—二聚体在泌尿系肿瘤中的应用

为了解Ｄ二聚体在泌尿系肿瘤患者中的应用价值，采用定量酶联免疫吸附法对４７例泌尿系肿瘤患者及７３例非肿瘤患者血浆Ｄ二聚体含量进行了测定。结果显示：肿瘤患者血浆Ｄ二聚体含量较非肿瘤患者及正常组明显增高（Ｐ＜０．００１），非肿瘤患者与正常组之间无显著性差异（Ｐ＞０．０５）。结果表明，泌尿系肿瘤患者体内处于继发性纤溶活性增高状态，Ｄ二聚体可作为监测泌尿系肿瘤患者的指标之一。     

Synthesis of side-chain to side-chain cyclized peptide analogs on solid supports

Cyclic peptide structures of the type -Lys-R₁—R_n-Glu- can be synthesized on the Merrifield resin by assembling the peptide chain using Nα-Fmoc-amino acids and Boc and tert.-butyl protection for the side-chains of Lys and Glu, respectively. If residues R₁ to R_n contain side-chain functional groups, TFA-resistant protection is required. After TFA treatment cyclization on the resin can be performed with appropriate coupling reagents. The formation of such cyclic structures may be preceded or followed by peptide chain assembly using Nα-Boc-amino acids and the entire peptide chain containing the cyclic portion is finally cleaved by HF treatment. Using this principle we synthesized the following opioid peptide related cyclic analogs: H-Tyr-d -Lys-Gly-Phe-Glu-NH₂ (I), H-Tyr-Lys-Gly-Phe-Glu-NH₂ (II), H-Tyr-d -Lys-Phe-Glu-NH₂ (III), H-Tyr-d -Glu-Gly-Phe-Lys-NH₂ (IV), H-Tyr-d -Glu-Phe-Lys-NH₂ (V), H-Tyr-d -Orn-Gly-Glu-NH₂ (VI) and H-Tyr-d -Ala-Lys-Phe-Glu-NH₂ (VII). Cyclic monomers were obtained in all cases, as demonstrated by mass spectrometry. Analysis of side-products revealed a slow-down of the HF deprotection of O-benzylated tyrosine as a consequence of hydrophobic interactions as well as the formation of a side-chain-linked antiparallel cyclic dimer in the case of compound VI. In conclusion, the described method permits the convenient preparation of peptide analogs cyclized via amide bond formation between side-chain amino and carboxyl groups in reasonable yield.     

Effects of Ginkgo biloba on haemostatic factors and inflammation in chronic peritoneal dialysis patients

Ginkgo biloba extract decreased plasma D-dimer concentration, a marker of intravascular coagulation, in chronic peritoneal dialysis patients. Blood levels of fibrinogen, von Willebrand factor, hs-CRP, albumin and liver enzyme levels were not significantly changed. No bleeding episode was reported. These results suggest that Ginkgo biloba extract was effective in partially reversing the thrombogenic coagulation profile without increasing the risk of bleeding.