Analytical Performance/Clinical Utility Of Laboratory Tests For Atherothrombosis

动脉粥样硬化血栓形成是一种多因素疾病，目前估计有数百种动脉粥样硬化血栓形成的危险因素。除了经典的危险因素如血脂（总胆固醇和甘油三酯）、脂蛋白和载脂蛋白外，各种炎症分子如c反应蛋白和血清淀粉样蛋白a、止血因子和各种特定细胞类型也被认为是风险和/或需要管理的因素。研究类风湿关节炎（RA）患者的心血管脂质谱，我们观察到诊断重要的实验室干扰，并研究其性质和来源。最初，我们尝试使用电泳酶胆固醇法（REP Vis cholesterol, Helena）定量这些患者先前冷冻的肝素化血浆中的脂蛋白-胆固醇组分。然而，我们的尝试失败了，因为所有54个样本的脂蛋白都没有完全分离，显然是由于冷冻现象。低温凝胶是含有额外结构域a的（细胞）纤维连接蛋白、血浆纤维连接蛋白、纤维蛋白原和肝素的共沉淀物。低温下存在肝素的情况下会发生冷冻，这在类风湿关节炎患者中很常见。我们在电泳脂蛋白-胆固醇测试中的失败促使我们研究冷冻可能与其他实验室测试的干扰。采用不同的采血和血液处理/储存方案制备6例RA患者的血清和肝素化血浆。这些方案包括从分离凝胶管中制备血清，在室温和37℃下制备肝素化血浆和EDTA血浆，并在实验室检测之前将标本存放在冰箱或冷冻在-70℃。使用Synchron LX-20自动化学分析仪（Beckman Coulter）进行20组常规化学试验和总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）和低密度脂蛋白胆固醇（LDL-C）试验。选择血清和血浆进行肌钙蛋白I、促甲状腺激素（TSH）、促肾上腺皮质激素或ACTH和皮质醇（Immulite 2500, DPC/Siemens）、肌钙蛋白T、前脑利钠肽（BNP）、肌酸激酶- mb或CK-MB同工酶、β -人绒毛膜促性腺激素或β - hcg （Elecsys 2010, Beckman Coulter）、脑利钠肽或BNP （Axsym, Abbott）、高敏c反应蛋白或hsCRP、促风湿因子或RF （imaging, Beckman Coulter）。血清淀粉样蛋白A或SAA （BN-II, Dade-Behring）和电泳蛋白组分（Hydragel, Sebia）。肝素化血浆标本中可见的低温凝胶需要在检测前进行高速离心。除了血清和血浆钾和总蛋白的预期差异外，大多数其他常规化学检查、TC、TG、各种免疫测定和蛋白质电泳的结果在不同方案之间的统计学或临床差异均不显著。与参考血清标本（在4℃下处理和储存的血清）相比，所有冷藏或冷冻的肝素化血浆标本的HDL-C结果较高（8%），LDL-C结果往往较低（4%）；在37℃下储存和处理的肝素化血浆标本效果更明显。除去可能导致取样问题的冷冻凝胶后，除了脂蛋白部分测试外，即使是冷藏或先前冷冻的肝素化血浆样本也适用于大多数常规和专门的化学测试。

Atherothrombosis is a multifactorial disease and risk factors for atherothrombosis are now estimated to be in the hundreds. In addition to classical risk factors like serum lipids (total cholesterol and triglycerides), lipoproteins, and apolipoproteins, a variety of inflammatory molecules such as C-reactive protein and serum amyloid A, hemostatic factors and various specific cell types have been also considered for risk and/or require management. Studying the cardiovascular lipid profile of patients with rheumatoid arthritis (RA), we observed diagnostically important laboratory interferences and studied their nature and source. Initially, we attempted to quantitate lipoprotein-cholesterol fractions in previously frozen heparinized plasma of these patients using an electrophoretic enzymatic cholesterol method (REP Vis Cholesterol, Helena). However, our attempt has failed because of incomplete separation of lipoproteins in all 54 specimens tested, apparently due to the phenomenon of cryogelation. Cryogel is a co-precipitate of extra domain A-containing (cellular) fibronectin, plasma fibronectin, fibrinogen and heparin. Cryogelation has been described to occur in the presence of heparin at low temperatures and has been found to be common in patients with RA. Our failure with the electrophoretic lipoprotein-cholesterol test prompted studying possible interference of cryogelation with other laboratory tests. Serum and heparinized plasma were prepared from 6 RA patients using different blood collection and blood processing/storage protocols. These protocols included preparation of serum from separator gel tube and preparation of heparinized and EDTA plasma at room temperature and at 37 oC and storage of the specimens in refrigerator or frozen at -70 oC prior to laboratory testing. A panel of 20 routine chemistry tests and tests for total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were performed using the Synchron LX-20 automated chemistry analyzer (Beckman Coulter). Select sera and plasmas were also analyzed for troponin I, thyroid-stimulating hormone (TSH), adrenocorticotropic hormone or ACTH and cortisol (Immulite 2500, DPC/Siemens), troponin T, pro-brain natriuretic peptide (BNP), creatine kine-MB or CK-MB isoenzyme, and beta-human chorionic gonadotropin or beta-HCG (Elecsys 2010, Beckman Coulter), brain natriuretic peptide or BNP (Axsym, Abbott), high-sensitivity C-reactive protein or hsCRP and rheumotaid factor or RF (Immage, Beckman Coulter), serum amyloid A or SAA (BN-II, Dade-Behring), and electrophoretic protein fractions (Hydragel, Sebia). The presence of visible cryogel in heparinized plasma specimens from the heparinized plasma specimens necessitated high-speed centrifugation prior to testing. Apart from expected differences between serum and plasma for potassium and total protein, the results for most other routine chemistry tests, TC, TG, various immunoassays and protein electrophoresis were insignificantly different, either statistically or clinically, among various protocols. Compared to the reference serum specimens (serum processed and stored at 4 oC), all refrigerated or frozen heparinized plasma specimens gave higher (8%) HDL-C results and tended to give lower (4%) LDL-C results; with more pronounced effects for heparinized plasma specimens stored and processed at 37oC. It appears that, apart from lipoprotein fraction testing, even refrigerated or previously frozen heparinized plasma samples are suitable for most routine and specialized chemistry tests after removal of the cryogel that may cause sampling problems. In a collaborative study, we found that cardiac rehabilitation in patients receiving stable statin therapy and with low-density lipoprotein cholesterol at goal increases endothelial progenitor cell number, endothelial progenitor cell survival, and endothelial differentiation potential, associated with increased nitric oxide in the blood. Although this response was observed in most patients, a significant minority showed neither endothelial progenitor cell mobilization nor increased nitric oxide in the blood. In another collaborative study, we found that both serum SAA and CRP are significantly elevated in unmedicated, remitted women with major depressive disorder (MDD) versus controls. These findings suggested a sustained low-grade pro-inflammatory state in women with MDD that may be related to the increased coronary artery disease (CAD) risk observed previously in this population. In still another collaborative study, we studied the ability of the rodent (mouse) and human scavenger receptors SR-BI (called CLA-1 in humans) and its splicing variant SRB-II (called CLA-2 in humans) to mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells. Our results indicate that these receptors, in addition to their already established function as high-density lipoprotein receptors, also are involved in the defense mechanism of mammalian host cells and, by facilitating bacterial adhesion and cytosolic invasion, they may play an important role in infection and sepsis.