Quantitative determination of bone marrow transplant engraftment using fluorescent polymerase chain reaction primers for human identity markers

We have developed a quantitative, nonisotopic method using variable number tandem repeat (VNTR) and short tandem repeat (STR) markers for monitoring donor cell engraftment in marrow transplant recipients. Posttransplant DNA from the recipient is amplified with fluorescent polymerase chain reaction (PCR) primers for polymorphic markers that distinguish donor alleles from recipient alleles. The fluorescent PCR products are then separated on agarose or acrylamide gels on the Applied Biosystems 373A Sequencer (Foster City, CA). Using GeneScan 672 software (Applied Biosystems) to analyze the separated alleles, we can correlate allele peak areas to the percentage of donor or recipient DNA. We quantitate engraftment in a mixed chimeric sample by mixing pretransplant recipient and donor DNAs in a range of percentages and amplifying the mixtures to produce a standard curve. By amplifying and analyzing the posttransplant sample DNA(s), we can determine the extent of engraftment by interpolating the percent peak area of the informative allele(s) from this standard curve. This approach provides a precision of measurement ranging, depending on the marker, from 3.5% to 8.0% (percent coefficient of variation) and an accuracy of engraftment determination ranging from 97% to 99%, with a sensitivity of detection of 1% donor or recipient DNA. We retrospectively analyzed a panel of 32 patients and found seven to be informative for some degree of mixed chimerism, indicative of either residual normal host cells or leukemic relapse. An analysis of different cell lineages obtained posttransplant showed different degrees of engraftment in myeloid and T-cell populations. In summary, this method can provide an accurate, quantitative assessment of mixed chimerism in patients posttransplant. Such information may be useful in the future in guiding early implementation of additional treatment designed to circumvent graft failure or suppress relapse.     

Multistix 10SG试纸在自发性细菌性腹膜炎快速诊断中的意义

目的探讨Multistix 10SG白细胞酯酶试纸在自发性细菌性腹膜炎快速诊断中的意义。方法收集来自74位肝硬化腹水患者的223份腹水样本。采用Multistix 10SG白细胞酯酶试纸检测腹水中的白细胞酯酶水平,并与腹水细胞手动计数、腹水培养结果进行比较。试纸诊断结果与自发性细菌性腹膜炎诊断标准行诊断一致性检验,并结合其他感染相关指标进行分析。结果 99份腹水符合自发性细菌性腹膜炎的诊断标准。Multistix 10SG白细胞酯酶检测结果以痕迹为阳性标准时,诊断自发性细菌性腹膜炎的敏感性、特异性、阳性预测值、阴性预测值、精确度、阳性结果似然比、阴性结果似然比、k值分别67.7%、71.1%、65.0%、73.3%、69.5%、2.3、0.5、0.451(P<0.01);以1+为阳性标准,诊断自发性细菌性腹膜炎的敏感性、特异性、阳性预测值、阴性预测值、精确度、阳性结果似然比、阴性结果似然比、k值分别21.2%、98.4%、91.3%、61.0%、64.1%、13.3、0.8、0.213(P<0.01)。结论Multistix 10SG白细胞酯酶试纸有助于SBP的快速诊断。     

Thrombospondin from activated platelets promotes sickle erythrocyte adherence to human microvascular endothelium under physiologic flow: a potential role for platelet activation in sickle cell vaso-occlusion

Adherence of erythrocytes to vascular endothelium likely contributes to the pathophysiology of episodic vascular occlusion in patients with sickle cell disease (SCD). In addition, coagulation activation has been reported in sickle patients during complications such as pain episodes. To test the hypothesis that platelet activation contributes to sickle erythrocyte binding, we investigated whether factors released from activated sickle platelets promote adherence of sickle erythrocytes to human microvascular endothelial cells (MEC) under flow conditions. Activated sickle platelet supernatant (ASPS) promoted high levels of sickle erythrocyte adherence to MEC (55.4 +/- 3.9 erythrocytes/mm2) but only moderate adherence of normal erythrocytes to MEC (14.1 +/- 0.7 erythrocytes/mm2). When MEC were incubated with an antibody (OKM5) against CD36 (a thrombospondin [TSP] receptor), platelet supernatant mediated sickle erythrocyte adherence was inhibited 86%, suggesting that TSP participated in the adherence. To further define the role of TSP in adherence, additional studies using purified TSP were performed. At a concentration of 0.2 micrograms/mL TSP in serum-free media (SFM), sickle erythrocyte adherence to MEC was 33.9 +/- 2.7 erythrocytes/mm2 and sixfold greater than either sickle erythrocyte adherence in the absence of TSP or normal erythrocyte adherence in the presence of TSP. Doubling the concentration of TSP to 0.4 micrograms/mL proportionally increased adherence of sickle erythrocytes. Incubation of MEC with OKM5 or anti-alpha v monoclonal antibodies inhibited TSP-mediated sickle erythrocyte adherence more than 95%. These data suggest that activated platelet release factors, including alpha-granule TSP, which promote receptor-mediated sickle erythrocyte adherence to microvascular endothelium. Such factors released during in vivo platelet activation could contribute to vaso-occlusive complications by promoting erythrocyte adherence and microvascular occlusion.     

Anticoagulation Bridging Around Device Surgery: Compliance with Guidelines

Background: Current guidelines recommend bridging anticoagulation in patients undergoing cardiac rhythm device surgery with a “moderate to high risk” of thromboembolism. Patients at “low risk” are advised to stop oral anticoagulation without bridging to the procedure. This study examines real world adherence to accepted guidelines and the clinical sequelae of nonadherence. Methods: We performed a review of all patients undergoing device surgery receiving chronic anticoagulation over a prespecified time period of 14 months. Patients were classified per American College of Chest Physician guidelines as “moderate/high risk” or “low risk” of thromboembolism. We then compared perioperative management of anticoagulation to guideline recommendations and assessed the rate of perioperative bleeding and thromboembolism. Results: One hundred and twenty‐nine patients were included in this study. Sixty‐two (48%) were classified as “moderate/high risk” and 67 (52%) “low risk.” In the “moderate/high risk” group 47/62 (76%) received perioperative anticoagulation but only 25/62 (40%) were bridged both pre‐ and postprocedure or maintained on uninterrupted warfarin. In the “low risk” group, 22/67 (33%) received bridging therapy. Device pocket hematoma or perioperative bleeding occurred in 10/129 (8%) with 4/10 receiving inappropriate bridging for a calculated low risk of thromboembolism. There were no perioperative thromboembolisms. Conclusions: Our study identified significant underutilization of bridging, particularly in the postoperative period, in patients at “moderate/high risk” of thromboembolism. Conversely, bridging was overused in “low risk” patients and associated with bleeding complications. Physicians should be urged to follow current expert guidelines in regard to bridging anticoagulation for cardiac rhythm device surgery. (PACE 2012;35:1480–1486)     

Quantitation of ferritin iron in plasma, an explanation for non- transferrin iron

In 33 patients with thalassemia and idiopathic hemochromatosis, plasma ferritin protein levels ranged from 36 to 5,850 micrograms/L. The iron content of this ferritin as determined by immunoprecipitation ranged from undetectable amounts to 507 micrograms/L. The mean iron content of ferritin protein in those and other subjects with plasma ferritin concentrations of over 1,000 was 6.8% +/- 2.7%. Plasma transferrin was usually saturated with iron in patients with measurable ferritin iron, but exceptions occurred. In studies using electrophoretic separation, it was shown that some ferritin iron moved to transferrin during in vitro incubation, whereas exchange in the opposite direction was extremely limited. Because some plasma ferritin iron was measured by the standard colorimetric plasma iron determination, these observations (a) indicate that plasma ferritin contains a significant amount of iron (b) indicate that a significant proportion of nontransferrin iron in individuals with nontransferrin iron as detected by standard plasma iron and total iron-binding capacity measurements is due to the presence of ferritin, and (c) suggest that large amounts of ferritin iron may affect the saturation of plasma transferrin.     

Blood use in patients undergoing repeat coronary artery bypass graft procedures: multivariate analysis

BACKGROUND : The prevailing clinical opinion is that patients undergoing repeat coronary artery bypass graft (CABG) operation require more blood transfusions than do patients undergoing primary CABG operation. To determine the extent of this increased demand and the variables responsible for it, the cases of 196 patients who had undergone primary procedures and 65 patients who had had repeat procedures at the same institution were reviewed. STUDY DESIGN AND METHODS : To analyze the differences in transfusion requirements for these two groups, the following data were obtained: number of transfusions given between the time of surgery and the time of hospital discharge; preoperative hemoglobin (Hb), hematocrit (Hct), prothrombin time, and platelet count; Hb and Hct at hospital discharge; time the patient was on cardiopulmonary bypass; number and type of grafts; estimates of intraoperative blood loss; and chest-tube blood shed during the first 48 hours after surgery. RESULTS : The groups were comparable with respect to age, body weight, preoperative Hb and Hct, number of grafts, and aspirin exposure. Patients in the repeat group had 35-percent greater blood loss and required 75-percent more blood components than did the patients undergoing primary procedures. The mean number of blood components transfused per patient was as follows: red cells, 3.8 +/? 0.5 units in repeat patients and 2.2 +/? 0.2 units in primary patients (p = 0.002); platelets, 2.9 +/? 0.9 vs. 1.1 +/? 0.2 (p = 0.043); fresh-frozen plasma, 1.6 +/? 0.4 vs. 0.8 +/? 0.1 (p = 0.044). Analysis of variables by regression method for repeat patients showed a predictive effect of blood loss (p < 0.0001), prolonged time on cardiopulmonary bypass (p < 0.0001), preoperative Hb (p = 0.0003), and aspirin exposure (p = 0.0094) on red cell transfusion rate in repeat patients (R-square = 0.7778, Prob > f = 0.0001). CONCLUSION : Repeat CABG patients have higher transfusion rates. These findings may be attributed to the increased microvascular bleeding, prolonged time on cardiopulmonary bypass, lower preoperative Hb, and the use of preoperative antiplatelet medications.     

The heritable nature of clonal characteristics in acute myeloblastic leukemia

Marked patient-to-patient variation is observed when blood or marrow from AML patients is examined using colony methods in culture. Concentrations of the progenitors of colonies change with time during the course of the disease. We asked whether blast progenitor properties were more stable. We measured blast cell self-renewal and drug sensitivity (adriamycin and cytosine arabinoside) repeatedly in the courses of seven AML patients. These properties were found to be stable or slowly evolving. We conclude that capacity for renewal and sensitivities to certain chemotherapeutic drugs are heritable characteristics in leukemic clones.     

过表达钙整合素结合蛋白CIB诱导SHG44胶质瘤细胞凋亡的实验研究

目的 观察CIB基因对人胶质瘤SHG44细胞体外生长和细胞凋亡的影响.方法 构建重组质粒pcDNA3-CIB,转染人胶质瘤细胞株SHG44,MTT观察各组细胞的体外生长情况,流式细胞术(FCM)测定各组细胞的细胞周期和凋亡细胞数量,Western-blot检测CIB转染后凋亡相关蛋白的表达变化.结果 RT-PCR、Western印迹显示pcDNA3-CIB转染组CIB的mRNA及CIB蛋白表达水平明显高于对照组;与对照组细胞相比,转染CIB的SHG44-CIB组细胞牛长明显减缓(P＜0.01),SHG44-CIB组细胞G_1、S期细胞减少,G_2期细胞增多,凋亡细胞增加至19.2%;凋亡相关蛋白Bcl-2表达下调,Bax表达上调.结论 CIB在体外明显抑制SHG44细胞的生长并诱导其发生凋亡,CIB诱导的凋亡可能与Bcl-2及Bax表达的变化相关.     

脂肪纤维瘤病的临床病理特点

目的了解脂肪纤维瘤病的临床病理。方法收集4例脂肪纤维瘤病临床资料,手术切除组织光镜切片观察,另作4项免疫组织化学单克隆标记,SMA,S100蛋白,Vimentin,Desmin。结栗3例为出生不久就发现肿块,1例为12岁。肿瘤1例位于左侧颞部眉弓下,3例位于躯干部。肿瘤由纤维组织和脂肪组织构成。免疫组织化学标记瘤组织S100蛋白和Vimentin阳性,SMA和Desmin阴性。结论脂肪纤维瘤病是一种罕见的肿瘤,它的诊断依靠病理组织学改变和免疫组织化学标记,肿瘤可多发和复发。     

卡维地洛对改善压力负荷大鼠心室肥厚的作用

1);与B组比较.C组上述指标均有所降低(P<0.05).结论:卡维地洛能减轻压力负荷大鼠的心窒肥厚,下调过度表达的MMP-2及升高的TNF-α水平是其防治心室重构的机制之一.