Application of a computer program to exclude additional unexpected antibodies

BACKGROUND : When a patient has produced red cell (RBC) antibodies in the past, he or she is at risk of producing additional antibodies after antigen challenge. The presence of these antibodies should be excluded before each transfusion. The following criteria are applied when using commercial test RBCs: RBCs should not express the antigen against which the previously documented antibody is directed. For other clinically significant antigens, at least one RBC sample should be from a donor who is homozygous for the encoding gene. The manual selection of such RBCs is tedious and requires experience. STUDY DESIGN AND METHODS : A computer program has been developed that generates exclusion panels (EPs) by selecting a minimum number of RBCs from commercial test panels complying with current criteria. When RBCs from a donor who is homozygous for the encoding gene are absent, the program selects, as a second-best option, RBCs from a donor who is heterozygous for that gene. The computer program developed for this study investigated the usefulness of commercially available panels from separate manufacturers in excluding the presence of additional antibodies. A list of 488 antibodies detected by a regional blood bank in 1994 was used as cases of antibodies documented in the past. RESULTS : In 61 percent of the cases, successful EPs (i.e., those complying with the criteria), consisting of three to four different phenotypes, were selected. In the remaining 39 percent of cases, it was impossible to generate successful EPs: 1 to 2 additional antibodies could not be excluded or could be excluded only by using RBCs from heterozygotes. Commercial panels differed only slightly in their efficiency in providing suitable RBCs. None of the commercial panels could provide suitable RBCs to exclude all additional antibodies in the presence of anti-c, anti-e, or anti-M. Increasing the number of RBCs from which to select EPs only slightly increased the percentage of success. CONCLUSION : Computer-aided construction of EPs quickly shows whether strict criteria can be met or whether alternative techniques should be used. It leads to a significant reduction in the number of RBC suspensions necessary to exclude additional antibodies. Results with various commercial panels differed only slightly.     

睡眠剥夺对力竭运动大鼠胸腺谷胱甘肽、丙二醛、超氧化物歧化酶的影响

目的:观察不同睡眠剥夺时间后力竭运动对大鼠胸腺谷胱甘肽、丙二醛含量和超氧化物歧化酶活性的变化,探讨睡眠剥夺对大鼠抗氧化能力的影响。方法:实验于2006-04/05在湖南师范大学体育学院运动生物化学实验室完成。实验分组:选择10周龄健康雄性SD大鼠30只,按随机数字表法分为5组,每组6只:睡眠非运动组,睡眠 力竭运动组和睡眠剥夺24,48,72h 力竭运动组。实验方法:①采用轻柔刺激法制备大鼠睡眠剥夺模型。②睡眠非运动组和睡眠 力竭运动组不进行睡眠剥夺。③睡眠 力竭运动组和睡眠剥夺各组大鼠运动方案:跑台坡度为10°,速度为19.3m/min(相当于76%VO2max),所有大鼠运动至力竭(运动末期,大鼠先后滞留跑道后1/3处达3次以上,各种刺激驱赶均无效,停跑后体征表现为呼吸急促,神情倦怠,腹卧位,对刺激反应迟钝,捕捉时,逃避反应较运动前减弱)。实验评估:①大鼠一般状态。②力竭时间。③大鼠力竭后麻醉处死,测定胸腺谷胱甘肽、丙二醛含量和超氧化物歧化酶活性。结果:纳入大鼠30只,均进入结果分析。①大鼠一般状态:睡眠 力竭运动组大鼠表现为形态正常,活泼好动,皮毛光亮,眼睛有神;睡眠剥夺48,h 力竭运动组大鼠均出现神态倦怠,眼神黯淡,四肢亦有不同程度的红肿;睡眠剥夺24h 力竭运动组大72鼠介于以上两者之间。②睡眠 力竭运动组和睡眠剥夺24,7248,h组大鼠的力竭时间分别为(232.36±37.67),(269.19±38.61),(162.42±35.70),(141.07±28.56)。③谷胱甘肽含量、超氧化物歧化酶活性:睡眠 力竭运动组谷胱甘肽含量和超氧min化物歧化酶活性均低于睡眠非运动组[分别为(25.54±0.79),(27.09±1.31)mg/g;(±0.21),(±0.10)mkat/g],差异有显4.594.88著性意义(P<0.05);睡眠剥夺24h 力竭运动组谷胱甘肽含量和超氧化物歧化酶活性均高于睡眠非运动组[分别为(28.60±0.96),(27.09±1.31)mg/g;(±0.10),(±0.10)5.234.88mkat/g],差异有显著性意义(P<0.05),睡眠剥夺48,h 力竭运动组P72均低于睡眠非运动组[分别为(23.74±1.19),(22.43±0.52),(27.09±1.31)mg/g;(±0.14),(±0.18),(±0.10)mkat/g],4.523.354.88差异均有非常显著性意义(P<0.01);睡眠剥夺各组与睡眠 力竭运动组间谷胱甘肽含量和超氧化物歧化酶活性差异均有非常P显著性意义(P<0.01);睡眠剥夺各组间比较差异有显著性意义(P<0.05)。④丙二醛浓度:睡眠 力竭运动组丙二醛浓度高于P睡眠非运动组[分别为(±0.27),(±0.24)μmol/L],差异有非常显著性意义(P<0.01);睡眠剥夺各组与睡眠非运动组之间6.565.35P差异均有非常显著性意义(P<0.01);睡眠剥夺24,48,72h 力竭运动组丙二醛含量均高于睡眠 力竭运动组[分别为(±0.12),(±P7.398.850.72),(10.89±0.82),(±0.27)μmol/L],差异有显著性意义(P<0.05;P<0.01);睡眠剥夺各组间比较,睡眠剥夺48h与睡眠剥夺24h差6.56异无显著性意义(P>0.05),睡眠剥夺72h与睡眠剥夺24h、睡眠剥夺72h与睡眠剥夺48h间比较差异有非常显著性意义(P<0.01)。结论:①睡眠剥夺24h可引起大鼠胸腺氧化应激,使氧自由基能力有所增强。②睡眠剥夺48,72h力竭运动后氧自由基能力降低。     

Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.     

Improvement in lipids after switch to boosted atazanavir or darunavir in children/adolescents with perinatally acquired HIV on older protease inhibitors: results from the Pediatric HIV/AIDS Cohort Study

Objectives

Dyslipidaemia is common in perinatally HIV‐infected (PHIV) youth receiving protease inhibitors (PIs). Few studies have evaluated longitudinal lipid changes in PHIV youth after switch to newer PIs.

Methods

We compared longitudinal changes in fasting lipids [total cholesterol (TC), triglycerides (TG), low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), and TC:HDL‐C ratio] in PHIV youth enrolled in the Pediatric HIV/AIDS Cohort Study (PHACS) Adolescent Master Protocol (AMP) study who switched to atazanavir/ritonavir (ATV/r)‐ or darunavir/ritonavir (DRV/r)‐based antiretroviral therapy (ART) from an older PI‐based ART and those remaining on an older PI. Generalized estimating equation models were fitted to assess the association of a switch to ATV/r‐ or DRV/r‐based ART with the rate of change in lipids, adjusted for potential confounders.

Results

From 2007 to 2014, 47 PHIV children/adolescents switched to ATV/r or DRV/r, while 120 remained on an older PI [primarily lopinavir/r (72%) and nelfinavir (24%)]. Baseline age ranged from 7 to 21 years. After adjustment for age, Tanner stage, race/ethnicity, and HIV RNA level, a switch to ATV/r or DRV/r was associated with a more rapid annual rate of decline in the ratio of TC:HDL‐C. (β = ?0.12; P = 0.039) than remaining on an older PI. On average, TC declined by 4.57 mg/dL/year (P = 0.057) more in the switch group. A switch to ATV/r or DRV/r was not associated with the rate of HDL‐C, LDL‐C, or TG change.

Conclusions

A switch to ATV/r or DRV/r may result in more rapid reduction in TC and the TC:HDL‐C ratio in PHIV youth, potentially impacting long‐term cardiovascular disease risk.

     

Human umbilical vein endothelial cells secrete transcobalamin II

Transcobalamin II (TC II) is essential for cellular uptake of cobalamin. However, the origin of this transport protein is controversial and many organ sources have been suggested. We studied human umbilical vein endothelial cells cultured in vitro. The cells contained TC II (2.3 pmol/10(8) cells) and released progressively increasing amounts of the protein into the surrounding medium during the 3-day incubation period. This release exceeded the starting intracellular content of TC II. In contrast, endothelial cells did not contain or elaborate R binder, the other major circulating binding protein for cobalamin, Cycloheximide inhibited the elaboration of TC II, suggesting that the endothelial cells synthesize the protein. Thrombin, which stimulates tissue plasminogen activator release, did not enhance TC II release, and neither did endotoxin or mellitin. However, thrombin did appear to partially protect TC II release from inhibition by cycloheximide. Among other cells studied, human fibroblasts also released TC II into the incubation medium, while K562 human leukemia cells, ARH-77 and HS Sultan human plasma cell lines, and Raji strain lymphoblasts did not. The data suggest that endothelial cells are an important source of the metabolically crucial TC II.     

BCR-ABL protein expression in peripheral blood cells of chronic myelogenous leukemia patients undergoing therapy

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome (Ph1) in more than 95% of these patients. The Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. In CML, the product of the fused BCR- ABL gene is typically a protein of approximately 2,000 amino acids termed P210 BCR-ABL. We have developed an assay for the BCR-ABL protein involving Western blotting of circulating white blood cells (WBC) with an anti-ABL monoclonal antibody that can detect P210 BCR-ABL and P145 ABL in peripheral blood cells from chronic phase Ph1-positive leukemia patients. This assay was used to analyze the BCR-ABL protein content of circulating WBC from CML patients before and after various treatments. In parallel to changes in percentages of Ph1-positive blood cells as determined by cytogenetic analyses of bone marrow samples, BCR-ABL protein expression in blood cells decreased or increased as patients entered remission or underwent relapse. Of interest, six Ph1-negative CML patients were BCR-ABL protein-positive. All except one had a rearrangement in the major breakpoint cluster region and that patient expressed P185 BCR-ABL and not P210. Our results indicate that the BCR- ABL Western blotting assay has clinical applications for both diagnosis and prospective evaluation of Ph1-positive and Ph1-negative CML patients.     

The polyadenylation site mutation in the alpha-globin gene cluster

In a previous study, we described a form of nondeletion alpha- thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.