Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient''s blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient''s blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 °C, the other sample represented the patient''s baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 °C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. ^2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.     

Neurohormonal and cytokine fluctuations following transcatheter closure for an atrial septal defect

Introduction

Inflammation and neurohormonal activation are considered to be involved in the development of earlier and/or later complications in congenital heart disease patients, even after a successful repair of the lesion. It is not yet clarified what is the role of the therapeutic interventions in the occurrence of such a response and how it could be associated with possible postoperative complications.

Aim

We sought to assess the inflammatory and neurohormonal response to transcatheter closure of secundum type atrial septal defects (ASD) over a six-month follow-up period. We also evaluated the association between the respective markers and catheterization data as well as echocardiographic measurements.

Methods

Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), N-terminal-proatrial natriuretic peptide (NT-proANP) and N-terminal-probrain natriuretic peptide (NT-proBNP) were assessed and echocardiographic measurements were performed in twenty-eight patients with atrial septal defect prior to, and at the first, second and sixth months post transcatheter closure. Thirty-three age-matched healthy volunteers were also enrolled.

Results

IL-6 plasma levels, although higher preoperatively, [physical logarithm (ln) IL-6: 3.37 ± 0.66 vs 2.92 ± 0.44 pg/ml, p = 0.015], reached control levels postoperatively, at the end of the third month, whereas TNF-α and IL-10 were not influenced by the procedure. NT-proANP levels were elevated preoperatively compared to the control group (ln NT-proANP 3.78 ± 0.572 vs 3.48 ± 0.30, p = 0.031), with a further significant increase during the 1st month (ln NT-proANP 3.78 ± 0.572 vs 4.2 ± 0.42, p = 0.006), following the pattern of the left atrial volume enlargement, and remained high even 6 months after the procedure .On the other hand, the initially normal concentrations of NT-proBNP, after a transient significant increase during the first month postoperatively (ln NT-proBNP 3.56 ± 0.94 vs 4.58 ± 0.91, p < 0.0001) returned to the controls’ levels at the end of the third month. Preoperative concentrations of NT-proANP positively correlated with NT-proBNP concentrations and pulmonary to systemic flow ratio (Qp/Qs).

Conclusions

Transcatheter closure could improve, on a mid- term basis, the inflammatory process but natriuretic peptides’ secretion continues in parallel with left atrial volume increase. Further follow up is required to determine the long-term progress of the inflammatory and neurohormonal response to the procedure.     

New approach to isolate mesenchymal stem cell (MSC) from human umbilical cord blood

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.     

Effects of blood meal source on the reproduction of Culex pipiens quinquefasciatus (Diptera: Culicidae)

Culex pipiens quinquefasciatus were fed blood meals from a live chicken (LC), chicken blood in Alsever's (AC) solution, defibrinated bovine blood (DB), or bovine blood in citrate (CB) and incubated at 28° C. The effects of different blood meal sources were evaluated with respect to rates of blood feeding and reproduction (i.e., fecundity and fertility) over two gonotrophic cycles. Mosquitoes that fed on the first blood meal were subjected to a second blood meal as follows (first blood meal / second blood meal): LC/LC, LC/DB, DB/DB, CB/CB, AC/AC. Fecundity and fertility of Cx. p. quinquefasciatus were significantly (P < 0.05) greater in mosquitoes fed LC blood; however, fecundity and fertility in different treatment groups varied by gonotrophic cycle. These results contribute to our understanding of the impact of blood meal source on feeding and reproduction in Cx. p. quinquefasciatus. The potential impacts of blood meal source on virus transmission experiments are discussed.     

Theileria,Babesia, and Anaplasma detected by PCR in ruminant herds at Bié Province,Angola

Distribution of Anaplasma spp., Babesia spp., Theileria spp., and Ehrlichia ruminantium, was for the first time studied in Bié Province, central Angola. We examined 76 blood samples of cattle originated from seven farms, and 13 blood samples of goats from two farms employing molecular genetic tools (PCR). Most prevalent was A. ovis-infection in goats (100%) and A. marginale-infection in cattle (38% of examined animals, and six out of seven farms). B. bigemina-infection was detected in only one specimen at Andulo, whereas B. bovis was not detected in Bié. We did not detected T. parva, the causative agent of serious diseases in cattle; nevertheless, infection by T. velifera was quite frequent (14% of examined animals, and five out of seven farms). Causative agent of heartwater disease – E. ruminantium, was not detected. Taking into account short-term perspective of PCR methods in monitoring of epidemiological status in herds, the number of infected animals and distribution of detected pathogens should not be ignored.     

OLFML3 expression is decreased during prenatal muscle development and regulated by microRNA-155 in pigs

The Olfactomedin-like 3 (OLFML3) gene has matrix-related function involved in embryonic development. MicroRNA-155 (miR-155), 21- to 23-nucleotides (nt) noncoding RNA, regulated myogenesis by target mRNA. Our LongSAGE analysis suggested that OLFML3 gene was differently expressed during muscle development in pig. In this study, we cloned the porcine OLFML3 gene and detected its tissues distribution in adult Tongcheng pigs and dynamical expression in developmental skeletal muscle (12 prenatal and 10 postnatal stages) from Landrace (lean-type) and Tongcheng (obese-type) pigs. Subsequently, we analyzed the interaction between OLFML3 and miR-155. The OLFML3 was abundantly expressed in liver and pancreas, moderately in lung, small intestine and placenta, and weakly in other tissues and postnatal muscle. There were different dynamical expression patterns between Landrace and Tongcheng pigs during prenatal skeletal muscle development. The OLFML3 was down-regulated (33-50 days post coitus, dpc), subsequently up-regulated (50-70 dpc), and then down-regulated (70-100 dpc) in Landrace pigs, while in Tongcheng pigs, it was down-regulated (33-50 dpc), subsequently up-regulated (50-55 dpc) and then down-regulated (55-100 dpc). There was higher expression in Tongcheng than Landrace in prenatal muscle from 33 to 60 dpc, and opposite situation from 65 to 100 dpc. Dual luciferase assay and real time PCR documented that OLFML3 expression was regulated by miR-155 at mRNA level. Our research indicated that OLFML3 gene may affect prenatal skeletal muscle development and was regulated by miR-155. These finding will help understanding biological function and expression regulation of OLFML3 gene in mammal animals.     

赤子爱胜蚓在不同猪粪和农作物秸杆混合物中生长繁殖状况

本文研究了赤子爱胜蚓在长春市城郊不同组合的粮食猪粪、饲料猪粪和玉米、水稻秸杆混合物中生长繁殖状况,结果发现赤子爱胜蚓的质量在0 ～30 d大幅度增加,30 ～ 60 d增加减缓,60～ 90 d呈负增长趋势.赤子爱胜蚓产茧量在试验期间的总趋势是递增的.猪粪种类、秸杆种类和两者的混合比例差异对赤子爱胜蚓的质量变化产生重要影响.粮食猪粪和玉米秸杆混合物的质量比例大致在1∶1时特别适宜赤子爱胜蚓生长和繁殖.赤子爱胜蚓在饲料猪粪和玉米秸杆混合物中的日增重倍数和日产茧量多于饲料猪粪和水稻秸杆混合物.本试验结论可为城郊猪粪和作物秸杆的大规模资源化利用提供参考.     

两个国内小型猪品种GH基因部分序列的多态性分析

目的对西藏小型猪和广西巴马小型猪生长激素基因（GH基因）部分序列的多态性进行分析。方法采用内切酶ApaI和Hin6I对西藏小型猪（108头）和广西巴马小型猪（132头）GH基因-119 bp～＋486 bp之间的区域进行PCR-RFLPs分析。结果 （1）从ApaI酶切产生的结果来看,ApaI酶切产生A（449 bp＋101 bp＋55bp）,B（316 bp＋133 bp＋101 bp＋55 bp）两种等位基因。等位基因A的频率高于等位基因B,等位基因A为优势基因。AA基因型频率高于AB和BB基因型频率;（2）从Hin6I酶切产生的结果来看,Hin6I酶切产生G1（605bp）、G2（498 bp＋107 bp）、G3（449 bp＋156 bp）、G4（449 bp＋107 bp＋49 bp）四种等位基因。等位基因G4的频率高于等位基因G1、G2、G3。等位基因G1频率很低。基因型G2G3、G2G4、G3G4、G4G4的频率较高。（3）由酶切产生的基因和基因型多态性,发现西藏小型猪与广西巴马小型猪在该基因部分序列差异不显著（P〉0.05）。结论国内的优质实验用小型猪,如西藏小型猪和广西巴马小型猪等位基因A频率均较高。     

手工和仪器两种血培养结果差异性分析

目的对手工法双相血培养瓶和BACTEC9120全自动血培养仪的阳性率作回顾性分析。方法将血液标本同时接种双相血培养基和BACTEC9120全自动血培养仪配套血瓶中,将阳性结果移种血平板,如为阴性再移种巧克力平板。结果370例血培养,双相血培养瓶阳性25例,阳性率为6．76％（25／370）,树脂需氧（儿童）瓶BACTEC9120报警显示阳性59例,阳性率为15．9％（59／370）,阳性标本移种到血平板及巧克力平板阳性54例,阳性率为14．6％（54／370）,假阳性5例,假阳性率为1．4％（5／370）,共有29例树脂需氧（儿童）瓶阳性,而双相血培养瓶为阴性,P〈0．001。结论BACTEC9120全自动血培养仪提高阳性率,缩短阳性的报告时间优于传统的双相血培养基。