Kentucky UST Field Manual

This study was undertaken to address the removal and closure of defective petroleum underground storage tanks in Kentucky. Goals for the study included: To address standards for levels of contamination requiring corrective action consistent with accepted scientific and technical principles. To recommend a matrix or scoring system to be used for (a) ranking sites as to actual or potential harm to human health and the environment caused by release of petroleum from a petroleum storage tank, and (2) establishing standards and procedures for corrective action that shall adequately protect human health and the environment. To address all compounds individually and collectively known as petroleum. To produce a report that shall be scientifically defensible

Status Report: Identification of Appropriate Standards for Corrective Action for a Release from Petroleum Underground Storage Tanks, Volume 1

This study was undertaken to address the removal and closure of defective petroleum underground storage tanks in Kentucky: To address standards for levels of contamination requiring corrective action consistent with accepted scientific and technical principles. To recommend a matrix or scoring system to be used for (a) ranking sites as to actual or potential harm to human health and the environment caused by a release of petroleum from a petroleum storage tank, and (b) establishing standards and procedures for corrective action that shall adequately protect human health and the environment. To address all compounds individually and collectively known as petroleum. To produce a report that shall be scientifically defensible

Loss of estrogen receptor β decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice

Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females

Biomechanical And Metabolic Changes Within Rabbit Articular Cartilage Following Treatment With Radiofrequency Energy

The effects caused to articular cartilage by the remote use of arthroscopically-delivered RF energy to soft tissues in the joint are unknown. This investigation reported the short and long-term effects of bRF and mRF energy on the biomechanical properties and metabolic activity of articular cartilage. In addition, the effect of Cosequin® therapy was addressed. Thirty New Zealand white rabbits were randomly assigned to one of two treatment groups (Group 1 - placebo; Group 2 - Cosequin®). Histopathology, cell viability, GAG synthesis, and mechanical function of the articular cartilage were compared between groups. Data were analyzed using a mixed model ANOVA (p=0.05). Immediate chondrocyte death was created by both RF devices. This damage was noted to be superficial and did not lead to the progressive deterioration of the extracellular matrix or mechanical function of the articular cartilage. Cosequin® therapy was unable to demonstrate significant differences compared to the control group

Interleukin-11 for treatment of hepatitis C-associated ITP

Immune thrombocytopenic purpura (ITP) is frequently associated with chronic hepatitis C (HpC-ITP). Recombinant interleukin-11 (rIL-11), which has both thrombopoietic and anti-inflammatory properties, was evaluated in 12 patients with HpC-ITP in this pilot study. Group 1 (7 patients) was treated at high dose (50 microg/kg daily) while group 2 (5 patients) at low dose (15-35 microg/kg three/week). In group 1, mean platelet counts rose from initial 54 x 10(9)/l to 103 x 10(9)/l (p = 0.02) and in group 2, from an initial 51 x 10(9)/l to 74 x 10(9)/l (p = 0.04). Antiplatelet antibody (aPlt-Ab) decreased in group 1. LFT improved in both groups. The mean HCV-RNA decreased in group 1 (p = 0.04), not in group 2. Side effects were common and troublesome, but were minimized with individualized dosing. One patient achieved good remission of both ITP and HpC lasting >2 years with low-dose maintenance. When used based on individual tolerance, rIL-11 appears useful in HpC-ITP

Interaction Of Platelets With Red Cell-Derived Microparticles (RMP): RMP Increase Platelet Aggregate Size In a Shear-Dependent Manner

Abstract Background Red cell microparticles (RMP) have come to recent attention as putative mediators of hemostasis. We reported that RMP improve hemostatic defect of blood samples of thrombocytopenia and thrombocytopathy and augment platelet function. To investigate possible mechanisms of this activity, we measured the effect of RMP on shear-dependent platelet adhesion and aggregation in whole blood. Methods RMP were produced by high-pressure extrusion of washed, packed RBC. The RMP produced in this way are similar to natural circulating RMP in phenotype and most functional assays. Blood was collected in citrate Vacutainers from normal healthy staff volunteers, and first 3 mL discarded to minimize artifact of platelet activation due to tissue factor. It was tested within 2 hours of drawing. Variable shearing rates were applied by a cone-and-plate device, the DiaMed Impact-R, which yields photomicrographs of objects adhering to the plate, and data including percent surface coverage (SC), number objects adhering (OBJ) per mm2, and mean aggregate size (AS) in μm2. Initially, 4μL of either RMP (1.0 x 108/μL) or saline were mixed with 126μL of whole blood obtained as above, and incubated in a microcentrifuge tube for 60s. The mixtures were pipetted to a well of the device and run at a selected shear rate (range 900s-1 to 2700s-1) for 2 minutes. Blood was then carefully drawn off and the wells were washed gently with deionized water. Wells were stained with May-Grünwald stain for 60s, and excess removed. When dry, micrographs of the well surfaces were taken. The most promising shear rates (1125 s-1, 1575s-1, 1800s-1, 2025s-1) were retested with higher numbers of RMP in the 130μL volume. Results Addition of RMP vs. saline (control) induced increased AS over a range of shear rates. For example, 4μL RMP (1x108/μL) running at 2250s-1 increased AS by 34.8%, from 59.2 ±25.1μm2 to 79.8 ±24.7μm2 (n=15, p=0.01). To investigate effect of RMP concentration at fixed shear rate of 1575s-1, we found that 8μL RMP induced increase of 12.0 μm2, 12μL RMP by 24.8 μm2, 16μL by 26.7 μm2, and 20μL by 26.3 μm2; p<0.05 and n=4 replicates for all. As seen by the trend, this effect on AS reached a plateau at 20 μL RMP. With fixed volume of RMP added and varying shear rate, we found the largest RMP-induced increase in AS occurred at 1125s-1 shear rate: 16μL of RMP increased aggregate size to 172% of control (from 41.3 ±10.2μm2 to 71.0 ±2.6μm2; n=3, p=0.02). With 1/4 as much RMP (4 μL), the peak effect occurred at 1125s-1. We did not observe any significant differences in SC at any shear rate or RMP volume. OBJ was generally lower as AS increased, but this did not reach significance. Conclusion/Discussion RMP increased the size of platelet aggregates under shear, indicating enhanced platelet adhesion by RMP. This effect varied with shear rate: larger amounts of RMP had maximum effect at lower shear rates; smaller amounts of RMP required higher shear to exert maximum effect. These effects are seen at physiologically relevant shear rates (200 - 2000s-1 or higher in pathology such as hypertension or valve defects). Due to the absence of endothelia from this model, high shear was required to induce maximum adhesion. Previous study has indicated the important role of GPIIb/IIIa in platelet adhesion and aggregate size under high shear [Varon, et al; Am Heart J, 1998]. We postulate that RMP may be acting in a GPIIb/IIIa dependent manner to enhance aggregate size. This was consistent with the findings that RMP-induced augmentation of platelet aggregation at relevant levels of ADP or AA concentrations in heparinized blood [Jy, et al; Thromb Haemost, in press]. We are continuing to investigate conditions affecting the interactions between RMP and platelets. Disclosures: No relevant conflicts of interest to declare

Exchange Plasmapheresis Is Effective In Removing Procoagulant Microparticles and Has Additional Therapeutic Benefit In The Treatment Of TTP

Abstract Introduction In thrombotic thrombocytopenic purpura (TTP), autoantibodies bind and deplete ADAMTS13, a vWF cleaving protease, leading to accumulation of large molecular weight vWF multimers, excessive platelet adhesion and aggregation in microvasculature, formation of platelet rich thrombi and disruption of microcirculation. This results in microangiopathic hemolytic anemia, severe thrombocytopenia and multi-organ dysfunction. Under higher shear stress, red cells undergo shape changes, fragmentation and release of red cell microparticles (RMP). Similarly, MP are released from activated platelets (PMP) and endothelium (EMP). It is now well established that cell derived MP play an important role in hemostasis and thrombosis. Roles of circulating MP in TTP are not well elucidated. Exchange plasmapheresis and infusion of FFP is now a standard therapy for TTP. It is assumed that plasmapheresis removes autoantibodies against ADAMTS 13 and plasma infusion supplies the deficient protease to resume normal hemostasis. It is possible that plasmapheresis also removes procoagulant MP, contributing to remission. We studied MP profiles in patients with acute TTP and investigated efficacy of exchange plasmapheresis to remove procoagulant MP. Methods Three patients presenting acute TTP were admitted for exchange plasmapheresis. In addition to routine CBC, platelet counts, reticulocyte counts, blood chemistry, LDH, special assays of MP derived from platelets (PMP), red cells (RMP), leukocytes (LMP) and endothelium (EMP) were performed daily and before and after each exchange plasmapheresis. Samples were drawn immediately before and immediately after plasmapheresis into citrated vacutainer tubes. Microparticles were assayed by flow cytometry using fluorophore conjugated mAb’s specific to endothelial, platelet, leukocyte, or erythrocyte lineage. Thromboelastography (TEG) was employed to study thrombus formation in vitroand correlate microparticles and clinical parameters. Results 1) Effects of plasmapheresis on MP levels: Circulating RMP and PMP levels fell rapidly following ExPh. Each round of plasmapheresis reduced RMP by 45% (p=0.001) from starting baselines. Repeated plasmapheresis over one week removed 74% of RMP from the peak level (p<0.01). By day 6, RMP were, on average, reduced from 4111/μL to a normal level of 1100/μL. Each round of plasmapheresis also reduced PMP by 53% from baseline (p=0.001). Over one week period, PMP were reduced by 69% from peak levels. 2) Effects of plasmapheresis on LDH, HGB, and platelets: LDH was reduced by 30% on average over a week period, from 845 ±211U/L to 545 ±33U/L, but this represented only 45% normalization. Platelet count normalized completely, increasing almost fivefold from an average of 46 ±22 to 219 ±68 after seven days (p<0.05). HGB fell an average of 10% over the course of treatment but this result was not significant. 3) Effect of plasmapheresis on TEG parameters: Exchange plasmaphresis tended to prolong the R time (8.4 ±2.2 minutes pre vs. 10.9 ±6.1 minutes post). It also increased K time by 38% and reduced CI by 42%. Although these changes did not reach statistical significance due to high degree of variation in the post-plasmapheresis group, the trend indicated plasmapheresis reduced procoagulance and hypercoagulability. Conclusion Exchange plasmapheresis is very effective in reducing procoagulant and proinflammatory RMP and PMP. Clearance of microparticles resulted in improved TEG parameters and reduced hypercoagulability in TTP. Benefits were pronounced even in the first round of plasmapheresis. Exchange plasmaphresis has additional therapeutic benefit in the treatment of TTP by removing procoagulant microparticles and reducing hypercoagulability in TTP. Disclosures: No relevant conflicts of interest to declare

Elevated Cholinesterase Activity in Patients with TIA and Other Thrombotic Disorders

Abstract Abstract 2991 Poster Board II-967 Background. The natural function of ACTH in blood is not well defined. However elevated plasma level was reported in patients with chronic inflammation, suggesting as one of inflammatory markers. Erythrocytes possess membrane-bound acetylcholinesterase (AChE) while plasma contains a less specific cholinesterase (ChE). We reported at a previous meeting [Blood 2008, 112(11) Abst #3849] that AChE activity of RBC-derived microparticles (RMP) is 6 times higher than those in PMP. In this study we measured plasma ACTH levels in several groups and encountered unexpectedly high plasma ChE activities in patients with TIA and other thrombosis. Methods. (i) Patient population. A series of n=108 consenting patients were recruited sequentially having various disorders For purpose of analysis, they were divided in 2 main groups: n=53 with thrombosis (TBS), F/M 27/26; and n=55 non-TBS, F/M 24/31. Thrombosis group consists of venous and arterial thrombosis including those with TIA. Non TBS group consists of anemias, thrombocytopenias including ITP, MDS. (ii) Assay was essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in unitsof mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma, = mean +2SD. (iii) Sample handling. Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate. Results: (i) Significantly higher ChE activity was observed in thrombosis (TBS) patients compared to non-TBS. Mean value ±SD in TBS was 2928 ±773, and in Non-TBS was 1897 ±713 (units as above). This difference was significant, p3000) in 30% of patients, while the Non-TBS had elevated ChE in 7.8%. (iii) Further analysis among thrombosis group revealed that the subgroup with neurological TBS (TIA, minor strokes) had the most consistent elevations of ACTH; 10 of15 with TIA group (66%) had ChE activities >3000. Summary/ Discussion. Although ChE and AChE in blood has long been studied, to our knowledge, this is the first report to show a relation between plasma ChE and thrombosis. Its elevation is very pronounce in thrombosis of the CNS, manifesting as TIA. Further study is warranted to elucidate how ChE is related to AChE of the CNS and on red cells. Disclosures: No relevant conflicts of interest to declare

Plasma Cholinesterase Activity in ITP Patients with/without Thrombosis

Abstract Abstract 3997 Poster Board III-933 Introduction In the process of immune mediated destruction, platelets may become activated and release procoagulant microparticles in ITP (Immune Thrombocytopenic Purpura). Although bleeding is common manifestation, some patients with ITP may suffer thrombotic complications, often presenting with TIA like syndromes. Brain MRI revealed findings consistent with ischemic small vessel disease in this subgroup (J Lab Clin Med 119: 334, 1992, Thromb Res 107:337, 2002). Recently acetylcoholinesterase (AChE) of RBC and/or non-specific plasma cholinesterase (ChE) was reported to be associated with the inflammation of blood vessels and may be a marker of some inflammatory states. In the present study we investigated plasma ChE activity in patients with ITP with or without thrombotic complication. Methods (i) Patients. We measured ChE activity in 49 patients with ITP. They were sub-grouped as having thrombosis (TBS), F/M (11/7), and non-thrombosis (Non-TBS), F/M (23/8). The TBS group included 9 with TIA like syndrome, 6 with CAD and 3 with venous thrombosis. (ii) Assay of ChE was essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in units of mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma (=mean +2SD). (iii) Sample handling. Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate. Results (i) We observed higher level of ChE in the TBS group compared to Non-TBS in ITP. The TBS group (+/- SD) had mean value 2859 ±866, while the Non-TBS group had 2267 ±777 (units as above). This difference was significant, p3000 (normal cutoff values) compared to only 2/9 (22%) of those with other TBS >3000. Conclusion (i) The ChE activity of ITP patients with TBS is significantly increased compared to Non-TBS in ITP patients. It has been suggested that Blood ChE/AChE may reflect some inflammatory, prothrombotic states. (ii) ITP patients with TIA exhibited higher ChE activity among TBS subgroups in ITP, suggesting that ChE activity may serve as a useful biomarker in TIA like syndrome associated with ITP. However, further study in a larger number of patients is needed to confirm these preliminary findings. Disclosures: No relevant conflicts of interest to declare