A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

INTRODUCTION: Standardization of BCR-ABL1 messenger RNA quantification by real-time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR-ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory-specific conversion factor (CF), co-ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR-ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results. METHODS: To overcome these difficulties, we have developed an alternative and simple approach of CF calculation, based on the retrospective analysis of existing external quality assessment (EQA) data. Our approach does not depend on the exchange of samples and is solely based on the mathematical CF calculation using EQA results. RESULTS AND CONCLUSION: We have demonstrated by thorough statistical validation that this approach performs well in converting BCR-ABL1 measurements to improve IS estimation. In expectation of a true golden standard method for BCR-ABL1 IS quantification, the proposed method is a valuable alternative.

Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5-20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics.

Contribution of postnatally formed small beta cell aggregates to functional beta cell mass in adult rat pancreas.

AIMS/HYPOTHESIS: Neogenesis of beta cells and their clustering to small aggregates is a key process in prenatal development of beta cell mass. We investigated the contribution of postnatally formed small aggregates to functional beta cell mass in adult rats. METHODS: Conditions were defined for (1) counting total beta cell number in pancreases with relative error of <10% and (2) determining their distribution over aggregates of different size and over functionally different subpopulations. RESULTS: Pancreases of 10-week-old male Wistar rats contained 2.8 ± 0.2 × 106 beta cells, of which >90% was generated postnatally, involving: (1) neo-formation of 30,000 aggregates with diameter <50 µm including single cells; and (2) growth of 5,500 aggregates to larger sizes, accounting for 90% of the increase in cell number, with number of growing aggregates in the tail 50% greater than elsewhere. At 10 weeks, 86% of aggregates were <50 µm; compared with aggregates >200 µm, their beta cells exhibited a higher basal insulin content that was also resistant to glibenclamide-induced degranulation. The pool of Ki67-positive beta cells was sixfold larger than at birth and distributed over all aggregate sizes. CONCLUSIONS/INTERPRETATION: We describe a method for in situ counting of beta cell numbers and subpopulations with low relative error. In adult rats, >90% of beta cells and beta cell aggregates are formed after birth. Aggregates <50 µm are more than 100-fold more abundant than aggregates >200 µm, which are selected for isolated islet studies. Their topographic and functional properties contribute to the functional heterogeneity of the beta cell population; their growth to larger aggregates with characteristic beta cell functions may serve future metabolic needs.

Restoring a functional beta-cell mass in diabetes.

Type 1 and type 2 diabetes have often been presented as disease forms that profoundly differ in the presence and pathogenic significance of a reduced beta-cell mass. We review evidence indicating that the beta-cell mass in type 1 diabetes is usually not decreased by at least 90% at clinical onset, and remains often detectable for years after diagnosis at age above 15 years. Clinical and experimental evidence also exists for a reduced beta-cell mass in type 2 diabetes where it can be the cause for and/or the consequence of dysregulated beta-cell functions. With beta-cell mass defined as number of beta-cells, these views face the limitation of insufficient data and methods for human organs. Because beta-cells can occur under different phenotypes that vary with age and with environmental conditions, we propose to use the term functional beta-cell mass as an assessment of a beta-cell population by the number of beta-cells and their phenotype or functional state. Assays exist to measure functional beta-cell mass in isolated preparations. We selected a glucose-clamp test to evaluate functional beta-cell mass in type 1 patients at clinical onset and in type 1 recipients following intraportal islet cell transplantation. Comparison of the data with those in non-diabetic controls helps targeting and monitoring of therapeutic interventions.

A medium-density genetic linkage map of the bovine genome.

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.

Comparison of exercise and normal variability on HDL cholesterol concentrations and lipolytic activity.

In order to compare the influence of a single bout of exercise on HDL-C metabolism with normal variability, 12 male runners (mean age: 24.9 +/- 4 yr) who ran 15-30 miles per week underwent exercise (E) and control (C) experimental conditions. During the E trial subjects ran on a motor driven treadmill at 75% (42.5 +/- 4.7 ml.kg-1.min-1) VO2max until 800 Kcals were expended. The C trial consisted of no exercise. Subjects were instructed to follow the same diet and keep a four d food diary during each experimental condition. Fasted blood samples were obtained at the same time of day in each condition at time points corresponding to 24 h pre-exercise (24 PRE), 6 h post- (6 h) and 24 h post-exercise (24 h). Plasma was analyzed for HDL-C, HDL2-C and HDL3-C (mg.dl-1). In addition post-heparin plasma samples were analyzed for lipoprotein lipase (LPL) and hepatic lipase (HL) activity (mumol.FFA-1.ml-1). All values were adjusted for changes in plasma volume and compared to Baseline. HDL-C levels were unaltered following the C trial. However, following the E trial, HDL-C increased (p < 0.01) above baseline values at 24 h. The increase in HDL-C was reflected in the HDL3-C subfraction (p < 0.05). Analysis of lipolytic activity revealed an overall greater LPL activity (p < 0.05) in the E trial vs the C trial. In addition, a decrease in HL was observed at 24 h (p < 0.05) but was not different between experimental conditions. These data suggest that exercise and not normal variability are responsible for alterations in lipolytic activity and corresponding increases in HDL-C levels.

Effect of carbohydrate substrate availability on ratings of perceived exertion during prolonged exercise of moderate intensity.

This investigation examined the effect of carbohydrate substrate availability on ratings of perceived exertion (RPE) during cycling at moderate intensity to exhaustion and the relation between submaximal endurance performance and RPE obtained following 2 hr. of cycling at moderate intensity. Seven male cyclists performed two exercise trials at power output corresponding to 70% of their peak oxygen uptake until exhaustion. Subjects ingested either a 6% glucose/sucrose solution at the rate of 0.6 g.kg-1 (Body Weight).hr.-1 or an equal volume of artificially flavored placebo every 20 min. throughout the exercise trials. RPE for the legs, chest, and over-all body, and oxygen consumption, expired ventilation, carbohydrate oxidation rate, and blood concentrations of glucose, glycerol, and lactate were measured every 20 min. throughout exercise and at exercise termination. Statistical analysis of these dependent variables indicates that (1) an exercise-induced decrease in blood-borne carbohydrate substrate intensifies leg and over-all perceptions of exertion during the later stages of prolonged cycling at 70% VO2peak. (2) Factors other than availability of blood-borne carbohydrate substrate may influence perceptual intensity at exhaustion. (3) Ratings of perceived exertion for the legs and over-all body obtained after 2 hr. of cycling at moderate intensity may be useful in predicting submaximal endurance performance.

Microsatellite mapping of the bovine roan locus: a major determinant of White Heifer disease.

In the Belgian Blue Cattle breed, coat color variation is mainly under the influence of a single autosomal locus, the roan locus, characterized by a pair of codominant alleles: r+ (black) and R (white). Heterozygous r+R animals have intermingled black and white hairs, yielding the "blue" phenotype typical of the breed. Major interest for the roan locus stems from its pleiotropic effect on fertility, owing to the critical role of the R allele in the determinism of White Heifer Disease. We describe the linkage mapping of the roan locus to bovine Chromosome (Chr) 5, in the interval between microsatellite markers BPI and AGLA293, with an associated lodscore of 11.2. Moreover, we map a candidate gene, the Steel locus coding for the mast cell growth factor, to bovine Chr 5.

Effects of exercise with varying energy expenditure on high-density lipoprotein-cholesterol.

To investigate the effect of varying energy expenditure on acute high-density lipoprotein-cholesterol (HDL-C) changes, 12 healthy endurance-trained men completed three- counterbalanced running trials at different energy expenditures: trial 1, 1690.3 (24.4) kJ [mean (SD)]; trial 2, 2529.1 (24.0) kJ; trial 3, 3384.3 (36.6) kJ, with exercise intensity at 75% of maximal oxygen consumption. For each trial, blood samples were collected at 24 h pre-exercise (24 h Pre), immediately post-exercise, 1 h post-exercise, 6 h post-exercise (6 h PE), and 24 h post-exercise (24 h PE). Plasma samples were analyzed for HDL-C, HDL2-C and HDL3-C subfractions, and triglycerides (TG). In addition, post-heparin plasma samples were analyzed at 24 h Pre, 6 h PE and 24 h PE for lipoprotein lipase activity (LPLA) and hepatic triglyceride lipase activity. All samples were corrected for plasma volume changes and compared to 24 h Pre (baseline). When trials were combined, an increase (P < 0.05) in HDL-C was observed 24 h PE, via an increase (P < 0.05) in HDL3-C. An increase (P < 0.05) in LPLA and decrease (P < 0.05) in TG at 24 h PE is suggested to be responsible for the increase in HDL3-C. In conclusion, no difference in HDL-C was observed among trials. However, when trials were combined, an increase in HDL-C was observed, suggesting that an energy expenditure of no greater than 3384 kJ is needed to promote favorable changes in HDL-C.

Effect of carbohydrate ingestion subsequent to carbohydrate supercompensation on endurance performance.

This investigation determined whether carbohydrate ingestion during prolonged moderate-intensity exercise enhanced endurance performance when the exercise was preceded by carbohydrate supercompensation. Seven male trained cyclists performed two trials at an initial power output corresponding to 71 +/- 1% of their peak oxygen consumption. During the trials, subjects ingested either a 6% glucose/sucrose (C) solution or an equal volume of artificially flavored and sweetened placebo (P) every 20 min throughout exercise. Both C and P were preceded by a 6-day carbohydrate supercompensation procedure in which subjects undertook a depletion-taper exercise sequence in conjunction with a moderate- and high-carbohydrate diet regimen. Statistical analysis of time to exhaustion, plasma glucose concentration, carbohydrate oxidation rate, fat oxidation rate, and plasma glycerol concentration indicated that in spite of a carbohydrate supercompensation procedure administered prior to exercise, carbohydrate ingestion during exercise can exert an additional ergogenic effect by preventing a decline in blood glucose levels and maintaining carbohydrate oxidation during the later stages of moderate-intensity exercise.

Predisposing risk factors and natural history of acute neurologic complications of left-sided cardiac catheterization.

The reported incidence of acute neurologic complications of left heart catheterization varies from 0.03% to 0.3%. The predisposing risk factors, clinical features, and natural history have not been well characterized. We retrospectively reviewed all cases of acute neurologic complications developing during or within 36 hours of diagnostic catheterization or angioplasty to determine the incidence, clinical features, and natural history, and (using a case-control methodology) the clinical variables associated with their development. During the 37-month study, 6,465 patients underwent diagnostic left-sided cardiac catheterization and balloon angioplasty or valvuloplasty, and 27 patients developed an acute neurologic complication (0.4%). The most common symptoms were visual disturbances (26%), hemiparesis (26%), and facial droop (26%). Deficits were localizable to the anterior or posterior circulation in 22 patients: posterior in 8 (36%), and anterior in 14 (64%). Long-term follow-up was available in all patients, with 17 of 27 (63%) having complete resolution with no residuum. With use of a case-control methodology and multiple logistic regression analysis, female gender, the presence of left ventricular hypertrophy, depressed ejection fraction, and the presence of > or = 2 coronary arteries with > 50% narrowing were independent predictors of a neurologic event.

Endogenous opioids may modulate catecholamine secretion during high intensity exercise.

To determine the effect of endogenous opioids on catecholamine response during intense exercise [80% maximal oxygen uptake (VO2max)], nine fit men [mean (SE) VO2max, 63.9 (1.7) ml.kg-1.min-1; age 27.6 (1.6) years] were studied during two treadmill exercise trials. A double-blind experimental design was used with subjects undertaking the two exercise trials in counterbalanced order. Exercise trials were 20 min in duration and were conducted 7 days apart. One exercise trial was undertaken following administration of naloxone (N; 1.2 mmol.l-1; 3 ml) and the other after receiving a placebo (P; 0.9% saline; 3 ml). Prior to each experimental trial a flexible catheter was placed into an antecubital vein and baseline blood samples were collected. Immediately afterwards, each subject received bolus injection of either N or P. Blood samples were also collected after 20 min of continuous exercise while running. Epinephrine and norepinephrine were higher (P < 0.05) in the N than P exercise trial with mean (SE) values of 1679 (196) versus 1196 (155) pmol.l-1 and 24 (2.2) versus 20 (1.7) nmol.l-1, respectively. Glucose and lactate were higher (P < 0.05) in the N than P exercise trial with values of 7 (0.37) versus 5.9 (0.31) mmol.l-1 and 6.9 (1.1) versus 5.3 (0.9) mmol.l-1 respectively. These data suggest an opioid inhibition in the release of catecholamines during intense exercise.

Angioscopic evaluation of incompletely obstructing coronary intraluminal filling defects: comparison to angiography.

At 66 sites in 40 patients, we evaluated the sensitivity and specificity of coronary angiography in detecting intraluminal filling defects of varying sizes and in characterizing the contents (thrombus, intimal flap, both) of such defects using coronary angioscopy as "the gold standard." Overall angiographic sensitivity for thrombus was 37% and for intimal flap 45%. Specificity for thrombus was 100% and intimal flaps 96%. Angioscopically small flaps were less frequently seen angiographically (28%) than larger sizes (65%, p = 0.03). Angioscopically small thrombi were seen less often angiographically (30%) than larger ones (75%, p = 0.13). Filling defects (intimal flaps, thrombus, both) were characterized correctly in only 37% of sites. Angiography is relatively insensitivity in detecting intraluminal filling defects. Angioscopy may be preferred to or adjunctive with angiography in detecting these lesions.

Automatic detection of ventricular tachycardia and fibrillation using ECG processing and intramyocardial pressure measurement.

Ventricular electrograms and intramyocardial pressure signals were recorded in 11 dogs during sinus rhythm, during paced ventricular tachycardia, and at the onset of and during ventricular fibrillation. The autocorrelation function and the probability density function of short episodes of the electrograms were analyzed off-line on a digital computer. Peak-to-peak values of the intramyocardial pressure were calculated during sinus rhythm and during ventricular tachycardia and fibrillation. An algorithm was developed to recognize tachycardia and fibrillation using the autocorrelation function, the probability density function, and the intramyocardial pressure as input signals. Results show that in case of sinus rhythm all detection methods are reliable (recognition rate of 100%). In case of ventricular tachycardia with hemodynamic impairment the autocorrelation function is slightly better (66.6%) than the probability density function (44.4%). The onset of ventricular fibrillation is sensed in 81.8% of all episodes with the autocorrelation function and in 63.6% with the probability density function. During ventricular fibrillation this improves, respectively, to 92.3 and 69.2%. In all previous cases the intramyocardial pressure signal was 100% reliable. It is concluded that in this arrhythmia model, the sensitivity of an automatic ventricular tachycardia/fibrillation detection system was increased by combining ECG processing with analysis of an hemodynamic parameter.

The acute effects of exercise intensity on HDL-C metabolism.

To determine whether exercise intensity influences acute HDL-C responses, 12 male recreational runners (24.8 +/- 4 yr) who ran 15-30 miles.wk-1 exercised on a motor driven treadmill at 60% (L) and 75% (H) VO2max. A counterbalanced experimental design was utilized and energy expenditure was 800 Kcal. Fasting blood samples were obtained 24 h before exercise (24 PRE), immediately post-(IPE), 1 h post- (1 h PE), 6 h post- (6 h PE), and 24 h post- (24 h PE) exercise and analyzed for HDL-C and HDL2&3-C. In addition, postheparin plasma samples, obtained 24 h PRE, 6 h PE, and 24 h PE were analyzed for lipolytic activity--LPLA and HTGLA. An exercise trial by time interaction was observed for HDL-C (P < 0.01). Post-hoc analysis revealed no change in HDL-C following the L trial. However, an increase in HDL-C was observed 24 h PE (P < 0.01) following the H trial. The increase in HDL-C was attributed to an elevated HDL3-C (P < 0.01), with no change in HDL2-C. Analysis of plasma lipolytic activity revealed an increase in LPLA 24 h PE (P < 0.05) which may be responsible for the postexercise alterations in HDL-C. However, HTGLA decreased 6 h PE (P < 0.01) and 24 h PE (P < 0.05). We conclude that increases in HDL-C levels following endurance activity are influenced, in part, by the exercise intensity.

Relaxation within the left ventricular myocardial wall.

To evaluate relaxation mechanics in the wall of the left ventricle needle mounted miniature pressure transducers were inserted at the subendocardial (ENDO) and subepicardial (EPI) level of the anterior wall of the left ventricle during acute open-chest experiments in 10 mongrel dogs. Pressures were recorded during control, volume load and after verapamil administration. The relaxation time constant (tau) was determined by fitting a monoexponential with offset to the isovolumic relaxation period of the ENDO, EPI and left ventricular pressure (LVP) tracings: p = p0e-t/tau + p1. Mean tau-values for LVP, ENDO and EPI during control were (mean +/- 1 SD, ms): 38 +/- 5, 60 +/- 12, 83 +/- 5; during volume overload: 55 +/- 10, 72 +/- 20, 85 +/- 31 and after verapamil administration: 58 +/- 13, 60 +/- 17 and 73 +/- 15, respectively. Relaxation time constants of ENDO and EPI were significantly longer than those of LVP during control and volume loading but not after verapamil when only EPI was significantly different from LVP. These results demonstrate that relaxation indices obtained from LVP may not always reflect intramyocardial mechanics.

Preoperative pulmonary hemodynamics and early mortality after orthotopic cardiac transplantation: the Pittsburgh experience.

The influence of preoperative transpulmonary pressure gradient (TPG) and pulmonary vascular resistance (PVR) on early post-transplant mortality was evaluated in 425 orthotopic transplant recipients. The overall 30-day post-transplant mortality rate was 12.5%; the majority of the deaths (52.8%) were due to primary allograft failure. The 0- to 2-day mortality rate was threefold higher in patients with severe preoperative pulmonary hypertension (TPG > or = 15 mm Hg or PVR > or = 5 Wood units), whereas the 3- to 7-day and 8- to 30-day mortality rates were similar. Early post-transplant mortality (0 to 2 days and 8 to 30 days) was also significantly higher (15.9% vs 3.9% and 9.9% vs 2.8%, respectively; p < 0.05) in women compared with men. Women with severe preoperative pulmonary hypertension had higher (p < 0.05) 0- to 2-day post-transplant mortality than comparable men. According to univariate analysis, recipients with preoperative TPG > or = 15 mm Hg had a significantly higher 30-day postoperative mortality rate, irrespective of their level of PVR. Furthermore, patients with severe preoperative pulmonary hypertension who underwent transplantation between 1980 and 1987 had a higher 0- to 2-day post-transplant mortality rate compared with patients operated on after that time. Multiple logistic regression analysis identified female recipient sex and preoperative TPG but not preoperative PVR, era of transplantation, or recipient age as significant (p < 0.001 and p < 0.01, respectively) independent predictors of early post-transplant mortality.

Ultrasound-assisted cannulation of the internal jugular vein. A prospective comparison to the external landmark-guided technique.

BACKGROUND: Central venous access is an essential part of patient management in many clinical settings and is usually achieved with a blinded, external landmark-guided technique. The purpose of this study is to evaluate whether an ultrasound technique can improve on the traditional method. METHODS AND RESULTS: We prospectively evaluated an ultrasound-guided method in 302 patients undergoing internal jugular venous cannulation and compared the results with 302 patients in whom an external landmark-guided technique was used. Ultrasound was used exclusively in an additional 626 patients. Cannulation of the internal jugular vein was achieved in all patients (100%) using ultrasound and in 266 patients (88.1%) using the landmark-guided technique (p < 0.001). The vein was entered on the first attempt in 78% of patients using ultrasound and in 38% using the landmark technique (p < 0.001). Average access time (skin to vein) was 9.8 seconds (2-68 seconds) by the ultrasound approach and 44.5 seconds (2-1,000 seconds) by the landmark approach (p < 0.001). Using ultrasound, puncture of the carotid artery occurred in 1.7% of patients, brachial plexus irritation in 0.4%, and hematoma in 0.2%. In the external landmark group, puncture of the carotid artery occurred in 8.3% of patients (p < 0.001), brachial plexus irritation in 1.7% (p < 0.001), and hematoma in 3.3% (p < 0.001). CONCLUSIONS: Ultrasound-guided cannulation of the internal jugular vein significantly improves success rate, decreases access time, and reduces complication rate. These results suggest that this technique may be preferred in complicated cases or when access problems are anticipated.