Comparison of the results from two different External Quality Assessment Schemes supports the utility of robust quality specifications.

BACKGROUND: The definition of quality goals to evaluate the performance of laboratories participating in External Quality Assessment Schemes (EQAS) is currently not homogeneous. The aim of the work was to verify the applicability of quality goals based on biological variation comparing the results from two different EQAS. METHODS: We evaluated the performance of the laboratories participating in two Italian EQAS, presenting similar characteristics in terms of number of participants, type of EQA-samples, and program organization. The results were obtained during 2007 for 27 components in the regional scheme of Lombardy (RGL) and in the national scheme of Prolarit (PRL). The percentage total error of single measurements was calculated for each reported EQA value. The total error values at the 68th percentile, at selected critical concentration values, were compared with maximum tolerable error derived from biological variation. RESULTS: The performance of laboratories participating in the RGL scheme was significantly better. The frequency of satisfactory performance at, respectively, minimum, desirable and optimum levels was 98%, 80% and 59% in the RGL scheme, and 73%, 56% and 22% in the PRL scheme. CONCLUSIONS: Due to the different performance shown in the two programs, objective analytical goals based on biological variability constitute the optimal solution.

Sleep related changes in blood pressure in hypocretin-deficient narcoleptic mice.

STUDY OBJECTIVES: Although blood pressure during sleep and the difference in blood pressure between sleep and wakefulness carry prognostic information, little is known on their central neural mechanisms. Hypothalamic neurons releasing hypocretin (orexin) peptides control wake-sleep behavior and autonomic functions and are lost in narcolepsy-cataplexy. We investigated whether chronic lack of hypocretin signaling alters blood pressure during sleep. DESIGN: Comparison of blood pressure as a function of the wake-sleep behavior between 2 different hypocretin-deficient mouse models and control mice with the same genetic background. SETTING: N/A. SUBJECTS: Hypocretin-ataxin3 transgenic mice with genetic ablation of hypocretin neurons (TG, n = 12); hypocretin gene knock-out mice (KO, n = 8); congenic wild-type controls (WT, n = 10). INTERVENTIONS: Instrumentation with electrodes for sleep recordings and a telemetric blood pressure transducer. MEASUREMENTS AND RESULTS: Blood pressure was significantly higher in either TG or KO than in WT during non-rapid eye movement sleep (NREMS; 4 ± 2 and 7 ± 2 mm Hg, respectively) and rapid eye movement sleep (REMS; 11 ± 2 and 12 ± 3 mm Hg, respectively), whereas it did not differ significantly between groups during wakefulness. Accordingly, the decrease in blood pressure between either NREMS or REMS and wakefulness was significantly blunted in TG and KO with respect to WT. CONCLUSIONS: Chronic lack of hypocretin signaling may entail consequences on blood pressure that are potentially adverse and that vary widely among wake-sleep states.

Dysregulation of heart rhythm during sleep in leptin-deficient obese mice.

STUDY OBJECTIVES: sleep deeply affects cardiac autonomic control, the impairment of which is associated with cardiovascular mortality. Obesity entails increased cardiovascular risk and derangements in sleep and cardiac autonomic control. We investigated whether cardiac autonomic control is impaired during sleep in ob/ob mice with morbid obesity caused by congenital leptin deficiency. DESIGN: indexes of cardiac autonomic control based on spontaneous cardiovascular fluctuations were compared between ob/ob and lean wild-type (+/+) mice during wakefulness, non-rapid eye movement sleep (NREMS), and rapid eye movement sleep (REMS). SETTING: N/A PATIENTS OR PARTICIPANTS: 7 ob/ob and 11 +/+ male mice. INTERVENTIONS: instrumentation with electrodes for sleep recordings and a telemetric transducer for measuring blood pressure and heart period. MEASUREMENTS AND RESULTS: In ob/ob mice, the variability of heart period and cardiac baroreflex sensitivity (sequence technique) were significantly lower than in +/+ mice during each wake-sleep state. The vagal modulation of heart period was significantly weaker in ob/ob than in +/+ mice during NREMS and REMS. In ob/ob mice, the cross-correlation function between heart period and blood pressure suggested that the baroreflex contribution to cardiac control was lower than in +/+ mice during wakefulness and NREMS, whereas the contribution of central autonomic commands was lower than in +/+ mice during NREMS and REMS. CONCLUSIONS: These data indicate a dysregulation of cardiac autonomic control during sleep in ob/ob mice. Ob/ob mice may represent a useful tool to understand the molecular pathways that lead to cardiac autonomic dysregulation during sleep in obesity.

Harmonization of values for serum alkaline phosphatase catalytic activity concentration employing commutable calibration materials.

BACKGROUND: Harmonization of results allows a more effective utilization of laboratory tests; we verified the feasibility of harmonizing serum alkaline phosphatase results by two methods. METHODS: Patient sera (n=106) and candidate calibration materials (n=8) were analyzed by two methods, employing either diethanolamine (DEA) or 2-amino-2-methyl-1-propanol (AMP) as phosphate-accepting buffers. Results for patient sera by the DEA method were recalculated, with either a commutable or a non-commutable calibration material, both with values assigned by the AMP method. RESULTS: After calibration with the commutable material, the median intermethod difference (DEA-AMP) and ratio (DEA/AMP) dropped from 195 U/l to 0 U/l and from 2.47 to 1.00, respectively. When a non-commutable material was used the former became 124 U/l and the latter 1.94. After recalibration with the commutable material, linear regression and correlation analysis of DEA vs AMP values for the set of 106 patient sera gave: intercept=0.8 U/l; slope=0.997; and nonparametric correlation coefficient r=0.9995. CONCLUSIONS: Harmonization of alkaline phosphatase results by AMP and DEA methods is feasible when commutable calibration materials are used in the trueness transfer process.

Central and baroreflex control of heart period during the wake-sleep cycle in consomic rats with different genetic susceptibility to hypertension.

1. In spontaneously hypertensive rats (SHR), the contributions of the baroreflex and central autonomic commands to the control of heart period (HP) vary among wake-sleep states and are impaired during quiet wakefulness and rapid eye movement sleep (REMS), respectively. 2. Dahl salt-sensitive (SS) rats are genetically susceptible to salt-sensitive hypertension, the development of which depends on diet. Substitution of chromosome 13 of SS rats with that of Brown Norway rats confers salt-resistance to consomic SS-13BN rats. 3. In the present study, we tested whether differences in the central and baroreflex contributions to HP control occur among wake-sleep states in SS and SS-13BN rats and reflect genetic susceptibility to hypertension. Rats (n = 5 per group) were fed a prohypertensive diet late during development to minimize hypertension in SS rats and were instrumented with an arterial catheter and electrodes for discriminating wake-sleep states. 4. The cross-correlation function between HP and blood pressure indicated that, in SS and SS-13BN rats, the contributions of the baroreflex and central commands to the control of HP differed significantly among wake-sleep states, with central commands outweighing the baroreflex in REMS. However, these contributions did not differ significantly between SS and SS-13BN rats in any wake-sleep state. 5. The data suggest that differences in the central and baroreflex contributions to HP control among wake-sleep states, which have been demonstrated in SHR, can be generalized to other rat models used in hypertension research. Impairments in the baroreflex and central autonomic control of HP during quiet wakefulness and REMS, respectively, cannot be generalized as an index of genetic susceptibility to hypertension.

Sleep modulates hypertension in leptin-deficient obese mice.

Leptin increases sympathetic activity, possibly contributing to hypertension in obese subjects. Hypertension increases cardiovascular mortality, with nighttime (sleep) blood pressure having a substantial prognostic value. We measured blood pressure in male leptin-deficient obese mice (ob/ob; n=7) and their lean wild-type littermates (+/+; n=11) during wakefulness, non-rapid-eye-movement sleep, and rapid-eye-movement sleep to investigate whether, in the absence of leptin, derangements of blood pressure are still associated with obesity and depend on the wake-sleep state. Mice were implanted with a telemetric pressure transducer and electrodes for discriminating wake-sleep states. Mean blood pressure was significantly higher in ob/ob than in +/+ mice during wakefulness (7.3+/-2.6 mm Hg) and non-rapid-eye-movement sleep (6.7+/-2.8 mm Hg) but not during rapid-eye-movement sleep (2.6+/-2.6 mm Hg). In ob/ob and +/+ mice, mean blood pressure was substantially higher during wakefulness than during non-rapid-eye-movement sleep. On passing from non-rapid-eye-movement sleep to rapid-eye-movement sleep, mean blood pressure decreased significantly in ob/ob but not in +/+ mice. The time spent during wakefulness was lower in ob/ob than in +/+ mice during the dark (active) period, whereas the opposite occurred during the light (rest) period. Consequently, mean blood pressure was significantly higher in ob/ob than in +/+ mice during the light (8.2+/-2.4 mm Hg) but not during the dark (3.0+/-2.9 mm Hg) period. These data suggest that, in the absence of leptin, obesity may entail hypertensive derangements of blood pressure, which are substantially modulated by the cardiovascular effects of the wake-sleep states.

Commutable calibrator with value assigned by the IFCC reference procedure to harmonize serum lactate dehydrogenase activity results measured by 2 different methods.

BACKGROUND: The availability of commutable calibrator materials may ease considerably the task of harmonizing assay results and ensuring their traceability to reference procedures. We sought to verify the commutability of potential calibrator materials and evaluate their effectiveness in harmonizing LDH results by 2 measurement methods. METHODS: We measured LDH in 109 serum samples and 31 materials, including frozen serum pools (with either normal or abnormal isoenzyme patterns), commercial stabilized materials, and the ERM-AD453/IFCC reference material. We assayed LDH activity with the IFCC reference procedure and with 2 commercial methods, 1 using the lactate-to-pyruvate (LP) reaction, and the other the pyruvate-to-lactate (PL) reaction. We selected a commutable material, with LDH value assigned by the reference procedure, as a calibrator for recalculating the results for patient sera by both LP and PL, thereby making them traceable to the IFCC reference procedure. RESULTS: Original values for patient sera (n = 109) by the 2 commercial methods showed a mean (SD) PL/LP ratio of 1.97 (0.03); this ratio changed to 1.06 (0.02) after recalculation of results. Linear regression of PL vs LP recalibrated values gave y = 1.108x - 9.7. At the clinically important concentration of 250 U/L (upper reference limit), the systematic difference between methods was 6.8%, which met our proposed quality specifications for inaccuracy and total error. CONCLUSIONS: By properly selecting the calibrator, the results of serum LDH measurement by 2 different methods may be harmonized and made traceable to the selected highest (reference) metrological level.

Surges of arterial pressure during REM sleep in spontaneously hypertensive rats.

STUDY OBJECTIVES: Rapid-eye-movement sleep (REM sleep) physiologically entails arterial pressure surges. Pressure surges may lead to acute cardiovascular events in risk conditions such as arterial hypertension. We investigated whether arterial hypertension alters the rate of occurrence and the characteristics of the pressure surges during REM sleep. DESIGN: Spontaneously hypertensive rats (SHR) were compared with Wistar-Kyoto normotensive controls (WKY) and a group of SHR, in which hypertension was prevented by long-term enalapril treatment (ena-SHR). SETTING: N/A. SUBJECTS: Seven male rats per group. INTERVENTIONS: Instrumentation with electrodes for polygraphic recordings, a nasal thermistor for measuring ventilatory period, and an arterial catheter for measuring arterial pressure and heart period. MEASUREMENTS AND RESULTS: SHR showed a significant increase in the rate of occurrence but a similar magnitude of the pressure surges during REM sleep, with respect to WKY and ena-SHR. The pressure surges were associated with a decrease of heart period and an increase of electroencephalographic theta frequency, which were significantly less pronounced in SHR than in either WKY or ena-SHR. The ventilatory period showed only a modest increase before the surges without significant differences among the groups. CONCLUSIONS: Pressure surges independent of sleep apnea occur during REM sleep at a rate increased in SHR with respect to their controls, supporting a potential role of REM sleep in triggering acute cardiovascular events in arterial hypertension. The characteristics of the pressure surges suggest that, in SHR, the underlying central autonomic commands are increased in frequency, but not in magnitude, by arterial hypertension.

The baroreflex contribution to spontaneous heart rhythm assessed with a mathematical model in rats.

In Spontaneously Hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats, we quantified the extent to which spontaneous fluctuations of heart period (HP) may be determined from arterial pressure based on linear baroreflex properties. We analyzed time series (30-s length) of low-frequency (<0.8 Hz) fluctuations of HP and mean arterial pressure obtained during quiet wakefulness, rapid-eye-movement sleep (REMS) and non-rapid-eye-movement sleep (NREMS) as well as a control set of surrogate isospectral data with random phase. HP was modeled as the summed output of two parallel linear transfer functions with arterial pressure as input. The mean square difference between modeled and recorded HP was minimized by varying model parameters. The percentage of time series, in which such difference was lower than half the measured HP variance was significantly lower in REMS (6+/-1%, SHR; 5+/-1%, WKY) than either in quiet wakefulness (25+/-2%, SHR; 35+/-3%, WKY) or NREMS (33+/-3%, SHR; 27+/-3%, WKY), and in quiet wakefulness, it was significantly lower in SHR than in WKY. In surrogate data, these percentages were significantly lower than in recorded data during quiet wakefulness and NREMS, but not during REMS. The extent to which linear baroreflex properties explain spontaneous heart rhythm thus depends on the interaction between the behavioral state and the hypertensive disease, and in REMS, may be accounted for by chance couplings between HP and arterial pressure.

Central and baroreflex control of heart period during the wake-sleep cycle in spontaneously hypertensive rats.

We investigated whether the relative contribution of the baroreflex and central commands to the control of heart period differs between spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) during physiological behavior. Rats were instrumented with an arterial catheter and with electrodes for discriminating wakefulness, nonrapid eye movement sleep (NREMS), and rapid eye movement sleep (REMS). The cross-correlation function (CCF) between spontaneous fluctuations of heart period and mean arterial pressure was computed at frequencies <0.2 Hz. The baroreflex determines a positive correlation between heart period and previous pressure values. This pattern was observed in the CCF during quiet wakefulness (QW) and NREMS, and in QW, it was accompanied by a pronounced negative correlation between heart period and subsequent pressure values. The relative baroreflex contribution to the control of heart period, estimated from the positive peak value of the CCF, was lower in SHR than in WKY during QW but not during NREMS. During REMS, the CCF showed a negative correlation between heart period and both previous and subsequent pressure values, reflecting the prevalence of central autonomic commands. The relative contribution of central commands to the control of heart period, estimated from the negative peak value of the CCF, was lower in SHR than in WKY during REMS. These results suggest that during QW and REMS, the control of heart period exerted by the baroreflex and central commands, respectively, is less effective in SHR than in WKY. This difference is not apparent in a behavioral state of autonomic stability such as NREMS.

Sleep-dependent changes in cerebral oxygen consumption in newborn lambs.

During rapid-eye-movement (REM) sleep in adult subjects, the cerebral metabolic rate of oxygen consumption (CMRO(2)) is as high as that during wakefulness. We investigated whether CMRO(2) during active sleep is already at the waking level in newborn life, to support the role of active sleep as a state of endogenous brain activation during early postnatal development. Newborn lambs, 2-5 days old (n = 6), were instrumented with electrodes for sleep-state scoring, catheters for blood sample withdrawal and pressure monitoring, and a transit-time ultrasonic blood-flow probe around the superior sagittal sinus. At the age of 19 +/- 3 days, blood samples were obtained simultaneously from the carotid artery and the superior sagittal sinus during uninterrupted epochs of wakefulness, quiet sleep, and active sleep. The arteriovenous difference in blood oxygen concentration was multiplied by cerebral blood flow to determine CMRO(2). CMRO(2) during active sleep (47 +/- 5 micromol min(-1)) was similar to the value in wakefulness (44 +/- 6 micromol min(-1)) and significantly higher than in quiet sleep (39 +/- 5 micromol min(-1), P < 0.05). These data show that active sleep provides newborn lambs with brain activity at a level similar to that in wakefulness in terms of cerebral oxygen metabolism. The high CMRO(2) during active sleep supports its functional role during early postnatal development, when time spent in active sleep is at a lifetime maximum, albeit constituting a metabolic challenge for newborns, because of the impairment of systemic and cerebral vascular regulation in this sleep state.

Trueness verification and traceability assessment of results from commercial systems for measurement of six enzyme activities in serum: an international study in the EC4 framework of the Calibration 2000 project.

BACKGROUND: The in vitro diagnostics directive of the European Union requires traceability to higher order reference measurement procedures and materials for analytes in assuring the result trueness and comparability of laboratory measurements. Manufacturers must ensure that the systems they market are calibrated against available reference systems. Validation of metrologically traceable calibrations is, however, required. METHODS: A commutable serum-based material was analyzed in three reference laboratories and target values for six enzymes (ALT, AST, CK, GGT, LD, amylase) were assigned using IFCC reference measurement procedures. 70 laboratories in Germany, Italy, and The Netherlands measured the same enzymes in the material using procedures from six commercial companies. A system for maximum allowable error was developed from the biological variation model and the results of the various procedures were tested on their compliance to trueness and between-laboratory and within-laboratory variations relative to the maximum allowable. RESULTS: For ALT results were relatively good. >95% of laboratories using systems from Dade, Olympus, Ortho and Roche are expected to comply traceability within the biologically derived limits, and 94% respectively 89% from Abbott and Beckman. For AST and GGT only Dade respectively Olympus fully complied. For CK all companies showed significant bias. Nevertheless >95% of laboratories applying Abbott, Beckman and Roche systems will comply. Finally, LD and amylase measurements require significant improvement. Some manufacturers continue to sell on the European market assays giving results which are not traceable to the internationally accepted reference systems. CONCLUSIONS: The traceability of enzyme measurements obtained with routine procedures to internationally accepted IFCC reference systems is not yet satisfactorily accomplished in clinical practice.

Sympathetic nervous control of the cerebral circulation in sleep.

Cerebral vessels are extensively innervated by sympathetic nerves arising from superior cervical ganglia, and these nerves might play a protective role during the large arterial pressure surges of active sleep (AS). We studied lambs (n=10) undergoing spontaneous sleep-wake cycles before and after bilateral removal of the superior cervical ganglia (SCGx, n=5) or sham ganglionectomy (n=5). Lambs were instrumented to record cerebral blood flow (CBF, flow probe on the superior sagittal sinus), carotid arterial pressure (P(ca)), intra-cranial pressure (P(ic)), cerebral perfusion pressure (Pcp=Pca-Pic) and cerebral vascular resistance (CVR). Prior to SCGx, CBF (mL min-1) was significantly higher in AS than in Quiet Sleep (QS) and Quiet Wakefulness (QW) (17+/-2, 13+/-3, and 14+/-3 respectively, mean+/-SD, P<0.05). Following SCGx, baseline CBF increased by 34, 31, and 29% respectively (P<0.05). CVR also decreased in all states by approximately 25% (P<0.05). During phasic AS, surges of Pca were associated with transient increases in Pcp, Pic and CBF. Following SCGx, peak CBF and Pic during surges became higher and more prolonged (P<0.05). Our study is the first to reveal that tonic sympathetic nerve activity (SNA) constricts the cerebral circulation and restrains baseline CBF in sleep. SNA is further incremented during arterial pressure surges of AS, limiting rises in CBF and Pic, possibly by opposing vascular distension as well as by constricting resistance vessels. Thus, SNA may protect cerebral microvessels from excessive distension during AS, when large arterial blood pressure surges are common.

The REALAB project: a new method for the formulation of reference intervals based on current data.

BACKGROUND: In a primary healthcare center concerned more with maintaining wellness than with diagnosing and monitoring illness, it is particularly important to compare patients' results with reference intervals derived from a matched population by use of defined statistical methods. METHODS: Laboratory results over a 3-year period (approximately 15,000,000 records; 197,350 individuals) were retrieved from our laboratory information system. An inclusion/exclusion procedure for individual patients was applied based on (a) presence of at least 1 of 23 previously defined "basic tests"; (b) only 1 measurement per test by the laboratory over the 3-year period; (c) for each test, absence of any abnormality in the correlated tests. Before the third step, correlations among quantities were assessed by a Spearman correlation matrix, comparing each of the 23 basic tests with all remaining tests by use of a novel multivariate algorithm. RESULTS: The initial sample group (n = 197,350) was reduced stepwise by the selection criteria outlined above to 166,027, then to 93,649, and finally to 61 246 individuals constituting our reference sample group. Results from the last 2 groups were used to calculate sex-specific, and in some cases age-related, reference limits for the 23 basic tests and for 13 additional quantities. Reference limits were calculated throughout this study by nonparametric estimation of percentiles. CONCLUSION: Reference values derived by retrospective analysis of large samples of data obtained at a given institution are particularly suitable for the evaluation of results for the presenting patient population at that institution.

Sleep-related brain activation does not increase the permeability of the blood-brain barrier to glucose.

We compared blood-brain barrier (BBB) permeability to glucose between quiet wakefulness and rapid-eye-movement (REM) sleep to assess whether changes in BBB permeability play a role in coupling glucose supply to the physiologic metabolic needs of the brain. Male Sprague-Dawley rats were prepared with electrodes for wake-sleep state scoring and with arterial and venous catheters. Using the single-pass, dual-label indicator method, unidirectional glucose extraction by the brain and cerebral blood flow (CBF) were simultaneously measured during states of quiet wakefulness (n=12) or REM sleep (n=7). The product of BBB surface area and permeability to glucose (PS product) was computed in each state. During REM sleep, CBF significantly exceeded that during quiet wakefulness in all regions but the cerebellum, whereas the difference in the PS product between quiet wakefulness and REM sleep was not statistically significant in any brain region. In the brain as a whole, CBF significantly increased 29% from quiet wakefulness to REM sleep, while a nonsignificant 0.8% increase occurred in the PS product. During REM sleep, the increase in CBF indicates a higher rate of brain glucose consumption than in quiet wakefulness, given the tight flow-metabolism coupling in the brain. Therefore, these data show that modulation of BBB permeability to glucose is not a mechanism that provides 'energy on demand' during the physiologic brain activation characterising REM sleep.

Autoregulation of the cerebral circulation during sleep in newborn lambs.

Autoregulation is a vital protective mechanism that maintains stable cerebral blood flow as cerebral perfusion pressure changes. We contrasted cerebral autoregulation across sleep-wake states, as little is known about its effectiveness during sleep. Newborn lambs (n= 9) were instrumented to measure cerebral blood flow (flow probe on the superior sagittal sinus) and cerebral perfusion pressure, then studied during active sleep (AS), quiet sleep (QS) and quiet wakefulness (QW). We generated cerebral autoregulation curves by inflating an occluder cuff around the brachiocephalic artery thereby lowering cerebral perfusion pressure. Baseline cerebral blood flow was higher (P < 0.05) and cerebral vascular resistance lower (P < 0.05) in AS than in QW (76 +/- 8% and 133 +/- 15%, respectively, of the AS value, mean +/-s.d.) and in QS (66 +/- 11% and 158 +/- 30%). The autoregulation curve in AS differed from that in QS and QW in three key respects: firstly, the plateau was elevated relative to QS and QW (P < 0.05); secondly, the lower limit of the curve (breakpoint) was higher (P < 0.05) in AS (50 mmHg) than QS (45 mmHg); and thirdly, the slope of the descending limb below the breakpoint was greater (P < 0.05) in AS than QS (56% of AS) or QW (56% of AS). Although autoregulation functions in AS, the higher breakpoint and greater slope of the descending limb may place the brain at risk for vascular compromise should hypotension occur.