Optimal back-extrapolation method for estimating plasma volume in humans using the indocyanine green dilution method

Background

The indocyanine green dilution method is one of the methods available to estimate plasma volume, although some researchers have questioned the accuracy of this method.

Methods

We developed a new, physiologically based mathematical model of indocyanine green kinetics that more accurately represents indocyanine green kinetics during the first few minutes postinjection than what is assumed when using the traditional mono-exponential back-extrapolation method. The mathematical model is used to develop an optimal back-extrapolation method for estimating plasma volume based on simulated indocyanine green kinetics obtained from the physiological model.

Results

Results from a clinical study using the indocyanine green dilution method in 36 subjects with type 2 diabetes indicate that the estimated plasma volumes are considerably lower when using the traditional back-extrapolation method than when using the proposed back-extrapolation method (mean (standard deviation) plasma volume?=?26.8 (5.4) mL/kg for the traditional method vs 35.1 (7.0) mL/kg for the proposed method). The results obtained using the proposed method are more consistent with previously reported plasma volume values.

Conclusions

Based on the more physiological representation of indocyanine green kinetics and greater consistency with previously reported plasma volume values, the new back-extrapolation method is proposed for use when estimating plasma volume using the indocyanine green dilution method.

     

Noninvasive Optical Measurement of Cerebral Blood Flow in Mice Using Molecular Dynamics Analysis of Indocyanine Green

In preclinical studies of ischemic brain disorders, it is crucial to measure cerebral blood flow (CBF); however, this requires radiological techniques with heavy instrumentation or invasive procedures. Here, we propose a noninvasive and easy-to-use optical imaging technique for measuring CBF in experimental small animals. Mice were injected with indocyanine green (ICG) via tail-vein catheterization. Time-series near-infrared fluorescence signals excited by 760 nm light-emitting diodes were imaged overhead by a charge-coupled device coupled with an 830 nm bandpass-filter. We calculated four CBF parameters including arrival time, rising time and mean transit time of a bolus and blood flow index based on time and intensity information of ICG fluorescence dynamics. CBF maps were generated using the parameters to estimate the status of CBF, and they dominantly represented intracerebral blood flows in mice even in the presence of an intact skull and scalp. We demonstrated that this noninvasive optical imaging technique successfully detected reduced local CBF during middle cerebral artery occlusion. We further showed that the proposed method is sufficiently sensitive to detect the differences between CBF status in mice anesthetized with either isoflurane or ketamine–xylazine, and monitor the dynamic changes in CBF after reperfusion during transient middle cerebral artery occlusion. The near-infrared optical imaging of ICG fluorescence combined with a time-series analysis of the molecular dynamics can be a useful noninvasive tool for preclinical studies of brain ischemia.     

Density indicator method to measure pulmonary blood flows

The injection of plasma, saline, or erythrocyte (RBC) concentrate into the pulmonary circulation produces a change in the gravimetric density of the blood outflow similar to the dilution curve of dye. We used an improved density-measuring system to assess the flow of these density indicators through the lung in vivo and in vitro perfused dog lobe. From the in vitro density-dilution curves of plasma and RBC concentrate we calculated the pulmonary flow rate and found it to be 1.04 +/- 0.02 (SD) times the measured one. The outflow-dilution curves of gravimetric density were not as broad as those of optical density following in vivo injection of plasma bolus containing indocyanine green, and the gravimetric measurements dipped to base line, whereas the optical measurement did not. The density-dilution curves of isotonic saline injection are similar to that of plasma. Following injection of RBC concentrates with the dye, density changes in the pulmonary outflow lag behind the emergence of the dye. This was presumably related to RBC aggregation in the concentrates. In reference to the injected plasma, no loss in the density indicators for saline and RBC injection was observed. Based on these results and the similarity of the density indicators to the blood, we conclude that the plasma and isotonic saline are good density indicators to be used for the determination of pulmonary blood flows.     

Cerebral blood flow assessment with indocyanine green bolus transit detection by near-infrared spectroscopy in the rat

In this study we evaluated the feasibility of measuring cerebral blood flow in rats by monitoring the transit of an indocyanine green bolus through the brain with multiwavelength near-infrared spectroscopy. Different volumes of a 1 mg/ml indocyanine green solution (5, 15, 25, 50 microl) were injected intravenously in the search for an optimal dose. Clear transit curves were obtained with all doses and a blood flow index could easily be determined. The indocyanine green signal obtained with the bolus of 5 microl rapidly returned to baseline and interfered minimally with the haemoglobin and cytochrome oxidase signals. This dose was used in a second study to evaluate the reproducibility of the signal and the effect of hypercapnia. Two groups of rats received 7 repetitive boli of indocyanine green. In one group, 7% CO(2) was added to the gas mixture before the second, fourth and sixth indocyanine green injection. Hypercapnia consistently caused a significant increase in blood flow index, cerebral haemoglobin concentration and O(2)-saturation. In the control group these variables remained stable in time. We conclude that monitoring of the transit of an indocyanine green bolus with multiwavelength near-infrared spectroscopy can be used to assess cerebral blood flow qualitatively in rats in combination with continuous monitoring of brain oxygenation.     

A method of sodium and potassium determination in red cells with a simple cell separation technique

Heparinized blood was centrifuged repeatedly in Eppendorf's test tubes at 7,500 g in the Unipan microcentrifuge type 320. Packed red cells were hemolysed, then sodium and potassium were determined by means of the flame photometer. The percentage of trapped plasma determined with indocyanine green amounted to on average 1 per cent. There was a good precision of the method controlled on 20 aliquots of the same blood sample. Results of red cell sodium and potassium in 80 healthy volunteers were 10.42 +/- 1.56 mmol/l and 87.8 +/- 4.03 mmol/l respectively. No significant changes in the red cell sodium and potassium concentration were observed in heparinized blood during 5 hours storage at room temperature. The method cannot be used interchangeably with the method of Helbock and Brown, since the correlation coefficients were too low in parallel examinations.     

Colorimetric device for measurement of transvascular fluid flux in blood-perfused organs

The aim of this study was to develop a device capable of measuring transvascular fluid flux in blood-perfused organs. For any given blood flow through the organ (QT), transvascular flux (QF) can be considered as the fraction of QT exchange. Presumably, QF would change the background concentration of an impermeable tracer residing in the perfusate. Thus QF could be calculated from the relative changes in tracer concentration for any given QT. We have used Blue Dextran (1 g/l of blood) as the reference tracer. Because the minimum molecular weight of Blue Dextran is 2 X 10(6), we anticipated it to behave as an impermeable tracer in most organs. QF was simulated with continuous infusions of plasma, normal saline solution, and a 50% mixture of both. Changes in Blue Dextran concentration were continuously followed colorimetrically by changes in transmission of specific light at a wavelength of 632 nm. Because 632-nm light is affected by hematocrit and O2 saturation changes, two additional wavelengths were used: 815-nm, which is not affected by saturation or Blue Dextran concentration changes, was used to account for changes in hematocrit, and 887-nm specific light, which is not affected by Blue Dextran, served to correct for saturation changes. Red cells could not be used as the reference tracer because of the possibility of hematocrit changes independent of fluid flux (Fahraeus effect). The device so constructed proved capable of measuring rates of fluid infusion in the order of 0.1% of QT with a variability of 10% around the mean.     

Photoacoustic Tomography of Human Hepatic Malignancies Using Intraoperative Indocyanine Green Fluorescence Imaging

Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green (0.5 mg/kg, intravenous) was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10) under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci (one intrahepatic cholangiocarcinoma and 4 colorectal liver metastases), photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical advances are needed to improve the visibility of photoacoustic signals emitted from deeply-located lesions.     

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.     

Evaluation of 515 expressed sequence tags obtained from guard cells of Brassica campestris

The ear shoot of maize (Zea mays L.) consists of the peduncle and reproductive tissues (ear). Genetic mosaics induced by the unstable allele of thech1 locus were used for cell lineage analysis of the ear shoot. The unstablech1-m1 allele, caused by the insertion of a transposable element, gives rise to yellow-green seedlings with many small revertant green stripes. Rare plants with large revertant sectors comprising 30–50% of the plant were selected. Nineteen plants showing large sectors on the main stem were subjected to sector boundary analysis. Sectoring was recorded for the main stem, leaf subtending the ear shoot, peduncle, prophyll and ear. The reproductive part of the ear shoot, the ear, was scored after removal of the husks and subsequent exposure to light. In 18 cases the ear was non-sectored yellow-green or green. In an additional four cases, peduncle cell lineages entered the ear, but only in the proximal part, while the tip of the ear was non-sectored. Two additional ears showed longitudinal sectors which reached the tip of the ear. These observations indicate that in the lateral meristem of the ear shoot two types of cellular clone exist. One will generate the peduncle, the other will found the ear. Sector boundary analysis indicates that for the vegetative part of the ear shoot the number of meristem founder cells is high, whereas only a few initials are recruited for the formation of the ear. The presence of ear sectors not starting in the peduncle and reaching the ear tip, and the finding that the ear is frequently non-sectored, suggest that this organ derives from an apical type of growth.     

Hepatic uptake of indocyanine green and perfusion rate in rats: effect of age and albumin concentration

Liver blood flow and hepatic uptake of some indicator substances have been reported to fall with age in both rats and humans. We used an isolated liver system, which was perfused in one pass with hemoglobin free buffer, to investigate the effect of albumin concentration, buffer flow rate, and age upon hepatic clearance of the dye, indocyanine green. We measured the half-life of a bolus of indocyanine green given intravenously to male Sprague-Dawley rats aged 10 and 24 months and then examined its clearance in vitro using their isolated perfused livers. After perfusion, the livers were homogenized and separated into subcellular fractions. The mean liver weight declined significantly (young, 19.7 +/- 2.9 g vs. old, 13.9 +/- 2.6 g; p less than 0.02). In vivo the indocyanine green clearance was reduced in the aged rats (3.2 +/- 1.0 vs. 5.1 +/- 1.7 mL/min; p less than 0.05). In the isolated perfused liver system, extraction ratio showed an inverse curvilinear correlation with albumin concentration and buffer flow rate, but did not differ with age. Hepatic protein content and dye subcellular localization did not differ between the two groups. In conclusion, the fall in indocyanine green clearance in vivo is not paralleled by the ability of the organs to extract the dye in vitro, and likely reflects a decline in hepatic mass and blood flow.     

Thermoacoustic molecular imaging of small animals

We have designed, constructed, and tested a thermoacoustic computed tomography (TCT) scanner for imaging optical absorption in small animals in three dimensions. The device utilizes pulsed laser irradiation (680-1064 nm) and a unique, 128-element transducer array. We quantified the isotropic spatial resolution of this scanner to be 0.35 mm. We describe a dual-wavelength subtraction technique for isolating optical dyes with TCT. Phantom experiments demonstrate that we can detect 5 fmol of a near-infrared dye (indocyanine green, ICG) in a 1-microL volume using dual-wavelength subtraction. Initial TCT imaging in phantoms and in two sacrificed mice suggests that three-dimensional, optical absorption patterns in small animals can be detected with an order of magnitude better spatial resolution and an order of magnitude better low-contrast detectability in small animals when compared to fluorescence imaging or diffusion optical tomography.     

Effects of partial hepatectomy on microtubules and hepatocellular transport of indocyanine green in rats

The effects of partial hepatectomy on plasma disappearance and biliary excretion of indocyanine green (ICG) have been studied in rats and correlated with morphometric changes of hepatocellular microtubules. The plasma disappearance rate of ICG was in good accord with recovery of liver weight after partial hepatectomy. Biliary excretion of ICG per 100 g liver significantly increased between 3 h and 7 days postoperatively. Colchicine significantly reduced plasma disappearance and biliary excretion of ICG, with no reduction in bile flow, in both intact and hepatectomized rats. Morphometrically, microtubules significantly increased from 3 h following partial hepatectomy and reached a maximum at 24 h with a gradual return to preoperative values at 5 days. These observations suggest that the increased hepatocellular transport of ICG after partial hepatectomy is related to an increase in the number of microtubules.     

Effect of hypoxemia on the renin-angiotensin-aldosterone system in humans

Hypoxemia was induced in five subjects older than 40 (group 1) and five younger than 35 yr (group 2) on normal and low-salt diets by having the subjects breathe hypoxic gas. The fractional inspired O2 of the hypoxic gas was regulated so that group 1 hemoglobin saturations fell to 90% for 1 h. Group 2 subjects had desaturation to 90% for 1 h followed by desaturation to 80% for a 2nd h. Plasma renin activity (PRA), angiotensin-converting enzyme activity (ACE), and plasma cortisol levels did not change during hypoxemia. Plasma aldosterone levels fell in both groups during the 1st h of hypoxemia. Decreases were greatest during salt restriction and were significant (P less than 0.01) for the combined groups. Plasma aldosterone levels plateaued during the 2nd h of more severe hypoxemia in group 2. Hepatic blood flow, measured by indocyanine green clearance, and the adrenal response to exogenous adrenocorticotropic hormone, measured by changes in plasma cortisol and aldosterone, were not changed by hypoxemia in group 2 subjects. These results indicate that plasma aldosterone falls during hypoxemia despite unchanged PRA, ACE, hepatic blood flow, and adrenal function.     

Semi-Quantitative Comparison of Infrared Thermography with Indocyanine Green Imaging in Porcine Intestinal Resection

BackgroundThe quality of intestinal blood supply is extremely important for healing of intestinal anastomoses. During the surgery the blood supply of the intestine may appear sufficient even though the microperfusion is not fully adequate. The degree of blood supply of remaining intestinal segment and the positioning of the resection margins is estimated subjectively by the surgeon's experience or objectively by means of indocyanine green fluorescence imaging. The subject of our study is the evaluation of the infrared thermal imaging as another supportive non-invasive imaging method in assessment of intestinal blood supply, and to compare surgeon's decisions of the position of resection line with information obtained by infrared thermal imaging and by indocyanine green fluorescence imaging.MethodsA pilot study on thermal imaging measurements was done in a porcine model. The infrared thermocamera Workswell WIC 640 was used in our study. The thermal imaging was correlated with the indocyanine green imaging method ICG NOVADAQ and with surgeon's subjective expert decisions of the places of resection lines. Fifteen monitored resections were performed on three experimental porcine models.ResultsBased on the data evaluated, experience of the surgical team and the post-operative conditions of the porcine models, we conclude that the thermal imaging is a useful tool for determination of the optimal intestinal resection margins, and thus contributes to lowering anastomotic complications rate in colorectal surgery. The results show relatively high consistency between the used imaging techniques. Both methods showed ischemic regions of the intestine at a comparable level. The IRT methods showed even a slightly higher match with the surgeon's rating.ConclusionBased on experiments, the infrared thermography can be considered as one of complementary imaging methods suitable for use during anastomosis surgery. The sensitivity and versatility of the infrared thermography method was demonstrated. There was confirmed agreement in obtained information about the position of area with maximal blood insufficiency obtained by IRT with the information obtained by using the ICG method.     

Oscillations of acetylcholine in oat seedlings

Using gas chromatography it was shown that acetylcholine (ACh) was present in both etiolated and green oat (Avena sativa L. cv. Diadem) seedlings. In etiolated seedlings the ACh level was low, but increased rapidly during exposure to sunlight and red light (RL). The stimulative influence of RL was reversed by far-red light (FRL). The RL- and FRL- changes in ACh level were correlated to changes in acetylcholinesterase (AChE) localization. Using Karnovsky's method, it was found that in the etiolated coleoptiles the products of enzymatic reaction showing AChE activity accumulated selectively on the external side of plasma membrane. After exposure of seedlings to RL AChE activity disappeared. Subsequent FRL made it reappear on the external side of the plasma membrane. When the plants became green, oscillations of ACh were clearly observable. For plants grown under a LD 16:8 cycle the changes were circadian.     

Thermoregulatory and acute-phase responses to endotoxin of full- term-pregnant rabbits

Fever, a fall in the plasma level of iron (Fe), and rises in the levels of certain plasma glycoproteins (indexed by protein-bound-N-acetylneuraminic acid [NANA]) normally occur during infection; they are thought to be mutually enhancing in host defense. It has been reported that fever is suppressed at full term of pregnancy; however, it is not known whether the blood chemical changes are similarly affected. Also, the mechanism for the suppression of fever is controversial. Since uteroplacental blood flow is at its maximum near term, competition between the demands of the fetoplacental unit and of thermoregulatory effectors might result in underperfusion of thermogenic tissues and therefore provide a basis for the lack of fever. To examine these questions, the changes in colonic temperature (Tco) and regional blood flow induced by Salmonella enteritidis endotoxin (LPS, 2 micrograms/kg iv) were compared in conscious nonpregnant and 30-day-pregnant rabbits 35 min after injection, using 15-microgram radiolabeled microspheres. In different rabbits, the effects of LPS on Tco and plasma Fe and NANA levels were measured before mating and at term. LPS induced fevers similar in heights and courses in both nonpregnant and full-term pregnant rabbits It caused decreases in the blood flows to brain, tongue, mammary gland, small intestine, and ear and increases in the blood flows to masseter muscle, bone, liver (hepatic artery), and left ventricle; blood flows to the kidneys, spleen, right ventricle, ovaries, and myometrium did not change. There were no significant differences in these vascular responses between nonpregnant and 30-day-pregnant rabbits, except a 28% reduction in the blood flow to the placentas.     

Alterations in insulin clearance and hepatic blood flow during the night do not contribute to the 'dawn phenomenon' in type 1 diabetes

To assess mechanisms leading to the 'dawn phenomenon' in type 1 diabetes mellitus, overnight insulin clearance, hepatic blood flow and insulin sensitivity of glucose metabolism were determined in 9 type 1 diabetic subjects treated with continuous subcutaneous insulin infusions. Glucose clamp studies were performed twice, once after midnight (from 24.00 to 02.00 h), and once in the early morning (from 06.00 to 08.00 h) during insulin infusion at 15 mU/m2/min. Insulin clearance was 482 +/- 57 ml/m2/min during the first, and 528 +/- 56 ml/m2/min during the second clamp (nonsignificant). Hepatic plasma flow assessed by measuring indocyanine green clearance was 984 +/- 115 and 1,040 +/- 163 ml/min, after the first and after the second clamp, respectively (nonsignificant). Glucose uptake during the two clamps was not significantly different. Since hepatic blood flow is known to influence insulin clearance and hepatic glucose metabolism, the data demonstrate that overnight changes in hepatic blood flow and insulin clearance do not contribute to the previously described early morning increase in insulin requirements in type 1 diabetic subjects (dawn phenomenon).     

Influence of Light on the Bioelectric Potential of the Bean (Phaseolus vulgaris) Hypocotyl Hook

By use of surface electrodes electropotenlial measurements were carried out on hypocotyl hooks of Phaseolus vulgaris seedlings. The hooks were illuminated with a small spot of white, blue, red or far red light. The potential changes in bean hypocotyl hooks do not show the red-far red reversible characteristics of phytochrome-mediated processes. By experimenting with inhibitors of photosynthesis we could demonstrate that the light-triggered potential changes in green bean hooks are correlated to photosynthetic electron transport phenomena. The red-light-induced transient is a depolarization, whereas blue light induces a hyperpolarization. Etiolated beans exhibit no bioelectric potential changes when subjected to red or far red irradiations. Blue light and white light induce a strong hyperpolarization in etiolated hooks cells. This transient seems to be an action potential induced by light. The action potential is influenced by inhibitors of electron transport and oxidative phosphorylation. By comparing the action spectrum of the action potential induced by light with the absorption spectra of extracted carotenoids and xanthophylls from etiolated bean hypocotyl hooks, we observed similarities.     

Dissociation of adrenal androgen and cortisol levels in acute stress

Patients recovering from acute surgical stress often excrete increased 17-OH corticosteroids with no change in 17-ketosteroids. The explanation for these findings is unclear. In order to investigate possible divergence between cortisol and adrenal androgen metabolism in acute stress, repeated morning cortisol and dehydroepiandrosterone (DHA) measurements were made in patients undergoing ACTH stimulation 48 to 96 hours preoperatively, followed by determinations before and during major surgery, also performed in the morning. Cortisol and DHA are largely metabolized by the liver, so liver blood flow under a constant general anesthetic regimen known not to affect cortisol metabolism was monitored by pre- and intraoperative indocyanine green dye clearance. Results indicated no difference between the cortisol and DHA stimulation resulting from two hours of ACTH stimulation or major surgery, and a small (14.4%) decline in hepatic blood flow during general anesthesia. However, while DHA concentrations remained constant immediately preceding surgery, cortisol concentrations increased by 61% (P less than 0.05). Previous studies have also demonstrated increased concentrations of cortisol before surgical procedures, presumably due to psychological stress. However, this is the first demonstration of a dissociation between concentrations of cortisol and an adrenal androgen due to psychological stress.     

The cardiovascular response to lower body negative pressure in humans depends on seal location

We tested whether seal location at iliac crest (IC) or upper abdomen (UA), before and during lower body negative pressure (LBNP), would affect thoracic electrical impedance, hepatic blood flow, and central cardiovascular responses to LBNP. After 30 min of supine rest, LBNP at -40 mm Hg was applied for 15 min, either at IC or UA, in 14 healthy males. Plasma density and indocyanine green concentrations assessed plasma volume changes and hepatic perfusion. With both sealing types, LBNP-induced effects remained unchanged for mean arterial pressure (-3.0+/-1.1 mm Hg), cardiac output (-1.0 l min(-1)), and plasma volume (-11 %). Heart rate was greater during UA (80.6+/-3.3 bpm) than IC (76.0+/-2.5 bpm) (p<0.01) and thoracic impedance increased more using UA (3.2+/-0.2 Omega) than IC (1.8+/-0.2 Omega) (p<0.0001). Furthermore, during supine rest, UA was accompanied by lower thoracic impedance (26.9+/-1.1 vs 29.0+/-0.8 Omega, p<0.001) and hepatic perfusion (1.6 vs 1.8 l.min(-1), p<0.05) compared to IC. The data suggest that the reduction in central blood volume in response to LBNP depends on location of the applied seal. The sealing in itself altered blood volume distribution and hepatic perfusion in supine resting humans. Finally, application of LBNP with the seal at the upper abdomen induced a markedly larger reduction in central blood volume and greater increases in heart rate than when the seal was located at the iliac crest.