Blood testing chamber

A. Testing Chamber. The blood testing chamber shown in Figure 2 consists of eight cylindrical wells machined into a Teflon block. Flat disks (22.2 mm X 3.2 mm) of the material of interest are placed in the bottom of the Teflon ports, then an inner sleeve of Tecoflex, a commercially available biocompatible polyurethane, is placed in the port to secure the disk and contain the test cell suspensions. The cylindrical sleeves were prepared by injection molding Tecoflex EG SOD purchased from Thermedics, Inc., Woburn, MA. The screw cap can be tightened with a torque wrench to a torque of 7 inch-ounces in order to provide a uniform pressure on the test disks so that distortion of the samples and leakage are minimized or prevented, respectively. Aliquots of suspensions of blood cellular constituents may be added to the chamber to assay interactions of the blood components with the test samples. 

Figure 2. The Teflon blood testing chamber with eight cylindrical sample wells. From left to right, a threaded cap, Tecoflex sleeve, and a sample disk are shown in front. 

The PNF-coated shafts were assembled into the test chambers. The test chambers were flushed with isotonic saline to displace the air interface. The blood flow through each of the chambers was adjusted to 200 mL/min using a Ward s doppler flow ultrasound cuff. The rotation of the shafts was maintained at 200 rpm. Under these conditions, a laminar flow regime was maintained with a shear rate of 150 s-1. Experiments were carried out for time periods of 60 min. After completion of each of the experiments, the chamber was flushed with isotonic saline. 

Blood collection from the tail vein is a simple and rapid, nonsurgical method which does not require anesthesia. A relatively large number of serial samples can be obtained within a short period of time. However, this method is limited to relatively small sample volumes (<250 pi per sample). Although larger volumes can be obtained by placing the rat in a wanning chamber, this procedure could significantly influence the disposition of the test compound and therefore is not recommended for routine studies. Blood collected from the cut tail has been shown to provide valid concentration data for numerous compounds. 

Carboxyhemoglobin Concentration [HbCO] This can be estimated with the method of Jones and co workers. The subject holds a deep breath for 20 s to allow equilibration of carbon monoxide between alveolar air and blood and then expires a sample of that air into a container. The air carbon monoxide concentration may be directly related to carboxyhemoglobin concentration [HbCO]. The test can be performed before exposure in an environmental chamber to help to verify that the subject has not received inordinate ambient pollutant exposure. 

Figure 2.5. Setup for in vitro measurement of blood-brain barrier permeability with a co-culture of bovine brain microvascular endothelial cells (BBMEC) and an astro ioma cell line, C6. The BBMEC are grown on top of a filter insert. The C6 cells are either grown on the opposite side of the filter or on the bottom of the wells. Transport across the BBMEC monolayer is measured by adding the test substance to the upper chamber and sampling from the lower chamber. The tightness of the monolayer is also characterized by the transendothelial electrical resistance (TEER). Courtesy of T. Abbruscato.

In order to integrate further some of the various reactions mentioned, and to detect its presence by other methods, the vessels of the rat s mesoappendix were employed as a test object (Chambers-Zweifach preparation, 12). A good correlation between the presence of a vasoexcitor material-like substance in the extracts and the presence of hypertension was found. When the whole rat was used for assay, a much cruder index, sizable quantities of active pressor material were isolated from the blood only of those patients showing at least a degree of renal impairment (lessened ability to concentrate urine, etc.). In general it may be stated unequivocally that patients with severe hypertension have in their arterial blood extractable substances which are pressor for the rat there are less or undemonstrable amounts in blood of less severe or neurogenic hypertensive patients there is little or none in blood of normotensive subjects a vasoexcitor material-like activity is exerted by blood from most hypertensive patients adenyl compounds, having a depressor action, present in extracts of blood are less prevalent in those from hypertensive patients the active rat pressor material (pherentasin) is probably aminelike in nature, is not a protein, but may be a simple peptide or an amine. 

To some extent, platelet interaction on surfaces is mediated by shear force. Therefore it is essential to screen antiplatelet dmgs under flow conditions. Two different techniques are used to monitor platelet interaction on surfaces. The classical Baumgartner technique employs denuded rabbit aorta to evaluate platelet interaction with basement membrane components (89). The flat chamber technique uses a chamber in which cover slips coated with various test materials can be exposed to flowing blood (90). One can get a layer of cell matrix components for testing, by growing endothelial cells on cover slips and stripping them off the glass surface after they reach confluency. 

Many researchers have attempted to determine mercury levels in the blood, urine, tissues, and hair of humans and animals. Most methods have used atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), or neutron activation analysis (NAA). In addition, methods based on mass spectrometry (MS), spectrophotometry, and anodic stripping voltametry (ASV) have also been tested. Of the available methods, cold vapor (CV) AAS is the most widely used. In most methods, mercury in the sample is reduced to the elemental state. Some methods require predigestion of the sample prior to reduction. At all phases of sample preparation and analysis, the possibility of contamination from mercury found naturally in the environment must be considered. Rigorous standards to prevent mercury contamination must be followed. Table 6-1 presents details of selected methods used to determine mercury in biological samples. Methods have been developed for the analysis of mercury in breath samples. These are based on AAS with either flameless (NIOSH 1994) or cold vapor release of the sample to the detection chamber (Rathje et al. 1974). Flameless AAS is the NIOSH-recommended method of determining levels of mercury in expired air (NIOSH 1994). No other current methods for analyzing breath were located. 

Formaldehyde is absorbed by the tissues of the respiratory traet during inhalation exposure in several species. Heck et al. (1985) determined the fate of inhaled formaldehyde in humans. Four men and two women were exposed to a 1.9 ppm air concentration of formaldehyde in a large walkin chamber for 40 minutes. Shortly before and shortly after the exposure, venous blood samples were taken from each person (each person served as his/her own control) and the blood was analyzed for formaldehyde content. Mean venous blood formaldehyde concentrations in humans prior to exposure showed a blood concentration of 2.61 0.41 g/g of blood. Individual variability was markedly present. Immediately after a 40-minute exposure, mean blood concentration of formaldehyde was 2.77 0.28 g/g of blood. There was no significant difference between pre- and postexposure blood concentrations of formaldehyde at the formaldehyde air concentrations tested in this study. This result suggests that formaldehyde was absorbed only into the tissues of the respiratory tract. The absence of increased formaldehyde concentrations in the blood is likely due to its rapid metabolism in these tissues and/or fast reaction with cellular macromolecules. 

An ex vivo test method consisting of a flow-through couette cylinder placed in an arteriovenous shunt of a dog was used to evaluate the effects of shear, surface properties, spatial relationships, and drugs on thrombus formation. In whole blood, thrombus formation does not appear to be a diffusion-limited process. Increasing shear rates from 150 to 260 s i resulted in a reduction in the rate of thrombus formation. When two surfaces were placed in tandem on the central rod of the chamber, thrombus formation was independent of blood flow direction. A reduction in thrombus formation by aspirin was found only for mildly (not strongly) throm-bogenic surfaces. 

In this laboratory, an ex vivo test system was developed and utilized to delineate some of the factors affecting thrombus formation on surfaces. The system consisted of a flow-through couette device (Figure 1) placed in an arteriovenous shunt in a dog. The device was designed to allow independent control of blood flow and shear by separate control of the flow rate through the chamber, and of the rotation speed of a central rod, which is coated with the material to be studied. The validation of the method with respect to flow conditions, hematological considerations, and reproducibility has been reported previously (2—4). 

---