Biomedical Engineering Reference
In-Depth Information
indicator for leukocyte activation. The production of cytokines is the best, most sensitive, and most
precise indicator for leukocyte activation, which can be measured in whole blood samples.
17.7.4 C
OMPLEMENT
A
CTIVATION
Activation of the complement system is an important as well as a quick response of the blood upon
contact with a biomedical implant (Figure 17.9).
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All complement activation assays are based
on the determination of one of the complement protein fragments. The complement system consists
of a number of proteases and scaffold proteins. The appearance of cleaved fragments is a direct
measure for the level of complement activation compared to a positive control lipopolysaccharides
(LPS). One of these complement proteins is C3b that is at the convergence point between classical
and alternative pathways. Another often determined component is the C5-9b, or also called the
TCC or the membrane attack complex. This is the endpoint of the complement cascade. Both are
determined using ELISA setups that are commercially available.
17.7.5 H
EMOLYSIS
Hemolysis is defi ned as the lysis of blood cells. In order to determine blood compatibility, hemolysis
is an important parameter that has to be determined.
106,122,123
Since over 99% of the blood's cells are
erythrocytes, the increase of free hemoglobin in plasma is taken as a direct measure for hemolysis.
Great care has to be taken in hemolysis measurements, since the basal level of free hemoglobin in
blood preparations can vary signifi cantly.
Hemolysis can be determined using a variety of different assays, but there is no generally accepted
method. The different assays are based upon two major principles. A number of assays depend on
direct optical measurement of hemoglobin. This means that the concentration of free hemoglobin
in blood plasma is determined by spectrometry, making use of the unique adsorption spectrum of
hemoglobin, with peaks at 415 nm, 541 nm, and 577 nm. The adsorption at these wavelengths can be
used to directly determine the concentration of hemoglobin by quantifying the absorbance peak.
Several other hemoglobin assays are based on the use of chemical techniques and the addition
of an agent that reacts with oxyhemoglobin. Especially benzidine chromogens are often used. The
oxidation of benzidine derivates, like tetramethylbenzidine, by hydrogen peroxide is catalyzed by
hemoglobin. The rate of the reaction is directly proportional to the concentration of hemoglobin.
The drawback of these chemical methods is that they make use of highly toxic chemicals that are
often expensive not only in purchase but also in disposal.
17.7.6 C
ELL
C
OMPATIBILITY
/E
NDOTHELIALIZATION
For blood-contacting devices that have only limited exposure time to the blood, the interaction with
cells is an unwanted phenomenon. However, for permanent implants that have to function for years
inside the circulation, the formation of a functional endothelial cell layer on its surface will greatly
improve the success rate of the device. For instance, for vascular prosthesis of heart valves, the endo-
thelial cell layer on the surface is the best guarantee for long-term hemostasis. The endothelium will
regulate coagulation and anticoagulation of the blood and the best part is that the layer can repair itself
upon damage. Therefore many attempts have been made to seed endothelial cells on synthetic vascular
grafts before implantation. Also the spontaneous endothelialization
in vivo
has been studied. The best
and easiest methods are based on microscopy. The parts of the implant for scanning electron micros-
copy can be prepared in a straightforward way, but the pictures will only be seen if cells are present
on the surface of the device (Figure 17.18). Fluorescence microscopy can be used to positively identify
the cells as endothelial cells by making use of specifi c endothelial markers of which the CD31 or
platelet-endothelial cell adhesion molecule-1 (PECAM-1) is most popular. There are more endothelial
specifi c markers, which can be demonstrated using specifi c monoclonal antibodies. The cells can then
be identifi ed by microscopy or by FACS analysis after detaching them from the surface.
