Products based on blood are of three distinct types: the cellular constituents of blood including erythrocytes, leukocytes, and platelets; the non-cellular constituents identified as either plasma or serum; and whole blood containing both the cellular and non-cellular constituents together.
Of major interest are the non-cellular constituents of blood, plasma and serum, which differ -rom one another only in the presence or absence of clotting factors. These non-cellular fluids are composed of water, dissolved salts, fats and lipids, proteins, enzymes, and a variety of subprotein groups. In particular, the chemical entities present in plasma and serum vary markedly in concentration, molecular weight, shape, size, chemical reactivity, and source of origin. In addition, the various fractions of plasma and serum have great value as therapeutic agents, as diagnostic indicators, and as a source of blood based products for in-vitro culture.
The separation of the non-cellular constituents from whole blood, particularly in volumes less than 0.5 milliliters, was previously and remains a tedious, time consuming, and complex process. The valuable components of plasma and serum, such as blood protein fractions, have a tendency to be fragile and are easily denatured; and unintended protein denaturization can result from shifts in pH, osmolality, temperature, and pressure. For these reasons, the separation of plasma and serum from whole blood is typically performed only in two ways: centrifugation and filtration.
Centrifugation (and its counterparts of sedimentation and precipitation) is primarily useful when relatively large volumes of whole blood are required to be separated. Typically, at least 10 milliliters of whole blood are required for effective centrifugation. This quantity of whole blood is required also when blood is allowed first to coagulate into a clotted mass before being able to draw off any useful quantity of serum as the separated fluid. Although a wide variety of apparatus and techniques are conventionally available, the centrifugation process of separation always relies exclusively upon the mass of the cellular constituents and the force of acceleration to achieve a separation of cellular and non-cellular constituents.
The alternative means for separating plasma or serum from whole blood is filtration. Unfortunately, the conventional materials and known mechanisms of action employed within general methods of filtration have proven not to be either useful or effective for separation of whole blood. This is best understood by a summary review of the three fundamental filtration mechanisms now generally available. These are: surface filtration (or two-dimensional sieving); depth filtration (or three-dimensional sieving); and adsorptive filtration.
Filters are typically classified according to the mechanism of filtration by which they operate, although most filters do not operate purely by a single mechanism of action; rather, the mechanism which dominates is frequently dictated by the size of the particles being filtered out. Surface filtration (or two-dimensional sieving) will invariably remove large particles (typically greater than 100 microns) at the surface. The requirement for this filter mechanism to function is simply that the pores or holes on the exterior surface or face of the filter be smaller than the particles which are to be removed. Some particles only a little smaller in size than the diameter of the pores will also be removed by surface filtration because of bridging effects. A thin perforated plate is an example of a filter which will operate only by surface filtration; however, if the perforated plate is composed of many layers of particles or fibers, then depth filtration may also occur to some degree in combination with surface filtration.
Alternatively, depth filtration (or three-dimensional sieving) is a filtration mechanism which occurs when the particles to be retained actually penetrate into the interior of the filter medium (fiber or particulate) and become entrapped in the tortuous internal passages within the thickness of the filter material itself. As the particle (or cell) travels through the many internal passages and pathways within the thickness of the filter material, the individual particle (or cell) becomes retained and removed from the carrying fluid when it tries to pass through an orifice smaller in size than itself. While surface filters require very fine fibers in order to remove medium (100-10 microns) and small (10.0-0.1 microns) particles or cells efficiently, depth filters can remove the same sized particles (or cells) using fibers rather less fine because an actual penetration of the filter material occurs. A depth filter is therefore like several inefficient surface filters in series, each layer removing only a proportion of the particles (or cells) from the liquid carrying them. Increasing the thickness of a depth filter thus generally increases its efficiency of separation.
Finally, adsorptive filtration is a mechanism which involves the removal of particles (or cells) from a liquid when the particles (or cells) come into contact with and become physically adsorbed to the internal surfaces of the filter material. This filtration mechanism does not rely upon sieving; rather, it removes particles (or cells) which are smaller in size than the pores within the filter material. There is thus no effective limit to the smallness of the particles (or cells) which can be removed; and very small particles (typically 0.1 um) are removed with high efficiency by filters employing this mechanism of action. Instead of sieving, adsorptive filtration requires an affinity by the particle (or cell) for the filter material such that adhesion of on.RTM.to the other occurs readily. The thickness of the adsorptive filter provides multiple layers, each of which physically adsorbs a proportion of the particles (or cells) and removes them from the carrying liquid. An efficient adsorptive filter will present a large internal surface area to the particles (or cells) such that the probability of adsorptive capture becomes very high.
There are two types of forces Which operate betWeen a particle (or cell) and the adsorptive filter medium which dictate whether the degree of adhesion necessary for adsorptive filtration to occur will take place: Van der Waal's forces which are relatively weak and only operative over very short distances (approximately 10 Angstroms); and electrostatic forces which are relatively strong because most solids acquire a surface electrical charge when brought into contact with polar liquids. The latter is by far the more important of the two.
Electrostatic forces and charges usually originate either by the preferential dissolution of a component ion from the solid phase, thus leaving an excess of the opposite charge on the surface of the solid; or by the preferential adsorption of a counter-ion from solution onto the surface of the solid. The overall net charge of the solid material can be either positive or negative in sign (polarity); and the magnitude of the polar charge is typically expressed as an electrical potential. The net electrical potential of a solid surface cannot be measured directly but a closely related quantity commonly known as the "zeta potential" can be determined empirically by any of four different, conventionally known, electrokinetic techniques. Regardless of how they are measured, surfaces with an electrostatic charge of the same sign (polarity) repel each other while surfaces of opposite electrical charge attract each other. The attraction of oppositely charged surfaces for each other is the constant requirement, underlying basis, and mechanism of action for adsorptive filtration. The adsorptive material comprising the filter itself always has a demonstrable surface electrical charge which is opposite in polarity to the surface electrical charge in comparison with the particle (or cell) to be separated and retained [Wnek, W., Filtration & Separation 11:237-242 (1974); Raistrick, J.H., Filtration & Separation 13:614-620 (1976); and Shackleton and Chem, Filtration & Separation 14:632-638 (1977)].
As regards the practice of filtering whole blood into cellular and non-cellular constituents, the presently available filters for this purpose conventionallY employ either a surface or depth filter mechanism. Filters representing the use of the surface filtration mechanism are described within U.S. Pat. Nos. 3,552,925 and 3,552,928. Filters representing the use of the depth filtration mechanism are disclosed by U.S. Pat. Nos. 4,246,107 and 3,448,041. Such filters and filtration mechanisms, however, are conventionally recognized as being ineffective for separating small volumes of whole blood. These filters clog easily and/or fail to separate cellular and non-cellular constituents effectively. The most recent advance in whole blood filtration is described by U.S. Pat. No. 4,477,575 which employs glass fibers within a prescribed diameter and density range for separating small volumes of whole blood. The filtration mechanism employed with these glass fiber filters is not described or characterized within the text of the patent. However, the capacity and utility of the glass fiber filter system appears to be limited by the requirement that the total volume of the plasma or serum to be separated from whole blood be at most 50% of the absorption volume of the glass fiber layer.
Accordingly, insofar as is presently known, there are no examples of either adsorptive filters or the adsorptive filtration mechanism being employed for separation of whole blood into constituents. Rather, it appears that the complexities and difficulties of controlling the overall net electrical surface charge for the filter material and the effects of purposefully imposing an electrical charge upon the cellular and non-cellular constituents of blood have been viewed as being too demanding, variable, and intricate for effective filtration to be achieved using an electrically charged filtration mechanism.