Therapeutic device

A therapeutic device for removing pathological effectors brom body fluids of a patient is disclosed. Said device includes a chamber for receiving the body fluids and positioned within the chamber is a biospecific polymer. Said biospecific polymer interacts with and binds specific pathological effectors carried by the body fluid that is passed through the chamber.

BACKGROUND OF THE INVENTION 
The present invention relates to a device for the extracorporeal treatment 
of disease. The course of many disease states is often reflected by 
elevated levels of specific blood proteins. This phenomenon is typically 
utilized as a diagnostic tool to define the pathology and to follow the 
course of clinical treatment. In many instances, these specific blood 
proteins are directly or indirectly responsible for the primary and 
secondary manifestations of the disease process. "Autoimmune" diseases can 
be described as diseases characterized by circulating antibodies to 
endogenous substrates and tissue proteins required by the body for normal 
growth and maintenance. "Neoplastic" diseases are typically characterized 
by uncontrolled growth of an undifferentiated transformed cell line which 
evades or compromises the body's natural defense mechanisms by producing 
immunosupressant blocking factors, surface antigen masking components 
and/or growth regulator constituents. Specific compartmentalization of 
these pathological effectors (i.e., causative agent) onto a biocompatible 
substrate is consistent with the restoration of "normal" body function by 
removal of the pathological effectors of the disease process. 
The basic function of the organs, cells and molecules that comprise the 
immune system is to recognize and to eliminate from the body foreign 
substances. These foreign substances are eliminated by reaction between 
the foreign substance and antibodies which are formed in response to the 
substance. In general, this function is performed efficiently and without 
detriment to the host. However, in certain instances, disturbances can 
occur which can lead to pathogenic disorders such as, for example, an 
uncontrolled response (allergic disorders) or an abnormal response 
(autoimmune disease). The pathogenesis of both of these disorders is 
related directly or indirectly to the production of antibodies with cross 
reactivities to either environmental antigens (allergens) or 
self-antigens. 
An autoimmune disease is a pathological condition arising when a host 
responds immunologically by production of antibodies with reactivity to a 
self-antigen. Autoimmunity can affect almost every part of the body, and 
generally involves a reaction between a self-antigen and an immunoglobulin 
(IgM or IgG) antibody. Representative autoimmune diseases can involve the 
thyroid, kidney, pancreas, neurons, gastric mucosa, adrenals, skin, red 
cells and synovial membranes as well as thyroglobulin, insulin, 
deoxyribonucleic acids and immunoglobulins. 
For some types of autoimmune and neoplastic diseases, non-specific 
immunosuppressant treatments, such as whole body X-irradiation or the 
administration of cytotoxic drugs, have been used with limited success. 
The disadvantages of such treatment include the toxicity of the agents 
used, and the increased incidence of various cancers, especially lymphomas 
and reticulum cell sarcomas, following such therapy. In addition, the use 
of non-specific agents for chronic cellular suppression greatly increases 
the susceptibility of the patient to serious infection from environmental 
fungi, bacteria and viruses which under ordinary circumstances would not 
cause problems. The invention disclosed herein is specific in that it 
removes only the pathological effector or those groups of pathological 
effectors which are related to and responsible for the manifestations of a 
particular disease. 
In viewing the state of the art, one finds that most recently there have 
been generally two approaches to therapeutic treatments for autoimmune 
and/or neoplastic diseases. The first of these is to introduce a material 
into the patient which causes a specific type of immunological tolerance 
to be produced. This suppression of antibody response would then effect a 
tolerance to the offending antigen. A typical example of this type of 
approach is U.S. Pat. No. 4,222,907 issued to Katz on Sept. 16, 1981. In 
this reference, the diseased patient is given a therapeutic treatment 
which consists of introducing conjugates of an antigen linked to a 
D-glutamic acid: D-lysine copolymer. 
The second approach has been the extracorporeal route. The procedures 
generally involve the removal of whole blood, separation of cellular and 
soluble blood substances, substitution or treatment of blood plasma and 
recombination-infusion of the treated whole blood. The first example of 
this approach would be plasma substitution or exchange with salt, sugar 
and/or protein solutions and is described by McCullough et al, 
"Therapeutic Plasma Exchange," Lab. Med. 12(12), p. 745 (1981). Plasma 
exchange is a rather crude technique that requires a large volume of 
replacement solution. A second example of this approach involves physical 
and/or biochemical modification of the plasma portion of whole blood. 
Typical of the state of the art of this therapeutic treatment are, for 
example, the Terman et al article "Extracorporeal Immunoadsorption: 
Initial Experience in Human Systemic Lupus Erythematosus," The Lancet, 
Oct. 20, 1979, pages 824-826. This article describes a hemodialysis type 
system utilizing two mechanical filters with a DNA collodian charcoal 
filter between said two mechanical filters. Typical of this state of the 
art, however, the adsorbent column is only semispecific for immune 
components because the charcoal substrate will nonspecifically delete many 
vital low molecular weight constituents from the treated plasma. A second 
application of this approach can be illustrated by the Terman et al 
article "Specific Removal of Circulated Antigen by Means of 
Immunoadsorption," FEBS Letters, Vol. 61, No. 1, Jan. 1976, pages 59-62. 
This reference teaches the specific removal of radiolabeled antigen by 
antibody treated cellulosic membranes. The author, however, demonstrates 
that control membranes have a significant capacity to non-specifically 
adsorb proteins. A third application of this approach is illustrated by 
the Bansal et al article "Ex vivo Removal of Serum IgG in a Patient With 
Colon Carcinoma," Cancer, 42(1), pp. 1-18 (1978). This report teaches the 
semi-specific absorption of immunoglobulin by ex vivo treatment of plasma 
with formalin and heat-killed Staphylococcus aureas. The biological 
activity of certain strains of S. aureas is attributed to a molecule 
present on the cell wall, called Protein A, which interacts and binds with 
the Fc portion of mammalian IgG. This treatment, because it interacts with 
the Fc moiety, does not discriminate between normal and pathological IgG 
components and experiments have shown the possibility of significant side 
effects. 
A fourth application of this approach can be illustrated by the Malchesky 
et al article "On-line Separation of Macromolecules by Membrane Filtration 
With Cryogelation," Artif. Organs 4:205, 1980. This publication teaches 
the semi-specific removal of cryoglobulin substances from plasma by the 
combination of filtration and cold treatment chambers. The incidence and 
composition of cryoglobular precipitates are not necessarily consistent 
with or indicative of many autoimmune or neoplastic diseases. 
Another problem associated with the current state of the art is that 
without systems using mechanical filtration, the specific pathological 
effectors desired to be removed have not been removed in large enough 
amounts to do much good for the diseased patient in that the columns do 
not specifically adsorb substantially only the desired specific 
pathological effectors. 
It has now been found that high specificity of pathological effector 
removal can be effectuated by treatment of body fluids in an economical 
and therapeutic procedure using the device of the present invention. 
SUMMARY OF THE INVENTION 
Broadly stated, this invention relates to a device for the extracorporeal 
treatment of disease comprising: means for withdrawing a body fluid from a 
patient, means for treating the body fluid including a chamber for 
receiving the body fluid and a biospecific polymer positioned within said 
chamber which will treat the body fluid by binding a specific pathological 
effector or specific group of pathological effectors carried by the body 
fluid that is passed through said chamber, and means for returning the 
body fluid to the patient. 
The biospecific polymer contains a fixed capacity, affinity and specificity 
for selective adsorption of pathological effectors. The pathological 
effectors are compartmentalized, e.g., are adsorbed onto the biospecific 
polymer and are thus diverted away from their endogenous receptors. This 
chemisorptive process ultimately consumes the biospecific polymer, serving 
to control and eliminate disease states by removing elevated pathological 
immune, nutritional and hormonal constituents from the diseased body 
fluid. Degenerative and metabolic disorders are diminished or eliminated 
by removing a key effector from a pathological process. 
As used herein and hereafter a biospecific polymer is a biocompatible 
polymer support which has a biological or biologicals attached to it which 
can specifically remove desired pathological effector or pathological 
effectors. 
These and other objects of the present invention are disclosed and 
described in the detailed description below and in the appended claims.

DETAILED DESCRIPTION 
I. Withdrawing Means 
Withdrawing means is defined herein as being a means of providing access to 
the body fluid of interest of the patient to be treated. In the majority 
of instances, the body fluid to be treated will be the blood or plasma of 
a patient. Thus, the manner of access and the type of access hereinbelow 
described is for the most common body fluid, i.e., the patient's blood. 
However, it is to be understood that access to any of the body fluids of 
interest may be provided by using well-known techniques and procedures in 
the medical arts. The access method is not critical with the caveat that 
it provides the required body fluid for the patient's treatment. In the 
case of vascular access an indwelling large bore cannula may be used 
intravenously or arterially. Examples of suitable veins and arteries 
include the antecubital vein, subclavian vein and brachial or radial 
arteries. It is further understood that an arterial-venous shunt or 
fistulae may also be used. In this case, the heart provides the pressure 
differential for fluid movement. If an AV shunt fistulae is not used, the 
preferred means for providing the pressure differential for fluid movement 
by a venous access is a roller-peristaltic pump capable of providing a 
flow rate of about 30 ml per minute to about 200 ml per minute. 
In cases where anticoagulants may be useful or necessary, suitable 
anticoagulants can be used utilizing well-known techniques and procedures 
in the medical arts. Suitable anticoagulants include, for example, acid 
citrate dextrose (approximately 1 ml to every 8 ml's of whole blood), 
heparin, heparin/acidic acid dextrose mixtures (e.g. 1250 IU heparin in 
125 ml acid citrate dextrose/L), and prostaglandin. It is to be 
appreciated that in using anticoagulants such as heparin and prostaglandin 
it is generally understood that a counteracting medication could be 
administered to the treated blood or plasma before returning or giving 
said blood or plasma to a patient. 
Further, in the case of treating plasma, it is understood that any 
conventional methods of removing the formed blood components may be used. 
Suitable examples of methods of separating plasma from formed blood 
components include, plasmapheresis, centrifugal cell separation, and cell 
sedimentation in a plasma bag. Where possible both continuous separation 
and intermittent (batch) separation are suitable--the aforementioned 
methods of separation are independent of the present invention and its 
use. 
II. Treating Means 
The treating means of the present invention comprises a chamber containing 
a biospecific polymer which may or may not be bound to a support member. 
Chambers suitable for use in the practice of the instant invention may take 
many forms. Referring to FIG. 1, a chamber 1 is of a box-like 
configuration consisting of top shell 2 and bottom shell 3 having upper 
and lower sealing flanges 4, 6 with sealing means 5 therebetween. Any 
means for sealing may be employed, i.e., gasket, TEFLON seal, heat 
sealing, etc., with the caveat that no leakage occurs. A pipe-like or 
nipple-like fluid inlet 7 and fluid outlet 8 ports protrude from the top 
and bottom shells of the chamber 1. Within the box-like chamber 1 are a 
plurality of alternating layers of biospecific polymer sheets 9 and inert 
separator plates 10. Referring to the biospecific polymer sheets and inert 
separator plates in greater detail, FIG. 2 illustrates the inert separator 
plates 10 with fluid flow channels 11 through which the body fluid may 
flow. The biospecific polymer sheet 9 is held therebetween whereby the 
fluid flow channels 11 perform to channel body fluid between the 
biospecific polymer sheet and inert separator plates. It should be 
apparent that a plurality of inert separator plates and biospecific 
polymer sheets could be stacked horizontally or placed side-by-side within 
a chamber. Depending on the amount of biospecific polymer surface area 
needed for treatment of the body fluid, the number of biospecific polymer 
sheets and inert separator plates employed may be reduced or increased. 
Referring back to FIG. 1, the body fluid to be treated enters the chamber 1 
through fluid inlet port 7 and passes into a head space (not shown). From 
the head space, the body fluid passes in contact with biospecific polymer 
9 by way of fluid flow channels 11. The body fluid then passes into a 
second head space (not shown) and finally exists the chamber through fluid 
outlet port 8. The head spaces function as a resevoir for the body fluid 
entering and exiting the chamber. 
The inert separator plates should be mechanically stable and sterilizable 
as well as compatible for use in a system which is in continual contact 
with body fluids. Examples of materials which are suitable for the 
practice of the present invention as inert separator plates are, for 
example, polypropylene, polyethylene, polyurethane, polycarbonate, ABS 
(acrylonitrile-butadiene-styrene), polysiloxane, and polystyrene. The 
configuration of the inert separator plates with fluid flow channels is 
not critical, with the caveat that a sufficient fluid flow configuration 
is provided eliminating shear forces on the body fluid while maintaining a 
fluid flow pathway allowing the pathological effectors carried within the 
body fluid to efficiently diffuse throughout, therefore, eliminating 
potential damages to the cellular components of the body fluid and 
maximizing the contact frequency between the pathological effectors and 
biospecific polymer. 
An equally preferred chamber configuration suitable for the practice of the 
present invention is illustrated in FIG. 3. FIG. 3a illustrates the top 
part 16 of a chamber 20 with an integral spiral fluid flow channel 18 
having fluid inlet port 19 and fluid outlet port 21. FIG. 3b illustrates 
the chamber 20 with top part 16 and a bottom part 17. The top part 16 has 
a protruding portion 22 which includes the integral spiral fluid flow 
channel 18. The bottom part 17 includes a recess 23 having a biospecific 
polymer sheet 24 received therein. The recess 23 and protrusion 22 are 
physically configured wherein the protrusion is closely received in the 
recess. The spiral fluid flow channel 18 performs to channel fluid between 
the biospecific polymer sheet and protrusion 22. 
Another chamber configuration useful in the practice of the present 
invention is illustrated in FIG. 4. A cylindrical chamber 26 with fluid 
inlet port 27 and fluid outlet port 28 is shown. The cylindrical chamber 
26 contains therein a porous reticulated foam 29 with biospecific polymer 
(not shown) coated thereon. The porous reticulated foam may be described 
for the purpose of the present invention as a polymer matrix of contiguous 
open cells forming fluid flow channels throughout said polymer matrix. The 
walls of the fluid flow channels are coated with a biospecific polymer 
whereby the body fluid flowing through such channels is in contact with 
the biospecific polymer. 
The reticulated foam may be any suitable polymeric foam which is curable to 
a porous, mechanically stable, and sterilizable polymer matrix. Such foams 
must also be biocompatible. 
It is to be appreciated that the configuration of the chamber used in the 
practice of the present invention does not limit the configuration of the 
biospecific polymer contained therein. Thus, for example, a box-like 
chamber may contain a biospecific polymer in a channeled cylindrical 
configuration or a channeled spiral cylinder configuration respectively 
illustrated in FIGS. 5 and 6. FIG. 5 illustrates a cylindrical biospecific 
polymer matrix 31 containing fluid flow channels 32 therethrough. FIG. 6 
illustrates a spirally configurated biospecific polymer cylinder 36 with 
integrally formed ribs 37 forming fluid flow channels 38. The ribs 37 may 
or may not be the same polymer composition as 36. 
In still another configuration, the biospecific polymer may be formed into 
a plurality of spherical beads that are situated within a chamber. As the 
body fluid passes into the chamber and up through these beads, the beads 
become fluidized, i.e., they separate from each other in the body 
fluid--maximizing channeling and effecting more efficient contact between 
the biospecific polymer and the body fluid. This configuration is known as 
a fluidized bed. 
The fluid flow channels through any of the foregoing embodiments of the 
biospecific polymer may be of any configuration, again with the caveat 
that a sufficient fluid flow configuration is provided eliminating shear 
forces on the body fluid while maintaining a fluid flow pathway allowing 
the pathological effectors carried within the body fluid to efficiently 
diffuse throughout, therefore, eliminating the damages to the cellular 
components of the body fluid and maximizing the contact frequency between 
the pathological effectors and biospecific polymer. 
III. Mechanical Support 
Most of the biocompatible polymer supports have very low mechanical 
stability. Most of these materials are, in fact, gels or gel-like as 
opposed to materials which have high mechanical stability, such as, for 
example, sheets of polypropylene. Thus, in most embodiments utilizing the 
present invention, a support member which is mechanically stable is 
necessary. This support member allows large surface areas to be utilized 
to insure rapid and medically, as well as commercially, acceptable levels 
of immune disease-associated component removal. The support member, 
besides being mechanically stable, should also be inexpensive and must be 
sterilizable so as to be made compatible for use in a system wherein the 
blood of a diseased patient is to be treated by the present invention. 
Examples of materials which are suitable for the present invention as 
support members include, for example, filter paper, cotton cloth, 
polyester fiber, reticulated polymeric foams, microporous polypropylene 
and other polymers including polycarbonate, polystyrene, ABS 
(acrylonitrile-butadiene-styrene), NORYL, a polyphenylene oxide polymer 
manufactured by the General Electric Company, and polysiloxanes. FIG. 7 
illustrates a mechanical supporting element (41) with fluid flow channels 
(42) and biospecific polymer (43) fixed thereon by any of the methods 
discussed hereinbelow. It should be evident that a plurality of mechanical 
supporting elements with biospecific polymer coatings could be 
horizontally stacked or placed side-by-side within the chamber providing 
an increased surface area contacting the body fluid. It should also be 
evident that the inert separator plates, discussed hereinabove, with the 
biospecific polymer clamped therebetween would also provide mechanical 
support for the flat sheet embodiments of the biospecific polymer. 
Many methods of fixing the biospecific polymer onto the mechanical support 
may be utilized. Thus, for example, methods such as spin coating-casting, 
horizontal casting, vacuum impregnating, dip coating, dip coating with 
later cross-linking, spray coating, and solution copolymerization may be 
used. 
IV. Biocompatible Polymer Support 
The biocompatible polymer supports useful in the present invention are 
materials which tend not to cause adverse effects when in contact with 
body fluids, while at the same time maintaining a reactive but immobilized 
biological oriented such that the biological is extending out from the 
surface of said polymer support. The materials which are suitable are 
those which may be cast into films and other physical forms, while at the 
same time being susceptible to having said biologicals covalently bound to 
them without damaging either themselves or the biologicals bound thereto. 
The types of materials generally contemplated to be suitable are those 
known in the art as hydrogels and may be either copolymers or 
homopolymers. 
Modified cellulose and cellulosic derivatives, particularly cellulose 
acetate, have also found utility as biocompatible supports useful in the 
present invention. By modified cellulosic derivatives what is meant is 
that the cellulosic polymer is surface modified by covalently linking 
pendant biocompatible surface groups to the cellulosic substrate polymer 
rendering it more biocompatible. Such surface groups are well known and 
need not be described here, however, for purposes of the present 
invention, albumin has shown particular utility as a modifying group. 
Methods of attaching such groups are described hereinbelow. 
Referring to the hydrogels, suitable polymers may either be regular 
homopolymers containing substantially nc other material in their matrices, 
or they may be copolymers which contain monomers such as styrene and vinyl 
acetate, for example. In certain instances, this type of tailoring of the 
copolymers with various monomers may enhance the desirable properties of 
the biocompatible polymer support material. Examples of suitable monomers 
which may be copolymerized include, for example, N-vinyl pyrrolidone and 
glycidyl methacrylate. 
Homopolymers may also be used as suitable biocompatible polymer supports in 
the present invention. It is to be understood, however, that when 
homopolymers are discussed, they include materials which can also be 
identified as "slightly cross-linked homopolymers." That is, they contain 
a minor amount of a second component either intrinsic in the production of 
the monomer or added purposely to insure enough cross-linking so as to 
protect the homopolymer from slowly dissolving away in an aqueous media, 
such as blood. An example of this type of homopolymer which is often 
slightly cross-linked is hydroxyethyl methacrylate (HEMA). 
Also useful are terpolymers which are a subclass of copolymers containing 
three monomers which are polymerized. An example of a suitable terpolymer 
is glycidyl methacrylate/N-vinyl pyrrolidone/hydroxyethyl methacrylate 
(GMA/NVP/HEMA). 
In addition to the specific copolymers and homopolymers listed above, 
copolymers and homopolymers suitable in the present invention may be 
polymerized from the following monomers: hydroxyalkyl acrylates and 
hydroxyalkyl methacrylates, for example, hydroxyethyl acrylate, 
hydroxypropyl acrylate, and hydroxybutyl methacrylate; epoxy acrylates and 
epoxy methacrylates, such as, for example, glycidyl methacrylate; amino 
alkyl acrylates and amino alkyl methacrylates; N-vinyl compounds, such as, 
for example, N-vinyl pyrrolidone, N-vinyl carbazole, N-vinyl acetamide, 
and N-vinyl succinimide; amino styrenes; polyvinyl alcohols and polyvinyl 
amines, which must be made from suitable polymeric precursors; 
polyacrylamide and various substituted polyacrylamides; vinyl pyridine; 
vinyl sulfonate and polyvinyl sulfate; vinylene carbonate; vinyl acetic 
acid, and vinyl crotonic acid; allyl amine and allyl alcohol; vinyl 
glycidyl ethers and allyl glycidyl ethers. Processes and procedures for 
creating copolymers and/or homopolymers from the above monomers are 
well-known and understood in that particular art. These parameters are not 
critical to the instant invention with the caveat that the final copolymer 
and/or homopolymer is nontoxic for animal, including human, use. 
The biocompatible polymers utilized in this invention are to be 
distinguished from those produced by radiation grafting techniques. The 
latter, which are generally synthesized using high energy radiation (gamma 
radiation, x-rays, electron beams and the like), are completely different 
in structure from those disclosed in out invention, particularly when more 
than one monomer is involved. Due to the nature of high energy radiation, 
radiation grafting and polymerization in general occur in an uncontrolled 
and indiscriminate fashion. Thus, when a polymeric substrate such as our 
mechanical support is bombarded with gamma radiation in the presence of 
one or more monomers, a multiplicity of structures is produced with very 
complex topography. This complexity is, of course, greatly enhanced when 
two or more monomers are available for reaction, and depending on such 
diverse factors as monomer relative reactivities and sensitivity of the 
various monomeric and polymeric species to radiation, the functional 
groups needed for bioactivation may not be available consistently in 
pracitcal concentration at the polymer surface. This imposes a 
considerable limitation on the choice of mechanical supports. It is also 
well known that may polymers are degraded under radiation grafting 
conditions and can, under certain conditions, evolve toxic moieties. 
Finally, it should be realized that radiation grafting creates an entirely 
new composition which is similar to a composite or an alloy, especially at 
the critical graft/substrate interface. A successful radiation graft 
copolymerization requires substantial penetration of the grafted polymer 
into the substrate with random covalent bonding among all available 
components. The resulting structure is chemically and physically different 
than any of the starting materials. For these reasons, application of 
radiation induced graft copolymerization to the manufacture of the 
biocompatible polymers of the present invention wound be commercially 
impracticable. 
In contrast, the biospecific polymers utilized in this invention are made 
by techniques which employ mild and controlled conditions. The synthesis 
of the biocompatible polymer support is not restricted to polymerization 
in the presence of the mechanical support, but may also be carried out as 
an entirely separate operation so that grafting is clearly unnecessary. 
The only major physical requirement is that the biocompatible polymer be 
in a state such that it can form a continuous and reasonably adherent 
coating on the mechanical support. As a result of these factors, the 
supporting polymeric structures perform only the separate functions 
implied by their names and, under suitable conditions, can even be 
stripped away from each other for analysis and physical studies if 
desired. Thus, the biocompatible polymeric system of the invention is well 
adapted to the tailoring demanded by the specificity of the ultimately 
attached biologicals. In addition, no problems are posed in device 
construction on any scale, since as has already been suggested, a variety 
of well known methods may be used to coat the mechanical support with the 
biocompatible support. 
V. Biologicals 
In the context of the present invention, biological and/or biologicals may 
be defined as a chemical compound which possesses an ability to covalently 
bond to the biocompatible polymer support or spacer (defined hereinbelow), 
while at the same time retaining an activity to bind a desired 
pathological-causing constituent. It is to be understood that, in 
addition, the biological or biologicals employed must be of such size that 
they covalently bond to the surface of the polymer support and are not 
small enough to penetrate the porous matrix of the polymer support and be 
chemically bonded therefore inside or in the interior of the support 
material. In this light, a spacer may be employed to insure that the 
reactive site of the biological, which remains and is susceptible to 
bonding with the desired pathological constituent, can in fact be 
presented to this constituent, i.e., that it is held outward away from the 
support so as to come into contact with the body fluid flowing over the 
support. It is obvious from the above that, of course, the reactivity for 
binding the desired pathological constituent is, in fact, retained after 
immobilization of the biological or biologicals onto the biocompatible 
polymer support. Examples of materials which may be used as biologicals 
include, for example: acetylcholine receptor proteins, histocompatibility 
antigens, ribonucleic acids, basement membrane proteins, immunoglobulin 
classes and subclasses, myeloma protein receptors, complement components, 
myelin proteins, and various hormones, vitamins and their receptor 
components. Particular examples are, for example, attaching insulin to a 
biocompatible polymer support to remove anti-insulin antibody which is 
associated with the autoimmune disease insulin resistance; attaching 
anti-Clq and/or Clq to a biocompatible polymer support to remove immune 
complexes which are associated with connective tissue and proliferative 
diseases such as, for example, rheumatoid arthritis and carcinomina. 
Any generally known method of chemical attachment will suffice for 
attaching the biologicals to the biocompatible polymer support, with the 
caveat that the biological still has at least one active site for the 
particular autoimmune disease-associated component. Generally, the methods 
of chemical attachment used fall into three classes or routes of 
attachment. These three routes are, (1) spontaneous attachment, (2) 
chemical activation of terminal functional groups, and (3) coupling 
reagent attachment. Spontaneous covalent attachment of biologicals to 
polymer support surface proceeds via chemically reactive groups extending 
from the polymer support. Thus, for example, reactive groups such as 
aldehyde and epoxy extending from the polymer support readily couple 
biologicals containing available hydroxyl, amino or thiol groups. Also, 
for example, free aldehyde groups on the polymer support couple via acetal 
linkages with hydroxyl-containing biologicals and via imide linkages with 
amino-containing molecules. Additionally, for example, free oxime groups 
couple via alkylamine, ether and thioether linkages with biologicals 
containing amine, hydroxyl and thio groups respectively. For purposes of 
convenience all said attachments and couplings are defined herein as 
immobilizations. More extensive discussions of these reactions may be 
found, for example, in "Chemical Procedures for Enzyme Immobilization of 
Porous Cellulose Beads," Chen, L. F. et al, Biotechnology and 
Bioengineering, Vol. XIX , pp. 1463-1473 (1977) and "Epoxy Activated 
Sepharose," 6B, Parmacia Fine Chemicals, Affinity Chromatography, pp. 
27-32 (1979). 
Chemical activation of terminal functional groups may be accomplished by 
activating polymer surface functional groups by chemical modification of 
their terminal components. This method can be exemplified by the oxidation 
of terminal epoxy functions with periodic acid to form active aldehyde 
groups. This method is further exemplified, for example, in 
"Immobilization of Amyloglucosidose on Poly [(Glycidyl Methacrylate) Co 
(Ethylene Dimethacrylate)] Carrier and Its Derivatives," Svec, F. et al, 
Biotechnology and Bioengineering, Vol. XX, pp. 1319-1328 (1978). The 
immobilization of the biologicals proceeds as described hereinabove. 
Condensation reactions may be accomplished between free carboxyl and amine 
groups via carbodiimide activation of the carboxy groups as is described, 
for example, in "New Approaches to Non-Thrombogenic Materials," Hoffman et 
al, Coagulation--Current Research and Clinical Applications, Academic 
Press, N.Y. (1973). Briefly the immobilization of the biologicals is 
effected by carbodiimide activation by either the polymer or biological 
carboxyl groups and condensation with a free amine to form a stable 
peptide bond. The final orientation of the biological is generally a 
factor as to whether an amine or a carboxyl containing polymer be 
utilized. 
Coupling reagent attachment can be accomplished using a variety of coupling 
agents to form covalent bridges between polymers and biologicals. Here 
free hydroxyl and/or amine containing polymers and biologicals are 
covalently coupled by reagents such as, for example, cyanogen bromide, 
diisocyanates, dialdehydes and trichloro -s-triazine. More exhaustive 
discussion of this technique may be found for example, in the Chen et al 
article cited hereinabove. 
The preferred method of immobilizing a reactive biological onto a 
biocompatible polymer substrate in a given case generally is dictated by 
the molecular locations of the reactive binding moiety of the biological 
and the functional groups on the biological and polymer substrate which 
can be covalently combined. For example, it is presently preferred in the 
case of polymer substrates containing terminal hydroxy functions to 
activate by treatment with an alkaline solution of cyanogen bromide (10 to 
20% w/v). Typically the reaction mixture is maintained at room temperature 
(20.degree. to 25.degree. C.) for about 30 minutes. The pH of the solution 
is maintained in a range of about 10 to 12, by the addition of alkaline 
material, e.g., KOH or NaOH. The polymer is extensively washed with 
physiological saline (0.9 gm%) and incubated with solutions of a purified 
biological dissolved in a slightly alkaline buffer solution for 12 to 16 
hours at 2.degree. to 8.degree. C. The polymer is extensively rinsed with 
physiological saline to remove unbound or nonspecifically bound biological 
components. 
Biologicals are immobilized on glycidyl containing polymers via ether, 
thioether or alkylamine bonds. Epoxy-activated polymer substrates are 
rinsed and swollen with aqueous neutral buffer solutions at room 
temperature. Purified biologicals, dissolved borate, carbonate or 
phosphate buffer solutions are incubated with the glycidyl polymer 
substrate for 12 to 20 hours at 4.degree. to 30.degree. C. Excess and 
nonspecifically bound biologicals are removed by rinsing the polymer with 
saline, acetic acid (0.2 to 1.0M) and phosphate-buffered (pH=7.2.+-.0.2) 
saline solutions. Activation of amine and carboxyl containing polymer 
matrices is effected by treatment with purified biologicals dissolved in 
slightly acidic (pH 4.5 to 6.5) buffer solutions of a water soluble 
carbodiimide. Biologicals are covalently coupled to polymer support 
substrates by incubation of polymer support, biological and carbodiimide 
reactants for 12 to 16 hours at 2.degree. to 8.degree. C. The 
polymer-biological conjugates are washed alternately in acid then alkaline 
rinses until the rinse solutions are clear of biological and carbodiimide 
reactants. 
In order to determine the specific binding characteristics of the polymer 
immobilized biologicals, physiological serum solutions of complementary 
biomolecules were treated with activated membranes. The amounts of 
biomolecule were measured spectrophotometrically and radiochemically. 
Significant reduction of specific biomolecules resulted following brief 
exposures to the biologically modified polymer substrates. 
VI. Spacers 
In the present invention, a spacer may be defined as a molecule or compound 
which is capable of attachment to the surface of a biospecific polymer 
support, is large enough to extend from the surface of said support and is 
capable of immobilizing a biological and/or biologicals. The spacer 
insures that the active site of the biological is held outward away from 
the support so as to contact the body fluid more efficiently. It is 
obvious from the above that, of course, the reactivity for binding with 
the desired disease complex is, in fact, retained after immobilization of 
the biological or biologicals onto the spacer and therefore onto the 
biocompatible polymer support. 
The spacers are derived from organic molecules having at least two reactive 
functional groups generally situated at opposing ends of the molecule. 
Such groups serve as attachment vehicles capable of coupling the spacer to 
the polymer support and to the biological. The reactive functional groups 
on the spacer may be the same or different with the caveat that they react 
with functional groups along the surface of the polymer support and 
functional groups extending from the biological forming covalent bonds. 
Any known method for carrying out such coupling reactions will suffice. 
For example, the methods described hereinabove outlining coupling routes 
for attaching a biological directly onto a polymer support may be used. 
Suitable examples of spacers which may be used in the present invention, 
where the reactive functional groups are the same, include, for example, 
1,6-diaminohexane, divinyl sulfone, glutaraldehyde, 
1,4-cyclohexanedicarboxylic acid, ethylenediamine tetraacetic acid, 
triethylene glycol, 1,4-butanediol diglycidyl ether, methylene-p-phenyl 
diisocyanate and succinic anhydride. Examples of spacers in which the 
reactive functional groups are not the same include, for example, 
6-aminocaproic acid, p-nitrobenzoyl chloride, 1,2-epoxy-3-(p-nitrophenoxy) 
propane, aminopropyltriethoxy-silane and homocysteine thiolactone. 
Polypeptides and more specifically proteins may also be used as spacers in 
the present invention. Albumin, a low affinity protein, for example, has 
been successfully employed as a spacer. In addition, albumin and other 
natural proteins serve to render the polymer support more biocompatible. 
Finally, it is understood that certain materials may act simultaneously as 
a spacer and as the activator in the reaction used to combine the spacer 
and the biocompatible support. Examples of these kinds of compounds, 
include, for example, gluteraldehyde and 1,4-butanediol diglycidyl ether. 
VII. Therapeutic Regimen 
Broadly stated, the contemplated therapeutic regimen of the present 
invention is for the treatment of (autoimmune and other) diseases by 
exposing a diseased patient's blood to a biospecific polymer having 
immobilized reactive biologicals, thereby removing the specific 
pathological effectors from said patient's blood and then returning said 
blood to said patient; characterized in that said biospecific polymer 
comprises: (a) a biocompatible polymer support, (b) a biological or 
biologicals immobilized on said biocompatible polymer support, via 
chemical bonding, characterized in that the biological or biologicals 
retain their reactivity for binding the specific pathological effectors or 
specific group of pathological effectors associated with said patient's 
particular disease or diseases. This therapeutic treatment may or may not 
necessitate the use of blood separation techniques. Thus the treatment is 
contemplated to be carried out in a manner similar to a dialysis treatment 
with the advantage that total blood separation may not be needed and that 
there is very little if any physical damaging of normal blood components. 
It is also possible, of course, to utilize the present invention and the 
process of the present invention in the treatment of plasma. The plasma 
may be obtained from whole blood by any of the currently known and 
practiced methods. Thus, for example plasma may be separated from a 
patient's blood by known methods, then treated by the present invention 
and then recombined with the other blood components and returned to the 
patient using currently known procedures. In addition plasma which is 
being used in known medical treatments may utilize the present invention 
to treat said plasma before being administered to a patient requiring 
plasma from a blood bank, for example. Obviously whole blood from a blood 
bank may also be treated by and benefit from the present invention. 
Because of the advantages of the present invention mentioned above as well 
as others which will be clear to a person skilled in this art many types 
of disease states are contemplated to respond to the present invention 
used in a therapeutic regimen. Broadly stated six groups of disease states 
could be advantageously treated. These six disease categories are 
disorders of immune components, drug excesses, toxin exposure, imbalances 
of body substances, infections, and neoplastic states. Many diseases are 
currently treated using plasmapheresis and cytopheresis where the desired 
result is removal of a specific substance. The present invention and the 
process of the invention would apply to these diseases currently treated 
by plasmapheresis and cytopheresis. 
Examples of immune complex diseases which can be treated are, for example, 
any disease states involving antibody, antigen, antibody-antigen, 
antigen-antigen and antibody-antibody interactions, cell surface 
complexes, cytoplasmic complexes, etc. 
Examples of drug overdoses which can be treated are, for example, overdoses 
of iron, dioxin, aspirin, TYLENOL.RTM., methotrexate and other tricyclics. 
Examples of toxins for which the present invention is suitable are, for 
example, lead, aluminum, mushrooms (Anatoxin) and organic phosphates. 
Body substances when present in excess can lead to disease. Examples of 
these which can be eliminated using the present invention include, for 
example, cholesterol, uric acid, immunoglobulins, sickle cells, uremic 
toxins, bilirubin, porphyrin, cortisol and prostaglandins. 
Some examples of infectious agents which may be treated are, for example, 
viral disorders such as cytomegalovirus; protozoan disorders such as 
malaria, trypanosomes and leishmanias; bacterial infections such as 
strepotococci; fungus infections such as tinea versicolor; mycoplasma such 
as pleuro-pneumonia-like organisms; rickettsia diseases such as typhus and 
spotted fevers; spirochetes such as syphilis and chlamydia-agents in the 
psittacosis lympho-granuloma-trachoma disease group. 
Neoplasms which are treatable using the present invention include, for 
example, the lymphomas, sarcomas, carcinomas and leukemias. These may be 
removed by specific removal of a cell line, inhibitors, initiators of the 
disease and combinations thereof. 
Further examples of disease states which may be treated using the present 
invention include, for example, the following: 
Infections such as; Post streptococcal glomerulonephritis, Subacute 
bacterial endocarditis, Secondary syphilis, Pneumococcal sepsis, 
Lepromatous leprosy, Ventricular shunt infection, Infectious 
mononucleosis, Typhoid fever, Subacute sclerosing encephalitis, 
Landry-Guillain-Barre syndrome, Hepatitis B infection, Quartan malaria, 
Schistosomiasis, and Trypanosomiasis. 
Neoplasmas such as; Hepatoma, Lymphoma and Hodgkins disease, Acute 
leukemia, Hypernephroma, Carcinoma of the colon, Bronchogenic carcinoma, 
and Burkitts lymphoma. 
Connective Tissue Disorders such as; Periarteritis nodosa, Chronic 
glomerulonephritis, Acute or subacute thyroiditis, Vinyl chloride 
poisoning, Chronic liver disease, Mixed cryoglobulinemias, Berger's 
disease or IgA nephropathy, Rapidly progressive glomerulonephritis, and 
Sickle cell anemia. 
Hematologic Diseases such as; Thrombic thrombocyto-penic purpura, 
Autoimmune hemolytic anemia, Idiopathic thrombocytopenic purpura, 
Idiopathic neutropenia, Cold hemagglutinin disease, Paroxysmal cold 
hemoglobinuria, Circulating anticoagulants, Acquired hemophilia, the 
leukemias, the lymphomas, Erythroblastosis fetalis, Pernicious anemia, and 
Rh diseases. 
Neurologic Diseases such as; Acute demyelinating encephalitis, Multiple 
Sclerosis, Landry's paralysis, Guillain-Barre syndrome, Peripheral 
neuritis, and Myasthenia gravis. 
Collagen Diseases such as; Raynaud's, Lupus Erythematosus, Polyarteritis 
nodosa, Scleroderma, Dermatomyositis, Sjogren's syndrome, Rheumatoid 
arthritis, Rheumatic fever, and Erythema nodosa. 
Endocrine Diseases such as, for example; Cushing's syndrome & disease, 
Thyroiditis, Thyrotoxicosis, Addison's disease, and Aspermatogenesis. 
Gastrointestinal Diseases such as; Portal cirrhosis, Acute hepatitis, 
Chronic active hepatitis, Lupoid hepatitis, Biliary cirrhosis, Ulcerative 
colitis, Regional enteritis, and Pancreatitis. 
Miscellaneous Diseases such as, for example; Hypercholesterolemia, 
Glomerulonephritis, Basement membrane disease, Psychogenic states--drugs, 
Postaortic valve prosthesis--hemolytic anemia, Exfoliative dermatitis, Id 
reaction, Psoriasis, Behcet's syndrome, Thrombotic thrombocytopenic 
purpura, Carcinoma, Subacute bacterial endocarditis, Hypertension, Asthma, 
Hereditary angioneurotic edema, Meningococcemia, Crohn disease, Hepatic 
encephalopathy and Raynaud disease. 
Further, Diseases characterized by Antibodies to Nuclear Antigens, 
Cytoplasmic Antigens, Cell Surface Antigens, and Subclasses may be treated 
by the present invention. Suitable examples include, for example; 
Antibodies to Native-DNA (double stranded) or single and double, stranded 
Antibodies to SS DNA, Antibodies to Deoxyribonucleoprotein, Antibodies to 
Histone, Antibodies to Sm, Antibodies to RNP, Antibodies to Sc 1--1 
-Scleroderma, Antibodies to SS-A Sjogren syndrome, Sicca complex, 
Antibodies to RAP - Rheumatoid Arthritis, Sjogren syndrome, Antibodies to 
PM-1 -Polymyositis-dermatomyositis, and Antibodies to nucleolarSystemic 
sclerosis, Sjogren syndrome. 
Also, Antibodies Associated With Specific Autoimmune Disorders such as; 
Antibodies to smooth muscle - Chronic Hepatitis, Antibodies to 
acetylcholine receptors Myasthenia gravis, Antibodies to basement membrane 
at the dermal-epidermal junction - Bullous pemphigoid, Antibodies to the 
mucopolysaccharide protein complex or intracellular cement substance 
Pemphigus, Antibodies to immunoglobulins - Rheumatoid arthritis, 
Antibodies to glomerular basement membrane Glomerulonephritis, 
Goodpasture's syndrome, Idiopathic primary hemasiderosis, Antibodies to 
erythrocytes - Autoimmune hemolytic anemia, Antibodies to the thyroid - 
Hashimoto's, Antibodies to intrinsic factor - Pernicious anemia, 
Antibodies to platelets - Idiopathic thrombocytopenic purpura, 
Alloimmunization, Antibodies to mitochondria - Primary biliary cirrhosis, 
Antibodies to salivary duct cells - Sjogren's syndrome, Antibodies to the 
adrenal - Idiopathic adrenal atropathy, Antibodies to thyroid microsomal 
Grave's Disease, Antibodies to thyroglobulin - Addison's Disease, and 
Antibodies to islet cells - Diabetes Mellitus. 
Paraproteinemias such as, for example, Multiple myeloma, Macroglobulinemia, 
Cryoglobulinemia, and Light chain disease; 
Hyperlipidemia such as, Primary biliary cirrhosis and Familial 
Hypercholesterolemia; 
Endocrinopathies such as, Grave disease and Diabetes mellitus; 
Alloimmunization such as, Hemolytic disease of the newborn and Renal 
homograft rejection. 
Also, suitable for treatment using the present invention include, for 
example, Post Transfusion Purpura and Autoantibody Diseases such as, 
Goodpasture's syndrome, Myasthenia gravis, Pemphigus vulgaris, 
Hematological disease, Idiopathic (autoimmune) thrombocytopenic purpura, 
Autoimmune hemolytic anemia, Inhibitor to factor VIII and 
Polyradiculopathy/Guillain-Barre Syndrome. 
Immune Complex Diseases may also be treated and include, for example; 
Systemic lupus erythematosus, Polyarteritis nodosa, Cutaneous vasculitis, 
Rheumatoid arthritis, Glomerulonephritis, and Dermatomyositis. 
While not subscribing to any one particular theory over another, a review 
of the probable progression of autoimmune pathology suggests that the 
pathological sequence is very likely initiated by a free antigen 
-challenge, followed by antibody evolution and complexing and finalized by 
antibody excess and complement fixation of formed complexes. Thus, for 
proper selection of the biospecific polymer formulation and provision for 
proper efficacy would require preliminary diagnostic procedures to 
determine the predominant form of the autoimmune effector. An illustrative 
example of this is described below for the treatment of rheumatoid 
disease. Briefly, rheumatoid disease can be characterized as following the 
progression from (a) free RF antigen (atypical Ig) (rheumatic 
condition),(b) free RF antibody evolution and RF complexing and finally 
(c) antibody excess and complement activated RF complex fixation. Thus 
treatment of rheumatoid disease in its early development could be 
determined by detection of atypical immunoglobulins by monoclonal 
rheumatoid factor (m-RF) antibodies. Treatment at this stage would be best 
effected by m-RF activated biospecific polymers to remove the offending 
antigen and thus prevent the evolution of endogenous RF (e-RF) antibodies. 
Diagnostic evidence of e-RF would indicate the utilization of biospecific 
polymers having both m-RF and aggregated gamma globulin active biologicals 
(RF antigen). Alternatively, two biospecific polymers in series, each 
having one type of active biological could be utilized. In either case 
this combination of m-RF and aggregated gamma globulin would adsorb both 
the offending antigen and antibody molecules to sequester the disease 
progression. In the case where significant levels of RF antigen-antibody 
complex is detected, biospecific polymers containing Clq and/or collagen 
effector molecules would be indicated. Finally, if the disease process has 
progressed to the stage of complement fixation of formed immune complexes, 
an effective biospecific polymer would contain one or more anti-complement 
antibodies such as, for example, anti-Clq, anti-C.sub.3 or anti-C.sub.4 
Again the biologicals, if more than one is desirable, can be immobilized 
on a single biocompatible support or each can be on a separate support and 
connected in series in relation to the blood or plasma flow. 
As has been proposed above, effective use of the present invention is 
realized by thorough definition of the dynamics and stage of the immune 
response for effective disease management. 
Today, plasmapheresis and cytopheresis are the treatments for disease by 
removal of noxious substances or cells from the blood. It is currently 
believed that any disease treated by plasmapheresis and/or cytopheresis, 
where the desired result is the removal of a specific substance, can be 
advantageously treated with the product and process of the present 
invention. 
More specifically, a presently contemplated therapeutic regimen for whole 
blood may be illustrated as follows: 
(a) a vascular access is provided which will allow for; 
(b) a blood flow from about 30 ml/min. to about 200 ml/min., 
(c) an anticoagulant is administered to the blood; and 
(d) a pumping means may or may not be provided; 
(e) the blood is passed into the chamber device containing therein 
biospecific polymer membrane(s); 
(f) treating the whole blood by passing it in contact with said biospecific 
membrane; 
(g) depending on the anticoagulant used, additional medication may be 
needed or desired to neutralize the anticoagulatory effect on said treated 
blood; 
(h) the treated blood is returned to the patient. 
The time frame presently contemplated for the above regimen is 
approximately 2 hours to 4 hours. It is realized, of course, that 
depending on the situation, such a time frame may be either shortened or 
lengthened. 
A presently contemplated therapeutic regimen for plasma may be illustrated 
as follows: 
(a) a vascular access is provided which will allow for; 
(b) a blood flow about 30 ml/min. to about 200 ml/min., 
(c) an anticoagulant is administered to the blood; and 
(d) a pumping means provided; 
(e) a plasma-formed blood component separation means is provided; 
(f) the plasma is passed into the chamber device containing therein 
biospecific polymer membrane(s); 
(g) treating the plasma by passing it in contact with said biospecific 
membrane; 
(h) filtration through a 0.2 micron filter to remove any microemboli, 
bacteria or fungi; 
(i) the treated plasma and the formed blood components are recombined; 
(j) depending on the anticoagulant used, additional medication may be 
needed or desired to neutralize the anticoagulating effect on said treated 
blood; 
(k) the treated blood is returned to the patient. 
The following example will serve to further illustrate the present 
invention. This example should not be considered, however, as a limitation 
upon the scope of the present invention. 
EXAMPLE 
This example illustrates how a biospecific polymer utilizing a spacer may 
be produced. It also demonstrates the effectiveness of a therapeutic 
device embodiment of the present invention for removing rheumatoid factor 
antibody from test sera. 
A. Spacer attachment. 
A polymer support consisting of 50 percent glycidyl methacrylate/46 percent 
n-vinyl pyrrolidone/4 percent hydroxyethyl methacrylate was hydrated by 
placing the polymer in deionized water for three hours. The hydrated 
polymer support was then placed in a therapeutic device similar to the 
device illustrated in FIG. 3. A 10 ml 1.0M ACA solution ph-7.2 was passed 
through the device contacting said polymer support at a flow rate of 0.33 
ml/min. The device was cleared of excess ACA solution and 40 ml of 0.1M 
(2[N-morpholine]ethanesulfonic acid) (MES) was then passed through the 
device at a flow rate of 0.33 ml/min allowing the polymer support to 
equilibrate. 
B. Polymer activation/biological immobilization. 
The polymer support with pendant ACA spacers contained in the device was 
treated with 10 ml solution of 1.0M 
1-ethyl-3(3-dimethylaminopropyl)carbodiimide (CDI). The CDI was recycled 
through the device in contact with said polymer support with pendant 
spacers at a flow rate of 0.5 ml/min. The reaction was allowed to proceed 
for 30 min. Excess CDI was rinsed from the polymer support by passing a 10 
ml solution of 0.2M MES through the device at a flow rate of 2 ml/min. 
A 10 ml heat aggregated human gamma globulin (HGG) solution was recycled 
through the device contacting the activated polymer support at a flow rate 
of 0.5 ml/min. Said activated polymer support was allowed to react with 
the aggregated HGG for 72 hours at room temperature giving a biospecific 
polymer. 
C. Evaluation of therapeutic device for removing rheumatoid factor 
antibody. 
Three trials were conducted using three sources of sera positive for 
rheumatoid factor antibody. For each trial, the device was placed in a 
fluid flow circuit with a resevoir, pump and in-line valve. The in-line 
valve was positioned so as to isolate the device from the circuit when 
engaged. Before each trial the whole circuit was rinsed with 0.05M PBS 
solution for approximately 24 hours. The device was then isolated from the 
circuit via the in-line valve. The circuit (excluding the device) was 
cleared of PBS solution and 6.0 ml of the respective rheumatoid positive 
control serum was then added to the circuit resevoir and recirculated for 
approximately 15 min. to prime the circuit. The in-line valve to the 
device was then disengaged allowing the test serum to recirculate through 
the full circuit at a flow rate of 0.414 ml/min..sup.1 At time intervals 
of 71/2, 15, 30, 60 and 120 min., 0.5ml aliquots of test serum was removed 
from the resevoir to determine rheumatoid factor concentration via 
nephelometric analysis. The analysis was conducted on a Beckman ICS.TM. 
Analyzer II nephelometer. Results are listed in Table I below. After each 
trial, two separate rinses of 5.0 ml of 1.0M acetic acid was circulated 
through the device to desorb the bound rheumatoid factor. 
FNT .sup.l The therapeutic device contained 1.5 ml of PBS solution which was 
allowed to mix with the test sera as the in-line valve was opened. 
TABLE I 
______________________________________ 
RF POSITIVE CONTROL SERA 
Time Re Beckman RF Clinical 
Circulated 
LAS-R .TM. Calibrator** Initial 
Patient 
(min.) Level I* Reading (Iu/ml) 
Serum*** 
______________________________________ 
335 400 211 
7.5 225**** 163**** 147**** 
15 234 166 154 
30 230 201 154 
60 226 210 155 
120 219 200 156 
______________________________________ 
*Product of Hyland Diagnostics 
**Product of Smith Kline/Beckman Instruments 
***Clinical patient (1185620) Serum diluted 1:6 with normal human plasma. 
****Initial sharp drop due in part to dilution effects.