Therapeutic Apheresis and Blood Fractionation

132.1

Plasmapheresis

Centrifugal Devices • Membrane Plasmapheresis

132.2

Plasma Perfusion

Andrew L. Zydney

132.3

Cytapheresis

University of Delaware

132.4

Summary

Apheresis is the process in which a specific component of blood (either plasma, a plasma component, white cells, platelets, or red cells) is separated and removed with the remainder of the blood returned to the patient (often in combination with some type of replacement fluid). Donor apheresis is used for the collection of specific blood cells or plasma components from blood donors, resulting in a much more effective use of limited blood-based resources. Donor apheresis developed during World War II as a means for increasing the supply of critically needed plasma, and clinical trials in 1944 demonstrated that it was possible to safely collect donations of a unit of plasma (approximately 300 ml) on a weekly basis if the cellular components of blood were returned to the donor. Therapeutic apheresis is used for the treatment of a variety of diseases and disorders characterized by the presence of abnormal proteins or blood cells in the circulation which are believed to be involved in the progression of that particular condition. Therapeutic apheresis thus has its roots in the ancient practice of bloodletting, which was used extensively well into the 19th century to remove “bad humors” from the patient’s body, thereby restoring the proper balance between the “blood, yellow bile, black bile, and phlegm.”

The term plasmapheresis was first used by Abel, Rowntree, and Turner in 1914 in their discussion of a treatment for toxemia involving the repeated removal of a large quantity of plasma, with the cellular components of blood returned to the patient along with a replacement fluid [Kambic & Nose, 1993]. The first successful therapeutic applications of plasmapheresis were reported in the late 1950s in the management of macroglobulinemia (a disorder characterized by a large increase in blood viscosity due to the accumulation of high-molecular-weight globulins in the blood) and in the treatment of multiple myeloma (a malignant tumor of the bone marrow characterized by the production of excessive amounts of immunoglobulins).

By 1990, there were well over 50 diseases treated by therapeutic apheresis [Sawada et al., 1990] with varying degrees of success. Plasmapheresis is used in the treatment of: (1) protein-related diseases involving excessive levels of specific proteins (e. g., macroglobulins in Waldenstrom’s syndrome and lipoproteins in familial hypercholesterolemia) or excessive amounts of protein-bound substances (e. g., toxins in hepatic failure and thyroid hormone in thyrotoxicosis), (2) antibody-related or autoimmune diseases (e. g., glomerulonephritis and myasthenia gravis), and (3) immune-complex-related diseases (e. g., rheumatoid arthritis and systemic lupus erythematosus).

Cytapheresis involves the selective removal of one (or more) of the cellular components of blood, and it has been used in the treatment of certain leukemias (for the removal of leukocytes) and in the treatment of polycythemia. Table 132.1 provides a more complete listing of some of the diseases and blood com­ponents that are removed during therapeutic apheresis. This list is not intended to be exhaustive, and there is still considerable debate over the actual clinical benefit of apheresis for a number of these diseases.

The required separation of blood into its basic components (red cells, white cells, platelets, and plasma) can be accomplished using centrifugation or membrane filtration; the more specific removal of one (or more) components from the separated plasma generally involves a second membrane filtration or use of an appropriate sorbent. The discussion that follows focuses primarily on the technical aspects of the different separation processes currently in use. Additional information on the clinical aspects of thera­peutic apheresis is available in the references listed at the end of this chapter.

Plasmapheresis

The therapeutic application of plasmapheresis can take one of two forms: plasma exchange or plasma perfusion. In plasma exchange therapy, a relatively large volume of plasma, containing the toxic or immunogenic species, is separated from the cellular components of blood and replaced with an equivalent volume of a replacement fluid (either fresh frozen plasma obtained from donated blood or an appropriate plasma substitute). In plasma perfusion, the separated plasma is treated by an adsorptive column or second membrane filtration to remove a specific component (or components) from the plasma. This treated plasma is then returned to the patient along with the blood cells, thereby eliminating the need for exogenous replacement fluids. The different techniques that can be used for plasma perfusion are discussed subsequently.

The reduction in the concentration (C) of any plasma component during the course of a plasma­pheresis treatment can be described using a single compartment pharmacokinetic model as

V—^ □□□ QC DG (132.1)

P dt i p i i

Where Vp is the volume of the patient’s plasma (which is assumed to remain constant over the course of therapy through the use of a replacement fluid or through the return of the bulk of the plasma after a plasma perfusion), Qp is the volumetric rate of plasma collection, Gt is the rate of component generation, and □i is a measure of the effectiveness of the removal process. In membrane plasmapheresis, □i, is equal to the observed membrane sieving coefficient, which is defined as the ratio of the solute concentration in the filtrate collected through the membrane to that in the plasma entering the device; □i is thus equal to 1 for a small protein that can pass unhindered through the membrane but can be less than 1 for large proteins and immune complexes. In plasma perfusion systems, □i is equal to the fraction of the particular component removed from the collected plasma by the secondary (selective) processing step. The gener­ation rate is typically negligible over the relatively short periods (fewer than 3 hours) involved in the actual plasmapheresis; thus the component concentration at the end of a single treatment is given as

C ^ a. Qt□ □ □ y □

—- □ expo – i p □□ expo – i exc□ (132.2)

Coo □ yp □ □ yp □

Where Vexc = Qpt is the actual volume of plasma removed (or exchanged) during the process. Plasma exchange thus reduces the concentration of a given component by 63% after an exchange of one plasma volume (for □i = 1) and by 86% after two plasma volumes. This simple single compartment model has been verified for a large number of plasma components, although immunoglobulin G actually appears to have about 50% extravascular distribution with the re-equilibration between these compartments occurring within 24-28 hr following the plasmapheresis.

Disease

Components Removed

Hematologic

Hemophilia

AntiFactor VIII Ab

Idiopathic Thrombocytopenia Purpura

Antiplatelet Ab, immune complexes

Thrombotic Thrombocytopenia Purpura

Antiplatelet Ab, immune complexes

AIDS, HIV

Antilymphocyte Ab, immune complexes

Autoimmune Hemolytic Anemia

Anti-red cell Ab, red cells

Rh Incompatibility

Anti-Rh Ab

Cryoglobulinemia

Cryoglobulins

Hyperviscosity Syndrome

Macroglobulins, immunglobulin M

Waldenstrom’s Syndrome

Immunoglobulin M

Paraproteinemia

Paraproteins

Sickle Cell Anemia

Red blood cells

Thrombocythemia

Platelets

Collagen/rheumatologic

Systemic Lupus Erythematosus

Anti-DNA Ab, immune complexes

Progressive Systemic Sclerosis

Antinonhistone nuclear Ab

CREST Syndrome

Anticentromere Ab

Sjorgen Syndrome

Antimitochondrial Ab

Rheumatoid Arthritis

Rheumatoid factor, cryoglobulins, immunoglobulins

Periarteritis Nodosa

Cryoglobulins, immune complexes

Raynaud’s Disease

Cryoglobulins, macroglobulins

Scleroderma

Immune complexes

Mixed Connective Tissue Diseases

Immunoglobulins

Neurologic

Myasthenia Gravis

Antiacetylcholine receptor Ab, cryoglobulins

Multiple Sclerosis

Antimyelin Ab

Guillain-Barre Syndrome

Antimyelin Ab

Polyneuropathy

Cryoglobulins, macroglobulins

Polyradiculoneuropathy

Antibodies

Lambert-Eaton Syndrome

Antibodies

Hepatic

Chronic Active Hepatitis

Antimitochondrial Ab

Hepatic Failure

Protein-bound toxins

Primary Biliary Cirrhosis

Protein-bound toxins, antimitochondrial Ab

Renal

Goodpasture’s Syndrome

Antiglomerular basement membrane Ab

Glomerulonephritis

Immune complexes

Lupus Nephritis

Immune complexes

Transplant Rejection

Immune complexes, anti-HLA Ab

Malignant diseases

Cancer

Tumor-specific Ab, immune complexes

Multiple Myeloma

Immunoglobulins

Leukemia

Leukocytes

Miscellaneous

Addison’s Disease

Antiadrenal Ab

Autoimmune Thyroiditis

Antimicrosomal Ab

Chronic Ulcerative Colitis

Anticolonic epithelial cell Ab

Diabetes Mellitus

Antiinsulin receptor Ab

Hashimoto’s Disease

Antithyroglobulin Ab

Insulin Autoimmune Syndrome

Antiinsulin Ab

Pemphigus

Antiepidermal cell membrane Ab

Ulcerative Colitis

Anticolonic lipopolysaccharide Ab

Asthma

Immunoglobulin E

Hypercholesterolemia

Cholesterol, lipoproteins

Hyperlipidemia

Low – and very low density lipoproteins

Thyrotoxicosis

Thyroid hormone

There is still considerable variability in the frequency and intensity of the plasmapheresis used in different therapeutic applications, and this is due in large part to uncertainties regarding the metabolism, pharmacokinetics, and pathogenicity of the different components that are removed during therapeutic apheresis. The typical plasma exchange therapy currently involves the removal of 2-3 L of plasma (approximately one plasma volume) at a frequency of 2-4 times per week, with the therapy continued for several weeks. There have also been a number of studies of the long-term treatment of several diseases via plasmapheresis, with the therapy performed on a periodic basis (ranging from once per week to once every few months) over as much as 5 years (e. g., for the removal of cholesterol and lipoproteins in the treatment of severe cases of hypercholesterolemia).

Centrifugal Devices

Initially, all plasmapheresis was performed using batch centrifuges. This involved the manual removal of approximately one unit (500 ml) of blood at a time, with the blood separated in a centrifuge so that the target components could be removed. The remaining blood was then returned to the patient before drawing another unit and repeating the entire process. This was enormously time-consuming and labor­intensive, requiring as much as 5 hours for the collection of only a single liter of plasma. Batch centrif­ugation is still the dominant method for off-line blood fractionation in most blood-banking applications, but almost all therapeutic plasmapheresis is performed using online (continuous) devices.

The first continuous flow centrifuge was developed in the late 1960s by IBM in conjunction with the National Cancer Institute, and this basic design was subsequently commercialized by the American Instrument Co. (now a division of Travenol) as the Aminco Celltrifuge [Nose et al., 1983]. A schematic diagram showing the general configuration of this, and most other continuous flow centrifuges is shown in Fig. 132.1. The blood is input at the bottom of the rotating device and passes through a chamber in

Plasma

1 White Cells

Red Cells

Therapeutic Apheresis and Blood Fractionation

&

I Feed Blood

Cp

Rotation

FIGURE 132.1 Schematic diagram of a generic continuous flow centrifuge for fractionation of blood into red cells, white cells/platelets, and plasma.

Which the actual separation into plasma, white cells/platelets, and red cells occurs. Three separate exit ports are located at different radial positions to remove the separated components continuously from the top of the chamber using individual roller pumps. The position of the buffy coat layer (which consists of the white cells and platelets) is controlled by adjusting the centrifugal speed and the relative plasma and red cell flow rates to obtain the desired separation. Probably the most difficult engineering problem in the development of the continuous flow centrifuge was the design of the rotating seals through which the whole blood and separated components must pass without damage. The seal design in the original NCI/IBM device used saline lubrication to prevent intrusion of the cells between the contacting surfaces.

In order to obtain effective cell separation in the continuous flow centrifuge, the residence time in the separation chamber must be sufficiently large to allow the red cells to migrate to the outer region of the device. The degree of separation can thus be characterized by the packing factor

GV t

P □ (132.3)

H

Where G is the g-force associated with the centrifugation, Vsed is the sedimentation velocity at 1 g, t is the residence time in the separation chamber, and h is the width of the separation chamber (i. e., the distance over which the sedimentation occurs). The packing factor thus provides a measure of the radial migration compared to the width of the centrifuge chamber, with adequate cell separation obtained when P > 1. The residence time in the separation chamber is inversely related to the blood flow rate (QB) as

AT

T □ (132.4)

QB

Where L and A are the length and cross-sectional area of the chamber, respectively.

Rotational speeds in most continuous flow centrifuges are maintained around 1500 rpm (about 100 g) to obtain a relatively clean separation between the red cells and buffy coat, to avoid the formation of a very highly packed (and therefore highly viscous) region of cells at the outer edge of the chamber, and to minimize excessive heating around the rotating seals [Rock, 1983]. The width of the separation chamber must be large enough to permit effective removal of the different blood components from the top of the device, while minimizing the overall extracorporeal blood volume. For example, a packing factor of 10 requires a residence time of about 20 s for a device operated at 100 g with a 0.1-cm-wide separation chamber. The blood flow rate for this device would need to be maintained below about 120 ml/min for a chamber volume of 40 ml. Most of the currently available devices operate at QB □ 50 ml/min and can thus collect a liter of plasma in about 30-40 minutes.

More recent models for the continuous flow centrifuge have modified the actual geometry of the separation chamber to enhance the cell separation and reduce the overall cost of the device [Sawada et al., 1990]. Examples include tapering the centrifuge bowl to improve flow patterns and optimizing the geometry of the collection region to obtain purer products. The IBM 2997 (commercialized by Cobe Laboratories) uses a disposable semirigid plastic rectangular channel for the separation chamber, which eliminates some of the difficulties involved in both sterilizing and setting up the device. Fenwal Labora­tories developed the CS-3000 Cell Separator (subsequently sold by Baxter Healthcare), which uses a continuous J-shaped mulitchannel tubing connected directly to the rotating element. This eliminates the need for a rotating seal, thereby minimizing the possibility of leaks. The tubing in this device actually rotates around the centrifuge bowl during operation, using a “jump rope” principle to prevent twisting of the flow lines during centrifugation.

In addition to the continuous flow centrifuge, Haemonetics has developed a series of intermittent flow centrifuges [Rock, 1983]. Blood flows into the bottom of a separation chamber similar to that shown in Fig. 132.1, But the red cells simply accumulate in this chamber while the plasma is drawn to the center of the rotating bowl and removed through an outlet port at the top of the device. When the process is complete, the pump action reverses, and the red cells are forced out of the bowl and reinfused into the patient (along with any replacement fluid). The entire process is then automatically repeated to obtain the desired level of plasma (or white cell) removal. This device was originally developed for the collection of leukocytes and platelets, but it is now used extensively for large-scale plasmapheresis as well. Maximum blood flow rates are typically about 70-80 ml/min, and the bowl rotates at about 5000 rpm. The device as a whole is more easily transported than most of the continuous flow centrifuges, but it requires almost 50% more time for the collection of an equivalent plasma volume due to the intermittent nature of the process. In addition, the total extracorporeal volume is quite high (about 500 ml compared to only 250 ml for most of the newer continuous flow devices) due to the larger chamber required for the red cell accumulation [Sawada et al., 1990].

All these centrifugal devices have the ability to carry out effective plasma exchange, although the rate of plasma collection tends to be somewhat slower than that for the membrane devices discussed in the next section. These devices can also be used for the collection of specific cell fractions, providing a degree of flexibility that is absent in the membrane systems. The primary disadvantage of the centrifugal units is the presence of a significant number of platelets in the collected plasma (typically about 105 platelets per □). Not only can this lead to considerable platelet depletion during repeated applications of plasma exchange, it can also interfere with many of the secondary processing steps employed in plasma perfusion.

Membrane Plasmapheresis

The general concept of blood filtration using porous membranes is quite old, and membranes with pores suitably sized to retain the cellular components of blood and pass the plasma proteins have been available since the late 1940s. Early attempts at this type of blood filtration were largely unsuccessful due to severe problems with membrane plugging (often referred to as fouling) and red cell lysis. Blatt and coworkers at Amicon recognized that these problems could be overcome using a cross-flow configuration [Solomon et al., 1978] in which the blood flow was parallel to the membrane and thus perpendicular to the plasma (filtrate) flow as shown schematically in Fig. 132.2. This geometry minimizes the accumulation of retained cells at the membrane surface, leading to much higher filtration rates and much less cell damage than could be obtained in conventional dead-end filtration devices [Solomon et al., 1978]. This led to the development of a large number of membrane devices using either flat sheet or hollow-fiber membranes made from a variety of polymers including polypropylene (Travenol Laboratories, Gambro, Fresenius), cellulose diacetate (Asahi Medical), polyvinyl alcohol (Kuraray), polymethylmethacrylate (Toray), and polyvinyl chloride (Cobe Laboratories).

Therapeutic Apheresis and Blood Fractionation

Plasma Filtrate Flux, J(z)

FIGURE 132.2 Schematic diagram of a parallel plate device for cross-flow membrane plasmapheresis.

Therapeutic Apheresis and Blood Fractionation

FIGURE 132.3 Experimental data for the filtrate flux as a function of the applied transmembrane pressure drop in a parallel plate membrane plasmaphersis device. Red cell hemolysis, defined as a filtrate hemoglobin concentration exceeding 20 mg/dl, occurs to the right of the dashed line. Data have been adapted from Zydney and Colton [1987].

These membrane devices all produce essentially cell-free plasma with minimal protein retention using membranes with pore sizes of 0.2-0.6 Dm. In addition, these devices must be operated under conditions that cause minimal red cell lysis, while maintaining a sufficiently high plasma filtrate flux to reduce the cost of the microporous membranes. Typical experimental data for a parallel plate membrane device are shown in Fig. 132.3 [Zydney & Colton, 1987]. The results are plotted as a function of the mean trans­membrane pressure drop (□□□□ ) at several values of the wall shear rate (Hy, where is directly propor­tional to the inlet blood flow rate (QB). The filtrate flux initially increases with increasing □□□□ reaching a maximum pressure independent value which increases with increasing shear rate. The flux under all conditions is substantially smaller than that obtained when filtering pure (cell-free) saline under identical conditions (dashed line in Fig. 132.3). No measurable hemolysis was observed at low □□□□ even when the flux was in the pressure-independent regime. Hemolysis does become significant at higher pressures, with the extent of hemolysis decreasing with increasing and with decreasing membrane pore size [Solomon et al., 1978]. The dashed diagonal line indicates the pressure at which the filtrate hemoglobin concentration exceeds 20 mg/dl.

The pressure-independent flux at high □Onn generally has been attributed to the formation of a concentration polarization boundary layer consisting of a high concentration of the formed elements in blood, mainly the red blood cells, which are retained by the microporous membrane (Fig. 132.2). This dynamic layer of cells provides an additional hydraulic resistance to flow, causing the flux to be substan­tially smaller than that obtained during filtration of a cell-free solution. At steady-state, this boundary layer is in dynamic equilibrium, with the rate of convection of formed elements toward the membrane balanced by the rate of mass transport back into the bulk suspension. There has been some debate in the literature over the actual mechanism of cell transport in these devices, and the different models that have been developed for the plasma flux during membrane plasmapheresis are discussed elsewhere [Zydney & Colton, 1986].

Zydney and Colton [1982] proposed that cell transport occurs by a shear-induced diffusion mechanism in which cell-cell interactions and collisions give rise to random cell motion during the shear flow of a concentrated suspension. This random motion can be characterized by a shear-included diffusion coef­ficient, which was evaluated from independent experimental measurements as

(132.5)

подпись: (132.5)D □ a2U f ([

Where □ is the local shear rate (velocity gradient), a is the cell radius (approximately 4.2 Dm for the red blood cells), and C is the local red cell concentration. The function f(C), which reflects the detailed concentration dependence of the shear-induced diffusion coefficient, is approximately equal to 0.03 for red cell suspensions over a broad range of cell concentrations.

The local filtrate flux can be evaluated using a stagnant film analysis in which the steady-state mass balance is integrated over the thickness of the concentration boundary layer yielding [Zydney & Colton, 1986]

J( □□ km ln – w (132.6)

Cb

Where z is the axial distance measured from the device inlet, and Cw and Cb are the concentrations of formed elements at the membrane surface and in the bulk suspension, respectively. The bulk mass transfer coefficient (km) can be evaluated using the Leveque approximation for laminar flow in either a parallel plate or hollow fiber device

Rf/3 □ a4 ri/3

Km □ 0516 □ “□ □ °.°47 ppn (I32.7)

z □ □ z □

Where the second expression has been developed using the shear-induced diffusion coefficient given by Eq (132.5) with f(C) = 0.03. The wall shear rate is directly proportional to the blood flow rate with

40

□ —^ (132.8)

NOR3

For a hollow-fiber device with N fibers of inner radius R and

W □ 0 (132.9)

Wh

For a parallel plate device with channel height h and total membrane width w.

At high pressures, Cw approaches its maximum value which is determined by the maximum packing density of the cells (about 95% under conditions typical of clinical plasmapheresis). The plasma flux under these conditions becomes independent of the transmembrane pressure drop, with this pressure – independent flux varying linearly with the wall shear rate and decreasing with increasing bulk cell concentration as described by Eqs. (132.6) and (132.7). A much more detailed numerical model for the flux [Zydney & Colton, 1987], which accounts for the concentration and shear rate dependence of both the blood viscosity and shear-induced cell diffusion coefficient as well as the compressibility of the blood cell layer that accumulates at the membrane, has confirmed the general behavior predicted by Eqs. (132.6) and (132.7).

The volumetric filtrate (plasma) flow rate (0p) in a hollow-fiber membrane filter can be evaluated by integrating Eqs. (132.6)-(132.8) along the length of the device accounting for the decrease in the blood flow rate (and thus H^) due to the plasma removal [Zydney & Colton, 1986]. The resulting expression for the fractional plasma yield is

^ □ n 2 rf2/3 □

0 1-1 n a 2 t r c n

(132.1°)

подпись: (132.1°)0a 1 – expо°.62lOT ln qH

An analogous expression can be evaluated for a parallel plate device with the channel height h replacing R and the coefficient 0.62 becoming 0.84. Even though the development leading to Eq. (132.10) neglects the detailed variations in the bulk cell concentration and velocity profiles along the fiber length, the final expression has been shown to be in good agreement with experimental data for the plasma flow rate in actual clinical devices [Zydney & Colton, 1986]. Equation (132.10) predicts that the volumetric plasma flow rate is independent of the number of hollow fibers (or the membrane width for a parallel plate membrane device), a result which is consistent with a number of independent experimental investigations.

According to Eq. (132.10), the plasma flow rate increases significantly with decreasing fiber radius. There are, however, a number of constraints on the smallest fiber radius that can actually be employed in these hollow-fiber membrane devices. For example, blood clotting and fiber blockage can become unacceptable in very narrow bore fibers. The blood flow in such narrow fibers also causes a very high bulk shear stress, which potentially can lead to unacceptable levels of blood cell damage (particularly for white cells and platelets). Finally, the hollow-fiber device must be operated under conditions which avoid hemolysis.

Zydney and Colton [1982] developed a model for red cell lysis during membrane plasmapheresis in which the red cells are assumed to rupture following their deformation into the porous structure of the membranes. A given red cell is assumed to lyse if it remains in a pore for a sufficient time for the strain induced in the red cell membrane to exceed the critical strain for cell lysis. Since the red cell can be dislodged from the pore by collisions with other cells moving in the vicinity of the membrane or by the fluid shear stress, the residence time in the pore will be inversely related to the wall shear rate.

The tension (□) in the red cell membrane caused by the deformation in the pore is evaluated using Laplace’s law [Zydney & Colton, 1987]

□Pn, Rp

□—(132.11)

2

Where Rp is the pore radius. Hemolysis is assumed to occur at a critical value of the strain in the red cell membrane (S); thus the time required for lysis is given implicitly by

S □□ #[}[□□ j 0.0010 □ 0.0012 [ □ exp(8l[ 4.5 10-6l} (132.12)

The function g(t) represents the temporal dependence of the lytic phenomenon and has been evaluated from independent experimental measurements [Zydney & Colton, 1982]. Cell lysis occurs when S □ 0.03 in Eq. (132.12) where □ is given in dyne/cm and 1 is in sec. This simple model has been shown to be in good agreement with experimental data for red cell lysis during cross-flow membrane plasmapheresis [Zydney & Colton, 1987].

This physical model for red cell lysis implies that hemolysis can be avoided by operating at sufficiently high shear rates to reduce the residence time in the membrane pores. However, operation at high shear rates also causes the inlet transmembrane pressure drop to increase due to the large axial pressure drop associated with the blood flow along the length of the device [Zydney & Colton, 1982]:

Ptm ([□□Ptm (□ 2D$L (132.13)

R

Where □Dcn(0) and □Ocn(L) are the inlet and exit transmembrane pressure drops, respectively, and D is the average blood viscosity. □Ocn(L) is typically maintained at a small positive value (about 20 mmHg) to ensure that there is a positive transmembrane pressure drop along the entire length of the device.

Since the increase in □0CD(0) with increasing has a greater effect on hemolysis than the reduction in

Therapeutic Apheresis and Blood Fractionation

FIGURE 132.4 Schematic representation of the safe operating regime for a clinical membrane plasmapheresis device.

The residence time for the red cells in the membrane pores, there is also an upper bound on the shear rate for the safe operation of any given clinical device.

The predicted safe operating regime for a clinical membrane plasmapheresis device can be determined using Eq. (132.11), with the maximum transmembrane pressure drop occurring at the device inlet, Eq. (132.13). The results are shown schematically in Fig. 132.4. HEmolysis occurs at very low shear rates due to the long residence time in the membrane pores, whereas lysis at high shear rates is due to the large value of the inlet transmembrane pressure drop associated with the axial flow. Note that there is a critical fiber length [at fixed values of the fiber radius and Шш (L)] above which there is no longer any safe operating condition.

To avoid some of the constraints associated with the design of both parallel plate and hollow-fiber membrane devices. Hemasciences developed a rotating membrane filter for use in both donor and therapeutic plasmapheresis. A nylon membrane is placed on an inner cylinder and rotated at about 3600 rpm inside a concentric outer cylindrical chamber using a magnetic coupling device. The rotating membrane causes a very high shear rate (on the order of 10,000 s-1) in the narrow gap between the cylinders. However, these high shear rates do not result in a large axial pressure drop, as found in the parallel plate and hollow-fiber devices, due to the decoupling of the axial blood flow and the shear rate in this system (the shear is now due almost entirely to the membrane rotation). The fluid flow in this rotating cylinder system also leads to the development of fluid instabilities known as Taylor vortices, and these vortices dramatically increase the rate of cell mass transport away from the membrane and back into the bulk suspension. This leads to a dramatic increase in the plasma filtrate flux and a dramatic reduction in the required membrane area. The Autopheresis-C (the rotating filter currently sold by Baxter Healthcare) uses only 70 cm2 of membrane, which is more than an order of magnitude less than that required in competitive hollow-fiber and parallel plate devices. The mathematical analysis of the plasma filtrate flux and the corresponding design equations for the rotating cylinder plasma filter are provided by Zeman and Zydney [1996].

Plasma Perfusion

In repeated applications of plasma exchange, it is necessary to use replacement fluids that contain proteins to avoid the risks associated with protein depletion. One approach to minimizing the cost of these protein – containing replacement fluids (either albumin solutions, fresh frozen plasma, or plasma protein fraction) is to use a saline or dextran solution during the initial stages of the process and to then switch to a protein-containing replacement fluid toward the end of the treatment. Alternatively, a number of tech­niques have been developed to selectively remove specific toxic or immunogenic components from the plasma, with this treated plasma returned to the patient along with the cellular components of blood. This effectively eliminates the need for any expensive protein-containing replacement fluids.

Plasma perfusion (also known as online plasma treatment) is typically performed using either mem­brane or sorbent-based systems. Membrane filtration separates proteins on the basis of size and is thus used to selectively remove the larger molecular weight proteins from albumin and the small plasma solutes (salts, sugars, amino acids, and so on). A variety of membranes have been employed for this type of plasma fractionation including cellulose acetate (Terumo), cellulose diacetate (Asahi Medical and Teijin), ethylene vinyl alcohol (Kuraray), and polymethylmetharcrylate (Toray). These membranes are generally hydrophilic to minimize the extent of irreversible protein adsorption, with pore sizes ranging from 100-600 A depending on the specific objectives of the membrane fractionation.

The selectivity that can be obtained with this type of plasma filtration can be examined using available theoretical expressions for the actual sieving coefficient (Sa) for a spherical solute in a uniform cylindrical pore:

Therapeutic Apheresis and Blood Fractionation

Where # is the ratio of the solute to pore radius. Equation (132.14) is actually an approximate expression which has been shown to be in good agreement with more rigorous theoretical analyses. This expression for the actual sieving coefficient, where Sa is defined as the ratio of the protein concentration in the filtrate to that at the upstream surface of the membrane, is valid at high values of the plasma filtrate flux, since it implicitly assumes that the diffusive contribution to protein transport is negligible. To avoid excessive albumin loss, it is desirable to have Sa > 0.8, which can be achieved using a membrane with an effective pore size greater than about 160 A (albumin has a molecular weight of 69,000 and a Stokes­Einstein radius of 36 A). This membrane would be able to retain about 80% of the immunglobulin M (which has a molecular weight of about 900,000 and a Stokes-Einstein radius of 98 A), but it would retain less than 40% of the immunglobulin G (with MW = 155,000 and a radius of 55 A).

The protein retention obtained during an actual plasma filtration is substantially more complex than indicated by the above discussion. The polymeric membranes used in these devices actually have a broad distribution of irregularly shaped (noncylindrical) pores. Likewise, the proteins can have very different (nonspherical) conformations, and their transport characteristics also can be affected by electrostatic, hydrophobic, and van der Waals interactions between the proteins and the polymeric membrane, in addition to the steric interactions that are accounted for in the development leading to Eq. (132.14). Protein-protein interactions can also significantly alter the observed protein retention. Finally, the partially retained proteins will tend to accumulate at the upstream surface of the membrane during filtration (analogous to the concentration polarization effects described previously in the context of blood cell filtration).

This type of secondary plasma filtration, which is generally referred to in the literature as cascade filtration, is primarily effective at removing large immune complexes (molecular weight of approximately 700,000) and immunglobulin M (MW of 900,000) from smaller proteins such as albumin. Several studies have, however, found a higher degree of albumin-immunoglobulin G separation than would be expected based on purely steric considerations [Eq. (132.14)]. This enhanced selectivity is probably due to some type of long-range (e. g., electrostatic) interaction between the proteins and the membrane.

A number of different techniques have been developed to enhance the selectivity of these plasma filtration devices. For example, Malchesky and coworkers at the Cleveland Clinic [Malchesky et al., 1980] developed the process of cryofiltration in which the temperature of the plasma is lowered to about 10°C prior to filtration. A number of diseases are known to be associated with the presence of large amounts of cryo – (cold-) precipitable substances in the plasma, including a number of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Lowering the plasma temperature causes the aggregation and/or gelation of these cryoproteins, making it much easier for these components to be removed by the membrane filtration. About 10 g of cryogel can be removed in a single cryofiltration, along with significant amounts of the larger-molecular-weight immune complexes and IgM. The actual extent of protein removal during cyrofiltration depends on the specific composition of the plasma and thus on the nature as well as the severity of the particular disease state [Sawada et al., 1990]. There is thus considerable uncertainty over the actual components that are removed during cryofiltration under different clinical and/or experimental conditions. The cryogel layer that accumulates on the surface of the membrane also affects the retention of other plasma proteins, which potentially could lead to unacceptable losses even of small proteins such as albumin.

It is also possible to alter the selectivity of the secondary membrane filtration by heating the plasma up to or even above physiologic temperatures. This type of thermofiltration has been shown to increase the retention of low- (LDL) and very low (VLDL) density lipoproteins, and this technique has been used for the online removal of these plasma proteins in the treatment of hypercholesterolemia. LDL removal can also be enhanced by addition of a heparin/acetate buffer to the plasma, which causes precipitation of LDL and fibrinogen with the heparin [Sawada et al., 1990]. These protein precipitates can then be removed relatively easily from the plasma by membrane filtration. The excess heparin is subsequently removed from the solution by adsorption, with the acetate and excess fluid removed using bicarbonate dialysis.

An attractive alternative to secondary membrane filtration for the selective removal of plasma com­ponents is the use of sorbent columns such as: (1) activated charcoal or anion exchange resins for the removal of exogenous toxins, bile acids, and bilirubin; (2) dextran sulfate cellulose for the selective removal of cholesterol, LDL, and VLDL; (3) immobilized protein A for the removal of immunoglobulins (particularly IgG) and immune complexes; and (4) specific immobilized ligands like DNA (for the removal of anti-DNA Ab), tryptophan (for the removal of antiacetylcholine receptor antibodies), and insulin (for the removal of anti-insulin antibodies). These sorbents provide a much more selective separation than is possible with any of the membrane processes; thus they have the potential to signifi­cantly reduce the side effects associated with the depletion of needed plasma components. The sorbent columns generally are used in combination with membrane plasmaphersis, since the platelets that are present in the plasma collected from available centrifugal devices can clog the columns and interfere with the subsequent protein separation.

The development of effective sorbent technology for online plasma treatment has been hindered by the uncertainties regarding the actual nature of the plasma components that must be removed for the clinical efficacy of therapeutic apheresis in the treatment of different disease states. In addition, the use of biologic materials in these sorbent systems (e. g., protein A or immobilized DNA) presents particular challenges, since these materials may be strongly immunogenic if they desorb from the column and enter the circulation.

Cytapheresis

Cytapheresis is used to selectively remove one (or more) of the cellular components of blood, with the other components (including the plasma) returned to the patient. For example, leukocyte (white cell) removal has been used in the treatment of leukemia, autoimmune diseases with a suspected cellular immune mechanism (e. g., rheumatoid arthritis and myasthenia gravis), and renal allograft rejection. Erythrocyte (red cell) removal has been used to treat sickle cell anemia, severe autoimmune hemolytic anemia, and severe parasitemia. Plateletapheresis has been used to treat patients with thrombocythemia.

Most cytapheresis is performed using either continuous or intermittent flow centrifuges, with appro­priate software and/or hardware modifications used to enhance the collection of the specific cell fraction. It is also possible to remove leukocytes from whole blood by depth filtration, which takes advantage of the strong adherence of leukocytes to a variety of polymeric materials (e. g., acrylic, cellulose acetate, polyester, or nylon fibers). Leukocyte adhesion to these fibers is strongly related to the configuration and the diameter of the fibers, with the most effective cell removal obtained with ultrafine fibers less than

Dm in diameter. Available leukocyte filters (Sepacel, Cellsora, and Cytofrac from Asahi Medical Co.) have packing densities of about 0.1-0.15 g fiber/cm3 and operate at blood flow rates of 20-50 ml/min, making it possible to process about 2 L of blood in 1.5 hr.

Leukocyte filtration is used most extensively in blood-banking applications to remove leukocytes from the blood prior to transfusion, thereby reducing the likelihood of antigenic reactions induced by donor leukocytes and minimizing the possible transmission of white-cell associated viral diseases such as cytomegalovirus. The absorbed leukocytes can also be eluted from these filters by appropriate choice of buffer solution pH, making it possible to use this technique for the collection of leukocytes from donated blood for use in the subsequent treatment of leukopenic recipients. Depth filtration has also been considered for online leukocyte removal from the extracorporeal circuit of patients undergoing cardio­pulmonary bypass as a means to reduce the likelihood of postoperative myocardial or pulmonary rep­erfusion injury which can be caused by activated leukocytes.

A new therapeutic technique that involves online cytapheresis is the use of extracorporeal photochemo­therapy, which is also known in the literature as photopheresis. Photopheresis can be used to treat a variety of disorders caused by aberrant T-lymphocytes [Edelson, 1989], and it has become an established therapy for the treatment of advanced cutaneous T-cell lymphoma in the U. S. and several European countries. In this case, the therapy involves the use of photoactivated 8-methoxypsoralen, which blocks DNA replication causing the eventual destruction of the immunoactive T-cells. The psoralen compound is taken orally prior to the phototherapy. Blood is drawn from a vein and separated by centrifugation. The white cells and plasma are collected, diluted with a saline solution, and then pumped through a thin plastic chamber in which the cells are irradiated with a high-intensity UV light that activates the psoralen. The treated white cells are then recombined with the red cells and returned to the patient. Since the photoactivated psoralen has a half-life of only several microseconds, all its activity is lost prior to reinfusion of the cells, thereby minimizing possible side effects on other organs. The removal of the red cells (which have a very high adsorptivity to UV light) makes it possible to use a much lower energy UV light, thereby minimizing the possible damage to normal white cells and platelets.

Photopheresis has also been used in the treatment of scleroderma, systemic lupus erythematosus, and pemphigus vulgaris. The exact mechanism for the suppression effect induced by the photo-therapy in these diseases is uncertain, although the T-cel! destruction seems to be highly specific for the immuno – active T-cells [Edelson, 1989]. The response is much more involved than simple direct photoinactivation of the white cells; instead, the photo-treated cells appear to undergo a delayed form of cell death which elicits an immunologic response possibly involving the production of anti-idiotypic antibodies or the generation of clone-specific suppressor T-cells. This allows for an effective “vaccination” against a par­ticular T-cell activity without the need for isolating or even identifying the particular cells that are responsible for that activity [Edelson, 1989].

Phototherapy has also been used for virus inactivation, particularly in blood-banking applications prior to transfusion. This can be done using high-intensity UV light alone or in combination with specific photoactive chemicals to enhance the virus inactivation. For example, hematoporphyrin derivatives have been shown to selectively destroy hepatitis and herpes viruses in contaminated blood. This technique shows a high degree of specificity toward this type of enveloped virus, which is apparently due to the affinity of the photoactive molecules for the lipids and glycolipids that form an integral part of the viral envelope.

Another interesting therapeutic application involving cytapheresis is the ex vivo activation of immu­nologically active white cells (lymphokine-activated killer cells, tumor-infiltrating lymphocytes, or acti­vated killer macrophages) for the treatment of cancer. The detailed protocols for this therapy are still being developed, and there is considerable disagreement regarding its actual clinical efficacy. A pool of activated cells is generated in vivo by several days of treatment with interleukin-2. These cells are then collected from the blood by centrifugal cytapheresis and further purified using density gradient centrif­ugation. The activated cells are cultured for several days in a growth media containing additional interleukin-2. These ex-vivo activated cells are then returned to the patient, where they have been shown to lyse existing tumor cells and cause regression of several different metastatic cancers.

Summary

Apheresis is unique in terms of the range of diseases and metabolic disorders which have been successfully treated by this therapeutic modality. This broad range of application is possible because apheresis directly alters the body” s immunologic system though the removal or alteration of specific immunologically active cells and/or proteins.

Although there are a number of adverse reactions that can develop during apheresis (e. g., fluid imbalance, pyrogenic reactions, depletion of important coagulation factors, and thrombocytopenia), the therapy is generally well tolerated even by patients with severely compromised immune systems. This has, in at least some instances, led to the somewhat indiscriminate use of therapeutic apheresis for the treatment of diseases in which there was little physiologic rationale for the application of this therapy. This was particularly true in the 1980s, where dramatic advances in the available technology for both membrane and centrifugal blood fractionation allowed for the relatively easy use of apheresis in the clinical milieu. In some ways, apheresis in the 1980s was a medical treatment that was still looking for a disease. Although apheresis is still evolving as a therapeutic modality it is now a fairly well-established procedure for the treatment of a significant number of diseases (most of which are relatively rare) in which the removal of specific plasma proteins or cellular components can have a beneficial effect on the progression of that particular disease. Furthermore, continued advances in the equipment and procedures used for blood fractionation and component removal have, as discussed in this chapter, provided a safe and effective technology for the delivery of this therapy.

The recent advances in sorbent-based systems for the removal of specific immunologically active proteins and in the development of treatment for the activation or inactivation of specific cellular components of the immune system has provided exciting new opportunities for the alteration and even control of the body’s immunologic response. This includes: (1) the direct removal of specific antibodies or immune complexes (using membrane plasmapheresis with appropriate immunosorbent columns), (2) the inactivation or removal of specific lymphocytes (using centrifugal cytapheresis in combination with appropriate extracorporeal phototherapy or chemotherapy), and/or (3) the activation of a disease – specific immunologic response (using cytapheresis and ex vivo cell culture with appropriate lymphokines and cell stimuli). New advances in our understanding of the immune system and in our ability to selectively manipulate and control the immunologic response should thus have a major impact on therapeutic apheresis and the future development of this important medical technology.

Defining Terms

Autoimmune diseases: A group of diseases in which pathological antibodies are produced that attack

The body’s own tissue. Examples include glomerulonephritis (characterized by inflammation of the capillary loops in the glomeruli of the kidney) and myasthenia gravis (characterized by an inflammation of the nerve/muscle junctions).

Cascade filtration: The combination of plasmapheresis with a second online membrane filtration of

The collected plasma to selectively remove specific toxic or immunogenic components from blood based primarily on their size.

Cytapheresis: A type of therapeutic apheresis involving the specific removal of red blood cells, white

Cells (also referred to as leukapheresis), or platelets (also referred to as plateletapheresis).

Donor apheresis: The collection of a specific component of blood (either plasma or one of the cellular

Fractions), with the return of the remaining blood components to the donor. Donor apheresis is used to significantly increase the amount of plasma (or a particular cell type) that can be donated for subsequent use in blood banking and/or plasma fractionation.

Immune complexes: Antigen-antibody complexes that can be deposited in tissue. In rheumatoid arthri­

Tis this deposition occurs primarily in the joints, leading to severe inflammation and tissue damage. Photopheresis: The extracorporeal treatment of diseases characterized by aberrant T-cell populations

Using visible or ultraviolet light therapy, possibly in combination with specific photoactive chemicals. Plasma exchange: The therapeutic process in which a large volume of plasma (typically 3 L) is removed

And replaced by an equivalent volume of a replacement fluid (typically fresh frozen plasma, a plasma substitute, or an albumin-containing saline solution).

Plasma perfusion: The therapeutic process in which a patient’s plasma is first isolated from the cellular

Elements in the blood and then subsequently treated to remove specific plasma components. This secondary treatment usually involves a sorbent column designed to selectively remove a specific plasma component or a membrane filtration designed to remove a broad class of plasma proteins. Plasmapheresis: The process in which plasma is separated from the cellular components of blood using

Either centrifugal or membrane-based devices. Plasmapheresis can be employed in donor applica­tions for the collection of source plasma for subsequent processing into serum fractions or in therapeutic applications for the treatment of a variety of disorders involving the presence of abnormal circulating components in the plasma.

Therapeutic apheresis: A process involving the separation and removal of a specific component of the

Blood (either plasma, a plasma component, or one of the cellular fractions) for the treatment of a metabolic disorder or disease state.

References

Edelson RL. 1989. Photopheresis: A new therapeutic concept. Yale J Biol Med 62:565.

Kambic HE, Nose Y. 1993. Plasmapheresis: Historical perspective, therapeutic applications, and new frontiers. Artif Organs 17(10):850.

Malchesky PS, Asanuma Y, Zawicki I, et al. 1980. On-line separation of macromolecules by membrane filtration with cryogelation. Artif Organs 400:205.

Nose Y, Kambic HE, Matsubara S. 1983. Introduction to therapeutic apheresis. In Y Nose, PS Malchesky, JW Smith, et al. (eds), Plasmapheresis: Therapeutic Applications and New Techniques, pp 1-22, New York, Raven Press.

Rock G. 1983. Centrifugal apheresis techniques. In Y Nose, PS Malchesky, JW Smith, et al. (eds), Plas­mapheresis: Therapeutic Applications and New Techniques, pp 75-80, New York, Raven Press. Sawada K, Malchesky P, Nose Y. 1990. Available removal systems: State of the art. IN UE Nydegger (ed), Therapeutic Hemapheresis in the 1990s, pp 51-113, New York, Karger.

Solomon BA, Castino F, Lysaght MJ, et al. 1978. Continuous flow membrane filtration of plasma from whole blood. Trans AM Soc Artif Intern Organs 24:21.

Zeman LJ, Zydney AL. 1986. Microfiltration and Ultrafiltration: Principles and Applications, pp. 471-489, New York, Marcel Dekker.

Zydney AL, Colton CK. 1982. Continuous flow membrane plasmaphersis: Theoretical models for flux and hemolysis prediction. Trans Am Soc Artif Intern Organs 28:408.

Zydney AL, Colton CK. 1986. A concentration polarization model for filtrate flux in cross-flow micro­filtration of particulate suspensions. Chem Eng Commun 47:1.

Zydney AL, Colton CK. 1987. Fundamental studies and design analyses of cross-flow membrane plas­mapheresis. In JD Andrade, JJ Brophy, DE Detmer (eds), Artificial Organs, pp 343-358, VCH Publishers.

Further Information

Several of the books listed above provide very effective overviews of both the technical and clinical aspects of therapeutic apheresis. In addition, the Office of Technology Assessment has published Health Tech­nology Case Study 23: The Safety Efficacy, and Cost Effectiveness of Therapeutic Apheresis, which has an excellent discussion of the early clinical development of apheresis. Several journals also provide more detailed discussions of current work in apheresis, including Artificial Organs and the Journal of Clinical Apheresis. The abstracts and proceedings from the meetings of the International Congress of the World Apheresis Association and the Japanese Society for Apheresis also provide useful sources for current research on both the technology and clinical applications of therapeutic apheresis.

Galletti, P. M., Jauregui, H. O. “Liver Support Systems.” The Biomedical Engineering Handbook: Second Edition. Ed. Joseph D. Bronzino Boca Raton: CRC Press LLC, 2000

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