Biocompatibility of extracorporeal immunoadsorption systems

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Tmnsfus. Sci. 1990; 11:223-239

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Biocompatibility of Extracorporeal Immunoadsorption Systems Janos G. Kadar, MD Helmut Borberg, MD

n Following the first publication of a technique aiming for clinical application in 1969, extracorporeal immunoadsorption is increasingly finding a place among haemapheresis techniques. The apparent advantages outweigh the technical difficulties and economical drawbacks still connected with this treatment modality. At present three different developments are under clinical investigation: Stuphylococcus Protein-A based immunoadsorption (SPA) the antibody-mediated adsorption (therapeutic aff3nity chromotography) and hydrophobic interactionbased immunoglobulin adsorption. The exact binding capacity and mechanism has not been completely elucidated for SpA and amino acid-based techniques. The lack of knowledge about pathogenic substrates exclusively responsible for the pathogenicity of many diseases, appears to be the major drawback for the prospective development of affinity chromatography-based extracorporeal systems. The practical application of molecular-biologic diagnostic procedures with a high grade of detection specificity (monoclonal antibodies) appears to be promising for the development of extracorporeal immunoadsorption. The immunomodulatory effect that results from the interaction of blood and plasma with artificial surfaces is an interesting subject of investigation, derived mainly from biocompatibility

From the Haemapheresis Kdn, F.R.G.

Unit,

University

studies. The careful clinical and laboratory investigation of the biocompatibility of extracorporeal immunoadsorption systems contribute considerably to the prevention of undesired side effects. n

INTRODUCTION Plasma exchange is characterized from several disadvantages. First, protein solutions are needed for the substitution. Second, the efficacy of the treatment is limited due to the increasing dilutions of the pathogenic substrate removed. Third, potential risks of transmittable viral diseases and/or cross reactive antibodies with allergic reactions exist. Treatment modalities that overcome these limits and hazards are of considerable interest. Extracorporeal immunoadsorption includes all therapeutic measures where: -either the specificity of the circulating pathogenic substrate-ligand interaction is based on immunologic regularities, or -the protein(s) to be removed from the circulation are immunologically active. Table 1 summarizes the clinically used immunoadsorption systems covered by this definition. From the classical point of view, some of them are selective and few specific. The term biocompatibility requires several features of the tissue/blood contacting material: the lack of a mechani-

of K6h-1, 223

Polyvinylalcohol

Sepharose-6MB Sepharose-CL4B

Colloidon charcoal Polyacrylamide beads Colloidon charcoal Silica

Microporous filter Silica Sepharose CL4B

Matrix

Bone marrow transplant Kidney transplant Haemophilia-B with inhibitor Familial hypercholesterolaemia

Rheumatoid arthritis Myasthenia gravis Chronic Guillain-Barre Syndrome SLE Multiple sclerosis

anti-A/B Abs anti-FIX Abs LDL-Cholesterol R. Factor anti-AchR Ab anti-Myelin Ab anti DNA Abs IgG, Complement

IIa

SLE

over

2 25 1 1 4

24 1 1 about 1OOOCl

4

25

HUS (mitomycin-C induced)

1

41 34 59 2 10 1 10 17

Colon carcinoma Miscellaneous malignant tumours Miscellaneous malignant tumours Haemophilia B Kidney graft rec. SLE ITP Breast adenocc.

Disorder(s) Treated

ssDNA,CIC

IgcI, 2,4 IgGl, 2.4 F-IX inhibitor anti-HLA-Abs ss DNA Ab CIC IgG

I~G, 2,4

Material Bound

Number of Reported Th Sessions

et a1.16

Shibuja et aZ.19

Tsuruta

Tsuruta et a1.16 Heininger et al.” Heininger et al.‘s

Bensinger et al.” Aeschbacher et al.l3 Nilsson et a1.14 Stoffel et a1.15

Terman et al. l1

Korec et al.”

Besa et al.’ Basal et al2 Bensinger et aI3 McIntosh et al4 Nilsson et a1.5 Palmer et aL6 Palmer et al.’ Guthrie et al.’ Temxm et a1.9

Reference First Clinical Report

Abbreviations:

SpA = Staphylococcus aureus Protein-A; AIHA = autoimmune haemolytic anaemia; ssDN Ab = antibodies against double stranded DNA; CIC = circulating immunecomplexes; HUS = haemolytic uremic syndrome; SLE = systemic lupus erythematosus; Anti AchR Ab antibodies against acetylcholine receptor.

Phenylalanine Polyvinylalcohol

Antigen(s) F-IX Anti-ApoB Ab Tryptophan

ABO group

DNA

SPA

SPA

SpA SpA

SPA

Ligand

Table 1. Immunoadsorbents for Clinical Use

Extracorporeal Immunwdsorption

cal cell damage, of plasma protein destruction, of thrombogenicity or toxicity, the prevention of initiation of immunological reactions, and the absence of an induction of inflammatory or allergic reactions.

STAPHYLOCOCCUS AUREUS PROTEIN-A-BASED IMMUNOADSORBENTS Clinical Observations of Side Effects using Staphylococcus Protein-A Containing Extracorporeal Immunoadsorbents GrtiO was the first to demonstrate the usefulness of immunoadsorption for the removal of circulating antibodies in a rabbit model in 1969. SpA could bind to human IgG by a reaction described as “pseudo-immune” because of an affinity similar to an immune reaction but without the specificity of the latter.21 This observation together with the “blocking factory theory” suggested that SpA may be of interest as a ligand for plasma adsorption in cancer patients.2 Purified Protein-A of Staphylococcus aureus (SPA) bound to a colloidon charcoal matrix has been extensively investigated for its antitumor effect.9,11 In four patients, clinical side effects were followed up by different repeated perfusion protocols and laboratory tests were performed to evaluate the pathophysiology of the observed complications.22 The main side effects were hypotension, noncardiogenit pulmonary oedema, bronchospasm, leukocytosis, hypocomplementaemia and fever. With modified perfusion conditions (less SpA used, diminished plasma perfusion rates, decreased amount of processed plasma) the rate and extent of symptoms could be reduced. The therapeutic effect (necrosis of the solid tumor) attributed to the SpA“blocking factor” binding could not be reproduced by the same investigators.23 Furthermore, Gordon et a1.24 reported that other staphyloccal strains lacking Protein-A effectively altered plasma to

Systems

225

inhibit tumor growth. Terman concluded that at least two other factors would be responsible for the clinical effect: leakage of Protein-A-IgG conjugates and enterotoxin-induced cytokine production and consecutive cytotoxic Tcell activation.25 Jonsson et a1.26reported about the clinical observations of 2 10 therapeutic sessions of 11 patients with cancer using SPA-SepharosedB columns for whole blood purification. In a study of Wallmark et ~1.~’who treated 6 patients with the same ligand-containing Sepharose4B columns anticoagulated with heparin side effects like fever, chills and respiratory distress occurred. C3 consumption in the effluent plasma of the primary membrane separator was also an important finding. Using different anticoagulation regimens, the behaviour of C3 conversion products could be effectively followed up with crossed immunoelectrophoresis. Korec et aLlo evaluated a polyacrylamide-based SpA column for cancer-associated haemolytic uremic syndrome. An average of 400 mL of plasma was perfused and returned to the patient; the number of therapeutic sessions per patient was between 2 and 15. No clinical side effects were attributable to the procedure. As a follow-up parameter, C3 and C4 (measured nephelometrically) increased at the end of the treatment course. Among the commercially available Protein-A columns one applies silica gel as a carrier material (Imre Corp, Seattle, USA). In a comparative analysis,28 it was concluded, that this Protein-A preparation is least contaminated with other staphylococcal components. According to the manufacturer, the columns can be used for on-line as well as for off-line perfusion. Clinical side effects evaluated during a multicenter study occurred in 43% of the 997 treatment sessions in tumor patients.28 The frequency of the “typical” Protein-A toxicity symptoms (chills, fever, pain) was relatively high, but in only three of the 113 patients the treatment was discon-

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tinued because of toxicity. The incidence of side effects was significantly higher if on-line perfusion was applied. Side effects attributable to complement activation such as respiratory symptoms and hypotension occurred in 28/l 13 and 20/ 113 patients respectively. A similar rate of side effects was observed in a study conducted in 42 patients for ITP of different origins .2* Certain laboratory parameters changed significantly during the procedure: monocyte count, haemoglobin, haematocrit, platelet-count, potassium, calcium, phosphate, albumin and total protein. The most recent publications using the Prosorba columns*~ 29130 report about extracorporeal treatment of thrombocytopenia of different origin. Guthrie* reported on the partial or complete response of 5/10 thrombocytopenic patients with manageable toxicities (urticaria, headaches, nausea, vomiting and hypotension). The other Protein-A preparation available for clinical use is a Sepharose4B-based immunoadsorbent based on the developmental work of Nilsson et ~l.~,14, 31 In the present form (with membrane separator for plasma collection, bolus heparin and citrate ,anticoagulation (ACD l:lO), two 62.5 mL Sepharose4B columns) it has been applied for antibody-mediated autoimmune diseases and alloimmunization.6~7 No side effects have been reported. The plasma flow rate is low (lo-35 mL/min). The small volume of the immunoadsorbent requires frequent working cycles within one treatment. (7-9.5 L plasma processed) with subsequently more rinsingsteps and unspecific protein loss. Biocompatibility of Protein-A hnmunoadsorbents The general characteristics of biocompatibility of the Protein-A containing immunoadsorbents (commercial and/or not commercial) can be summarized as follows: (1) The clinical side effects appear to correlate with the purification of the Protein-A preparations used, especially if

the SpA is not covalently bound to the matrix. (2) Several important molecules have been identified which are either contaminating commercial Protein-A preparations or found in post-perfusion p1asma,3234 suggesting that Protein-A was not an absolute requirement for the antitumor effect. (3) Hypocomplementemia was due to complement activation by at least two different mechanisms: first, solid phase generation of alternative pathway convertase (colloidon, Sepharose, polyacrylamide, etc.) second: leakage of the column and in vivo immunocomplexmediated activation of the classical pathway of complement. (4) For antibody-mediated diseases the high binding capacity of the ProteinA for IgGl, 2 and 4 is essential and bioincompatibility has to be minimized. Terman elaborated out recommendations for the investigation of each batch of Protein-A preparation prior to the use with Protein-A colloidon charcoal (PACC) method for further clinical research.25 It is not clear to what extent these safety tests are necessary now before commercially-available Protein-A columns. In the light of the clinical side effects discussed above, the wide variability of the clinical responses, and the lack of clear recommendations for online or off-line perfusion, the present level of safety seems to be not fully acceptable. The potentially advantageous role of a certain bioincompatibility in the effective treatment of malignancies through Protein-A immunoadsorption has to be mentioned here.‘0,32,34”” Nakanishi demonstrated that the type of anticoagulation which permits the ex vivo alternative pathway activation by different Sepharose preparations plays a role in the consumption of complement factor 3 and may be essential for the perfused effect of the antitumor plasma.36 In ITP29 the amount of IgG and/ or circulating immunocomplexes (CIC) removed during one perfusion is not likely to be responsible for the clinical efficacy, The role of the perfusion-

Extracorporeal

induced complement activation in the complement-mediated immunecomplex solubilization has been suggested as a possible mechanism for the observed elevation of platelet counts.

AFFINITY CHROMATOGRAPHYBASED IMMUNOADSORBENTS Clinical and Laboratory Investigation of Therapeutic Af%nity Chromatography Systems Bensinger et ~1.~reported about the clinical efficacy of a whole blood immunoadsorption system using colloidon as a carrier material for the columns. The ligand was a synthetic oligosaccharid with the antigenic properties of either A or B blood group substance. During the clinical evaluation, a combined anticoagulation [heparin, approx. 2U/mL blood and citrate (ACD-A 16-20:1)] was used at a 30-90 mL/min whole blood flow rate. The sole significant clinical problem during the initial study was leakage of the system. Foreign-body granulomas could be histologically demonstrated in the lung of one patient treated with regenerated columns among 40 patients treated (some of them with reused columns]. It has been claimed, that during the regeneration process small silica fragments are generated that may not be completely washed off the column before reuse. Complement haemolytic function declined during the session down to 55% in some cases (median 15%) Subsequently, Osterwalder et a1.37used a modified setting with precedent separation of the plasma by a centrifugal blood cell separator. No clinical side effects were described. In 198 1 Nilsson et al. 5reported of the treatment of a haemophiliac with acquired inhibitor against factor IX who required urgent surgical intervention. SPA-Sepharose 4BCL was used for differential plasma separation, No significant side effects occurred. The same author reported about the clinical applications

Immunoadaorption

Systems

227

of a modified technique,14 using factor IX-Sepharose 6MB immunoadsorbent columns for whole blood immunoadsorption. Using sodium citrate as an anticoagulant, whole blood was pumped through the column at a rate of 20 mL/ min. After the therapeutic session, the C3 and C4 levels were unchanged, and no decrease in coagulation factors was observed. Stoffel et ~21.‘~introduced a continuous flow extracorporeal system for the removal of low density lipoproteins. Since 1981, more than 80 patients have undergone a total of more than 10,000 therapeutic procedures in Germany, the U.S.A. and in the U.S.S.R. The system consists of two 300 mL Sepharose-4BCL columns38 carrying a sheep anti-human APO-B antibody. The same or similar type of column was recently also produced by Baxter, Germany and Immuno, Austria. For primary separation, continuous flow blood cell separators are exclusively used. The clinical effectiveness of the method has been reported elsewhere.39 The columns remain with the patient for about 1 yr, undergoing 100-300 adsorption-desorption cycles on a weekly treatment basis (l-3 adsorptions/week/therapeutic session). The attractiveness of the method is the extent of the decrease of the LDLcholesterol limited only if at all from the length of the treatment session. For anticoagulation heparin (40 IU/ min) and citrate solution (ACD-Formula B 1:20 to 1: 16) are used. The frequency of side effects has been evaluated [Table 2). Antibody against sheep IgG was found from the New York group using columns of different origin from the Cologne group. According to the ELISA developed in New York, IgG anti-sheep antibodies could be related to shaking chills and a positive skin-prick test,@ whereas according to the Cologne experience such a relation did not exist. The presence of prepheresis-IgG with the above mentioned cross-specificity could be detected in one case.

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Table 2. Frequency of Side Effect of LDL-Apheresis (Analysis of Clinical Data, Cologne 1981-19891 Therapies with Side Reactions/ Treatments Analyzed

Frequency of Side Reaction

1980 to 1983 (March] March 1983 to October 1983 1985-1987 1987-1989

611290 251175 163/1455 1301892

20.1% 14.0% 11.2% 14.6%

Overall side effect rate

37912812

13.2%

Analyzed Periods

Biocompatibility of Affinity Chromatography-based Immunoadsorbents In Cologne the discrepancy between the complement activating matrix and the low rate of side effects (Table 3) was recognized and a systematic study was started to clarify the causes of the good clinical tolerance.26t28 Characteristic leukocyte count changes could be demonstrated from samples of the inlet line during the LDL-apheresis session. The site of PMN-derived elastase-a-l inhibitor complex generation within the extracorporeal system was also detected.41 Subsequently, the site of the biologically-active anaphylatoxin generation could be demonstrated. Figure 1 shows anaphylatoxin levels derived from pre- and post-column plasma samples of a routine therapeutic session. Within the Table 3.

Analysis

of Clinical Complications

Total Number of Treatments

first 30 min of the column loading, a significant post column-anaphylatoxin elevation can be demonstrated using commercially-available radioimmunoassays (Ua(desarg), C4a(desarg), C5 (desarg)).9 The pre-column anaphylatoxin levels remain within the normal range, which proves the biological inertness of the primary separation (centrifugal blood cell separator). Due to the cell free ex viva conditions, to our knowledge these are the first data with an absolute amount of on-line generated C5a (desarg). The causal relationship between the anaphylatoxin generation and the drop of PMN/monocyte counts is further strengthened by the data in Table 4. During the so called “bioconditioning”therapeutic sessions, the immunoadsorbent columns are loaded with the patient’s plasma which is subsequently discarded instead of being returned to the of 2812 LDL-Apheresis Therapies 2812

Analyzed

Side Reactions 1. Due to plasma separation

Moderate No. %

Severe No.

%

Hypocalcaemia Volume challenge Shaking chills Technical problems 2.Due to plasma immunoadsorption Dyspnea 3. Nonclassifiable

40 187 28 79

1.4 6.7

4 1

0.14 0.03

;::

s

0,14

7 28

0.28 1.0

-

-

Total Number of Complications

369

Moderate complication: Means immediate control following medication. Severe complication: Required termination of the treatment.

10

ExtracorporealImmunoadso@on Systems 229

70Q0r(1) +\+ 6000

-

5000

-

0

10 15 20

25 30 35 40

0

r

I

I

10 15

I

I

20 25

1 30

1 I 35 40

Time ( min) Figure 1. Anaphylatoxiu generation of an LDL-immunoadsorbent column. Legends: n I + post-column plasma. (1) C3a (desarg); (2) C4a (desarg); (3) C5a pre-column plasma. + jdesarg). patient.

Human albumin (5%) is substituted isovolumetrically and retransfused with the blood cells. The data obtained from these sessions served as controls. The granulocyte and monocyte cell count changes expressed as percentage of the pretreatment level differ significantly (CSa-receptor positive cells), whereas the lymphocyte cell counts do not change in any case. After an initial decrease only the granulocyte counts are found to demonstrate an overshoot, and during the loading of the next column within the same therapeutic session the fall of PMN/monocyte count can be repetitively observed, suggesting a different regulatory mechanism, when comwith haemodialysis pared the leukopenia.8142 The large restorative capacity of the regulation of individual complement protein level and of the total haemolytic function of the complement is demonstrated in Table 5. During routine therapies no significant changes could be observed except for a decrease of Cls and Factor P levels. Chenoweth@ described an experimental setting for in vitro evaluation of the complement activating potential of

different biomaterials. We reconstructed his experimental conditions in order to find out whether the antibody coupled to the Sepharose-4B with and without Fc part has an influence on the complement activation pattern. We incubated an equal amount of Sepharose 4BCL with and without IgC antibody as well as F(abh with normal human serum. In vitro, during 30 min of incubation at 37°C there is no difference between the conversion-inducing capacity of the different columns. (Fig 2a-d). Here, the Sepharose 4B can be characterized as a strong alternative pathway activator with a special temporal profile of conversion similar to the particulate activator insulin. Using post-column plasma levels derived from anaphylatoxin routine treatments, we calculated again the anaphylatoxin/native protein conversions: a different distribution became apparent: The C4 conversion became more pronounced and the C5 conversion remained very low (data not shown). This different behaviour could be at least partially attributed to the different conditions of the in vivo loading process of the columns: room temperature, 8 min

P S

X

S

a **

18 10

l

78 19

PMN-G

**

27 6

l

73 17

Monoeytes

17 9

29 15

Lymphocytes

Maximal Decrease of Cell Count During Therapy (% Fall as Compared to Pretreatment Level)

4

15

n

4821 974

5325 2177

C3a desarg

3297 575

1167 846

C4a desarg

66 9

92 34

C5a desarg

2

11

n

Maximal Auaphylatoxin Level (From Post-column Cell-free Plasma,+ ng/mL)

LDL-poor Plasma on the Inlet Line Leukocyte Counts

l + P <0.015. Legend: l ** P co.001; t Samples were drawn before reconstitution of plasma with the patient’s own blood cells. $ Columns were loaded with cell-poor plasma which thereafter is discarded. The blood cells were reinfused with an equal volume of 5% human albumin solution.

Control* (off-line loaded)

lst-10th session (new1

Column Used by No. of Sessions

Table 4. Influence of Reinfusion of Anaphylatoxin-containing during Specific Extracorporeal Therapy

Extracorporeal Immunoadsorption Systems

231

Table 5. Effect of Extracorporeal Immunoadsorption (LDL-apheresis) on In Vivo Complement Factor Levels Protein

Before X

Total protein CIq Clr

17

6.2

Cls c3 c4 c5 C6 c7 C8 c9 Factor B Factor P Cl-Esterase-inhibitor C4-Binding-protein Factor H Factor I C3dg CHlOO CH50

20 20 13 18 20 20 17 20 20 13 19 15 15 19 1.3 13 52

15

After X

S

S

0.5

5.2

0.6

1.4 1.8 3.4 2.2 3.5 4.9 1.6 1.7 1.8 1.8 3.1 1.9 2.2 0.7 2.5 1.7 0.5 1.7 11.6

14 14 l16 16 13 21 18 19 16 20 20 l*8 19 11 14 19 1.4 14 49

2.2 1.3 3.4 2.9 3.3 2.9 1.7 2.7 2.5 1.3 2.9 2.5 3.4 2.2 2.8 1.9 0.6 3.5 10.9

Complement levels were measured in blood drawn prior to and after routinely performed therapeutic apheresis sessions with several loading cycles. Absolute complement values (% of normal) were divided by total plasma protein concentration. Relative values are given in % of total protein and compared by Student’s paired t-test. N=5. 70 -

60

-

50

-

(a)

(b)

o\" .c Q) '; h f

40-

z

.r

I! 8

30-

..

b E 'G is! 20

I ,/

-

:, u 10

-

.N.

/

4 /

/&

,,+Lccf-;

0

i_rr 5

10 15 20

pj(tJ .

25 30

.

0

5

IO

15 20

/+-+-+ I,+

‘-+

25 30

Time

*/hII 0 5 10 15 20

25

30

( min 1

Figure 2. Time-dependent anaphylatoxin generation in human serum induced by Sepharose-4B at

37°C (expressed as % of the native protein). Legends: (a) F (ab’),-Sepharose-4B-CL; (b) anti-ApoB IgG Sepharose-4B-CL; (c) CNBR-activated chemically-blocked Sepharose-4B-CL; (d) spontaneous activation of the serum. C---m C3a (desarg); + -+ C4a (desarg); A -A C5a (desarg).

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maximal incubation time for the plasma within the column, and finally a combined anticoagulation. The role of the combined anticoagulation has also extensively been studied using the same in vitro setting. 41The optimal inhibition of anaphylatoxin generation was seen when ACD-Formula A at a ratio of whole blood to anticoagulant 9:1, and heparin 10 I/ml was used. The anaphylatoxin generation was comparably low of the EDTA-plasma. Summarizing the obtained and only partially outlined data, we observed a consistent PMN and monocyte count drop with significant elevation of resulting from the anaphylatoxins immunoadsorbent-induced alternative and classical pathway complementactivation using new immunoadsorbent columns. No functional impairment of the complement system could be demonstrated at the end of the clinically uneventful LDL-apheresis sessions. The extent of C-activation remains beyond clinical relevance. The treatments are because of well tolerated chosen and well defined treatment conditions. The longest observation period with this treatment modality is 9 yr without apparent side effects.

HYDROPHOBIC INTERACTIONBASED IMMUNOSORBENTS Clinical and Laboratory Observations a new Yamazaki et al.@ introduced polyvinyl-alcohol beaded sorbent with covalently linked tryptophan or phenylalanine amino acids as ligands. These ligands bind with IgG and/or IgGimmunecontaining circulating complexes-especially antiacetylcholine receptor antibodies pathogenetically involved in myasthenia gravis. The nature of the binding is of noncovalent hydrophobic interaction. However, the exact mechanism explaining the high affinity between tryptophan residues and AchR antibodies is not yet fully clarified.” During in vivo treatment sessions, using combined anticoagulation,

centrifugal separation of plasma and reuse after regeneration of the immunoadsorbent, Heininger found no C3a (desarg) and/or C5a (desarg) in the post-column plasma, but a consequent post-treatment leukocytosis.” Nonspecific adsorption of fibrinogen (up to 40% of the initial level) limited the intensity of the perfusion. Until 1988, no serious clinical side effects have been reported. Motomura et a1.36were the first to claim the occurrence of shock in patients who underwent membrane plasma separation and IM-T adsorption using heparin anticoagulation. In further clinical experiments, the authors substituted heparin with nafamostat mesilate45 and no further shock was seen with concomittant disappearance of C5a (desarg) elevation (suggested inhibition of alternative pathway activation).

Biocompatibility of the Hydrophobic Interaction-based ixumunosorbents The chemical composition of the matrix (free OH groups) is anticipated to represent strong contact activation potency.46 Shin investigated in 1980 the first PVAbased membrane dialysators and found a marked leukopenic effect without apparent complement activation.47 The presence of free amino groups on a polymer surface potentiates complement activation.48 Based mainly on in vitro batch experiments, Behm et a1.49 concluded that the observed decrease of the haemolytic activity of complement (CH-50) after incubation with the column material was caused by the selective absorption of Clq rather than by consumption due to C-activation. In these preliminary experiments the plasma used contained very high amounts of C3a (desarg), therefore the net effect of the IM-T or IM-P column on the further activation and/or adsosrption can hardly be reproduced. The same group demonstrated the in vitro C3a (desarg) binding potency of the IM-T and IM-P column.50 The type of the primary separation device and the anticoagulation can markedly influence the

Extracorporeal Immunoadsorption Systems split product content of the retransfused plasma.45 PRIMARY SEPARATION SYSTEMS Biocompatibility is influenced not only from the secondary separation systems. The effect of primary separation on the interaction between the blood/plasma and the artificial surface is also important. Centrifugal blood cell separation are usually considered to be rather biocompatible, as for example assessed by C3d generation potency51 but do very rarely lead to respiratory distress symptoms. Using granulocyte aggregometry as a functional assay for the anaphylatoxin generation the responsible part of the extracorporeal system seems to be the upper seal of the centrifugal bowl. Pfaeffl et ~1.~~investigated the anaphylatoxin generating potency of several centrifugal and membrane apheresis devices, and demonstrated an unexpected slight increase of plasma C4a (desarg) level. It could be prevented by an initial loading dose of heparin. Our own experience, using leukocyte cell count changes as an indicator for the complement activation compares well with these observations. 53 Different membrane plasma separators all activate complement in a wide variety, however the total amount of split products generated is very much dependent on different treatment conditions.‘0,54 During plasma exchange the bulk of the released anaphylatoxin is found in the discarded plasma but might be of importance if a secondary purification system like immunoadsorption is concomitantly used.

OTHER FACTORS INFLUENCING BIOCOMPATIBILITY AND CLINICAL TOLERANCE OF THE IMMUNOADSORPTION TREATMENT We try to summarize the separation parameters, technical data and disease and/or patient-related individual factors 1s11:2-w

233

which all influence the probability of side effects and biocompatibility of the known extracorporeal immunoadsorption systems (Table 6). Some of the listed items are analyzed furthermore. Anticoagulation The administration of heparin with well characterized preventive effects of alternative pathway activation54 together with citrate is of benefit for the amount of C3a (desarg) generated as suggested in previous reports on blood cell separators,55 membrane plasma flltration53,56 and during in vitro studies of SPA-containing immunoadsorbents.34 If the primary separation system consists of contact activating surface and the anticoagulation permits the generation of bioactive split products, the side effect rate can increase.36 With careful selection of the different parts of the system, the anticomplementary and anticoagulation activities of the low dose heparin/ citrate combination can be optimalized. Relationship Between Cellular Changes and Pulmonary Difficulties Based on considerable evidence Craddock reasoned that during haemodialysis PMN sequestration in the lung results from two different mechanisms: first, after direct solid phase activation of all C3b receptor bearing cells and second, complement-induced PMN aggregation and consequent leukoembolization (reviewed).57 Wegmuller et ~1.~~found no causal relationship between the frequent pulmonary problems during dialysis and complement-and granulocyte-mediated pulmonary dysfunctions. Hakim demonstrated individual differences in the frequency of hypersensitivity reaction of chronic dialysis patients with correlation of in vitro capacity of their serum to be activated by zymosan.59 The presence of anti-dextran antibodies as a result of cross-reactivity against bacterial antigens may be related to differences in the individual reactivity and can influence the extent of complement activation.60

234 Transfus.Sci. Vol. 11, No. 2

Table 6.

Factors Influencing Biocompatibility of Existing Extracorporeal Immunoadsorbents

1. Method of primary separation 1.1. None (whole-blood immunoadsorption) 1.2.1. Continuous-flow blood cell separator 1.2.2. Discontinuous-flow blood cell separator 1.3. Membrane, on-line 1.4. Off-line separation 2. Type of access 2.1 Arteriovenous 2.2. Venovenous 3. Type and amount of anticoagulation 3.1. Heparin only 3.2 Citrate only + bolus heparin 3.3 Combined (citrate and heparin) 4. Size of extracorporeal volume 5. Temperature of secondary separation 6. Flow rate of retransfusion 7. Matrix characteristics 7.1. Surface area 7.2. Complement (alternative pathway] activation potency influenced by: 7.2.1. Type of exposed free groups 7.2.2. Density of active groups 7.2.3. Efficiency C5a convertase formation relative to C3a convertase formation 7.2.4. Activated protein binding properties of the matrix 7.2.5. hnmunogenicity of the matrix

No data is available about any individual susceptibility differences however, on patients treated with either of extracorporeal immunoadsorption systems. Her-1 et aL6’ showed the lack of causal relationship between complement activating potency and PMN-derived enzyme release of certain membranes and concluded that the degranulation occurs on the surface of the membrane without the contribution of complement. No direct contact of the adsorbent surface and the cells occurs during extracorporeal immunoadsorption except for whole blood immunoadsorption, therefore the above listed causes of complement activation and cell-triggering mechanism has no clinical relevance. In the extracorporeal circulation of the LDL-apheresis,

8. Ligand characteristics 8.1 Type of the ligand (antibody, antigen, amino acid, bacterial product) 8.2. Strength of binding to the matrix 8.3. Immunogenicity of the ligand 8.4. Purity of the ligand 8.5. Complement activation potency of the ligand 9. Sterility of the system 9.1. Disposable parts of the primary and of the secondary system 9.2. Sterility of solutions used for priming, rinsing, elution, regeneration and storage 9.2.1. Endotoxin content (Limulus test) 9.3. Methods of sterilization used 9.3.1. Manufacturer’s 9.3.2. Maintaining sterility during and between therapeutic sessions 10. Patient-dependent factors of biocompatibility 10.1. Preformed alteration of cellular reactivity 10.2. Preformed activation enhancing antibodies 10.3. Deficiency of an enzyme participating in the active pathway 10.4. Regulator protein deficiency (Carboxipeptidase-B, Cl Inhibitor, etc.) 11, Non-specific loss of plasma proteins 12. Length of a treatment session and a treatment period

in the tube segment of reconstruction of cells and LDL-poor plasma the generated anaphylatoxins are very probably already desarginated with significant loss of biological activity and specific receptor deactivation potency (reviewed58,62). Other cellular elements can be triggered also during immunoadsorption: centrifugal separators cause elevation of @thromboglobulin 1eveF and platelets aggregate and release serotonin in the presence of C3a (desarg).45 The demonstrated granulocyto/and or monocytopenia in the SPA-based systems,35 the observed post-treatment leukocytosis’* and the temporal disappearance of the C5a (desarg) receptor-bearing cells from the circulation of the patients with familial hypercholesterinemia treated with

Extracotpored

the LDL-apheresis suggests cell activation of different degree.

235

Acknowledgements

PVC plasticizers are known to be removed from the tube during haemodialysis and are adsorbed with the lipid fraction of the plasma.64 For the immunoadsorbents the leaching can be of clinical importance as discussed above, 12,24,32,40 furthermore the elegant solution of Sato can be recommended65 using a secondary sorbent with antigen specificity.65

FUTURE CONSIDERATIONS The technical development and basic scientific activity will enable the realization of most of the wishes of extracorporeal immunoadsorption systems as listed in Table 7. The long-term effect of chronic complement activation on the immune system is not yet clarified. Recent data suggest however, that in certain conditions bioincompatibility can represent not only drawbacks but also benefits for the patient.38,45 The immunomodulatory effect of complement activation and/or ex vivo adsorption of pathologically elevated levels of complement split products with/or without specific elimination of a given pathogenic substrate is an exciting possibility

of Extracorporeal

System6

especially for the treatment of autoimmune diseases.&

Leaching

Table 7. Optimation

Immunoadsorption

This work was partially supported by the Federal Minist of Research and Technolo..gK Bonn, FRG. The valuab7e advice of PhD Peter 1. Spat , Bern, Switzerland is gratefully acknowledged.

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-electronically integrated primary and secondary separation modules -low and constant extracorporeal volume -low anticoagulation need -minimal or no loss of blood cells during the separation process -separated plasma without cell contamination -veno-venous access sufficient for primary separation -no contact activation within the primary system -y-sterilized disposables

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