Clin. exp. Immunol. (1991) 84, 535-538
ADONIS 0009910491001834
Activation of complement during apheresis G. HETLAND, T. E. MOLLNES* & P. GARRED* Blood Bank and Department of Immunology, Ullevaal Hospital, and *Institute of Immunology and Rheumatology, The National Hospital, Oslo, Norway (Acceptedfor publication 7 January 1991)
SUMMARY C3 activation products and the terminal complement complex (TCC) were examined in plasma during plasmapheresis of patients with Guillain-Barre Syndrome (GBS) (n=4), Waldenstrom's syndrome (n = 4), and hypercholesterolaemia (n = 1), or during cytapheresis of platelet (n = 10) and granulocyte (n =2) donors. Blood specimens were taken before, during and after the procedures. There was a significant activation of complement after apheresis in the GBS patients and one of the patients with Waldenstr6m's syndrome, but not in the other patients. There were no significant differences in complement activation products before compared with after cytapheresis in the healthy donors. This demonstrates the biocompatibility with respect to complement activation of the materials used. The observed complement activation in some of the patients during plasma exchange is probably caused by activation products in the replacement plasma. Keywords C3 activation products terminal complement complex apheresis
INTRODUCTION
Cytapheresis and plasmapheresis (plasma exchange) performed with cell separators are generally safe. Plasmapheresis is commonly used for symptomatic treatment of various diseases, including hypercholesterolaemia, Guillain-Barr& syndrome (GBS) (Raphael et al., 1987), and the hyperviscosity state in myeloma and Waldenstr6m's syndrome (Russel, Toy & Powles, 1977). However, there have been deaths attributed to plasma exchange (Heustis, 1985), and complement activation has probably contributed to the fatal outcome in some of the cases (Rubenstein et al., 1983; Anonymous, 1988). The alternative pathway of complement is activated during extracorporal circulation in cardiopulmonary bypass (Chenoweth et al., 1981; Videm et al., 1989) and haemodialysis (Kazatchkine & Carreno, 1988). The generated anaphylatoxins C3a and C5a participate in the pathogenesis of the adult respiratory distress syndrome (Ing et al., 1983) and have other inflammatory effects (Kirklin et al., 1983; Kazatchkine & Carreno, 1988). However, except for one report on C3 activation after plasma exchange in myasthenia gravis (Rosenkvist et al., 1984), little is known about complement activation during extracorporal circulation in apheresis. The objective of the present study was therefore to investigate whether both the early and late phase of complement are activated during such procedures. This was done by examining the plasma levels of C3 activation products and the terminal complement complex (TCC) before, Correspondence: Geir Hetland, IMM 18, Department of Immunology, Scrys Clinic & Research Foundation, 10666 N. Torrey Pines Road, La Jolla, CA 92037, USA.
under and after apheresis in patients undergoing plasma exchange and in healthy donors of platelets and granulocytes
(PMN). MATERIALS AND METHODS
Subjects The study included four patients with GBS, four with Waldenstr6m's syndrome and one with hypercholesterolaemia, who were referred to the Blood Bank, Ullevaal Hospital, Oslo, for plasmapheresis, and healthy donors of MHC-matched platelets (n = 10) or PMN (n = 2) to leukaemic patients or granulocytopenic children with refractory Gram-negative septicaemia, re-
spectively. Plasmapheresis and cytapheresis All plasmaphereses and cytaphereses were carried out on a Haemonetics model 30 cell separator (Haemonetics, Natick, MA) for 90-120 min according to standard protocol. Normally 2-6 1 of the patients' plasma were exchanged with 8 U of fresh frozen plasma heated to approximately 37°C, except for one patient with hypercholesterolaemia who received a 40 mg/ml albumin solution as replacement fluid. Anti-coagulant was usually 4-8 mg/ml citric acid solution, but PMN donors received 60 mg/ml hydroxyethylglycogen (Plasmasterilo, McGaw Lab., Evanston, IL). Prior to apheresis, the PMN donors were subjected to 5 min of physical exercise and given corticosteroids (Decadron') intravenously to mobilize marginating PMN. Blood specimens Venous blood samples were taken immediately before the apheresis, after the third and the last sixth or eighth operation
535
536
G. Hetland, T. E. Mollnes & P. Garred Table 1. Terminal complement complex (TCC) in plasma from patients before, during, and after plasma exchange (medians and ranges)
Concentration of TCC (AU/ml) Diagnosis
No. of exchanges
GBS (n=4) Waldenstr6m's syndrome (n= 1) Waldenstr6m's syndrome (n=3) Hypercholesterolaemia (n= 1)
7 3 3 2
Before
During
After
5-8 (2-8-7 9) 4 5 (2 3-6 8) 6-2 (2-8-10-8)* 5-9 (4-1-6-5) 56 (5-4-5-7) 7-5 (7-4-10-1) 7-1 (2-4-7-6) 6-3 (6-1-6-5) 6-0 (1-3-6-4) ND 4-4 (2-2-6 5) 4-5 (2-2-6 7)
*P = 0 05 compared with before plasma exchange; otherwise P > 0 05. AU, arbitrary units; GBS, Guillain-Barr6 Syndrome; ND, not determined.
with cell donors or patients, respectively, and stored at 40C in EDTA before separation and storage of the plasma at - 700C. With the GBS patients, specimens were also taken from the units of withdrawn plasma and treated similarly. Some of these patients and donors were subjected to apheresis more than once. Fresh frozen citrate plasma from two donors stored at -400C for I month and thawed, and plasma from three blood donors stored at 40C for 1, 10, 20, 30 and 35 days were also examined for complement activation. In additon, serum from two GBS patients was incubated with AB Rh-matched donor serum or 15 g/lgammaglobulin (Kabi Pharmacia, Stockholm, Sweden) at 370C and examined before, immediately after mixing and I h later. Agarose gel double-diffusion of serum from these patients gave no precipitation lines against dilutions of donor serum or gammaglobulin. Gammaglobulin served as positive controls since it is produced from large plasma pools and therefore contains many idiotype specificities.
Analyses of complement activation products Complement activation was assessed in two enzyme immunoassays (EIA) specific to neoepitopes on activation products not expressed in native complement factors. One was specific for C3 activation products, thus evaluating the initial part of the complement cascade (Garred, Mollnes & Lea, 1988). In brief, polystyrene plates were coated with a mouse monoclonal antibody (bH6) specific for a C3 neoepitope expressed on C3b, iC3b and C3c, but not on native C3. A rabbit anti-human C3c antiserum (Behring, Marburg, Germany) was used in the secondary antibody layer. Finally, a peroxidase-conjugated anti-rabbit immunoglobulin was added (Amersham International, Amersham, UK). The substrate was 2,2'-azino-di-(3ethylbenzthiazoline sulphonic acid) (ABTS). The results were read on a Dynatech MR 580 (Alexandria, VA) and referred to a standard of zymosan-activated serum defined to contain 1000 arbitrary units (AU)/ml. The results were analysed by an Apple II computer with Dynatech Immunosoft program. The other assay detected the fluid-phase TCC, SC5b-9 (Mollnes et al., 1985). TCC is an indirect indicator of C5a release since it can not be formed in vivo without activation of C5 and release of C5a. In brief, polystyrene plates were coated with a mouse monoclonal antibody specific for a neoepitope expressed on activated C9. A rabbit anti-human C5 antiserum (Dakopatts Immunoglobulins, Copenhagen, Denmark) was used in the subsequent step. The rest of the assay was performed as described for the C3 activation assay.
Statistical analysis The significance of the results was tested by Wilcoxon's rank sum test for paired observations, and P < 0 05 was considered to be statistically significant. Student's t-test was used for some calculations.
RESULTS Complement activation during plasma exchange Plasmapheresis was performed on patients with GBS or Waldenstr6m's disease, and plasma samples for complement activation analyses were taken immediately before, during or after the treatment. Plasma was also exchanged in one hypercholesterolaemia patient, but with albumin solution instead of donor plasma. We found significantly higher plasma levels of TCC (Table 1), but not C3 activation products (Table 2) in GBS patients after compared with before plasmapheresis. However, the median values for both C3 activation products and TCC increased significantly (P < 0 05) from 10-6 to 20-9 and from 5-7 to 6-6 AU/ml, respectively, from the first unit to the last three pooled units of the withdrawn plasma from these patients. In one patient with Waldenstrom's syndrome there was modest increase in TCC and a substantial increase in C3 activation products after plasma exchange (Tables 1 and 2). The small number of samples did not enable statistical evaluation in this case. Significant changes in the plasma levels of these products were not observed during plasma exchange in the other patients (Tables 1 and 2).
Complement activation during cytapheresis We found no statistical differences in the plasma levels of C3 activation products or TCC before and after apheresis in the healthy donors of platelets or PMN (Table 3).
Complement activation during in vitro incubation of plasma! serum The values for C3 activation products and TCC in fresh frozen citrate-plasma stored at -40°C for 1 month and then thawed were 16-7 and 17-3, and 5-1 and 6-0 AU/ml from two donors, respectively. In comparison, plasma stored at 4°C up to 35 days showed an exponential increase in C3 activation products reaching 22 AU/ml after 10-14 days, whereas the levels of TCC were unaltered and close to the upper normal value (data not shown).
Activation of complement during apheresis
537
Table 2. C3 activation products in plasma from patients, before, during, and after plasma exchange (medians and ranges)
Concentration of C3 activation products (AU/ml) No. of
exchanges
Diagnosis GBS (n=4)
7 3 3 2
Waldenstrdm's syndrome (n= 1) Waldenstr6m's syndrome (n = 3) Hypercholesterolaemia (n= 1)
Before
During
After
12-0 (8 4-36 4) 10-6 (7 8-25 8) 13-0 (7-2-51-8) 13-6 (8 3-18-7) 17 7 (11-7-23-7) 35-0 (31-0-69-9) 13-4 (12-4-19-7) 10-4 (9-4-11-4) 13 4 (7 4-16 9)
10-9 (7-8-13-9)
ND
3-9 (3-7-41)
GBS, Guillain-Barr6 syndrome; ND, not determined. Table 3. Complement activation in plasma during cytapheresis of platelet and granulocyte (PMN) donors (medians and ranges)
Concentrations (AU/ml)
DISCUSSION
Donors of
of
Before
During
After
Platelets PMN Platelets PMN
C3 act. C3 act. TCC TCC
7-6 (4-8-13-8) 9.1 (10-0-13-4)
6-0 (4-1-15-7) ND 6-1 (2-7-14-3) ND
5 9 (4-0-16-9) 7 5 (5 0-9 9)
5-6(2-5-4-8) 3-7 (2 5-4-8)
5-4(2-6-11-0) 27 (2-5-29)
Results of 12 or two cytaphereses of 10 donors of platelet and two donors of PMN, respectively (P > 0-05). C3 act., C3 activation products; ND, not determined.
Table 4. Complement activation products in serum from GBS patients and donors before, during, and after co-incubation in vitro
Concentrations (AU/ml) Source of sample
of
Before
During
After
Donors GBS patients Gammaglobulin
C3 act. 42 2+ 11 -7 C3 act. 156-5+58-8 C3 act.
93 9+ 19-5 137-3+46-5 91-2+25-4 115-0+49-1
Donors GBS patients
TCC TCC TCC
179+26 1 2[+2± 1
Gammaglobulin
18-0+5-1 24-6+6.4
subsequent testing had antibodies to leucocyte antigens or irregular blood type antibodies.
225+86
35-0+2-5
Results are mean + 1 s.d. of sera from two GBS patients and four donors (two for each patient). P > 0-05 for the GBS patients (Student's
Mtest). To test whether the activation products arise because of reacting antibodies serum from two GBS patients was incubated with donor serum or gammaglobulin. We found no increase in the products after compared with before mixing the sera (Table 4). The higher (not significant) initial C3 values of the patients' than donors' sera probably reflect storage at -20'C and additional freeze-thawing of the former sera. Neither the four GBS patients nor the three donors who were available for
We conclude that the initial and terminal complement pathway is activated during plasmapheresis of patients with GBS, but not in cytapheresis of healthy platelet or PMN donors. Since C3 activation is suggested to be a prerequisite for terminal pathway activation in general, the significant increase in the generated TCC indicates that also the initial pathway is activated in the GBS patients after plasma exchange. This is supported by the significant rise in C3 activation products in the withdrawn plasma from these subjects. One of the GBS patients also had respiratory problems after the treatment, possibly caused by complement activation products (Ing et al., 1983). The observed decrease in plasma levels of C3 split products in the hypercholesterolaemia patient who received albumin solution instead of cholesterol-containing donor plasma, is consistent with the dilution of other plasma proteins after albumin administration. The finding of no significant changes in C3 activation products or TCC during 14 cytaphereses of healthy donors clearly demonstrates the biocompatibility with respect to complement activation of the materials used in this procedure.
The observed complement activation after plasmapheresis in some patients is partly caused by complement activation products in the donor plasma, which is supported by the following: (i) lack of complement activation during cytapheresis; (ii) usually higher levels of such products in thawed fresh frozen plasma than the initial C3 values of the patients; (iii) increase in C3 products and TCC in the withdrawn plasma from the patients after administration of donor plasma; and (iv) the reported C3 activation in myasthenia gravis after exchange of the patients' plasma with fresh frozen plasma, but not albumin (Rosenkvist et al., 1984). Fresh frozen plasma is nevertheless preferable as replacement fluid in plasmapheresis to plasma stored at 4 C because of the far stronger C3 activation in the latter. The plasma was heated to 370C prior to the exchange which was previously shown to increase complement activation rapidly (Mollnes, Garred & Bergseth, 1988). Thus, when up to 31 of heated plasma are exchanged during 2 h, administration of plasma containing activated complement could very well explain our findings. The lack of anaphylatoxin-related symptoms in most patients after plasmapheresis despite the high content of C3 activation products in donor plasma could be due to rapid transformation
538
G. Hetland, T. E. Mollnes & P. Garred
of C3a to the inactive derivative C3a-desArg (Chenoweth, 1986; Kazatchkine & Carreno, 1988). Moreover, C5a is present only in trace amounts in donor plasma due to the relative inefficiency of the fluid-phase C5 convertase compared with the C3 convertase (Bhakdi et al., 1988). An unknown 'patient factor' in GBS may also be responsible for the observed discrepancy in complement activation after plasma exchange between these patients and the majority of those with Waldenstr6m's syndrome. A probable candidate could be GBS autoantibodies in complement activating immuncomplexes (Cook & Dowling, 1981) which activate both the supplied and own complement components during apheresis. However, our negative in vitro results after mixing sera from GBS patients and donors did not indicate that the activation products arise through idiotype-anti-idiotype reactivity. This possibility is also most unlikely due to the fact that plasma from eight different donors was used in each plasma exchange. Immunodiffusion of the patient's sera against donors' sera did not give precipitation lines, and other alloantibodies that might react with cellular antigens were not demonstrated. Activation of complement in only one out of four patients with Waldenstr6m's syndrome could be due to an individually different reaction to this treatment. Unexpectedly, cryoagglutinins were found in one of the latter patients and not in the Waldenstrdm's syndrome patient with rise in complement activation products. This may indicate that cryoagglutinins do not promote complement activation during plasmapheresis. Although we found that plasma exchange induced complement activation in patients with GBS and occasionally in Waldenstrdm's syndrome clinical manifestations probably caused by this activation were observed only in one of these subjects. The median levels of the activation products were below the upper reference values in all patients, except for the Waldenstrdm's syndrome patient. In this case, treatment with longer intervals should be considered. Otherwise, plasmapheresis of such and other patient groups should be continued when there is a documented clinical effect of the treatment. However, donor plasma should be replaced with virus-inactivated albumin which is now commonly used because of the risk of H IV and other serious infections and recommended for plasmapheresis in GBS (Raphael et al., 1987) and myasthenia gravis (Rosenkvist et al., 1984). Moreover, additional gammaglobulin substitution should be considered (Raphael et al., 1987).
ACKNOWLEDGMENTS We thank Ms Grethe Bergseth and Ms Bente Falang for excellent technical assistance. Financial support was provided by the Nordic Insulin Foundation and the Norwegian Research Council for Science and the Humanities.
REFERENCES ANONYMOUS. (1988) Complement activation in plasma exchange (Editorial). Lancet, ;;, 1464. BHAKDI, S., FASSBENDER, W., HUGO, F., CARRENO, M.P., BERSTECHER, C., MALASIT, P. & KAZATCHKINE, M.D. (1988) Relative insufficiency of terminal complement activation. J. Immunol. 141, 3117. CHENOWETH, D.E. (1986) Complement mediators of inflammation. In Inmmunobiologv of the Complement System (ed. by E.D. Ross) p 63. Academic Press, London. CHENOWETH, D.E., COOPER, S.W., HUGLI, T.E., STEWARD, R.W., BLACKSTONE, E.H. & KIRKLIN, J.W. (1981) Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylatoxins. N. Engl. Med. 304, 497. COOK, S.D. & DOWLING, P.C. (1981) The role of autoantibodies and immune complexes in the pathogenesis of Guillain-Barr& Syndrome. Ann. Neurol. 9, 70. GARRED, P., MOLLNES, T.E. & LEA, T. (1988) Quantification in enzymelinked immunosorbent assay of a C3 neoepitope expressed on activated human complement factor C3. Scand. J. Immunol. 27, 329. HEUSTIS, D.W. (1985) Complications of frequent donor cytapheresis and plasma exchange. Plasma Ther. Tranfus. Technol. 6, 541. ING, T.S., DAUGIRDAS, J.T., POPLI, S. & GANDHI, V.C. (1983) First-use syndrome with cuprammonium cellulose dialysers. Int. J. artif. Organs, 6, 235. KAZATCHKINE, M.D. & CARRENO, M.P. (1988) Activation of the complement system at the interface between blood and artificial surfaces. Biomaterials, 8, 30. KIRKLIN, J.K., WESTABY, S., BLACKSTONE, E.H., KIRKLIN, J.W., CHENOWETH, D.E. & PACIFICO, A.D. (1983) Complement and damaging effects of cardiopulmonary bypass. J. thorac. cardioclasc. Surg. 86, 845. MOLLNES, T.E., GARRED, P. & BERGSETH, G. (1988) Effect of time, temperature and anticoagulants on in vitro complement activation: consequences for collection and preservation of samples to be examined for complement activation. Clin. exp. Immunol. 73, 484. MOLLNES, T.E., LEA, T., FROLAND, S.S. & HARBOE, M. (1985) Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against neoantigen of the complex. Scand. J. Immunol. 22, 197. RAPHAEL, J.C., CHASTANG, C., JAIS, J.P., LARIBOISIfRE, V.E.R. & BRUNEL, D. IN THE FRENCH COOPERATIVE GROUP ON PLASMA EXCHANGE IN GUILLAIN-BARRt SYNDROME (1987) Efficiency of plasma exchange in Guillain-Barr& Syndrome: Role of replacement fluids. Ann. Neurol. 22, 753. ROSENKVIST, J., BERKOWICZ, A., HOLSOE, E., SORENSEN, H. & TAANING, E. (1984) Plasma exchange in myastenia gravis complicated with complement activation and urticarial reactions using fresh-frozen plasma as replacement solution. Vox sang. 46, 13. RUBENSTEIN, M.D., WALL, R.T., WOOD, G.S. & EDWARDS, M.A. (1983) Complications of therapeutic apheresis, including a fatal case with pulmonary vascular occlusion. Am. J. Med. 75, 171. RUSSEL, J.A., TOY, J.L. & POWLES, R.L. (1977) Plasma exchange in malignant paraproteinaemias. Exp. hematol. 5, 105. VIDEM, V., FOSSE, E., MOLLNES, T.E., ELLINGSEN, 0., PEDERSEN, T. & KARLSEN, H. (1989) Different oxygenators for cardiopulmonary bypass lead to varying degrees of human complement activation in vitro. J. thorac. cardiorasc. Sur. 97, 764.
ADONIS 0009910491001834
Activation of complement during apheresis G. HETLAND, T. E. MOLLNES* & P. GARRED* Blood Bank and Department of Immunology, Ullevaal Hospital, and *Institute of Immunology and Rheumatology, The National Hospital, Oslo, Norway (Acceptedfor publication 7 January 1991)
SUMMARY C3 activation products and the terminal complement complex (TCC) were examined in plasma during plasmapheresis of patients with Guillain-Barre Syndrome (GBS) (n=4), Waldenstrom's syndrome (n = 4), and hypercholesterolaemia (n = 1), or during cytapheresis of platelet (n = 10) and granulocyte (n =2) donors. Blood specimens were taken before, during and after the procedures. There was a significant activation of complement after apheresis in the GBS patients and one of the patients with Waldenstr6m's syndrome, but not in the other patients. There were no significant differences in complement activation products before compared with after cytapheresis in the healthy donors. This demonstrates the biocompatibility with respect to complement activation of the materials used. The observed complement activation in some of the patients during plasma exchange is probably caused by activation products in the replacement plasma. Keywords C3 activation products terminal complement complex apheresis
INTRODUCTION
Cytapheresis and plasmapheresis (plasma exchange) performed with cell separators are generally safe. Plasmapheresis is commonly used for symptomatic treatment of various diseases, including hypercholesterolaemia, Guillain-Barr& syndrome (GBS) (Raphael et al., 1987), and the hyperviscosity state in myeloma and Waldenstr6m's syndrome (Russel, Toy & Powles, 1977). However, there have been deaths attributed to plasma exchange (Heustis, 1985), and complement activation has probably contributed to the fatal outcome in some of the cases (Rubenstein et al., 1983; Anonymous, 1988). The alternative pathway of complement is activated during extracorporal circulation in cardiopulmonary bypass (Chenoweth et al., 1981; Videm et al., 1989) and haemodialysis (Kazatchkine & Carreno, 1988). The generated anaphylatoxins C3a and C5a participate in the pathogenesis of the adult respiratory distress syndrome (Ing et al., 1983) and have other inflammatory effects (Kirklin et al., 1983; Kazatchkine & Carreno, 1988). However, except for one report on C3 activation after plasma exchange in myasthenia gravis (Rosenkvist et al., 1984), little is known about complement activation during extracorporal circulation in apheresis. The objective of the present study was therefore to investigate whether both the early and late phase of complement are activated during such procedures. This was done by examining the plasma levels of C3 activation products and the terminal complement complex (TCC) before, Correspondence: Geir Hetland, IMM 18, Department of Immunology, Scrys Clinic & Research Foundation, 10666 N. Torrey Pines Road, La Jolla, CA 92037, USA.
under and after apheresis in patients undergoing plasma exchange and in healthy donors of platelets and granulocytes
(PMN). MATERIALS AND METHODS
Subjects The study included four patients with GBS, four with Waldenstr6m's syndrome and one with hypercholesterolaemia, who were referred to the Blood Bank, Ullevaal Hospital, Oslo, for plasmapheresis, and healthy donors of MHC-matched platelets (n = 10) or PMN (n = 2) to leukaemic patients or granulocytopenic children with refractory Gram-negative septicaemia, re-
spectively. Plasmapheresis and cytapheresis All plasmaphereses and cytaphereses were carried out on a Haemonetics model 30 cell separator (Haemonetics, Natick, MA) for 90-120 min according to standard protocol. Normally 2-6 1 of the patients' plasma were exchanged with 8 U of fresh frozen plasma heated to approximately 37°C, except for one patient with hypercholesterolaemia who received a 40 mg/ml albumin solution as replacement fluid. Anti-coagulant was usually 4-8 mg/ml citric acid solution, but PMN donors received 60 mg/ml hydroxyethylglycogen (Plasmasterilo, McGaw Lab., Evanston, IL). Prior to apheresis, the PMN donors were subjected to 5 min of physical exercise and given corticosteroids (Decadron') intravenously to mobilize marginating PMN. Blood specimens Venous blood samples were taken immediately before the apheresis, after the third and the last sixth or eighth operation
535
536
G. Hetland, T. E. Mollnes & P. Garred Table 1. Terminal complement complex (TCC) in plasma from patients before, during, and after plasma exchange (medians and ranges)
Concentration of TCC (AU/ml) Diagnosis
No. of exchanges
GBS (n=4) Waldenstr6m's syndrome (n= 1) Waldenstr6m's syndrome (n=3) Hypercholesterolaemia (n= 1)
7 3 3 2
Before
During
After
5-8 (2-8-7 9) 4 5 (2 3-6 8) 6-2 (2-8-10-8)* 5-9 (4-1-6-5) 56 (5-4-5-7) 7-5 (7-4-10-1) 7-1 (2-4-7-6) 6-3 (6-1-6-5) 6-0 (1-3-6-4) ND 4-4 (2-2-6 5) 4-5 (2-2-6 7)
*P = 0 05 compared with before plasma exchange; otherwise P > 0 05. AU, arbitrary units; GBS, Guillain-Barr6 Syndrome; ND, not determined.
with cell donors or patients, respectively, and stored at 40C in EDTA before separation and storage of the plasma at - 700C. With the GBS patients, specimens were also taken from the units of withdrawn plasma and treated similarly. Some of these patients and donors were subjected to apheresis more than once. Fresh frozen citrate plasma from two donors stored at -400C for I month and thawed, and plasma from three blood donors stored at 40C for 1, 10, 20, 30 and 35 days were also examined for complement activation. In additon, serum from two GBS patients was incubated with AB Rh-matched donor serum or 15 g/lgammaglobulin (Kabi Pharmacia, Stockholm, Sweden) at 370C and examined before, immediately after mixing and I h later. Agarose gel double-diffusion of serum from these patients gave no precipitation lines against dilutions of donor serum or gammaglobulin. Gammaglobulin served as positive controls since it is produced from large plasma pools and therefore contains many idiotype specificities.
Analyses of complement activation products Complement activation was assessed in two enzyme immunoassays (EIA) specific to neoepitopes on activation products not expressed in native complement factors. One was specific for C3 activation products, thus evaluating the initial part of the complement cascade (Garred, Mollnes & Lea, 1988). In brief, polystyrene plates were coated with a mouse monoclonal antibody (bH6) specific for a C3 neoepitope expressed on C3b, iC3b and C3c, but not on native C3. A rabbit anti-human C3c antiserum (Behring, Marburg, Germany) was used in the secondary antibody layer. Finally, a peroxidase-conjugated anti-rabbit immunoglobulin was added (Amersham International, Amersham, UK). The substrate was 2,2'-azino-di-(3ethylbenzthiazoline sulphonic acid) (ABTS). The results were read on a Dynatech MR 580 (Alexandria, VA) and referred to a standard of zymosan-activated serum defined to contain 1000 arbitrary units (AU)/ml. The results were analysed by an Apple II computer with Dynatech Immunosoft program. The other assay detected the fluid-phase TCC, SC5b-9 (Mollnes et al., 1985). TCC is an indirect indicator of C5a release since it can not be formed in vivo without activation of C5 and release of C5a. In brief, polystyrene plates were coated with a mouse monoclonal antibody specific for a neoepitope expressed on activated C9. A rabbit anti-human C5 antiserum (Dakopatts Immunoglobulins, Copenhagen, Denmark) was used in the subsequent step. The rest of the assay was performed as described for the C3 activation assay.
Statistical analysis The significance of the results was tested by Wilcoxon's rank sum test for paired observations, and P < 0 05 was considered to be statistically significant. Student's t-test was used for some calculations.
RESULTS Complement activation during plasma exchange Plasmapheresis was performed on patients with GBS or Waldenstr6m's disease, and plasma samples for complement activation analyses were taken immediately before, during or after the treatment. Plasma was also exchanged in one hypercholesterolaemia patient, but with albumin solution instead of donor plasma. We found significantly higher plasma levels of TCC (Table 1), but not C3 activation products (Table 2) in GBS patients after compared with before plasmapheresis. However, the median values for both C3 activation products and TCC increased significantly (P < 0 05) from 10-6 to 20-9 and from 5-7 to 6-6 AU/ml, respectively, from the first unit to the last three pooled units of the withdrawn plasma from these patients. In one patient with Waldenstrom's syndrome there was modest increase in TCC and a substantial increase in C3 activation products after plasma exchange (Tables 1 and 2). The small number of samples did not enable statistical evaluation in this case. Significant changes in the plasma levels of these products were not observed during plasma exchange in the other patients (Tables 1 and 2).
Complement activation during cytapheresis We found no statistical differences in the plasma levels of C3 activation products or TCC before and after apheresis in the healthy donors of platelets or PMN (Table 3).
Complement activation during in vitro incubation of plasma! serum The values for C3 activation products and TCC in fresh frozen citrate-plasma stored at -40°C for 1 month and then thawed were 16-7 and 17-3, and 5-1 and 6-0 AU/ml from two donors, respectively. In comparison, plasma stored at 4°C up to 35 days showed an exponential increase in C3 activation products reaching 22 AU/ml after 10-14 days, whereas the levels of TCC were unaltered and close to the upper normal value (data not shown).
Activation of complement during apheresis
537
Table 2. C3 activation products in plasma from patients, before, during, and after plasma exchange (medians and ranges)
Concentration of C3 activation products (AU/ml) No. of
exchanges
Diagnosis GBS (n=4)
7 3 3 2
Waldenstrdm's syndrome (n= 1) Waldenstr6m's syndrome (n = 3) Hypercholesterolaemia (n= 1)
Before
During
After
12-0 (8 4-36 4) 10-6 (7 8-25 8) 13-0 (7-2-51-8) 13-6 (8 3-18-7) 17 7 (11-7-23-7) 35-0 (31-0-69-9) 13-4 (12-4-19-7) 10-4 (9-4-11-4) 13 4 (7 4-16 9)
10-9 (7-8-13-9)
ND
3-9 (3-7-41)
GBS, Guillain-Barr6 syndrome; ND, not determined. Table 3. Complement activation in plasma during cytapheresis of platelet and granulocyte (PMN) donors (medians and ranges)
Concentrations (AU/ml)
DISCUSSION
Donors of
of
Before
During
After
Platelets PMN Platelets PMN
C3 act. C3 act. TCC TCC
7-6 (4-8-13-8) 9.1 (10-0-13-4)
6-0 (4-1-15-7) ND 6-1 (2-7-14-3) ND
5 9 (4-0-16-9) 7 5 (5 0-9 9)
5-6(2-5-4-8) 3-7 (2 5-4-8)
5-4(2-6-11-0) 27 (2-5-29)
Results of 12 or two cytaphereses of 10 donors of platelet and two donors of PMN, respectively (P > 0-05). C3 act., C3 activation products; ND, not determined.
Table 4. Complement activation products in serum from GBS patients and donors before, during, and after co-incubation in vitro
Concentrations (AU/ml) Source of sample
of
Before
During
After
Donors GBS patients Gammaglobulin
C3 act. 42 2+ 11 -7 C3 act. 156-5+58-8 C3 act.
93 9+ 19-5 137-3+46-5 91-2+25-4 115-0+49-1
Donors GBS patients
TCC TCC TCC
179+26 1 2[+2± 1
Gammaglobulin
18-0+5-1 24-6+6.4
subsequent testing had antibodies to leucocyte antigens or irregular blood type antibodies.
225+86
35-0+2-5
Results are mean + 1 s.d. of sera from two GBS patients and four donors (two for each patient). P > 0-05 for the GBS patients (Student's
Mtest). To test whether the activation products arise because of reacting antibodies serum from two GBS patients was incubated with donor serum or gammaglobulin. We found no increase in the products after compared with before mixing the sera (Table 4). The higher (not significant) initial C3 values of the patients' than donors' sera probably reflect storage at -20'C and additional freeze-thawing of the former sera. Neither the four GBS patients nor the three donors who were available for
We conclude that the initial and terminal complement pathway is activated during plasmapheresis of patients with GBS, but not in cytapheresis of healthy platelet or PMN donors. Since C3 activation is suggested to be a prerequisite for terminal pathway activation in general, the significant increase in the generated TCC indicates that also the initial pathway is activated in the GBS patients after plasma exchange. This is supported by the significant rise in C3 activation products in the withdrawn plasma from these subjects. One of the GBS patients also had respiratory problems after the treatment, possibly caused by complement activation products (Ing et al., 1983). The observed decrease in plasma levels of C3 split products in the hypercholesterolaemia patient who received albumin solution instead of cholesterol-containing donor plasma, is consistent with the dilution of other plasma proteins after albumin administration. The finding of no significant changes in C3 activation products or TCC during 14 cytaphereses of healthy donors clearly demonstrates the biocompatibility with respect to complement activation of the materials used in this procedure.
The observed complement activation after plasmapheresis in some patients is partly caused by complement activation products in the donor plasma, which is supported by the following: (i) lack of complement activation during cytapheresis; (ii) usually higher levels of such products in thawed fresh frozen plasma than the initial C3 values of the patients; (iii) increase in C3 products and TCC in the withdrawn plasma from the patients after administration of donor plasma; and (iv) the reported C3 activation in myasthenia gravis after exchange of the patients' plasma with fresh frozen plasma, but not albumin (Rosenkvist et al., 1984). Fresh frozen plasma is nevertheless preferable as replacement fluid in plasmapheresis to plasma stored at 4 C because of the far stronger C3 activation in the latter. The plasma was heated to 370C prior to the exchange which was previously shown to increase complement activation rapidly (Mollnes, Garred & Bergseth, 1988). Thus, when up to 31 of heated plasma are exchanged during 2 h, administration of plasma containing activated complement could very well explain our findings. The lack of anaphylatoxin-related symptoms in most patients after plasmapheresis despite the high content of C3 activation products in donor plasma could be due to rapid transformation
538
G. Hetland, T. E. Mollnes & P. Garred
of C3a to the inactive derivative C3a-desArg (Chenoweth, 1986; Kazatchkine & Carreno, 1988). Moreover, C5a is present only in trace amounts in donor plasma due to the relative inefficiency of the fluid-phase C5 convertase compared with the C3 convertase (Bhakdi et al., 1988). An unknown 'patient factor' in GBS may also be responsible for the observed discrepancy in complement activation after plasma exchange between these patients and the majority of those with Waldenstr6m's syndrome. A probable candidate could be GBS autoantibodies in complement activating immuncomplexes (Cook & Dowling, 1981) which activate both the supplied and own complement components during apheresis. However, our negative in vitro results after mixing sera from GBS patients and donors did not indicate that the activation products arise through idiotype-anti-idiotype reactivity. This possibility is also most unlikely due to the fact that plasma from eight different donors was used in each plasma exchange. Immunodiffusion of the patient's sera against donors' sera did not give precipitation lines, and other alloantibodies that might react with cellular antigens were not demonstrated. Activation of complement in only one out of four patients with Waldenstr6m's syndrome could be due to an individually different reaction to this treatment. Unexpectedly, cryoagglutinins were found in one of the latter patients and not in the Waldenstrdm's syndrome patient with rise in complement activation products. This may indicate that cryoagglutinins do not promote complement activation during plasmapheresis. Although we found that plasma exchange induced complement activation in patients with GBS and occasionally in Waldenstrdm's syndrome clinical manifestations probably caused by this activation were observed only in one of these subjects. The median levels of the activation products were below the upper reference values in all patients, except for the Waldenstrdm's syndrome patient. In this case, treatment with longer intervals should be considered. Otherwise, plasmapheresis of such and other patient groups should be continued when there is a documented clinical effect of the treatment. However, donor plasma should be replaced with virus-inactivated albumin which is now commonly used because of the risk of H IV and other serious infections and recommended for plasmapheresis in GBS (Raphael et al., 1987) and myasthenia gravis (Rosenkvist et al., 1984). Moreover, additional gammaglobulin substitution should be considered (Raphael et al., 1987).
ACKNOWLEDGMENTS We thank Ms Grethe Bergseth and Ms Bente Falang for excellent technical assistance. Financial support was provided by the Nordic Insulin Foundation and the Norwegian Research Council for Science and the Humanities.
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