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Immune response to collagen impregnated Dacron double velour grafts for aortic and aorto-femoral reconstructions
Eur J VascSurg 4, 379-384 (1990)
Immune Response to Collagen Impregnated Dacron Double Velour Grafts for Aortic and Aorto-femoral Reconstructions Lars N o r g r e n 1, Stig Holtas 2, G u n n a r P e r s s o n 4, Else Ribbe I , T o r e S a x n e 3 and J o h a n T h 6 r n e 1
Departments of lSurgery, 2Diagnostic Radiology, 3Rheumatology and 4Internal Medicine, Lund University, Sweden This study presents 20 patients, randomised to receive either a collagen-treated or an ordinary Dacron graft for aortic reconstructions, and the results of a skin-prick test, blood parameters and ELISA for anti-collagen antibodies as well as NMR pictures during a 6 weekfollow-up period. Forty per cent (4/11) of those receiving a collagen impregnated graft had a significantly increased titre of antibodies and NMR revealed in two out ofll patients either a slightly increased amount ofJ:luid or fibrosis around the graft, both collagen impregnated. No differences werefound between the graft groups concerning body temperature and leucocyte or platelet counts. The skin-prick testfor collagen was negative in all cases. Key Words: Dacron grafts; Collagen impregnation; Immune response.
Introduction
Material and M e t h o d s
Since 1959, w h e n the first Dacron graft was inserted in the aortic position, efforts have been made to improve grafts in order to simplify the surgical procedure. In elective operations, preclotting of the knitted Dacron velour graft usually obviates bleeding problems; however, in an emergency situation as well as w h e n larger grafts are used, e.g. for thoraco-abdominal aneurysms, minimising leakage of blood becomes important. For this purpose, impregnation a n d coating of the graft with different biological materials, such as collagen, albumin and gelatin has become common. Impregnation with collagen has not caused an increased thrombogenicity, ~ but the possibility that this biological material can induce an increased febrile reaction or a perigraft reaction remains.2 Anecdotally seromas have been described a r o u n d axillo-femoral collagen-treated Dacron grafts, but no definitive description of them has appeared in the literature. Both PTFE and Dacron grafts have been described giving rise to perigraft seromas.3" 4 The aim of this study was to compare the systemic response to a collagen-treated and a non-treated graft in patients with occlusive aorto-iliac disease or aortic aneurysm.
T w e n t y patients with either abdominal aortic aneury s m or aortic occlusive disease were randomised to receive either Microvel ® Dacron double velour, or Microvel ® double velour with Hemashield ® (impregnated collagen) grafts (Meadox Medicals Inc, USA). Approval from the Ethics Committee of L u n d University and informed consent from the patients were obtained. The study included 13 m e n and seven w o m e n with a m e a n age of 62 years (range 42-77 years). Seven of the patients h a d an aortic a n e u r y s m and 13 had occlusive disease. Nine patients received a Microvel ® and 11 a Microvel ® with Hemashield ® graft. None of the patients h a d any immunological or allergic disease. The study protocol is described in Table 1.
Please address all correspondence to: Lars Norgren, Department of Surgery, Lund University, $22185 Lund, Sweden. 0950-821X/90/040379+06 $03.00/0© 1990Grune & Stratton Ltd.
Skin prick test The graft collagen in its pre-impregnation un-crosslinked state was supplied by Meadox Medicals Inc, USA, as a paste and was s u s p e n d e d in pure water at a concentration of 2 mg/ml. Skin prick tests were performed with this suspension and with histamine (3 mg/ml) as a positive control. A solution of sodium chloride was used as a negative control.
L. Norgren etaL
380
Table 1. Investigative procedure
Preoperatively Skin prick test againstcollagen
X
ELISAfor collagenantibodies
X
1 day
Postoperatively 2 days
1 week
6 weeks
X
X
X
X
X
X
X
X X X
X X X
Magneticresonancetomography Bloodparameters: Platelet count Leucocytecount Differentialcount
X X X
X X X
X X X
Bodytemperature
X
Every second hour
Twice dailyfor I week
Enzyme linked immunosorbent assay (ELISA) Preparation of antigens The graft collagen (see above) was supplied by Meadox Medicals Inc, USA, in the form of a paste suspended in pure water at a concentration of 13-15%. According to the manufacturer the preparation contains bovine skin collagen, mainly type I and some of type III; the detailed process used for producing the graft collagen is, however, a trade secret u n k n o w n to us. About 100 mg of this paste was freeze dried and yielded 11 mg dry substance. Sodium dodecylpolyacrylamide-get electrophoresis of a soluble sample of this material confirmed the presence of the two collagen types and no other detectable proteins. The bovine type I collagen used for comparison was prepared from bovine tendon by acid extraction using the method described by Chandrakasan and coworkers.S Before use for coating in the ELISA, the graft collagen dry substance was solubilised in 0.5 M acetic acid to a desired final concentration of 5 p~g/ml. The solution was mixed by continuous shaking on a shake board for 24h at room temperature (22°C). Despite this treatment the collagen was not completely solubilised which means that the final concentration was somewhat variable. The bovine type I collagen was handled in a similar way and was completely solubilised.
Measurement of antibodies An ELISA for quantification of circulating anti-collagen antibodies was performed by adapting the techniques of Engvall and Perlmann.6 The assay was originally developed for measuring antibodies against type I and type II collagen. The intra- and interassay variations were, in previous studies, found to be less than 10% (Hed, Saxne, Heineg~rd, unpublished). Polyvinyl microtitre plates (Falcon 3912) were Eur J VascSurgVol4, August1990
incubated with 200 ~l of a solution of graft collagen (approximately 5 ~g/ml in 0.5 M acetic acid) for 24 h at room temperature (22°C) in a moist chamber. The wells were then rinsed three times with 0.9% sodium chloride containing 0.05% Tween 20. After incubation with washing solution for I h they were finally rinsed again. A 200 p~l aliquot of serum diluted 1/50 in phosphate buffered saline, pH 7.4, was then incubated in the coated plates in a moist chamber overnight. After rinsing, as described above, 200 p~l of a dilution of alkaline phosphatase conjugated rabbit antihuman immunoglobulins A, G, M (Dakopatts, Denmark) in 0.1M sodium chloride, 0.05M sodium phosphate, 0.05% Tween 20 and 2 mg/ml of bovine serum albumin (pH 7.5) was added. After I h at room temperature the plates were again rinsed and 200 p~l of enzyme substrate, I mg/ml of p-nitrophenyl phosphate in 1 M diethanolamine, pH 9.8, containing 0.5mMMgC12, was added. The absorbance at 405 nm was measured in a Multiscan filter photometer (Flow laboratories, USA) immediately and after incubation for I h at room temperature. The increase in absorbance was used for calculations. All samples were analysed in triplicate, and the mean value was used for calculations. A positive and a negative reference sample were included in all plates. The results are given as a percentage of the positive control in order to facilitate comparisons between patients. Thus, the absorbance of each serum was divided by the absorbance of the control and the result was multiplied by 100. All patients who were found to be positive were also tested against the type I collagen prepared in the laboratory. Furthermore the analyses for these patients were also run separately with alkaline phosphatase conjugated antibodies specific for IgG, IgA and IgM (Orin Diagnostica, Helsinki, Finland). All analyses were performed without knowledge about
Immune Response to Grafts
the t y p e of graft u s e d in the individual patient. All s a m p l e s f r o m a given patient w e r e a n a l y s e d in the s a m e plate.
Magnetic resonance imaging Eleven of the patients o p e r a t e d on for occlusive disease w e r e e x a m i n e d with m a g n e t i c r e s o n a n c e (MR) i m a g i n g o n a 0.3 Tesla Fonar Scanner 1 a n d 6 w e e k s after surgery. Solenoid surface coils w e r e u s e d a n d i m a g i n g w a s p e r f o r m e d in axial projection c o v e r i n g the l o w e r a b d o m e n u s i n g 10 m m thick slices a n d T1-W a n d T2-W spin echo (SE) sequences. Five of these patients h a d received a Microvel, ® six a Microvel ® with H e m a s h i e l d ® graft. The total a n d differential counts of leucocytes as well as platelet c o u n t s w e r e r e c o r d e d according to Table 1. Details of surgery w e r e r e c o r d e d a n d the b o d y t e m p e r a t u r e w a s m e a s u r e d e v e r y second h o u r d u r i n g the first 2 4 h p o s t o p e r a t i v e l y a n d t h e n twice daily during a week.
Results
381
5 m i n for the Microvel ® a n d Microvel ® w i t h H e m a shield ® g r o u p s respectively. All patients s u r v i v e d the operation, one h a d a severe m y o c a r d i a l infarction o n the first p o s t o p e r a t i v e d a y a n d died of a second infarct 5 w e e k s later. The r e m a i n i n g 19 patients w e r e m o n i t o r e d as scheduled. All patients h a d a raised b o d y t e m p e r a t u r e d u r i n g the first 24 p o s t o p e r a t i v e hours. There w a s no significant difference b e t w e e n the highest t e m p e r a t u r e s r e c o r d e d for the Microvel ® a n d H e m a s h i e l d ® g r o u p s respectively (39.4 + 0.2 a n d 39.6 + 0.2 °C). There w e r e no differences b e t w e e n leucocyte differential or platelet c o u n t s in the two graft groups. All patients, h o w ever, h a d a reduction in circulating platelets a n d a n increase of circulating leucocytes d u r i n g the early p o s t o p e r a t i v e p h a s e (Table 3). Table 3. Blood parameters Mean ± SE
Microvel® Preoperative leucocyte count
9.1 + 0.4
7.4 + 0.3
Second day leucocyte count
12.6 + 1.3
10:6 + 1.0
Sixth day leucocyte count
11.8 + 1.1
9.9 + 0.9
8.8 + 0.7
8.0 + 0.8
<
Six week leucocyte count Thirteen of the patients received a bifurcated p r o s t h e sis a n d s e v e n h a d a straight tube (Table 2). The preclotting p r o c e d u r e of Microvel ® grafts g a v e rise to difficulties in o n e patient. Bleeding f r o m the graft w a s Table 2. The use of graft with consideration to size
Graft size (mm)
Microvel ®
12x6 14x7
3
3
16x8
3
3 1"
14
1
16
1
18
1
Preoperative platelet count
269 ± 25
235 4 15
Second day platelet count
120 ± 10
129 ± 11
Sixth day platelet count
244 ± 24
246 ± 13
Six week platelet count
257 ± 15
261 ± 15
Microvel®with Hemashield® 1
8
Microvel® with Hemashield ®
2
Skin prick test T w o of the patients did n o t a t t e n d for a skin prick test at 6 weeks. There w a s no w h e a l reaction in a n y of the patients w h e n tested w i t h collagen a n d there w a s no significant c h a n g e in the size of the h i s t a m i n e w h e a l d u r i n g the study.
1
* Unilateral aorto-femoral reconstruction. e s t i m a t e d to be 100-800ml ( m e a n 290ml) b u t no b l e e d i n g w a s recorded for the H e m a s h i e l d ® grafts. The total b l o o d loss w a s 1733 + 339 ml for the Microvel ~. g r o u p , 1118 + 149ml for the Microvel ® w i t h H e m a s h i e l d ® group, w h i c h w a s not significantly different. The clamp time w a s 65 + 8 m i n a n d 57 +
Development of antibodies against the graft collagen All s a m p l e s w e r e a n a l y s e d at least twice a n d reproducible results w e r e o b t a i n e d w h e n the results w e r e correlated to the reference samples. There w a s s o m e variability in the results d u e to difficulties in solubilising the graft collagen. W h e n the reactivity against the purified b o v i n e collagen w a s tested the intra- a n d Eur J Vasc Surg Vol 4, August 1990
382
L. Norgren et al.
was due to formation of all three types of immunoglobulins with IgM and IgG predominating.
200~
Magnetic resonance imaging
o
MR visualised the graft in all 11 patients operated on for occlusive disease - - no reaction was observed a r o u n d the graft in nine of them. In one patient, a signal reduction was noted around the graft both on T1-W and T2-W sequences 6 weeks after surgery. The signal pattern was thus consistent with fibrosis. In one patient a low signal was noted a r o u n d the graft on T1W sequences and a high signal on T2-W sequences 1 w e e k after surgery, consistent with fluid. This finding was normalised 6 weeks after surgery (Fig. 2).
I00
@
o
o
Discussion
o o
0 o
M
MH
Fig. 1. Comparison of the rise in concentrations of circulatingantibodies against the Hemashield®collagenbetween day 0 (preoperatively) and day 42 (postoperatively) in patients having received Microvel®grafts (M) or Microvel®with Hemashield®collagengrafts (MH) respectively. The values are expressed as percentage of a positive control (for details see text). interassay variation was low and similar to that found in previous studies with ELISA. Fig. I shows the change in antibody concentration from d a y 0 to d a y 42 comparing the two treatment groups. Four of the 11 patients in the Hemashield ® group s h o w e d a significant rise in antibody concentrations. The difference between the groups was highly significant (P < 0.01, M a n n - W h i t n e y test for unpaired variables, 2-tailed). N o n e of the eight patients w h o received a Microvel ® graft w i t h o u t collagen s h o w e d any change in antibody concentrations. The patient developing a fatal postoperative myocardial infarction belonged to the Microvel ® group and was excluded, since only preoperative and day 6 samples were available. On day 6 after the operation a slight decrease in antibody concentrations was seen in the majority of patients, probably due to haemodilution. The four patients with antibody response to the graft collagen s h o w e d a similar, although less pronounced, rise in antibody reactivity against purified type I collagen, emphasising that most, but not all, reactivity was due to formation of antibodies against this collagen type. The increased antibody reactivity Eur J VascSurg Vol 4, August 1990
The bovine collagen used for treatment of the Microvel ® grafts with Hemashield ® is mostly type I but also contains some type III collagen. Febrile reactions after the use of this graft have been suspected but no proof that collagen was the cause has been given. 2 Furthermore, reactions around the graft have been anecdotally reported, especially in the axillo-femoral position but this p h e n o m e n o n has not been objectively ascribed to the Hemashield ® process. This study shows that a temporary increase of b o d y temperature during the first or first and second postoperative days to a m a x i m u m level a r o u n d 39 °C is common. However, no difference between the patients receiving collagen treated and those receiving non-treated grafts was observed. Neither was there a difference due to the a m o u n t of graft material used, since both patients with aorto-femoral and straight grafts reacted in the same way. There was an expected decrease of platelet counts postoperatively due to haemodilution and blood transfusions. An increase of leucocyte counts without a n y specific reaction in the differential counts was f o u n d for all patients. Both the temperature reaction a n d the changes in leucocyte counts were most probably due to the surgical procedure per se. No positive skin prick tests to collagen were f o u n d in this study, neither pre- nor postoperatively. It should be emphasised that even if this result was expected, there is a slight risk that the preparation of the bovine collagen material for testing m a y have reduced the validity of test. All patients r e s p o n d e d to histamine with a typical wheal and flare reaction. A l t h o u g h modified to diminish antigenicity as
Immune Responseto Grafts
383
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EurJ Vasc Surg Vol 4, August 1990
384
L. Norgren et aL
stated by the manufacturer, the graft collagen can apparently elicit an asymptomatic antibody response in a significant proportion of individuals. This is in line with previous reports of antibody formation against injectable collagen in patients undergoing correction of dermal defects. 7 It is also in keeping with the results of Nimni and co-workers who showed retained, but diminished, antigenicity of a collagen preparation after cross-linking with glutaraldehyde and diamine.8 The procedure for preparing the Hemashield ® collagen is a trade secret, not revealed to us. Therefore any comparison to previous studies is less informative, although it appears likely that the graft collagen was prepared in a similar fashion. The finding of similar antibody response both to Hemashield ® collagen and native type I collagen in these patients makes nonspecific reactions unlikely. Magnetic resonance tomography was only performed in patients with occlusive disease in order not to create visualising disturbances from the aneurysm capsule "sutured around the grafts. Two patients showed a pattern different from the remaining nine. Both patients had Microvel ®with Hemashield ® grafts. In one case a limited amount of fluid was found around the graft after 1 week, disappearing within 6 weeks. This patient showed an increase in circulating antibodies against the collagen. A fibrotic reaction after 6 weeks was found in another patient receiving a Hemashield ® graft but this patient showed no increased antibody formation. The presented patient material is limited, but in all other aspects representative for the population operated on for aortic occlusive disease and aortic aneurysms. No acute reactions to any of the graft materials could be revealed, but it seems evident that the collagen preparation used in the Hemashield ®
Eur J Vasc Surg Vol 4, August 1990
graft is antigenic. The differences in response between patients is probably due to genetic factors currently unknown. As no patient had abnormal levels of circulating antibodies prior to surgery and little is known about the pathological significance of these antibodies it seems unlikely that a preoperative screening procedure would be worthwhile.
Acknowledgements This study was supported by Meadox Medicals Inc, USA. Preparation of the graft collagen for the skin prick test was done by Pharmacia AB, Uppsala, Sweden.
References 1 QUINONES-BALDRICH WJ, MOORE WS, ZIOMEK S, CHVAPIL M. Development of a leak proof knitted Dacron vascular prosthesis. ] Vasc Surg 1986; 3: 895-903. 2 NORGREN L, BERGGRENU, PXRSSON H, RIBBE E, THORNE J. How does Hemashield ® affect surgical technique? Clinical results in aortic surgery. Angio Archiv 1985; 9: 39-43. 3 BORREROE, DOSCHERW. Chronic perigraft seroma in PTFE grafts. J Cardiovasc Surg 1988; 29: 46-49. 4 KAuPPHA, MATULEWICZTJ,KREMENJE, CELANIVJ. Graftinfection or graft reaction? Arch Surg 1979; 11: 1419-1422. 5 CHANDRAKASANG, TORCHIA DA, PIEZ KA. Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the non helical ends in solution. J Biol Chem 1976; 251: 6062-6067. 6 ENGVALLE. Enzyme immunoassay ELISA and EMIT. Methods of Enzymology 1988; 70: 419-439. 7 COOPERMAN L, MICHAELI D. The immunogenicity of injectable collagen. I. A 1-year prospective study. J Am Acad Derm 1984; 10: 638-646. 8 NIMNI ME, CHEUNGD, STRATESB, KODAMAM, SHEIKH K. Chemically modified collagen: A natural biomaterial for tissue replacement. J BiomedMat Res 1987; 21: 741-771.
Accepted 27 November 1989
Immune Response to Collagen Impregnated Dacron Double Velour Grafts for Aortic and Aorto-femoral Reconstructions Lars N o r g r e n 1, Stig Holtas 2, G u n n a r P e r s s o n 4, Else Ribbe I , T o r e S a x n e 3 and J o h a n T h 6 r n e 1
Departments of lSurgery, 2Diagnostic Radiology, 3Rheumatology and 4Internal Medicine, Lund University, Sweden This study presents 20 patients, randomised to receive either a collagen-treated or an ordinary Dacron graft for aortic reconstructions, and the results of a skin-prick test, blood parameters and ELISA for anti-collagen antibodies as well as NMR pictures during a 6 weekfollow-up period. Forty per cent (4/11) of those receiving a collagen impregnated graft had a significantly increased titre of antibodies and NMR revealed in two out ofll patients either a slightly increased amount ofJ:luid or fibrosis around the graft, both collagen impregnated. No differences werefound between the graft groups concerning body temperature and leucocyte or platelet counts. The skin-prick testfor collagen was negative in all cases. Key Words: Dacron grafts; Collagen impregnation; Immune response.
Introduction
Material and M e t h o d s
Since 1959, w h e n the first Dacron graft was inserted in the aortic position, efforts have been made to improve grafts in order to simplify the surgical procedure. In elective operations, preclotting of the knitted Dacron velour graft usually obviates bleeding problems; however, in an emergency situation as well as w h e n larger grafts are used, e.g. for thoraco-abdominal aneurysms, minimising leakage of blood becomes important. For this purpose, impregnation a n d coating of the graft with different biological materials, such as collagen, albumin and gelatin has become common. Impregnation with collagen has not caused an increased thrombogenicity, ~ but the possibility that this biological material can induce an increased febrile reaction or a perigraft reaction remains.2 Anecdotally seromas have been described a r o u n d axillo-femoral collagen-treated Dacron grafts, but no definitive description of them has appeared in the literature. Both PTFE and Dacron grafts have been described giving rise to perigraft seromas.3" 4 The aim of this study was to compare the systemic response to a collagen-treated and a non-treated graft in patients with occlusive aorto-iliac disease or aortic aneurysm.
T w e n t y patients with either abdominal aortic aneury s m or aortic occlusive disease were randomised to receive either Microvel ® Dacron double velour, or Microvel ® double velour with Hemashield ® (impregnated collagen) grafts (Meadox Medicals Inc, USA). Approval from the Ethics Committee of L u n d University and informed consent from the patients were obtained. The study included 13 m e n and seven w o m e n with a m e a n age of 62 years (range 42-77 years). Seven of the patients h a d an aortic a n e u r y s m and 13 had occlusive disease. Nine patients received a Microvel ® and 11 a Microvel ® with Hemashield ® graft. None of the patients h a d any immunological or allergic disease. The study protocol is described in Table 1.
Please address all correspondence to: Lars Norgren, Department of Surgery, Lund University, $22185 Lund, Sweden. 0950-821X/90/040379+06 $03.00/0© 1990Grune & Stratton Ltd.
Skin prick test The graft collagen in its pre-impregnation un-crosslinked state was supplied by Meadox Medicals Inc, USA, as a paste and was s u s p e n d e d in pure water at a concentration of 2 mg/ml. Skin prick tests were performed with this suspension and with histamine (3 mg/ml) as a positive control. A solution of sodium chloride was used as a negative control.
L. Norgren etaL
380
Table 1. Investigative procedure
Preoperatively Skin prick test againstcollagen
X
ELISAfor collagenantibodies
X
1 day
Postoperatively 2 days
1 week
6 weeks
X
X
X
X
X
X
X
X X X
X X X
Magneticresonancetomography Bloodparameters: Platelet count Leucocytecount Differentialcount
X X X
X X X
X X X
Bodytemperature
X
Every second hour
Twice dailyfor I week
Enzyme linked immunosorbent assay (ELISA) Preparation of antigens The graft collagen (see above) was supplied by Meadox Medicals Inc, USA, in the form of a paste suspended in pure water at a concentration of 13-15%. According to the manufacturer the preparation contains bovine skin collagen, mainly type I and some of type III; the detailed process used for producing the graft collagen is, however, a trade secret u n k n o w n to us. About 100 mg of this paste was freeze dried and yielded 11 mg dry substance. Sodium dodecylpolyacrylamide-get electrophoresis of a soluble sample of this material confirmed the presence of the two collagen types and no other detectable proteins. The bovine type I collagen used for comparison was prepared from bovine tendon by acid extraction using the method described by Chandrakasan and coworkers.S Before use for coating in the ELISA, the graft collagen dry substance was solubilised in 0.5 M acetic acid to a desired final concentration of 5 p~g/ml. The solution was mixed by continuous shaking on a shake board for 24h at room temperature (22°C). Despite this treatment the collagen was not completely solubilised which means that the final concentration was somewhat variable. The bovine type I collagen was handled in a similar way and was completely solubilised.
Measurement of antibodies An ELISA for quantification of circulating anti-collagen antibodies was performed by adapting the techniques of Engvall and Perlmann.6 The assay was originally developed for measuring antibodies against type I and type II collagen. The intra- and interassay variations were, in previous studies, found to be less than 10% (Hed, Saxne, Heineg~rd, unpublished). Polyvinyl microtitre plates (Falcon 3912) were Eur J VascSurgVol4, August1990
incubated with 200 ~l of a solution of graft collagen (approximately 5 ~g/ml in 0.5 M acetic acid) for 24 h at room temperature (22°C) in a moist chamber. The wells were then rinsed three times with 0.9% sodium chloride containing 0.05% Tween 20. After incubation with washing solution for I h they were finally rinsed again. A 200 p~l aliquot of serum diluted 1/50 in phosphate buffered saline, pH 7.4, was then incubated in the coated plates in a moist chamber overnight. After rinsing, as described above, 200 p~l of a dilution of alkaline phosphatase conjugated rabbit antihuman immunoglobulins A, G, M (Dakopatts, Denmark) in 0.1M sodium chloride, 0.05M sodium phosphate, 0.05% Tween 20 and 2 mg/ml of bovine serum albumin (pH 7.5) was added. After I h at room temperature the plates were again rinsed and 200 p~l of enzyme substrate, I mg/ml of p-nitrophenyl phosphate in 1 M diethanolamine, pH 9.8, containing 0.5mMMgC12, was added. The absorbance at 405 nm was measured in a Multiscan filter photometer (Flow laboratories, USA) immediately and after incubation for I h at room temperature. The increase in absorbance was used for calculations. All samples were analysed in triplicate, and the mean value was used for calculations. A positive and a negative reference sample were included in all plates. The results are given as a percentage of the positive control in order to facilitate comparisons between patients. Thus, the absorbance of each serum was divided by the absorbance of the control and the result was multiplied by 100. All patients who were found to be positive were also tested against the type I collagen prepared in the laboratory. Furthermore the analyses for these patients were also run separately with alkaline phosphatase conjugated antibodies specific for IgG, IgA and IgM (Orin Diagnostica, Helsinki, Finland). All analyses were performed without knowledge about
Immune Response to Grafts
the t y p e of graft u s e d in the individual patient. All s a m p l e s f r o m a given patient w e r e a n a l y s e d in the s a m e plate.
Magnetic resonance imaging Eleven of the patients o p e r a t e d on for occlusive disease w e r e e x a m i n e d with m a g n e t i c r e s o n a n c e (MR) i m a g i n g o n a 0.3 Tesla Fonar Scanner 1 a n d 6 w e e k s after surgery. Solenoid surface coils w e r e u s e d a n d i m a g i n g w a s p e r f o r m e d in axial projection c o v e r i n g the l o w e r a b d o m e n u s i n g 10 m m thick slices a n d T1-W a n d T2-W spin echo (SE) sequences. Five of these patients h a d received a Microvel, ® six a Microvel ® with H e m a s h i e l d ® graft. The total a n d differential counts of leucocytes as well as platelet c o u n t s w e r e r e c o r d e d according to Table 1. Details of surgery w e r e r e c o r d e d a n d the b o d y t e m p e r a t u r e w a s m e a s u r e d e v e r y second h o u r d u r i n g the first 2 4 h p o s t o p e r a t i v e l y a n d t h e n twice daily during a week.
Results
381
5 m i n for the Microvel ® a n d Microvel ® w i t h H e m a shield ® g r o u p s respectively. All patients s u r v i v e d the operation, one h a d a severe m y o c a r d i a l infarction o n the first p o s t o p e r a t i v e d a y a n d died of a second infarct 5 w e e k s later. The r e m a i n i n g 19 patients w e r e m o n i t o r e d as scheduled. All patients h a d a raised b o d y t e m p e r a t u r e d u r i n g the first 24 p o s t o p e r a t i v e hours. There w a s no significant difference b e t w e e n the highest t e m p e r a t u r e s r e c o r d e d for the Microvel ® a n d H e m a s h i e l d ® g r o u p s respectively (39.4 + 0.2 a n d 39.6 + 0.2 °C). There w e r e no differences b e t w e e n leucocyte differential or platelet c o u n t s in the two graft groups. All patients, h o w ever, h a d a reduction in circulating platelets a n d a n increase of circulating leucocytes d u r i n g the early p o s t o p e r a t i v e p h a s e (Table 3). Table 3. Blood parameters Mean ± SE
Microvel® Preoperative leucocyte count
9.1 + 0.4
7.4 + 0.3
Second day leucocyte count
12.6 + 1.3
10:6 + 1.0
Sixth day leucocyte count
11.8 + 1.1
9.9 + 0.9
8.8 + 0.7
8.0 + 0.8
<
Six week leucocyte count Thirteen of the patients received a bifurcated p r o s t h e sis a n d s e v e n h a d a straight tube (Table 2). The preclotting p r o c e d u r e of Microvel ® grafts g a v e rise to difficulties in o n e patient. Bleeding f r o m the graft w a s Table 2. The use of graft with consideration to size
Graft size (mm)
Microvel ®
12x6 14x7
3
3
16x8
3
3 1"
14
1
16
1
18
1
Preoperative platelet count
269 ± 25
235 4 15
Second day platelet count
120 ± 10
129 ± 11
Sixth day platelet count
244 ± 24
246 ± 13
Six week platelet count
257 ± 15
261 ± 15
Microvel®with Hemashield® 1
8
Microvel® with Hemashield ®
2
Skin prick test T w o of the patients did n o t a t t e n d for a skin prick test at 6 weeks. There w a s no w h e a l reaction in a n y of the patients w h e n tested w i t h collagen a n d there w a s no significant c h a n g e in the size of the h i s t a m i n e w h e a l d u r i n g the study.
1
* Unilateral aorto-femoral reconstruction. e s t i m a t e d to be 100-800ml ( m e a n 290ml) b u t no b l e e d i n g w a s recorded for the H e m a s h i e l d ® grafts. The total b l o o d loss w a s 1733 + 339 ml for the Microvel ~. g r o u p , 1118 + 149ml for the Microvel ® w i t h H e m a s h i e l d ® group, w h i c h w a s not significantly different. The clamp time w a s 65 + 8 m i n a n d 57 +
Development of antibodies against the graft collagen All s a m p l e s w e r e a n a l y s e d at least twice a n d reproducible results w e r e o b t a i n e d w h e n the results w e r e correlated to the reference samples. There w a s s o m e variability in the results d u e to difficulties in solubilising the graft collagen. W h e n the reactivity against the purified b o v i n e collagen w a s tested the intra- a n d Eur J Vasc Surg Vol 4, August 1990
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was due to formation of all three types of immunoglobulins with IgM and IgG predominating.
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Magnetic resonance imaging
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MR visualised the graft in all 11 patients operated on for occlusive disease - - no reaction was observed a r o u n d the graft in nine of them. In one patient, a signal reduction was noted around the graft both on T1-W and T2-W sequences 6 weeks after surgery. The signal pattern was thus consistent with fibrosis. In one patient a low signal was noted a r o u n d the graft on T1W sequences and a high signal on T2-W sequences 1 w e e k after surgery, consistent with fluid. This finding was normalised 6 weeks after surgery (Fig. 2).
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Discussion
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M
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Fig. 1. Comparison of the rise in concentrations of circulatingantibodies against the Hemashield®collagenbetween day 0 (preoperatively) and day 42 (postoperatively) in patients having received Microvel®grafts (M) or Microvel®with Hemashield®collagengrafts (MH) respectively. The values are expressed as percentage of a positive control (for details see text). interassay variation was low and similar to that found in previous studies with ELISA. Fig. I shows the change in antibody concentration from d a y 0 to d a y 42 comparing the two treatment groups. Four of the 11 patients in the Hemashield ® group s h o w e d a significant rise in antibody concentrations. The difference between the groups was highly significant (P < 0.01, M a n n - W h i t n e y test for unpaired variables, 2-tailed). N o n e of the eight patients w h o received a Microvel ® graft w i t h o u t collagen s h o w e d any change in antibody concentrations. The patient developing a fatal postoperative myocardial infarction belonged to the Microvel ® group and was excluded, since only preoperative and day 6 samples were available. On day 6 after the operation a slight decrease in antibody concentrations was seen in the majority of patients, probably due to haemodilution. The four patients with antibody response to the graft collagen s h o w e d a similar, although less pronounced, rise in antibody reactivity against purified type I collagen, emphasising that most, but not all, reactivity was due to formation of antibodies against this collagen type. The increased antibody reactivity Eur J VascSurg Vol 4, August 1990
The bovine collagen used for treatment of the Microvel ® grafts with Hemashield ® is mostly type I but also contains some type III collagen. Febrile reactions after the use of this graft have been suspected but no proof that collagen was the cause has been given. 2 Furthermore, reactions around the graft have been anecdotally reported, especially in the axillo-femoral position but this p h e n o m e n o n has not been objectively ascribed to the Hemashield ® process. This study shows that a temporary increase of b o d y temperature during the first or first and second postoperative days to a m a x i m u m level a r o u n d 39 °C is common. However, no difference between the patients receiving collagen treated and those receiving non-treated grafts was observed. Neither was there a difference due to the a m o u n t of graft material used, since both patients with aorto-femoral and straight grafts reacted in the same way. There was an expected decrease of platelet counts postoperatively due to haemodilution and blood transfusions. An increase of leucocyte counts without a n y specific reaction in the differential counts was f o u n d for all patients. Both the temperature reaction a n d the changes in leucocyte counts were most probably due to the surgical procedure per se. No positive skin prick tests to collagen were f o u n d in this study, neither pre- nor postoperatively. It should be emphasised that even if this result was expected, there is a slight risk that the preparation of the bovine collagen material for testing m a y have reduced the validity of test. All patients r e s p o n d e d to histamine with a typical wheal and flare reaction. A l t h o u g h modified to diminish antigenicity as
Immune Responseto Grafts
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stated by the manufacturer, the graft collagen can apparently elicit an asymptomatic antibody response in a significant proportion of individuals. This is in line with previous reports of antibody formation against injectable collagen in patients undergoing correction of dermal defects. 7 It is also in keeping with the results of Nimni and co-workers who showed retained, but diminished, antigenicity of a collagen preparation after cross-linking with glutaraldehyde and diamine.8 The procedure for preparing the Hemashield ® collagen is a trade secret, not revealed to us. Therefore any comparison to previous studies is less informative, although it appears likely that the graft collagen was prepared in a similar fashion. The finding of similar antibody response both to Hemashield ® collagen and native type I collagen in these patients makes nonspecific reactions unlikely. Magnetic resonance tomography was only performed in patients with occlusive disease in order not to create visualising disturbances from the aneurysm capsule "sutured around the grafts. Two patients showed a pattern different from the remaining nine. Both patients had Microvel ®with Hemashield ® grafts. In one case a limited amount of fluid was found around the graft after 1 week, disappearing within 6 weeks. This patient showed an increase in circulating antibodies against the collagen. A fibrotic reaction after 6 weeks was found in another patient receiving a Hemashield ® graft but this patient showed no increased antibody formation. The presented patient material is limited, but in all other aspects representative for the population operated on for aortic occlusive disease and aortic aneurysms. No acute reactions to any of the graft materials could be revealed, but it seems evident that the collagen preparation used in the Hemashield ®
Eur J Vasc Surg Vol 4, August 1990
graft is antigenic. The differences in response between patients is probably due to genetic factors currently unknown. As no patient had abnormal levels of circulating antibodies prior to surgery and little is known about the pathological significance of these antibodies it seems unlikely that a preoperative screening procedure would be worthwhile.
Acknowledgements This study was supported by Meadox Medicals Inc, USA. Preparation of the graft collagen for the skin prick test was done by Pharmacia AB, Uppsala, Sweden.
References 1 QUINONES-BALDRICH WJ, MOORE WS, ZIOMEK S, CHVAPIL M. Development of a leak proof knitted Dacron vascular prosthesis. ] Vasc Surg 1986; 3: 895-903. 2 NORGREN L, BERGGRENU, PXRSSON H, RIBBE E, THORNE J. How does Hemashield ® affect surgical technique? Clinical results in aortic surgery. Angio Archiv 1985; 9: 39-43. 3 BORREROE, DOSCHERW. Chronic perigraft seroma in PTFE grafts. J Cardiovasc Surg 1988; 29: 46-49. 4 KAuPPHA, MATULEWICZTJ,KREMENJE, CELANIVJ. Graftinfection or graft reaction? Arch Surg 1979; 11: 1419-1422. 5 CHANDRAKASANG, TORCHIA DA, PIEZ KA. Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the non helical ends in solution. J Biol Chem 1976; 251: 6062-6067. 6 ENGVALLE. Enzyme immunoassay ELISA and EMIT. Methods of Enzymology 1988; 70: 419-439. 7 COOPERMAN L, MICHAELI D. The immunogenicity of injectable collagen. I. A 1-year prospective study. J Am Acad Derm 1984; 10: 638-646. 8 NIMNI ME, CHEUNGD, STRATESB, KODAMAM, SHEIKH K. Chemically modified collagen: A natural biomaterial for tissue replacement. J BiomedMat Res 1987; 21: 741-771.
Accepted 27 November 1989