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
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
