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Immune responses to bovine collagen implants
Significance of pretreatment serology
J. Philip McCoy, Jr., Ph.D.,* William Schade, B.S.,* Ronald 1. Siegle, M.D.,*** Evelyn E. Vanderveen, M.D.,** Christopher B. Zachary, M.D.,** Thomas P. Waldinger, M.D. ,** and Neff A. Swagson, M.D.** Ann Arbor, MI, and / Columbus, OH
Bovine collagen implants are in widespread use for the correction of soft tissue deficiencies. A small portion of the population responds adversely to these implants, evidenced by either positive skin tests or treatment site reactions. Antibodies against bovine collagen implants have been detected in the sera of virtually all patients with adverse treatment reactions and in small percentages of untreated individuals and treated individuals without adverse reactions. In this study we examined the levels of anti-bovine collagen implant antibodies in 150 individuals prior to bovine collagen implant therapy and then monitored these patients for adverse reactions to skin tests and bovine collagen implant treatments. Statistical analyses were performed to determine the value of pretreatment serology in predicting positive skin tests or adverse reactions. It was determined that individuals with greatly elevated levels (greater than 2 standard deviations [SDs] above the mean reactivity of pretreatment sera) of anti-bovine collagen implant antibodies prior to treatment were approximately sixfold more likely to suffer adverse reactions than patients with no elevation of antibodies prior to treatment. (J AM ACAD DERMATOL 1987;16:955-60.)

The widespread use of bovine collagen implants to correct dermal contour deformities has led to numerous reports in the literature concerning the immunogenicity of these implants, l"~ While it is clear that these bovine collagen preparations are of low immunogenicity, due to the selective removal of the nonhelical ends of the collagen mol-

From the Departments of Pathology* and Dermatology,** The University of Michigan Medical School, Ann Arbor, and the Division of Dermatology, Department of Internal Medicine,*** The Ohio State University Medical School, Columbus. Accepted for publication Nov. 6, 1986. Reprint requests to: Dr. J. Philip McCoy, Jr., The University Hospital, Department of Pathology, Rm 2245, Box 0602, 1500 East Medical Center Dr., Ann Arbor, MI 48109-0602.

ecule, it is unclear why some individuals react adversely to these implants whereas the majority of patients receive the implants without immune recognition. The nature of the immune reactions against bovine coUagen implants appears to be quite varied and to include mild erythema, urticaria, localized swelling and/or redness, granulomas, and specific antibody production. The significance of these responses, particularly the development of anti-bovine collagen implant antibodies, is not known. The observation that some individuals react to skin tests with bovine collagen implants and/or have antibodies that react with bovine collagen implant prior to treatment while other individuals become sensitized only after treatment indicates the high degree of heteroge955

to bovine collagen implant treatments. 7 Many of the characteristics of these antibodies have been described previously. 120 0. Measurement of anti-bovine collagen implant antibodies Circulating antibodies against the bovine collagen implants were measured using an enzyme-linked immunosorbent assay (ELISA) described in a previous publication. . The blood was allowed to clot overnight at 4 ° C and the serum was separated by low-speed centrifugation. the present investigation involved screening a large number of patients for anti-bovine collagen implant antibodies prior to bovine collagen implant therapy and then monitoring the responses. all values expressed as A. Clinically. To address the tatter question. and the significance of circulating anti-bovine collagen implant antibodies before treatment requires elucidation. In a previous study. both clinically and serologically. Bovine collagen implants The bovine collagen implants are commercially available suspensions of purified bovine dermal collagen (35 rag/m1) in phosphate-buffered saline. The number and extent of bovine collagen implant treatments received by each patient depended upon need and patient desire and was therefore quite varied. lo The fact that some individuals are presensitized to bovine collagen implants is well established and is the reason patients are subjected to skin tests prior to treatment. 2 × 2 . . .0-7. Suggestions have been offered concerning the involvement of diet or histocompatibility antigens in these immune reactions. these reactions are characterized by the development of edema. Clinical observations Throughout the study. were skin-tested with bovine collagen implant and screened for preexisting antibodies in the circulation against bovine collagen.956 McCoy et al Journal of the American Academy of Dermatology Table I. pH 7.090 0. *n = 150. . The sera were then divided into aliquots and stored at . although a previous study failed to demonstrate a correlation between human lymphocyte antigen (HLA) type and the occurrence of adverse reactions. A total of 150 individuals were screened for the presence of anti-bovine collagen implant antibodies prior to administration of implants. erythema.064 therapy ~ and.2 0 ° C.212 0. All patients were free of any contraindications for bovine collagen implant Statistical analyses Positive sera were defined as those yielding A405values in ELISA greater than 2 SDs above the mean of the pretreatment sera at two consecutive dilutions.4% of the untreated patients. 11 The purpose of the current study was to examine the prognostic significance of anti-bovine collagen implant antibodies in untreated individuals. ELISA: Enzyme-linked immunosorbent assay. The cause or route of this presensitization has yet to be determined. Blood was drawn prior to implantation of the skin test and before each bovine collagen implant treatment. induration. with type III collagen comprising the majority of the remaining material.036 0.009 0. . m Sera were assayed in tenfold dilutions ranging from undilute to 1 : 1000.092 0. and/or urticaria at implantation sites that lasted longer than 24 hours. neity involved in immune responses to the bovine collagen implants.~ measurements. .516 0. patients were closely observed for the onset of adverse reactions to bovine collagen implant that might be of an immune nature. Summary of ELISA data from pretreatment sera* Dilution o f sera .300 0. MATERIALS AND METHODS Serum samples Blood was obtained from patients by routine venipuncture under aseptic conditions into tubes free of heparin.028 0.3% lidocaine.4 with 0.018 0. Undiluted 10 102 liP Mean SD Mean ± 2 SD 0. positive serology for anti-bovine collagen implant antibodies was observed in 8. prior to treatment. Approximately 95% of the collagen present in these implants is type I.060 O. SD: standard deviation. Patients and bovine collagen implant treatments Individuals selected for participation in this study were patients seeking bovine collagen implant therapy at the Dermatology Outpatient Clinics at the University of Michigan or Duke University.

Relative risks were calculated using the method of Kahn. six had positive skin tests and were excluded from receiving bovine collagen implant therapy. Another 21 did not receive treatment for a variety of reasons. Antibody levels Greater than N+2SD Less than g + 2SD n = 129 2 109 1 5 2 10 p = 0. It should be emphasized that the correlation is not absolute--half (2 of 4) o f the patients with pretreatment anti-bovine co!lagen implant antibodies and negative skin tests did not develop adverse reactions. As can be seen in Tables IV and V. Thus 123 patients received therapeutic injections of bo~)ine collagen implant. Based upon the observed frequencies of subsequent adverse treatment reactions (excluding patients with positive skin tests). although followup blood was obtained from five of these patients.6 RESULTS Table 1I. Subsequent injections were given as necessary in each case. Initial treatments typically consisted of ~ 1. five pretreatment sera were defined as positive. . The results of the ELISAs on pretreatment sera and skin tests are summarized in Table I.047).0 ml given approximately 4 weeks after skin testing. adverse reactions were most often accompanied by anti-bovine collagen implant antibodies (even if the patient's test results were negative for antibodies prior to treatment). Contingency table indicating the prognostic value of pretreatment levels of circulating anti-BCI antibodies Positive reactions No reaction Skin test Treatment reaction Immune responses prior to bovine collagen implant therapy A total of 150 patients were bled and skin-tested prior to potential bovine collagen implant therapy. Three of the five patients with positive pretreatment serology suffered adverse reactions. ~ T tests were done to test for differences between mean age and mean amount of Zyderm injected in the different groups. Table II indicates the correlation between positive pretreatment antibodies and positive skin tests or treatment reactions.Volume 16 Number 5. thus the number and volume of injections. as well as interval between injections. blood was obtained from 8! patients during the course of treatment. Twelve of these 123 patients suffered adverse reactions during the course of therapy (Table III). Immune responses during bovine collagen implant therapy Of the initial 150 patients. In other words. Statistical analyses were performed to determine the risk ratios of patients eventually suffering adverse treatment reactions based upon their pretreatment levels of anti-bovine collagen implant antibodies. Of the 123 patients receiving bovine collagen implant therapy. Positive sera were then defined as those that yielded ELISA values greater than the mean and 2 SDs at two consecutive dilutions of all of the pretreatment sera. Part 1 May 1987 Immune responses to bovine collagen implants 957 contingency tables were used to analyze whether any association existed between pretreatment serology and subsequent clinical reactions. one had a positive skin test and two suffered adverse treatment reactions. The existence of pretreatment antibodies did lead to an increased incidence of adverse reactions. varied from patient to patient. In this manner.'4 p values were calculated from Fisher's t test to determine statistical significance.019 (Fisher's exact tes0 BCI: Bovine collagen implant. Elevation of posttreatment levels of circulating anti-bovine collage n implant antibodies was observed in individuals receiving bovine collagen implant therapy without overt adverse reactions as Well as in those patients with adverse . it was determined that comparison of patients with pretreatment serology values greater than the mean plus 2 SDs at two consecutive dilutions with the remaining patients yielded a risk ratio of 5. Using BMDP statistical software. patients with elevated pretreatrnent antibovine collagen implant antibodies and negative skin tests were approximately six times more likely subsequently to suffer hdverse treatment reactions than the rest of the patients with negative skin tests.95 (p = 0. Levels of anti-bovine collagen implant circulating antibod- ies were monitored by ELISA.

erythematous. swells with direct exposure to sun Persistent (>2 wk). swollen. persistent (>4 wk). Anti-bovine collagen implant antibodies may also be found in patients prior to treatment or patients may develop elevated levels of anti-bovine collagen implant antibodies without concomitant overt adverse reactions.958 McCoy et aI Journal of the American Academy of Dermatology Table III. swollen red lump. edema. Most often these reactions occur after the first or second injection and are accompanied by the appearance of antibodies against the bovine collagen implants. however. swollen Localized. erythematous. firmness over injection site. erythematous Localized. Demographic information A survey was conducted to determine if significant associations existed between positive serology or adverse reactions and any of the following parameters: age. blood type. swollen Erythematous. localized BCI: Bovine collagen implant. reactions. and race. erythematous. The use of partially digested collagen lacking the nonhelical amino and carboxy terminals undoubtedly reduces the antigenicity of the implants. the increased frequency of elevated antibodies in patients with adverse reactions was statistically significant compared with treated patients not suffering adverse reactions. finn. erythematous. urticaria. erythematous. leading to this heterogeneity in immune responsiveness to bovine collagen implants remains to be delineated.l"3 These reactions include erythema. pain. persistent (>6 wk). induration. volume of bovine collagen implant received. and granulomatous reactions at the site of injection. sex. Only a small percentage of bovine collagen implant-treated patients suffer overt adverse reactions.'° The underlying factor. pruritus. test site involvement Localized. No association was found between any of these parameters and serologic or treatment reactions. erythematous. erythematous. erythematous. firm. swollen Intermittent. erythematous. of BCI Patient injections Interval between last injection and treatment reaction Interval between initial [ BCI injection and treatment reaction Description of reaction 1 1 7 wk 7 wk 2 3 4 5 6 7 8 9 10 11 12 1 2 4 2 2 1 6 1 2 3 2 9 wk 4 wk 4 wk 1 day 1 wk 6 wk 2 rno 4 wk 7 wk 3 wk 8 wk 9 wk 8 wk 11 mo 18 mo 3V2 mo 6 wk 2 yr 4 wk 9 wk 3 mo 4 mo Localized. swollen. continuous. Our data indicate the heterogeneity of immune responsiveness to bovine collagen implants among a large patient population. DISCUSSION The therapeutic utility of bovine collagen implants is clear. or factors. swelling. numerous reports appear in the literature describing adverse treatment reactions that are apparently of an immune nature. palpable lump Localized. swollen Persistent (several wk). intermittent Persistent (> 1 mo). swelling. granulomatous Localized. Characteristics of clinical reactions No. swollen. localized. granulomatous Persistent (14 wk). soft tissue augmentation can be accomplished with minimal risk and discomfort. Previous work found no apparent differences in the nature or specificity between anti-bovine collagen implant antibodies from patients with adverse reactions and from serologically positive patients not suffering overt reactions. however. Recent data from our laboratory indicated no precise human lymphocyte antigen (HLA) association with .

and seven of 18 patients with elevated anti-bovine collagen implant antibodies following treatment did not have any evidence of an adverse clinical reaction. Part 1 May 1987 Immune responses to bovine collagen implants 959 the overt adverse reactions nor with the appearance of circulating anti-bovine collagen implant antibodies. most adverse reactions occurred early in the course of therapy. This indicates that examination of the humoral responses does not accurately reflect cellular immunity in all instances. diet has been suggested as one of the factors underlying the heterogeneity in immune responses to bovine collagen implant. BCI: bovine collagen implants.17 Among other possibilities. As stated above. and vice versa. yet the measurement of anti-bovine collagen implant antibodies reflects the humoral arm of the immune system. Thus.0001 (Fisher's exact test) ment reaction. n: number of samples.Volume 16 Number 5. ELISA: enzyme-linked immunosorbent assay. Skin test reactions and adverse clinical reactions are primarily manifestations of the cellular arm of the immune system.and posttreatment anti-BCI antibody in patients with or without adverse treatment reactions Table Pretreatment Posttreatment Ab Clinical response (11) determinations {n) Ab determinations (n) + - Nonreactors (69) Reactors (12) + - (1) (68) (7) (62) + - (2) (lo) + - (11) (1) Ab: Antibody. although statistically significant differences in frequency of loci were observed between reactors and nonreactors. In the present study. While of great value in reducing the incidence of adverse treatment reactions. Previous work in our laboratorf indicated that some individuals have elevated levels of anti-bovine collagen implant antibodies prior to exposure to bovine collagen implants. Increased length of treatment or volume of bovine collagen implants did not influence the rate of adverse reactions." No requirement for HLA-DR4 to respond to bovine collagen implant was observed as could be suggested by previous work. The significance of these antibodies is unclear. Contingency table indicating the correlation between posttreatment levels of circulating anti-BCI antibodies and clinical response Table Adverse clinical reaction No 1 Yes Antibody levels Greater than ~ + 2 SD Less than ~ + 2 SD n = 81 BCI: Bovine collagen implants. the . Currently no data exist to substantiate this possibility. ELISA determination of pre. In contrast. both branches of the immune system appear to be involvedqas in this study in which 11 of 12 patients with adverse reactions also displayed elevated anti-bovine collagen implant antibodies. skin testing does not eliminate the occurrence of treatment reactions. it would seem that most patients suffeting adverse treatment reactions had in some way been "primed" to bovine collagen since characteristics associated with immunization (such as repeated injection with small amounts of antigen) did not appear pertinent to this study. no demographic parameters were found that were associated with increased risk of adverse reactions. Pretreatment skin testing is one method of identifying patients who are presensitized to bovine collagen implants. five of six patients with positive skin tests did not have elevated pretreatment anti-bovine collagen implant antibodies. In cases of adverse treatment reactions. V. Although the number of patients in the current study was not as large as would be desired. Humoral responses may occur without overt evidence of cellular responses. 7 11 62 l p < 0. The pivotal concern Of this study was whether there was sufficient overlap between the two arms of the immune response in recognizing bovine collagen implants for the serologic studies to be of value in identifying patients at higher risk of suffering an adverse reaction. two of five patients with positive pretreatment serology did not develop a positive skin test nor an adverse treat- IV.

McCoy JP. I. Siegle RJ. . New York: Oxford University. Ban" RJ. McPherson J. less than 2% of the patients would have been erroneously denied treatment. Sawamura S. 12.8:111-4. five of 129 patients would not have received therapy based upon serology. McCoy JP. In the current study.119:533-4. 4. Delayed skin test reaction to injectable collagen implant (Zyderm). Arch Dermatol 1986. J AM Acho DERMATOL 1984. Necrobiotic granulomas associated with bovine collagen test site injections. 7. J Dermatol Surg Oneol 1982. Department of Biomathematics. A retrospective review of 72 tested and treated patients. two eventually suffered adverse treatment reactions. McCoy JP.. REFERENCES 1.136:877-82. The nonrandom nature of the study may also have contributed to the apparent incidence of adverse reactions. Characterization of the humoral immune response to bovine collagen implants. Stobo JD. Vanderveen EE. particularly pretreatment serology. 1983. BMDP statistical software. 5. Medenhall W. Elevated pretreatment antibodies identified only two of the 12 patients with subseqtient adverse treatment reactions. Intradermal implantation of bovine collagen: humoral immune responses associated with clinical reactions. Kamer FM. Arch Dermatol 1985. Immune response gene control of collagen reactivity in man: collagen unresponsiveness in HLA-DR4 negative nonresponders is due to the presence of T-dependent suppressive influences. Zyderm Collagen Implant--Physician reference guide. 9. Swanson NA. 8. An introduction to epidemiologic methods. CA: Collagen Corp. Vanderveen EE. Clearly it could serve only as an adjunct to skin testing in the conservative management of patients. Stegman SJ. Berkley: University of Califomia Press. Michaeli D. The association of HLA and immune responses to bovine coll~tgen implants. while the number of adverse treatment reactions would have been reduced by almost 20%. Hanke CW. although it must be remembered that skin testing missed all twelve. but not eliminate. Patients participating in the study were interested in close evaluation of their bovine collagen implant therapy and may have been more likely to report self-observed adverse reactions that were later confirmed in a clinic visit. Injectable collagen implants. the incidence of adverse treatment reactions. Stoner JG. Of these five. 1975. 1981. Waldinger TP. 15. Thus in the current study. I6. King DF. This is reflected by the rather substantial attrition rate in this study. 10:638-46. Brooks N. Dixon WJ.6:867-9. Siegle RJ. 1982.6%) would have been denied treatment unnecessarily.120:183-7. Brennan JE.110:93-8. Arch Dermatol 1983. has a place in the management of patients undergoing bovine collagen implant therapy.129:1916-20. The human immune response to reconstituted bovine collagen. The immunogenieity of injectable collagen. Kahn HA. 121:990-4. II. Palo Alto. J Immunol 1982. Schade WJ. Solomon AR. Swanson NA. Of these four. Bartlow GA. Churukian MM. J Dermatol Surg Oncol 1983. Thus prescreening prospective pa- tients desiring bovine collagen implant therapy by skin testing and pretreatment serology should reduce. Ellingsworth LR. It would th~is appear that serology. MA: Duxbury Press. 14. J AM ACAD DERMA'rOt. UCLA. 1981.10:652-8. if positive pretreatment serology were used as a contraindication of treatment. A l-year prospective study. Cooperman L. 10. Cooperman L. Solinger AM. et al. J AM ACAD DERMATOL 1984. Treatment site reactions to Zyderm collagen implantation.9:377-80. 17. 6. 13. J Immunol 1986. Arch Otolaryngol 1984. Robinson JK. 11. 3. while two Of 12 reactors (17%) would have been identified prior to treatment. one suffered a positive skin test and would not have received treatment On that basis. Swanson NA. Introduction to probability and statistics.122:650-4. North Soltuate. Schade W. Patient participation was strictly voluntary and there was great variability in patient cooperation and interest. Siegle RJ.10: 647-51. McDonald RM. Thus only i~wo patients of 129 (1.960 McCoy et al Journal of the American Academy of Dermatology data show that patients with elevated pretreatment levels of anti-bovine collagen implant antibodies were approximately sixfold more likely to suffer an adverse treatment reaction. DeLustro F. J AM ACAD D~RMATOL 1984. R should be noted that this study was not orchestrated in a truly randomized fashion and the incidetlce of adverse clinical reactions should not be considered representative of our actual overall experience. Therefore four patients would have been excluded from receiving bovine collagen implaiat therapy who normally would have received treatment. Barr RJ. The immunogenicity of injectable collagen. 2. Schade W. A foreign body granuloma produced by an injectable collagen implant at a test site. Arch Dermatol 1984. Clinical use of injectable collagen: a three year retrospective review. Michaeli D.