ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 16, No. 4 Copyright © 1986, Institu te for Clinical Science, Inc.

Avitene Granulomas of Colonic Serosa*DOUGLAS H. McGREGOR, M D .t RICHARD I. MacARTHUR, M D ,t and TONY CARTER, MD§

Laboratoryf and Surgicalt§ Services, Veterans Administration Medical Center,

Kansas City, MO 64128 and Department of Pathology and Oncologyt

and Department of Surgery,$ University of Kansas Medical Center,

Kansas City, KS 66103

ABSTRACTA vitene, m icrofibrillar collagen hem ostat, is an absorbable topical

hem ostatic agent prepared from purified bovine corium collagen. A case is reported of a 58-year-old man who, one m onth after sigmoid colectomy for volvulus in which Avitene was used to control bleeding from a splenic capsular tear, developed symptoms of intestinal obstruction. On explor­atory laparotomy, the transverse and descending colon had num erous 0.1 to 2.0 cm nodules, adherent to the serosa of the bowel and mesentery, with kinking and partial obstruction of the splenic flexure. Light micro­scopically, the nodules w ere found to be composed of chronic inflamma­tory and granulom atous reaction with num erous deposits of eosinophilic fibrillar m aterial consistent with Avitene. Electron microscopic examina­tion of the lesions and of Avitene confirmed the nature of this material. The practical and pathobiological implications of utilization of Avitene as a hem ostatic agent are discussed.

IntroductionA v ite n e , m ic ro f ib r i l la r c o lla g e n

h e m o s ta t, is an a b so rb a b le to p ica l hemostatic agent p repared as a dry, ster­ile , f ib ro u s, w a te r in so lu b le p a rtia l hydrochloric acid salt of purified bovine corium co llagen .2,5,12 This substance, when in contact with a bleeding surface, attracts p la te le ts w hich ad h ere to the

* Address for correspondence: Dr. D. H. McGre­gor, Laboratory Service, Veterans Administration Medical Center, 4801 Linwood Boulevard, Kansas City, MO 64128.

fibrils and undergo the release phenom ­enon to trigger aggregation of platelets into throm bi in the interstices of the col­lagenous m ass.7 Avitene is indicated for use in surgical procedures as an adjunct to hemostasis w hen control of bleeding by ligature or conventional procedures is ineffective or im practica l.6,12,14 It has been found especially useful in control­ling generalized b leeding from raw or friable tissues, bleeding from highly vas­cularized organs, b leeding from cancel­lous bone, and bleeding around vascular anastomoses.

2960091-7370/86/0700-0296 $01.20 © Institute for Clinical Science, Inc.

A V IT E N E G R A N U L O M A S O F C O L O N 2 9 7

The case reported here is an unusual example of intestinal obstruction caused in p a rt by reac tio n to A vitene. This m icrofib rilla r co llagen h em o sta t had been used one m onth previously during surgery on this patien t to control b leed­ing from a small splenic capsular tear. Pathologically, the cause of the marked foreign body granulom atous reaction, chronic inflam m ation and fibrosis in the colonic serosa was determ ined by light and electron m icroscopic study of the colonic serosal lesions, in com parison w ith lig h t an d e le c tro n m icroscop ic study of commercially available Avitene.

Case HistoryThe patient was a 58-year-old black male. One

month prior to admission, he had a reduction of sig­moid volvulus and a sigmoid colectomy with end-to- end colocolostomy. During this procedure, a small splenic capsular tear had been noted, and bleeding from this tear had been successfully controlled with Avitene and pressure. The Avitene had been applied with forceps and packed with a sponge. On the day of admission the p a tien t developed acute onset of abdominal pain and vomiting. Physical examination demonstrated moderate abdominal distention with tenderness on p ressu re and hyperactive bowel sounds. Upright abdominal x-ray showed distention of small bowel loops.

Exploratory laparotomy was performed, and two problems were identified. Firstly, the ascending colon was attached by adhesions to the sigmoid colon anastomatic region resulting in an internal hernia and consequent small bowel obstruction; these adhesions were taken down, and one was biopsied. Secondly, the mid-transverse to m id-descending colon had numerous 0.1 to 2.0 cm nodules, adherent to the serosa of the bowel and mesentery, with kinking and partial obstruction of the splenic flexure. Three of these nodules were biopsied. The splenic capsular tear was noted to be well healed. Postoperative course was uneventfu l and th e pa tien t was dis­charged three weeks after surgery free of abdominal or gastrointestinal symptoms. There has been no fur­ther evidence of intestinal obstruction during the subsequent four years.

Materials and MethodsSpecimens for light microscopic study,

in c lu d in g p o rtio n s of one ascen d in g colonic adhesion, th ree splenic flexure nodu les, and com m ercia lly o b ta ined A vitene, w ere fixed in form alin, ro u ­

tinely processed for histologic sectioning and s ta in ed w ith hem atoxy lin -eosin , M asson’s trich ro m e, phospho tungstic ac id -h em a to x y lin , an d p e rio d ic acid Sell ill {PAS).

Specim ens for e lec tron m icroscopic study, including portions of ascending colonic adhesion, splenic flexure nod­ules, and com m ercially ob ta ined Avi­tene, w ere fixed in four percent glutaral- dehyde in 0.1 M phosphate buffer at pH 7.4. These w ere postfixed in one percent osmium tetroxide in 0.1 M phosphate buffer, routinely processed, and em bed­ded in epoxy resin. U ltrathin sections were stained with a saturated solution of uranyl acetate and lead citrate and exam­ined in an electron microscope.

ResultsG r o s s Pa t h o l o g y

The specim en consisted of th ree ovoid nodules of soft tissue and one irregular elongated piece of soft tissue (figure 1). The th ree ovoid nodules (obtained from transverse colonic serosa) m easured 2 X l x l cm ., 1.5 X 1 x 1 cm. and 0.9 X 0.6 x 0.5 cm in greatest dim ensions. They had an irregular m ottled pale red ­dish brown and yellow external surface, and a rubbery hom ogeneous pale tan cut su rface . T h e o n e e lo n g a te d n o d u le (obtained from the sigmoid colon anasto­motic region) m easured 1.3 X 0.5 X 0.4 cm and was similar in color.

L ig h t M ic r o s c o p y

Hematoxylin-eosin stained sections of the th ree transverse colonic specimens (figures 2, 3, and 4) showed serosal adi­pose tissue extensively involved by large and sm all n o d u la r p ro life ra tio n s of fibrous tissue with m oderate to marked chronic inflam m atory cell infiltrate and num erous sm all and large deposits of eosinophilic m ateria l frequen tly asso-

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ciated with foreign body giant cells. The inflam m atory cell infiltrate was mainly com posed of eosinoph ils w ith in te r ­spersed plasm a cells, lym phocytes and histiocytes. The eosinophilic m aterial was variably am orphous and coarsely fibrillar and occurred as m inute to large irregular accumulations. The color of this m aterial varied from in tensely eosino­philic (similar to normal collagen) to less eosinophilic to somewhat pale gray and faintly basophilic. The m ore intensely eosinophilic areas tended to appear blue on trichrom e stain and under polarized light to have a pink birefrigence similar to normal collagen, while the less eosin­ophilic areas tended to lack these collag­enous ch arac teris tics . No fib rin was identified in PTAH-stained sections.

Hematoxylin-eosin stained sections of the sigmoid colon anastomotic specimen show ed serosal ad ipose tissu e e x ten ­sively involved by fibrosis with scattered capillaries, patchy recen t hem orrhage, minimal focal lymphocytic infiltrate and one suture granuloma. The intercellular m aterial in this specim en consistently dem onstrated a blue color on trichrom e s ta in and p in k b ire fr in g e n c e u n d e r polarized light, typical of normal colla­gen.

Hematoxylin-eosin stained sections of commercially available Avitene (figure 5) showed material similar to that seen in the transverse colonic specimens, except that it was less am orphous and m ore consistently composed of thick strands of intensely eosinophilic m aterial and nar­row coarse fibers of less intensely eosin­ophilic material with a faintly basophilic hue. This m aterial foeally stained blue w ith tr ic h ro m e s ta in an d th e th ick

intensely eosinophilic m aterial showed a pink birefringence under polarized light.

E l e c t r o n M ic r o s c o p y

On electron microscopic study (figures 6, 7, and 8), the transverse colonic gran­u lom atous le s io n s w ere fo und to be m ainly com posed of h istiocy tes w ith sc a tte red eosinoph ils and occasional lymphocytes. The histiocytes generally had a m oderate to m arked increase in rough endoplasmic reticulum . The most notable feature of the lesions, however, was the num erous small and large irreg­ularly shaped accumulations of electron dense material, which was adjacent to, partially engulfed by and occasionally phagocytized by the histiocytes. This material was composed of regular cylin­drical, m oderately electron dense fibers, approximately four times the d iam eter of typical collagen, within a small am ount of highly electron dense matrix.

U ltrastructural study of commercially o b ta in e d A v iten e (figure 9) d e m o n ­strated it to be com posed of two main forms of fibrillary material: (1) discrete b und les of cy lind rica l p a ra lle l fibers about four times the d iam eter of normal collagen, on cross section having a dark central core and pale outer layer and on longitudinal section having a periodicity much finer than that of normal collagen; and (2) loosely a rra n g e d cy lin d rica l fibers, sim ilar to those com posing the above discrete bundles except for being more electron-dense and less parallel in arrangem ent. There was focal transition betw een the d iscrete bundles of fibers and th e loosely a rran g ed fibers. T he fibrillary material which had been seen

F i g u r e 1. External (a) and cut (b) surfaces of three nodules removed from transverse colonic serosa. The fourth tissue (lower right) is fibrous tissue removed from sigmoid colon anastomotic region.

F i g u r e s 2 , 3 AND 4 . Medium, low, and high power views of nodule from transverse colonic serosa. Multiple irregular deposits of amorphous and coarsely fibrillar material are dispersed about fibrous tissue with granulomatous reaction and chronic inflammation.

F i g u r e 5 . H i g h p o w e r v i e w o f s e c t i o n o f c o m m e r c i a l l y a v a i l a b l e A v i t e n e , s i m i l a r in a p p e a r a n c e t o t h e m a t e r i a l i n f ig u r e 4 .

300 M cG R E G O R , M acA R T H U R , A N D C A R T E R

FIG U R ES 6, 7, AND 8. Electron microscopic appearance of nodule from transverse colonic serosa. Histio­cytes with prom inent rough endoplasmic reticulum are associated with intracellular and extracellular deposits of cylindrical fibrillary material. The fibers have a diam eter approximately four times that of typical collagen, (figure 6, x 2810; figure 7, x 8960; figure 8, x 14180.

Figure 9. E lectron microscopic appearance of commerically available Avitene. The fibers have dim en­sions similar to those in figures 7 and 8, but also have some features of typical collagen fibers. (X 8820).

A V IT E N E G R A N U L O M A S O F C O L O N 30 1

in the pericolonic granulomas was simi­lar to the loosely arranged fibers of Avi- tene, except for the former fibers being em bedded in a small am ount of highly e le c tro n -d e n se m atrix and h av ing a finely m icrofibrillary tex ture w ith very little evidence of periodicity on longitu­d inal section . T he p rev ious findings w ere felt to be consistent with the con­clusion that the pericolonic granulomas rep resen ted a foreign body reaction to Avitene.

Electron microscopic study of the sig­moid colonic anastomotic specimen (per­icolonic fibrotic area separate from the transverse colonic granulomas) dem on­stra ted reactive fibrocyte proliferation and collagen deposition. The collagen fibers w ere typical in diam eter and peri­odicity and had a d iam eter about four times less than Avitene and the material in the granulomas. This was felt to indi­cate that the nature of the collagenous m aterial com posing A vitene (and the material in the transverse colonic granu­lomas) was different from normal colla­gen present in the patien t’s sigmoid p er­icolonic fibrosis.

DiscussionIn a d v e rta n t sp len ic laceratio n is a

com m on co m p lica tio n of le ft u p p e r quadrant surgery, and this has frequently been successfully treated by the topical application of Avitene9,10, as in the case reported here. Adverse reactions to Avi­tene have been reported, and the most serious of these appear to be potentiation of infection (abscesses, etc.), hem atom a and wound dehiscence. O ther reported a d v e rs e re a c t io n s in c lu d e a lle rg ic responses, adhesions and foreign body reactions.3,11,13,14 These last three, par­ticularly foreign body reaction, appear to have b e e n in v o lv e d in th e p re s e n t reported case. In one reported series of 811 patients treated with Avitene, there w ere 103 instances of adverse responses,

including two w ith foreign body reac­tions and three with adhesions.14 Thus it can be seen that such complications are relatively infrequent.

The cause of the unexpected adverse response to Avitene which occurred in this case is difficult to ascertain exactly b u t is likely re la ted to severa l factors such as immunologic reactivity and in tra­o p e ra tiv e te c h n iq u e . Im m u n o lo g ic h y p e rre a c tiv ity is su g g e s te d by th e marked infiltration of eosinophilic leuko­cytes involving all of the biopsied Avi­tene granulomas. Such an eosinophilic infiltrate has not been rep o rted to be characteristic of th e usual local tissue response to A vitene,15 or, for that m at­ter, to o ther injectable bovine collagen preparations.1 This peculiar response in the present case appears to represen t a local manifestation of a systemic im m u­nologic resp o n se since rev iew of th e patien t’s laboratory data revealed that h is p e r i p h e r a l b lo o d e o s in o p h i l s increased from zero to one percent at the tim e of th e first surgery and A vitene tre a tm e n t to five to 15 p e rc e n t one month later. The few apparent allergic reactions to Avitene reported in patients have sim ply consisted of e ry th em a or p r u r i t i s .14 F u r th e rm o re , e x te n s iv e immunologic studies in animals (guinea pigs, rabbits and dogs) and hum ans have indicated that allergic reactions to Avi­tene should be rare and of little signifi­cance. It is unknow n why th e patien t reported here had developed an im m u­nologic re a c tiv ity w h ich a p p a re n tly resulted in the local and systemic eosin- ophilia.

A n o th er cause of th e u n e x p e c te d a d v e rse re a c tio n to A v ite n e w h ich occurred in this case was likely related to the technique involved in Avitene appli­cation. The num erous nodu les no ted during the second operation w ere adher­en t to the serosa and m esentery from m id-transverse to m id-descending colon. This w ould indicate a m ore extensive

3 0 2 M c G R E G O R , M acA R T H U R , A N D C A R T E R

application of Avitene than was required to trea t the splenic capsular tear, p e r­haps in part due to colonic serosa and m esentery inadvertently coming in con­tact with the site of splenic application. The prevention or lessening of the occur­rence of this problem appears to now be available, w ith th e d ev elopm en t of a m ethod of preparing Avitene into a dis­crete firm pledget using a syringe.4

O ne im p o rtan t aspect of th is case involves the identification of a material which appears to be eliciting a foreign body g ranu lom atous reac tio n . R efer­ences such as th e ex ce llen t Particle Atlas8 are one available approach bu t in the p resen t case w ere of little help in solving this problem . In this case the steps which yielded the identification of the material included: (1) review of the o p era tiv e re p o rt and com m unication betw een the pathologist and surgeon, resulting in inform ation which circum ­stantially incrim inated Avitene; (2) light m icroscopic exam ination w ith rou tine a n d sp e c ia l s ta in s , c o m p a r in g th e patient’s biopsy m aterial with com m er­cially available Avitene; and (3) electron microscopic examination, comparing the patient’s biopsy m aterial and Avitene.

In sum m ary, A vitene (m icrofibrillar collagen hemostat) can be a highly b ene­ficial agent as an adjunct to hemostasis when control of bleeding by ligature or conventional procedures is ineffective or impractical. Surgeons, however, should be aw are of p o ten tia l com plications, which in the present case w ere probably re la ted to im m unologic reactiv ity and o p e ra tiv e te c h n iq u e . F u r th e rm o re , pathologists should be aware of the histo- morphologic appearance of this material which may resu lt in bowel obstruction and other complications.Acknowledgments

Thanks are extended to William J. Bopp for assis­tance in electron microscopic processing, the Histol­ogy Section of the Laboratory Service for assistance in histologic processing, and Brenda Adams, Irene Gray and Angie Allen for secretarial assistance.

American Critical Care provided the Proceedings ofa Symposium on Avitene. Ultrastructural studieswere performed using the facilities of the ElectronM icroscopy Unit, L aboratory Service, VeteransAdministration Medical Center, Kansas City, MO.

References1. B a r r , R. J ., K i n g , D . E , M c D o n a l d , R. M .,

and B a r t l o w , G. A.: Necrobiotic granulomas associated with bovine collagen test site injec­tions. Am. J. Acad. Dermatol. 6:867-869, 1982.

2. B a t t is t a , O. A.: The chemistry and preparation microfibrillar collagen hemostat. Proceedings o f Symposium on Avitene Microfibrillar Collagen Hemostat. San Francisco, Am erican Critical Care, 1975, pp. 38-40.

3. C h u n g , A. F., M e n o n , J., and D i l l o n , T. F.: Acute postoperative retroperitoneal fibrosis and ureteral obstruction secondary to the use of Avi­tene. Am. J. Obstet. Gynecol. i32:908-909, 1978.

4 . G o r d o n , L . A., S h a p i r o , S . J . , and F e i n b e r g , S. E.: Microfibrillar collagen hemostatic pled­gets. Arch. Surg. 116:359-360, 1981.

5. H a it , M. R.: Microcrystalline collagen: A new hemostatic agent. Am. J. Surg. 120:330, 1970.

6. JO H N SO N , L. P.: A review of the use of Avitene in otolaryngologic surgery. Otolaryngol. Head Neck Surg. 88:8-9, 1980.

7. M a s o n , R .: Hemostatic mechanisms of microfi­brillar collagen. Proceedings of Symposium on Avitene Microfibrillar Collagen Hemostat. San Francisco, American Critical Care, 1975, pp. 32-35.

8 . M c C r o n e , W . C . and D e l l y , J. C .: The Particle Atlas: Encyclopedia of Techniques for Small Par­ticle Identification, 2nd ed. Ann Arbor, Ann Arbor Science Publishers, 1973.

9. M O R G EN STERN , L .: Splenic and general surgery. Proceedings of Symposium on Avitene Microfi­b rilla r C ollagen H em osta t. San Francisco, American Critical Care, 1975, pp. 8—10.

10. M ORGEN STERN , L .: The avoidable complications o f sp len e c to m y . S urg . G y n eco l. O b s te t. 145:515-528, 1977.

11. P a r k , S. A., G i a n n a t t a s i o , C. and T a n c e r , M. L.: Foreign body reaction to the intraperi- toneal use of Avitene. Obstet. Gynecol. 58:664— 667, 1981.

12. Physicians Desk Reference, 37th ed.: Medical Economics Company, Oradell, NJ, 1983, p. 578.

13. R a o , P. S., C a v e n a u g h , D., R i c h m a n , A. V., and F r e n c h m a n , S. M.: An evaluation of the tendency of Avitene to produce periureteral fibrosis in the dog. Am. J. Obstet. Gynecol. 745:87-91, 1983.

14. T e n e r y , J. H.: A summary. Proceedings of Sym­posium on A vitene M icro fib rillar C ollagen Hemostat. San Francisco, American Critical Care, 1975, pp. 46-47.

15. Z e l e z n i c k , L. D.: Toxicity and antigenicity of microfibrillar collagen hemostat. Proceedings of Symposium on Avitene Microfibrillar Collagen Hemostat. San Francisco, American Critical Care, 1975, pp. 41—44.