Upsala J Med Sci 90: 163-168, 1985 Fine-needle Biopsies of Renal Tkansplants in Clinical Rejection Monitoring Jan Wahlberg,' Ulla Backman,2 Lars Frodin,' Bjorn Stenkvist3 and Gunnar Tufveson' Departments of 'Urology, 2Nephrology and 3Pathology, University Hospital, Uppsala, Sweden ABSTRACT F i n e - n e e d l e a s p i r a t i o n b i o p s y (FNAB) o f r e n a l a l l o g r a f t t r a n s p l a n t s has been used a t Uppsala U n i v e r s i t y H o s p i t a l f o r 3 y e a r s . E x p e r i e n c e from 5 1 c o n s e c u t i v e p a t i e n t s ( f r o m 1 4 y e a r s ) w i t h 333 FNAB was r e v i e w e d . R e p r e s e n t a t i v e m a t e r i a l was o b t a i n e d i n more t h a n 70 5 o f t h e b i o p s i e s . E l e v e n r e j e c t i o n e p i s o d e s i n 1 2 p a t i e n t s were c o n f i r m e d w i t h t h i s method. One was n o t r e c o g n i z e d . S i g n i f i c a n t i n f l a m m a t i o n i n t h e k i d n e y w i t h o u t c l i n i c a l r e j e c t i o n was found i n 22 p a t i e n t s . The p o s s i b l e causes o f such i n f l a m m a t i o n a r e d i s c u s s e d . Repeatedly r e c o r d e d i n - f l a m m a t i o n i n t h e k i d n e y w i t h m i n o r or no e f f e c t on g r a f t f u n c t i o n may some- t i m e s be caused b y v i r a l i n f e c t i o n . The c l i n i c a l v a l u e o f FNAB i n v a r i o u s i m - munosuppressive regimens i s d i s c u s s e d . INTRODUCTION F i n e - n e e d l e a s p i r a t i o n b i o p s y (FNAB) as a d i a g n o s t i c t o o l i n c l i n i c a l r e n a l t r a n s p l a n t a t i o n was f i r s t r e p o r t e d b y P a s t e r n a c k e t a l . i n 1973 ( 9 ) , and t h e method was r e f i n e d by Hayry & von W i l l e b r a n d ( 4 ) . I n 1 9 8 1 Uppsala became one o f t h e f i r s t f i v e t r a n s p l a n t c e n t r e s t o use t h i s method, w h i c h has g a i n e d c l i n i c a l acceptance and c u r r e n t l y i s used i n a t l e a s t 50 t r a n s p l a n t c e n t r e s t h r o u g h o u t t h e w o r l d . Two i n t e r n a t i o n a l workshops have been c o n d u c t e d on t h e i s s u e ( 7 ) . Much e f f o r t has been d e v o t e d t o i n v e s t i g a t i n g t h e r e l i a b i l i t y o f FNAB i n r e n a l t r a n s p l a n t s and t o r e f i n e m e n t o f t h e c y t o l o g i c d i a g n o s i s , e.g. u s i n g immune h i s t o c h e m i c a l t e c h n i q u e s ( 3 ) . L e s s i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g t h e i m - p a c t o f d i f f e r e n t immunosuppressive regimens on t h e method's c l i n i c a l u s e f u l - ness. I n s t e a d o f comparing FNAB r e s u l t s w i t h t h e h i s t o l o g i c r e p o r t s , we have re- l a t e d them t o t h e c l i n i c a l s t a t u s o f t h e p a t i e n t s . A r e t r o s p e c t i v e r e v i e w o f o u r own e x p e r i e n c e i s now p r e s e n t e d . The i m p a c t o f immunosuppression w i t h l o w - dose s t e r o i d s and c y c l o s p o r i n or a z a t h i o p r i n e on FNAB was s t u d i e d . 163 MATERIAL AND METHODS P a t i e n t s The s t u d y c o m p r i s e d 5 1 c o n s e c u t i v e p a t i e n t s who had r e c e i v e d k i d n e y a l o , g r a f t and were i n v e s t i g a t e d w i t h FNAB d u r i n g t h e i r i n i t i a l h o s p i t a l s t a y R immunosuppressive r e g i m e n c o n s i s t e d i n 1 8 cases o f a z a t h i o p r i n e ( I m u r a n , Wellcome, London, UK) and low-dose s t e r o i d s (Aza) as o u t l i n e d by McGeown e t a l . ( 8 ) and 30 p a t i e n t s r e c e i v e d C y c l o s p o r i n R (Sandoz, B a s l e , S w i t z e r l a n d ) and low-dose s t e r o i d s (CyA) a c c o r d i n g t o t h e p r o t o c o l o f t h e S c a n d i n a v i a n M u l t i - c e n t e r T r a n s p l a n t Study ( 6 ) . Three p a t i e n t s were s w i t c h e d f r o m one r e g i m e n t o t h e o t h e r (Aza/CyA). C l i n i c a l r e e v a l u a t i o n The The c l i n i c a l c o u r s e was r e t r o s p e c t i v e l y r e e v a l u a t e d . A d i a g n o s i s o f r e j e c t - i o n was based on c l i n i c a l o b s e r v a t i o n s o f w e i g h t g a i n , f e v e r and p a i n o v e r t h e g r a f t c o n c o m i t a n t w i t h r i s i n g serum c r e a t i n i n e . I n a d d i t i o n , most o f t h e p a t - i e n t s responded w i t h f a l l o f serum c r e a t i n i n e t o supplementary s t e r o i d t r e a t - ment. F i n e - n e e d l e a s p i r a t i o n FNAB was p e r f o r m e d as d e s c r i b e d by Hayry & von W i l l e b r a n d ( 5 ) . B r i e f l y , a s p i n a l n e e d l e was i n t r o d u c e d under s t e r i l e c o n d i t i o n s i n t o t h e g r a f t and cells f r o m t h i s t i s s u e were a s p i r a t e d i n t o a s y r i n g e c o n t a ~ n ~ n g t i s s u e c u l t u r e medium. A c a p i l l a r y b l o o d sample was c o l l e c t e d a t t h e same t i m e t o p e r m i t c a l c u l a t i o n o f a c o r r e c t e d i n c r e m e n t . These b i o p s i e s u s u a l l y were p e r f o r m e d a t r e g u l a r i n t e r v a l s ( t h r i c e w e e k l y ) d u r i n g t h e i n i t i a l p m t o p e r a t i v e s t a y . Some b i o p s i e s were p e r f o r m e d l a t e r , a s need arose. T a b l e 1. Number o f b i o p s i e s a c c o r d i n g t o iwmunosuppressive r e g i m e n No o f T o t a l n o No o f n o n r e p r e s e n t - Treatment * p a t i e n t s o f FNAB a t i v e FNAB C Y A Aza Aza/CyA 30 190 56 18 106 31 3 37 5 T o t a l 5 1 333 92 * D e f i n i t i o n s i n M a t e r i a l and Methods 164 Evaluation of FNAB Cytologic evaluation of the biopsy specimens was performed as previously de- scribed (11). Essentially this method transposes the observed occurrence of in- flammatory cells within the graft to a numerical value called the corrected increment. A corrected increment higher than 2.9 was classified as significant inflammation. Only biopsies containing 10 o r more kidney tissue cells per 100 inflammatory cells were regarded as representative. RESULTS Table 1 summarizes the total number of biopsies and the number of nonrepre- sentative specimens in the three treatment groups. More than 70 per cent of the biopsies were representative. In this respect there was no clear difference ac- cording to immunosuppressive regimen. The number of biopsies per patient was highest in the Aza/CyA group, the reason being that these three patients had a more difficult and protracted postoperative course than the average. Table 2 . Number of patients with ( > 2 . 9 ) o r without ( < 2 . 9 ) inflamnation in FNAB at the start of clinical rejection Corrected increment Treatment > 2 . 9 < 2.9 CYA 0 0 Aza/CyA 2 1 ** Total with rejection 8 4 Aza 6 3 * * next biopsy positive in 2 of these patients ** next biopsy positive Table 2 presents the corrected increment in the cases with clinical signs of rejection. In 11 of the 12 episodes of rejection there was significant rise in corrected increnent. In three patients, however, the increment was below the significance level ( > 2 . 9 ) in the morning of the day when rejection started, but had risen when the next FNAB was taken. F o r the fourth patient without significant corrected increment no further FNAB information was available. Not- ably, there was no rejection episode in the CyA group. The three patients on Aza/CyA had rejection only when on the Aza regimen. 165 T a b l e 3. c o r r e c t e d i n c r e m e n t b u t w i t h o u t c l i n i c a l r e j e c t i o n s i g n s Number o f p a t i e n t s and number o f FNABs w i t h h i g h No o f h i g h i n c r e m e n t s / p a t i e n t No o f Treatment 1 2 3 4 5 >6 p a t i e n t s Aza 4 1 C Y A 5 6 Aza/CyA 1 2 a 1 12 1 2 The number o f h i g h c o r r e c t e d i n c r e m e n t s ( > 2.9) i n t h e absence o f r e j e c t i o n s i g n s i s shown i n T a b l e 3. A l t o g e t h e r 22 o f t h e 51 p a t i e n t s t h u s had a t l e a s t one such FNAB b u t no c l i n i c a l s i g n s o f g r a f t r e j e c t i o n . R e t r o s p e c t i v e a n a l y s i s o f a l l t h e s e o c c a s i o n s was n o t w i t h i n t h e scope o f t h e p r e s e n t s t u d y . However, c o n c o m i t a n t c y t o m e g a l o v i r u s i n f e c t i o n was known f r o m v i r a l i s o l a t i o n or con- v e n t i o n a l s e r o l o g y t o have been p r e s e n t i n t w o o f t h e f i v e p a t i e n t s w i t h a t l e a s t f o u r h i g h i n c r e m e n t s . F o r t h e o t h e r t h r e e p a t i e n t s no such i n f o r m a t i o n was a v a i l a b l e . DISCUSSION FNAB was e q u a l l y r e l i a b l e i n p a t i e n t s w i t h CyA and i n t h o s e w i t h Aza i m - munosuppression. More t h a n 70 p e r c e n t o f t h e r o u t i n e l y p e r f o r m e d b i o p s i e s y i e l d e d r e p r e s e n t a t i v e specimens. T h i s p e r c e n t a g e s h o u l d be i m p r o v a b l e w i t h use o f u l t r a s o n i c g u i d i n g , e s p e c i a l l y i f t h e k i d n e y i s d i f f i c u l t t o p s l p a t e . I f a r e p r e s e n t a t i v e specimen i s p a r t i c u l a r l y d e s i r a b l e , t h e p r o b a b i l i t y o f success can be i n c r e a s e d by making s e v e r a l b i o p s i e s . The p r o c e d u r e is i n no way h a r m f u l t o t h e p a t i e n t , i n o u r e x p e r i e n c e o r i n t h a t f r o m o t h e r c e n t r e s ( 7 ) . We have p e r f o r m e d more t h a n 700 b i o p s i e s w i t h o u t c o m p l i c a t i o n s a p a r t from b r i e f , m i l d h a e m a t u r i a . The main advantage o f a s p i r a t i o n o v e r " t r u - c u t " b i o p s y i s , i n f a c t , t h a t FNAB can be p e r f o r m e d as o f t e n as n e c e s s a r y , thus p e r m i t t i n g immunologic m o n i t - o r i n g o f t h e p a t i e n t . The c l i n i c a l r e l e v a n c e o f t h i s m o n i t o r i n g can p e r h a p s be debated. When t h e Aza im-nunosuppressive r e g i m e n was used, 11 o f 12 c l i n i c a l re- j e c t i o n e p i s o d e s were diagnosed. S i g n s o f i n f l a m m a t i o n i n t h e k i d n e y a r e n o t pathognomonic f o r r e j e c t i o n . Thus c o r r e c t e d i n c r e m e n t s o f > 2.9 were o f t e n seen w i t h o u t c l i n i c a l s i g n s o f r e j e c t i o n . The r e a s o n was n o t c l e a r , t h o u g h s e v e r a l e x p l a n a t i o n s may be proposed. F i r s t , n o t a l l immune r e a c t i o n s t o t h e g r a f t may damage g r a f t f u n c t i o n . I n a d d i t i o n , subc i n i c a l r e j e c t i o n e p i s o d e s may o c c u r . 166 T h i r d l y , v i r u s i n f e c t i o n s w i t h i n t h e g r a f t may cause immune r e a c t i o n s t o v i r a l a n t i g e n s w i t h l i t t l e e f f e c t on k i d n e y f u n c t i o n b u t e v o k i n g an i n f l a m m a t o r y re- sponse. F o u r t h l y , t r a n s p l a n t a t i o n o f organs always i n d u c e s i s c h a e m i c damage t o t h e g r a f t t h a t may l e a d t o some i n t r a g r a f t i n f l a m m a t i o n . Thus, s y n g e n e i c r a t a l l o g r a f t s a r e i n f i l t r a t e d w i t h l y m p h o c y t e s , b u t t h e g r a f t s a r e n o t r e j e c t e d (1). I r r e s p e c t i v e o f t h e cause o f i n f l a m m a t i o n o b s e r v e d i n FNAB from n o n r e j e c t i n g p a t i e n t s , i t s f r e q u e n t o c c u r r e n c e must l e a d t o c a u t i o n i n a d m i n i s t e r i n g a n t i - r e j e c t i o n t h e r a p y t o p a t i e n t s w i t h h i g h c o r r e c t e d i n c r e m e n t s u n l e s s t h e r e a r e c l i n i c a l s i g n s o f r e j e c t i o n . I f h i g h i n c r e m e n t s a r e r e p e a t e d l y found i n a p a t - i e n t w i t h o u t c l i n i c a l s i g n s o f r e j e c t i o n , a s e a r c h f o r v i r u s i n f e c t i o n is ad- v i s a b l e ( u n p u b l i s h e d r e s u l t s ) . From o u r t o t a l e x p e r i e n c e o f FNAB i n A z a - t r e a t e d p a t i e n t s , i t seems w a r r a n t a b l e t o i n c l u d e t h e s e b i o p s i e s i n t h e p o s t o p e r a t i v e ( f i r s t 3 weeks) m o n i t o r i n g o f r e n a l t r a n s p l a n t p a t i e n t s . I f r e j e c t i o n i s sus- p e c t e d , a t l e a s t two b i o p s i e s s h o u l d be p e r f o r m e d , i f necessary g u i d e d by u l t r a s o u n d s c a n n i n g , t o i n c r e a s e t h e p o s s i b i l i t y o f a w e l l r e p r e s e n t a t i v e b i o p s y . T h i s s t u d y d e m o n s t r a t e s t h e r a r i t y o f c l i n i c a l r e j e c t i o n e p i s o d e s w i t h o u r p r e s e n t CyA regimen d u r i n g t h e p o s t o p e r a t i v e p e r i o d . I n c l u s i o n o f FNAB i n t h e r o u t i n e m o n i t o r i n g o f C y A - t r e a t e d p a t i e n t s is t h e r e f o r e o f q u e s t i o n a b l e j u s t i - f i c a t i o n . We s h a l l c o n t i n u e t o use t h e p r o c e d u r e f o r s e v e r a l reasons, however. C y c l o s p o r i n i s a n e p h r o t o x i c d r u g and t h e serum l e v e l o n l y p a r t l y r e f l e c t s i t s t o x i c i t y . Some i n f o r m a t i o n o f t h e n e p h r o t o x i c a c t i o n can be g a i n e d f r o m s t u d y - i n g t h e morphology o f t h e t u b u l a r c e l l s i n t h e a s p i r a t e s ( 1 2 ) . As e a r l i e r s t a t e d , r e p e a t e d l y h i g h i n c r e m z n t s have l e d us t o i n t e n s i f y s e a r c h f o r v i r a l i n f e c t i o n s . Use o f FNAB s h o u l d f a c i l i t a t e comparisons w i t h some o t h e r , r e c e n t l y suggested m o n i t o r i n g a l t e r n a t i v e s ( 2 , l O ) . ACKNOWLEDGEMENTS We w i s h t o t h a n k Mrs I n g e g e r d H a k i n g and Mrs Norma Jansson f o r e x p e r t t e c h n i c a l a s s i s t a n c e . The c l i n i c a l r e s e a r c h was s u p p o r t e d by t h e Tore N i l s s o n and Maud & Birger Gustavsson F o u n d a t i o n s and by t h e Swedish M e d i c a l Research C o u n c i l . REFERENCES 1. 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