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Simplification of the antiglobulin-augmented lymphocytotoxicity test: Addition of AHG to the complement
136
Abstracts
C-610 #155
B-7.1 #156
ASSOCIATION OF SOLUBLE CLASS I HLA ANTIGENS IN SERUM WITH REJECTION FOLLOWING LIVER TRANSPLANTATION. P Abbasalizadeh, D Cluff, G Klintmalm, I Watemberg, A Nikaein. Baylor University Medical Center, Dallas, TX. It has been suggested that serum levels of soluble class I HLA antigens (sHLA) may be associated with rejection in renal transplant recipients and therefore mgiht be useful in monitoring transplant (Tx) recipients. We, therefore, studied the expression of sHLA in sera of 33 liver Tx recipients within the first month post-Tx, during which time the majority of rejection episodes occur. EDTA-blood samples were obtained prior and 3 times/week postTx. 18/33 (55%) patients had a rejection episode. Pre-Tx level of sHLA ranged from 1003700 (mean 1 2 9 7 + 1 0 7 5 ) . The level of sHLA in 17/18 (94%) significantly rose from a lowest level mean of 736 + 657 prior to the rejection to 1326 + 9 6 9 on the day of rejection and in 5 patients continued to rise to reach the mean of 1 9 6 8 + 1 4 7 7 . Eight/18 (44%) patients with rejection episodes received bolus methylprednisolone for treatment, sHLA level decreased in all 8 patients, and in 4/8 (50%) rose again for as yet an unknown reason. In 8/18 (44%) patients, rejection persisted as indicated by treatment with OKT3 (5), FK506 (2) or retransplantation (1). In all 8 cases, the sHLA level remained high or continued to rise until after the treatment. Interestingly, sHLA level rose in 13/33 (39%) patients who had no rejection, as well. We have not yet been able to correlate the increase of sHLA to any clinical outcome in this group. However, in 7/12 (58%), the level of sHLA increased above the level prior to Tx and in the remaining 5/12 (42%) stayed below the pre-Tx level. In addition, 3/12 (25%) developed viral infection, in 2 of whom sHLA increased and in 1 declined. The above study shows that the rejection episode was significantly correlated with the increased sHLA class I level, sHLA level remained high if rejection persisted and declined following rejection treatment. This test, therefore, may be a very useful tool for monitoring liver transplant recipients.
S I M P L I F I C A T I O N OF THE A N T I G L O B U L I N - A U G M E N T E D L Y M P H O C Y T O T O X I C I T Y TEST: A D D I T I O N OF AHG TO THE COMPLEMENT. $I Steen, CY Cheng, A Ting, T Vayntrub, S Dunn, and FC Grumet, Histocompatibility Laboratories, California Pacific Medical Center, San Francisco, CA and Stanford University, Palo Alto, CA. The a n t i g l o b u l i n - a u g m e n t e d (AHG) l y m p h o c y t o t o x i c i t y test is w i d e l y used for PRA s c r e e n i n g and crossmatching. However, its m a i n d r a w b a c k is the c r i t i c a l t i m i n g after the a d d i t i o n of AHG, w h i c h m a k e s it c u m b e r s o m e for p e r f o r m a n c e of large numbers of tests. In this study we i n v e s t i g a t e d an a l t e r n a t i v e m e t h o d of d i l u t i n g the AHG r e a g e n t in c o m p l e m e n t b e f o r e a d d i t i o n to the w a s h e d cells. Initially, sera from 128 p a t i e n t s were s c r e e n e d a g a i n s t a panel of 47 T - c e l l s by the standard NIH, m o d i f i e d Amos, and c o n v e n t i o n a l AHG methods. T h i r t y sera were found to have a n t i b o d i e s d e t e c t a b l e only by the AHG technique. T h e s e sera were d i l u t e d from i:i to 1:32 and s c r e e n e d against a panel of I0 T - l y m p h o c y t e s u s i n g 3 m o d i f i c a t i o n s of the A H G technique: (i) a d d i t i o n of lul AHG d i l u t e d 1:50 w i t h RPMI for e x a c t l y 2 minutes f o l l o w e d by 5ul c o m p l e m e n t (method c u r r e n t l y used), (2) a d d i t i o n of 5ul A H G d i l u t e d 1:50 in the complement, and (3) a d d i t i o n of 5ul A H G diluted 1:8 in the complement. In all m e t h o d s cells and s e r u m w e r e incubated for 30 m i n at 22C then w a s h e d 4 times w i t h 5ul RPMI b e f o r e the a d d d i t i o n of AHG. A f t e r the a d d i t i o n of complement, trays w e r e i n c u b a t e d for 60 minutes at 22C b e f o r e reading. We found no d i f f e r e n c e in the d i l u t i o n end-point and in the strength of reactions among the 3 methods. In a second study we t e s t e d ROP c r o s s m a t c h trays (representing d u p l i c a t e s of sera from 97 patients) a g a i n s t 2 T - c e l l s u s i n g the same 3 m e t h o d s d e s c r i b e d above. We found no d i f f e r e n c e in the s t r e n g t h of the r e a c t i o n s among the 3 methods. More importantly, the c o m p a t i b l e candidates list was the same r e g a r d l e s s of the m e t h o d used. We c o n c l u d e that m o d i f i c a t i o n to the c o n v e n t i o n a l A H G m e t h o d by addition of A H G d i l u t e d in the c o m p l e m e n t p r o v i d e s a m e t h o d w h i c h is more simple to p e r f o r m w i t h o u t s a c r i f i c i n g the a c c u r a c y of test results. Its use will e n a b l e t e s t i n g of larger numbers of PRA s c r e e n s or c r o s s m a t c h e s w i t h ease.
Abstracts
C-610 #155
B-7.1 #156
ASSOCIATION OF SOLUBLE CLASS I HLA ANTIGENS IN SERUM WITH REJECTION FOLLOWING LIVER TRANSPLANTATION. P Abbasalizadeh, D Cluff, G Klintmalm, I Watemberg, A Nikaein. Baylor University Medical Center, Dallas, TX. It has been suggested that serum levels of soluble class I HLA antigens (sHLA) may be associated with rejection in renal transplant recipients and therefore mgiht be useful in monitoring transplant (Tx) recipients. We, therefore, studied the expression of sHLA in sera of 33 liver Tx recipients within the first month post-Tx, during which time the majority of rejection episodes occur. EDTA-blood samples were obtained prior and 3 times/week postTx. 18/33 (55%) patients had a rejection episode. Pre-Tx level of sHLA ranged from 1003700 (mean 1 2 9 7 + 1 0 7 5 ) . The level of sHLA in 17/18 (94%) significantly rose from a lowest level mean of 736 + 657 prior to the rejection to 1326 + 9 6 9 on the day of rejection and in 5 patients continued to rise to reach the mean of 1 9 6 8 + 1 4 7 7 . Eight/18 (44%) patients with rejection episodes received bolus methylprednisolone for treatment, sHLA level decreased in all 8 patients, and in 4/8 (50%) rose again for as yet an unknown reason. In 8/18 (44%) patients, rejection persisted as indicated by treatment with OKT3 (5), FK506 (2) or retransplantation (1). In all 8 cases, the sHLA level remained high or continued to rise until after the treatment. Interestingly, sHLA level rose in 13/33 (39%) patients who had no rejection, as well. We have not yet been able to correlate the increase of sHLA to any clinical outcome in this group. However, in 7/12 (58%), the level of sHLA increased above the level prior to Tx and in the remaining 5/12 (42%) stayed below the pre-Tx level. In addition, 3/12 (25%) developed viral infection, in 2 of whom sHLA increased and in 1 declined. The above study shows that the rejection episode was significantly correlated with the increased sHLA class I level, sHLA level remained high if rejection persisted and declined following rejection treatment. This test, therefore, may be a very useful tool for monitoring liver transplant recipients.
S I M P L I F I C A T I O N OF THE A N T I G L O B U L I N - A U G M E N T E D L Y M P H O C Y T O T O X I C I T Y TEST: A D D I T I O N OF AHG TO THE COMPLEMENT. $I Steen, CY Cheng, A Ting, T Vayntrub, S Dunn, and FC Grumet, Histocompatibility Laboratories, California Pacific Medical Center, San Francisco, CA and Stanford University, Palo Alto, CA. The a n t i g l o b u l i n - a u g m e n t e d (AHG) l y m p h o c y t o t o x i c i t y test is w i d e l y used for PRA s c r e e n i n g and crossmatching. However, its m a i n d r a w b a c k is the c r i t i c a l t i m i n g after the a d d i t i o n of AHG, w h i c h m a k e s it c u m b e r s o m e for p e r f o r m a n c e of large numbers of tests. In this study we i n v e s t i g a t e d an a l t e r n a t i v e m e t h o d of d i l u t i n g the AHG r e a g e n t in c o m p l e m e n t b e f o r e a d d i t i o n to the w a s h e d cells. Initially, sera from 128 p a t i e n t s were s c r e e n e d a g a i n s t a panel of 47 T - c e l l s by the standard NIH, m o d i f i e d Amos, and c o n v e n t i o n a l AHG methods. T h i r t y sera were found to have a n t i b o d i e s d e t e c t a b l e only by the AHG technique. T h e s e sera were d i l u t e d from i:i to 1:32 and s c r e e n e d against a panel of I0 T - l y m p h o c y t e s u s i n g 3 m o d i f i c a t i o n s of the A H G technique: (i) a d d i t i o n of lul AHG d i l u t e d 1:50 w i t h RPMI for e x a c t l y 2 minutes f o l l o w e d by 5ul c o m p l e m e n t (method c u r r e n t l y used), (2) a d d i t i o n of 5ul A H G d i l u t e d 1:50 in the complement, and (3) a d d i t i o n of 5ul A H G diluted 1:8 in the complement. In all m e t h o d s cells and s e r u m w e r e incubated for 30 m i n at 22C then w a s h e d 4 times w i t h 5ul RPMI b e f o r e the a d d d i t i o n of AHG. A f t e r the a d d i t i o n of complement, trays w e r e i n c u b a t e d for 60 minutes at 22C b e f o r e reading. We found no d i f f e r e n c e in the d i l u t i o n end-point and in the strength of reactions among the 3 methods. In a second study we t e s t e d ROP c r o s s m a t c h trays (representing d u p l i c a t e s of sera from 97 patients) a g a i n s t 2 T - c e l l s u s i n g the same 3 m e t h o d s d e s c r i b e d above. We found no d i f f e r e n c e in the s t r e n g t h of the r e a c t i o n s among the 3 methods. More importantly, the c o m p a t i b l e candidates list was the same r e g a r d l e s s of the m e t h o d used. We c o n c l u d e that m o d i f i c a t i o n to the c o n v e n t i o n a l A H G m e t h o d by addition of A H G d i l u t e d in the c o m p l e m e n t p r o v i d e s a m e t h o d w h i c h is more simple to p e r f o r m w i t h o u t s a c r i f i c i n g the a c c u r a c y of test results. Its use will e n a b l e t e s t i n g of larger numbers of PRA s c r e e n s or c r o s s m a t c h e s w i t h ease.