Automated reading of cytotoxicity with the contrast fluorescence test (CFI)

384

Annual A A C ~ T Meeting, 1981

AUTO~qTEDREADING OF CYTOTOXICITYWITHTHECONTRASTFLUORESCENCE TEST (CFT). P. R u b i n s t e l n t H . Walker and N . . H o l l e n t Immunogenetlcs, The New York Blood C e n t e r . Thls t e s t i s based on FI)A fluorochromasxa ( f o r l i v l n g c e l l s ) and e t h l d i u m bromide ( f o r dead o n e s ) . I t has been a p p l i e d t o HLA t y p l n s u s l n g e x a c t l y the same conditions r e q u i r e d by t h e NIH t e s t ( I u l o f c e l l s u s p e n s i o n , 1 ul o f a n t i s e r u m , $ u l o f r a b b i t C and 90' t o t a l I n c u b a t l o n a t 22C o f ~ h i c h 60' i s i n the p r e s e n c e o f C) i n m l c r o t ~ t e r (Teranakl) t r a y s . I t may be read v l s u a l l y o r by an automated mlcroscope-photoDJltlpller-computer system. The Instrm~ent i s based on 4 Z e l s s i n v e r t e d microscope w i t h E p l i l l u m i n a t l o n (Ploem) f i t t e d w i t h an automated s c a n n i n g s t a g e , a p h o L o m u l t l p l l e r and e l e c t r o n i c a l l y c o n t r o l e d m o t o r - d r i v e n f i l t e r changer. Two r e a d i n g s a r e o b t a i n e d from each w e l l {one w l t h each f i l t e r combination) and a r a t i o o f red e m i s s i o n (dead c e i l s ) o v e r t o t a l e m i t t e d l i g h t i s co~cputed a f t e r t h e e l i m i n a t i o n o f o u t l i e r s . The ratlos a r e t h e n a l l o c a t e d by t h e l l n e a r c l u s t e r a n a l y s i s (LCA) a l g o r i t h m t o one o f four c l u s t e r s . Reading o f a t r a y i s c u r r e n t l y performed i n f o u r minutes and t h e r e s u l t s a r e Immedlately a v a i l a b l e i n computer comp a t l b l e form f o r s t o r a g e and o t h e r p u r p o s e s . Because t h e r a t i o i s used, r a t h e r than a s l n g l e - c o l o r m e a s u r e m e n t , t h e accuracy o f t h e I n s t r u = e n t e d r e a d i n g i s not dependent on h e v l n s e x a c t l y t h e same t o t a l ntmber o f c e i l s p e r ~ e l l a t t h e end o f the s e r o l o g i c a l p r o c e s s . Use o f l o v p o w e r o b j e c t i v e s and high q u a l i t y phot~tubes reduce f h e n e e d f o r a c c u r a t e c e n t £ r l n g o f each w e l l . A simple d e v i c e f o r t h e e l l m l n a ~ I o n o f e x t r a - c e l L u l a r fluornchromes p r o v i d e s n e a r b l a c k backgrounds. The r e s u l t s o t t h e HLA-typlng t e s t s performed w i t h p a r a l l e l v i s u a l r e a d i n g o f t h e same t r a y s w i l l be p r e s e n t e d .

GENETICS OF HI.A-DISEASE ASSOCIATIONS. THE USE OF THE HAPLOI'YPEBELATIVE RISK (HRR) AND THE "HAPLO-DELTA" (Dh) ESTTHATES IN JUVENILE DIABETES ~ THREE RACIAL CROUPS. P. R u b l n s t e l n , N. Walker, C. C a r p e n t e r , C. C a r r i e r , J . Kransner, C. F a l k , and F. Ginsberg, :mmunogenetlca, The New York ISlood Center and P e d l a c r f c Endocrinology, )ft. S i n a i School o f H e d l c l n e , N.Y., N.Y, The BRR i s an e s t i m a t e o f the a s s o c i a t i o n hetween a d i s e a s e and m marker s y s tem a t t h e g e n e t i c r a t h e r than t h e p h e n o t y p l c l e v e l . I t i s computed i n HLAtyped f a m i l i e s o f p a t i e n t s by t a k i n g a s p u t a t i v e l y " a f f e c t e d " t h o s e h o p l o t y p e s i n h e r i t e d by and a s " u n a f f e c t e d " t h o s e n o t i n h e r i t e d by t h e probsnd. The HRR e l i m i n a t e s u n c e r t a l n t i e s a s t o t h e c o m p a r a b i l i t ~ o f " c o n t r o l " and p a t i e n t samples. ; e r e p o r t on 76 "me#' JDH f a m i l i e s i n c l u d i n g 23 o f Hispanic o r , g l n , 24 Ashkenazi Jewish and 29 American c a u c a s i a n . The HRit i s shove t o d e v i a t e s i g n l f l c a n t l y from u n i t y f o r some a n t i g e n s p r e t l o u s l y not known t o he a s s o c i a t e d w i t h JDH, including BIT and DR5 (reduced in f r e q u e n c y ) . The c a l c u l a t i o n o f HRR a l l o w s a method f o r e s t i m a t i n g t h e s t r e n g t h o f t h e a s s o c i a t i o n , d e s i g n s t e c l)h, in terms o f t h e e x c e s s o r d e f i c i t o f " a f f e c t e d " baplotypes carrying a specific allele. Dh i s d e f i n e d f o r each a n t i g e n a s DhObs-Exp/Exp, where Obs. sad Exp. r e f e r co " a f f e c t e d " h e p l o t y p e s ; when Obs= Exp, Dh-0. HRR a n t Ph ~ay a l l o ~ a more r i g o r o u s e s t i m a t i o n o f t h e r i s k t o b e a r e r s o f s p e c i f i c h a p l o t y p e s than d o ~ Wo,,If'~ Kit. AFF. UNAFF. HRR Dis X2 AFF. L~AFF. HRR Dh X2 N-161 B"159 N'I61 N-159 B7 10 16 ..59 - . 2 3 1.59 DR2 & 25 .l& - . 7 2 17.01 B8 21 9 2.50 .40 .5.13 1~3 39 10 4.76 .59 19.84 BIT 3 13 .21 - . 6 3 b.71 DI~ $3 30 2.11 .28 8.2Z HLA ANTIGENS AND ISLET CELL ANTIBODIES (ICA) lN GESTATIO~AL DIABETES (GD). £ . R u b i n s t e l n , H. Walker, J._~ra!.snero C. C a r r i e r , C. C a r p e n t e r , H. Dobergen t A. .Notkins s and P . Ginsberg, I m u n o g e n e t i c s , The New York Blood C e n t e r , P e d i a t r i c Endoc r i n o l o g y , N t . ~ S i n a l H o s p l t a l , N.Y., and I n s t . oz Oral l l e a l t h , NIH., k t h e s d a , Hd. GD o c c u r s i n 1-2Z o f a l l p r e g n a n c i e s an~, though g l u c o s e t o l e r a n c e u s u a l l y r e t u r n s t o normal a f t e r parturition, GD p a t i e n t s a r e a t an i n c r e a s e d r i s k o t d e v e l o p i n g I n s u l l n - d e p e n d e n t d i a b e t e s m e l l l t o s . We r e p o r t h e r e on the HLA A, B, C and DR a n t i g e n s o f J sample (N-86) o f GD p . , t l e n t s b e i n g s t u d i e d p r o s p e c t i v e l y i n o r d e r t o a s c e r t a i n whether pregnancy i s a t r i g g e r f o r c l l n -