16
M e d . Sci. Law ( 1 9 7 8 ) Vol. 1 8 , No. 1
Printed
in Great
Britain
Increased Sensitivity of Tests for the Detection of Blood Group Antigens in Stains Using a Low Ionic Strength Medium M . J . M c D O W A L L * , P. J . L I N C O L N and Β. E. D O D D Departments of Forensic Medicine and Haematology. The London Hospital Medical College SUMMARY T h e incorporation of a low ionic strength solution (LISS) in the micro-elution technique used for the detection of blood group antigens in stains markedly improves the test's sensitivity. This is because LISS increases the amount of antibody taken up by the antigen in the stain which results in a greater yield of antibody recovered from the slain by elution. LISS also enhances the activity of the eluted antibody if it is introduced as a suspension medium for the red cells used to detect the antibody. T h e introduction of suitably diluted AB serum as diluent when testing the eluates is an additional advantage. T h e improvement in the sensitivity of the microelution technique is great enough in some instances to allow the detection of an antigen in a stain which is undetectable in the absence of LISS. Moreover some doubtful positive reactions are enhanced sufficiently for the presence of an antigen to be definitely estab lished.
INTRODUCTION I t is w e l l k n o w n t h a t t h e i o n i c s t r e n g t h o f t h e m e d i u m is o n e o f t h e factors w h i c h influences antigen-antibody reaction. H u g h e s - J o n e s et a l . ( 1 9 6 4 a ) s h o w e d t h a t t h e r a t e of a s s o c i a t i o n o f a n t i - D w i t h D p o s i t i v e cells c a n b e i n c r e a s e d 1000-fold b y l o w e r i n g t h e i o n i c s t r e n g t h o f t h e m e d i u m from 0-17 M t o 0 · 0 3 M . H o w e v e r it h a s also b e e n n o t e d t h a t t h e r e is a t e n d e n c y for n o n specific u p t a k e o f a n t i b o d y t o o c c u r w h e n t h e i o n i c s t r e n g t h o f t h e m e d i u m is r e d u c e d , l e a d i n g t o false p o s i t i v e r e a c t i o n s ( H u g h e s J o n e s et a l . , 1 9 6 4 b ; E l l i o t et a l . , 1 9 6 4 ) . I n 1974, L o w a n d Messeter, described a
* M . J. M c D o w a l l gratefully acknowledges from the Sir Halley Stewart Trust.
a grant
m e t h o d using 0-03 M N a C l w i t h glycine, by w h i c h t h e r a t e of u p t a k e o f a n t i b o d y b y r e d cells h a v i n g t h e c o r r e s p o n d i n g a n t i g e n w a s increased but without the accompanying non-specific reactions. W e h a v e n o w a d a p t e d t h e i r m e t h o d for u s e i n c o n j u n c t i o n w i t h a m i c r o - e l u t i o n t e c h n i q u e for t h e d e t e c t i o n of r e d cell a n t i g e n s i n b l o o d s t a i n s b e c a u s e n o t o n l y d o c s t h e m e t h o d i n c r e a s e t h e rate of a n t i b o d y u p t a k e b u t t h e total amount o f a n t i b o d y t a k e n u p b y t h e s t a i n is i n c r e a s e d a n d t h i s is reflected i n t h e a c t i v i t y of t h e e l u a t e . T h i s is p a r t i c u l a r l y a d v a n t a g e o u s for t h e t y p i n g of s t a i n s w h e r e t h e a m o u n t of a n t i g e n m a y b e e i t h e r v e r y s m a l l o r its a c t i v i t y m a y b e i m p a i r e d for v a r i o u s r e a s o n s . T e s t s h a v e b e e n p e r f o r m e d o n a l a r g e n u m b e r of e x p e r i m e n t a l stains, some m a n y m o n t h s old, u s i n g a n t i b o d i e s o f different R h specificities a n d also a n t i - S . W e h a v e s t u d i e d t h e effect of i n t r o d u c i n g a l o w i o n i c s t r e n g t h m e d i u m first a t t h e a b s o r p t i o n s t a g e of t h e m i c r o - e l u t i o n t e c h n i q u e , t h e n for t h e t e s t i n g of t h e e l u t e d a n t i b o d y a n d finally at b o t h s t a g e s of t h e tests. A series of e x p e r i m e n t s s h o w i n g t h e a d d e d a d v a n t a g e of using a suitable dilution of A B s e r u m a s t i t r a t i o n d i l u e n t is also included.
MATERIALS T h e anti-sera a n d A B s e r u m used in t h e investigation were generously supplied by D r K . L I . R o g e r s of t h e S o u t h L o n d o n Blood Transfusion Centre. Bovine a l b u m i n w a s o b t a i n e d e i t h e r from O r t h o D i a g n o s t i c s o r Biotest F o l c x L t d . A s a fluid for t h e s u s p e n -
M c D o w a l l et al. : Detection of Blood Group Antigens
17
^ble I. Tests showing the effect of LISS on the Rh typing of blood stains of various ages T
Specificity of anti-serum Anti-D
Rh type of stain
Age of stain in weeks
ddccee DCcEe DccEe DccEe ddccee DCcee DCCee DCcEe DccEE DCCee DCcEe ddccee DCCee ddccee DCCee DccEe DccEe DccEe DccEE DCcEe ddccee DCcEe DCCee DCcEe
Anti-C
Anti-c
Anti-E
Activity of eluate from stain Normal LISS
3 8 12 32 32 60 1 1 1 2J 2i 2i 2 2 15 20 24 32
M e d . Sci. Law ( 1 9 7 8 ) Vol. 1 8 , No. 1
Printed
in Great
Britain
Increased Sensitivity of Tests for the Detection of Blood Group Antigens in Stains Using a Low Ionic Strength Medium M . J . M c D O W A L L * , P. J . L I N C O L N and Β. E. D O D D Departments of Forensic Medicine and Haematology. The London Hospital Medical College SUMMARY T h e incorporation of a low ionic strength solution (LISS) in the micro-elution technique used for the detection of blood group antigens in stains markedly improves the test's sensitivity. This is because LISS increases the amount of antibody taken up by the antigen in the stain which results in a greater yield of antibody recovered from the slain by elution. LISS also enhances the activity of the eluted antibody if it is introduced as a suspension medium for the red cells used to detect the antibody. T h e introduction of suitably diluted AB serum as diluent when testing the eluates is an additional advantage. T h e improvement in the sensitivity of the microelution technique is great enough in some instances to allow the detection of an antigen in a stain which is undetectable in the absence of LISS. Moreover some doubtful positive reactions are enhanced sufficiently for the presence of an antigen to be definitely estab lished.
INTRODUCTION I t is w e l l k n o w n t h a t t h e i o n i c s t r e n g t h o f t h e m e d i u m is o n e o f t h e factors w h i c h influences antigen-antibody reaction. H u g h e s - J o n e s et a l . ( 1 9 6 4 a ) s h o w e d t h a t t h e r a t e of a s s o c i a t i o n o f a n t i - D w i t h D p o s i t i v e cells c a n b e i n c r e a s e d 1000-fold b y l o w e r i n g t h e i o n i c s t r e n g t h o f t h e m e d i u m from 0-17 M t o 0 · 0 3 M . H o w e v e r it h a s also b e e n n o t e d t h a t t h e r e is a t e n d e n c y for n o n specific u p t a k e o f a n t i b o d y t o o c c u r w h e n t h e i o n i c s t r e n g t h o f t h e m e d i u m is r e d u c e d , l e a d i n g t o false p o s i t i v e r e a c t i o n s ( H u g h e s J o n e s et a l . , 1 9 6 4 b ; E l l i o t et a l . , 1 9 6 4 ) . I n 1974, L o w a n d Messeter, described a
* M . J. M c D o w a l l gratefully acknowledges from the Sir Halley Stewart Trust.
a grant
m e t h o d using 0-03 M N a C l w i t h glycine, by w h i c h t h e r a t e of u p t a k e o f a n t i b o d y b y r e d cells h a v i n g t h e c o r r e s p o n d i n g a n t i g e n w a s increased but without the accompanying non-specific reactions. W e h a v e n o w a d a p t e d t h e i r m e t h o d for u s e i n c o n j u n c t i o n w i t h a m i c r o - e l u t i o n t e c h n i q u e for t h e d e t e c t i o n of r e d cell a n t i g e n s i n b l o o d s t a i n s b e c a u s e n o t o n l y d o c s t h e m e t h o d i n c r e a s e t h e rate of a n t i b o d y u p t a k e b u t t h e total amount o f a n t i b o d y t a k e n u p b y t h e s t a i n is i n c r e a s e d a n d t h i s is reflected i n t h e a c t i v i t y of t h e e l u a t e . T h i s is p a r t i c u l a r l y a d v a n t a g e o u s for t h e t y p i n g of s t a i n s w h e r e t h e a m o u n t of a n t i g e n m a y b e e i t h e r v e r y s m a l l o r its a c t i v i t y m a y b e i m p a i r e d for v a r i o u s r e a s o n s . T e s t s h a v e b e e n p e r f o r m e d o n a l a r g e n u m b e r of e x p e r i m e n t a l stains, some m a n y m o n t h s old, u s i n g a n t i b o d i e s o f different R h specificities a n d also a n t i - S . W e h a v e s t u d i e d t h e effect of i n t r o d u c i n g a l o w i o n i c s t r e n g t h m e d i u m first a t t h e a b s o r p t i o n s t a g e of t h e m i c r o - e l u t i o n t e c h n i q u e , t h e n for t h e t e s t i n g of t h e e l u t e d a n t i b o d y a n d finally at b o t h s t a g e s of t h e tests. A series of e x p e r i m e n t s s h o w i n g t h e a d d e d a d v a n t a g e of using a suitable dilution of A B s e r u m a s t i t r a t i o n d i l u e n t is also included.
MATERIALS T h e anti-sera a n d A B s e r u m used in t h e investigation were generously supplied by D r K . L I . R o g e r s of t h e S o u t h L o n d o n Blood Transfusion Centre. Bovine a l b u m i n w a s o b t a i n e d e i t h e r from O r t h o D i a g n o s t i c s o r Biotest F o l c x L t d . A s a fluid for t h e s u s p e n -
M c D o w a l l et al. : Detection of Blood Group Antigens
17
^ble I. Tests showing the effect of LISS on the Rh typing of blood stains of various ages T
Specificity of anti-serum Anti-D
Rh type of stain
Age of stain in weeks
ddccee DCcEe DccEe DccEe ddccee DCcee DCCee DCcEe DccEE DCCee DCcEe ddccee DCCee ddccee DCCee DccEe DccEe DccEe DccEE DCcEe ddccee DCcEe DCCee DCcEe
Anti-C
Anti-c
Anti-E
Activity of eluate from stain Normal LISS
3 8 12 32 32 60 1 1 1 2J 2i 2i 2 2 15 20 24 32
