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Detection of Duffy and Kidd antibodies by Groupamatic 360
Revue Franqaise de Transfusion et d'Immuno-h6matologie
Tome XXI. -- N° 2. - - 1978
451
Detection of Duffy and Kidd antibodies by Groupamatic 360 b y P. R U B I N S T E I N , M.E. W A L K E R a n d F.H. A L L E N Jr
New York Blood Center, NEW YORK.
UTOMATION of d o n o r blood processing is i m p o r t a n t to us in New York. We serve a population of 18 million people, and process 600 t h o u s a n d units of blood yearly. Our first G r o u p a m a t i c 360 was obtained in 1973. It was the hard-wired S model. We have had an FDA license for routine use of this e q u i p m e n t for 2 years, and the m a c h i n e has been in constant use for routine blood processing ever since then. We acquired a second G r o u p a m a t i c 360 in 1975. This m a c h i n e was, in the beginning, on loan f r o m Roche for the development of antibody screening m e t h o d s and for initial trials of a new sample-identification label. The second G r o u p a m a t i c was a model C, and it n o w is also in routine daily use in processing donor bloods, the two machines being operated simultaneously.
A
A substantial p o r t i o n of o u r donations is obtained by sub-centers, but all pilot tubes are sent to the central l a b o r a t o r y for processing. Up to the present time we a r e s t i l l doing the tests for syphilis, hepatitis, and antibody detection by hand, although we expect to have all of these p r o c e d u r e s done by the G r o u p a m a t i c eventually. What I shall present now is w o r k done by Dr. P. RUBINSTEIN and M. WALKER at the New York Blood Center in their efforts to perfect a m e t h o d that could be used in the G r o u p a m a t i c for routine detection of anti-Duffy and anti-Kidd antibodies in d o n o r plasma. They did a lot of w o r k on the conditions affecting antibody u p t a k e
452
SYMPOSIUM SUR LES SYSTEMES GROUPAMAT1C
a n d agglutination - - e l e c t r o p h o r e t i c m o b i l i t y of the r e d cells, viscosity of the m e d i u m , ionic strength, e n z y m a t i c t r e a t m e n t of cells, and various c o n c e n t r a t i o n s of p o l y m e r s . I a m s o r r y t h a t Dr. R u b i n s t e i n w a s unable to be at this m e e t i n g to p r e s e n t the m a t e r i a l himself. W h a t I shall p r e s e n t is a m e t h o d , not yet fully refined a n d not yet t h o r o u g h l y tested, which we t h i n k will be s a t i s f a c t o r y f o r the detection of Duffy and Kidd antibodies.
METHOD The m e t h o d uses 5 lines, f o u r of t h e m being at the filling station a n d the 5th at a n e w position b e t w e e n the c e n t r i f u g a t i o n a n d shaking stages. These 5 lines are used for the following p r e p a r a t i o n s : line 1, polybrene, a b o u t 1 : 5000 . . . . . . . . . . . . . . . . . . .
055 m l
line 2, 6-8 % trypsinized r e d cells s u s p e n d e d in dialyzed p l a s m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
055 m l
line 3, PVP K90 0.75 % . . . . . . . . . . . . . . . . . . . . . . . . . . .
055 ml
line 4, u n k n o w n p l a s m a . . . . . . . . . . . . . . . . . . . . . . . . . . .
125 m l
line 5, t r i s o d i u m citrate 2 %
055 m l
.....................
Low-ionic Strength Saline (LIS). Low-ionic saline is u s e d f o r diluting nearly everything. It consists of s o d i u m chloride 0.2 % a n d glycine 2 %. The glycine serves b o t h as a b u f f e r a n d as a red cell preservative, as well as m a i n t a i n i n g o s m o l a r i t y of the solution. This is p r e p a r e d daily. 1. POLYBRENE SOLUTION.
P o l y b r e n e is diluted in LIS. The c o n c e n t r a t i o n varies w i t h the b a t c h of polybrene, a n d w i t h the p l a s m a t h a t is u s e d f o r cell suspension. C o n c e n t r a t i o n is usually b e t w e e n 1 : 2000 a n d 1 : 10,000. Daily b a t c h e s of p o l y b r e n e solution are p r e p a r e d f r o m a 1 : 5 0 0 stock dilution w h i c h is stable f o r long periods of time. The final dilution is p r e p a r e d in such a w a y t h a t it will p r o d u c e a tight c l u m p of red cells t h a t is d i s p e r s e d (in the a b s e n c e of a n t i b o d y ) b y the citrate solution. N o r m a l p l a s m a s differ c o n s i d e r a b l y in their inhibition of the p o l y b r e n e effect. This leads to too m u c h variability in test
D E T E R M I N A T I O N DES A N T I C O R P S I R R E G U L I E R S
453
results unless s o m e t h i n g is done a b o u t it. W h a t doctor R u b i n s t e i n did was to select a p l a s m a w i t h high inhibitory p r o p e r t i e s in which to suspend the test red cells. The p r e s e n c e of this p l a s m a d a m p s the effect of individual variations of the u n k n o w n p l a s m a s b u t also m e a n s t h a t m o r e p o l y b r e n e m u s t be used. I t is i m p o r t a n t in using p o l y b r e n e t h a t n e u t r a l plastic c o n t a i n e r s and p i p e t t e s are used. P o l y p r o p y l e n e is s a t i s f a c t o r y a n d tygon is satisfactory. The usual p u m p tubing used in G r o u p a m a t i c m a y not be neutral, and it m a y be i m p o r t a n t to change the type of tubing used in the p u m p , at least for this line. 2. CELL SUSPENSION. The selected diagnostic cells are p r e p a r e d in the following fashion. Crystalline t r y p s i n is p r e p a r e d freshly in a c o n c e n t r a t i o n of 1 : 10,000 in n o r m a l saline. The r e d cells are i n c u b a t e d at 37 ° for only 15 minutes, t h e n w a s h e d in LIS a n d s u s p e n d e d to a c o n c e n t r a t i o n of 6-8 % in dialyzed p l a s m a . P l a s m a (selected f o r high-polybrene i n h i b i t o r y activity) is dialyzed overnight against 100 v o l u m e s of L I S in the cold. W h a t is not used i m m e d i a t e l y can be k e p t frozen at m i n u s 30 ° . 3. PVP. A 0.75 % solution of PVP (Kg0) is m a d e in LIS, a n d m u s t be p r e p a r e d daily. PVP is disgustingly sticky. E v e r y t h i n g t h a t it c o m e s in contact w i t h m u s t be rinsed i m m e d i a t e l y a f t e r use, especially the cuvettes. 4. UNKNOWN PLASMA. The a m o u n t of p l a s m a u s e d is 0.125 ml. washing of this line be done w i t h LIS.
I t is n e c e s s a r y t h a t the
5. SODIUM CITRATE. T r i s o d i u m citrate is used at a c o n c e n t r a t i o n of 2 % in n o r m a l saline with no a d d e d buffer. This solution will have a p H of a b o u t 8.4. 0.055 m l is used in each cuvette. The citrate solution is delivered b y a s e p a r a t e p u m p . We h a d a special one built, using 12 commercially-available m i c r o syringes that deliver b e t w e e n 0 and 500 m i c r o l i t e r s of fluid into channel 6.
454
SYMPOSIUM SUR LES SYSTEMES GROUPAMAT1C
This channel was selected arbitrarily for our own convenience in working. The a m o u n t delivered by this p u m p is adjustable. Delivery is mechanically triggered by the disk itself as it moves t o w a r d the agitation station. The p u r p o s e of the sodium citrate is, of course, to disaggregate non-specifically agglutinated red cells in the presence of polybrene and PVP. CO?qCLUSlON : The features of this system that seem to me to be particularly n o t e w o r t h y are the use of glycine as a preservative, the variation in polybrene inhibition by different n o r m a l plasmas and the use of a p l a s m a with high polybrene-inhibiting activity as a red-cell suspension m e d i u m and the dialysis of this p l a s m a to reduce its ionic strength. Trypsinization of the red cells involves the use of a very dilute solution of crystalline trypsin for a relatively very short time.
RESUME
Ddtection des anticorps anti Dutfy et anti Kidd sur Groupamatic 560 Cette technique utilise une buse 4 voies au niveau de la p o m p e de c h a r g e m e n t r6actifs-dchantillons : 1) Polybr~ne 1/20.000 g 1/10.000 en LIS . . . . . . . . . .
0,55 ml
2) Globules rouges trait6s p a r la trypsine en suspension 6 g 8 % dans du p l a s m a dialys6 contre du LIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0,55 ml
3) PVP, Kg0, 0,75 % en L I E . . . . . . . . . . . . . . . . . . . . . .
0,55 ml
4) Plasma 6chantillon ~ tester
1,25 ml
..................
Une p o m p e sp6ciale est n6cessaire entre les postes de centrifugation et d'agitation afin de d61ivrer 0,55 ml de citrate de sodium en solut6 sal6 physiologique. - - LIS (Low Ionic Saline), milieu de faible force ionique, NaC1 0,2 % ; -
-
Plasma utilis6 p o u r la m i s e en suspension des globules rouges tests, sdlectionn6 en fonction de son action inhibitrice sur le polybr6ne, dialys6 toute une nuit c o n t r e 100 volumes de LIS ;
D E T E R M I N A T I O N DES ANT1CORPS 1RREGULIERS
455
- - Solution mbre de polybr6ne (1/500), port6e h une dilution perm e t t a n t d ' o b t e n i r une masse c o m p a c t e d'6rythrocytes p o u v a n t ~tre remise en suspension p a r la solution de c i t r a t e ; - - T r y p s i n e cristallis6e 1/10.000 en LIS, utilis6e ~ 37 ° p e n d a n t 15 minutes ; - - PVP, K90, 0,75 % en LIS, pr6par6 quotidiennement. Les godets doivent 6tre laves r a p i d e m e n t apr6s leur utilisation ; Citrate de sodium, 2 % en NaC1 0,15 M, p H _+ 8,4, utilis6 sans tampon.
-
-
Dr P. RUBIRSTEIN, New York Blood Center, 310 East 67th street New York, N.Y. 10021 - U.S.A.
Tome XXI. -- N° 2. - - 1978
451
Detection of Duffy and Kidd antibodies by Groupamatic 360 b y P. R U B I N S T E I N , M.E. W A L K E R a n d F.H. A L L E N Jr
New York Blood Center, NEW YORK.
UTOMATION of d o n o r blood processing is i m p o r t a n t to us in New York. We serve a population of 18 million people, and process 600 t h o u s a n d units of blood yearly. Our first G r o u p a m a t i c 360 was obtained in 1973. It was the hard-wired S model. We have had an FDA license for routine use of this e q u i p m e n t for 2 years, and the m a c h i n e has been in constant use for routine blood processing ever since then. We acquired a second G r o u p a m a t i c 360 in 1975. This m a c h i n e was, in the beginning, on loan f r o m Roche for the development of antibody screening m e t h o d s and for initial trials of a new sample-identification label. The second G r o u p a m a t i c was a model C, and it n o w is also in routine daily use in processing donor bloods, the two machines being operated simultaneously.
A
A substantial p o r t i o n of o u r donations is obtained by sub-centers, but all pilot tubes are sent to the central l a b o r a t o r y for processing. Up to the present time we a r e s t i l l doing the tests for syphilis, hepatitis, and antibody detection by hand, although we expect to have all of these p r o c e d u r e s done by the G r o u p a m a t i c eventually. What I shall present now is w o r k done by Dr. P. RUBINSTEIN and M. WALKER at the New York Blood Center in their efforts to perfect a m e t h o d that could be used in the G r o u p a m a t i c for routine detection of anti-Duffy and anti-Kidd antibodies in d o n o r plasma. They did a lot of w o r k on the conditions affecting antibody u p t a k e
452
SYMPOSIUM SUR LES SYSTEMES GROUPAMAT1C
a n d agglutination - - e l e c t r o p h o r e t i c m o b i l i t y of the r e d cells, viscosity of the m e d i u m , ionic strength, e n z y m a t i c t r e a t m e n t of cells, and various c o n c e n t r a t i o n s of p o l y m e r s . I a m s o r r y t h a t Dr. R u b i n s t e i n w a s unable to be at this m e e t i n g to p r e s e n t the m a t e r i a l himself. W h a t I shall p r e s e n t is a m e t h o d , not yet fully refined a n d not yet t h o r o u g h l y tested, which we t h i n k will be s a t i s f a c t o r y f o r the detection of Duffy and Kidd antibodies.
METHOD The m e t h o d uses 5 lines, f o u r of t h e m being at the filling station a n d the 5th at a n e w position b e t w e e n the c e n t r i f u g a t i o n a n d shaking stages. These 5 lines are used for the following p r e p a r a t i o n s : line 1, polybrene, a b o u t 1 : 5000 . . . . . . . . . . . . . . . . . . .
055 m l
line 2, 6-8 % trypsinized r e d cells s u s p e n d e d in dialyzed p l a s m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
055 m l
line 3, PVP K90 0.75 % . . . . . . . . . . . . . . . . . . . . . . . . . . .
055 ml
line 4, u n k n o w n p l a s m a . . . . . . . . . . . . . . . . . . . . . . . . . . .
125 m l
line 5, t r i s o d i u m citrate 2 %
055 m l
.....................
Low-ionic Strength Saline (LIS). Low-ionic saline is u s e d f o r diluting nearly everything. It consists of s o d i u m chloride 0.2 % a n d glycine 2 %. The glycine serves b o t h as a b u f f e r a n d as a red cell preservative, as well as m a i n t a i n i n g o s m o l a r i t y of the solution. This is p r e p a r e d daily. 1. POLYBRENE SOLUTION.
P o l y b r e n e is diluted in LIS. The c o n c e n t r a t i o n varies w i t h the b a t c h of polybrene, a n d w i t h the p l a s m a t h a t is u s e d f o r cell suspension. C o n c e n t r a t i o n is usually b e t w e e n 1 : 2000 a n d 1 : 10,000. Daily b a t c h e s of p o l y b r e n e solution are p r e p a r e d f r o m a 1 : 5 0 0 stock dilution w h i c h is stable f o r long periods of time. The final dilution is p r e p a r e d in such a w a y t h a t it will p r o d u c e a tight c l u m p of red cells t h a t is d i s p e r s e d (in the a b s e n c e of a n t i b o d y ) b y the citrate solution. N o r m a l p l a s m a s differ c o n s i d e r a b l y in their inhibition of the p o l y b r e n e effect. This leads to too m u c h variability in test
D E T E R M I N A T I O N DES A N T I C O R P S I R R E G U L I E R S
453
results unless s o m e t h i n g is done a b o u t it. W h a t doctor R u b i n s t e i n did was to select a p l a s m a w i t h high inhibitory p r o p e r t i e s in which to suspend the test red cells. The p r e s e n c e of this p l a s m a d a m p s the effect of individual variations of the u n k n o w n p l a s m a s b u t also m e a n s t h a t m o r e p o l y b r e n e m u s t be used. I t is i m p o r t a n t in using p o l y b r e n e t h a t n e u t r a l plastic c o n t a i n e r s and p i p e t t e s are used. P o l y p r o p y l e n e is s a t i s f a c t o r y a n d tygon is satisfactory. The usual p u m p tubing used in G r o u p a m a t i c m a y not be neutral, and it m a y be i m p o r t a n t to change the type of tubing used in the p u m p , at least for this line. 2. CELL SUSPENSION. The selected diagnostic cells are p r e p a r e d in the following fashion. Crystalline t r y p s i n is p r e p a r e d freshly in a c o n c e n t r a t i o n of 1 : 10,000 in n o r m a l saline. The r e d cells are i n c u b a t e d at 37 ° for only 15 minutes, t h e n w a s h e d in LIS a n d s u s p e n d e d to a c o n c e n t r a t i o n of 6-8 % in dialyzed p l a s m a . P l a s m a (selected f o r high-polybrene i n h i b i t o r y activity) is dialyzed overnight against 100 v o l u m e s of L I S in the cold. W h a t is not used i m m e d i a t e l y can be k e p t frozen at m i n u s 30 ° . 3. PVP. A 0.75 % solution of PVP (Kg0) is m a d e in LIS, a n d m u s t be p r e p a r e d daily. PVP is disgustingly sticky. E v e r y t h i n g t h a t it c o m e s in contact w i t h m u s t be rinsed i m m e d i a t e l y a f t e r use, especially the cuvettes. 4. UNKNOWN PLASMA. The a m o u n t of p l a s m a u s e d is 0.125 ml. washing of this line be done w i t h LIS.
I t is n e c e s s a r y t h a t the
5. SODIUM CITRATE. T r i s o d i u m citrate is used at a c o n c e n t r a t i o n of 2 % in n o r m a l saline with no a d d e d buffer. This solution will have a p H of a b o u t 8.4. 0.055 m l is used in each cuvette. The citrate solution is delivered b y a s e p a r a t e p u m p . We h a d a special one built, using 12 commercially-available m i c r o syringes that deliver b e t w e e n 0 and 500 m i c r o l i t e r s of fluid into channel 6.
454
SYMPOSIUM SUR LES SYSTEMES GROUPAMAT1C
This channel was selected arbitrarily for our own convenience in working. The a m o u n t delivered by this p u m p is adjustable. Delivery is mechanically triggered by the disk itself as it moves t o w a r d the agitation station. The p u r p o s e of the sodium citrate is, of course, to disaggregate non-specifically agglutinated red cells in the presence of polybrene and PVP. CO?qCLUSlON : The features of this system that seem to me to be particularly n o t e w o r t h y are the use of glycine as a preservative, the variation in polybrene inhibition by different n o r m a l plasmas and the use of a p l a s m a with high polybrene-inhibiting activity as a red-cell suspension m e d i u m and the dialysis of this p l a s m a to reduce its ionic strength. Trypsinization of the red cells involves the use of a very dilute solution of crystalline trypsin for a relatively very short time.
RESUME
Ddtection des anticorps anti Dutfy et anti Kidd sur Groupamatic 560 Cette technique utilise une buse 4 voies au niveau de la p o m p e de c h a r g e m e n t r6actifs-dchantillons : 1) Polybr~ne 1/20.000 g 1/10.000 en LIS . . . . . . . . . .
0,55 ml
2) Globules rouges trait6s p a r la trypsine en suspension 6 g 8 % dans du p l a s m a dialys6 contre du LIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0,55 ml
3) PVP, Kg0, 0,75 % en L I E . . . . . . . . . . . . . . . . . . . . . .
0,55 ml
4) Plasma 6chantillon ~ tester
1,25 ml
..................
Une p o m p e sp6ciale est n6cessaire entre les postes de centrifugation et d'agitation afin de d61ivrer 0,55 ml de citrate de sodium en solut6 sal6 physiologique. - - LIS (Low Ionic Saline), milieu de faible force ionique, NaC1 0,2 % ; -
-
Plasma utilis6 p o u r la m i s e en suspension des globules rouges tests, sdlectionn6 en fonction de son action inhibitrice sur le polybr6ne, dialys6 toute une nuit c o n t r e 100 volumes de LIS ;
D E T E R M I N A T I O N DES ANT1CORPS 1RREGULIERS
455
- - Solution mbre de polybr6ne (1/500), port6e h une dilution perm e t t a n t d ' o b t e n i r une masse c o m p a c t e d'6rythrocytes p o u v a n t ~tre remise en suspension p a r la solution de c i t r a t e ; - - T r y p s i n e cristallis6e 1/10.000 en LIS, utilis6e ~ 37 ° p e n d a n t 15 minutes ; - - PVP, K90, 0,75 % en LIS, pr6par6 quotidiennement. Les godets doivent 6tre laves r a p i d e m e n t apr6s leur utilisation ; Citrate de sodium, 2 % en NaC1 0,15 M, p H _+ 8,4, utilis6 sans tampon.
-
-
Dr P. RUBIRSTEIN, New York Blood Center, 310 East 67th street New York, N.Y. 10021 - U.S.A.