Clin. Biochem. 9, (3) 109-110 (1976)
Proficiency Testing of Radioimmunoassay W. J. LONGLEY
Division of Clinical Chemistry, Nova Scotia Pathology Institute and Department of Pathology, Dalhousie University, Halifax, Nova Scotia B3H 4H7 (Accepted March 1976)
CLBIA, 9, (3) 109-110 (1976) Clin. Biochem. Longley, W. J.
Division of Clinical Chemistry, Nova Scotia Pathology Institute, and Department o/ Pathology, Dalhousie University, Halifax, N.S. P R O F I C I E N C Y T E S T I N G OF RADIOIMMUNOASSAY 1. Quality control is essential during the setting up and routine application of radioimmunoassay. 2. Aspects discussed' include the 50% binding intercept, slope of standard curve, and inter- and intra-assay variance.
RADIOIMMUNOASSAY ( R I A ) was i n t r o d u c e d by B e r s o n and Yalow in the mid 1950s f o r m e a s u r i n g insulin. I t s use has r a p i d l y i n c r e a s e d to include o t h e r h o r mones and such s u b s t a n c e s as c a r c i n o e m b r y o n i c a n t i b o d i e s , h e p a t i t i s a n t i g e n s a n d d r u g s (aee r e v i e w s , ref. 3, 5, and 6). R I A is a f o r m of s a t u r a t i o n analysis"-", a g r o u p of methods t h a t includes c o m p e t i t i v e - p r o t e i n - b i n d i n g analysis. T h e following comments a r e equally a p p r o p r i a t e to t h i s t e c h n i q u e and R I A . G r a p h i c a l r e p r e s e n t a t i o n of RIA data engenders considerable difficulties, mainly because (a) the t y p i c a l R I A d o s e - - r e s p o n s e curve is n o n l i n e a r ; a n d (b) the ' s c a t t e r ' of r e s i d u a l v a r i a n c e a r o u n d t h e curve is not constant, d e p e n d i n g r a t h e r on t h e dose level c''4~. L o g i t t r a n s f o r m a t i o n , w i t h w e i g h t i n g a c c o r d i n g to t h e r e c i p r o c a l of t h e v a r i a n c e of logit ( Y ) , a p p e a r s to be an acceptable g r a p h i c a l m e a n s of h a n d l i n g d a t a g e n e r a t e d by R I A c''4~, b u t n e c e s s i t a t e s t h e use of a t l e a s t a p r o g r a m m a b l e c a l c u l a t o r . To obtain the weighting factors, each assay system must be e x a m i n e d a n d v a r i a n c e s o f t h e l o g i t Y e s t i m a t e d . S t r i c t c r i t e r i a , to m a i n t a i n q u a l i t y control, m u s t be applied, b o t h d u r i n g d e v e l o p m e n t o f t h e a s s a y and d u r i n g its r o u t i n e use.
Criteria to be followed w h e n s e t t i n g up an a s s a y M a n y a n t i b o d i e s can be o b t a i n e d commercially, a n d m o s t of t h e m p e r f o r m s a t i s f a c t o r i l y . However, r e g a r d less of the source, p r e l i m i n a r y t e s t i n g should be done to d e t e r m i n e t h e s e n s i t i v i t y , precision, and s p e c i f i c i t y (as d e f i n e d b y M i d g l e y et al ~') of t h e a s s a y - - even w h e n u s i n g a p p a r e n t l y i d e n t i c a l procedures, d i f f e r e n t l a b o r a t o r i e s o b t a i n d i f f e r e n t values f o r t h e s e v a r i a b l e s . F u r t h e r m o r e , t h e s t a n d a r d curves should be shown to be p a r a l l e l to t h e u n k n o w n s ; f o r example, when s e r u m is used, its s e r i a l d i l u t i o n s should r e s u l t in a curve p a r a l l e l to t h e s t a n d a r d curve. Also, t h e c o n c e n t r a t i o n
of the s u b s t a n c e p e r unit volume should be shown to be t h e same when v a r i o u s volumes of the unknown solut i o n a r e used. I f possible, one should also m e a s u r e t h e r e c o v e r y of a known q u a n t i t y of the s u b s t a n c e u n d e r test, added to unknown samples. The above c o n s t i t u t e only the b a r e m i n i m u m of r e q u i r e m e n t s to be m e t b e f o r e i n s t i t u t i n g r o u t i n e R I A of a substance. These p a r a m e t e r s should be checked periodically, m e a s u r e m e n t of s e n s i t i v i t y and p r e c i s i o n b e i n g noted on each run. F o r ease of c a l c u l a t i n g valid s t a t i s t i c s , each a s s a y r u n should follow a set protocol. Ideally, s t a n d a r d s and unknowns should be r a n d o m i s e d , b u t t h i s is c u m b e r some a n d often r e s u l t s ill t e c h n i c i a n e r r o r . To e f f e c t a s a t i s f a c t o r y compromise, in our l a b o r a t o r y we r u n t h e t u b e s for t h e f i r s t s t a n d a r d curve, followed by t h e f i r s t s e r i e s of unknowns, then the second s t a n d a r d curve followed b y a second set of the same unknowns, and f i n a l l y the t h i r d s t a n d a r d curve. Each s t a n d a r d curve c o n t a i n s t h r e e O - s t a n d a r d t u b e s (total, 9) : t h i s is imp o r t a n t , as the c o u n t i n g r a t e s of the o t h e r a s s a y t u b e s a r e e x p r e s s e d as a p r o p o r t i o n of the O - s t a n d a r d t u b e s . Some l a b o r a t o r i e s do not duplicate and t r i p l i c a t e to this extent, b u t in my opinion t h i s p r o c e d u r e is essential f o r several e a r l y runs of the a s s a y ; later, provided precision is s a t i s f a c t o r y , it m a y be possible to reduce t h e degree of replication.
Quality control in routine RIA a s s a y F i g . 1 shows most of the m e a s u r e m e n t s t h a t can be used. Note t h a t no l i m i t s of v a r i a t i o n a r e i n d i c a t e d on t h i s g r a p h , a l t h o u g h some a s s a y s a r e r e j e c t e d m a i n l y on t h e basis of b e t w e e n - a s s a y v a r i a n c e . Q u a l i t y control c h a r t s should be constructed, u s i n g a t l e a s t some of t h e s e p a r a m e t e r s , and l i m i t s of v a r i a t i o n should be s t r i c t l y a d h e r e d to. The use of s t a n d a r d d e v i a tions can lead to problems, in t h a t d i f f e r e n c e s m a y n o t be detected unless the c h a n g e s a r e f a i r l y large. The C U S U M plot a p p e a r s to have a d v a n t a g e s , b u t w i t h t h i s also one m u s t a p p l y a l i m i t to the change. The c r i t e r i a t h a t have proved most u s e f u l in r o u t i n e q u a l i t y c o n t r o l o f R I A a r e : 1) t h e 50% b i n d i n g i n t e r cept, 2) slope, 3) w i t h i n - a s s a y v a r i a n c e , and 4) bet w e e n - a s s a y variance. The 50% binding intercept (the h o r m o n e concent r a t i o n when the counts bound equal 50% of t h e counts b o u n d in t h e O - s t a n d a r d t u b e ) is m o r e u s e f u l t h a n o t h e r i n t e r c e p t values because i t n o r m a l l y falls on t h e p o r t i o n of t h e curve h a v i n g the s t e e p e s t slope; t h e r e fore, its value is influenced b y such f a c t o r s as t h e slope of the c u r v e a n d the p r e c i s i o n of t h e assay. Thus, small v a r i a t i o n s in t h i s value i n d i c a t e t h e a s s a y ' s overall v a r i a t i o n .
LONGLEY
110
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Fig. 1. Paramete~s 1o1" quality control of radioimmunoassay results. (Reproduced, with permission of the author and publishers, from Rodbard's article4.) Varialwzs labelled R are rejected oahws.
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ASSAY NU~2~R
The slope is i m p o r t a n t , even when the 50% b i n d i n g value is u n i f o r m , because a curve with little slope restilts in an assay t h a t lacks precision. Generally, the g r e a t e r the slope the better the assay, b u t sometimes iL is necessary to compromise between slope and r a n g e of the assay. W i t h i n - a s s a y variance can be calculated f r o m t r i plicate and duplicate d e t e r m i n a t i o n s w i t h i n a part i c u l a r r u n , or by u s i n g s e r u m pool3 r u n in t r i p l i c a t e or quadruplicate in each assay run. Between-assay variance is based upo:l pools r u n repeatedly in each assay run. Its d e t e r m i n a t i o n is essential in r o u t i n e clinical laboratorie~ when f r e q u e n t comparison is made from day to day or even m o n t h to month. REFERENCES 1. Midgley, A. R., Jr., Niswender, G. D. and Rebar, R. W. (1969). Principles for the assessment of the reliability of radioimmTtnoassay methods (precision, acauraey, sensitivity, specificity). In Immunoassay of Gonadotrophins (E. Diczfalusy, ed.), Trans. 1st Karolinska Symposium on Research Methods in Reproductive Endocrinology, Stockhohn, 1969. Karolinska Sjukhuset, Stockho!m, pp. 163-~80.
2. Murphy, B. E. P. (1970} Methodological problems i,~ competitive protein-binding techniqnes; the use of Sephadex cob~mn chromatography to separate steroids. In Steroid Assay by Protein Binding (E. Diezfalusy, ed.), Trans. 2nd Karolinska Symposium on Research Methods in Reproductive Endocrinology, Geneva, 1970. Karolinska Sjukhuset, Stockholm, pp. 37-56. 3. Odel[, W. D. and Daughaday, W. H., eds. (1971} Principles of Competitive Proteimbi~tdi~g Assays. Lipp.Lncott, Philadelphia and Toronto. 4. Rodbard, D. (1971) Statistical aspects of radioimmunoass:tys. In Principles of Competitive Protein-binding Assays (W. D. Odell and W. H. Daughaday, eds.), Lippincott, Philadelphia and Toronto, pp. 204-253. 5. Skelley, D. S., Brown, L. P. and Besch, P. K. (1973). Clin. Ch~-m., 19, 146-186. ~. Yalow, R. S. and Berson, S. A. (1971) Problems of •validation of radioi~lmunoa~says. In Principles of Competitive Protein-binding Assays (W. D. Odell and W. H. Daughaday, eds.), Lippincott, Philadelphia and Toronto, pp. 374-400.
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Proficiency Testing of Radioimmunoassay W. J. LONGLEY
Division of Clinical Chemistry, Nova Scotia Pathology Institute and Department of Pathology, Dalhousie University, Halifax, Nova Scotia B3H 4H7 (Accepted March 1976)
CLBIA, 9, (3) 109-110 (1976) Clin. Biochem. Longley, W. J.
Division of Clinical Chemistry, Nova Scotia Pathology Institute, and Department o/ Pathology, Dalhousie University, Halifax, N.S. P R O F I C I E N C Y T E S T I N G OF RADIOIMMUNOASSAY 1. Quality control is essential during the setting up and routine application of radioimmunoassay. 2. Aspects discussed' include the 50% binding intercept, slope of standard curve, and inter- and intra-assay variance.
RADIOIMMUNOASSAY ( R I A ) was i n t r o d u c e d by B e r s o n and Yalow in the mid 1950s f o r m e a s u r i n g insulin. I t s use has r a p i d l y i n c r e a s e d to include o t h e r h o r mones and such s u b s t a n c e s as c a r c i n o e m b r y o n i c a n t i b o d i e s , h e p a t i t i s a n t i g e n s a n d d r u g s (aee r e v i e w s , ref. 3, 5, and 6). R I A is a f o r m of s a t u r a t i o n analysis"-", a g r o u p of methods t h a t includes c o m p e t i t i v e - p r o t e i n - b i n d i n g analysis. T h e following comments a r e equally a p p r o p r i a t e to t h i s t e c h n i q u e and R I A . G r a p h i c a l r e p r e s e n t a t i o n of RIA data engenders considerable difficulties, mainly because (a) the t y p i c a l R I A d o s e - - r e s p o n s e curve is n o n l i n e a r ; a n d (b) the ' s c a t t e r ' of r e s i d u a l v a r i a n c e a r o u n d t h e curve is not constant, d e p e n d i n g r a t h e r on t h e dose level c''4~. L o g i t t r a n s f o r m a t i o n , w i t h w e i g h t i n g a c c o r d i n g to t h e r e c i p r o c a l of t h e v a r i a n c e of logit ( Y ) , a p p e a r s to be an acceptable g r a p h i c a l m e a n s of h a n d l i n g d a t a g e n e r a t e d by R I A c''4~, b u t n e c e s s i t a t e s t h e use of a t l e a s t a p r o g r a m m a b l e c a l c u l a t o r . To obtain the weighting factors, each assay system must be e x a m i n e d a n d v a r i a n c e s o f t h e l o g i t Y e s t i m a t e d . S t r i c t c r i t e r i a , to m a i n t a i n q u a l i t y control, m u s t be applied, b o t h d u r i n g d e v e l o p m e n t o f t h e a s s a y and d u r i n g its r o u t i n e use.
Criteria to be followed w h e n s e t t i n g up an a s s a y M a n y a n t i b o d i e s can be o b t a i n e d commercially, a n d m o s t of t h e m p e r f o r m s a t i s f a c t o r i l y . However, r e g a r d less of the source, p r e l i m i n a r y t e s t i n g should be done to d e t e r m i n e t h e s e n s i t i v i t y , precision, and s p e c i f i c i t y (as d e f i n e d b y M i d g l e y et al ~') of t h e a s s a y - - even w h e n u s i n g a p p a r e n t l y i d e n t i c a l procedures, d i f f e r e n t l a b o r a t o r i e s o b t a i n d i f f e r e n t values f o r t h e s e v a r i a b l e s . F u r t h e r m o r e , t h e s t a n d a r d curves should be shown to be p a r a l l e l to t h e u n k n o w n s ; f o r example, when s e r u m is used, its s e r i a l d i l u t i o n s should r e s u l t in a curve p a r a l l e l to t h e s t a n d a r d curve. Also, t h e c o n c e n t r a t i o n
of the s u b s t a n c e p e r unit volume should be shown to be t h e same when v a r i o u s volumes of the unknown solut i o n a r e used. I f possible, one should also m e a s u r e t h e r e c o v e r y of a known q u a n t i t y of the s u b s t a n c e u n d e r test, added to unknown samples. The above c o n s t i t u t e only the b a r e m i n i m u m of r e q u i r e m e n t s to be m e t b e f o r e i n s t i t u t i n g r o u t i n e R I A of a substance. These p a r a m e t e r s should be checked periodically, m e a s u r e m e n t of s e n s i t i v i t y and p r e c i s i o n b e i n g noted on each run. F o r ease of c a l c u l a t i n g valid s t a t i s t i c s , each a s s a y r u n should follow a set protocol. Ideally, s t a n d a r d s and unknowns should be r a n d o m i s e d , b u t t h i s is c u m b e r some a n d often r e s u l t s ill t e c h n i c i a n e r r o r . To e f f e c t a s a t i s f a c t o r y compromise, in our l a b o r a t o r y we r u n t h e t u b e s for t h e f i r s t s t a n d a r d curve, followed by t h e f i r s t s e r i e s of unknowns, then the second s t a n d a r d curve followed b y a second set of the same unknowns, and f i n a l l y the t h i r d s t a n d a r d curve. Each s t a n d a r d curve c o n t a i n s t h r e e O - s t a n d a r d t u b e s (total, 9) : t h i s is imp o r t a n t , as the c o u n t i n g r a t e s of the o t h e r a s s a y t u b e s a r e e x p r e s s e d as a p r o p o r t i o n of the O - s t a n d a r d t u b e s . Some l a b o r a t o r i e s do not duplicate and t r i p l i c a t e to this extent, b u t in my opinion t h i s p r o c e d u r e is essential f o r several e a r l y runs of the a s s a y ; later, provided precision is s a t i s f a c t o r y , it m a y be possible to reduce t h e degree of replication.
Quality control in routine RIA a s s a y F i g . 1 shows most of the m e a s u r e m e n t s t h a t can be used. Note t h a t no l i m i t s of v a r i a t i o n a r e i n d i c a t e d on t h i s g r a p h , a l t h o u g h some a s s a y s a r e r e j e c t e d m a i n l y on t h e basis of b e t w e e n - a s s a y v a r i a n c e . Q u a l i t y control c h a r t s should be constructed, u s i n g a t l e a s t some of t h e s e p a r a m e t e r s , and l i m i t s of v a r i a t i o n should be s t r i c t l y a d h e r e d to. The use of s t a n d a r d d e v i a tions can lead to problems, in t h a t d i f f e r e n c e s m a y n o t be detected unless the c h a n g e s a r e f a i r l y large. The C U S U M plot a p p e a r s to have a d v a n t a g e s , b u t w i t h t h i s also one m u s t a p p l y a l i m i t to the change. The c r i t e r i a t h a t have proved most u s e f u l in r o u t i n e q u a l i t y c o n t r o l o f R I A a r e : 1) t h e 50% b i n d i n g i n t e r cept, 2) slope, 3) w i t h i n - a s s a y v a r i a n c e , and 4) bet w e e n - a s s a y variance. The 50% binding intercept (the h o r m o n e concent r a t i o n when the counts bound equal 50% of t h e counts b o u n d in t h e O - s t a n d a r d t u b e ) is m o r e u s e f u l t h a n o t h e r i n t e r c e p t values because i t n o r m a l l y falls on t h e p o r t i o n of t h e curve h a v i n g the s t e e p e s t slope; t h e r e fore, its value is influenced b y such f a c t o r s as t h e slope of the c u r v e a n d the p r e c i s i o n of t h e assay. Thus, small v a r i a t i o n s in t h i s value i n d i c a t e t h e a s s a y ' s overall v a r i a t i o n .
LONGLEY
110
t ~ I de
I
2
3
4
.5
t
i
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i
6
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L~ A.~SAYCONTROL CHART 8 9 I0 II 12 13 14 15 16 17 18 19 20 i
i
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i
i
l
i
i
i
e I T ~ 5°° 250 t P.¢//~ 0L ~.r TviN 0
20,O00[-
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~0,000t0L O,O00r
llm:k~vnd CFm i 5o00~
Fig. 1. Paramete~s 1o1" quality control of radioimmunoassay results. (Reproduced, with permission of the author and publishers, from Rodbard's article4.) Varialwzs labelled R are rejected oahws.
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ASSAY NU~2~R
The slope is i m p o r t a n t , even when the 50% b i n d i n g value is u n i f o r m , because a curve with little slope restilts in an assay t h a t lacks precision. Generally, the g r e a t e r the slope the better the assay, b u t sometimes iL is necessary to compromise between slope and r a n g e of the assay. W i t h i n - a s s a y variance can be calculated f r o m t r i plicate and duplicate d e t e r m i n a t i o n s w i t h i n a part i c u l a r r u n , or by u s i n g s e r u m pool3 r u n in t r i p l i c a t e or quadruplicate in each assay run. Between-assay variance is based upo:l pools r u n repeatedly in each assay run. Its d e t e r m i n a t i o n is essential in r o u t i n e clinical laboratorie~ when f r e q u e n t comparison is made from day to day or even m o n t h to month. REFERENCES 1. Midgley, A. R., Jr., Niswender, G. D. and Rebar, R. W. (1969). Principles for the assessment of the reliability of radioimmTtnoassay methods (precision, acauraey, sensitivity, specificity). In Immunoassay of Gonadotrophins (E. Diczfalusy, ed.), Trans. 1st Karolinska Symposium on Research Methods in Reproductive Endocrinology, Stockhohn, 1969. Karolinska Sjukhuset, Stockho!m, pp. 163-~80.
2. Murphy, B. E. P. (1970} Methodological problems i,~ competitive protein-binding techniqnes; the use of Sephadex cob~mn chromatography to separate steroids. In Steroid Assay by Protein Binding (E. Diezfalusy, ed.), Trans. 2nd Karolinska Symposium on Research Methods in Reproductive Endocrinology, Geneva, 1970. Karolinska Sjukhuset, Stockholm, pp. 37-56. 3. Odel[, W. D. and Daughaday, W. H., eds. (1971} Principles of Competitive Proteimbi~tdi~g Assays. Lipp.Lncott, Philadelphia and Toronto. 4. Rodbard, D. (1971) Statistical aspects of radioimmunoass:tys. In Principles of Competitive Protein-binding Assays (W. D. Odell and W. H. Daughaday, eds.), Lippincott, Philadelphia and Toronto, pp. 204-253. 5. Skelley, D. S., Brown, L. P. and Besch, P. K. (1973). Clin. Ch~-m., 19, 146-186. ~. Yalow, R. S. and Berson, S. A. (1971) Problems of •validation of radioi~lmunoa~says. In Principles of Competitive Protein-binding Assays (W. D. Odell and W. H. Daughaday, eds.), Lippincott, Philadelphia and Toronto, pp. 374-400.
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