Clin. Biochem. 9, (4) 225-227 (1976)
LABORATORY NOTE
Kinetic Urine Creatinine Determination With the Gemsaec Analyzer P. W A R D , Department
M. E W E N ,
J.A.
of C l i n i c a l B i o c h e m i s t r y ,
POMEROY
and F.Y.
LEUNG
University Hospital, London, Ontario
( A c c e p t e d A p r i l 30, 1976) Reagents CLBIA 9, (4) 225-227 (1976) Cli~. Bioche,n. Ward, P., Ewen, M., Pomeroy, J. A. and Leung, F. Y.
Department of Clinical Biochemistry, Hospital, Londo~l, Ontario.
University
KINETIC URINE CREATININE DETERMINATION WITH THE GEMSAEC ANALYZER A rapid automated method for u r i n a r y creatinine with the Jaff4 reaction is described. Interferences by protein, glucose and ketones ,~re circumvented by the reaction rate kinetics. The precision and accuracy of this centrifugal method on the ENIGemsaec analyzer are comparable to the nmnual end-point Jaff6 reaction with deproteinization and to the common Auto-Analyzer technique.
Manual and Gemsaec Sodium tungstate 10 g/dl aqueous solution, sulphuric acid '0.66N, sodium hydroxide 0.75N, and picric acid 0.04M, were prepared from reagent grade chemicals (Fisher Scientific Co., Toronto, Ontario). Creatinine standards 5 nag, 8 nag, and 100 mg/dl in distilled water (Sigma Chemical Co., St. Louis, Missouri, 63178). A u to-A nalyzer Brij-35 (30% solution), 0.1% V/V solution in distilled water, picric acid 0.115 M (1.3% W / V ) , sodium hydroxide 0.5N (2% W/V) and sodium chloride, 1.8% were Technicon prepared reagents. PROCEDURE
Manual
Sample Preparation URINARY CREATININE DETERMINATIONS are r o u t i n e l y requested in a clinical c h e m i s t r y l a b o r a t o r y to monitor collection accuracy of 24-hour u r i n e specimens, to m e a s u r e c r e a t i n i n e clearance, and to d e t e r m i n e u r i n e c o n s t i t u e n t q u a n t i t i e s as c r e a t i n i n e ratios "). I n order to meet a steadily g r o w i n g workload of these requests and to provide a rapid b u t accurate procedure for u r i n a r y c r e a t i n i n e , an a u t o m a t e d procedure was adopted which uses the c e n t r i f u g a l analyzer. A m a n u a l Jaff~ procedure'-" was modified for application on the Gemsaec batch-parallel analyzer. The cent r i f u g a l analyzer r e a c t i o n - r a t e method was compared to a c o n t i n u o u s f l o w A u t o - A n a l y z e r procedure as well as to the m a n u a l end-point technique. MATERIALS AND METHODS
Urine samples are diluted 1:100 with distilled water, and 3 ml are used for the assay. Urine samples having a positive Labstix (Ames) test for protein are treated with 1 ml sodium tungstate and 1 ml sulphuric acid to obtain a protein-free filtrate of the 1:]00 diluted urine.
Method To each 3 ml sample for analysis, 1 ml picric acid and 1 Inl 0.75N sodium hydroxide are added and mixed thoroughly. In exactly 20 minutes, the samples are measured using a Gilford spectrophotometer at 520 nm equipped with a flow-through cuvette. The optical density of the blank is subtracted from each standard and test before calculation of the values. Auto-Analyzer
Sample Preparatiou Urines are diluted 1:20 with distilled water before analysis.
Method Instruments Micro-Sample Spectrophotometer, 300-N, for sample volumes less than 500 ~d, Gilford I n s t r u m e n t Laboratories Inc., Oberlin, Ohio, 44074, USA, Auto Analyzer II system equipped with a dual pen recorder, a modular digital printer, and a Creatinine Analytical Cartridge (PN157A066-01), from Technicon Instruments Corp., Tarrytown, N.Y., 10591, USA, and Gemsaec centrifugal ~nalyzer from Electro-Nucleonics Inc., Fairfield, New Jersey, 07006, USA.
A standard Technicon method is used for the Auto Analyzer II in which protein is removed by dialysis. Specifications are described in Technicon Bulletin SF40011FH4, August 1974. Gemsaec
Reagent Preparation Picric acid solution 0.04M, 7.5 ml is mixed with sodium hydroxide, 0.75N, 7.5 ml and 15 ml distilled water. This reagent mixture is stable for one day.
Sample Preparation Correspondence: Dr. F. Y. Leung, Department of Clinical Biochemistry, University Hospital, London, Ontario
Urines are diluted 1:20 with distilled water. Deproteinization is unnecessary as proteins do not interfere with this rate-reaction method. Creatinine standard 100 rag/ dl is diluted 1:20 as for the samples.
WARD, EWEN, POMEROY AND LEUNG
226
TABLE 1
P a r a m eters
Reaction temperature 30 °, wavelength 515nm, filter position 385-560 nm, reaction mode = rate, r u n n i n g mode = auto, first reading = 10 seconds, reading interval = 40 seconds, number of readings = 2. Method
The rotor head is loaded with 25 ~l aliquots of standard or sample into the sample cavities. A 75 ~tl water rinse is used on the rotor loader, and added to the sample wells. Alkaline picrate reagent, 400 ltl is loaded into all the innermost cavities. A reagent blank and a standard reference solution are placed in positions 1 and 2, respectively. The assay is run under the above parameter modes.
PRECISIONOF GEMSAECKINETICMETHOD Within rotor runs
mg/dl
Sample
Number
Hyland 'Q-Pak' * Urine a Urine b Between rotor runs Urine c Hyland 'Q-Pak' * Urine d
Mean
4- S.D.
C.V. %
14 14 14
56.3 57.9 105.4
0.98 1.05 1.26
1.7 1.8 1.2
121 215 129
22.4 51.9 104.6
L63 1.48 2.52
7.2 2.8 2.4
*Hyland, Division of Travenol Laboratories, Lot No. 0401K004A1 & 4A2
RESULTS The time on the Gemsaec for the reaction kinetics was chosen to read at a p p r o x i m a t e l y 76 seconds which includes an i n s t r u m e n t delay of a p p r o x i m a t e l y 26 seconds from the i n i t i a t i o n of the run, 10 seconds m i n i m u m .for f i r s t r e a d i n g and a 40 second r e a d i n g interval. These s e t t i n g s were f o u n d to give identical kinetics for aqueous c r e a t i n i n e s t a n d a r d s and u r i n e c r e a t i n i n e samples. The absorbance was l i n e a r to 20 m g / d l (equivalent to 400 m g / d l , u r i n e s diluted 1:20) u s i n g p r i m a r y s t a n d a r d solutions. The precision of the Gemsaec procedure for u r i n e c r e a t i n i n e s and lyophilized control u r i n e s is shown in Table 1. Single rotor r u n s of 14 samples plus a blank and a s t a n d a r d gave coefficient of v a r i a t i o n s less t h a n 2 G . Between rotor analyses and day-to-day v a r i a t i o n s ranged between 2 and 8%. Redovery studies of added c r e a t i n i n e s t a n d a r d s to u r i n e samples were compared for the reaction rate (Gemsaec) to the continuous-flow (Auto A n a l y z e r ) procedure. As shown in Table 2, the average recoveries for the r a n g e between 60 and 430 m g / d l were 102 -- 29~ and 100 ---+2c)~- respectively. A comparison of 87 p a t i e n t u r i n e c r e a t i n i n e results by the m a n u a l and the Gemsaec procedures gave a linear regression line, y = 1.004x-0.84 with a correlation coefficient of r = 0.999. A close correlation is similarly found for the r e g r e s s i o n a n a l y s i s of the Gemsaec and the Auto A n a l y z e r results for 83 u r i n e c r e a t i n i n e samples y = 0.984x-0.92, r = 0.998. A p p l y i n g the S t u d e n t t-test on the paired results showed a s i g n i f i c a n t r e l a t i o n s h i p exists for these correlations. The values f o u n d were t = 2.4, 0.05 > p > 0.01 for the m a n u a l / G e m s a e c and t ~ 7.4, 0.05 > p < 0.01 for the Auto A n a l y z e r / G e m s a e c comparisons. The influence of known c o n s t i t u e n t s t h a t i n t e r f e r e with the J a f f 6 picrate reaction in u r i n e u n d e r pathological conditions was tested with the kinetic reaction method. As noted in Table 3, no a p p a r e n t effects on the level of e r e a t i n i n e were found. DISCUSSION L a r s e n ~") has d e m o n s t r a t e d with the L K B 8600 Reaction Rate A n a l y z e r t h a t the second m i n u t e of the Jaff6 picrate reaction for c r e a t i n i n e is as specific as c r e a t i n i n e methods u s i n g Lloyd's reagent. A t this time of the reaction, i n t e r f e r e n c e by a n o n - c r e a t i n i n e pic r a t e - r e a c t i n g s u b s t a n c e s is at a m i n i m u m . I n p a t i e n t s with p r o t e i n u r i a , d e p r o t e i n i z a t i o n was un-
TABLE 2 RECOVERY OF STANDARDCREATININEADDEDTO URINES Expected
Found Gemsaec
Auto Analyzer II
mg/dl
mg/I
%
mg/dl
63 88 113 138 163 288 431
66 92 112 145 169 288 446
104 104 99 105 103 100 103
64 86 112 138 164 286 452
102 4- 2%
% 101 98 99 100 101 99 i05 100 4- 2%
TABLE 3 EFFECT OF CONSTITUENTS ON URINARY CREATININE VALUES BY KINETIC GEMSAEC M E T H O D
Substance
Concentration mg/dl.
Multistix Reaction*
Apparent Creatinine mg/dl.
Acetone
0 800
0 3W
28.9 28.6
Glucose
0 1500
0 3+
52.3 52.4
Protein
0 1000
0 4+
85.0 85.1
*Ames Company Dipstik
necessary as in s i m i l a r k i n e t i c s e r u m c r e a t i n i n e assays '~'~'. The m a n u a l or Auto A n a l y z e r J a f f 6 reaction both of which r e q u i r e p r i o r p r o t e i n removal is considered the most n e a r l y specific procedure for u r i n a r y c r e a t i n i n e when tested by a c o u n t e r - c u r r e n t d i s t r i b u t i o n technique 'e'. These l a t e r studies also showed t h a t no a d v a n t a g e was gained by t r e a t i n g u r i n e with Lloyd's r e a g e n t or t h r o u g h p r i o r extraction with ether. I n the p r e s e n t study, very close correlation of the r e s u l t s were obtained by the k i n e t i c Gemsaec method with the m a n u a l and the A u t o - A n alyzer II techniques. By a comparison of the Gemsaec to the m a n u a l and Auto Analyzer I I mean values for u r i n e crea t i n i n e , the r e a c t i o n - r a t e results show a n e g a t i v e bias of 0.04 and 0.07 mg/dl, respectively. A s i m i l a r bias has been reported by Cook c') who compared the
URINE CREATININE DETERMINATION findings of a quality control p r o g r a m m e of creatinine in serum samples using discrete analyzer (-0.15) and Lloyd's reagent (-0.08) methods. It was suggested that these procedures probably avoid measuring the contribution of i n t e r f e r i n g substances from the alkaline picrate reaction 'TJ. The main advantages for the use of a reaction-rate procedure for urine creatinine measurements a r e : unnecessary deproteinization, absence of interference by usual u r i n a r y constituents such as glucose and ketones in pathological urines, rapid generation of results, and specificity in comparison to the Auto Analyzer or more time consuming manual procedure. The method's simplicity and reagent s t a b i l i t y have proven to be reliable features of this procedure over the past y e a r ' s routine use in the laboratory.
227 REFERENCES
1. Henry, R. J., Cannon, D. C. and Winkelman, J.W., eds., Clinical Chemistry P~qnciples and Technics, 2nd Ed., Harper and Row, N.Y., p. 541, 1974. 2. Owen, J. A., Iggo B., Scandrett, F. J., and Stewart, C. P. (1954). Bioche~ J., 58, 426-437. 3. Larsen, K. (1972). Clin. Chim. Acts, 41, 209-217. 4. Fabiny, D. L. and Ertingshausen, G. (1971). Cli~t. Chem., 17, 696-700. 5. Cook, J. G. H. (1971). Cli~t. Chichi. Acts, 32, 485-486. 6. Narayanan, S. and Appleton, H. D. (1972). CIbt. Chem., 18, 270-274. 7. Cook, J. G. H. {1975). A~t~. Clbl. Biochem., 12, 219-232.
LABORATORY NOTE
Kinetic Urine Creatinine Determination With the Gemsaec Analyzer P. W A R D , Department
M. E W E N ,
J.A.
of C l i n i c a l B i o c h e m i s t r y ,
POMEROY
and F.Y.
LEUNG
University Hospital, London, Ontario
( A c c e p t e d A p r i l 30, 1976) Reagents CLBIA 9, (4) 225-227 (1976) Cli~. Bioche,n. Ward, P., Ewen, M., Pomeroy, J. A. and Leung, F. Y.
Department of Clinical Biochemistry, Hospital, Londo~l, Ontario.
University
KINETIC URINE CREATININE DETERMINATION WITH THE GEMSAEC ANALYZER A rapid automated method for u r i n a r y creatinine with the Jaff4 reaction is described. Interferences by protein, glucose and ketones ,~re circumvented by the reaction rate kinetics. The precision and accuracy of this centrifugal method on the ENIGemsaec analyzer are comparable to the nmnual end-point Jaff6 reaction with deproteinization and to the common Auto-Analyzer technique.
Manual and Gemsaec Sodium tungstate 10 g/dl aqueous solution, sulphuric acid '0.66N, sodium hydroxide 0.75N, and picric acid 0.04M, were prepared from reagent grade chemicals (Fisher Scientific Co., Toronto, Ontario). Creatinine standards 5 nag, 8 nag, and 100 mg/dl in distilled water (Sigma Chemical Co., St. Louis, Missouri, 63178). A u to-A nalyzer Brij-35 (30% solution), 0.1% V/V solution in distilled water, picric acid 0.115 M (1.3% W / V ) , sodium hydroxide 0.5N (2% W/V) and sodium chloride, 1.8% were Technicon prepared reagents. PROCEDURE
Manual
Sample Preparation URINARY CREATININE DETERMINATIONS are r o u t i n e l y requested in a clinical c h e m i s t r y l a b o r a t o r y to monitor collection accuracy of 24-hour u r i n e specimens, to m e a s u r e c r e a t i n i n e clearance, and to d e t e r m i n e u r i n e c o n s t i t u e n t q u a n t i t i e s as c r e a t i n i n e ratios "). I n order to meet a steadily g r o w i n g workload of these requests and to provide a rapid b u t accurate procedure for u r i n a r y c r e a t i n i n e , an a u t o m a t e d procedure was adopted which uses the c e n t r i f u g a l analyzer. A m a n u a l Jaff~ procedure'-" was modified for application on the Gemsaec batch-parallel analyzer. The cent r i f u g a l analyzer r e a c t i o n - r a t e method was compared to a c o n t i n u o u s f l o w A u t o - A n a l y z e r procedure as well as to the m a n u a l end-point technique. MATERIALS AND METHODS
Urine samples are diluted 1:100 with distilled water, and 3 ml are used for the assay. Urine samples having a positive Labstix (Ames) test for protein are treated with 1 ml sodium tungstate and 1 ml sulphuric acid to obtain a protein-free filtrate of the 1:]00 diluted urine.
Method To each 3 ml sample for analysis, 1 ml picric acid and 1 Inl 0.75N sodium hydroxide are added and mixed thoroughly. In exactly 20 minutes, the samples are measured using a Gilford spectrophotometer at 520 nm equipped with a flow-through cuvette. The optical density of the blank is subtracted from each standard and test before calculation of the values. Auto-Analyzer
Sample Preparatiou Urines are diluted 1:20 with distilled water before analysis.
Method Instruments Micro-Sample Spectrophotometer, 300-N, for sample volumes less than 500 ~d, Gilford I n s t r u m e n t Laboratories Inc., Oberlin, Ohio, 44074, USA, Auto Analyzer II system equipped with a dual pen recorder, a modular digital printer, and a Creatinine Analytical Cartridge (PN157A066-01), from Technicon Instruments Corp., Tarrytown, N.Y., 10591, USA, and Gemsaec centrifugal ~nalyzer from Electro-Nucleonics Inc., Fairfield, New Jersey, 07006, USA.
A standard Technicon method is used for the Auto Analyzer II in which protein is removed by dialysis. Specifications are described in Technicon Bulletin SF40011FH4, August 1974. Gemsaec
Reagent Preparation Picric acid solution 0.04M, 7.5 ml is mixed with sodium hydroxide, 0.75N, 7.5 ml and 15 ml distilled water. This reagent mixture is stable for one day.
Sample Preparation Correspondence: Dr. F. Y. Leung, Department of Clinical Biochemistry, University Hospital, London, Ontario
Urines are diluted 1:20 with distilled water. Deproteinization is unnecessary as proteins do not interfere with this rate-reaction method. Creatinine standard 100 rag/ dl is diluted 1:20 as for the samples.
WARD, EWEN, POMEROY AND LEUNG
226
TABLE 1
P a r a m eters
Reaction temperature 30 °, wavelength 515nm, filter position 385-560 nm, reaction mode = rate, r u n n i n g mode = auto, first reading = 10 seconds, reading interval = 40 seconds, number of readings = 2. Method
The rotor head is loaded with 25 ~l aliquots of standard or sample into the sample cavities. A 75 ~tl water rinse is used on the rotor loader, and added to the sample wells. Alkaline picrate reagent, 400 ltl is loaded into all the innermost cavities. A reagent blank and a standard reference solution are placed in positions 1 and 2, respectively. The assay is run under the above parameter modes.
PRECISIONOF GEMSAECKINETICMETHOD Within rotor runs
mg/dl
Sample
Number
Hyland 'Q-Pak' * Urine a Urine b Between rotor runs Urine c Hyland 'Q-Pak' * Urine d
Mean
4- S.D.
C.V. %
14 14 14
56.3 57.9 105.4
0.98 1.05 1.26
1.7 1.8 1.2
121 215 129
22.4 51.9 104.6
L63 1.48 2.52
7.2 2.8 2.4
*Hyland, Division of Travenol Laboratories, Lot No. 0401K004A1 & 4A2
RESULTS The time on the Gemsaec for the reaction kinetics was chosen to read at a p p r o x i m a t e l y 76 seconds which includes an i n s t r u m e n t delay of a p p r o x i m a t e l y 26 seconds from the i n i t i a t i o n of the run, 10 seconds m i n i m u m .for f i r s t r e a d i n g and a 40 second r e a d i n g interval. These s e t t i n g s were f o u n d to give identical kinetics for aqueous c r e a t i n i n e s t a n d a r d s and u r i n e c r e a t i n i n e samples. The absorbance was l i n e a r to 20 m g / d l (equivalent to 400 m g / d l , u r i n e s diluted 1:20) u s i n g p r i m a r y s t a n d a r d solutions. The precision of the Gemsaec procedure for u r i n e c r e a t i n i n e s and lyophilized control u r i n e s is shown in Table 1. Single rotor r u n s of 14 samples plus a blank and a s t a n d a r d gave coefficient of v a r i a t i o n s less t h a n 2 G . Between rotor analyses and day-to-day v a r i a t i o n s ranged between 2 and 8%. Redovery studies of added c r e a t i n i n e s t a n d a r d s to u r i n e samples were compared for the reaction rate (Gemsaec) to the continuous-flow (Auto A n a l y z e r ) procedure. As shown in Table 2, the average recoveries for the r a n g e between 60 and 430 m g / d l were 102 -- 29~ and 100 ---+2c)~- respectively. A comparison of 87 p a t i e n t u r i n e c r e a t i n i n e results by the m a n u a l and the Gemsaec procedures gave a linear regression line, y = 1.004x-0.84 with a correlation coefficient of r = 0.999. A close correlation is similarly found for the r e g r e s s i o n a n a l y s i s of the Gemsaec and the Auto A n a l y z e r results for 83 u r i n e c r e a t i n i n e samples y = 0.984x-0.92, r = 0.998. A p p l y i n g the S t u d e n t t-test on the paired results showed a s i g n i f i c a n t r e l a t i o n s h i p exists for these correlations. The values f o u n d were t = 2.4, 0.05 > p > 0.01 for the m a n u a l / G e m s a e c and t ~ 7.4, 0.05 > p < 0.01 for the Auto A n a l y z e r / G e m s a e c comparisons. The influence of known c o n s t i t u e n t s t h a t i n t e r f e r e with the J a f f 6 picrate reaction in u r i n e u n d e r pathological conditions was tested with the kinetic reaction method. As noted in Table 3, no a p p a r e n t effects on the level of e r e a t i n i n e were found. DISCUSSION L a r s e n ~") has d e m o n s t r a t e d with the L K B 8600 Reaction Rate A n a l y z e r t h a t the second m i n u t e of the Jaff6 picrate reaction for c r e a t i n i n e is as specific as c r e a t i n i n e methods u s i n g Lloyd's reagent. A t this time of the reaction, i n t e r f e r e n c e by a n o n - c r e a t i n i n e pic r a t e - r e a c t i n g s u b s t a n c e s is at a m i n i m u m . I n p a t i e n t s with p r o t e i n u r i a , d e p r o t e i n i z a t i o n was un-
TABLE 2 RECOVERY OF STANDARDCREATININEADDEDTO URINES Expected
Found Gemsaec
Auto Analyzer II
mg/dl
mg/I
%
mg/dl
63 88 113 138 163 288 431
66 92 112 145 169 288 446
104 104 99 105 103 100 103
64 86 112 138 164 286 452
102 4- 2%
% 101 98 99 100 101 99 i05 100 4- 2%
TABLE 3 EFFECT OF CONSTITUENTS ON URINARY CREATININE VALUES BY KINETIC GEMSAEC M E T H O D
Substance
Concentration mg/dl.
Multistix Reaction*
Apparent Creatinine mg/dl.
Acetone
0 800
0 3W
28.9 28.6
Glucose
0 1500
0 3+
52.3 52.4
Protein
0 1000
0 4+
85.0 85.1
*Ames Company Dipstik
necessary as in s i m i l a r k i n e t i c s e r u m c r e a t i n i n e assays '~'~'. The m a n u a l or Auto A n a l y z e r J a f f 6 reaction both of which r e q u i r e p r i o r p r o t e i n removal is considered the most n e a r l y specific procedure for u r i n a r y c r e a t i n i n e when tested by a c o u n t e r - c u r r e n t d i s t r i b u t i o n technique 'e'. These l a t e r studies also showed t h a t no a d v a n t a g e was gained by t r e a t i n g u r i n e with Lloyd's r e a g e n t or t h r o u g h p r i o r extraction with ether. I n the p r e s e n t study, very close correlation of the r e s u l t s were obtained by the k i n e t i c Gemsaec method with the m a n u a l and the A u t o - A n alyzer II techniques. By a comparison of the Gemsaec to the m a n u a l and Auto Analyzer I I mean values for u r i n e crea t i n i n e , the r e a c t i o n - r a t e results show a n e g a t i v e bias of 0.04 and 0.07 mg/dl, respectively. A s i m i l a r bias has been reported by Cook c') who compared the
URINE CREATININE DETERMINATION findings of a quality control p r o g r a m m e of creatinine in serum samples using discrete analyzer (-0.15) and Lloyd's reagent (-0.08) methods. It was suggested that these procedures probably avoid measuring the contribution of i n t e r f e r i n g substances from the alkaline picrate reaction 'TJ. The main advantages for the use of a reaction-rate procedure for urine creatinine measurements a r e : unnecessary deproteinization, absence of interference by usual u r i n a r y constituents such as glucose and ketones in pathological urines, rapid generation of results, and specificity in comparison to the Auto Analyzer or more time consuming manual procedure. The method's simplicity and reagent s t a b i l i t y have proven to be reliable features of this procedure over the past y e a r ' s routine use in the laboratory.
227 REFERENCES
1. Henry, R. J., Cannon, D. C. and Winkelman, J.W., eds., Clinical Chemistry P~qnciples and Technics, 2nd Ed., Harper and Row, N.Y., p. 541, 1974. 2. Owen, J. A., Iggo B., Scandrett, F. J., and Stewart, C. P. (1954). Bioche~ J., 58, 426-437. 3. Larsen, K. (1972). Clin. Chim. Acts, 41, 209-217. 4. Fabiny, D. L. and Ertingshausen, G. (1971). Cli~t. Chem., 17, 696-700. 5. Cook, J. G. H. (1971). Cli~t. Chichi. Acts, 32, 485-486. 6. Narayanan, S. and Appleton, H. D. (1972). CIbt. Chem., 18, 270-274. 7. Cook, J. G. H. {1975). A~t~. Clbl. Biochem., 12, 219-232.
