Clin Biochem, Vol. 24, pp. 59-62, 1991 Printed in Canada. All rights reserved.
0009-9120/91 $3.00 + .00 Copyright c 1991 The Canadian Society of Clinical Chemists.
Quality Assessment of Cyclosporine Monitoring by 32 Canadian Laboratories P. Y. WONG, 1 A. V. MEE, ~ J. GLENN, 2 and P. A. KEOWN 2 1The Toronto Hospital, Toronto, Ontario, Canada and 2Vancouver General Hospital, Vancouver, British Columbia, Canada The Canadian Quality Assurance Program was initiated in June 1989, and is a voluntary program which currently encompasses all 32 laboratories involved in the measurement of cyclosporine (CsA) across Canada. Two whole blood samples from control or clinical patients (kidney, liver and heart) containing unknown concentrations of CsA are circulated to each participating laboratory monthly, and analyzed by all techniques employed within that laboratory. Four analytical methods are currently employed: HPLC (n = 4), Sandimmun SP (n = 3), CycloTrac SP (n = 27) and TDx (n = 3). Four laboratories reported survey results in more than one methodology. Results from all participating centers are analyzed monthly. The mean, SD, standard deviation index and range are reported to each laboratory with information coded to preserve confidentiality. Accuracy, precision, recovery, analytical specificity, linearity and blank studies have been performed. This report covers the period from June 1989 to April 1990.
KEY WORDS: cyclosporine monitoring; quality assurance; accuracy; precision. Introduction
the Fall of 1988, a small group of clinical and investigators was formed to address Ithe nlaboratory difficulties in cyclosporine (CsA) monitoring. The principal objectives were to establish standard methods for measurement of CsA, to develop a national quality assurance program, and to define the optimal therapeutic range according to immunosuppressive protocol, organ graft, analytical technique and matrix. The Canadian Quality Assurance Program is similar to the U K CsA Scheme organized by Holt and Johnston (1). It is voluntary, and consists of two blood samples containing unknown concentrations of CsA circulated monthly from The Toronto Hospital (Sample Distribution Center). Results from all participating centers are reported monthly, and all information is coded to preserve confidentiality (Vancouver General Hospital is the Data Manage-
Correspondence: Dr. P. Y. Wong, Department of Clinical Biochemistry, The Toronto Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4. Manuscript received May 30, 1990; revised July 9, 1990; accepted August 21, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
ment Center). The first survey started in J u n e 1989. Every one of the 32 Canadian laboratories involved in the measurement of CsA has participated in this program. This report covers the period from J u n e 1989 to April 1990, during which 3 accuracy, 6 precision, 1 recovery, 1 analytical specificity, 1 linearity and 1 blank studies have been performed. Materials
and
methods
Whole blood testing materials were obtained either by spiking CsA in time-expired blood obtained from the Red Cross or by mixing EDTA blood samples from renal, heart and liver transplant patients. All pools were negative for hepatitis B surface antigen and HIV antibody. For accuracy and recovery studies, 129.2 mg of pure CsA (Sandoz Batch No. 84097) was dissolved in high-performance liquid chromatography (HPLC) grade methanol in a 100 mL volumetric flask. The working CsA standard concentration was 12.92 tLg/ mL in 70% (v/v) aqueous methanol. It was then spiked into blood pools from the Blood Bank and from kidney transplant patients. The final methanol concentration was less than 2.5%. Three accuracy and 1 recovery studies were performed. For precision studies, same blood pools were sent to participating laboratories in separate surveys. Blood pools from kidney, heart and liver transplant patients were used. For the linearity study, a renal transplant whole blood pool was diluted 1:1 with the Blood Bank pool. For the analytical specificity study, CsA metaboTABLE 1
Stability of Testing Materials Storage Temp.
Method
0 too. b
CsA (~g/L) 3 mo.
4 too.
RT~ RT - 20 °C - 20 °C
CycloTrac SP CycloTrac SP HPLC HPLC
150 289 113 240
162 290 114 235
142 287 120 --
aRT, room t e m p e r a t u r e ; bmo., months. 59
WONG, MEE, GLENN, AND KEOWN
TABLE 3
TABLE 2 Summary of Three Accuracy Studies
Method HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
Recovery Study
Distribution of Results as Compared to Target Values 0-10% 10-20% >20% 9 7
2 2
1 0
39
22
8
7 7
1 2
1 0
Three accuracy studies with CsA target values of 388, 258 and 129 ~Lg/L, respectively, were performed. This table shows the cumulative distribution of laboratory results expressed as the percent difference compared to target values. The percent difference = [laboratory's result - target value] x 100 / target value. The number of laboratories using HPLC, Sandimmun, CycloTrac SP with kit standards, CycloTrac SP with in-house standards and TDx are 4, 3, 23, 3, and 3, respectively. Four laboratories reported survey results in more than one methodology.
Method
N
HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
Range of Recovery
4 3
95-110% 87-103%
23
88-129%
3 3
84-109% 95-113%
tibody kits (6). Four laboratories use the HPLC method (7,8). Results were analyzed monthly. The mean, SD, median, r a n g e , n u m b e r of l a b o r a t o r i e s u s i n g a part i c u l a r methodology, s t a n d a r d d e v i a t i o n index (SDI = [result - SD]/SD) and g r a p h i c d i s t r i b u t i o n of l a b o r a t o r y r e s u l t s were r e p o r t e d to each l a b o r a t o r y w i t h i n f o r m a t i o n coded to p r e s e r v e confidentiality.
Results and discussion STABILITY OF TESTING MATERIALS
lites which were isolated and purified from the bile of liver t r a n s p l a n t p a t i e n t s on CsA (100 ~Lg/L each of M1, M8, M17, M18 and M21) were spiked in a blood pool c o n t a i n i n g 258 ~tg/L of p u r e CsA. For t h e b l a n k study, CsA-free E D T A blood was o b t a i n e d from n o r m a l volunteers. F o u r different methodologies are b e i n g used by 32 laboratories. T h e y are H P L C , r a d i o i m m u n o a s s a y (RIA) u s i n g 12sI-tracer, RIA u s i n g 3H-tracer a n d fluorescence polarization i m m u n o a s s a y (polyclonal antibody) (FPIA). F o u r l a b o r a t o r i e s r e p o r t e d s u r v e y r e s u l t s in m o r e t h a n one methodology. T w e n t y - f i v e l a b o r a t o r i e s use CycloTrac SP kits (2,3) (Incstar, Stillwater, MN, USA, 12SI-CsA w i t h specific monoclonal antibody). T h r e e laboratories use CycloTrac SP kits w i t h in-house whole blood standards. T h r e e l a b o r a t o r i e s use S a n d i m m u n SP kits (4,5) (Sandoz, Basel, Switzerland, 3H-CsA w i t h specific monoclonal antibody). T h r e e l a b o r a t o r i e s use T D x F P I A (Abbott, Chicago, IL, USA) nonselective polyclonal an-
Two pools, stored at room temperature (RT) and a t - 2 0 °C for a period of t h r e e m o n t h s were t e s t e d by u s i n g CycloTrac SP kits a n d the H P L C m e t h o d (8). T h e r e s u l t s are s u m m a r i z e d in T a b l e 1. T h e d a t a show t h a t the t e s t i n g m a t e r i a l s are stable w h e n stored e i t h e r a t RT or a t - 2 0 °C. ACCURACY
Three accuracy studies with CsA target values of 388, 258 and 129 ~g/L, respectively, were performed. Table 2 summarizes the results of all 3 studies, expressed as the percent difference of a laboratory's result compared to the target value. Over 90% of laboratories reported results within 20% of target values. RECOVERY
In the recovery study, Vial A obtained from a kidney transplant patient was the baseline sample.
TABLE4 Linearity Study Method HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
N
Vial A
Vial B*
B/A
4 3
129 +- 23.9 144 _+ 1.0
64.6 - 5.1 70.3 +- 5.5
0.50 0.49
24
173 -- 16.7
86.1 -+ 11.0
0.50
3 3
151 -+ 27.6 396 -+ 15.0
79.3 -+ 8.4 214 - 5.3
0.53 0.54
*B = 1 part A + 1 Part normal blood (unspiked). 60
CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
QUALITY ASSURANCE OF CYCLOSPORINE
TABLE 5 Summary of Six Precision Studies
Method HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
TABLE 6 CsA Concentrations of a CsA-Free Whole Blood Sample Determined by 4 Methods
Percent Difference Between Duplicate Results 0-10% 10-20% >20% 14 13
1 4
7 1
97
28
17
13 16
4 0
2 0
For the precision study, the same blood pool was sent to participating laboratories in two separate surveys. Six precision studies were performed. This table shows the cumulative distribution of laboratory results expressed as the percent difference between two results. The percent difference = [result 1 - result 2] × 100 / lower result. Four laboratories reported survey results in more than one methodology.
Vial B was obtained by spiking 258 ~g/L CsA to Vial A. The majority of laboratories (29 out of 32) recovered 90 to 110% of added CsA (Table 3). L~NEARrrY All four methods showed good linearity at a parent CsA level below 200 ~g/L (Table 4).
Method
N
HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
Range (~Lg/L)
4 3
0-15 16-25
25
0-37
3 3
0-46 10-
0009-9120/91 $3.00 + .00 Copyright c 1991 The Canadian Society of Clinical Chemists.
Quality Assessment of Cyclosporine Monitoring by 32 Canadian Laboratories P. Y. WONG, 1 A. V. MEE, ~ J. GLENN, 2 and P. A. KEOWN 2 1The Toronto Hospital, Toronto, Ontario, Canada and 2Vancouver General Hospital, Vancouver, British Columbia, Canada The Canadian Quality Assurance Program was initiated in June 1989, and is a voluntary program which currently encompasses all 32 laboratories involved in the measurement of cyclosporine (CsA) across Canada. Two whole blood samples from control or clinical patients (kidney, liver and heart) containing unknown concentrations of CsA are circulated to each participating laboratory monthly, and analyzed by all techniques employed within that laboratory. Four analytical methods are currently employed: HPLC (n = 4), Sandimmun SP (n = 3), CycloTrac SP (n = 27) and TDx (n = 3). Four laboratories reported survey results in more than one methodology. Results from all participating centers are analyzed monthly. The mean, SD, standard deviation index and range are reported to each laboratory with information coded to preserve confidentiality. Accuracy, precision, recovery, analytical specificity, linearity and blank studies have been performed. This report covers the period from June 1989 to April 1990.
KEY WORDS: cyclosporine monitoring; quality assurance; accuracy; precision. Introduction
the Fall of 1988, a small group of clinical and investigators was formed to address Ithe nlaboratory difficulties in cyclosporine (CsA) monitoring. The principal objectives were to establish standard methods for measurement of CsA, to develop a national quality assurance program, and to define the optimal therapeutic range according to immunosuppressive protocol, organ graft, analytical technique and matrix. The Canadian Quality Assurance Program is similar to the U K CsA Scheme organized by Holt and Johnston (1). It is voluntary, and consists of two blood samples containing unknown concentrations of CsA circulated monthly from The Toronto Hospital (Sample Distribution Center). Results from all participating centers are reported monthly, and all information is coded to preserve confidentiality (Vancouver General Hospital is the Data Manage-
Correspondence: Dr. P. Y. Wong, Department of Clinical Biochemistry, The Toronto Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4. Manuscript received May 30, 1990; revised July 9, 1990; accepted August 21, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
ment Center). The first survey started in J u n e 1989. Every one of the 32 Canadian laboratories involved in the measurement of CsA has participated in this program. This report covers the period from J u n e 1989 to April 1990, during which 3 accuracy, 6 precision, 1 recovery, 1 analytical specificity, 1 linearity and 1 blank studies have been performed. Materials
and
methods
Whole blood testing materials were obtained either by spiking CsA in time-expired blood obtained from the Red Cross or by mixing EDTA blood samples from renal, heart and liver transplant patients. All pools were negative for hepatitis B surface antigen and HIV antibody. For accuracy and recovery studies, 129.2 mg of pure CsA (Sandoz Batch No. 84097) was dissolved in high-performance liquid chromatography (HPLC) grade methanol in a 100 mL volumetric flask. The working CsA standard concentration was 12.92 tLg/ mL in 70% (v/v) aqueous methanol. It was then spiked into blood pools from the Blood Bank and from kidney transplant patients. The final methanol concentration was less than 2.5%. Three accuracy and 1 recovery studies were performed. For precision studies, same blood pools were sent to participating laboratories in separate surveys. Blood pools from kidney, heart and liver transplant patients were used. For the linearity study, a renal transplant whole blood pool was diluted 1:1 with the Blood Bank pool. For the analytical specificity study, CsA metaboTABLE 1
Stability of Testing Materials Storage Temp.
Method
0 too. b
CsA (~g/L) 3 mo.
4 too.
RT~ RT - 20 °C - 20 °C
CycloTrac SP CycloTrac SP HPLC HPLC
150 289 113 240
162 290 114 235
142 287 120 --
aRT, room t e m p e r a t u r e ; bmo., months. 59
WONG, MEE, GLENN, AND KEOWN
TABLE 3
TABLE 2 Summary of Three Accuracy Studies
Method HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
Recovery Study
Distribution of Results as Compared to Target Values 0-10% 10-20% >20% 9 7
2 2
1 0
39
22
8
7 7
1 2
1 0
Three accuracy studies with CsA target values of 388, 258 and 129 ~Lg/L, respectively, were performed. This table shows the cumulative distribution of laboratory results expressed as the percent difference compared to target values. The percent difference = [laboratory's result - target value] x 100 / target value. The number of laboratories using HPLC, Sandimmun, CycloTrac SP with kit standards, CycloTrac SP with in-house standards and TDx are 4, 3, 23, 3, and 3, respectively. Four laboratories reported survey results in more than one methodology.
Method
N
HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
Range of Recovery
4 3
95-110% 87-103%
23
88-129%
3 3
84-109% 95-113%
tibody kits (6). Four laboratories use the HPLC method (7,8). Results were analyzed monthly. The mean, SD, median, r a n g e , n u m b e r of l a b o r a t o r i e s u s i n g a part i c u l a r methodology, s t a n d a r d d e v i a t i o n index (SDI = [result - SD]/SD) and g r a p h i c d i s t r i b u t i o n of l a b o r a t o r y r e s u l t s were r e p o r t e d to each l a b o r a t o r y w i t h i n f o r m a t i o n coded to p r e s e r v e confidentiality.
Results and discussion STABILITY OF TESTING MATERIALS
lites which were isolated and purified from the bile of liver t r a n s p l a n t p a t i e n t s on CsA (100 ~Lg/L each of M1, M8, M17, M18 and M21) were spiked in a blood pool c o n t a i n i n g 258 ~tg/L of p u r e CsA. For t h e b l a n k study, CsA-free E D T A blood was o b t a i n e d from n o r m a l volunteers. F o u r different methodologies are b e i n g used by 32 laboratories. T h e y are H P L C , r a d i o i m m u n o a s s a y (RIA) u s i n g 12sI-tracer, RIA u s i n g 3H-tracer a n d fluorescence polarization i m m u n o a s s a y (polyclonal antibody) (FPIA). F o u r l a b o r a t o r i e s r e p o r t e d s u r v e y r e s u l t s in m o r e t h a n one methodology. T w e n t y - f i v e l a b o r a t o r i e s use CycloTrac SP kits (2,3) (Incstar, Stillwater, MN, USA, 12SI-CsA w i t h specific monoclonal antibody). T h r e e laboratories use CycloTrac SP kits w i t h in-house whole blood standards. T h r e e l a b o r a t o r i e s use S a n d i m m u n SP kits (4,5) (Sandoz, Basel, Switzerland, 3H-CsA w i t h specific monoclonal antibody). T h r e e l a b o r a t o r i e s use T D x F P I A (Abbott, Chicago, IL, USA) nonselective polyclonal an-
Two pools, stored at room temperature (RT) and a t - 2 0 °C for a period of t h r e e m o n t h s were t e s t e d by u s i n g CycloTrac SP kits a n d the H P L C m e t h o d (8). T h e r e s u l t s are s u m m a r i z e d in T a b l e 1. T h e d a t a show t h a t the t e s t i n g m a t e r i a l s are stable w h e n stored e i t h e r a t RT or a t - 2 0 °C. ACCURACY
Three accuracy studies with CsA target values of 388, 258 and 129 ~g/L, respectively, were performed. Table 2 summarizes the results of all 3 studies, expressed as the percent difference of a laboratory's result compared to the target value. Over 90% of laboratories reported results within 20% of target values. RECOVERY
In the recovery study, Vial A obtained from a kidney transplant patient was the baseline sample.
TABLE4 Linearity Study Method HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
N
Vial A
Vial B*
B/A
4 3
129 +- 23.9 144 _+ 1.0
64.6 - 5.1 70.3 +- 5.5
0.50 0.49
24
173 -- 16.7
86.1 -+ 11.0
0.50
3 3
151 -+ 27.6 396 -+ 15.0
79.3 -+ 8.4 214 - 5.3
0.53 0.54
*B = 1 part A + 1 Part normal blood (unspiked). 60
CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
QUALITY ASSURANCE OF CYCLOSPORINE
TABLE 5 Summary of Six Precision Studies
Method HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
TABLE 6 CsA Concentrations of a CsA-Free Whole Blood Sample Determined by 4 Methods
Percent Difference Between Duplicate Results 0-10% 10-20% >20% 14 13
1 4
7 1
97
28
17
13 16
4 0
2 0
For the precision study, the same blood pool was sent to participating laboratories in two separate surveys. Six precision studies were performed. This table shows the cumulative distribution of laboratory results expressed as the percent difference between two results. The percent difference = [result 1 - result 2] × 100 / lower result. Four laboratories reported survey results in more than one methodology.
Vial B was obtained by spiking 258 ~g/L CsA to Vial A. The majority of laboratories (29 out of 32) recovered 90 to 110% of added CsA (Table 3). L~NEARrrY All four methods showed good linearity at a parent CsA level below 200 ~g/L (Table 4).
Method
N
HPLC Sandimmun CycloTrac SP (kit standards) CycloTrac SP (in-house standards) TDx
Range (~Lg/L)
4 3
0-15 16-25
25
0-37
3 3
0-46 10-
