Nov 15, 1979 - Reagents, Union Carbide. 'Staizyme CPK,, Worthington. JCPK,, Worthington. k AGENT CPK Test,. Abbott.'CK-Enztrate for TR. Beckman.
CLIN.CHEM. 26/3,513-519(1980)
p#{174}@fiill#{174}Ip©
An Interlaboratory Study of Creatine Kinase and Creatine Kinase Isoenzymes Joe Boone, E. J. Sampson, S. Lewis, V. Whitner, S. McKneally,
and B. Houston
Over 400 laboratories participated in an interlaboratory CK-MB activity has led to a number of method comparisons study of creatine kinase (EC 2.7.3.2) in which they were (22-38), and conclusions based on the data from studiesof requested to analyze five lyophilized samples for total methods forMB differentiation have oftenbeen in conflict. The purposeof our survey was to determinethe “stateof the creatine kinase and creatine kinase isoenzymes and to art” forMB activity differentiation and total CK activity. complete a questionnaire regarding methodology. The Interlaboratory data were compiled in variousways so that specimens were prepared from an homogenate of human the results obtainedwith different instruments, kits, reaction myocardium that had been centrifuged, dialyzed, and added temperatures, a nd reagent compositions could be examined. to a matrix containing bovine serum albumin, buffer, and Although an interlaboratory study involving porcine-heart N-acetylcysteine. Stability studies indicated a half-life for enzyme for determinationof totalCK activity has recently the samples of two to seven years at 37 #{176}C. The been reported (39), we report on the use of stable, humanamong-vial coefficient of variation was 1.4% or less. For source CK samplessuitable for measurement of totalCK actotalcreatinekinase most laboratories used an NADP+ tivity and CK isoenzymes. reduction method monitored at 340 nm (86%) and reported results in U/L (87%) at either 30 #{176}C (42%) or 37 #{176}C Materials and Methods (48%). The average of the 37 #{176}C results reported for each sample was within 10% of the results obtained by the Materials2 Center for Disease Control with the Scandinavian Society’s Imidazole, hexokinase (EC 2.7.1.1), glucose-6-phosphate Recommended Method (Scand. J. C/in. Lab. Invest. 36: dehydrogenase (EC 1.1.1.49), tris(hydroxymethyl)amino711, 1976), adapted to a centrifugal analyzer. To separate methane (Tris), and Tris hydrochloride were obtained from the creatine kinase isoenzymes, 280 laboratories used 26 Sigma Chemical Co., St. Louis, MO 63178; 2-mercaptoethanol, different methods or kits, including electrophoresis on acetic acid, and magnesium acetate from J. T. Baker Chemical cellulose acetate (45%) and agarose (39%), ion-exchange Co., Phillipsburg NJ 08865; N-acetylcysteine (grade A) from chromatography (13%), and selective activation (3%). Calbiochem, San Diego, CA 92112; bovine serum albumin, Twenty-nine different methods or kits were used to fraction V (Pentex), from Miles Laboratories Inc., Kankakee, quantitate the isoenzymes, with about 21 % of the laboIL 60901; adenosine-5’-diphosphate (ADP), adenosine-5’ratories using visual examination (ultraviolet irradiation). monophosphate (AMP), P’,P5-di(adenosine-5’)-pentaQuantitative values reported for the MB isoenzyme ranged phosphate (AP5A), nicotinamide adenine dinucleotide from within normal limits to 19-fold greater for each phosphate (NADP), and creatine phosphate from sample. A few participants (14%) reported BB isoenzyme Boehringer Mannheim Biochemicals, Indianapolis, IN 46250; in every sample, although most laboratories indicated BB and D-glucose from the National Bureau of Standards, was absent. Washington, DC 20234. electrophoAdditional Keyphrases: proficiency testing ion-exchange resis, agarose gel and cellulose acetate state of the chromatography selective activation art “kit” methods .
.
Different analytical techniques have been developed to differentiate and quantitate creatine kinase (CK)t isoenzymes, including electrophoretic separation (1-8); ion-exchange chromatography (9-11); kinetics (12); “high-performance” liquid chromatography (13, 14); and immunological (15-20) and selective activation (21). The importance of having sensitive, specific, inexpensive, and timely methods of evaluating
Bureau of Laboratories, Center for Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare, Atlanta, GA 30333. Presented in part at the 31st national meeting of the AACC, New Orleans, LA, July 1979. An expanded account of this study was published by the Center for Disease Control (see ref. 41). Nonstandard abbreviations used: CK, creatine kinase (ATP: creatine N-phosphotransferase; EC 2.7.3.2); BB, MB, MM, isoenzymes of creatine kinase; Tris, tris(hydroxymethyl)aminomethane; CDC, Center for Disease Control. Received Nov. 15, 1979, accepted Dec. 5, 1979.
Creatine Kinase Measurements The method used at the CDC to measure CK activity is the Scandinavian Society’s Recommended Method (40), adopted to a centrifugal analyzer (GEMSAEC; Electro-Nucleonics, Inc., Fairfield, NJ 07006) (41). All reagents and samples were dispensed with automatic pipets (Micromedic Systems, Inc., Horsham, PA 19044).
Enzyme Preparations Human heart was diced and then homogenized in 70 Tris.HCI buffer, pH 8.8, and 5 mmolfL 2-mercaptoethanol. The homogenate was centrifuged (1000 X g) and the supernate filtered and dialyzed at 4 #{176}C against Tris.HCI buffer, pH 8.8. This extract, with a CK activity of 160 kU/L, was stored at -70 #{176}C until pool preparation. mmolfL
Pool Preparation A matrix containing, per liter, 30 g of bovine serum albumin, of Tris.HC1, and 10 mmol of N-acetylcysteine, was
50 mmol
2 Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health, Education and Welfare.
CLINICAL CHEMISTRY,
Vol. 26, No. 3, 1980
513
Table 1. Composition, Among-Vial, and Within-Vial Variability of Enzyme Activity of Creatine Kinase poolsa Volume of Heart Extract (mL)
Pool A B C D E
Volume of Matrix (mL) Added
2000 1500 800 400 200
0 500 1200 1600 1800
MEAN Within-Vial IJ/L S.D.(U/L) 978 745 409 207 107
14.4 6.8 3.8 1.9 1.0
‘Using Scandinavian Society’s Recommended Method (Scand. J. Clin. Lab. Invest. 36, 711(1976)) per vial, 20 vials for each pool. b The mean square within-vials was greater than the mean square among-vials. C The degrees of freedom were approximated by using Satterthwaite’sapproximation.
by initially dissolving the bovine serum albumin (Fraction V) in distilled water (250 g/L) and dialyzing overnight at 4 #{176}C against distilled water; the other components were then added to the dialysate and the pH was adjusted to 7.5. A portion of the heart extract was thawed and 3 mL was added to a 4.9-L pool of the bovine serum albumin matrix to obtain approximately 1000 U of CK activity per liter. The latter pool was diluted further with the matrix to provide four additional pools as indicated in Table 1. Two liters of each of the different pools was filtered through stacked Millipore filters with a final filter of 0.22 tm average pore size. Each pool was dispensed in 2-mL aliquots into amber-colored vials (Wheaton Scientific, Millville, NJ 08322) with automatic pipets that had been calibrated with de-ionized, degassed water. All pools were lyophilized simultaneously and sealed under reduced pressure, a metal crimp was applied, and the vials were stored at -20 #{176}C. prepared
Among-Vial
Variability
Twenty vials were randomly selected from each of the five pools and three CK measurements were made on each specimen after it had been reconstituted with 2 mL of distilled water. Means, within-vial standard deviations, among-vial standard deviations, and among-vial coefficients of variation were calculated and are listed in Table 1.
Specimen Distribution Participants were asked to analyze five lyophilized samples for total CK and CK isoenzymes and to complete a ques-
Among-Vial S.D.(U/L)
d.fc 40 40 40 40 40
Among-Vial C.V. ()
d.f.
4.8 2.0
5.4 4.7
0.5 0.3
-
-
1.0 1.5
9.7 49.6
0.5 1.4
adapted to the GEMSAEC at 37 #{176}C. Based on 3 determinations
all pools except E (which contained the lowest CK activity) analytical errors were greater than variations among vials. Temperature-accelerated degradation experiments were performed on the lyophilized CK materials (Figure 1) to assure that the CK in the samples could withstand nationwide shipping to the participants and storage in their laboratories until they were assayed. Using the Arrhenius data derived from these studies (Figure 1, inset) with the rate of degradation at high temperatures treated as first-order reactions, we have estimated that the half-life of the CK would be 2 to 7 years if the vials were stored at 37 #{176}C, and 1.5 X 10 years at -20 #{176}C. Therefore, the specimens were stable during the time required for this interlaboratory study. An examination of the number of participating laboratories in which a particular method kit was used for measuring CK indicated that 269 (86.7%) used NADP reduction methods, nine (2.9%) used NADPH oxidation methods, and 32 (10.4%) used a creatine measurement method. Laboratories using measurement methods based on the detection of NADPH monitored the reaction at 339, 340, or 340/380 nm and accounted for 89.0% of the participants. Other participants monitored the reaction at 500 nm (1.6%) or 520 nro (9.4%). Participants reported results at several temperatures, including ambient (0.1%), 25 (4.2%), 30 (41.9%), 32 (0.1%), 37 (47.7%), and 37.5 #{176}C (3.2%), and other temperatures (2.8%). In many instances, the reporting temperature appeared to be determined by the particular kit or instrument the laboratory used. Means, standard deviations, and coefficients of variation
tionnaire regarding their methodology. The reported results for total CK were grouped by method and temperature. Results for isoenzymes were separated into two groups: those providing qualitative estimates and those providing quantitative determinations of MB and BE activity. Quantitative results for isoenzymes were further subdivided into electrophoretic, ion-exchange chromatography, and selective activation categories. When five or more participants used the same method and temperature, the results were tabulated and are included in this report.
10001 900
I:
800
700
600 N
500
Results and Discussion
> F-
400
Total CK activity 300
Our mean
in the five pools are given in Table 1. These data verify the expected relationship between predicted enzyme activity for each pool, based on the extent of dilution of Pool A. To verify that vials from all pools were sufficiently uniform in CK content for distribution as samples in this study, we examined the vial-to-vial variation of each pool. The variability expressed as within-vial standard deviations in Table 1 represented total analytical errors, and the vial-to-vial variation indicated the degree of inter-vial homogeneity. For 514
CLINICAL
values for total CK activity
CHEMISTRY,
Vol. 26, No. 3, 1980
200
100
0
10
20
30 TIME
40
50
60
70
60
(DAYS)
Fig.1.Temperature-accelerated degradation ofCK activity in Pool A and Arrhenius plot (inset) Temperature (#{176}c) indicated for each line by circled number
Table 2. Interlaboratory Analytical Results for Total CK CategorIzed by Method and Temperature No
Reportins (‘C) 25 30
-
l’ethod particor kit pants Total8 13
272
21 5 6 8 Roche 7 5. Kline9 16 U. Carbide”. 5 Uorthinqton1 6 Worthinqton 20 Totala
CalbiochemC Calbiochemd
37
Abbott Becman1
21 7 8 WorthingtOni 10 DuPont’1’ 41
37.5
b
122
-
A
S.D. 70.6
C.V. 2S9
681 676 458 669 517 447 527 488 507
64.8 62.9 56.0 68.0 33.8 52.7 51.7
9.5 9.3 12.2 10.1 6.5 11,9 9.8
37.0 36.7
7.6
527
109
20.8
415
1017 794 965 783 942
63.2 69.5 114 75.9 42.9
6.2 8.7 11.8 9.7 4.6
747 577 749
I
7.2
I
20 518 522 393 516 381) 354 401
8 S.D.
JIJCt
I
C
426
20U
I 1T
48.8 37.6 37.4 50.0 27.2 43.8
9.4 7.0 9.5 9.7 7.2 12.4 9.8 15.7
285 289 208 277 211 192 216 209
22.0 33.2 33.1 31.0 25.3 24.5 15.9
6.3
211
16.9
39.4 404 63.2 399 25.2
C.V.
D
S.D. 26T4
CV. 2T
30.5
I T
S.D. 8.8
CV. T1
t S.D. 3472 10.0
7.7 11.5 15.9 11.2 12.0 12.8 7.4 14.6
143 146 101 133 103 101 110 106
16.0 11.1 11.0 15.6 7.8 25.8 10.2 12.5
11.2 7.6 10.9 11.8 7.6 25.7 9.3 11.8
74.8 74.2 53.2 69.6 52.4 50.3 54.7 54.2
8.0
113
14.2
12.6
23.1
115
26.5
23.1
CV.
29.4
6.90 5.9 4.5 8.3 6.5 8.1 7.8 5.5
9.2 7.0 11.9 12.5 16.1 13.5 10.2
57.2
6.9
12.1
59.4
12.1
20.3
8.5
20.1
230 53.2
83.7 94.8 85.2 639 68.9 718 110
11.2 16.4 11.3 10.8 15.4
408 332 418 324 405
21.2 26.0 40.6 27.4 15.4
5.4 7.8 9.7 8.5 3.8
204 165 209 160 199
10.5 13.5 18.4 14.2 8.6
8.1 8.2 8.8 8.9 4.3
102 85 122 83 104
8.0 6.7 24.4 7.7 6.8
7.9 7.9 20.0 9.4 6.6
694
16.5
379
67.5
17.8
186
33.2
19.8
99
18.9
19.2
65.5
17.5
180
24 -
13.3
86
16.5
19.1
83.3
Total8
124
891
164
18.4
Technicon”
18
915
139
15.270611215.9374
115
Includes all results at a given temperature regardless of method after outliers (results greater than ±3 S.D. from originalmean) UV - CK - NAC, Boehringer Mannheim Corp. “CPK-SVR, Calbiochem. CPK Reagents, Calbiochem. CPK-UV, Dow. CPK-Total, Hoffmann-La Roche.
Spin Chem CPK, SmithKline. “Centrifichem System-CPK Reagents, Union Carbide. Abbott.‘CK-Enztrate for TR. Beckman. “CPK-ACA, DuPont. “CPK, Technicon.
for analytical data submitted by participants are shown in Table 2 for all method groups for which there were five or more laboratories. Data from six laboratories were excluded on the basis of outlier exclusion (results greater than 3 SD from the original mean). The results obtained with the Scandinavian Society’s Recommended Method (40) adapted to the GEMSAEC are listed in Table 1. Because human myocardium was the source of the CK enzyme, artifacts seen with enzymes from nonhuman sources are not the cause of the analytical differences observed between groups of laboratories in Table 2. Most method group results were apparently linear in response to increasing CK activity; the differences between method group means resulted primarily from the different sulfhydryl activators and temperatures used for analysis. Several other factors such as the formulation of reagents, instrumentation, calibration materials, and the omission of reagent blanks may disperse the data in Table 2. In some methods and reagent sets,thiolcompounds have been included in the reactionmixture to prevent inactivation of creatinekinase. Lost CK activity, however, is not only restored but actually enhanced (42). The most potent activators (in kits or reagents), indicated by data in Table 2, appeared to be N-acetylcysteine (UV-CK-NAC, Boehringer Mannheim Corp., Indianapolis, IN 46250; CPK-SVR, Calbiochem, San Diego, CA 92112; CPK-UV, Dow Chemical Co., Indianapolis, IN 46268), and dithiothreitol or dithioerythritol (CPK-aca, DuPont Instruments, Wilmington, DE 19898; CPK-total, Roche Diagnostics, Nutley, NJ 07110; CPK-CentrifiChem System, Union Carbide Clinical Diagnostics, Rye, NY 10580), followed by glutathione (CPK Reagent, Calbiochem, San Diego, CA 92112; Spin Chem CPK, SmithKline, Inc., Sunnyvale, CA 94086). rphis pattern of activation by thiol agents is illustrated in a plot of values from users of Dow, Roche, and SmithKline reagents vs the CDC values (Figure 2). Creatine
Kinase
‘Staizyme CPK,, Worthington. JCPK,, Worthington.
k
AGENT CPK Test,
electrophoresis; however, ion-exchange chromatography (13%) and selective activation (3%) were also used. Twenty-nine different quantitation or development methods or kits were used to determine the isoenzyme distribution in the samples. Approximately 21% of the participants determined the presence or absence of MB or BB isoenzyme by visual examination after the isoenzymes had been separated. Results reported for isoenzymes were expressed as a percentage of total CK activity, as activity by fraction, and as a determination of the presence or absence of the MB or BB isoenzyme. Listed in Table 3 are the means or medians and ranges of results whenever five or more participants using a particular method reported results as either a percentage or as CK activity. Table 4 contains data from participants who indicated the presence or absence of MB or BE isoenzyme in each sample by visual examination. Participants were also asked whether the separation of the isoenzymes in their control materials and the CDC samples closely resembled that observed for fresh human sera; approximately 78% indicated that separation of their control material and of the CDC samples was similar to the separation of fresh human sera, seven laboratories indicated an increase
lsoenzymes
Of the 310 laboratories that provided methodology information, 280 reported making CK isoenzyme determinations. Participants used 26 different separation methods or kits involving five different separation techniques. Most laboratories used either cellulose acetate (45%) or agarose (39%)
COC
Voiw
(U/Li
Fig. 2. Effect of different thiol activators on the measured CK activity Mean activity of N.acetylcysteine. 0; dithioerythritol, 0; glutathione. . at 30 #{176}C are plotted vs CDC values at 37 #{176}C
Results
CLINICALCHEMISTRY,Vol.26,No. 3, 1980
515
Table 3. QUantitative Results for Creatine Kinase Isoenzymes No. participants
Separation Method Own reagents
Beckman
6
14
Calbioche,n
5
Pool No. A B C 0 E
96
8
Roche
Worthington
U/liter
‘
91.5 83.2 93.6 75.2 70.4
(628) (530) (277) (145) (100)
88- 95 60- 92 88-100 0-100 0- 92
A
87.4
(645)
B C 0
88.6 91.3 91.9 94.7
(516) (294) (133) ( 81)
74- 95 645-998 21.3 80- 93 479-685 10.9 83- 95 268-368 11.2 87-100 133-190 9.0 75-102 5.3 84-100
A B C D
392 329 193 97 51
-
-
A B
-
-
A B C
83.3 85.0 90.0
-
66- 96
-
-
73-
-
-
0
94.3
-
C
94.0
-
86- 96 88-99 89- 97
(508) (369) (213) (130) ( 54)
-
A B C 0 C
94
-
-
70-99 41-96
-
48-97
-
64-100 25-100
84.8 84.7 84.8
-
83.9 80.5
-
-
284-592 225-378 128-263 59-206 29-66
-
1.4
(0)
0-7
-
1.6
(0)
0-8
(88) (61) (24) (16) (0)
5-88 7-16 5-30 0-15 0-16
9-96 57-61 19-24 7-18 0-2
0.1 0.6 0.0 0.4 0.0
(0) (0.7) (0) (0) (0)
0-1 0-5 0 0-4 0
o
-
46-187
56-117 37-60 18-47 10-18
-
0
-
-
0 U 0
-
0 0
-
0.5 0.9 0:8 1.0 0.7
2 7 2 3 2
0-6 0-13 0-13 0-11 0-10
3-11 0-67 0-18 0-17 0-14
0 0 0 0 0
-
0 0 0 0 0
-
-
4-61 6-48 4-25 1-12 3-11
-
(129)
-
-
(93)
(53) (25) (13)
-
27-259 15-208 14-68 19-29 0-40 -
-
-
69-134 49-115
-
24-70
-
1-20 8-51 2-32 0-31 1-75
9-66 2-18
-
0-2 1)
0 0
0
0 0
-
)ig)
2-26
(is)
1.21
) t) 1 s)
-
( 4)
3-13 4-7 1-11
1.6
-
0-10
-
-
0-8 0-18
-
-
0-14 0-20
-
-
was used if more than five laboratories reportedin the same units; if less thanfive used thesame units the median was used. Includes participants
in background on electrophoresis plates, and six laboratories reported that migration rates for the CDC samples differed from those for fresh human sera. The quantitative isoenzyme results for MB and BB were expressed as a ratio of each participant’s results (in either percentage of total CK activity or units of isoenzyme activity) to the upper limit of normal indicated by the participant. To calculate a ratio for isoenzyme results for laboratories that indicated an upper limit of normal of zero, we assumed that the upper limit of normal was one (1). Therefore, all ratios of 0 to 1 are within normal limits for the isoenzyme. The distributions of the calculated ratios are listed in Table 5 by the separation method for MB and RB isoenzymes for each sample. Because only human myocardium was used to prepare these samples, the amount of BB isoenzyme present should be negligible (43). Most ratios for both qualitative and quantitative results support the conclusion that the BB isoenzyme was not present in any sample. Some laboratories using electrophoresis or ion-exchange chromatography separation techniques reported the presence of BB isoenzyme, but none of the laboratories using selective activation reported RB isoenzyme. The mode of sample preparation should have produced a constant proportion of MM and MB isoenzyme in every sample. Therefore, an increase in total CK activity should be reflected by a proportional increase in both MM and MB activity. The order of decreasing total CK activity was Pool A>B>C>D>E. The same order should have been observed for MB activity for both qualitative and quantitative results.
CLINICAL
-
-
-
-
-
-
-
-
-
0
-
0.6 0.7 1.0 1.5
-
that reported results in either percentage or activity units. CPK Isozyme kit and Agarose Gelkit, Beckman Instruments, Inc., electrophoresis at 37 #{176}C. CPK/MB-UV, Calbiochem,selective activation. CPK-lsoenzymes.Helena. electrophoresis, generally at 37 #{176}C. CPK-CS (cardiac specific), Hoffmann-LaRoche. DEAE Sephadex. CPKrn,Worthington, DEAE Sephadex. CK Isozyme, Corning.agarose electrophoresis. (In “Corning”rows,dashes are not quite aligned under ‘UIL’
516
Wlie_sr
0-9
-
-
(o)
Ii/litr
(4)
-
14.4 17.3 13.1 13.1 16.4
(o)
0.4 0.6
23.7 19.4 11.7 6.4 4.4
-
0.4
-
0-23 0-34 0-21 0-20 0-60
-
:
-
0-2 0-2 0-3
100 70 42 25 7
102-958 10.0 331-790 9.8 198-411 9.4 104-197 8.6 47-123 7.3
0 E
-
-
Mean or (Median)
5-36 9-40 0-15 0-8
103 78 40 26 14
-
--
J1.ljj.ar
) ) ) ) )
(7 (8 (8 (6 (8
43-60
-
R40
U/liter
85-116
73-100 61-100 51-100 74-100 71-100
-
a The mean
-
639 535 293 147 75
A
96
-
309-439 265-387 162-231
-
‘
13.6 20.6 6.4 3.8 3.2
-
89.5 88.9 89.3 91.1 94.8
-
Corning
U/liter
“
-
Helena
Mean or (Median)
Ranqe
Mean8 or (Median)
CHEMISTRY,
Vol.26,No.3,1980
columns.)
Generally,the data presented
in Tables 4 and 5 do followa pattern of decreasing MB activity from Pool A to Pool E. The order for MB activity, however, was not as sharply defined by users of electrophoresis as by users of ion-exchange chromatography or of selective activation techniques. Ten laboratories that used electrophoretic separation reported results within normal limits for MB isoenzyme for both Pools A and B, whereas no participant using either ion-change chromatography or selected activation reported results within normal limits for both of these samples. None of the participants used an immunoassay for isoenzymes. Of those participants quantitating the isoenzymes, 69% used a reaction temperature of 37 #{176}C for the CK activity measurement; 13% used 30 #{176}C, 6.9% used room temperature; 5.0% each used 25 #{176}C or 32 #{176}C; and 2% used 37.5 #{176}C. Participants were asked to provide the upper limit of their normal range for CK isoenzymes. These limits were sorted according to the reaction temperature, and means and ranges were determined for both the MB and BB isoenzymes fractions (Table 6). Eight laboratories may have reported results as being within normal limits for MB isoenzyme activity for above-normal MB pools because of an inappropriately high value for the upper limit of normal for MB activity. Most participants reported an increase in MB activity in Pools A, B, and C, a marginal increase in Pool D, and no increase in Pool E for both qualitative (Table 4) and quantitative results (Table 5). Most participants were in agreement that the RB isoenzyme was absent from all samples. Because
Table 4. Qualitative Responses for Creatine Kinase lsoenzymes M8 Reported
BB Reported
participants No. U
participants No.
U
B C 0 E
5 5 5 2 0
100 100 100 40 0
0 0 0 0 0
0 0 0 0 0
Helena8
A B C 0 E
20 20 19 12 4
100 100 95 60 20
1 0 1 1 1
5 0 5 5 5
Corni
A B C 0 E
18 18 14 11 5
100 100 78 61 28
1 O 1 0 O
6 0 6 0 0
Other
A B C 0
13 12 11 4 3
100 92 85 31 23
0 0 0 0 0
0 0 0 0 0
56 55 49 29 12
100 98 88 52 21
2 0 2 1 1
4 0 4 1 2
Separation Method
Pool No.
Dade
A
C Total
A B C 0 E a CPK-isoenzymes, Helena, cellulose acetate electrophoresis. I) CI( Isozyrne, Corning, agarose electrophoresis. Laboratory reports isoenzyme,
were considered positive for the respective
indicating
trace or moderate
amounts,
terms denoting
the presence
ofeither MB or SB
isoenzyme.
Table 5. Quantitative lsoenzyme Results, Expressed as a Ratio of the Results to the Upper Limit of Normal for the lsoenzyme BB
Separation Method Electrophoresis 121 participants
Ratio8 01.0 1.1- 2.0 2.1- 3.0 3.1- 4.0 4.1- 5.0 5.1-10.0 10 .1-15 .0 15. 1-20 .0 >20.1
no. participants within ratio limits Pool A 8 C D E 12 22 15 12 7 29 16 4
13 25 14 7 11 27 10 8
4
3
Ion-exchange
0- 1 .0
1
1
32 participants
1.1- 2.0 2.1- 3.0 3.1- 4.0 4.1- 5.0 5. 1-10 .0 10. 1-15 .0
2
3
Selective
1
0
1
1
O 9
3 14 4
15.1-20.0
9 3
>20.1
6
1 5
0- 1.0 O 0 activation 1.1- 2.0 0 0 5 1 0 2.1- 3.0 partici pants 3.1- 4.0 1 4 4.1- 5.0 0 0 5.1-10 .0 3 1 10.1-15.0 O 0 15. 1-20 .0 0 0 >20.1 0 0 a All ratios of 0-1 are within normal limits for theisoenzyme.
no. participants within ratio limits Pool A
8
C
0
E
125
124
128
3 3
1 1
1 2
123 1
124 5
3
3
1
4
1
4
0
4 2 0 0 2
2 3 5 0 1
3 3 2 1 0
1 2 3
1 4 1
1 0
0 2
13 10 4 4 1
13 1 2 1
16 1 2 0 0
11 2 3 1 1
18 0 0 1 0
13 1 1 2 0
5
0
2
0
0
0
0
O 4 O
0 1 0
0 0 0
O 0 O
0 0 0
0 0 0
0 0 0
0 0 0
0 0 5 0 O 0
0 5 0 0
5 0 0 0
0
0
0
o
o
O O
0 0
0 0 0 0
22 20 17 19 6 17 12 4 1
38 25 14 10 4 9 7 7 0
67 17 6 5 3 17 2 3
2 3 4 4 6
5 9 5 6 1
9
BB was probably absent from all samples, we could not determine what impact the presence of RB isoenzyme might have on the results reported for MB. It is apparent, however, that most laboratories using electrophoresis, ion-exchange chromatography, or selective activation techniques can detect increases in MB isoenzyme in the absence of RB isoenzyme.
2
O
5
5
5
5
5
O
0
0
0
0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Data reported in an interlaboratory study represent both a laboratory’s ability to perform its method and the accuracy and precision of the method itself. An interlaboratory survey of proficiency in clinical enzymology presents unique interpretation problems (44); however, when a relatively large number of observations can be combined into a given method group, the effects of poor laboratory performance are diminCLINICAL CHEMISTRY,
Vol. 26, No. 3, 1980
517
Table 6. Average and Range of Upper Limits for Creatine Kinase lsoenzymes by Temperature of the Detection Reaction for Creatine Kinase MM
_____
mean8 % U/liter
Temperature room 30#{176}C
a
MB
mean
range
%
U/liter
%
%
U/liter
50-188
3
7
0-5
0-10
0.1
0
0-1
0
97
79
83-100
64-293
3
5
0-15
2-15
0.3
0.2
0-1
0-2
0.4
0-21
0-5
antibody.
We gratefully acknowledge the sonnel: Dr. Jim Barbaree, Dr. Sam Deenie Dudley, Ms. Bobbie Taylor, Mr. Randy Kuykendall, Ms. Nelle
assistance of the following perCaudill, Mr. Nelson Clement, Ms. Mr. Tim Groza, Mr. Tom Hearn, Ponder, and Mr. Jim Woesht.
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CHEMISTRY,
Ulliter
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After completing this study, we received a personal communication from Ying Foo and Sidney Rosalki of the Royal Free Hospital in London, England, in which they outlined their findings with the Merck inomunoinhibition technique. They concluded that, in contrast to patients’ sera, the samples distributed in this study yielded different MB contents; that is, MB estimates by the immunoinhibition technique were higher than by a cellulose-acetate electrophoretic technique. They postulate that the lyophilized MM CK in these samples was abnormally resistant to immunoinhibition by anti-M
4. Smith, A. F., Separation
‘
70
37#{176}C 100 132 35-100 67-225 4 10 0-44 0-20 0.5 One participant apparently reversed the MM and BB upper limits,and these values were not included inthetable.
of serum creatine
range
U/liter
100
ished and conclusions about method performance can be drawn. The evidence in this study support the conclusion that laboratories can detect above-normal MB isoenzyme in samples that contain no RB isoenzyme, regardless of the method used. Laboratories that provide visual estimates of MB activity performed as well as those that provide quantitative values.
518
U/liter
Rn
mean
range
Vol.26,No.3,1980
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