Clinical Chemistry 51, No. 2, 2005
Table 1. The 95% interfractile reference intervals for all
analytes, ratios, and subgroups meriting stratification.
4.
n
95% interfractile
reference interval
5.
205
90
115
105
100
205
205
205
205
90
115
0.46–1.78 ␮mol/L
0.45–2.05 ␮mol/L
0.46–1.71 ␮mol/L
0.43–1.61 ␮mol/L
0.57–1.95 ␮mol/L
1.50–4.98 mmol/L
158–522 ␮mol/mol
3.57–8.40 mmol/L
101–265 ␮mol/mol
121–284 ␮mol/mol
88–244 ␮mol/mol
10.
These subgroups are not statistically required to be stratified based on
Harris and Boyd criteria (11 ), but they may be of interest.
11.
Total CoQ10
Malesa
Femalesa
Age 18–44 years
Age 45–83 years
LDL-C
Ratio of total CoQ10 to LDL-C
TC
Ratio of total CoQ10 to TC
Males
Females
6.
7.
8.
9.
a
12.
vidual to interindividual variation (16 ). When the index is
low, particularly when it is ⬍0.6, the dispersion of values
for any individual will span only a small part of the
reference interval. Reference values will thus be of little
use, in particular for deciding whether a significant
change has occurred. Conversely, when the index is high,
particularly when it is ⬎1.4, values from a single individual will cover much of the entire distribution of the
reference interval. In this context, reference values will be
of significant value for clinical interpretation. For total
CoQ10, the index of individuality is 12.2/29.0 ⫽ 0.42; thus,
it is low.
In conclusion, we have derived data for the biological
variation of CoQ10 together with reference values for the
New Zealand population. The low index of individuality
for CoQ10 indicates that individual CoQ10 concentrations
are tightly distributed around a homeostatic setpoint and
that significant changes can occur within the reference
interval. From a statistical perspective, seven samples
should be evaluated to ensure that the estimate of the
homeostatic setpoint is within 10% of the true value, with
95% probability (15 ). From a clinical perspective, serial
changes in CoQ10 should be evaluated against the RCV to
allow for both biological variation and analytical imprecision.
We would like to acknowledge the Tertiary Education
Commission and the Health Research Council of New
Zealand for financial assistance, Associate Professor
Christopher Frampton for statistical advice, Core Biochemistry for lipid analyses, and Endolab for collaboration in the reference interval studies.
References
1. Shults CW, Beal MF, Song D, Fontaine D. Pilot trial of high dosages of
coenzyme Q10 in patients with Parkinson’s disease. Exp Neurol 2004;188:
491– 4.
2. Watts GF, Playford DA, Croft KD, Ward NC, Mori TA, Burke V. Coenzyme Q10
improves endothelial dysfunction of the brachial artery in type II diabetes
mellitus. Diabetologia 2002;45:420 – 6.
3. Viña J, Lloret A, Ortı́ R, Alonso D. Molecular bases of the treatment of
13.
14.
15.
16.
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Alzheimer’s disease with antioxidants: prevention of oxidative stress [Review]. Mol Aspects Med 2004;25:117–23.
Ishii N, Senoo-Matsuda N, Miyake K, Yasuda K, Ishii T, Hartman PS, et al.
Coenzyme Q10 can prolong C. elegans lifespan by lowering oxidative stress.
Mech Ageing Dev 2004;125:41– 6.
Hara KS, Yamashita S, Fujisawa A, Ishiwa S, Ogawa T, Yamamoto Y.
Oxidative stress in newborn infants with and without asphyxia as measured
by plasma antioxidants and free fatty acids. Biochem Biophys Res Commun
1999;257:244 – 8.
Laaksonen R, Ojala J-P, Tikkanen MJ, Himberg J-J. Serum ubiquinone
concentrations after short- and long-term treatment with HMG-CoA reductase
inhibitors. Eur J Clin Pharmacol 1994;46:313–7.
Baker SK, Tarnopolsky MA. Statin myopathies: pathophysiologic and clinical
perspectives [Review]. Clin Invest Med 2001;24:258 –72.
Chopra RK, Goldman R, Sinatra ST, Bhagavan HN. Relative bioavailability of
coenzyme Q10 formulations in human subjects. Int J Vitam Nutr Res
1998;68:109 –13.
Kurowska EM, Dresser G, Deutsch L, Bassoo E, Freeman DJ. Relative
bioavailability and antioxidant potential of two coenzyme Q10 preparations.
Ann Nutr Metab 2003;47:16 –21.
Tang PH, Miles MV, DeGrauw A, Hershey A, Pesce A. HPLC analysis of
reduced and oxidized coenzyme Q10 in human plasma. Clin Chem 2001;
47:256 – 65.
Harris EK, Boyd JC. On dividing reference range data into sub-groups to
produce separate reference ranges. Clin Chem 1990;36:265–70.
National Committee for Clinical Laboratory Standards. How to define and
determine reference intervals in the clinical laboratory: approved guideline.
NCCLS document C28-A. Villanova, PA: NCCLS, 2001;20:1–31.
Miles MV, Horn PS, Morrison JA, Tang P, DeGrauw T, Pesce AJ. Plasma
coenzyme Q10 reference intervals, but not redox status, are affected by
gender and race in self-reported healthy adults. Clin Chim Acta 2003;332:
123–32.
Kaikkonen J, Tuomainen TP, Nyyssönen K, Salonen JT. Coenzyme Q10:
absorption, antioxidative properties, determinants, and plasma levels. Free
Radic Res 2002;36:389 –97.
Fraser CG. Biological variation: from principles to practice. Washington DC:
AACC Press, 2001:67–90.
Harris EK. Statistical aspects of reference values in clinical pathology. Prog
Clin Pathol 1981;8:45– 66.
DOI: 10.1373/clinchem.2004.043653
Application of Commercial Calibrators for the Analysis
of Immunosuppressant Drugs in Whole Blood, Thomas
M. Annesley (University of Michigan Health Systems, 1500
East Medical Center Dr., Ann Arbor, MI 48109-0054;
e-mail [email protected])
Multiple immunosuppressant drugs are now available to
prevent organ rejection. Because of their different modes
of action, these drugs are often prescribed together as part
of a multidrug protocol. This has created a challenge for
clinical laboratories to analyze multiple immunosuppressant drugs in the same blood specimen. HPLC–mass
spectrometry (HPLC-MS) or -tandem mass spectrometry
(HPLC-MS/MS) (1– 6 ) assays can measure all of the
common drugs.
A practical issue with the use of HPLC-MS/MS is the
necessity of preparing whole-blood calibrators containing
cyclosporine, tacrolimus, and sirolimus. With the introduction of everolimus, yet another set of calibrators will
be needed. As with any assay for which in-house calibrators are prepared, this added variable will contribute to
the interlaboratory differences observed in proficiency
testing programs. The availability of either standard reference materials (SRMs) or uniform calibrators for use
458
Technical Briefs
among different laboratories would help with this problem.
Recently, the Thermo Electron and Waters Corporations announced alliances to distribute Chromsystems
products. Among the Chromsystems products are wholeblood calibrators and controls for cyclosporine, tacrolimus, and sirolimus, with everolimus to be added in future
lots of material. The availability of multiconstituent
whole-blood calibrators or controls would address the
issues stated above, if validation of their performance
were to be performed in actual laboratory use. With this
in mind I undertook a set of experiments to answer
several important questions: (a) Do the assigned concentrations in these whole-blood materials appear to be
accurate? (b) When they are used as the calibrators, do the
assays yield results for patient specimens that are similar
to those obtained when validated in-house calibrators are
used for the assays? (c) Do they yield expected results for
separate commercial whole-blood controls and for proficiency testing samples? (d) Are there any apparent matrix
effects associated with these materials that could affect the
accuracy or reproducibility of results?
The cyclosporine for in-house whole-blood calibrators
was obtained from Novartis, and sirolimus was from LC
Laboratories. For tacrolimus, I used Abbott Tacrolimus II
calibrators. For internal standards, the cyclosporin D was
from Novartis, the 32-desmethoxyrapamycin was from
Wyeth, and the ascomycin was from Sigma. Lyphochek
whole-blood controls were purchased from Bio-Rad, and
external proficiency testing specimens were obtained
from the College of American Pathologists (CAP). The
commercial calibrator and control materials were from
Chromsystems. These consisted of a set containing a
whole-blood blank, a single whole-blood calibrator, and
four quality-control (QC) whole-blood samples covering
the range of concentrations that would be expected in
patient specimens. The target (assigned) concentrations
for cyclosporine, tacrolimus, and sirolimus have been
verified for the company by an independent set of reference laboratories. Assigned concentrations are provided
for immunoassay (cyclosporine and tacrolimus), HPLC
with ultraviolet detection (cyclosporine), and HPLC-MS
(sirolimus and tacrolimus). The assigned concentrations
used for comparison of results were the HPLC-ultraviolet
detection (cyclosporine) and HPLC-MS/MS (sirolimus
and tacrolimus) values provided by Chromsystems.
HPLC-MS/MS analyses were performed with the instrumentation, chromatographic conditions, and multiple-reaction monitoring transitions monitored for the immunosuppressants described previously (7 ). Extraction of
whole-blood calibrators, controls, proficiency samples,
and human specimens was performed according to a
recently published protocol (7 ). Because matrix effects or
ion suppression could contribute to the performance of
the commercial calibrators or controls (8 ), additional
solid-phase extraction (SPE) using a 25-mg (1-mL) Varian
LMS crossed-linked styrene-divinylbenzene column (7 )
was performed.
For the “in-house” calibrators, I prepared 6 and 30
␮g/L whole-blood calibrators for sirolimus and 100, 200
and 400 ␮g/L whole-blood calibrators for cyclosporine,
and used the Abbott 6 and 30 ␮g/L calibrators for
tacrolimus. These calibrators and the Chromsystems
whole-blood calibrator and controls were extracted and
analyzed within a single run to avoid any day-to-day
variation in instrument response. The in-house and
Chromsystems samples were analyzed in three separate
runs, and the mean measured concentrations were determined. Also included in runs were deidentified patient
specimens, Bio-Rad Lyphochek controls, or CAP proficiency testing samples.
Using the in-house calibrators in my laboratory’s assay,
I quantified the immunosuppressants in the Chromsystems calibrator and controls to see how closely the assigned values agreed with those measured. I then used
the Chromsystems one-point calibrator (plus the zero
blank sample) as the calibrator and measured the inhouse calibrators and Chromsystems controls. Lastly, I
used the four Chromsystems controls as the calibrators
and measured the immunosuppressant concentrations in
the other two materials. I also measured the concentrations of the immunosuppressant drugs in the Bio-Rad
controls and CAP survey samples, using the in-house
calibrators, the Chromsystems one-point calibration, or a
four-point calibration using the Chromsystems controls as
assigned calibrators.
The mean values for sirolimus calculated with use of
the Chromsystems materials, the Bio-Rad controls, CAP
proficiency samples CS-01 and CS-02, and the in-house
calibrators are shown in Table 1. In the case of sirolimus,
some differences ⬎10% were noted (bold font in Table 1)
when the calculated concentrations of sirolimus were
compared for the Chromsystems materials and in-house
whole-blood calibrators. This occurred for specimens prepared by zinc sulfate/methanol protein precipitation, but
was not evident when the more extensive SPE preparation
was performed. These results point toward a potential
matrix effect (7, 8 ) rather than differences in the accuracy
of the concentration assignments for the calibrators. This
is also supported by the fact that the agreement of results
for the CAP specimens and Bio-Rad controls was improved after the SPE cleanup.
For tacrolimus (see Table 1 in the Data Supplement that
accompanies the online version of this Technical Brief at
http://www.clinchem.org/content/vol51/issue2/), use
of the Chromsystems materials or in-house whole-blood
calibrators yielded acceptable agreement of results,
whether the simple protein precipitation or the more
complete SPE protocol was used for specimen preparation. Using any of the calibrator sets, I found acceptable
results for the CAP samples.
For cyclosporine, the performance of the Chromsystems
or in-house calibrator sets was also comparable (Table 2 in
the online Data Supplement). The calculated results for
the CAP proficiency specimens and Bio-Rad controls were
in slightly closer agreement with target values when SPE
was added to further clean up the whole-blood specimens
and to presumably remove additional constituents that
Clinical Chemistry 51, No. 2, 2005
Table 1. Cross-comparison of observed
sirolimus concentrations.a
Sirolimus, ␮g/L
Sample
No SPE
Chrom Cal 1
Chrom QC 1
Chrom QC 2
Chrom QC 3
Chrom QC 4
CS-01
CS-02
Bio-Rad Low
Bio-Rad High
In-house 6.0
In-house 30.0
Plus SPE
Chrom Cal 1
Chrom QC 1
Chrom QC 2
Chrom QC 3
Chrom QC 4
CS-01
CS-02
Bio-Rad Low
Bio-Rad High
In-house 6.0
In-house 30.0
Targetb
14.5
3.4
9.3
18.3
39.0
7.0
17.7
6.0
30.0
14.5
3.4
9.3
18.3
39.0
7.0
17.7
6.0
30.0
Chrom 1
Pt Calc
3.6
9.2
18.8
40.9
7.2
18.3
11.9
24.9
6.0
36.3
Chrom 4
Pt Cal
In-house
Cal
14.0
12.5
3.5
8.1
16.1
35.9
6.7
16.3
10.3
21.2
7.1
17.6
11.5
24.0
5.8
34.9
14.4
3.8
9.1
18.0
39.4
7.7
18.4
11.6
25.6
6.0
32.4
7.5
17.8
11.5
25.5
5.9
32.2
13.6
3.7
8.6
16.9
38.5
7.3
17.8
11.0
24.0
a
Concentrations listed are the means of three values obtained for the
Chromsystems materials and in-house calibrators, using either a single precipitation extraction (no SPE) or additional SPE (plus SPE) (7 ).
b
Targets for CS-01 and CS-02 are participant MS mean values.
c
Chrom, Chromsystems; Pt, point; Cal, calibration/calibrator.
could cause matrix effects. One would have to perform a
separate study with different “artificial” or lyophilized
specimens to verify whether a significant matrix effect
exists for different HPLC-MS/MS cyclosporine assays.
To determine whether the comparability of the Chromsystems and in-house calibrators found above was a
reproducible phenomenon, I evaluated a new lot of the
Chromsystems four QC bloods, using these in a fourpoint calibration as in the experiments described above.
The quantitative results obtained for four CAP proficiency samples and Bio-Rad controls when the two lots of
the Chromsystems QC materials were used as the calibrators are shown in Table 3 of the online Data Supplement.
The two calibrator sets provided similar results for tacrolimus, cyclosporine, and sirolimus.
The sirolimus linear regression data for patient wholeblood specimens, with and without SPE cleanup, are
shown in Fig. 1, in which the results obtained with the
Chromsystems materials are compared with the in-house
calibrators. The intercepts in Fig. 1 are nearly the same,
but the slopes are closer to 1.0 after SPE cleanup; the
regression data therefore indicated that the addition of
SPE cleanup improved the agreement of results. Because
459
the same patient samples were used for all experiments in
Fig. 1, a paired t-test was performed to verify this observation (Table 4 in the online Data Supplement). The P
values in supplemental Table 4 demonstrate that when
the Chromsystems materials were compared with inhouse calibrators, the differences in patient results for
paired samples were statistically significant if the simple
precipitation protocol was used but not significant if
additional SPE cleanup was included. The results for
patient specimens quantified by use of the in-house
calibrators, with or without SPE, were not statistically
different. This improvement in agreement for patient
specimens after SPE purification also parallels the data in
Table 1, in which the agreement for sirolimus in CAP
surveys and Bio-Rad controls was also better after additional SPE cleanup.
For tacrolimus, Fig. 1 in the online Data Supplement
shows the excellent correlation and agreement for patient
results with all selected calibrator sets. All slopes were
close to 1.0, and no intercept (background) effects were
present whether simple protein precipitation or additional SPE cleanup was performed. The comparisons for
cyclosporine (Fig. 2 in the online Data Supplement)
showed statistically equivalent quantitative results (P ⫽
0.187– 0.704) for patient specimens when the Chromsystems material was used vs in-house calibrators. Because
trough cyclosporine concentrations are routinely monitored at my institution, no random patient specimens
with higher concentrations (e.g., c2 values) were selected;
the study was therefore limited to specimens with concentrations ranging from 50 to 400 ␮g/L.
Several conclusions can be drawn from these data. The
first conclusion is that the assignments for the immunosuppressant drugs for the Chromsystems materials agree
with other in-house or commercial whole-blood calibrators for cyclosporine, tacrolimus, and sirolimus. The
agreement extended to more than one lot of material.
Comparable values were obtained for three types of
samples: a commercial whole-blood control, CAP proficiency testing specimens, and actual human whole-blood
specimens. In many laboratories, the regulatory agencies
require multiple-point calibrations; the Chromsystems
four QC materials can be used for this purpose. QC
materials from other sources (e.g., Bio-Rad) can then be
used.
The second conclusion is that although all of the calibrators tested performed acceptably with proficiency testing, QC, and patient samples, the agreement among
values was slightly better when a more extensive SPE
cleanup was used. This might be expected because previous work has shown, for these immunosuppressant drugs
in whole blood, that ion suppression and assay imprecision are dependent on the type of specimen extraction
protocol used before liquid chromatography–MS/MS
analysis (7 ). Nonetheless, the Chromsystems calibrator
materials do not appear to be associated with, or subject
to, severe matrix effects when used with the water hemolysis precipitation method that was used in this study.
Because these are lyophilized materials and may contain
460
Technical Briefs
Fig. 1. Patient sirolimus whole-blood specimens analyzed without (A and C) and with (B and D) SPE cleanup: comparison of results obtained by
calibration with the Chromsystems materials vs in-house calibrators.
1 Pt Cal and 4 Pt Cal, one- and four-point calibration, respectively; Cals, calibrators.
unspecified stabilizers, plasticizers, and other components, matrix effects could still be a possibility if other
specimen preparation protocols are used.
References
1. Volosov A, Napoli KL, Soldin SJ. Simultaneous simple and fast quantification
of three major immunosuppressants by liquid chromatography-tandem massspectrometry. Clin Biochem 2001;34:285–90.
2. Christians U, Jacobsen W, Servoka N, Benet LZ, Vidal C, Sewing K, et al.
Automated, fast and sensitive quantification of drugs in blood by liquid
chromatography-mass spectrometry with on-line extraction: immunosuppressants. J Chromatogr B 2000;748:41–53.
3. Deters M, Kirchner G, Resch K, Kaever V. Simultaneous quantification of
sirolimus, everolimus, tacrolimus and cyclosporine by liquid chromatographymass spectrometry (LC-MS). Clin Chem Lab Med 2002;40:285–92.
4. Keevil BG, McCann SJ, Cooper DP, Morris MR. Evaluation of a rapid
microscale assay for tacrolimus by liquid chromatography-tandem mass
spectrometry. Ann Clin Biochem 2002;39:487–92.
5. Taylor PJ, Salm P, Lynch SV, Pillans PI. Simultaneous quantification of
tacrolimus and sirolimus, in human blood, by high-performance liquid chromatography-tandem mass spectrometry. Ther Drug Monitor 2000;22:608 –
12.
6. Streit F, Armstrong VW, Oellerich M. Rapid liquid chromatography-tandem
mass spectrometry routine method for simultaneous determination of sirolimus, everolimus, tacrolimus, and cyclosporine A in whole blood. Clin Chem
2002;48:955– 8.
7. Annesley TM, Clayton, L. Simple extraction protocol for analysis of immunosuppressant drugs in whole blood. Clin Chem 2004;50:1845– 8.
8. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49:
1041– 4.
Previously published online at DOI: 10.1373/clinchem.2004.043992
Cardiac Troponin and Creatine Kinase MB Monitoring
during In-Hospital Myocardial Reinfarction, Fred S.
Apple* and MaryAnn M. Murakami (Department of Laboratory Medicine and Pathology, Hennepin County Medical Center and University of Minnesota School of Medicine, Minneapolis, MN; * address correspondence to this
author at: Hennepin County Medical Center, Clinical
Laboratories P4, 701 Park Ave., Minneapolis, MN 55415;
fax 612-904-4229, e-mail [email protected])
Cardiac troponin monitoring for detection of myocardial
injury has been designated the new standard for differ-