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Technical Briefs |
1
Lab. de Toxicol., and
2
Réanimation Toxicol., Hôpital Fernand Widal, 200 rue du Fg-St-Denis, 75475 Paris Cedex 10, France;
3
C.N.E.H., 9 rue Antoine Chantin, 75014 Paris, France;
a author for correspondence: fax +33 1 40 05 48 78
Methylene blue (MB) is frequently used as an antidote
in treating methemoglobinemia (1) because it facilitates
the reducing activity of the NADPH-dependent methemoglobin reductase
system in erythrocytes (2). However, MB absorbs strongly
between 550 and 700 nm (Fig. 1
), the same spectrophotometric region as that of the various
hemoglobin derivatives: oxyhemoglobin (O2Hb),
deoxyhemoglobin (HHb), methemoglobin (MetHb), and carboxyhemoglobin
(COHb). To evaluate the potential magnitude and direction of errors
linked to the presence of MB for the results for total hemoglobin (tHb)
and its derivatives, we evaluated six CO-Oximeters. The wavelengths
used by each instrument for these determinations are as follows: IL 482
(Instrumentation Laboratory, Lexington, MA), 535, 585.2, 594.5, and
626.6 nm; CCD 270 (Chiron Diagnostics, Medfield, MA), 557, 577, 597,
605, 624, 635, and 650 nm; CCD 835 (Chiron; wavelengths not
communicated); OSM3 (Radiometer, Copenhagen, Denmark), 535, 560, 577,
622, 636, and 670 nm; ABL 520 (Radiometer; same wavelengths as OSM3);
AVL 912 (AVL Scientific Corp., Roswell, GA), 530, 536, 542, 548, 554,
560, 566, 572, 578, 584, 590, 604, 612, 622, 630, 640, and 648 nm.
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Blood was collected from five healthy volunteers with informed consent. Because the study involved only blood sampling, Institutional Ethics Committee Review was not required in France. The five samples were combined to obtain 120 mL of pooled blood, which were then separated into three 40-mL fractions:
–Fraction N, which had no enrichment in CO or MetHb.
–Fraction CO, which was enriched in CO by tonometry with use of an IL 237 tonometer (Instrumentation Laboratory) and CO in nitrogen, 10 mL/L (Société Cosma, Igny 91430, France). Because of the time required to analyze a large number of samples, the tonometry was carried out separately on four 10-mL specimens just before analysis.
–Fraction Met, which was treated with 4 mg of hydroquinone (Prolabo, Paris, France) to obtain samples enriched in MetHb. Hydroquinone, a known inducer of MetHb, was selected for its lack of absorbance in the spectral range of hemoglobin. Again, this enrichment step was carried out on four 10-mL specimens just before analysis.
Each fraction was separated into aliquots. Four aliquots were adulterated with MB by dilution with a stock 10 g/L solution (Pharmacie Centrale des Hôpitaux de Paris, Paris, France) to obtain final concentrations of 0.1, 0.25, 0.50, and 1 g/L. We then added 1.0 mL of one of these solutions to 9.0 mL of blood to obtain a blood MB concentration of 10, 25, 50, or 100 mg/L. These MB concentrations were chosen to correspond to the plasma concentrations clinically anticipated when MB is slowly injected intravenously as 5–25 mL of a 10 g/L solution (the 100 mg/L concentration is rarely attained). Four control aliquots were prepared for each fraction as well, the MB being replaced with NaCl, 9 g/L. Each adulterated sample was compared with its own control, and measurement was performed immediately after treatment of the blood with either MB or NaCl. Measurements were performed in triplicate with all six CO-Oximeters.
The tHb concentrations of controls measured by CO-Oximetry were ~146 g/L. The COHb and MetHb percentages of control specimens obtained from CO-Oximeters were respectively 1.05–1.55% and 0.50–0.55% for the N samples, 23.1–36.6% and 0.1% for the CO samples, and 0.9–1.2% and 4.6–14.5% for the Met samples.
Using uncorrected data, we calculated the difference, negative or
positive, between the mean of three values of the adulterated specimen
and the mean for the corresponding control. The results (Table 1
) are the absolute differences in values, as reported by the
instruments. The values for tHb are reported in g/L, the values for
hemoglobin derivatives in % of tHb. The relative errors for the
derivatives are thus much greater than the absolute differences in
reported percentage. For example, comparing the IL 482 results for an
untreated N control (i.e., physiological COHb and MetHb; no MB) with
those for a sample to which MB (100 mg/L) had been added shows that the
percentage of MetHb increased from 0.5% to 42.9%, an absolute
difference of +42.4% MetHb. Given that the known concentration of
MetHb in these specimens is ~0.5%, this would mean a relative error
of 8500%!
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Large negative differences are observed as well. For instance, in samples with small percentages of COHb or MetHb, negative readings for samples adulterated with MB result in negative tHb percentages. In the CCD 270 results for the untreated (N) control and the specimen adulterated with MB (100 mg/L), the reported MetHb decreased from 0.5% to -8.5%—giving an absolute difference of -9%, but a relative error of 1800%. Furthermore, one sample adulterated with 100 mg/L MB gave a result >100% (CCD 270 O2Hb result for specimen Met) with a difference of +26.5%. Several of the AVL 912 data points are missing, because the software for that analyzer eliminates most of the unreasonable results.
In interpreting the clinical significance of these findings, the
relative errors are of greater importance, with each laboratory
determining what constitutes an acceptable deviation. If, for example,
relative errors of 20% for MetHb, HHb, and COHb; 3% for
O2Hb; and 2 g/L for tHb are considered "acceptable,"
only a handful of the values reported in Table 1
would be retained. The
"acceptable" relative errors are indicated in bold type in Table 1
.
Thus, we consider the majority of the values in this study clinically
unacceptable.
The presence of MB in the samples perturbs most of the measurements
(Table 1
). The results obtained for unadulterated samples or for
samples enriched with CO or MetHb vary greatly not only from one
instrument to another, but also in direction. A precise interpretation
of the errors is difficult. Indeed, one must take into account several
types of problems of analysis, i.e., spectral order and software.
Regarding spectral order, analysis of a mixture of the four major
derivatives of hemoglobin presupposes a spectral measurement of four
wavelengths followed by a mathematical treatment of the signals. To
correct for any eventual interference, one must make these measurements
at other wavelengths and integrate into the system of calculation the
absorption data specific to the interfering substance. MB absorbs
strongly at 600–700 nm and more weakly at 550–600 nm (Fig. 1
). Thus,
its presence mainly affects the determination of MetHb near 620 nm and,
more weakly, determinations of the other hemoglobin derivatives near
that wavelength. This interference may be observed with the IL 482,
which works uniquely at four wavelengths: MB absorbance at 626 nm
simulates the presence of MetHb, resulting in a large increase for this
derivative and consequently for tHb. This positive error for MetHb
results in overcorrection for the other derivatives in the calculation
system, so the values reported for them are too small.
The importance of software-related errors can be appreciated by
comparing the results reported by the OSM3 and ABL 520, which are
identical instruments; in the absence of an alternative explanation
from the manufacturer, we believe the differences (error) in reported
results may be attributed to differences in the software, which
calculates the contributions of the various hemoglobin species. All
CO-Oximeters tested other than the IL 482 measure at several
wavelengths in the 630–670-nm region, which might be used to correct
for MB. Generally, however, these wavelengths are used to correct for
turbidity, and no instrument takes into account the absorption of MB in
their calculation program. The corrections are therefore poorly adapted
to the presence of MB and may produce errors of potentially significant
magnitude (Table 1
). The AVL 912 system of calculation eliminates the
most perturbed results, explaining the great number of missing values
in Table 1
.
The effect of MB on measured tHb concentrations deserves further comment. The IL 482 apparently detects MB as an increase in the peak at 626 nm, registering it as an increase in MetHb and, consequently, in tHb. The other instruments, which measure near the maximum for MB absorption (650–700 nm), apparently recognize this peak outside the maximum range of hemoglobin as "nonhemoglobin" and correct the tHb downward.
We conclude, therefore, that these six CO-Oximeters should not be used
to determine the concentration of hemoglobin derivatives in blood
samples containing MB. This is particularly important in cases of
methemoglobinemia, because the samples with high proportions of MetHb
give the most affected results (Table 1
). Furthermore, pending further
refinements, CO-Oximetry cannot be safely used clinically to evaluate
the efficacy of treatment of methemoglobinemia with MB.
Acknowledgments
We thank Instrumentation Laboratory, Lexington, MA; Radiometer, Copenhagen, Denmark; Chiron Diagnostics, Medfield, MA; and AVL Scientific Corporation, Roswell, GA, for the loan of CO-Oximeters during the course of this study.
References
The following articles in journals at HighWire Press have cited this article:
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  B. H. A. Maas, A. Buursma, R. A. J. Ernst, A. H. J. Maas, and W. G. Zijlstra Lyophilized bovine hemoglobin as a possible reference material for the determination of hemoglobin derivatives in human blood Clin. Chem., November 1, 1998; 44(11): 2331 - 2339. [Abstract] [Full Text] [PDF]
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