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Technical Briefs |
1
Pathology and
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Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908;
a author for correspondence: fax 804-924-2574, e-mail pll8n{at}avery.med.virginia.edu
Inhaled nitric oxide (NO) is an established treatment for persistent pulmonary hypertension of the newborn (1). NO can oxidize erythrocyte hemoglobin Fe(II) to Fe(III), however, and can lead to potentially toxic accumulation of methemoglobin (MetHb) (2). Because MetHb fractions >5% can lead to toxicity, monitoring of MetHb throughout therapy is required. In one exceptional case, excessive dosing of NO led to a MetHb fraction of 40% (3). Because neonatal blood contains a high proportion of fetal hemoglobin (HbF), typically 75–90% at term (4), accurate measurement of MetHb in the presence of HbF is essential. We wished to ascertain the accuracy of MetHb determination by the Chiron 800 system CO-oximeter module (Chiron Diagnostics), part of their 865 analyzer, in the presence of high HbF. A similar study of the Corning 270 CO-oximeter (Ciba-Corning) was performed recently (5). The Chiron 800 CO-oximeter is an overdetermined system that uses 10 wavelengths to directly measure total hemoglobin, oxyhemoglobin, carboxyhemoglobin, deoxyhemoglobin, and MetHb. For comparison, MetHb concentrations were also measured with the Corning 270 CO-oximeter, an overdetermined system that uses seven wavelengths, and the Evelyn-Malloy KCN addition manual method (6) on a Shimadzu UV-1201 spectrophotometer (Shimadzu Scientific Instruments).
Two ~40-mL pools of blood were collected into lithium heparin
tubes: adult blood and fetal umbilical cord blood with 85.5% HbF. By
preparing 100% MetHb samples with potassium nitrite, as outlined
previously (5), samples with 0%, 5%, 10%, 15%, 25%,
50%, 75%, and 100% MetHb were produced for each pool. These samples
were then analyzed by the Chiron 800, the Corning 270, and the manual
method. The results are shown in Table 1
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Analysis of covariance of the results was carried out using the SAS general linear model procedure with a homogeneity of slopes model (SAS Institute) to compare results for adult and umbilical cord blood. For MetHb concentrations of 0–50%, the Corning 270 showed only a small, although statistically significant, proportional error (P = 0.0013), and the Chiron 800 showed a significant constant error (P = 0.0001) to higher MetHb values in the presence of HbF. For MetHb concentrations of 15% or less, i.e., values most likely to be encountered clinically with NO therapy, the Corning 270, in agreement with the study carried out previously (5), was unaffected by the presence of HbF in the umbilical cord sample (P = 0.7380). By contrast, the Chiron 800 showed a constant error to significantly higher MetHb (P = 0.0047) for MetHb concentrations of 15% or less. The manual method was unaffected by the presence of HbF in the cord blood samples.
These results indicate that, in the presence of HbF, the Chiron 800 CO-oximeter produces falsely increased MetHb values in the clinically significant range. Overestimation of MetHb in the presence of HbF may have serious implications, with the possibility of NO therapy being discontinued prematurely. The proportional bias of the Corning 270 is apparent only at values >15% and hence would seem clinically insignificant.
The absorbance peak at 632 nm is the optimal region for measuring MetHb if we consider 100% adult or 100% fetal hemoglobin (HbF). Unfortunately, this 632-nm region also corresponds to a considerable difference between the absorbance spectra of adult hemoglobin and HbF (7). This difference is minimal at 610 nm, an isosbestic point. Below 610 nm, however, the absorbance curves become very steep, indicating that a compromise using wavelengths between 610 and 625 nm, for example, may prove better. According to Chiron Diagnostics, the 10 wavelengths chosen for the 800 series CO-oximeter avoid the region between 580 and 610 nm (8); in contrast, the Corning 270 uses two wavelengths in this region (597 and 605 nm).
For neonates receiving NO therapy, we recommend continued use of the model 270 CO-oximeter for monitoring of MetHb.
References
The following articles in journals at HighWire Press have cited this article:
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  P. L. M. Lynch and M. J. O'Kane Methemoglobin Interferes with the HemoCue B-Glucose Analyzer Clin. Chem., March 1, 2002; 48(3): 581 - 583. [Full Text] [PDF]
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M. H. de Keijzer, R. Brandts, and B. A.J. Giesendorf Comment on the Overestimation of Methemoglobin Concentrations in Neonatal Samples with the Chiron 800 System CO-Oximeter Module Clin. Chem., August 1, 1999; 45(8): 1313 - 1314. [Full Text]
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