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Methemoglobin Concentrations in Neonatal Samples. Containing Fetal Hemoglobin, Patrick L.M. Lynch,1* David. E. Bruns,1 James C. Boyd,1 and John Savory1 ...
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Clinical Chemistry 44, No. 7, 1998

We are grateful to B. Rohland for skillful assistance and S. Powarzynski for providing us with patient samples. We also thank Dr. M. Page for reading the manuscript and Dr. P. Luppa for valuable advice. References 1. Wall RT, Harlan JM, Harker LA, Striker GE. Homocysteine-induced endothelial cell injury in vitro: a model for the study of vascular injury. Thromb Res 1980;18:113–21. 2. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997;4:230 – 6. 3. Perry IJ, Refsum H, Morris RW, Ebrahim SB, Ueland PM, Shaper AG. Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men. Lancet 1995;346:1395– 8. 4. Fermo I, D’Angelo SV, Paroni R, Mazzola G, Calori G, D’Angelo A. Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease. Ann Intern Med 1995;123:747–53. 5. Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Lindenbaum J. Elevation of total homocysteine in the serum of patients with cobalamin and folate deficiency detected by capillary gas chromatography-mass spectrometry. J Clin Invest 1988;81:466 –74. 6. Chauveau P, Chadefaux B, Coude M, Aupetit J, Hannedouche T, Kamoun P, Jungers P. Increased plasma homocysteine concentration in patients with chronic renal failure. Miner Electrolyte Metab 1992;18:196 – 8. 7. Andersson A, Isaksson A, Hultberg B. Homocysteine export from erythrocytes and its implication for plasma sampling. Clin Chem 1992;38:1311–5. 8. Fiskerstrand T, Refsum H, Kvalheim G, Ueland PM. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem 1993;39:263–71. 9. Ubbink JB, Vermaak WJH, Van der Merwe A, Becker PJ. The effect of blood sample aging and food consumption on plasma total homocysteine levels. Clin Chim Acta 1992;207:119 –28. 10. Moller J, Rasmussen K. Homocysteine in plasma: stabilization of blood samples with fluoride. Clin Chem 1995;41:758 –9. 11. Vester B, Rasmussen K. High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum. Clin Chem 1991;29:549 – 4. 12. Passing H, Bablok W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. J Clin Chem Clin Biochem 1983;21:709 –20. 13. Nehler MR, Taylor ML, Porter JM. Homocysteinemia as a risk factor for atherosclerosis: a review. Cardiovasc Pathol 1997;6:1–9. 14. Refsum H, Fiskerstrand T, Guttormsen AB, Ueland PM. Assessment of homocysteine status. J Inher Metab Dis 1997;20:286 –94. 15. Rasmussen K, Moller J, Lyngbak M, Holm Pedersen AM, Dybkjaer L. Ageand gender-specific reference intervals for total homocysteine and methylmalonic acid in plasma before and after vitamin supplementation. Clin Chem 1996;42:630 – 6.

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 (CibaCorning) 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. 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 5 0.0013), and the Chiron 800

Table 1. MetHb results (%) for adult and umbilical cord blood samples treated with potassium nitrite. Nominal

Chiron 800 System CO-oximeter Module Overestimates Methemoglobin Concentrations in Neonatal Samples Containing Fetal Hemoglobin, Patrick L.M. Lynch,1* David E. Bruns,1 James C. Boyd,1 and John Savory1,2 (Departments of 1 Pathology and 2 Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908; * author for correspondence: fax 804-924-2574, e-mail [email protected]) 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

Adult blood 0 5 10 15 25 50 75 100 Umbilical cord blood 0 5 10 15 25 50 75 100 a

Manual

Chiron 800a

Corning 270

0.6 5.6 10.5 15.3 24.7 50.8 76.0 99.7

0.1 5.6 10.9 15.9 25.8 53.3 80.2 100.0

0.1 4.9 9.9 14.7 23.7 48.3 72.5 95.3

0.7 6.3 10.9 15.3 24.8 50.5 72.8 98.4

2.2 8.4 13.2 18.1 28.9 55.4 80.0 100.0

0.0 5.2 9.8 14.6 25.0 50.2 72.4 97.0

Both the 100.0% Chiron 800 are cutoff values.

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Technical Briefs

showed a significant constant error (P 5 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 5 0.7380). By contrast, the Chiron 800 showed a constant error to significantly higher MetHb (P 5 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 1. Roberts JD Jr, Fineman JR, Morin FC III, Shaul PW, Rimar S, Schreiber MD, et al. Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. N Engl J Med 1997;336:605–10. 2. Ignarro LJ. Nitric oxide. A novel signal transduction mechanism for transcellular communication. Hypertension 1990;16:477– 83. 3. Nakajima W, Ishida A, Arai H, Takada G. Methaemoglobinaemia after inhalation of nitric oxide in infant with pulmonary hypertension. Lancet 1997;350:1002–3. 4. Bard H. The postnatal decline of hemoglobin F synthesis in normal full-term infants. J Clin Invest 1975;55:395– 8. 5. Speakman ED, Boyd JC, Bruns DE. Measurement of methemoglobin in neonatal samples containing fetal hemoglobin. Clin Chem 1995;41:458 – 61. 6. Evelyn KA, Malloy HT. Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J Biol Chem 1938; 126:655– 62. 7. Zijlstra WG, Buursma A, Meeuwsen-van der Roest WP. Absorption spectra of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin and methemoglobin. Clin Chem 1991;37:1633– 8. 8. Brunelle JA, Moran RF. Data processing in CO-oximeters that use overdetermined systems [Reply]. Clin Chem 1997;43:190 –1.

Detection of H-ras Mutations in Urine Sediments by a Mutant-enriched PCR Technique, Jose´ Ramo´n Conejo,1*† Trinidad Parra,1* Miguel Cantero,1 Agustı´n Jime´nez,2 Vicente Granizo,2 Gabriel de Arriba,1 and Fernando Carballo1 (1Unidad de Investigacio´n and 2 Seccio´n de Bioquı´mica, Hospital General Universitario de Guadalajara, 19002 Guadalajara, Spain; *J.R.C. and T.P. contributed equally to this work; † author for correspondence: Hospital General Universitario de Guadalajara, Unidad de Investigacio´n, C/Donantes de Sangre, sn., 19002 Guadalajara, Spain; fax 34-49-209216, e-mail [email protected]) Identification of DNA mutations in urine sediments has been proposed as a noninvasive and early indicator of urinary tract cancer (1– 4). The sensitivity of this approach is limited by the proportion of DNA containing the mutant allele, because the resolution of the technique used to detect oncogenes or suppressor gene mutations (mainly single-strand conformational polymorphism) requires ;10% representation of mutant product (4, 5), whereas estimates to the proportion of tumor cells in the urine of bladder cancer patients were not .7% (1). H-ras represents a single member of a family of genes coding for a 21-kDa protein involved in the regulation of cell growth and differentiation (6). Most activation events of this protooncogene to the oncogenic form found in human bladder cancer occur via a point mutation at codon 12 (7, 8). To provide a more sensitive means for detecting mutations, we have devised a sensitive, nonradioactive procedure based on two consecutive PCRs with intermediate restriction digestion, which leads to effective enrichment of mutant alleles for further amplification and elimination of wild-type alleles by digestion (5, 9 –11). Wild-type DNA was prepared from the urine sediments of three healthy volunteers, and two DNA samples containing known mutations in H-ras codon 12 were mixed with an equal quantity (200 ng) of that wild-type DNA. These mutant-type samples were extracted from tumor specimens after surgical resection. The mutations were known through direct sequencing. Amplifications in the first round were carried out in a final volume of 100 mL in 13 PCR buffer containing 2 mmol/L MgCl2, 50 pmol of each primer, dNTPs at 40 mmol/L each, and 2.5 units of UlTmat DNA Polymerase (Perkin–Elmer–Cetus). After an initial denaturation step at 94 °C for 2 min, samples were subjected to 10 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, which were followed by 12 more cycles in which 20 s was added to the extension time after each consecutive cycle, with a final extension at 72 °C for 7 min. Primers were as follows: F (59-GACGGAATATAAGCTGGTGGTGG-39) and R (59TGGATGGTCAGCGCACTCTT-39). The 308-bp fragment amplified with these primers contains two naturally occurring MspI sites located at codon 12, which are destroyed by mutations in either of the first two positions and 55 bp upstream of codon 12, which provided a positive control for MspI cleavage. Intermediate restriction digestion was carried out with 8 mL of PCR product with 8 U of MspI (Fermentas AB) at