DOI: 10.1097/01.POC.0000335928.99030.a6

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Issn Print: 1533-029X

Publication Date: 2008/09/01


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Stable Reference Material for Bilirubin and Hemoglobin

M. Pistorino; M. De Abreu; D. Conlon; P. V. A. Pamidi

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Bilirubin, a principal component of bile pigment, is produced by the decomposition of heme from hemoglobin in red blood cells (RBCs). Increased serum bilirubin levels indicate the clinical condition known as hyperbilirubinemia. In adults, bilirubin excretion or metabolism may be impaired due to obstructive jaundice or various liver conditions, whereas in neonates, hyperbilirubinemia is caused by an increased hemoglobin turnover paired with immature liver function. Severe neonatal jaundice may cause Kernicterus, a serious disease leading to brain damage. Therefore, accurate bilirubin measurements are essential for the diagnosis and treatment of hyperbilirubinemia.
Stat critical care whole blood analyzers, such as the GEM® Premier™ 4000 (Instrumentation Laboratory; Lexington, MA, USA; a Werfen Group company) and the ABL 735 (Radiometer America; Westlake, OH, USA; a Danaher company), have either been developed or are in development to quantify total bilirubin (tBil), as well as total hemoglobin (tHb) and hemoglobin fractions in whole blood using spectrophotometric assay and multi-component analysis over a range of wavelengths. In order to validate the performance of these diagnostic instruments, reference materials are used. However, there are no quality control or reference materials available that include physiological and/or pathological levels of hemoglobin, hemoglobin fractions, and bilirubin in one sample without the use of manufacturer specific secondary algorithms. The lack of availability may be due to inadequate useful shelf-life caused by instability of bilirubin in the presence of oxygen, which is always present with oxyhemoglobin.
In order to overcome this limitation, a reference material comprised of lysed whole blood, with a predetermined mixture of oxyhemoglobin (O2Hb) and carboxyhemoglobin (COHb), was combined with one or more dyes (chrysophenine, metanil yellow, and/or flavianic acid), pre-dissolved in a protein solution. The dyes are stable in solution, closely simulate the absorbance spectra of bilirubin, and do not interfere with other hemoglobin measurements or electrochemical sensors. The O2Hb is stabilized through the use of native methemoglobin reductase, where this reduction of methemoglobin to O2Hb requires NADH as a substrate. To satisfy this necessity, the reference materials were formulated with lactate and NAD, which in the presence of the native lactate dehydrogenase, provided a constant supply of NADH. Due to the strong binding of carbon monoxide to hemoglobin, no additional stabilization for COHb was necessary.
In comparing 7 GEM Premier 4000s for their recovery of tBil, the instrument-to-instrument variability was low (SD < 0.9 mg dL−1) and average within instrument SD was 0.2 mg dL−1 at 15 mg dL−1 measured tBil. Due to the similarity of the dye spectra to that of native bilirubin, the GEM 4000 and ABL 735 tBil measured values were in close agreement (y = 0.960x + 1.395, R2 = 0.999 and y = 0.906x + 1.609, R2 = 0.997 for chrysophenine and metanil yellow, respectively). When these dyes are used in combination with hemoglobin, they provide the means to evaluate tHb, hemoglobin fractions, and tBil across instrument models from multiple manufacturers across a clinically relevant range, and are not expected to require analytical algorithms separate from those used to analyze clinical samples. Testing in progress indicates stability of tHb and tBil (±3% and ±4%, respectively) at refrigerated temperatures for at least 2 months.
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