| |
| author |
Laila Andaloussi Serraj
| | title |
Investigating and Improving the Accuracy of Bilirubin Measurement in Neonates
| | abstract |
Icterus or newborn jaundice can progress to severe hyperbilirubinemia in 8-10% of healthy
newborn infants and may result in kernicterus or irreversible brain damage. Although recent
studies suggest the usefulness of bilirubin measurements in managing high-risk neonates including
premature infants, to date, no validated clinical chemistry methods for the accurate and precise
determination of neonatal bilirubin are available. Our study aimed to establish a methodology for
clinical use through a combination of analytic techniques. A state-of-the-art system of size
exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography
(RP-HPLC) with visible (Vis) and ultraviolet (UV) detection was utilized. Acquisition
wavelengths were configured to measure at ๐=460 nm, consistent with the yellow-orange pigment
of bilirubin, and ๐=280 nm, consistent with the absorption of serum proteins, most notably
albumin, bilirubin's carrier protein. Patient samples were diluted with an aqueous mobile phase,
spun, and filtered using a 0.2 ยตm membrane. This method enabled the determination of total
bilirubin (TBIL) concentrations through SEC, along with a baseline separation of all three species
of bilirubin (Bu, mono, and di Bc) and the respective degradation product (lumirubin) in a 30-
minute gradient elution run excelling in ultra-high sensitivity through RP-HPLC. Since bilirubin
binds to the carrier protein albumin, TBIL concentrations were determined from a single
chromatographic peak in a record time of 11.54 minutes using SEC. Results of several
chromatographic separations of patient samples indicated a wide variation in the relative
proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of
bilirubin peaks produced by RP-HPLC was found with the total bilirubin value obtained by a
standard reference procedure using 97% Alfa Aesar. Both techniques afford high precision and
accuracy with an SEC linearity test yielding an R2 value of 1 over a targeted concentration range
(0 - 32 mg/dL) and an RP-HPLC relative standard deviation (RSD) of 0.35% over a concentration
range of clinical interest (0 - 100 mg/dL). Thus, this analytical methodology could identify the
components of bilirubin in its various forms exceeding the precision and accuracy of recent clinical
techniques cost-effectively. Because the effects of bilirubin toxicity are often devastating and
irreversible, quickly determining TBIL concentrations in newborns is essential for the appropriate
diagnosis and management of hyperbilirubinemia. Thus, laboratory testing can play a pivotal role
in treating affected neonates.
| | school |
The College of Liberal Arts, Drew University
| | degree |
B.S. (2023)
|
| advisor |
Dr. Jonathan Crowther
|
| full text | LSerraj.pdf |
| |
