© 2006 American Association for Clinical Chemistry, Inc.
Letters to the Editor |
Evaluation of the Efficacy of Chloroform Extraction of Amniotic Fluid Bilirubin
1 Department of Pathology, University of Utah, Health Sciences Center, Salt Lake City, UT
2 Department of Pathology, University of Arkansas, for Medical Sciences, Little Rock, AR
3 ARUP Laboratories, Salt Lake City, UT
4 ARUP Institute for Clinical, and Experimental Pathology, Salt Lake City, UT
aAddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108. Fax 801- 584-5207; e-mail Ashwood{at}aruplab.com.
To the Editor:
The presence of increased concentrations of bilirubin in amniotic fluid is an indicator of fetal hemolysis (1)(2). In the 
A450 (or OD450) method, the absorbance at 450 nm is used as a surrogate for concentration of amniotic bilirubin. Chloroform extraction of bilirubin from amniotic fluid samples can be used to eliminate spectral interference from hemoglobin, which absorbs at a maximum at 410 nm. However, clinical interpretation of the A450 often relies on studies in which chloroform extraction was not used (3)(4).
We studied the extraction of bilirubin from 37 amniotic fluid samples submitted for bilirubin scanning. All studies were in accordance with the guidelines approved by the Institutional Review Board of the University of Utah. Frozen (–70 °C), light-protected samples were thawed and centrifuged at 900g for 5 min at 2–6 °C. Absorbances of samples were scanned (Beckman DU-800) from 350 to 550 nm in 1-mL air-blanked quartz cuvettes. The use of water or chloroform blanks increased A450 <0.01 in both native and chloroform-extracted samples (n = 4). The A450 was determined as the difference between the absorbance at 450 nm and a logarithmic baseline between 365 and 550 nm, calculated as: log(A450) = m (450 nm) + b, where m = [log(A550) – log(A365)]/(550nm – 365nm), and b = log(A365) – m(365 nm). This can be rearranged to yield the equation: A450 = A450 – 10 [0.541 log(A365) + 0.459 log (A550)]. Hemoglobin concentrations in 24 consecutive samples were determined from the net absorbance of the oxyhemoglobin peak at 578 nm and a molar hemoglobin absorptivity of 44 000 mol–1 cm–1.
Chloroform was added (2 mL, HPLC grade, Fischer Scientific) to an equal volume of amniotic fluid in a closed 12-mL screw-top glass tube. Samples were shaken vigorously by hand for 1 min and centrifuged (at 2–6 °C) for 5 min at 900g. The A450 was measured as the difference in absorbance between the bilirubin peak at 450 nm, and a baseline was established between the absorbance of wavelengths flanking the bilirubin peak (
370 nm and 525 nm) on a linear scale. We also calculated A450 for the chloroform extract with a logarithmic baseline. Quality control specimens consisting of pooled amniotic fluid samples had A450 means (SD) of 0.0360 (0.0030), and 0.1223 (0.0073). Finally, the proportion of bilirubin remaining in the clarified extracted aqueous layer was estimated in 24 consecutive samples as for native fluid.
For 36 of the 37 amniotic fluid samples examined, mean (SD) recovery of A450 in the chloroform fraction was 88 (4)% as determined by regression analysis (Fig. 1
). After A410 nm correction in which 5% of the absorbance of the hemoglobin peak at 410 nm was subtracted from the A450, mean (SD) recovery of A450 increased to 93 (4)% (1). A mean (SD) of 12 (11)% of the A450 in the native sample was observed in the aqueous phase after extraction in samples (n = 24) with an original A450 of >0.02. The mean hemoglobin concentration in unextracted samples was 0.093 g/L (range 0–0.12 g/L).
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Figure 1. The correlation of measured A450 in patient samples before and after chloroform extraction.
Comparison of the
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A450 in the chloroform extract increased the mean bilirubin recovery to 89 (4)%. Thus, although use of a logarithmic baseline is imperative for A450 determination in native amniotic fluid samples (2), the use of a linear baseline vs a logarithmic baseline did not notably affect (1% change) measured A450 in chloroform extract.
In a single sample, the apparent bilirubin recovery after chloroform extraction was low (A450 = 0.17 vs 0.31 in the unextracted sample). The remaining bilirubin was visible in the aqueous fraction (A450 = 0.16). Use of the A410 correction did not resolve the unexpectedly low recovery. A second chloroform extraction did not substantially decrease the amount of bilirubin remaining in the aqueous layer. Thus, the amniotic fluid bilirubin appears to be sequestered in the aqueous layer, possibly covalently bound to albumin.
For almost all native amniotic fluid samples studied, A450 results with and without chloroform extraction were similar. The observed 12% mean decrease after extraction is consistent with previous studies that reported a mean decrease of 20% (5). Thus, the Liley plot can be used in conjunction with chloroform extraction of amniotic bilirubin, although caution should be used in classifying patient samples that exhibit A450 measurements close to Liley zone boundaries.
In contrast to our findings, unconjugated bilirubin added to pooled amniotic fluid samples is poorly extracted by chloroform (<50%) (3)(4). Incomplete chloroform extraction in some patient samples has also been reported by others (3). The aqueous fraction of the amniotic fluid sample should be analyzed to detect such cases. In these situations, the use of an unextracted sample, or the use of an alternative method such as the observation of photometric degradation, may be more appropriate (6).
Acknowledgments
We thank Jacquelyn McCowen-Rose for her assistance in preparing this manuscript. The University of Utah Department of Pathology and the ARUP Institute of Clinical and Experimental Pathology provided financial support for this work.
References
- Oepkes D, Seaward PG, Vandenbussche F, Windrim R, Kingdom J, Beyene J, et al. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med 2006 Jul 13;355:156-64.
- Grenache DG. Hemolytic Disease of the Newborn. Gronowski AM eds. Current Clinical Pathology: Handbook of Clinical Laboratory Testing During Pregnancy. Totowa 2004:219-244 Humana Press Totowa, NJ. .
- Foster K, Moore J, Hankins K, Parvin CA, Gronowski AM. Effect of blood contamination on delta 450 bilirubin measurement: an in vitro comparison of two corrective methods. Clin Chem 2004;50:1420-1422.
[Free Full Text] - Bailey DN, Briggs JR. Studies of extraction of bilirubin from human amniotic fluid. Am J Clin Pathol 2006;125:771-773.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
- Spinnato JA, Ralston KK, Greenwell ER, Marcell CA, Spinnato JA, III. Amniotic fluid bilirubin and fetal hemolytic disease. Am J Obstet Gynecol 1991;165:1030-1035.[Web of Science][Medline] [Order article via Infotrieve]
- Dubin SB. Determination of amniotic fluid bilirubin absorbance in specimens contaminated by blood. Clin Chem 2005;51:2213-2214.
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