Bilirubin estimation in amniotic fluid obtained by amniocentesis is effective in predicting the severity of hemolytic disease of the newborn (HDN), as it gives the best indication of the degree of intrauterine hemolysis. It can be used to predict fetal survival and can aid in making management decisions for affected infants.


Indications for measurement:

(1) when there is a past obstetric history of an infant affected by HDN, and/or

(2) when there are antibodies titers indicative of possible severe hemolytic disease

(3) when ultrasonography shows fetal hydrops or other signs suggestive of hemolytic disease


Specimen requirements:

(1) Serial readings are usually more informative than single values; this is because trends are more evident.

(2) Single readings, if the values are high, can provide sufficient evidence for the need of therapy.

(3) Specimens should be protected from exposure to light. Light may degrade the bilirubin, resulting in a falsely low reading.



(1) Some method of quantitation is required. While gross inspection of the fluid can recognize unaffected or minimally affected infants, due to the fact that the fluid is colorless, visual inspection is incapable of discriminating accurately between different amounts of bilirubin if it is present. This is particularly important if serial specimens from the same infant are examined.

(2) Usually the amniotic fluid is scanned in a spectrophotometer from at least 300 to 700 nanometers and the absorbance recorded using semilogarithmic paper. A tangent drawn to each curve corrects for background absorbance. The peak at 450 millimicrons above the background line ("deltaOD450", or ?OD450) is directly related to the severity of the disease affecting the fetus in utero.


?OD450 =

= (absorbance of specimen at 450 nm) - (projected baseline from scan)



• Normally the absorbance of amniotic fluid decreases as pregnancy progresses, due to dilution of bilirubin. A stable absorbance reading on serial testing indicates increasing severity of HDN, as normally the values should fall.

• Before 28 weeks and after 35 weeks only the moderately and severely affected infants can be detected accurately.

• A single tap after 35 weeks is difficult to interpret because of the decrease in ?OD in almost all fluids after this time.

• Before 30 weeks of gestation, the risk of neonatal death following premature delivery is very high, so that intrauterine transfusion is usually performed.

• If the results suggest severe disease and the infant is at least 32-34 weeks of gestation, the infant is usually delivered. Determination of the lecithin-to- sphingomyelin (L/S) ratio on the amniotic fluid may assist in assessing the degree of fetal lung maturity and thereby predicting the ability of the infant to survive premature delivery.

• The variability in testing is relatively high, especially between institutions. Decision points developed at one institution may not be transferable to another unless care is made to standardize equipment, specimen handling, and technical processing between the institutions.


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