Amniotic fluid becomes more dilute as pregnancy progresses. This means that the bilirubin concentration will tend to decrease as the gestational age increases. Thus a given absorbance reading will have different significance depending on the gestational age of the fetus.


This means that:

1) a given ?OD450 value later in pregnancy is more serious than it is at an earlier stage.

2) a constant value is indicative of more severe disease.


Test Performance:

(1) Liley measured the optical density of the fluid over the 350-700 nm range and plotted the readings on semilogarithmic paper so that the wavelength is the x-axis (linear) and the optical density is the y-axis (logarithmic).

(2) The assigned baseline is determined by drawing a line to connect the readings at 360 nm and 550 nm.

(3) 450 nm is the absorption maximum for bilirubin. The height of the peak at 450 nm above the baseline is termed the ?OD-450 and is a rough measure of the amount of bilirubin present.

(4) If oxyhemoglobin is present, as indicated by a peak at 410 nm, then an additional correction is needed. The ?OD410 is determined by subtracting the baseline value for 410 nm from the measured value. This is multiplied by 5% to generate a value which is subtracted from the raw ?OD450 to give the corrected ?OD450.


Analysis: Liley plotted semilogarithmically the ?OD-450 vs. week of gestation such that the weeks of gestation is the x-axis (linear) and the ?OD-450 is the y-axis (logarithmic).


Initial Work:

(1) Liley initially divided the results into 3 main zones based upon the degree of fetal involvement:



Zone III (top)

severe disease

Zone II (middle)

intermediate (mild-moderate) disease

Zone I (bottom)

Rh-negative, unaffected, or mildly affected infant


(2) Based on diagrams in paper (Am. J. Obst. Gynecol. 82:1359 (1961), the following estimations as to the curves were made (not exact, due to inaccuracy of diagrams):

(3) Plot: semilogarithmic, with x-axis (linear) weeks 27-37 and y-axis (logarithmic) of ?OD450

(4) Top line estimate: y intercept at 27 weeks shows O.D. 0.25; y value at 37 weeks estimated as O.D. 0.115

(5) Lower line estimate: y intercept at 27 weeks shows O.D. 0.065; y value at 37 weeks estimated as O.D. 0.025

(6) Slopes based on estimates (ideally these should be parallel but the slopes based on estimates are not):

(6a) upper line: -0.041

(6b) lower line: -0.034


Later Work:

(1) Later Liley subdivided the middle (moderate) and top (severe) zones. Each zone is associated with a different degree of disease.

(2) The upper severe (top) zone: intrauterine death, neonatal death, severe neonatal anemia (cord hemoglobin < 7.9 g/dL)

(3) The lower severe (top) zone: cord hemoglobin 8.0-10.9 g/dL.

(4) The upper moderate (middle) zone: values in this zone usually were followed by values of equal or greater value, which can be interpreted as evidence of increasing hemolysis as gestation progresses.

(5) The lower moderate (middle) zone: values in this zone are followed by values which often decrease with time, which can be interpreted as indicating steady or decreasing hemolysis.


Interpretation of the amniotic fluid analysis depends on sequential studies repeated at intervals; these intervals are typically 2 weeks but may be variable depending on the circumstances.

(1) Although a single reading may be accurate, serial readings give a more accurate prediction of the severity of the disease unless the initial reading was in zone 3.

(2) Rapid increases in the ?OD-450 can occur in severe D-incompatibility. This readings in zone 2 may increase to zone 3 within 2 weeks.



• One disadvantage of this method is that it merely discriminates unaffected infants from those that are severely affected and does not measure fetal maturity in any way.


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