In a newborn infant with rectal bleeding or a blood streaked gastric aspirate, we need to determine whether the blood originated from infant’s gut or whether it’s the swallowed maternal blood, which may have been ingested during delivery or during breast feeding from cracked nipples.
This is accomplished using the APT TEST (hemoglobin alkaline denaturation test), which detects fetal hemoglobin. Infant HbF resists denaturation with alkali better than adult HbA. Fresh stool or GI aspirates is mixed with water (1:5 dilution) and centrifuged to lyse the RBCs and release the Hemoglobin. Then sodium hydroxide then is added to the supernatant (1 mL of 1% NaOH for each 5 mL of supernatant). Adult hemoglobin changes to brown-yellow within two minutes (alkaline hematin), while fetal hemoglobin resists denaturation and retains its pink color.
Exposure of the stool sample to air for more than 30 minutes will cause fetal hemoglobin to have the same color change as adult hemoglobin, resulting in a false-positive result.
In cases in which the Apt test is inconclusive, a spectrophotometric assay can be used to quantify the color change. HbF >50 percent indicates fetal blood and HbF <10 percent suggests maternal blood.
Interesting Fact: The APT test was developed by Leonard Apt, an American Pediatric ophthalmologist
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