The alkali denaturation test, also known as A or Apt test, is a medical test used to differentiate fetal or

neonatal An infant or baby is the very young offspring of human beings. ''Infant'' (from the Latin word ''infans'', meaning 'unable to speak' or 'speechless') is a formal or specialised synonym for the common term ''baby''. The terms may also be used to ...

blood from maternal blood found in a newborn's stool or vomit, or from maternal vaginal blood.

History

The test was developed by

Leonard Apt Leonard Apt (June 28, 1922 – February 1, 2013) was an American pediatrician and ophthalmologist. He was one of the first U.S. physicians to complete a fellowship in pediatric ophthalmology. Apt identified that povidone-iodine could be safety us ...

(1922–2013), an American pediatric ophthalmologist. The test was originally used to identify the source of bloody stools in newborn infants. It has been modified to distinguish fetal from maternal hemoglobin in blood samples from any source.

Uses

The Apt test is most commonly used in cases of vaginal bleeding late during pregnancy ( antepartum haemorrhage) to determine if the bleeding is from the mother or the fetus. * A positive test would indicate that blood is of fetal origin, and could be due to

vasa previa Vasa praevia is a condition in which fetal blood vessels cross or run near the internal opening of the uterus. These vessels are at risk of rupture when the supporting membranes rupture, as they are unsupported by the umbilical cord or placental ...

. * A negative test indicates that the blood is of maternal origin. In practice, the Apt test may not be done when there is suspicion of vasa previa, because the time to fetal collapse with bleeding from vasa previa is often very short. The Apt test can also be used to detect the presence of fetal blood in the maternal circulation in cases of suspected

fetal-maternal hemorrhage Fetal-maternal haemorrhage is the loss of fetal blood cells into the maternal circulation. It takes place in normal pregnancies as well as when there are obstetric or trauma related complications to pregnancy. Normally the maternal circulation an ...

. Since the test is only a qualitative determination of the presence of fetal hemoglobin in maternal blood, the quantitative Kleihauer-Betke test is more commonly used. Finally, the Apt test can be used after birth (

postpartum hemorrhage Postpartum bleeding or postpartum hemorrhage (PPH) is often defined as the loss of more than 500 ml or 1,000 ml of blood following childbirth. Some have added the requirement that there also be signs or symptoms of low blood volume fo ...

) if the newborn has bloody vomiting, bloody stool, or active bleeding from the nasogastric tube. A positive apt test would mean that the blood is either due to gastrointestinal or pulmonary bleeding from the neonate. A negative Apt test would indicate that the blood is of maternal origin, suggesting that the neonate swallowed or aspirated maternal blood, either during delivery or during breastfeeding (e.g., from breast fissures).

Theory

The test is based on differences between maternal and fetal

hemoglobin Hemoglobin (haemoglobin BrE) (from the Greek word αἷμα, ''haîma'' 'blood' + Latin ''globus'' 'ball, sphere' + ''-in'') (), abbreviated Hb or Hgb, is the iron-containing oxygen-transport metalloprotein present in red blood cells (erythrocyt ...

. Maternal blood contains adult hemoglobin composed of two alpha and two beta subunits (aka hemoglobin A or HbA; i.e., normal adult hemoglobin). Fetal blood contains fetal hemoglobin composed of two alpha and two gamma subunits (aka hemoglobin F or HbF; i.e., normal fetal hemoglobin). This difference in composition gives the different types of hemoglobin different chemical properties (in addition to the higher affinity HbF has for dissolved blood oxygen over HbA, allowing baby to extract oxygen from the mother's blood). Fetal hemoglobin is resistant to alkali (basic) denaturation, whereas adult hemoglobin is susceptible to such denaturation. Therefore, exposing the blood specimen to sodium hydroxide (

NaOH Sodium hydroxide, also known as lye and caustic soda, is an inorganic compound with the formula NaOH. It is a white solid ionic compound consisting of sodium cations and hydroxide anions . Sodium hydroxide is a highly caustic base and alkali ...

) will denature the adult but not the fetal hemoglobin. The fetal hemoglobin will appear as a pinkish color under the microscope while the adult hemoglobin will appear as a yellow-brownish color.

Method

The blood is mixed with a small amount of sterile water to cause

hemolysis Hemolysis or haemolysis (), also known by several other names, is the rupturing ( lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid (e.g. blood plasma). Hemolysis may occur in vivo ...

of the

RBCs Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek language, Greek ''erythros'' for "red" and ''k ...

, yielding free

. The sample is next centrifuged for several minutes. The pink hemoglobin-containing supernatant is then mixed with 1 mL of 1%

for each 5 mL of supernatant. The color of the fluid is assessed after 2 minutes. Fetal hemoglobin will stay pink and adult hemoglobin will turn yellow-brown since adult hemoglobin is less stable and will convert to

hematin Haematin (also known as hematin, ferriheme, hematosin, hydroxyhemin, oxyheme, phenodin, or oxyhemochromogen) is a dark bluish or brownish pigment containing iron in the ferric state, obtained by the oxidation of haem. Haematin inhibits the synth ...

which has a hydroxide ligand.

History

Uses

Theory

Method

See also

References

Further reading