Alkali denaturation test

Alkali denaturation test
Purpose	differentiate neonatal from maternal blood

The alkali denaturation test, also known as A or Apt test, is a medical test used to differentiate fetal or neonatal blood from maternal blood found in a newborn's stool or vomit, or from maternal vaginal blood.^[1]^[2]

History

The test was developed by Leonard Apt (1922–2013),^[3] 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.^[4]

Uses

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It is used to differentiate whether the blood coming out of vagina is of the mother or fetus during delivery i.e, placenta previa versus vasa previa. It is based on the principle that fetal HbF is resistant to acids and alkali whereas the mother's blood which has HbA is sensitive to acid and alkaline.

Theory

The test is based on differences between maternal and fetal hemoglobin. 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) 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 of the RBCs, yielding free hemoglobin. The sample is next centrifuged for several minutes. The pink hemoglobin-containing supernatant is then mixed with 1 mL of 1% NaOH 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 which has a hydroxide ligand.^[5]

References

^ Moustafa MH, Taylor M, Fletcher L (August 2005). "My two-week-old daughter is throwing up blood". Acad Emerg Med. 12 (8): 775–7. doi:10.1197/j.aem.2005.03.524. PMID 16079432.
^ APT L, DOWNEY WS (July 1955). "Melena neonatorum: the swallowed blood syndrome; a simple test for the differentiation of adult and fetal hemoglobin in bloody stools". J. Pediatr. 47 (1): 6–12. doi:10.1016/S0022-3476(55)80118-7. PMID 14392548.
^ Elaine Schmidt, Obituary: Leonard Apt, 90, doctor-scientist who gave gift of vision to millions of children, UCLA Newsroom, February 7, 2013
^ Venes, D. Taber's Cyclopedic Medical Dictionary - 20th Ed. F. A. Davis Company, Philadelphia, PA. 2005.
^ Pediatrics, Gastrointestinal Bleeding

Heath, K. Abruptio Placentae. in 5-Minute Clinical Consult, 15th ed. Domino, FJ editor. Lipponcott Williams & Wilkins, Philadelphia, PA. 2007.
McRury JM, Barry RC (June 1994). "A modified Apt test: a new look at an old test". Pediatr Emerg Care. 10 (3): 189–91. doi:10.1097/00006565-199406000-00018. PMID 7520161.
Lindqvist PG, Gren P (April 2006). "An easy-to-use method for detecting fetal hemoglobin-- a test to identify bleedingfrom vasa previa". Eur J Obstet Gynecol Reprod Biol. 131 (2): 151–153. doi:10.1016/j.ejogrb.2006.05.011. PMID 16797824.
Lab Tests and Results: Apt-Downey test and [1]

v t e Signs and symptoms in obstetrics
Pregnancy	Abderhalden reaction Chadwick sign Goodell's sign Hegar's sign Ladin's sign Piskacek's sign Von Braun-Fernwald's sign
Other	Apgar score Braxton Hicks contractions hydrops fetalis (Hemoglobin Barts) ectopic pregnancy (Spiegelberg criteria) fetal death (Spalding's sign) Leopold's maneuvers

Tests and procedures relating to pregnancy and childbirth

Maternal tests

Fetal tests

Imaging	Obstetric ultrasonography Nuchal scan Anomaly scan Crown-rump length Fetal movement counting Contraction stress test Nonstress test Vibroacoustic stimulation Biophysical profile Amniotic fluid index
Sampling	Chorionic villus sampling Amniocentesis Triple test Quad test Fetoscopy Fetal scalp blood testing Fetal scalp stimulation test Percutaneous umbilical cord blood sampling Apt test Kleihauer–Betke test Lung maturity Lecithin–sphingomyelin ratio Lamellar body count Fetal fibronectin test
Other	Cardiotocography

Interventions

Delivery

Vaginal delivery

Induction	Assisted vaginal delivery Artificial rupture of membranes Episiotomy Symphysiotomy Obstetrical forceps Vacuum extraction Odon device
Dystocia management	McRoberts maneuver Wood's screw maneuver Zavanelli maneuver
Third stage of labor	Manual placenta removal

Caesarean section

Postpartum bleeding

v t e Hematology blood tests
Complete blood count	Hemoglobin Hematocrit Red blood cell count Red blood cell indices Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Mean corpuscular volume Red blood cell distribution width White blood cell count White blood cell differential Absolute neutrophil count Platelet count Mean platelet volume Reticulocyte count Reticulocyte index
Other tests of red blood cells	Blood volume Total blood volume (TBV) Plasma volume (PV) Red cell volume (RCV) Fetal hemoglobin Apt–Downey test Kleihauer–Betke test Hemoglobinopathy testing Hemoglobin electrophoresis Sickle solubility test Mentzer index Erythrocyte sedimentation rate Haptoglobin Monocyte distribution width (MDW) Osmotic fragility
Coagulation	Clotting factors Prothrombin time Partial thromboplastin time Thrombin time Activated clotting time Fibrinogen Bleeding time animal enzyme Reptilase time Ecarin clotting time Dilute Russell's viper venom time Thrombin generation assay Calibrated automated thrombogram ST Genesia-based test Thromboelastography Thrombodynamics test Overall hemostatic potential Coagulation activation markers Prothrombin fragments 1+2 Thrombin–antithrombin complex Fibrinopeptide A Fibrin monomers Activated protein C–protein C inhibitor Fibrinolysis Euglobulin lysis time D-Dimer Plasmin-α₂-antiplasmin complex Von Willebrand factor Ristocetin-induced platelet aggregation Activated protein C resistance test aPTT-based activated protein C resistance test ETP-based activated protein C resistance test
Other	Blood film Blood viscosity Nitro blue tetrazolium chloride test Flow cytometry Immunophenotyping

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