c. Normal Values
A3 Hemoglobin: One and three tenths to 3 percent.
F Hemoglobin: Zero to 2 percent (except in infants).
6-14. FETAL HEMOGLOBIN (ALKALI DENATURATION TEST)
a. Principle. Fetal hemoglobin (HbF) is more resistant to denaturation in
alkaline solution than adult hemoglobin (HbA). Alkali converts HbA to alkaline hematin.
Alkaline hematin is insoluble and precipitates. HbF is quantitated by measuring the
hemoglobin concentration before and after denaturation.
Drabkin's reagent. Available in pellets or powder in the Federal Supply
(2) Sodium hydroxide (NaOH) 0.10 N. Dilute 5.55 saturated NaOH to 1-liter
with distilled water. Mix well and store in a polyethylene bottle at room temperature.
(3) Ammonium sulfate, saturated. Add 400 g ammonium sulfate, AR, to 500
ml of distilled water. Stir mechanically for 10 minutes. Heat until all the salt has
dissolved and filter rapidly through a Whatman number one filter paper. Store in
c. Preparation of Hemolysate. The procedure is as follows:
Dilute 0.2 ml of hemolysate with 1.8 of Drabkin's reagent and mix gently.
(2) Place 2.5 ml of 0.1 N NaOH in a 13 X 100 mm test tube. At zero time,
add. 0.5 ml of diluted hemolysate and mix rapidly.
(3) Exactly 2 minutes after addition of the hemolysate, add 2.0 ml of
saturated ammonium sulfate. Mix by inverting six times. DO NOT SHAKE!
(4) Filter through a 7 or 9 cm Whatman number 42 filter paper. If the filtrate
is not crystal clear, filter again. This is the fetal hemoglobin solution.
(5) Prepare a total hemoglobin solution by adding 0.05 ml of diluted
hemolysate to 5.0 ml distilled water. Mix well by inversion. DO NOT SHAKE!
(6) Prepare a fetal hemoglobin blank by adding 0.5 ml of Drabkin's reagent
to 4.5 ml of distilled water. Mix as above.