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Quantifying HbF Using a Modified Kleihauer‐Betke Assay
Author(s) -
Singh Rhena,
Randolph Tim R
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1082.8
Subject(s) - medicine , cord blood , hematocrit , sickle cell anemia , anemia , fetal hemoglobin , population , disease , fetus , pregnancy , biology , environmental health , genetics
Sickle cell anemia is one of the most commonly inherited diseases, annually affecting approximately 275,000 births worldwide. The disease is most prevalent in African countries and countries in which a large proportion of the population is of African descent, such as the Caribbean nation of Haiti. Hydroxyurea is the only FDA approved drug that is used to mitigate sickle cell symptoms by reducing the percentage of HbS and increasing HbF. Since hydroxyurea is also mildly carcinogenic, HbF levels must be monitored during treatment to determine the minimum effective dosage or to stop the drug in non‐responders. The objective of this study is to develop and validate an inexpensive and simple method of measuring HbF levels in sickle cell patients undergoing Hydroyurea treatment. This method uses a modified Kleihauer‐Betke (KB) principle to elute HbA and HbS in a weak citric acid solution while retaining HbF. Normal blood samples (source of HbA) were collected from the research team, de‐identified cord blood samples (source of HbF) were provided by the St. Louis Cord Blood Bank at Cardinal Glennon's Children's Hospital and de‐identified sickle cell blood samples (source of HbS) were provided by Cardinal Glennon Children's Hospital. The procedure involves the mixing of 20 uL of type specific cord blood/normal blood, or cord blood/sickle blood mixtures that were standardized to 35% hematocrit to 4 mL of citrate solution (0.07 M sodium citrate in a 0.06 M sodium phosphate buffer solution). Samples were incubated at 37 0 C for 6 minutes, centrifuged for 30 seconds and the supernatant read spectrophotometrically at 395 nm. As HbA or HbS levels increase (decreasing levels of HbF), the absorbance also increases showing a strong positive linear correlation (r 2 = 0.99, N=5). This simple and cost‐effective method shows promise to be used in underdeveloped countries for HbF quantification. Support or Funding Information RS is supported by the DeNardo Education and Research Foundation.