Division of Chemistry and Toxicology
	Sickle Cell Disease Unit

		Sickle cell disease is a hemoglobin mutation (variant) which causes severe pain and sometimes death. The pain is a result of the malformed red blood cells; they are rigid, unable to flex in order to make passage through capillaries and can build up in places as the hands and feet.  The patient with sickle cell disease is very susceptible to bacterial pneumonia, and with early detection, penicillin prophylaxis can begin to prevent this.  Other hemoglobin variants can produce more mild diseases and are more rare but just as easily detected by this test.

 

	The Principles of the Test
		Hemoglobin variants are detected in this test using high performance liquid chromatography (HPLC) manufactured by BIO-RAD. During this procedure, the hemoglobins are separated by ionic strength. The blood spot eluate is injected into the carrier solvent, which flows to the column. The different hemoglobins are attracted to the column for different lengths of time as an increasingly cationic solution is passed over them.  The released hemoglobins are detected by a visible wavelength absorbance detector.  By comparing variants to controls, different hemoglobins are identified.   Those patients with hemoglobin variants detected by HPLC are retested via isoelectric focusing (IEF) manufactured by Isolab-Wallac.   In this method, the variants migrate characteristic distances based on their isoelectric points (pI's).  This migration takes place in the presence of an electric current through an ampholyte-filled agarose gel.  After resolution is achieved, the hemoglobins are affixed to the gel ("fixed") and stained.  Comparison with controls gives identification of major hemoglobins. Sue Goodness MT(ASCP) 517-335-9754 goodnesss@state.mi.us Chromatogram from BioRad Variant showing a normal infant with phenotype FA . Chromatogram from BioRad Variant showing an FS phenotype, indicating a likelihood of sickle cell disease.