Hemoglobin Electropheresis

Description

Electropheresis can be used to separate different hemoglobins based on their electrical charge, which can be useful in the diagnosis of the different hemoglobinopathies.

Testing:

(1) electropheresis at alkaline pH (cellulose acetate at pH 8.6)

(2) electropheresis at acid pH (citrate agar at 6.2)

Key bands on electropheresis at alkaline pH

Normal Pattern	Abnormal Hemoglobins
cathode
point of application
carbonic anhydrase
A2	C, C-Harlem, E, O
	S, D, G
	Lepore
	F
A1
	Barts
	H
anode

Key bands on electropheresis at acid pH

Normal	Abnormal Hemoglobins
cathode
	F
A1, A2	E, D, G, Lepore, Barts
	O
	S, C-Harlem
	H
point of application
	C
anode

Note:

(1) There is some variability between the sources for the band patterns, especially for electropheresis at acid pH.

(2) The precise location of S and O hemoglobins relative to the application point at acid pH appears to vary with conditions, with some showing the point of application between O and A1, while others between S and O.

(3) Hemoglobin E may be masked by A2 in both acid and alkaline conditions.

Additional tests:

(1) solubility tests to confirm hemoglobin S

(2) hemoglobin A2 by DEAE cellulose column

(3) alkali denaturation of hemoglobin F

(4) testing for iron deficiency

If iron deficient, repeat testing after iron replacement complete

• Iron deficiency can mask beta-thalassemia.

Hemoglobin	A1	A2	Other
normal	almost all	0-3% in adults	< 2% F in adults
newborn			> 60% F during 1st week; no abnormal bands
beta thalassemia		normal or increased	microcytic despite normal iron stores
alpha thalassemia		normal	microcytic despite normal iron stores
AS		normal	34-42% S; positive solubility test
AS + alpha thalassemia		normal	< 34% S; positive solubility test; microcytic
AS + beta thalassemia		normal or increased	> 34% S; positive solubility test; microcytic
SS	none unless transfused		predominantly S; positive solubility test
AC			30-42% C
AC + alpha thalassemia			< 30% C; microcytic
AC + beta thalassemia			> 30% C; microcytic
CC	none unless transfused		predominantly C
AE			30-35% E
AE + alpha thalassemia			< 30% E; microcytic
AE + beta thalassemia			microcytic

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