This last month, I rotated through our Children’s hospital, which included reviewing hemoglobin electrophoresis tests. I’d learned about them before in residency, but they can be quite more interesting (complicated) than I expected.
Hemoglobin electrophoresis is a blood test to look at different types of hemoglobin to determine if there are any abnormalities. In a children’s hospital it is frequently ordered as a reflex for an abnormal newborn screen or when a child is incidentally found to be anemic. The test is performed in 2 stages. 1st lysed blood samples are run on gel electrophoresis and different types of hemoglobin are separated as they move at different speeds. Several types of hemoglobin will run within the same region, so a secondary method of separation is always employed.
Below, you can see how some bands in the same area of an acidic gel (agarose) are actually very different on the alkaline gel (cellulose acetate) and vice versa.
At our hospital, we use HPLC and measure retention times of the hemolysate to quantify and identify different hemoglobin types present. As a basic primer you should recall that hemoglobin is a tetramer with a pair of alpha globin + a pair of either beta, delta or gamma globin (each separate genes).
Alternative hemoglobins are enriched in populations where malaria is endemic as these variants may provide improved fitness by promoting resistance to the malarial parasite that reproduces inside red blood cells. Thus, many people of African or south east Asian descent may carry these variants.
Our case is that of a 2 year old girl with anemia who had testing sent by her primary care doctor for the following CBC:
This is indicative of microcytic anemia, but unlike some Thalessemias the RBC isn’t very high. More on this later.
Looking at the gel result, there is a large band in the area coinciding with Hgb C. We also see the normal Hgb A2 and a small amount of Hgb F. We know Hgb F can be increased in Hgb SS and thus could also be present if she had Hgb C trait or disease.
Looking at the next HPLC result, we see there is a similar very high level of Hgb C (68%) with corresponding levels of Hgb F and Hgb A2 (note: acetylated Hgb F and Hgb F are added together). Thus, this fits with a homozygous C with some compensatory A1 and F, right?
Remember Hgb C is a β -globin variant and you only have 2 β -globin genes, so if you are homozygous for the C variant on the β-globin gene (HBB), then Hgb A1, which is made of normal β-globin would be impossible to produce. Also you might be bothered by all of these small peaks. However, there are often small peaks that can’t be definitively identified and are likely post-translationally modified hemoglobin. But in the context of an abnormal Hgb A1 that shouldn’t be there, we dug deeper.
One of the most common hemoglobinopathies is Beta Thalassemia (β-Thal), which clinically manifests when less of the beta hemoglobin protein is produced. Heterozygous mutations lead to Beta Thalassemia minor with minimal symptoms, while homozygous mutations lead to β-thal major with symptoms of anemia. Mutations in the β -globin gene, HBB, can lead to complete loss of β-globin (β0 variant) or partial of β-globin (β+ variant).
As this patient has less than 50% of Hgb A present (expected amount), they could also have a β+ variant as well. This would make them compound heterozygous for C and β+.
One of the hallmarks of Thalassemia is an increase in Hgb A2 (normal 2.5-3.5%). Hemoglobin A2 is a normal variant of A that is composed of two alpha and two delta chains (δ2α2). We see in our case that the Hgb A2 is normal at 2.5%. So it seems the patient doesn’t display a typical Thalassemia picture.
One condition that could create this scenario is if there is a variant in the delta chain of A2 that causes it to elute differently. Indeed, there is a delta variant that creates hemoglobin A2 prime (A2’) that moves near the S region of the HPLC. And when we look back at our unknown hemoglobins, Hgb X is marked at 1.03 of the S region and has an abundance of 3.9%. This supports it being the Hgb A2’ and if we add this together with the Hgb A2 we get an elevated 6.6% A2 total, which would be consistent with Beta Thalassemia. Lastly, one would wonder if we could find this third hemoglobin variant A2’ on the alkaline gel. Previous studies have shown the A2’ variant is more negatively charged, so on a basic gel, it should move further from the negative anode than the other hemoglobins. We don’t see anything to the left of the HgbC, but if we flip the gel over and look under the patient label, you can see a faint band that is likely the A2’!
In summary this case arose from 3 separate mutations in a single patient. She was compound heterozygous for a Hgb C and β+ variants in the β-globin gene and she was heterozygous for an A2’ variant on the delta-globin gene. This was certainly a case where paying close attention mattered.
- Abdel-Gadir D, Phelan L, and Bain BJ. Haemoglobin A2′ and its significance in beta thalassaemia diagnosis. Int J Lab Hematol. 2009 Jun;31(3):315-9. doi: 10.1111/j.1751-553X.2008.01038.x. Epub 2008 Feb 21.
-Dr. Charles Timmons MD PhD is a pediatric pathologist at Children’s Medical Center in Dallas, TX. His responsibilities include signing out hemoglobin electrophoresis, HPLC and globin sequencing, and has been residency director for 17 years.
-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.