This week our blog post is from the 2013 George Grannis Award Winner, Dina Greene. Congratulations to Dr. Greene on this great accomplishment!
When I began working at my current institution I was surprised to learn that we perform all of our hemoglobinopathy screens using both HPLC and isoelectric focusing electrophoresis (IEF). Yes, you read that right: all of them. This is in contrast to the workflow in most laboratories, where the general approach is to screen by HPLC (or a similar high throughput, automated assay like capillary electrophoresis) and to confirm only those specimens that initially screen abnormal with a slab-gel based technique.
I curiously asked, “Why do we do this? Isn’t it redundant?”
The astute supervisor answered, “Well, what about the patients with low concentrations (<2%) of Hb H? We only detect the Hb H using IEF; it is not present on the HPLC chromatogram. Aren’t those low concentrations of Hb H clinically relevant?”
Hmm. I didn’t know. So, I did what I usually do when I don’t know something - I googled it. Google confirmed that Hb H is a hemoglobin tetramer composed of only beta subunits. These subunits associate when there is a decreased amount of the alpha subunit, usually as a result of alpha gene deletion(s). What was still missing was the clinical significance of very low Hb H concentrations. The only documentation I could find recognized that Hb H concentrations are >5% in patients with three alpha gene deletions and that four alpha gene deletions was incompatible with life [1]. I was also reminded that early in life alpha-thalassemia patients with any number of alpha gene deletions usually have detectable Hb Bart’s (gamma tetramers; sometimes absent in silent carriers). However, the vast majority of the samples which we detected and reported the low concentrations of Hb H were adult samples submitted for unexplained mild anemia or at risk pre-natal screening.
I found myself still confused about the clinical significance of these samples, so I did my second favorite thing to do when I don’t know something – I phoned a friend. The call recipient was a hematopathologist (who is responsible for hemoglobinopathies at a large reference lab) and upon asking her she was puzzled. She responded with, “You have samples like this?” The light bulb went off in both of our heads, and we knew we were about to collaborate on a fun project.
The hypothesis was that these samples were from alpha thalassemia patients with only one or two alpha gene deletions. It’s currently thought that once these patients (silent carriers or those with alpha thalssemia trait) reach adulthood they can only be distinguished from wild type individuals using molecular-based assays. Our study, which will be published in the September 2013 issue of the American Journal of Clinical Pathology (AJCP), refutes this dogma and encourages that IEF has high specificity for detecting patients with two alpha gene deletions [2].
In the age of technology, it’s often easy to overlook classical techniques, like those based on electrophoresis, for those that are sexier (and by sexy, I mean involve molecular or mass spectroscopy detection). It might just be nostalgia, but it makes me happy to know that these classical techniques still have a valuable place in the clinical laboratory, and that we can still further our learning about how their utilization can be optimized to better patient care.
- Galanello R, Cao A. Gene test review. Alpha-thalassemia. Genetics in medicine : official journal of the American College of Medical Genetics. 2011; 13:83-88.
- Archana M Agarwal, Roberto H Nussenzveig, Carolyn Hoke, Thomas S Lorey, and Dina N. Greene. Identification of One or Two Αlpha-Globin Gene Deletions by Isoelectric Focusing Electrophoresis. AJCP. Scheduled for press September, 2013.