American Association for Clinical Chemistry
Better health through laboratory medicine
NACB - Scientific Shorts
NACB - Scientific Shorts (formerly NACB Blog)
By William E. Winter, MD, Lindsay Bazydlo, PhD, Neil S. Harris, MD

Case: The patient is a 30 year old male. CBC showed a normal hemoglobin concentration, hematocrit and mean corpuscular volume (MCV). Hemoglobin electrophoresis was ordered because the patient had been told that he had sickle cell trait. On alkaline gel electrophoresis, the patient's sample is in the second complete lane (see below).


Alkaline gel electrophoresis showed 2 major hemoglobins: an anodal peak of 71.5% and a cathodal peak of 27.4%. Hemoglobin A2 was not elevated. The anodal peak migrated in the normal location of hemoglobin A whereas the cathodal peak migrated in the normal location for hemoglobin S. Is this sickle cell trait? How should you proceed?





In the case of sickle cell trait, hemoglobin S is confirmed by either a positive sickle solubility test (e.g., mixing the reducing agent sodium hydrosulfite with blood inducing sickling in cells containing hemoglobin S) or by performing an acid gel electrophoresis where hemoglobin S migrates separately from hemoglobin A.


In this case, the sickle solubility test was negative ruling against the non-A peak being hemoglobin S. As well, the A to S ratio in sickle cell trait is ~60:40 and the ratio in this patient is higher than 60:40 (i.e., ~70:30).

Acid gel electrophorsis confirmed that the non-A peak was not hemoglobin S as the acid gel did not reveal an S peak, but did reveal a single large peak migrating in the location of hemoglobin A (middle lane on the acid gel below). The differential diagnosis of this alkaline gel second peak are hemoglobins D, G-Philadelphia, Lepore and Korle-Bu.

response image.JPG
Because hemoglobin Lepore produces a thalassemic phenotype, A/Lepore is unlikely. Therefore this patient could be heterozygous for A and D, A and G, or A and Korle-Bu. Hemoglobin D in the heterozygous state with A (i.e., AD) or homozygous state (i.e., DD) is benign; however, heterozygosity of D with S (i.e., SD), produces a sickle cell anemia-like-phenotype that can produce crises. In order to differentiate D from G from Korle-Bu, molecular analysis was recommended.



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Posted by Sutirtha Chakraborty
On 8/19/2012

The previous lane shows only Hb S , I hope there has been no carryover/contamination from the pipetting tips to this lane

Posted by Sutirtha Chakraborty
On 8/15/2012

Considering the electrophoresis to be done at alkaline pH it is difficult to confirm this case as Sickle Cell trait.As because Hb D can comigrate along with HbS arriving at a diagnosis based on electrophoresis result might be erroneous.Considering an absolutely normal complete hemogram with lack of any symptoms I strongly suspect possibility of D trait(Hb D Los Angeles/Punjab etc).Hence I would like to perform an Isoelectric focusing.An HPLC could also be helpful.And ofcourse mutation analysis is confirmatory. Dr Sutirtha Chakraborty,MD Peerless Hospital, Kolkata,INDIA email: