(P-HC-7) Quantitative Hemoglobin S Testing by Cation Exchange High-Performance Liquid Chromatography

Background/Case Studies: Hemoglobin S (HbS) is an abnormal hemoglobin variant resulting from a beta-globin protein gene mutation. HbS deoxygenation leads to the deformation of red blood cells (RBCs), causing the microvasculature occlusion and anemia characteristic of sickle cell disease (SCD). SCD treatment employs various robust procedures and pharmaceuticals to manage patients. HbS measurement is used to monitor disease progression, risk stratification, and response to treatment, especially for patients requiring RBC exchange, making measurement ease and availability critical to SCD management. Methods for HbS quantification, such as capillary electrophoresis, are not widely available. Having a method to rapidly quantify HbS is critical in providing necessary care.

Study

Design/Methods: At our institution, the laboratory implemented HbS quantification by cation-exchange high-performance liquid chromatography (HPLC) via the Bio-Rad D100 platform. This instrument is used for HbA1c quantification, and was selected for non-diagnostic HbS quantification because of its ease of use, rapidity, precision and ability to already separate and quantify HbS. Inter- and intra-day precision, accuracy, analytical sensitivity and specificity were validated. For accuracy the D100 method was correlated to a send-out method for HbS by hemoglobin electrophoresis.

Results/Findings: Inter- and intra-day precision acceptability were set at < 4% and CVs of 2.7% and 1.1% were achieved, respectively. Accuracy and linearity were analyzed over a range of 8.2% to 83.6% and all results were sufficiently accurate (i.e., within systematic error budget of 10% and total allowable error of 20%) and linear within the specified reportable range.

Conclusions: HbS quantification is a necessary tool for management of patients with SCD, especially for patients requiring RBC exchange. Capillary electrophoresis has long been used as the primary method of HbS quantification, but requires a dedicated platform, and most instruments are not run at all times due to specimen volume, lab personnel availability, and maintenance issues. Due to the acuity of patient presentation, development of a rapid, easy, method for HbS quantification has become essential. Our validation of the BioRad D100 system for HbS takes advantage of the inherent ability of cation-exchange methods to separate most Hb variants and quantify them. We took the necessary steps to validate this capability as a rapid lab developed test for HbS quantification and recommend this to other hospitals with the same need.