A Comparative Study of Solubility Based Sickle Cell Screening Versus Genomic Method in Blood Donors

Background:

Sickle cell trait (SCT) is more common in minority African-Americans (AA) and Hispanic blood donors. Screening donors for Sickle Hemoglobin (HbS) carrier status (SCT) can be useful, especially in managing the transfusion needs of neonates and patients with sickle cell disease. Currently, the Sickledex test, based on the principle of relative insolubility of HbS in phosphate buffers, is routinely used for HbS donor screening. However, there are several limitations including the inability to perform high-throughput testing by automation as donor centers target larger numbers of minority blood donors. False positives results due to hyperlipidemia, dysglobulinemia and higher peripheral blood hematologic counts have also been reported. Hence alternative methods to identify SCT donors are highly desirable in blood centers. The purpose of this study was to validate testing, and to identify rates of False Positive (FP) and False Negative (FN) sickledex screening in blood donors by verifying the presence or absence of the most common mutation seen with HbS production using molecular methods. The comparison could offer evidence to use molecular methods as a test of record to manage HbS carriers at blood centers.

Methods and Results

Between Jan 2012 till Dec 2014, 200 sickledex screening tests were done daily, totaling approximately 90,000 donors self-identifying as Caucasians and 60,000 donors identifying as African American or Hispanic tested during the study period. Amongst Caucasians tested, 154 had a positive sickledex screen result.  Since the prevalence of SCT is less common in the Caucasian population, we hypothesized several of these Caucasian donors could have a false positive screening test. All 154 sickledex positive Caucasian donor samples were prospectively tested for the HbS mutation detection with molecular methods. BioArray Human Erythrocyte Antigen (HEA) Bead Chip DNA assay with PCR multiplex based technology was used to detect the HbS mutation (β-globin change -173 A>T, more commonly designated 20A>T). Of the 154 sickledex + Caucasians, 108 (70%) donors tested negative (FP Screen) and 46 (30%) tested positive for the HbS mutation. A retrospective review of sickledex results in our electronic database for minority donors (n-3431) who also had HEA BeadChip assay was performed to determine concordance. Of 3431 minority donors tested, only 2 (0.0006%) had a positive sickledex screening and were  negative by HEA, with the remaining negative by both the sickledex screen and HEA tests. Among minority donors (n= 254) testing + for HbS by HEA, 3% (8/254) were negative by sickledex screening. To establish concordance of HEA assay with high performance liquid chromatographic (HPLC) assay, two separate cohorts were evaluated.  Initially 12 sickle screen and HEA positive donors was subject to HPLC analysis, which confirmed SCT status in all 12 samples. Subsequently sickledex screen positive but HEA negative samples (n= 40), were subject to HPLC.  All 40 showed absence of SCT by HPLC. Finally, 58 random sickledex + samples had gene sequencing and HEA performed in parallel. Out of 58 samples, 54 showed a positive HbS mutation by HEA with A>T (SCT) nucleotide change noted with gene sequencing. In the remaining 4 samples, HEA was negative with gene sequencing showing (3 T>T and in 1C>T) change at nucleotide 163 associated with Hemoglobin Okayama.

Conclusion:

The HEA bead chip assay testing for the HbS mutation showed 100% concordance with HPLC and gene sequencing in samples studied. Molecular methods can process multiple samples in a single setting to detect SCT. FP sickledex screening results in Caucasians needs confirmatory testing for the HbS mutation to avoid unnecessary donor notification and deferral.  The association, if any, of false positive sickledex screening in Caucasian donors with the Hgb Okayama mutation is under further investigation.