Decoding Sickle Cell Disorders Using Flow Injection Analysis Mass Spectrometry

Chromatographic and electrophoretic methods reliably distinguish the heterozygous and homozygous sickle cell states with hemoglobin variants appearing at consistent percentages with specific retention or migration times. However, co-migrating variants alter the overall hemoglobin profile, complicating interpretation in compound heterozygous conditions such as β^S/β^D-Punjab since these variants electrophoretically migrate close to each other. These cases often require hematological correlation or molecular confirmation. As molecular testing is not available in all laboratories, mass spectrometry (MS) may serve as an alternate approach for examining complex compound heterozygous sickle cell disorders.

Hemoglobin extracted from K2 EDTA blood samples (n = 154) was analyzed using flow-injection triple quadrupole MS. Summed intensities of three charge states each of β^S and β globin monomers enabled calculation of the β^S/β ratio to differentiate sickle cell trait (SCT) from sickle cell disease (SCD). Additional globin ratios (α/β, γ/β, δ/α (%)) were evaluated to subclassify SCD into Group 1 (β^Sβ^E, β^Sβ^D-Punjab, β^Sβ^C, β^SLepore-BW), Group 2 (β^Sβ⁰), and Group 3 (β^Sβ^S). Statistical analysis was performed using SPSS v27.

The β^S/β ratio effectively differentiated SCT from SCD with an optimal cut-off value of 0.71 yielding 90.3% sensitivity and 98.4% specificity. The ratios α/β, γ/β, δ/α (%), β^S/β, significantly discriminated against Group 1 from Group 3 (p < 0.001). While γ/β ratio significantly differentiated Group 1 from Group 2 (p < 0.001), the δ/α (%) ratio was superior for distinguishing Group 2 from Group 3 (p < 0.001).

A rapid, cost-effective MS approach distinguishes SCT from SCD while γ/β and δ/α (%), ratios refine SCD subclassification alongside β^S/β. Combined with capillary electrophoresis (CE), this approach enhances diagnostic resolution and may reduce the need for molecular testing.