Application of flow cytometry-based genotyping for rapid detection of hemoglobin variants.
The hemoglobinopathies represent a genetically heterogeneous group of disorders. Clinically important hemoglobin variants have been increasingly reported in the USA. Consequently, rapid and accurate testing methods are needed to address the growing diagnostic challenges of identifying these variants. To evaluate the utility of the Luminex LabMAP system for hemoglobinopathy testing, we adapted single base primer extension (SBPE) to this platform to detect 11 clinically important hemoglobin variants. Clinical samples from 11 individuals were tested for five beta-globin mutations (C-Harlem, D-Iran, Fannin-Lubbock and Hope) and six alpha-globin mutations (J-Toronto, Hasharon, G-Philadelphia, G-Norfolk, Constant-Spring and Quong-Sze). Two separate multiplexed SBPE assays were developed. Biotinylated amplification products were hybridized to fluorescent microspheres tagged with allele-specific capture probes and analyzed by flow cytometry on the Luminex100 instrument. The median fluorescent intensity (MFI) ranged from 1255 to 7478 fluorescence units (FU) and from 282 to 2609 FU above background for all positive beta-globin and alpha-globin alleles, respectively. Using the highest background MFI + 3 SD as a conservative threshold, MFI values uniformly discriminated wild type from mutant alleles, and genotypes were correctly identified in all samples tested. This pilot study demonstrates the potential application of the Luminex LabMAP genotyping platform to newborn screening for definitive hemoglobinopathy testing.
Aslanian
S
S
Hemoglobinopathy Reference Laboratory, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
Azimi
M
M
Noble
J
J
Hoppe
C
C
D005434:Flow Cytometry / Q000379:methods; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D005914:Globins / Q000235:genetics; D006453:Hemoglobinopathies / Q000097:blood / Q000175:diagnosis*; D006455:Hemoglobins, Abnormal / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction
D006455:Hemoglobins, Abnormal; D005914:Globins