Application of flow cytometry‐based genotyping for rapid detection of hemoglobin variants

Abstract

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.

This article has been retracted: A gene‐specific primer extension and liquid bead array system for killer cell immunoglobulin‐like receptor genotyping

“…Data were collected from a minimum of 50 microspheres of each type and the median fluorescence intensity (MFI) of any signals that is >660 was considered for the presence of KIR genes. The cut‐off value for minimal signal was calculated by the average of the negative MFI value of 16 types of KIR genes plus three standard deviation (SD) values (17).…”

Variable Clinical Phenotypes of α-Thalassemia Syndromes

“…Screening of newborns for Hb H disease was implemented in California in 1999 [65] and expanded to include confirmatory testing by DNA analysis for detection of Hb H-CS [43]. Initial screening has expanded to include a program aimed to resolve ambiguous results from State Newborn Hemoglobinopathy Screening [66]. The determination of an early diagnosis allows proper care for these infants and raises the awareness of screening for the prevention of homozygous α-thalassemia.…”

Newborn screening for spinal muscular atrophy: Anticipating an imminent need

“…129,130 These methods involve PCR of the target sequence with synthetic nucleobases, allele-specific target extension of the unique base in SMN1 exon 7, microbeads that hybridize to tags on the extension region, and fluorescent signal detection. An advantage of using these assays is the large multiplex capability of 80-100 targets.…”