HCS should be recognized as a distinct thalassemia syndrome with a high risk of life-threatening anemia during febrile illnesses. HbH was not associated with an increased rate of severe anemia with infections and was managed without blood transfusions. Many patients with these disorders had mixed ethnic backgrounds, which highlights the need for extended newborn screening in populations that are traditionally considered to be at low risk for hemoglobin H disease.

Background Newborn screening (NBS) for hemoglobinopathies facilitates early identification of affected individuals to ensure the prompt institution of comprehensive medical care for affected newborns in California. When linked to extensive follow-up and education, NBS has been shown to significantly reduce mortality in children with sickle cell disease. Due to changing immigration patterns from Asia and Latin America, the State of California has witnessed an increased prevalence of clinically significant hemoglobin (Hb) disorders, including those resulting from novel genotypes. In 1999, newborn screening for Hb H disorders was incorporated in the statewide hemoglobinopathy screening program. Procedure Primary screening for hemoglobin variants was performed using high performance liquid chromatography. Confirmatory testing on hemoglobinopathy mutations was performed by electropheresis techniques and genotyping methods. Results Of 530,000 newborn samples screened annually in California, 2,118 samples were referred to the Hemoglobin Reference Laboratory (HRL) for confirmatory testing between January 1, 1998 and June 30, 2006 (0.05%). Sickle cell disease was most frequently observed (1 in 6,600 births) followed by α-thalassemia (1 in 9,000 births) and β-thalassemia disease (1 in 55,000 births). The confirmatory analysis modified the initial screening in 5% of cases and revealed 25 rare or new genotypes. Diverse ethnicities were associated with hemoglobin mutations including Southeast Asian, Black, Indian/Asian, Middle Eastern, and Hispanic. Conclusions The California hemoglobinopathy screening program provides accurate diagnosis of hemoglobinopathies. Increasing incidence of diverse mutations require new strategies of laboratory screening, counseling, and patient management.

Newborn screening is an accepted public health measure to ensure that appropriate health care is provided in a timely manner to infants with hereditary/metabolic disorders. Alpha-thalassemia is a common hemoglobin (Hb) disorder, and causes Hb H (beta4) disease, and usually fatal homozygous alpha(0)-thalassemia, also known as Hb Bart's (gamma4) hydrops fetalis syndrome. In 1996, the State of California began to investigate the feasibility of universal newborn screening for Hb H disease. Initial screening was done on blood samples obtained by heel pricks from newborns, and stored as dried blood spots on filter paper. Hb Bart's levels were measured as fast-moving Hb by automated high-performance liquid chromatography (HPLC) identical to that currently used in newborn screening for sickle cell disease. Subsequent confirmation of Hb H disease was done by DNA-based diagnostics for alpha-globin genotyping. A criterion of 25% or more Hb Bart's as determined by HPLC detects most, if not all cases of Hb H disease, and few cases of alpha-thalassemia trait. From January, 1998, through June, 2000, 89 newborns were found to have Hb H disease. The overall prevalence for Hb H disease among all newborns in California is approximately 1 per 15,000. Implementation of this program to existing newborn hemoglobinopathy screening in populations with significant proportions of southeast Asians is recommended. The correct diagnosis would allow affected infants to be properly cared for, and would also raise awareness for the prevention of homozygous alpha(0)-thalassemia or Hb Bart's hydrops fetalis syndrome.

Hemoglobin Constant Spring (HbCS) is the most common nondeletional alpha-thalassemia variant causing HbH disease, making its detection crucial in populations at risk. Universal newborn screening for HbH is carried out in California. Identification of alpha-thalassemia genotypes responsible for HbH and HbH-CS requires rapid, accurate and cost-effective genotyping methods suitable for population screening. We incorporated the HbCS mutation into our existing seven-plex genotyping assay for common alpha-thalassemia deletions. To assess the feasibility and diagnostic utility of this expanded multiplex gap-PCR assay, we determined genotypic frequencies of HbCS in samples referred for alpha-thalassemia testing between 1 January 2006 and 31 December 2008. During the 3-year study period, 1436 samples were genotyped for alpha-thalassemia. HbH-CS accounted for 23 (13%) of the 176 cases of HbH disease identified. In a subset of 145 newborns referred by the California NBS program with an elevated Hb Bart's level at birth, HbH disease was confirmed in 134 (93%) and HbH-CS identified in 13 (10%) of these. This expanded genotyping assay has proven to be a rapid, reliable and clinically useful diagnostic tool for the detection of HbH-CS disease.

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.