Detection of Hemoglobin (Hb) Variants By HPLC Screening in Cord Blood Units (CBU) Donated to the National Cord Blood Program (NCBP)

Purpose. To evaluate the frequency and types of Hb abnormalities in CBU collected by the NCBP at the New York Blood Center.

Background and Methods. Established in 1992, NCBP has collected and tested over 76,000 ethnically diverse CBU and manages an inventory of over 60,000 clinical grade CB products, including the first FDA-licensed (HEMACORD®). NCBP has released over 5,600 CBU for transplantation worldwide. To prevent transplants from donors affected with clinically significant hemoglobinopathy (HP), current Good Tissue Practices (cGTP) require Hb screening of all CBU before they are accepted to the public CB inventories. Clinically significant Hb disorders are caused by either structurally abnormal Hb variants (e.g., Hb S, Hb C, Hb E) or by decreased or absent production of α- or β-globin chains (thalassemia). Evaluation of NCBP CBU for possible HP includes: a) detailed family history for HP; b) complete blood count (CBC) and RBC indices, particularly mean corpuscular volume (MCV); c) screening using High-Performance Liquid Chromatography (HPLC) and d) confirmatory diagnostic testing using molecular methods (Hemoglobinopathy Reference Laboratory, Oakland, CA). To date, 76,312 CBU have been screened at the NCBP with the Bio-Rad Variant HPLC system using the Sickle Cell Short Program, a 3-min assay, specifically designed and FDA approved to provide a qualitative result for the presence of Hbs A, F, S, C, D and/or E in the neonate.

Results.The HPLC chromatographic patterns were normal (Hb FA, where F is fetal and A is adult Hb) in 73,163 CBU (95.87%), and revealed an abnormal Hb in the remaining 3,149 CBU (4.13%), the majority of which were not clinically significant.

The most frequent abnormal Hb phenotypes were, sickle cell trait (FAS): 1,755 CBU (57%), Hb E trait (FAE): 562 (18%) and Hb C trait (FAC): 506 (17%). Among the 3,149 CBU with abnormal Hb, 87 carried two genes for Hb disorders of clinical relevance, of which 54 were homozygous for sickle hemoglobin (Hb FS), 6 for Hb C (Hb FC) and 19 were double heterozygotes (Hb FSC). The HPLC results of all CBU with Hb traits and homozygous patterns were confirmed on repeat testing. In addition, all homozygous HPLC results were confirmed by molecular testing.

Thalassemia and other abnormalities. Hb Barts (γ4) elutes "Fast" in the HPLC chromatogram. Its normally low concentration in CB increases when γ-chains replace non-functional or deleted α-globin chains in α-thalassemia (α-thal). Testing of all 76,312 NCBP CBU showed levels of Hb Barts from 0.5% to 34% of total Hb: 69.3% of the samples had Hb Barts level below 2.5%, 29.9% between 2.6 and 6.0% and 0.8% greater than 6.1%.

Accordingly, for presumptive identification of α- or β-thalassemia, HPLC results must be interpreted together with red cell indices and family history. Of 788 CBU samples submitted for molecular (DNA) analyses as "possible thalassemia carriers", one was confirmed as Hb H disease (three α-globin genes deleted), 270 had α-thal trait (two α-globin gene deletions) and 517 had one or no α-globin gene deletion. The 270 CBU with α-thal trait had Hb Barts levels between 4.0% (above 6% in most cases) and 18.9% of total Hb. Further, 258 (95.6%) of the CBU with α-thal trait had MCV below 105 fL (range: 79.9 - 104.8 fL); only 12 cases (4.4%) had MCV >105 fL, all of which had levels of Hb Barts > 5%. The Hb H disease case had Fast Hb 34.2% (by HPLC) and MCV was 76.1 fL.

β-thalassemia (β-thal): 4 CBU had β-thal major and in those, only Hb F (no Hb A) was seen in the HPLC result. Among 26 CBU found to have β-thal trait by the reference lab's DNA analysis, 16 (61.5%) were tested because of positive family history and 10 (38.5%) because of MCV <105 fL (seven CBU had both, low MCV and family history of thalassemia). Another CBU with a still-uncharacterized β-globin gene mutation had MCV <105 fL.

HPLC patterns disclosing "Unknown Hbs" as "traits" in 5 CBU, were confirmed by molecular testing.

Conclusions.

HPLC is a simple to perform, accurate, comprehensive, inexpensive and fast screening method for HP in cord blood.

All homozygous Hb detected by HPLC were confirmed by the reference lab. This is particularly relevant for sickle cell disease, as it is the most common HP in ethnic minority donors.

Screening with HPLC, when used in combination with CBC and family history, is a useful tool to identify possible α- and β-thalassemia carriers in CBU.

Molecular DNA testing is diagnostic in confirming and identifying globin gene deletions and mutations.