Identification of the Hb Lepore phenotype by HPLC

Area of Science:

• Hematology
• Molecular Biology
• Biochemistry

Background:

• Hb Lepore is a structurally abnormal hemoglobin resulting from a fusion of delta (db) and beta (b) globin genes.
• Three variants exist: Hb Lepore Hollandia, Hb Lepore Baltimore, and Hb Lepore Boston, with Spain primarily identifying the latter two.
• Distinguishing Hb Lepore types traditionally requires complex peptide chromatography, posing challenges for some laboratories.

Purpose of the Study:

• To develop and validate a simpler method for identifying Hb Lepore phenotypes in Spanish patients.
• To compare the efficacy of reversed-phase HPLC with traditional molecular techniques for Hb Lepore typing.

Main Methods:

• Studied 23 patients from 13 Spanish families with suspected Hb Lepore.
• Quantified Hb Lepore using ion-exchange HPLC.
• Analyzed globin chain phenotypes using reversed-phase HPLC.
• Corroborated results with molecular biology techniques (PCR and restriction enzyme digestion).

Main Results:

• Reversed-phase HPLC successfully identified distinct globin chain peaks (a, b, db) in 14 patients, corresponding to Hb Lepore Boston via molecular analysis.
• The remaining 9 patients showed no hybrid db chain peak and were identified as Hb Lepore Baltimore heterozygotes through molecular studies.
• The study demonstrated a clear correlation between HPLC findings and molecularly confirmed Hb Lepore types.

Conclusions:

• Reversed-phase HPLC of globin chains is sufficient for routine Hb Lepore phenotype identification.
• This method simplifies diagnosis, especially for labs lacking advanced molecular biology capabilities.
• Molecular testing remains crucial for prenatal diagnosis due to its precision and reproducibility.