The Diagnosis of α-Thalassaemia: A Case of Hemoglobin H -α Deletion

Abstract

We report a case of hemolytic anemia that was subsequently identified to be a case of α-thalassaemia harboring the common rightward 3.7 kb deletion/HbH. The diagnosis was based on sequential analyses using BioRad D10 HPLC, Alkaline gel electrophoresis, GPO α THAL-IC strips and the identification of the specific genetic lesion using an α Globin reverse dot blot hybridization assay. Supravital stain of RBCs helped in identifying classical HbH inclusions. In a background of a variable clinical presentation, lack of definitive hematological markers, and general under-diagnosis of α-thalassaemias we have used this case to highlight the features and sequence of techniques involved in identifying and characterizing an α-globin chain mutation, starting from a diffuse clinical history and presentation up to the identification of a specific genetic lesion involved.

Keywords: Alkaline hemoglobin electrophoresis; HPLC; HbH; Reverse dot blot hybridization; α THAL-IC strip.

Fig. 1

Schematic representation of the Test Principle using the Vienna Lab Reverse Dot Blot Hybridization technique (Courtesy: Vienna Lab, Labordiagnostika, GmbH)

Fig. 2

Printed output for the analysis of EDTA-Blood sample from patient on a Sysmex XT 1800i Hematology analyzer

Fig. 3

Print out-put from the BioRad D10 HPLC machine showing (arrow) unidentified proteins eluting at retention times of 0.17 and 0.38 min. HbA2 levels are also reduced (at 1%)

Fig. 4

Electropherogram of the alkaline hemoglobin electrophoresis (pH 8.6) conducted on the HELENA SASII system for the patient sample. Lane 1: AFSA2 pack Control, Identities of the migrating bands are indicated on the figure. Lane 2: Patient sample shows the presence of a putative HbH band migrating ahead of HbA

Fig. 5

a Results for the GPO α THAL-IC strip test conducted on control and patient samples. Strip 1: Normal sample, Strip 2: Patient sample showing positive for an extra band (arrow) indicating the presence of α-thalassaemia disorder. b 1% brilliant cresyl blue (BCB) precipitated HbH inclusions within typical ‘golf ball’ cells (arrows) confirming the presence of HbH

Fig. 6

Vienna Lab α-thal mutation screening strips for the deletion (A) and non-deletion (B) mutations reported for α-thalassaemia. A Strip 1: normal control sample, Strip 2: patient sample showing the −3.7 kb gene deletion positive band, Strip 3: Mutation key strip. B Strip 1: No mutations detected in the non-deletional set of mutations for the patient sample, Strip 2: Mutation key strip