Accidental detection of clinically silent compound heterozygous Hb D Punjab/Hb Q India while analyzing HbA1c by high performance liquid chromatography

HbA1c is routinely prescribed investigations for diagnosing and monitoring diabetes and high-performance liquid chromatography (HPLC) is preferred method which is also able to identify presence of hemoglobin variant. A case was encountered where presence of variant hemoglobin was indicated. On further investigation with three different instruments, diagnosis of compound heterozygous Hb D Punjab/Hb Q India was made. The chromatogram on Bio-Rad D10 showed Hb D Punjab (ααββHbD Punjab)-29.89% at 3.96 minutes retention time (RT), Hb Q India (ααHbQ Indiaββ) -9.5% with 4.45 minutes RT, hybrid of HbQ India/Hb D Punjab (ααHbQ IndiaββHbD Punjab)-6% with 4.66 minutes RT, Hb A2 (ααδδ) was 2.5% and Hb A (ααββ) was 52.2%. Analysis done on Bio-Rad variant V-II confirmed these findings. Analysis done on Sebia capillary electrophoresis revealed major peak of 50.9% in zone 9/Z(A) constituted by Hb A, second peak of 39.8% in zone 6/Z(D) constituted by co-elution of Hb D and Hb Q India, third peak of 8.8% in zone 3-4/Z(A2-C) constituted by co-elution of Hb A2 and hybrid of Hb D Punjab/Hb Q India and a fourth peak of 0.5% in zone 1 representing Hb A2HbQ India (ααHbQ Indiaδδ). Ideally variants detected while analyzing HbA1c should be further investigated for confirmation and result of which should be shared, discussed and the patient should be encouraged for screening of available family members for relevant variant hemoglobin. Combination of cation exchange HPLC and capillary electrophoresis in this case was sufficient to arrive at conclusion.

Hemoglobin Yamagata [β132(H10)Lys→Asn; (HBB: c.399A>T)]: a mosaic to be put together

Objectives Artifactually altered glycated hemoglobin (HbA1c) concentrations are frequently linked to hemoglobin (Hb) variants. Their expression and detection require in-depth analysis. Methods Cation exchange high performance liquid chromatography (HPLC) (Bio-Rad Variant™ II; Trinity Biotech Premier Hb9210 Resolution), capillary electrophoresis (CE) (Sebia Capillarys 2 Flex Piercing) and mass spectrometry (MS) (Waters) were used for variant detection; Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) were used for DNA analysis; HbA1c was measured with cation exchange HPLC (Bio-Rad Variant™ II; Arkray Adams HA-8180V; Tosoh HLC-723 G7), CE (Sebia Capillarys 2 Flex Piercing), boronate affinity HPLC (Trinity Biotech Hb9210 Premier), immunoassay (Cobas c501 Tina-quant HbA1c Gen. 3; Nihon Kohden CHM-4100 Celltac chemi HbA1c HA-411V) and enzymatic assay (Abbott Architect c 8000 HbA1c). Results Hb Yamagata [β132(H10)Lys→Asn; (HBB: c.399A>T)] was identified in the proband by MS after the observation of an abnormal peak in HPLC and CE. A mosaic expression of this variant was detected by NGS (mutant: 8%; wild type: 92%), after negative results in Sanger sequencing. Hb Yamagata interfered with HbA1c measurements by cation exchange HPLC and CE whereas immuno and enzymatic assay values showed good agreement with boronate affinity HPLC measurement. Conclusions A mosaicism of Hb Yamagata was found in a patient with altered HbA1c values. This rare gene variant was detected only by advanced technologies as MS and NGS. The variant interfered with common HbA1c determination methods.