False HbA1cValue due to a Rare Variant of Hemoglobin J-Cubujuqui.

BACKGROUND: Hemoglobin (Hb) J-Cubujuqui is a rare Hb variant, and reports about it are very limited. There are no descriptions that it affects the results of glycated Hb. METHODS: In this study, we describe a rare variant discovered during newborn screening. Both high-performance liquid chromatography (HPLC) and capillary electrophoresis for hemoglobin analysis displayed abnormal peaks. The Hb variant was confirmed by Sanger sequencing. RESULTS: The pedigree study shows the variant was inherited from the newborn's father. His fasting blood glucose (FBG) level was 5.5 mmol/L. HbA1c measured by HPLC was falsely low in her father (2.41%), whereas that measured by immunoassay was normal (5.11%). Sanger sequencing revealed a heterozygous mutation (CGT˃AGT) at amino acid position 141 of the α1 gene, corresponding to Hb J-Cubujuqui [α1 141(HC3) Arg→Ser (CGT˃AGT); HBA1:c.424C˃A (or HBA2)]. CONCLUSIONS: This is the first report that Hb J-Cubujuqui interferes with the measurement of HbA1cand prompts clinicians to pay attention to the accuracy of glycated Hb results.

Evaluation of the Interference of Hemoglobin Variant J-Bangkok on Glycated Hemoglobin (HbA1c) Measurement by Five Different Methods.

Objective The interference of the hemoglobin variant (Hb J-Bangkok) was evaluated on 4 different glycated hemoglobin assays and compared with a reference immuno assay. Methods An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of Hb J-Bangkok caused a statistically significant difference in HbA1c results compared with a reference immuno assay. Statistical analysis was performed on the difference of the estimated average glucose calculated from HbA1c values and fasting plasma glucose in the Hb J-Bangkok variant group using the different detection systems. Deming regression analysis was used to determinate whether Hb J-Bangkok had a significant interference on HbA1c results using an HbA1c±10% relative bias at 6% and 9% HbA1c as evaluation limits. Results Turbidimetric inhibition immunoassay method, and enzymatic methods were not affected by Hb J-Bangkok. However, Hb J-Bangkok showed statistically significant interference to the two ion-exchange high-performance liquid chromatography methods. Conclusion When performing HbA1c tests, clinical laboratory personnel should identify the Hb variant and select the appropriate methods or use alternative indicators.

Hemoglobin Variants Acquired Post-Exchange Transfusion in Pediatric Sickle Cell Disease (SCD) Patients.

BACKGROUND: Many SCD patients receive chronic transfusions for prevention or treatment of disease related complications. Complications of the chronic transfusion noted in these patients include allergic reactions, transfusion transmitted infections, iron overload, and alloantibody formation. Even though hemoglobin (Hb) variants are prevalent in the general population, reports of transfusion-acquired Hb variants are rare. We performed a retrospective analysis on all SCD patients who underwent red cell exchange (RBCEx) transfusions at our institution during 2011-2013 to identify the presence of Hb variants acquired as a result of RBCEx. RESULTS: We found 66 occurrences of acquired Hb variants in 30 SCD patients during the period examined. The most commonly acquired Hb variant was Hemoglobin C (HbC) (64/66 occurrences). More than half of the patients (19/30) acquired HbC on multiple occasions (2-6 times). One patient acquired HbJ and another patient acquired HbD/G in addition to HbC. The segments from donor units were available in some of these cases and hemoglobin electrophoresis (HBE) was performed to confirm the presence of the variant Hb in the donor segments corresponding to that seen on the post-RBCEx sample. CONCLUSIONS: Heterozygous donors are asymptomatic and show no abnormalities during donor screening. Since HBE is not routinely performed on the donor specimen, it may go unrecognized until the post-transfusion recipient results pose diagnostic difficulties. There are no definitive guidelines on deferring these donors; hence one should be cognizant of these findings to prevent misdiagnosis. In a population where HbS negative blood is routinely requested, the effect of other Hb variants remains unknown. None of the patients in our study showed any adverse events due to the acquired Hb variants; however, this is of special concern in the pediatric population where a single RBC unit can contribute a significant portion of the exchanged blood volume. Additionally, donor centers may need mechanisms to confirm the findings and counsel the donors as needed.

Ion mobility augments the utility of mass spectrometry in the identification of human hemoglobin variants.

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.

Conundrum of elevated HbA1C and hypoglycemia-a rare cause.

A white diabetic patient on insulin therapy presented with recurrent hypoglycemia despite very high glycosylated hemoglobin (HbA1c) values. Hemoglobin (Hb) variants, chemically modified Hb, and abnormalities of red cell turnover cause errors in HbA1c measurement. Widely prevalent Hb variants affecting HbA1c estimation include HbS and HbC in African Americans, HbE in southeast Asians, and carbamyl-Hb in uremic patients. In addition, there are at least 893 other Hb variants as of 2005, many of which affect HbA1c estimation. HbA1c values are also affected by methodology of estimation. Our patient had HbJ, which is rare amongst whites. The relationship between HbA1c values and mean plasma glucose allows estimation of expected HbA1c. Significant discrepancy between expected and measured HbA1c should be evaluated. Considering Hb variants, evaluating for the same and estimating HbA1c with the appropriate method under such circumstances are described. Numerous new or rare Hb variants will be diagnosed if suspicion is appropriately entertained.

Beta thalassemia IVS-I-5(G-->C) heterozygosity masked by the presence of HbJ-Meerut in a Dutch-Indian patient.

We describe the genotype/phenotype correlation in a 35 year old anemic female referred to our laboratory because a fast eluting minor fraction on HPLC, mild hemolysis and hematological parameters suggesting a Thalassemia trait, eventually in combination with iron depletion. Direct sequencing of the alpha globin genes revealed heterozygosity for HbJ-Meerut, a Glu-->Ala substitution at residue 120 not justifying the hematological parameters. No other point mutations were found on the alpha genes and Gap-PCR excluded the 6 common deletion defects. Direct sequencing of the beta-globin genes revealed the IVS-I-5 (G-->C) transversion in absence of the elevated HbA2 levels usually measured in carriers of this beta-Thalassemia mutation. The HbA2 tetramer in the presence of HbJ-Meerut divides in two parts. One alphaN2/delta2 migrating on the right spot on HPLC. The other alphaJ2/delta2 migrating under the HbA fraction. Classic alkaline electrophoresis and the modern capillary electrophoresis CE showed these two tetramers and the reduction of the elevated HbA2 level of the beta-Thalassemia trait by at least 20% due to HbA2 Meerut.

Molecular and haematological characterization of compound Hb E/Hb Pyrgos and Hb E/Hb J-Bangkok in Thai patients.

We describe haematological and DNA characterization of haemoglobinopathies in Thai adolescents caused by compound heterozygosities for Hb E [beta26(B8) Glu-Lys] and two other beta-globin chain variants, Hb Pyrgos [beta83(EF7) Gly-Asp] and Hb J Bangkok [beta56(D7) Gly-Asp]. Hb analysis demonstrated that although these two beta-chain variants have separated elution profiles on liquid chromatography-based Hb analysis, they have similar alkaline electrophoretic mobilities on cellulose acetate electrophoresis. Haematological data associated with these two previously undescribed conditions were compared with those of pure carriers of the variants found in other unrelated Thai individuals. beta-Globin gene haplotypes linked to these two beta-chain variants and a simple DNA testing based on multiplex allele-specific polymerized chain reaction for differential diagnosis are presented.

Analytical evaluation of the Bio-Rad variant II automated HbA(1C) analyzer.

OBJECTIVES: To evaluate the analytical performance of the Bio-Rad Variant II HbA(1C) analyzer (a completely automated system for the quantification of glycohemoglobin [HbA(1C)] in blood). DESIGN AND METHODS: The analytical parameters of precision, linearity and analytical range were assessed and HbA(1C) results from the Variant II were compared to HbA(1C) results from the Bio-Rad Variant (a method certified by the National Glycohemoglobin Standardization Program). The effect of a variety of hemoglobin variants on HbA(1C) obtained on the system was investigated. RESULTS: Total imprecision was less than 5% and the results compared well with those from an established method. The method has a wide analytical range with no carryover between specimens. CONCLUSION: The HbA(1C) method on the Variant II gives acceptable analytical performance.

A patient with a hemoglobin variant (Hb JLome) unexpectedly detected by HPLC for glycated hemoglobin (Hb A1c).

A rare hemoglobin variant, Hb JLome, was identified by chance in a male patient with diabetes mellitus (DM). The patient had no evidence of anemia or hemolysis. However, when his glycated hemoglobin (Hb A1c) was examined by high-performance liquid chromatography (HPLC) to assess the state of his DM, an abnormal Hb was unexpectedly detected on the chromatogram. The morphology of the red blood cells was normal. A fast-moving band as well as a normally moving Hb band, of roughly equal intensities, were observed by cellulose acetate membrane electrophoresis. The oxygen equilibrium curve was essentially normal (P50 = 3.59 kPa). In other words, the ability of the patient's Hb to carry oxygen was nearly the same as that of typical Hb A. The stability of his Hb in isopropanol was normal, and all the functions of his Hb that were tested were essentially normal. The identity of the abnormal Hb was finally determined, by sequencing the globin gene, to be Hb JLome, which is produced by a point mutation changing AAG to AAC at the 59th codon in exon 2 of the Hb beta chain. As previously reported, replacing the beta 59 lysine with asparagine does not affect the function of Hb or the red blood cells. There have been only five documented cases of Hb JLome in Japan. Interestingly, all these cases are from Kyushu Island. When an abnormal chromatogram for Hb A1c is unexpectedly obtained, it is worthwhile searching for an abnormal Hb, even if there are no signs that suggest its existence, such as anemia, hemolysis, erythrocytosis, or cyanosis.

Resistance to recombinant erythropoietin in a hemodialysis patient with heterozygous hemoglobinopathy J-Meinung.

The mechanism of resistance to recombinant human erythropoietin (EPO) in hemodialysis patients with hemoglobinopathy is not yet fully understood. Poor responses to EPO have been reported in anemic dialysis patients with sickle cell disease and thalassemia. We present the first case of a hemodialysis patient with EPO resistance and hemoglobin J-Meinung, which is initially found by hemoglobin electrophoresis and finally proven by molecular genetic analysis. Additionally, the patient was diagnosed as having chronic active hemolysis with hallmarks of splenomegaly, an increased serum bilirubin and reticulocyte index, and a reduced haptoglobin level. We discuss the possible mechanisms and proper treatment options in such patients with a poor response to EPO.

[A case of abnormal hemoglobin (HbJ Cape Town) with high serum levels of HbAlc].

We describe a case of hemoglobinopathy detected on admission for examination for high blood glucose levels and abnormal liver function. In 1991, it was pointed out that he had postprandial hyperglycemia. In 1994, at age 60, he had lassitude and anorexia. He was admitted to our hospital on the suspicion of diabetes mellitus and liver disease. Glycosylated hemoglobin levels was very high, but the 75 gram oral glucose tolerance test result was within the normal range. After abstinence from alcohol, his glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and gamma glutamyl traspeptidase became normal. Diabetes was excluded and abnormal hemoglobinopathy had been suspected. We analyzed his abnormal hemoglobin. In isoelectro-phoresis a fast moving variant was detected suggesting the presence of abnormal hemoglobin at the cathode. We fractionated hemolytic globin by CM-chromatography and detected an abnormal peak before the alpha chain band. Subsequently, we sequenced isolated abnormal alpha chain and detected the substitution of Ariginine for Glutamamine at position 92 (Hb J Cape Town). So far he has not demonstrated any symptoms or signs of HbJ Cape Town. Hemoglobinopathy is not uncommon in aged people.

Hemoglobin variant detection from dried blood specimens by high performance liquid chromatography.

The convenience of dried blood filter paper specimens for genetic screening programs has prompted us to test the stability of these specimens for hemoglobin identification by cation exchange high performance liquid chromatography. This report shows that identification of Hb AA, Hb AF, Hb AS, Hb FAS, Hb AJ, Hb FJ, Hb EF, and Hb SS can be achieved by high performance liquid chromatography even after six weeks of storage at room temperature. Also, accurate hemoglobin quantitation can be obtained from the same samples within three weeks of storage at room temperature. The combination of dried blood samples and high performance liquid chromatography provides an accurate system to screen for hemoglobinopathies, even after long periods of sample storage at ambient conditions.

Rare beta chain hemoglobin variants found in Swedish patients during HBA1c analysis.

Here we report the occurrence of five different beta chain hemoglobin variants not previously described in Sweden. The variants were found during quantification of HbA1c using ion exchange high performance liquid chromatography (HPLC) or isoelectrofocusing. Samples were examined either at protein level by separation of globin chains on C8 reversed phase HPLC, digestion with trypsin or lysylendopeptidase and separation of peptides by C18 reversed phase HPLC, or at DNA level by direct nucleotide sequencing of double-stranded DNA fragments amplified from exon 1 + 2 of the beta-globin gene. The variants were: Hb Raleigh [beta 1 (NA1)Val-->Ac-Ala], Hb J-Baltimore [beta 16(A13)Gly-->Asp], Hb Tacoma [beta 30(B12)Arg-->Ser], Hb K-Ibadan [beta 46(CD5)Gly-->Glu], and Hb Fukuyama [beta 77(EF1)His-->Tyr]. Hb Tacoma, Hb K-Ibadan, and Hb Fukuyama were slightly unstable in the isopropanol test, but no signs of hemolysis were found in the patients who all had normal hematological findings.

Enzyme immunoassay for the identification of hemoglobin variants.

We have prepared monospecific antibodies to Hbs D-Los Angeles, J-Baltimore, O-Arab and J-Paris-I and developed an enzyme immunoassay (ELISA) for their identification in hemolysates. Hbs in adult or cord blood hemolysates were coated to the wells of microtiter plates and reacted with the appropriate antisera followed by the detection system which contains anti-rabbit IgG/peroxidase conjugate and the substrate tetramethylbenzidine. Sixty-nine samples were tentatively considered to contain the above hemoglobin variants by isoelectrofocusing and the identity of 83% of them was confirmed by ELISA. Some of the non-reacting hemolysates were shown by amino acid sequence analysis to contain Hbs Korle-Bu, D-Ibadan, G-Copenhagen and the new variant Chandigarh. This ELISA offers specificity and simplicity for the confirmatory identification of hemoglobin variants.

Structural analysis of protein variants by mass spectrometry: characterization of haemoglobin providence using a grand-scale mass spectrometer.

A grand-scale mass spectrometer with high mass resolution and high transmission was employed for the analysis of haemoglobin variant. Two variants were isolated from a haemolysate by chromatography. Secondary ion mass spectrometry of complex peptide mixtures derived from these variants precisely determined the molecular weight of abnormal peptides. The molecular weight, 2857.4 and 2858.4, indicated the amino acid substitutions of asparagine and aspartic acid, respectively, for lysine at position 82 of beta globin chain. The mutations had been reported in haemoglobin Providence.