Hemoglobin A2 and Heterogeneous Diagnostic Relevance Observed in Eight New Variants of the Delta Globin Gene

Background: Hemoglobin A (Hb A) (α2β2) in the normal adult subject constitutes 96–98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the β-thalassemia carrier. Methods: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. Results: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1–2% during the screening tests in our laboratories. Conclusions: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

Screening for hemoglobinopathy with capillary electrophoresis in adult patients

Hemoglobinopathy screening is frequently needed in adult patients, including prenatal carrier screen, workup of unexplained anemia, and bone marrow donor and recipient screening. However, the preferred test method for screening of hemoglobinopathy is not well established due to limited guidance from professional societies. American College of Obstetricians and Gynecologists’ Committee on Genetics recommended hemoglobin electrophoresis as the screening method of hemoglobinopathy in pregnancy; nevertheless, electrophoresis employs various methodologies, including acid gel electrophoresis, alkaline gel electrophoresis, and capillary electrophoresis (CE) with alkaline buffer. For other adult patient populations who need hemoglobinopathy screening, no clear guidelines dictate the method of choice. A previous study has shown that CE captures major hemoglobinopathies with comparable performance to high-performance liquid chromatography (HPLC) in pediatric patients but no study has investigated using CE alone in adult patient screening. In this retrospective study, we evaluated the utility of CE as a screening method to rule out clinically significant hemoglobin variants. During eight months, 312 adult patients without previously identified hemoglobin variants had hemoglobinopathy screening performed using a comprehensive testing algorithm. This cascade algorithm screens for hemoglobinopathy using both CE (Capillarys, Sebia, Paris, France) and HPLC (laboratory-developed test) with reflex to more advanced variant identification such as mass spectrometry and genetic analyses. Categories of abnormal findings were reviewed to determine if hemoglobinopathy can be identified by using CE only. The patient population mainly consists of pregnant women and anemic patients with hematologic malignancies with an average age of 42. Out of the 312 screened patients, 47 had abnormal results. The most frequent condition was elevated hemoglobin F (N=25) ranging 2-5% seen in leukemia patients on chemotherapy attributed to bone marrow stress. Eight cases of beta plus thalassemia (featuring hemoglobin A2 >4%) and 3 cases of hemoglobin C trait were identified in patients with little to mild clinical manifestations (red blood cell indices suggesting anemia). Decreased hemoglobin A2 fraction was observed in 7 patients, and potential causes were alpha thalassemia or iron deficiency. Other less common hemoglobinopathies included heterozygote A2 prime (N=3, a benign delta chain variant that migrates separately from hemoglobin A2 on CE) and hemoglobin G-Philadelphia (N=1). All of the abnormal results are identifiable by CE alone, although HPLC and more advanced methods help confirm the diagnosis. Our study shows that CE as the first line of screening method would rule out major hemoglobinopathies in adults. There have been reports that rare but clinically significant hemoglobin variants like hemoglobin Malmo may not be detected by CE, and therefore, certain pre-test probability factors need to be considered when testing for hemoglobinopathies, such as race/ethnicity background, family history, red blood cell indices, and iron deficiency status.

Difference in hemoglobin %A2 between homozygous hemoglobin A and hemoglobin S-trait patients as measured by capillary electrophoresis

Introduction/Objective Quantitation by high-performance liquid chromatography (HPLC) of hemoglobin %A2 is often not used in evaluation for thalassemia of hemoglobin S-trait patients, due to analytical interference from glycated hemoglobin S1d to increase %A2. In contrast, an increase in %A2 for S-trait when measured by capillary electrophoresis (CE) has been reported, without known analytical interference. This observation has not been re- evaluated in modern versions of CE, however. For validation exercises associated with startup of CE at our institution, we compared distributions of %A2 among A patients and S-trait patients using Sebia “Capillarys® 2” CE. Methods/Case Report %A2 is provided in two Sebia “Capillarys® 2” methods: analysis of A1c (method 1, M1) and analysis of hemoglobin variants (method 2, M2). To minimize effect of potential preselection for thalassemia among M2 samples, we first evaluated distributions of %A2 for A and S-trait among M1 samples. We then evaluated correlation of A2 measurements between M1 and M2. Statistical analyses were conducted using R programming. Results (if a Case Study enter NA) Using M1, %A2 for S-trait patients (2.61±0.31%, n=116) was higher than for A patients (2.11±0.27%, n=108) (p<0.001), with difference=0.42-0.57 %A2 (95% confidence interval, CI). %A2 by M1 was consistently less than %A2 by M2, for both A and S-trait (p>0.25): for A, M1/M2=0.89±0.05 (n=35); for S-trait, M1/M2=0.88±0.05 (n=32). Decreased %A2 by M1 compared to M2 may in part be due to separation in M1 of a glycated form of A2. Using M2, %A2 for S-trait patients (3.05±0.29%, n=32) was higher than for A patients (2.41±0.29%, n=35) (p<0.001), with difference=0.50-0.76 %A2 (CI). M2 results were consistent with M1 data when combined with the observed M1/M2 ratios. Conclusion Results suggest a physiological increase in %A2 in S-trait patients compared to A patients, not likely to be attributable to thalassemia. The average increase is ~0.6 %A2 for hemoglobin variant analysis by CE.