Hemoglobin C Variant Affecting Glycated Hemoglobin Test Results: A Rare Case Report
摘要: 血红蛋白（hemoglobin, Hb）变异体是影响糖化血红蛋白A1C（glycated hemoglobin, A1C）检测结果的常见因素。不同的血红蛋白变异体对检测结果的影响各不相同。本文首次报道了1例中国人中血红蛋白C（Hb C）对A1C测定结果的影响。本研究分别使用高效液相色谱法（high performance liquid chromatography, HPLC）和毛细管电泳法检测A1C；采用血红蛋白电泳识别血红蛋白变异体；Hb测序确定β链上的突变位点。HPLC检测A1C结果降低，可通过电泳法纠正，但电泳图谱仍存在异常峰。Hb电泳结果提示存在Hb变异，测序结果显示为Hb C。因此，我们建议对A1C常规开展解释性报告模式，对每份报告中的检测方法进行注释，并对图谱进行分析，避免单一检验结果影响临床决策。Abstract: Hemoglobin (Hb) variants are common factors that affect the results of glycosylated hemoglobin (A1C) tests. Hemoglobin variants react differently to different testing methods. Herein, we presented the first ever report of the effect of hemoglobin C (Hb C) on the test results of A1C in the Chinese population. High performance liquid chromatography (HPLC) and capillary electrophoresis were performed to measure A1C. Hemoglobin electrophoresis was conducted to identify the hemoglobin variants. Hb sequencing was performed to determine the mutation sites on the β chain. HPLC showed decreased A1C results, which could be corrected by electrophoresis, but the electrophoresis graph still showed abnormal peaks. The hemoglobin electrophoresis results suggested that there were hemoglobin variants, which hemoglobin sequencing results revealed to be Hb C. Uncommon variations in a specific population tend to be overlooked. To avoid clinical decision-making being affected by the results of a single test, we recommend that an explanatory reporting model be routinely adopted for A1C tests so that all reports always contain explanatory notes for the testing methodology and analysis of the graphs.
图 1 使用不同方法测定的A1C结果
Figure 1. A1C results determined with different methods
A: HPLC showed an extra band (black band); B: capillary electrophoresis showed an abnormal band in A2; C: Hb variant identified by hemoglobin electrophoresis (fraction); D: gene sequencing of the patient showed Hb C mutation sites. * The coincidence of the bands of A2 and Hb variant.
表 1 A1C检验报告关键要素
Table 1. Key reporting elements for A1C
Test Results Methodology Notes Quantitation %, mmol/mol HPLC, electrophoresis, immunoassay or ion-exchange For example, comigration of multiple bands and interferences due to hemoglobin variants Abnormal band(s) presence Yes/no/equivocal Variant mode Yes/no Previous history Yes/no For example, describing changes in the quantity of plasma glucose in comparison with what it was before Recommendation Follow-up testing For example, glycated albumin recommended (if A1C does not reflect the glucose)
 American Diabetes Association. 2. Classification and diagnosis of diabetes: standards of medical care in diabetes-2021. Diabetes Care,2021,44(Suppl 1): S15–S33. doi: 10.2337/dc21-S002  中华医学会糖尿病学分会. 中国2型糖尿病防治指南(2020年版). 中华内分泌代谢杂志,2021,37(4): 311–398. doi: 10.3760/cma.j.cn115791-20210221-00095  JOHN W G. Global standardisation of haemoglobin A(1c) using metrological principles. Clin Biochem,2012,45(13/14): 1048–1050. doi: 10.1016/j.clinbiochem.2012.07.105  ROHLFING C, HANSON S, WEYKAMP C, et al. Effects of hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by twelve methods. Clin Chim Acta,2016,455: 80–83. doi: 10.1016/j.cca.2016.01.031  XIA Y, YE Y L, TIAN Y H, et al. Molecular epidemiological investigation of Thalassemia in the Chengdu Region, Sichuan Province, Southwest China. Hemoglobin,2015,39(6): 393–397. doi: 10.3109/03630269.2015.1070733  WANG W D, HU F, ZHOU D H, et al. Thalassaemia in China. Blood Rev,2023,17: 101074. doi: 10.1038/s41598-017-00967-2  LI Q, XIAO Y, SHAH A D, et al. Visual inspection of chromatograms assists interpretation of HbA1c: a case report. Diabetes Care,2018,41(8): 1829–1830. doi: 10.2337/dc18-0378  GIARDINE B, JOLY P, PISSARD S, et al. Clinically relevant updates of the HbVar database of human hemoglobin variants and thalassemia mutations. Nucleic Acids Res,2021,49(D1): 1192–1196. doi: 10.1093/nar/gkaa959  PIEL F, HOWES R, PATIL A, et al. The distribution of haemoglobin C and its prevalence in newborns in Africa. Sci Rep,2013,3: 1–18. doi: 10.1038/srep01671  HOELZEL W, WEYKAMP C, JEPPSSON J O, et al. IFCC reference system for measurement of hemoglobin A1c in human blood and the national standardization schemes in the United States, Japan, and Sweden: a method-comparison study. Clin Chem,2004,50(1): 166–174. doi: 10.1373/clinchem.2003.024802  ZENG Y, HE H, ZHOU J, et al. The association and discordance between glycated hemoglobin A1c and glycated albumin, assessed using a blend of multiple linear regression and random forest regression. Clin Chim Acta,2020,506: 44–49. doi: 10.1016/j.cca.2020.03.019  ROHLFING C, HANSON S, ESTEY MP, et al. Evaluation of interference from hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by fifteen methods. Clin Chim Acta,2021,522: 31–35. doi: 10.1016/j.cca.2021.07.027  ZHAO Z, BASILIO J, HANSON S, et al. Evaluation of hemoglobin A1c measurement by Capillarys 2 electrophoresis for detection of abnormal glucose tolerance in African immigrants to the United States. Clinica Chimica Acta,2015,446: 54–60. doi: 10.1016/j.cca.2015.03.025  MOSCA A, PALEARI R, HARTEVELD C. A roadmap for the standardization of hemoglobin A. Clin Chim Acta,2021,512: 185–190. doi: 10.1016/j.cca.2020.11.008  BERAL L, LEMONNE N, ROMANA M, et al. Proliferative retinopathy and maculopathy are two independent conditions in sickle cell disease: is there a role of blood rheology? Clin Hemorheol Microcirc,2019,71(3): 337–345. doi: 10.3233/CH-180412