| Objective To identify common types of hemoglobin variants during glycosylated hemoglobin(HbA1c)assays and compare the consistency of glycosylated hemoglobin results of common hemoglobin variant specimens by ion exchange high-pressure liquid chromatography(IE-HPLC)and capillary electrophoresis methods.The glycosylation levels of HbA,Hb G-Coushatta and Hb G-Taipei hemoglobin chains were detected separately by liquid chromatography tandem mass spectrometry(LC-MS/MS),and the differences in chain glycation levels as well as overall hemoglobin glycation levels were compared among the three.Methods A total of 149 clinical residual samples with abnormal separation peaks in HbA1c detected by IE-HPLC method in Peking Union Medical College Hospital from January 2017 to November 2019 were collected,and glycated hemoglobin was detected by capillary electrophoresis method to initially identify the presence of hemoglobin variants.The hemoglobin variants were reconfirmed using hemoglobin electrophoresis,and the ratio of hemoglobin variants to normal hemoglobin content was also obtained.Finally,Sanger sequencing was used to sequence the genes of the specimens containing hemoglobin variants to determine the type of hemoglobin variants.The concordance between IE-HPLC and capillary electrophoresis methods was compared in detecting the HbAlc assay results of hemoglobin variants Hb G-Coushatta heterozygote,HbG-Taipei heterozygote and HbE heterozygote(chain hemoglobin variants specified in Chapter 2).The mean glucose levels(eAG)estimated from the HbA1c assay results of the two methods were calculated separately,and the correlation between eAG and patients’ fasting plasma glucose(FPG)during the same period was explored.To establish a method for the detection of glycosylation levels of hemoglobin chains by LC-MS/MS.The erythrocytes in the hemoglobin-containing variant specimens were washed,separated,and disrupted using deionized water,tetrachloroethylene was added and shaken,and the supernatant was centrifuged for the assay.The glycosylation ratios of normal and variant hemoglobin chains were detected by electrospray ionization in positive ionization mode and in multiple ion reaction monitoring mode.The precision of the method was evaluated using Bio-Rad Lyphochek? Diabetes Control glycosylated hemoglobin high and low levels of quality controls.Each level of QC was measured five times per day and repeated for five days,and intra-batch and total variation was calculated for each level.The linear range of the method was evaluated using one low concentration fresh whole blood specimen(L)and one high concentration fresh whole blood specimen(H),prepared in five concentration gradients of mixed whole blood specimens at 1.0L,0.75L:0.25H,0.5L:0.5H,0.25L:0.75H,and 1.0H.Their expected chain glycation levels were calculated according to Eq:x=β glycatedL×VL×HbL+β glycatedH×VH×HbH/VL×HbL+VH×HbH.The correlation between the chain glycosylation levels detected by LC-MS/MS and HbAlc was evaluated using the primary reference substance pcal-2014 designated by the IFCC glycated hemoglobin reference laboratory network and 20 fresh whole blood specimens,respectively.The glycosylation levels of the most common Hb G-Coushatta and Hb GTaipei hemoglobin variant heterozygous specimens from our laboratory were examined,and the differences in glycosylation levels of β G-Coushatta,β G-Taipei and β Ain the variants were compared.The overall glycosylation levels of Hb G-Coushatta and Hb GTaipei hemoglobin variant heterozygotes were calculated and compared with the overall glycosylation differences between them and HbAA,based on the conclusion reported in previous studies that the percentage of glycosylation of α chains was about 64%of that of β chains.Results A total of 149 specimens with suspected hemoglobin variants were found,and DNA sequence analysis of the hemoglobin chains showed that there were 128 cases of chain hemoglobin variants,all of which were heterozygous mutations.The most common chain hemoglobin variants were Hb G-Coushatta and Hb G-Taipei,accounting for 31.3%and 33.3%,respectively.chain hemoglobin variants such as HbE,HbS,and HbJ-Bangkok were less common,accounting for a total of 21.3%.Among the remaining 21 specimens,DNA sequencing failed in 9 cases(bimodal and disordered peaks)and no gene mutations were found in 12 cases,which were likely to be hemoglobin variants generated by chain gene mutations.In the two HbAlc assays,IE-HPLC method and capillary electrophoresis,the Hb GCoushatta patient group test statistic Z=-1.73,P=0.08>0.05,,the assay results were not statistically different.The HbAlc test results of the two assays were significantly correlated,r=0.81,P<0.001 by the two-sided Spearman test.The HbA1c test statistic in the Hb G-Taipei patient group Z=-0.499,P=0.618>0.05,and there was no statistical difference between the results of the two assays;the HbAlc test results of the two assays were significantly correlated,r=0.83,P<0.001 by bilateral Spearman test.In the group of patients with HbE variants,t=6.965,P<0.0001 in the two-sided paired ttest,and there was a statistically significant difference between the two assays.The correlation coefficient of HbAlc results between the two assays was r=0.822,P<0.001 in the two-sided significance test,and there was a correlation.Applying Bland Altman plots to analyze the deviation of HbA1c assay results between IEHPLC and capillary electrophoresis methods,8.7%(4/46)and 4.0%(2/50)of samples in the Hb G-Coushatta and Hb G-Taipei variant heterozygous groups,respectively,fell within the 95%agreement limits of IE-HPLC and capillary electrophoresis methods.Within the limits of agreement of the two methods,the absolute maximum value of the difference was 0.84%,and the mean values of the differences were-0.10%and 0.04%,respectively.The linear regression equation between HbA1c and assessed mean glucose derived from the ADAG study was applied:assessed mean glucose eAG(mmol/L)=1.5944 × A1C2.5944 to calculate the assessed mean glucose predicted by HbAlc measured by IE-HPLC and capillary electrophoresis methods,respectively.The results of the correlation between the assessed mean glucose(eAG)and fasting glucose(FPG)predicted by both methods using Spearman analysis showed that the Hb G-Coushatta variant affected both HbAlc assay methods to a similar extent,with r=0.677 and 0.679 correlation coefficients between eAG and FPG,respectively;the Hb G-Taipei variant affected both The Hb G-Taipei variant had a greater impact on the two HbAlc assays,with r=0.698 and 0.787 correlation coefficients between eAG and FPG,respectively.The results of LC-MS/MS detection of glycosylation levels of the chains showed that βG-Coushatta glycosylation level=1.36±0.11 βA glycosylation level(95%CI 1.311.39),β G-Taipei glycosylation level=0.1.34±0.12 β A glycosylation level(95%CI 1.29-1.38).Hb AG-Coushatta heterozygotes and Hb AG-Taipei heterozygotes had overall glycosylation levels 1.09 and 1.08 times higher than the normal hemoglobin HbAA glycosylation levels,respectively.Conclusion 1.the common chain hemoglobin variants in glycosylated hemoglobin specimens detected in our laboratory were Hb G-Coushatta and Hb G-Taipei.2.the effects of Hb G-Coushatta and Hb G-Taipei variants on the detection of HbAlc by IE-HPLC method and capillary electrophoresis were similar.3.an LC-MS/MS method was established to detect hemoglobin glycosylation level by LC-MS/MS,and the accuracy and reproducibility of the method meet the needs of related studies.The overall glycosylation levels of patients with the common hemoglobin variants Hb G-Coushatta and Hb G-Taipei heterozygotes in the northern region of China were 1.09 and 1.08 times the glycosylation levels of normal hemoglobin HbAA,respectively,with similar glycosylation rates.Without considering the erythrocyte lifespan,this group of patients can monitor the mean glucose level using HbA1c. |
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