TY - JOUR
AU - Dalton, R, Neil
AB - Abstract Background: Peptide-based analysis of whole blood using electrospray tandem mass spectrometry (MSMS) in multiple reaction monitoring (MRM) mode enables rapid detection and sequence confirmation of clinically significant hemoglobin (Hb) variants. We applied a similar, quantitative approach to the measurement of δ:β-globin peptide ratios as potential surrogate markers of HbA2, a biomarker used in population screening for β-thalassemia trait. Methods: We studied 163 blood samples with normal HbA2 (%), 105 with increased HbA2, 43 with δ-chain variants, and 8 with Hb Lepore. All were tested by HPLC. The samples were also incubated with trypsin for 30 min at 37 °C for MSMS with flow injection analysis. MRMs for the δ- (T2, T3, and T14) and β- (T2, T3, and T13) globin tryptic peptides were acquired for 1 min, and δ:β peptide ratios were calculated. We used HPLC and MSMS to analyze 26 paired whole blood and dried blood spot samples after storage for 1, 8, and 29 days. Results: Within- and between-assay imprecision values (CVs) were <6.1% and <8.4%, respectively, for the δ:β peptide ratios. Digests were stable at 10 °C for 6 days. Significant correlations (P <0.0001) between MSMS δ:β-globin peptide ratios and HPLC HbA2 allowed differentiation between increased HbA2 concentrations and concentrations within the reference interval and identification of Hb Lepore. This differentiation was repeatable by MSMS, but not by HPLC, after blood spot samples had been stored for 1 month. Conclusion: This study validates the quantitative δ:β-globin peptide ratio as a surrogate marker of HbA2 and demonstrates the potential of rapid peptide-based MSMS for multiplexed, high-throughput protein biomarker characterization and quantification. We recently described a rapid and specific tandem mass spectrometry (MSMS)1 method using multiple reaction monitoring (MRM) targeting of tryptic peptides for the simultaneous detection of the clinically significant β-chain hemoglobinopathies: hemoglobin (Hb) S, HbC, HbE, HbDPunjab, and HbOArab (1). This multiplex mutation detection and confirmation approach to the diagnosis of hemoglobinopathies is suitable for newborn screening. However, antenatal screening requires β-thalassemia trait detection, for which HbA2 is the validated biomarker (2). HbA2 was identified more than 50 years ago, the 2nd Hb in the blood of healthy adults (3), representing ∼3% of total Hb. Later studies demonstrated that HbA2 was increased in thalassemia trait (4) and that HbA2 comprised 2 α- and 2 δ-globin chains, leading Ingram and Stretton to postulate that, because β-globin synthesis was decreased, increase of HbA2 was likely in β-thalassemia (5). Such an increase is not specific, because increased HbA2 concentrations have also been reported in other conditions (6)(7)(8)(9). In conjunction with erythrocyte indices, however, HbA2 (%) remains the standard biomarker for β-thalassemia screening in adults. Thalassemia is also observed with Hb Lepore (10), a series of δ-β fusion proteins (11). Hb Lepore trait is characterized by decreased erythrocyte indices and, by HPLC, of undetectable HbA2 and a variant (Hb Lepore) at 10%–15% of total Hb. Detection is essential in population screening programs since interaction with sickle and β-thalassemia traits produces clinically significant morbidity. Initially, population screening programs were restricted to the use of erythrocyte indices (2), but screening for HbA2 has been facilitated by use of automated HPLC of intact Hbs. Although MSMS appears an unlikely technique for accurately measuring HbA2, an α2δ2 tetramer, we postulated that the relative proportion of δ- and β-globins should be equivalent to the proportion of HbA2. Our aim was to extend our MSMS approach to include the quantitative measurement of the relative amounts of δ- and β-globins to determine the utility of the ratio as a surrogate biomarker of HbA2 in the detection of β-thalassemia trait and Hb Lepore. Materials and Methods msms strategy The β- and δ-chains of Hb exhibit strong sequence homology. Tryptic digestion of the β-chain produces 15 defined peptides (12) and the δ-chain produces 16 peptides (T1–16), differing from β in T2, T3, T5, T10, T12, T13, and T14. In the T2 peptide, amino acids 9–17 in both the β- and δ-chains, the respective sequences are SAVTALWGK, monoisotopic mass 931.5 Da, and TAVNALWGK, monoisotopic mass 958.5 Da. In MRM mode (1), the respective observed T2 [M + 2H]2+ ions, mass-to-charge ratio (m/z) β, 466.8, and δ, 480.3, were selected and fragmented, and the most informative singly-charged peptide fragments VTALWGK, 675.4, and VNALWGK, 688.4, targeted. The theoretical advantage of measuring the δ T2 peptide is that any Lepore variant will have a high signal. The disadvantage is that the most common δ-chain variant HbA′2 (Hb B2) occurs at position 16 (13). To ensure detection of β-thalassemia trait in the presence of a concomitant δ-chain variant, we included the next informative peptide, T3, β sequence VNVDEVGGEALGR, monoisotopic mass 1313.7 Da, and δ sequence VNVDAVGGEALGR, monoisotopic mass 1255.7 Da. In MRM mode, the respective observed T3 [M + 2H]2+ ions m/z β, 657.9, and δ, 628.9, were selected and fragmented, and the most informative singly-charged peptide fragments, EVGGEALGR, m/z 887.5, and AVGGEALGR, m/z 829.5 (T3i), targeted. We also included the informative C-terminal peptides β T13 and δ T14. This inclusion allows for the rare problem of 2 δ-chain mutations and encompasses both ends of the δ-chain. The T13 β peptide, amino acids 121–132, sequence EFTPPVQAAYQK, has a monoisotopic mass 1377.7 Da, and the T14 δ peptide, amino acids 121–132, sequence EFTPQMQAAYQK, 1440.7 Da. In MRM mode, the β T13 [M + 2H]2+ ions, m/z 689.9, and δ T14 [M + 2H]2+ ions, m/z 721.4, were selected and fragmented, and the doubly-charged ions of the respective informative peptide fragments PPVQAAYQK, m/z 501.3, and PQMQAAYQK, m/z 532.9, were targeted. patient group We obtained 319 whole-blood EDTA samples from patients who consented for hemoglobinopathy diagnosis. The samples, selected to be informative, were analyzed routinely using established methods and then anonymized for MSMS. The samples comprised 163 with HbA2 within the reference interval (1.8%–3.4%), 105 with increased HbA2 (4 compound heterozygotes, 3 HbS, 1 HbC), 43 δ-chain variants (1 homozygous), and 8 Hb Lepore (6 Boston-Washington, 2 Baltimore). The dried blood spot study (see below) comprised 26 anonymized whole-blood EDTA samples: 13 with HbA2 within the reference range, 11 with increased HbA2, and 2 δ-chain variants. msms controls and standards The WHO 1st international reference reagent for HbA2 (89/666, National Institute of Biological Standards and Control) was used as a calibrator in each batch analysis. Controls were donated by Canterbury Scientific (Christchurch, New Zealand): HbA2 normal, assigned value 2.9% (range 2.5%–3.3%) and increased, assigned value 5.5% (range 4.6%–6.4%). Calibrators and controls were reconstituted and stored per manufacturers’ instructions and treated as whole-blood samples for analysis. standard methods for hba2 quantification and hb lepore identification whole blood The guideline “Laboratory Diagnosis of Hemoglobinopathies” (British Committee for Standards in Hematology, 1998) was taken as minimum standard. We performed the hemoglobinopathy screen and HbA2 quantitations by HPLC using a Variant™ II operating with HbA2/HbA1c Dual Program reagent set, including calibrators and controls (Bio-Rad Laboratories). Hb Lepore variants initially detected by HPLC were confirmed by mass spectrometry (14). blood spots We analyzed whole-blood EDTA specimens by HPLC and MSMS. We prepared blood spots by pipetting 35 μL blood/spot onto Schleicher and Schuell 903 filter paper (Whatman), drying at room temperature overnight, and storing at room temperature. We analyzed spots (3.2 mm) by HPLC and MSMS at days 1, 8, and 29. For HPLC, 2 spots were eluted for 90 min into 1 mL Bio-Rad wash buffer (listed as “water”; Bio-Rad Laboratories), approximating to the standard 1:201 dilution. sample preparation for msms whole blood We prepared samples as described (1). We diluted EDTA whole blood 1:50 in deionized water. We added acetonitrile (10 μL) and 10 mL/L formic acid (10 μL) to 100 μL of the dilution and mixed to denature the Hb. After 5 min at room temperature, we added 1 mol/L ammonium bicarbonate (6 μL) and N-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin (5 μL of 5 g/L trypsin in deionized water). When the initially cloudy solution had cleared, it was pulse centrifuged and incubated for 30 min at 37 °C. After digestion, we diluted 40 μL of the solution in 360 μL acetonitrile:water (1:1) with 2 mL/L formic acid and performed MSMS. blood spots We prepared a working solution by eluting 1 spot with 200 μL deionized water for 30 min on an automated shaker. We processed 100 μL of this solution as for whole blood. mass spectrometry Samples (2 μL) were introduced in acetonitrile:water (1:1) containing 0.025% formic acid flowing at 75 μL/min (Agilent 1100 series) into a SCIEX API 4000 (Applied Biosystems) MSMS with an electrospray source in positive ion mode at 5500 V and 250 °C. The interface heater was on, declustering potential 81.0 V, and entrance potential 10 V. The collision gas setting (6.0), collision energy (30 V), and exit potential (15.0 V) were the same for all MRMs. Acquisitions were as described (1), with the additional δ MRMs 150 ms/MRM. Total acquisition time remained 60 s. data analysis We derived peak areas using Analyst 1.4.0 (Applied Biosystems) and exported to Excel v. 2003 (Microsoft) for analysis. For each peptide, the δ-chain percentage ratio was calculated as 100 × δ-chain peptide area/(δ-chain peptide area + corresponding β-chain peptide area) and corrected using the WHO 1st international reference reagent HbA2, nominal value of 5.3% (by weight) of total Hb. Statistical analyses used Analyze-It™ (Analyze-It Software). We compared groups using correlation, paired, and unpaired t tests. Results When the WHO international reference reagent was analyzed in 12 separate assays, the mean peptide δ-chain ratios were T2 4.48%, T3i 8.50%, and T13 2.70%. Consequently, the mean (range) correction factors were 1.19 (1.06–1.27), 0.63 (0.56–0.79), and 1.98 (1.70–2.36). HPLC measurement in the samples within the reference interval revealed a mean (range) HbA2 of 2.77% (2.1%–3.4%). In the samples with increased HbA2, the mean (range) HbA2 was 5.34% (4.1%–7.9%). The corresponding corrected T2 peptide δ chain ratios were significantly lower (P <0.0001): 1.91% (1.31%–2.75%) and 4.04% (2.92%–5.77%) but highly significantly correlated, y = 0.8043x − 0.2911 (r2 = 0.9387, P <0.0001; Fig. 11 ). The lines marked on the graph correspond to 3.4%, the upper limit of our reference interval for HbA2 measured by HPLC, and 2.4%, an equivalent value for the δ-chain ratio measured by MSMS calculated from the regression equation. Compared to HPLC, the corrected T3i peptide δ-chain ratios were also significantly lower (P <0.0001), 2.05% (1.40%–2.97%) and 4.27% (3.25%–5.78%), but significantly correlated, y = 0.8041x − 0.2502 (r2 = 0.9344, P <0.0001; Fig. 22 ). The estimated upper limit of normal for the T3i δ-chain ratio, calculated as above, was 2.5. The corrected T13 peptide δ-chain ratios were also in accord; significantly lower (P <0.0001), 1.98% (1.29%–3.24%) and 4.28% (3.21%–6.17%), but highly significantly correlated, y = 0.8682x − 0.3953 (r2 = 0.9295, P <0.0001; Fig. 33 ). The estimated upper limit of normal for the T13 δ-chain ratio was 2.5. We used repeated injections (n = 6) to assess the within-assay variability (as CV) of the δ/β ratios for the controls and 10 individual patient samples. The mean ratio (CV) of the control for the T2 peptide was 2.57% (5.3%), T3i peptide, 2.64% (4.9%), and T13 peptide, 2.41% (3.8%). For the increased control the equivalent results were T2 peptide, 4.83% (2.0%), T3i peptide, 4.88% (4.0%), and T13 peptide, 4.85% (2.2%). Patient samples were not significantly different from controls. Between-assay imprecision was assessed using the controls, n = 8. The mean (CV) of the normal control for the T2 peptide was 2.35% (7.0%), T3i, 2.53% (7.4%), and T13 peptide, 2.40% (6.5%). For the increased control, the equivalent results were T2, 4.55% (6.5%), T3i, 4.87% (8.1%), and T13, 4.83% (6.9%). To assess the stability of prepared tryptic digests, 17 samples were prepared, analyzed by MSMS within 4 h, stored at 10 °C for 6 days, and reanalyzed by MSMS. Results did not differ significantly. δ-Chain variants measured by HPLC are identified by the presence of 2 approximately equivalent peaks, 1 eluting as HbA2. Accepted practice is to sum the peaks to provide an equivalent HbA2. MSMS analysis of the 43 samples (42 heterozygous, 1 homozygous) identified as δ-chain variants by HPLC demonstrated, compared to normal, significantly decreased T2 and T3 peptide ratios: T2 1.05% vs 1.91% (P <0.0001) and T3i 1.63% vs 2.05% (P <0.0001). In contrast, the T13 peptide ratio was significantly increased, 2.17% vs 1.98% (P = 0.0004). The correlation between the T13 peptide ratio and the calculated HPLC HbA2 was only r2 = 0.2 (P = 0.0015). The homozygous δ-chain variant produced virtually no signal (0.07%) for the T2 peptide, decreased signal for T3i (0.9%), and normal for T13 (2.6%). Eight δ-β fusion variants, 6 Lepore Boston-Washington and 2 Lepore Baltimore detected by HPLC and confirmed by MS and MSMS of intact β-chains, had highly significantly increased (P <0.0001) T2 and T3 peptide ratios, mean (range): T2 14.3% (12.6%–15.6%) and T3i 13.5% (11.9%–15.1%). The T13 peptide ratios were only slightly increased, 2.3% (1.8%–2.9%, P = 0.015). The results of the 2 Lepore Baltimore samples were within the Lepore Boston-Washington ranges. Blood spot samples analyzed by HPLC produced significantly different traces, compared with the equivalent whole blood, even within 1 day (Fig. 44 ). HbA2 was decreased and continued to decline over the 29 days for both normal and increased samples (Fig. 55 , Table 11 ). Using the whole-blood HbA2 cutoff of 3.4%, of the 11 initially increased HbA2 samples, 2 moved within the normal range and 1 within the equivocal range (>3.4 and <4.0) at day 1. By day 8, 5 moved within the normal range and 6 within the equivocal range. At day 29, all were in the normal range. The decrease in HbA2 was accompanied by the appearance of an increasing number of secondary peaks (Fig. 44 ). This chromatographic deterioration was exacerbated in δ-chain variant blood spots. In contrast, using MSMS, the results of whole blood and 24-h blood spot samples were comparable and remained stable for a period of 29 days (Fig. 55 , Table 11 ). Discussion Peptide-based MSMS is a relatively new development in the measurement of clinically significant proteins, offering cost-effective, high-throughput, multiplexed mutation analysis (1) and quantification (15)(16)(17), with potential for combining measurement of small molecules (18) and proteins on a single technology platform (17). The characterization of this model for the clinically significant biomarker HbA2 highlights both the advantages and pitfalls associated with this approach. Population screening for hemoglobinopathies was revolutionized by the introduction of automated HPLC packages combining hardware, consumables, and automated reporting. However, differences in program requirements result in technical differences between newborn and adult schemes. In the UK, HPLC is the only approved method for adult screening, but both HPLC and isoelectric focusing are approved for newborn blood spot sickle cell disease and thalassemia major screening (19)(20). The newborn blood spot HPLC platform is optimized for detection of sickle Hb and sample throughput. We have previously published a simple peptide-based strategy for MSMS screening of clinically significant hemoglobinopathies, based on tryptic digestion of whole blood and MRM peptide mutation targeting, that is faster and more specific than conventional methods (1). It requires no chromatography and uses the same solvent for amino acid and acylcarnitine screening, allowing the 2 methods to be run sequentially on the same MSMS instrument. The method offered considerable advantages with respect to newborn screening but did not fulfill the requirements for adult screening, for which thalassemia carrier detection is essential. In this study, we have added additional MRM acquisitions to demonstrate the potential for quantitative MSMS in the diagnosis of β-thalassemia. In contrast to existing techniques of Hb analysis measuring intact tetramers, MSMS measures individual denatured globin proteins or, after tryptic digestion, specific peptides. HbA2 is a tetramer of equal α- and δ-globins, suggesting the possibility that measuring specific δ protein peptides by MSMS might provide a useful surrogate biomarker. The results presented in Figs. 1–3123 demonstrate the validity of this approach; δ-chain percentage ratios that are highly correlated with HbA2 measured by HPLC. However, the results for each peptide are numerically different, primarily because of differences in ionization relative to control peptides. Even after calibration against the WHO international HbA2 reference reagent, the MSMS peptide results are not identical with HPLC, confirming that the 2 methods are measuring fundamentally different molecular targets. However, the results for each peptide are highly consistent and, more importantly, differentiation between normal and increased HbA2 is maintained. A further correction could be applied to MSMS δ:β peptide ratio results to align them with HbA2. In our previous publication (1), we were primarily concerned with qualitative mutation detection. However, the application of MSMS to quantitative plasma protein analysis has been elegantly demonstrated (15)(16). The current study is essentially quantitative, using the corresponding β peptide MRM signal as an internal calibrator. Although this works well in the current context, a more rigorous approach to peptide quantification, using synthetic stable isotope peptides as internal calibrators (15)(16), is the next logical step. As with any quantitative method, the analytical criteria are more stringent. The within- and between-assay imprecision data demonstrate that, despite the lack of sample cleanup, the performance of the 30-min tryptic digestion and rapid flow injection MSMS analysis is sufficiently robust for clinical application. The prepared peptides are stable for at least 6 days at 10 °C. A further advantage of directly targeting δ peptides by MSMS, in addition to speed and sensitivity, is the ability to detect and accurately quantify δ protein even in the presence of confounding factors which may prevent or interfere with HbA2 quantification using traditional methods (2). These include variants that coelute with HbA2 (e.g., HbE) or, in some formats, give rise to falsely increased HbA2 values (e.g., HbS). We have analyzed several such samples and obtained δ values consistent with phenotypic and genotypic information (complete comparison data not shown). A potentially critical disadvantage of MSMS quantification of any protein using a single peptide is the importance that sequence variations (mutations/polymorphisms) assume. Consequently, in protein quantification by MSMS, it is essential that multiple peptides—at least 3—are included to prevent underestimation. Our results on δ-chain mutations accentuate the necessity for understanding the genetics of any protein to be measured. δ-Chain variants were first described more than 40 years ago with the recognition of HbA′2 (HbB2), the result of a glycine-to-arginine substitution at position 16 (21). This hematologically silent mutation has a high frequency in Herero populations (22) and African Americans (23). It is usually identified using HPLC as a reduction in HbA2 and the presence of a later running peak of approximately equal concentration (HbA′2). The clinical importance of HbA′2 lies in the possibility that HbA2 may be underestimated and a diagnosis of β-thalassemia trait missed. It is essential to obtain a total HbA2 by addition of HbA′2 and HbA2. We investigated 1 homozygous and 42 heterozygous δ-chain variants identified by HPLC. With MSMS, the T2 δ-chain ratio in the heterozygous samples was approximately half the control values and absent in the homozygous sample. The results confirm that the mutation in our δ-chain variant population is in the T2 peptide. Interestingly, in heterozygous samples, the T3 δ-chain ratio was decreased by ∼25%. In the homozygous sample, the reduction was ∼50%. In retrospect, this is probably the result of the glycine-to-arginine substitution introducing a new trypsin digestion site directly proximal to the existing lysine at position 17. The data are consistent with an initial 50:50 chance of scission at either the arginine or lysine residues. Scission at the arginine site results in a peptide, KVNVDAVGGEALGR, that cannot bind to trypsin (24) for removal of the N-terminal lysine. This observation negates the value of the T3 peptide for ensuring detection of β-thalassemia trait in the presence of our most common δ-chain variant, which is confirmed as HbA′2. Demonstration of this phenomenon in the current study emphasizes the precision of the quantitative approach. The T13 δ-chain ratios were normal for the δ-chain variant heterozygotes and homozygote, providing a true estimation of the total δ chain present in this group. Selection of 2 δ-peptide transitions will ensure that δ-chain variant heterozygotes and homozygotes are not missed provided the peptides are located in sufficiently remote positions. It is, however, possible for a person to inherit 2 different δ-chain mutations (25). This is rare, but indicates the necessity for multiple peptide analysis in quantitative proteomics. This will apply to the quantification of any protein, a point not emphasized in previous studies (15)(16). We analyzed 3 peptides to measure the δ-chain, so that even in compound heterozygote δ-chain mutations, 1 of the transitions should still give the correct value. This might appear excessive; nonetheless, it is worth noting that increasing the number of acquisitions does not increase the analysis time but allows further specificity and sensitivity. In retrospect, the combination of the T2 and T3 peptides is not ideal, because HbA′2 is relatively common and affects T3 digestion. It might be argued, because the most common δ-chain mutation is clinically silent and occurs in the T2 peptide, that the T2 peptide should not be included. However, it is essential for the detection of all 3 reported Hb Lepore variants; the result of δ-β fusion processes with sequences as follows: Hb Lepore Hollandia, δ 1–22; Hb Lepore Baltimore, δ 1–50; and Hb Lepore Boston-Washington, δ 1–87. We predicted that we should measure an increased proportion of δ-chain peptides before the fusion point. The T2 peptide (9)(10)(11)(12)(13)(14)(15)(16)(17) is the only peptide with a difference from the β sequence before amino acid 22. The effect on the respective δ-chain ratios is impressive, with significant increases, to 12%–17%, in the T2 and T3 peptides and essentially normal results for T13. The pattern is diagnostic of an Hb Lepore but does not distinguish between the 3 variants. An unequivocal characterization may be obtained using whole-molecule MSMS (14). Antenatal screening for hemoglobinopathies is usually a local service, owing to the requirement for fresh blood samples for HbA2 measurement and interpretation together with erythrocyte indices. In contrast, newborn screening is centralized, using blood spot cards that are easily transported. Hb spotted onto filter paper is oxidized rapidly to form methemoglobin, which has a more positive charge than that of the original oxyhemoglobin (26) and is a major problem for the quantification of HbA2 by HPLC. The methemoglobin appears as a secondary peak, similar to that of a δ-chain variant, and renders the chromatogram difficult to interpret. In contrast, using MSMS measurement of δ-chain ratios, it is possible to reliably quantify an equivalent HbA2 in blood spots up to a month old. It remains essential that results be interpreted in conjunction with erythrocyte indices and ethnic origin. In the current health economic environment, a method offering centralization of antenatal screening is of considerable interest. Development of an automated package for trypsin digestion, MSMS, and data analysis for combined adult and newborn screening (1) should facilitate efficient and cost-effective delivery of population hemoglobinopathy screening programs. In conclusion, this study demonstrates the feasibility of quantitative MSMS analysis to measure the δ:β-globin ratio as a surrogate marker of HbA2. It also highlights the criteria that must be considered in the development of quantitative clinical proteomics, in which both silent and clinically significant polymorphisms are relatively common. Figure 1. Open in new tabDownload slide MSMS corrected T2 δ:β peptide ratio compared with HPLC HbA2. The dotted lines represent the upper limits of the HPLC reference range and the equivalent calculated δ:β peptide ratio. Figure 1. Open in new tabDownload slide MSMS corrected T2 δ:β peptide ratio compared with HPLC HbA2. The dotted lines represent the upper limits of the HPLC reference range and the equivalent calculated δ:β peptide ratio. Figure 2. Open in new tabDownload slide MSMS corrected T3i δ:β peptide ratio compared with HPLC HbA2. The dotted lines represent the upper limits of the HPLC reference range and the equivalent calculated δ:β peptide ratio. Figure 2. Open in new tabDownload slide MSMS corrected T3i δ:β peptide ratio compared with HPLC HbA2. The dotted lines represent the upper limits of the HPLC reference range and the equivalent calculated δ:β peptide ratio. Figure 3. Open in new tabDownload slide MSMS corrected T13 δ:β peptide ratio compared with HPLC HbA2. The dotted lines represent the upper limits of the HPLC reference range and the equivalent calculated δ:β peptide ratio. Figure 3. Open in new tabDownload slide MSMS corrected T13 δ:β peptide ratio compared with HPLC HbA2. The dotted lines represent the upper limits of the HPLC reference range and the equivalent calculated δ:β peptide ratio. Figure 4. Open in new tabDownload slide HPLC chromatograms of a whole blood sample (A) and dried blood spots prepared from the same sample after storage at room temperature for 1 (B), 8 (C), and 29 (D) days. Note the decline in the HbA2 peak and the appearance and increase in the peak at 3.3 min (arrowed). Figure 4. Open in new tabDownload slide HPLC chromatograms of a whole blood sample (A) and dried blood spots prepared from the same sample after storage at room temperature for 1 (B), 8 (C), and 29 (D) days. Note the decline in the HbA2 peak and the appearance and increase in the peak at 3.3 min (arrowed). Figure 5. Open in new tabDownload slide Stability of dried blood spot MSMS corrected T2 δ:β peptide ratio, mean (SE) for normal and increased HbA2 samples compared with decreases in HPLC HbA2. Figure 5. Open in new tabDownload slide Stability of dried blood spot MSMS corrected T2 δ:β peptide ratio, mean (SE) for normal and increased HbA2 samples compared with decreases in HPLC HbA2. Table 1. Stability of dried blood spot HbA2 and corrected δ:β peptide ratios.1 . Day 0 (whole blood) . Day 1 (blood spot) . Day 8 (blood spot) . Day 29 (blood spot) . Normal A2  HPLC 2.8 (2.4–3.2) 2.1 (1.7–2.4) 1.7 (1.2–2.2) 1.3 (0.7–1.6)  T2 1.96 (1.61–2.34) 1.76 (1.17–2.38) 1.96 (1.68–2.26) 2.17 (1.62–2.71)  T3i 2.25 (1.64–2.64) 1.94 (1.12–2.44) 2.14 (1.62–2.78) 2.29 (1.50–2.84)  T13 2.21 (1.75–2.54) 1.87 (1.11–2.44) 1.94 (1.62–2.18) 2.01 (1.55–2.51) Increased A2  HPLC 5.6 (4.6–6.7) 4.2 (3.3–5.1) 3.4 (2.6–3.9) 2.4 (1.8–2.9)  T2 3.89 (3.07–4.54) 3.92 (3.17–4.48) 4.11 (3.27–4.60) 4.28 (3.18–4.94)  T3i 4.44 (3.82–5.36) 4.29 (3.77–4.96) 4.07 (3.36–4.89) 4.35 (3.49–4.96)  T13 4.55 (3.59–5.76) 4.22 (3.50–4.74) 4.06 (3.37–4.62) 3.93 (2.73–4.83) . Day 0 (whole blood) . Day 1 (blood spot) . Day 8 (blood spot) . Day 29 (blood spot) . Normal A2  HPLC 2.8 (2.4–3.2) 2.1 (1.7–2.4) 1.7 (1.2–2.2) 1.3 (0.7–1.6)  T2 1.96 (1.61–2.34) 1.76 (1.17–2.38) 1.96 (1.68–2.26) 2.17 (1.62–2.71)  T3i 2.25 (1.64–2.64) 1.94 (1.12–2.44) 2.14 (1.62–2.78) 2.29 (1.50–2.84)  T13 2.21 (1.75–2.54) 1.87 (1.11–2.44) 1.94 (1.62–2.18) 2.01 (1.55–2.51) Increased A2  HPLC 5.6 (4.6–6.7) 4.2 (3.3–5.1) 3.4 (2.6–3.9) 2.4 (1.8–2.9)  T2 3.89 (3.07–4.54) 3.92 (3.17–4.48) 4.11 (3.27–4.60) 4.28 (3.18–4.94)  T3i 4.44 (3.82–5.36) 4.29 (3.77–4.96) 4.07 (3.36–4.89) 4.35 (3.49–4.96)  T13 4.55 (3.59–5.76) 4.22 (3.50–4.74) 4.06 (3.37–4.62) 3.93 (2.73–4.83) 1 Data are mean (range). Table 1. Stability of dried blood spot HbA2 and corrected δ:β peptide ratios.1 . Day 0 (whole blood) . Day 1 (blood spot) . Day 8 (blood spot) . Day 29 (blood spot) . Normal A2  HPLC 2.8 (2.4–3.2) 2.1 (1.7–2.4) 1.7 (1.2–2.2) 1.3 (0.7–1.6)  T2 1.96 (1.61–2.34) 1.76 (1.17–2.38) 1.96 (1.68–2.26) 2.17 (1.62–2.71)  T3i 2.25 (1.64–2.64) 1.94 (1.12–2.44) 2.14 (1.62–2.78) 2.29 (1.50–2.84)  T13 2.21 (1.75–2.54) 1.87 (1.11–2.44) 1.94 (1.62–2.18) 2.01 (1.55–2.51) Increased A2  HPLC 5.6 (4.6–6.7) 4.2 (3.3–5.1) 3.4 (2.6–3.9) 2.4 (1.8–2.9)  T2 3.89 (3.07–4.54) 3.92 (3.17–4.48) 4.11 (3.27–4.60) 4.28 (3.18–4.94)  T3i 4.44 (3.82–5.36) 4.29 (3.77–4.96) 4.07 (3.36–4.89) 4.35 (3.49–4.96)  T13 4.55 (3.59–5.76) 4.22 (3.50–4.74) 4.06 (3.37–4.62) 3.93 (2.73–4.83) . Day 0 (whole blood) . Day 1 (blood spot) . Day 8 (blood spot) . Day 29 (blood spot) . Normal A2  HPLC 2.8 (2.4–3.2) 2.1 (1.7–2.4) 1.7 (1.2–2.2) 1.3 (0.7–1.6)  T2 1.96 (1.61–2.34) 1.76 (1.17–2.38) 1.96 (1.68–2.26) 2.17 (1.62–2.71)  T3i 2.25 (1.64–2.64) 1.94 (1.12–2.44) 2.14 (1.62–2.78) 2.29 (1.50–2.84)  T13 2.21 (1.75–2.54) 1.87 (1.11–2.44) 1.94 (1.62–2.18) 2.01 (1.55–2.51) Increased A2  HPLC 5.6 (4.6–6.7) 4.2 (3.3–5.1) 3.4 (2.6–3.9) 2.4 (1.8–2.9)  T2 3.89 (3.07–4.54) 3.92 (3.17–4.48) 4.11 (3.27–4.60) 4.28 (3.18–4.94)  T3i 4.44 (3.82–5.36) 4.29 (3.77–4.96) 4.07 (3.36–4.89) 4.35 (3.49–4.96)  T13 4.55 (3.59–5.76) 4.22 (3.50–4.74) 4.06 (3.37–4.62) 3.93 (2.73–4.83) 1 Data are mean (range). 1 Nonstandard abbreviations: MSMS, tandem mass spectrometry; MRM, multiple reaction monitoring; Hb, hemoglobin. Grant/funding support: Y.A.D. is the recipient of a Benefaction Grant from the Guy’s and St. Thomas’ Charity. R.N.D. is the recipient of a WellChild Trust endowment. 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Saunders Company Philadelphia. . © 2007 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Quantification of Hemoglobin A2 by Tandem Mass Spectrometry
JF - Clinical Chemistry
DO - 10.1373/clinchem.2007.088682
DA - 2007-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/quantification-of-hemoglobin-a2-by-tandem-mass-spectrometry-b32ySnSoRL
SP - 1448
VL - 53
IS - 8
DP - DeepDyve
ER -