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Short-term changes in plasma glucose affect glycated hemoglobin measurement
diabetes research and clinical practice 100 (2013) e17–e19
Contents available at Sciverse ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Brief report
Short-term changes in plasma glucose affect glycated hemoglobin measurement Richeek Pradhan a, Sandip Kumar Batabyal b, Subrata Maiti b, Sudip Chatterjee b,* a b
Department of Pharmacology, Institute of Post-Graduate Medical Education and Research, Kolkata, India The Park Clinic, Kolkata, India
article info
abstract
Article history:
The study aimed to investigate the impact of a delay of 12 h between sample collection and
Received 8 July 2012
HbA1c measurement. We propose an easy method to determine the adequacy of removal of
Received in revised form
HbA1d and in case of insufficient removal suggest measurement of HbA1c 12 h after
5 November 2012
collection.
Accepted 3 January 2013
# 2013 Elsevier Ireland Ltd. All rights reserved.
Published on line 29 January 2013 Keywords: HbA1c HbA1d
1.
Introduction
Since the 1970s glycated hemoglobin (GHb) has become the most widely used measure of glycemic control [1]. The degree of glycation of the terminal amino acid residue of the beta chain of hemoglobin over the lifetime of the red blood cell is a measure of glycemic control over that period. We hypothesized that a delay of 12 h between sample collection and analysis of GHb could have an impact on the result.
2.
Materials and methods
Blood was collected in EDTA vials from 8 healthy volunteers (3 males), without reference to their last meal intake. The study was approved by the institution’s ethics committee. The samples were separated into 4 aliquots each. One aliquot was
kept unaltered (Set A). Dextrose solution (10%) was added to the 3 other aliquots in varying amounts in order to have 3 different ambient glucose concentrations, i.e., high (10.5– 11.6 mmol/l) (Set B); moderately high (15.5–17.7 mmol/l) (Set C); and very high (22.2–24.7 mmol/l) (Set D). The 3 different ranges of glucose values were achieved using a glucometer by trial and error method. For each subject 4 different glucose ranges were thus obtained. From each of the 4 aliquots per subject, glucose was measured by autoanalyzer using the GOD/POD method and GHb using the DS5 Analyzer (Drew Scientific Group). Measurements were made within 20–25 min of sample collection. This time point was denoted as 0 h. A mean of two values was taken as the baseline GHb for each subject. The samples were kept at a temperature of 20–25 8C (293–298 K) for 12 h after which the assays were repeated. Samples were also obtained from 6 people with diabetes (2 males) and GHb was measured in these unaltered samples at 0 and 12 h.
* Corresponding author at: The Park Clinic, 4 Gorky Terrace, Kolkata 700017, India. Tel.: +91 3322808424; fax: +91 3322801807. E-mail addresses: [email protected], [email protected] (S. Chatterjee). 0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.diabres.2013.01.004
e18
diabetes research and clinical practice 100 (2013) e17–e19
4.
Fig. 1 – Mean(WSD) change in measured GHb values from baseline (0 h) after 12 h in Sets A, B, C and D, respectively, in samples from healthy volunteers (n = 8). **p < 0.001 and ***p < 0.0001 by paired t test.
3.
Results
Changes of GHb between 0 and 12 h in the unaltered and each of the 3 altered samples obtained from 8 volunteers without reference to their last meals is shown in Table 1. There was a mean decrease in HbA1c value of 0.2% (2.5 mmol/mol) in Set A and a mean increase of 0.2% (2.5 mmol/mol), 0.4% (5.1 mmol/mol), 0.9% (10.3 mmol/mol) in Sets B, C and D, respectively, when measured after 12 h. The 12 h values were significantly different from the 0 h values in Sets A, C and D by paired t test with p values 0.009, 0.005 and 0.0002, respectively (Fig. 1). The baseline glucose values of samples from the diabetic patients were 7.1, 7.1, 10.7, 7.6, 10.2 and 11.2 mmol/l, respectively. A similar trend of decreased GHb was seen in these samples where a mean(SD) reduction of 0.2 0.09% (2.2 0.1 mmol/mol) was observed over 12 h. The 0–12 h changes in GHb values were significant with a p value of 0.002.
Discussion
Before the formation of the stable ketamine, the HbA1c form of glycated hemoglobin, formation of an aldamine form (HbA1d) takes place [2]. Unlike the stable fraction, HbA1d varies with ambient glucose level both in vitro and in vivo with a small phase delay of about 2 h [3]. This fraction does not help in the assessment of long-term glycemic control and if not removed from the total glycated hemoglobin, gives a falsely elevated value of HbA1c. Since older chromatographic methods cannot effectively separate the two fractions, various methods have been devised to eliminate the HbA1d fraction. They include saline incubation, dialysis, lowering the pH, increasing the temperature or applying certain buffers like bis-tris phosphate [4]. The procedure used in our laboratory to measure HbA1c is low pressure cation exchange chromatography followed by gradient elution. The manufacturer’s manual states that 90% of labile GHb is destroyed by raising the hemolyzer/diluent temperature in a patented procedure [5]. The results of our study show that there is a decrease in measured HbA1c level in unaltered samples after 12 h compared with the value at 0 h which we suggest is due to the measurement of HbA1d as well as HbA1c due to inadequate removal of the former from the 0 h sample. Subsequent decrease is due to dissociation of HbA1d due to the falling ambient plasma glucose level. At 12 h, there is an increase in measured HbA1c in samples where 10% dextrose was added confirming that the labile fraction was being measured as it is unlikely that alteration of the HbA1c level would occur over 12 h as this process takes at least 7 days [3]. It can be inferred that the 12 h value with lower ambient glucose, and thus with minimal interference from HbA1d, more closely represents the glycemic control of the individual. The reference method of the DCCT is about 60% specific for HbA1c [2]. The National Glycohemoglobin Standardization Program requires laboratories to produce ‘DCCT-traceable’ results. Again, the International Federation of Clinical Chemistry approach is that of establishing an international reference standard for HbA1c [6]. Despite these efforts the reality remains that many different methods for GHb measurement are still in use worldwide, especially in developing countries [7]. Multiple
Table 1 – Showing change of GHb after 12 h. All the values are significantly different from baseline (0 h) values. Sl. No.
Change of GHb after 12 h in unaltered sample in % (mmol/mol)
1 2 3 4 5 6 7 8
0.2 ( 0.2 ( 0 (0) 0.3 ( 0.4 ( 0.5 ( 0.2 ( 0 (0)
Mean SD
0.2 0.17 ( 2.5 2)
3) 2) 3) 4) 6) 2)
Change of GHb after 12 h in sample with ambient glucose made up to 10.5–11.6 mmol/l in % (mmol/mol) 0.8 0.2 0.2 0.3 0.4 0.3 0.1 0.2
(9) (2) (3) (4) (4) ( 3) ( 1) (2)
0.2 0.3 (2.5 3.5)
Change of GHb after 12 h in sample with ambient glucose made up to 15.5–17.7 mmol/l in % (mmol/mol) 0.5 0.6 0.7 0.4 0.8 0.2 0.4 0.6
(6) (7) (8) (4) (9) ( 3) (4) (6)
0.4 0.3 (5.1 3.7)
Change of GHb after 12 h in sample with ambient glucose made up to 22.2–24.7 mmol/l in % (mmol/mol) 1.6 0.9 0.7 1.3 1.0 0.5 0.8 0.7
(17) (10) (8) (15) (11) (5) (9) (7)
0.9 0.35 (10.25 4)
diabetes research and clinical practice 100 (2013) e17–e19
methods and their lack of consistency in GHb values obtained from fresh and stored samples make interpretation of glycemic control difficult [8]. Discrepancy in GHb measurement depending on the time elapsed between collection of sample and performance of the assay will be of special concern in scenarios where blood needs to be transported to reference laboratories at ambient temperatures, e.g. multicentric trials. This is likely to result in a new level of uncertainty in the HbA1c measurement, particularly if the ambient glucose is high in the sample. Methods like TOSOH and Arkray, which do not need a preincubation step and have an in-built mechanism to remove the labile fraction, are likely to be free of this discrepancy [9]. While our finding seems to be assay specific, the adequacy of HbA1d removal by any GHb measurement technique can be checked by the simple addition of glucose to test samples, as shown in our study. In case of inadequate removal of labile GHb, we suggest that performing the assays after a delay of 12 h would give a closer estimate of the subject’s glycemic control.
[2] [3]
[4]
[5]
[6]
[7]
Conflict of interest The authors declare that they have no conflict of interest.
[8]
references [9] [1] The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on
e19
the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329(September (14)): 977–86. Bunn HF. Evaluation of glycosylated hemoglobin diabetic patients. Diabetes 1981;30(July (7)):613–7. Mortensen HB, Volund A. Application of a biokinetic model for prediction and assessment of glycated haemoglobins in diabetic patients. Scand J Clin Lab Invest 1988;48:595402. Nathan DM, Avezzano E, Palmer JL. Rapid method for eliminating labile glycosylated hemoglobin from the assay of hemoglobin A1. Clin Chem 1982;28:512–5. DS5. Quick and easy HbA1c measurement; 2012, Retrieved on June 13, 2012, from http://www.drew-scientific.com/ techinfo/brochures/ds5brochure.pdf. Hoelzel A, Weykamp C, Jeppsson JO, 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;50:166–74. Report of a World Health Organization Consultation. Use of glycated haemoglobin (HbA1c) in the diagnosis of diabetes mellitus. Diabetes Res Clin Pract 2011;93: 299–309. Little RR, Rohlfing CL, Tennill AL, Connolly S, Hanson S. Effects of sample storage conditions on glycated hemoglobin measurement: evaluation of five different high performance liquid chromatography methods. Diabetes Technol Ther 2007;9(1):36–42. Analysis of HbA1c on ion exchange HPLC; 2012, Retrieved on October 17, 2012, from http://www.canterburyscientific.com/ products/technical-information/hba1c-on-ion-exchangehplc.
Contents available at Sciverse ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Brief report
Short-term changes in plasma glucose affect glycated hemoglobin measurement Richeek Pradhan a, Sandip Kumar Batabyal b, Subrata Maiti b, Sudip Chatterjee b,* a b
Department of Pharmacology, Institute of Post-Graduate Medical Education and Research, Kolkata, India The Park Clinic, Kolkata, India
article info
abstract
Article history:
The study aimed to investigate the impact of a delay of 12 h between sample collection and
Received 8 July 2012
HbA1c measurement. We propose an easy method to determine the adequacy of removal of
Received in revised form
HbA1d and in case of insufficient removal suggest measurement of HbA1c 12 h after
5 November 2012
collection.
Accepted 3 January 2013
# 2013 Elsevier Ireland Ltd. All rights reserved.
Published on line 29 January 2013 Keywords: HbA1c HbA1d
1.
Introduction
Since the 1970s glycated hemoglobin (GHb) has become the most widely used measure of glycemic control [1]. The degree of glycation of the terminal amino acid residue of the beta chain of hemoglobin over the lifetime of the red blood cell is a measure of glycemic control over that period. We hypothesized that a delay of 12 h between sample collection and analysis of GHb could have an impact on the result.
2.
Materials and methods
Blood was collected in EDTA vials from 8 healthy volunteers (3 males), without reference to their last meal intake. The study was approved by the institution’s ethics committee. The samples were separated into 4 aliquots each. One aliquot was
kept unaltered (Set A). Dextrose solution (10%) was added to the 3 other aliquots in varying amounts in order to have 3 different ambient glucose concentrations, i.e., high (10.5– 11.6 mmol/l) (Set B); moderately high (15.5–17.7 mmol/l) (Set C); and very high (22.2–24.7 mmol/l) (Set D). The 3 different ranges of glucose values were achieved using a glucometer by trial and error method. For each subject 4 different glucose ranges were thus obtained. From each of the 4 aliquots per subject, glucose was measured by autoanalyzer using the GOD/POD method and GHb using the DS5 Analyzer (Drew Scientific Group). Measurements were made within 20–25 min of sample collection. This time point was denoted as 0 h. A mean of two values was taken as the baseline GHb for each subject. The samples were kept at a temperature of 20–25 8C (293–298 K) for 12 h after which the assays were repeated. Samples were also obtained from 6 people with diabetes (2 males) and GHb was measured in these unaltered samples at 0 and 12 h.
* Corresponding author at: The Park Clinic, 4 Gorky Terrace, Kolkata 700017, India. Tel.: +91 3322808424; fax: +91 3322801807. E-mail addresses: [email protected], [email protected] (S. Chatterjee). 0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.diabres.2013.01.004
e18
diabetes research and clinical practice 100 (2013) e17–e19
4.
Fig. 1 – Mean(WSD) change in measured GHb values from baseline (0 h) after 12 h in Sets A, B, C and D, respectively, in samples from healthy volunteers (n = 8). **p < 0.001 and ***p < 0.0001 by paired t test.
3.
Results
Changes of GHb between 0 and 12 h in the unaltered and each of the 3 altered samples obtained from 8 volunteers without reference to their last meals is shown in Table 1. There was a mean decrease in HbA1c value of 0.2% (2.5 mmol/mol) in Set A and a mean increase of 0.2% (2.5 mmol/mol), 0.4% (5.1 mmol/mol), 0.9% (10.3 mmol/mol) in Sets B, C and D, respectively, when measured after 12 h. The 12 h values were significantly different from the 0 h values in Sets A, C and D by paired t test with p values 0.009, 0.005 and 0.0002, respectively (Fig. 1). The baseline glucose values of samples from the diabetic patients were 7.1, 7.1, 10.7, 7.6, 10.2 and 11.2 mmol/l, respectively. A similar trend of decreased GHb was seen in these samples where a mean(SD) reduction of 0.2 0.09% (2.2 0.1 mmol/mol) was observed over 12 h. The 0–12 h changes in GHb values were significant with a p value of 0.002.
Discussion
Before the formation of the stable ketamine, the HbA1c form of glycated hemoglobin, formation of an aldamine form (HbA1d) takes place [2]. Unlike the stable fraction, HbA1d varies with ambient glucose level both in vitro and in vivo with a small phase delay of about 2 h [3]. This fraction does not help in the assessment of long-term glycemic control and if not removed from the total glycated hemoglobin, gives a falsely elevated value of HbA1c. Since older chromatographic methods cannot effectively separate the two fractions, various methods have been devised to eliminate the HbA1d fraction. They include saline incubation, dialysis, lowering the pH, increasing the temperature or applying certain buffers like bis-tris phosphate [4]. The procedure used in our laboratory to measure HbA1c is low pressure cation exchange chromatography followed by gradient elution. The manufacturer’s manual states that 90% of labile GHb is destroyed by raising the hemolyzer/diluent temperature in a patented procedure [5]. The results of our study show that there is a decrease in measured HbA1c level in unaltered samples after 12 h compared with the value at 0 h which we suggest is due to the measurement of HbA1d as well as HbA1c due to inadequate removal of the former from the 0 h sample. Subsequent decrease is due to dissociation of HbA1d due to the falling ambient plasma glucose level. At 12 h, there is an increase in measured HbA1c in samples where 10% dextrose was added confirming that the labile fraction was being measured as it is unlikely that alteration of the HbA1c level would occur over 12 h as this process takes at least 7 days [3]. It can be inferred that the 12 h value with lower ambient glucose, and thus with minimal interference from HbA1d, more closely represents the glycemic control of the individual. The reference method of the DCCT is about 60% specific for HbA1c [2]. The National Glycohemoglobin Standardization Program requires laboratories to produce ‘DCCT-traceable’ results. Again, the International Federation of Clinical Chemistry approach is that of establishing an international reference standard for HbA1c [6]. Despite these efforts the reality remains that many different methods for GHb measurement are still in use worldwide, especially in developing countries [7]. Multiple
Table 1 – Showing change of GHb after 12 h. All the values are significantly different from baseline (0 h) values. Sl. No.
Change of GHb after 12 h in unaltered sample in % (mmol/mol)
1 2 3 4 5 6 7 8
0.2 ( 0.2 ( 0 (0) 0.3 ( 0.4 ( 0.5 ( 0.2 ( 0 (0)
Mean SD
0.2 0.17 ( 2.5 2)
3) 2) 3) 4) 6) 2)
Change of GHb after 12 h in sample with ambient glucose made up to 10.5–11.6 mmol/l in % (mmol/mol) 0.8 0.2 0.2 0.3 0.4 0.3 0.1 0.2
(9) (2) (3) (4) (4) ( 3) ( 1) (2)
0.2 0.3 (2.5 3.5)
Change of GHb after 12 h in sample with ambient glucose made up to 15.5–17.7 mmol/l in % (mmol/mol) 0.5 0.6 0.7 0.4 0.8 0.2 0.4 0.6
(6) (7) (8) (4) (9) ( 3) (4) (6)
0.4 0.3 (5.1 3.7)
Change of GHb after 12 h in sample with ambient glucose made up to 22.2–24.7 mmol/l in % (mmol/mol) 1.6 0.9 0.7 1.3 1.0 0.5 0.8 0.7
(17) (10) (8) (15) (11) (5) (9) (7)
0.9 0.35 (10.25 4)
diabetes research and clinical practice 100 (2013) e17–e19
methods and their lack of consistency in GHb values obtained from fresh and stored samples make interpretation of glycemic control difficult [8]. Discrepancy in GHb measurement depending on the time elapsed between collection of sample and performance of the assay will be of special concern in scenarios where blood needs to be transported to reference laboratories at ambient temperatures, e.g. multicentric trials. This is likely to result in a new level of uncertainty in the HbA1c measurement, particularly if the ambient glucose is high in the sample. Methods like TOSOH and Arkray, which do not need a preincubation step and have an in-built mechanism to remove the labile fraction, are likely to be free of this discrepancy [9]. While our finding seems to be assay specific, the adequacy of HbA1d removal by any GHb measurement technique can be checked by the simple addition of glucose to test samples, as shown in our study. In case of inadequate removal of labile GHb, we suggest that performing the assays after a delay of 12 h would give a closer estimate of the subject’s glycemic control.
[2] [3]
[4]
[5]
[6]
[7]
Conflict of interest The authors declare that they have no conflict of interest.
[8]
references [9] [1] The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on
e19
the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329(September (14)): 977–86. Bunn HF. Evaluation of glycosylated hemoglobin diabetic patients. Diabetes 1981;30(July (7)):613–7. Mortensen HB, Volund A. Application of a biokinetic model for prediction and assessment of glycated haemoglobins in diabetic patients. Scand J Clin Lab Invest 1988;48:595402. Nathan DM, Avezzano E, Palmer JL. Rapid method for eliminating labile glycosylated hemoglobin from the assay of hemoglobin A1. Clin Chem 1982;28:512–5. DS5. Quick and easy HbA1c measurement; 2012, Retrieved on June 13, 2012, from http://www.drew-scientific.com/ techinfo/brochures/ds5brochure.pdf. Hoelzel A, Weykamp C, Jeppsson JO, 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;50:166–74. Report of a World Health Organization Consultation. Use of glycated haemoglobin (HbA1c) in the diagnosis of diabetes mellitus. Diabetes Res Clin Pract 2011;93: 299–309. Little RR, Rohlfing CL, Tennill AL, Connolly S, Hanson S. Effects of sample storage conditions on glycated hemoglobin measurement: evaluation of five different high performance liquid chromatography methods. Diabetes Technol Ther 2007;9(1):36–42. Analysis of HbA1c on ion exchange HPLC; 2012, Retrieved on October 17, 2012, from http://www.canterburyscientific.com/ products/technical-information/hba1c-on-ion-exchangehplc.