Biological variation components of glycated hemoglobin HbA1c on tosoh G7 HPLC

  • Abstract
  • FullTEXT
  • Additional info
  • Attachments
  • Related items
  • Video

Aim: Biological variation (BV) of the HbA1c test can be affected by analysis method, sampling period, and disease activity. The aim of the present study was to

examine the impact of CVI, CVA, and II for Tosoh G7 system. Material and Method: 96 samples from 12 healthy volunteers were collected for every two weeks

during a two-month period. HbA1c levels of all samples were measured by G7 HPLC (non-porous ion-exchange) Analyser. Biological variation parameters CVI,

CVA, CVG, RCV, and II were calculated. Results: The mean of HbA1c was 5.10% (32.3 mmol/mol). CVA was calculated as 2.81%. The CVI and CVG for HbA1c

were 1.69% and 7.56%, respectively. RCV and II values were 9.08% and 0.22. Discussion: According to our results, the sampling period and study method were

fundamental to determining biological variation parameters. When using the Tosoh G7 system, we observed similar results to other methods, e.g. boronate

affinity or immunoturbidimetry. Low individuality index of HbA1c presented that the analyte had marked individuality and that the population-based reference

values had low utility for disease monitoring. Thus, estimation and inclusion of RCV in laboratory reports for HbA1c will guide the clinicians in evaluating the

importance of difference between serial results and glycemic status of the patients.

Biological variation components of HbA1c

Müjgan Ercan1, Esin Avcı2, Emiş Deniz Akbulut3, Esra Fırat Oğuz4, Canan Topçuoğlu4, Çiğdem Yücel4, Sedat Abuşoğlu5, Turan Turhan4

1Department of Biochemistry, Bozok University Faculty of Medicine, Yozgat, 2Department of Biochemistry, Pamukkale University Faculty of Medicine, Denizli,

3Biochemistry Laboratory, Çukurova Dr. Aşkım Tüfekçi State Hospital, Adana, 4Biochemistry Laboratory, Ankara Numune Training and Research Hospital, Ankara,

5Department of Biochemistry, Selçuk University Faculty of Medicine, Konya, Turkey

Abstract

Aim: Biological variation (BV) of the HbA1c test can be affected by analysis method, sampling period, and disease activity. The aim of the present study was to examine the impact of CVI, CVA, and II for Tosoh G7 system. Material and Method: 96 samples from 12 healthy volunteers were collected for every two weeks during a two-month period. HbA1c levels of all samples were measured by G7 HPLC (non-porous ion-exchange) Analyser. Biological variation parameters CVI, CVA, CVG, RCV, and II were calculated. Results: The mean of HbA1c was 5.10% (32.3 mmol/mol). CVA was calculated as 2.81%. The CVI and CVG for HbA1c were 1.69% and 7.56%, respectively. RCV and II values were 9.08% and 0.22. Discussion: According to our results, the sampling period and study method were fundamental to determining biological variation parameters. When using the Tosoh G7 system, we observed similar results to other methods, e.g. boronate affinity or immunoturbidimetry. Low individuality index of HbA1c presented that the analyte had marked individuality and that the population-based reference values had low utility for disease monitoring. Thus, estimation and inclusion of RCV in laboratory reports for HbA1c will guide the clinicians in evaluating the importance of difference between serial results and glycemic status of the patients.

Keywords

HbA1c; Biological Variation Parameters; Tosoh G7 System

DOI: 10.4328/JCAM.5761 Received: 15.02.2018 Accepted: 26.03.2018 Published Online: 28.03.2018 Printed: 01.11.2018 J Clin Anal Med 2018;9(6): 469-73

Corresponding Author: Müjgan Ercan, Bozok University Faculty of Medicine, Biochemistry Laboratory, 66200, Yozgat, Turkey.

T.: +90 3542123801 E-Mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

ORCID ID: 0000-0002-9291-4197

Biological variation components of glycated hemoglobin HbA1c on tosoh G7 HPLC

Introduction

The American Diabetes Association (ADA) has recommended glycated hemoglobin (HbA1c) as a possible alternative for fasting blood glucose in diagnosis of diabetes [1] According to the suggestions of the ADA, generally HbA1c, fasting plasma glucose, and 2-h plasma glucose during 75-g oral glucose tolerance test are equally appropriate tests for both diabetes screening and diagnosis [2]. After providing the traceability of the existing commercial HbA1c measurement methods to the reference system, analytical targets must be met to ensure the clinical safety of the HbA1c test [3]. The generally accepted method for this purpose is determination of analytical performance target using the biological variation of the analyte of interest. For this reason, it is important to obtain reliable data on biological variation [4].

Biological variation (BV) is defined as an inherent fluctuation that can be described as random variation around the mean concentration during steady-state periods [4]. Biological variation has generally been defined as random fluctuation around the homeostatic set point for each individual (within-subject or intra-individual biological variation) and the differences between the set points of different individuals (between-subject or inter-individual biological variation). These are usually considered as coefficients of variation (CV) and termed CVI and CVG, respectively [5]. Determining the biological variation values and subsequently estimating quality goals of HbA1c are of high importance for optimal patient care [6].

Information about BV is also a prerequisite for the calculation of reference change value (RCV), which is defined as the minimal difference that must be exceeded for a change in two consecutive results in the same individual to become clinically relevant, which depends on both analytical variations (CVA) and within-subject biological variations (CVI) [4]. The clinician will be guided by determining the reference change value (RCV) calculated by considering the analytical systems of HbA1c and variations due to normal biological fluctuations to determine whether the difference between consecutive samples of the individual is indicative of a clinically significant difference.

In the literature there is a wide heterogeneity in data on components of biological variation for HbA1c. In prospective studies, biological variation values of HPLC methods in different devices have been calculated but, to our knowledge, no studies have been performed with the analyser Tosoh G7 (HPLC) to date.

The aim of this study was to estimate the components of biological variation for HbA1c measured by G7 HPLC analyser (Tosoh Bioscience, USA).

Materials And Method

Study subjects

The study was initially planned to include 17 healthy participants, but 5 of them were excluded from the study during follow-up due to discontinuing blood sampling. Therefore, the study group consisted of 12 healthy volunteers (9 male, 3 female). The study protocol was approved by the local ethics committee, and written informed consent was obtained from all participants. The inclusion criteria were that the subjects had no predispositions for diabetes, thalassemia syndrome, or other hemoglobinopathies and, for women, to have regular menstrual cycles and to not be using hormonal contraceptives. Smokers and subjects taking any medication were excluded from the study. Furthermore, fasting plasma glucose and body mass index (BMI) were < 6.0 mmol/L and <30.0 kg/m2, respectively, in all subjects.

Venous blood samples were collected on the same day once every two weeks for two months (January to March). Venous blood samples were collected between 09.00 and 09.30 a.m. after 8 h fasting in the sitting position for 1–5 min with minimal stasis by the same skilled phlebotomist to minimize procedural variations. Whole blood specimens were taken into evacuated collection tubes containing K3 EDTA (BD vacutainer) for HbA1c and stored at -800C until the time of analysis.

Analytical procedure

HbA1c was measured by G7 HPLC (non-porous ion-exchange) Analyser (Tosoh Bioscience, USA). Analysis was performed without off-line sample pretreatment or interference from Schiff base. The analyser dilutes the whole blood with Hemolysis & Wash Solution, and then injects a small volume of this specimen onto the analytical column. Separation is achieved by utilizing differences in ionic interactions between the cation exchange group on the column resin surface and the hemoglobin components. The hemoglobin fractions are subsequently removed from the column by performing a step-wise elution using the different salt concentrations in the Elution Buffers HSi Variant 1,2, and 3. The time that passes from injection of the sample to the time the specific peak elutes off the column is called “retention time.” The G7 software has been written so that each of the expected fractions has a window of acceptable retention times. When the designated peak falls within the expected window, the chromatogram peaks will be properly identified. If a peak elutes at a retention time not within a specified window, an unknown peak (POO) is detected. The separated hemoglobin components pass through the LED photometer flow cell where the analyser measures changes in absorbance at 415 nm. The analyser integrates and reduces the raw data, and then calculates the relative percentages of each hemoglobin fraction. The area of the S- Alcis is divided by the sum of the total areas of all peaks up to and including the Ao to obtain a raw S-A1c percentage. This uncorrected result is substituted as the “x” value in the linear regression formula determined during calibration. The analyser prints the final numerical results and plots a chromatogram showing changes in absorbance versus retention time for each peak fraction. The G7 Variant Analysis Mode is NGSP* certified and the final reportable result is traceable to the Diabetes Control and Complications Trial (DCCT).

All samples were assayed in duplicate, using the analytical system in accordance with the manufacturer’s instructions and checking its alignment before the run by HbA1c control materials according to the manufacturer’s established validation range. The same lots of calibrators, reagent lot- and quality-control materials were used throughout the study and analyses were performed by a single analyst.

Statistical analysis

Microsoft Office Excel 2007 and ANOVA tests were used for the statistical analysis. Before performing calculations with the patients’ results, the Cochrane test was applied to exclude outlying values from the individual subjects, and the Reed test to eliminate mean outlying values. Analytical variation (CVA) was calculated from the duplicate results of each sample. Mean and standard deviation (SD) of the duplicate results were calculated, and the following formula, CVA = SD*100/mean, was used to calculate the within-run CVA%.

ANOVA test was used to estimate CVA plus CVI (CVA+I). CVI was calculated by a subtraction step with the two previous variables (CVA+I – CVA).

CVI =(CVA+I2 -CVA2)1/2

Between-subject BV (CVG) was obtained by subtracting the CVA+I from the total variation (CVT) found by using all data from all patients.

CVG = (CVT2– CVI2 – CVA2)1/2

The index of individuality (II) was calculated using the CVI/CVG ratio. The RCVs for the analytes at various probabilities, which were applied to either a single quantity or to two combined quantities, were calculated using the following formula:

RCV = 21/2 x Z x (CVA2 +CVI 2 )1/2

Z; probability selected for statistical significance (Z = 1.96 at 95% confidence interval, CI).

Results

The demographic characteristics and laboratory parameters of healthy subjects are summarized in Table 1. The volunteers’ mean of HbA1c was 5.10% (32.3 mmol/mol). The CVI and CVG for HbA1c were 1.69% and 7.56% respectively. CVA was calculated as 2.81%. These results indicated that our data was reliable for calculating BV. The II of the HbA1c was 0.22. When the index is less than 1, two consecutive results from a subject could be outside the RCV but may lie within the population-based reference interval. Thus, the reference values would be of little use, in particular, for deciding whether a significant change had occurred. For this reason, RCV was calculated and found to be 9.08% for HbA1c (at 95% CI). The results of the HbA1c calculations for CVA, CVI, CVG, RCV, and II (CVI/CVG) are presented in Table 2. Additionally, Figure 1 and Figure 2 present the median and absolute (minimum maximum) range of the HbA1c concentrations for each study participant as % and mmol/mol units.

Discussion

Although many studies have been performed with the ion-exchange method, it is important to calculate the device-specific biological variation, because CVA affects all CVI and CVG. The present study is the first one to determine the BV of HbA1c values measured by the Tosoh G7 system. In order to improve analytical targets in HbA1c monitoring, the patterns revealed by Fraser and Harris [7] must be accurately observed and traceability of the HbA1c test with reference material should be ensured. From the analytical point of view, the determination of HbA1c was carried out by the ion-exchange method accepted as the gold standard [8].

Studies conducted with ion-exchange methods revealed similar CV values to our study. The data are presented in Table 3. Kolatkar et al. [6] designed a study in 29 insulin-dependent diabetics for three months sampling interval revealed CVI 2.4%. In another study using ion-exchange method, Carlsen et al. [9] designed a group consisting of 6 female and 9 male between 27 and 59 years of age with a mean time from tip 1 DM diagnosis of 151 months (range 21–408). Researchers evaluated CVI and CVG values of 1.2% and 5.7%, respectively. For a one-year period, Desmeules et al. [10] conducted a study in 38 (24 male, 14 female) pediatric cases with cystic fibrosis but without diabetes or impaired glucose tolerance with the ion-exchange method. In this period they collected five samples from each patient and calculated CVI and CVG values of 4.8% and 12.8%. For this study, we can see that as the sampling period increases, the CVA increases too, which means that CVI and CVG are affected. The CVI calculated for HbA1c was 4.8% and the between-subject variation (CVG) was 12.8%. In the present study we collected samples from healthy volunteers at intervals of two weeks over the course of two months. As in the previous studies we calculated CVI as 1.69%. Our CVG values were higher than in the other studies (7.56%). Studies that are conducted with the boronate affinity method had different results than those designed with ion-exchange. Ucar et al. [11] calculated CVI and CVG as 2.15% and 4.03%. During 7 days and for 12 samples in 45 healthy men CVI and CVG values were reported as 1.7% and 4%. With the boronate affinity method CVG values are lower than with ion-exchange chromatography. A design similar to our study was conducted by Braga et al. [12] which used the competitive immunoassay method. They followed 18 healthy volunteers for two months and collected five samples from each subject and calculated CVI as 2.5% and CVG as 7.1%. When we analyzed these robust data, it is clear that the BV of the HbA1c test was affected by sampling interval and periods.

The index of individuality (II) parameter gives information about the appropriateness of the evaluation of test results according to reference ranges. A value of II <1 points out high individuality which means that the test results of an individual should be interpreted according to his/her own previous test results instead of pre-set reference values. The individuality indices were found to be 0.35 and 0.93 for ion-exchange chromatography and boronate affinity chromatography, respectively, by Ucar et al.[11] Other studies of individuality indices for ion-exchange chromatography found II values as 0.27 and 0.96 by Kilpatric et al.[13] and Garde et al.[14], respectively. Similarly, the calculated II values for HbA1c in our study were 0.22 (Table 3). This value demonstrated that evaluation of HbA1c test results should not be made according to population-based reference values.

RCV, also an important parameter in BV for many analytes, could be calculated using BV data from a healthy population. RCV is defined as the critical difference that must be exceeded between two sequential analyses from the same patient. RCV is used when the results from consecutive measurements from the same patient fall within the reference range but represent a considerable change, which indicates a change in the patient’s condition [4]. Ucar et al.[11] estimated RCV (critical difference) as 5.45% for ion-exchange chromatography and 10.40% for boronate affinity chromatography; another study conducted by Braga et al. [12] found RCV (critical difference) as 9.5%. These studies investigated how RCV and results are affected by different methods. RCV values measured by HPLC were found to be about 5% lower than by boronate affinity. For the immunoturbidimetric method, these values vary by up to 30% (Table 3). These results indicate that when calculating RCV we should carefully notice the method that was used.

All of the estimations [minimum, desirable and optimal analytical goals for imprecision, bias, total error] were calculated based on data about CVI, CVG, and CVA for the ion-exchange method (Table 4).

In conclusion, knowledge about BV parameters and RCV values for HbA1c might be important for the optimum use of this test in clinical laboratories and reliable calculation of analytical performance. This preliminary research contributes to the literature on BV data and calculation of RCV for HbA1c measured by the G7 HPLC analyser and may be improved with a follow-up study including a larger, gender-matched participant group.

Scientific Responsibility Statement

The authors declare that they are responsible for the article’s scientific content including study design, data collection, analysis and interpretation, writing, some of the main line, or all of the preparation and scientific review of the contents and approval of the final version of the article.

Animal and human rights statement

All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article.

Funding: None

Conflict of interest

None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.

References

1. Sherwani SI, Khan HA, Ekhzaimy A, Masood A, Sakharkar MK. Significance of HbA1c test in diagnosis and prognosis of diabetic patients. Biomark Insights. 2016;11:95-104.

2. American Diabetes Association. 2. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes-2018. Diabetes care. 2018;41(Suppl 1):S13-s27

3. Braga F, Dolci A, Mosca A, Panteghini M. Biological variability of glycated hemoglobin. Clin Chim Acta. 2010;411(21-22):1606-10

4. Ricos C, Perich C, Minchinela J, Alvarez V, Simons S et al. Application of biological variation- a review. Biochemia Medica 2009; 19(3):250-9

5. Biosca C, Ricós C, Jiménez CV, Lauzurica R, Galimany R. Are equally spaced specimen collections necessary to assess biological variation? Evidence from renal transplant recipients. Clin Chim Acta. 2000;301(1-2):79-85.

6. Kolatkar NS, Cembrowski GS, Callahan PL, Etzwiler D. Intensive diabetes management requires very precise testing of glycohemoglobin. Clin Chem 1994;40(8): 1608–10

7. Fraser GG, Harris EK. Generation and application of data on biological variation in clinical chemistry. Crit Rev Clin Lab Sci.1989;27(5):409-37.

8. Karami A, Baradaran A. Comparative evaluation of three different methods for HbA1c measurement with High-performance liquid chromatography in diabetic patients. Advanced biomedical research. 2014;3:94.

9. Carlsen S, Petersen PH, Skeie S, Øyvind S, Sandberg S. Within-subject biological variation of glucose and HbA1c in healthy persons and in type 1 diabetes patients. Clin Chem Lab Med. 2011;49(9):1501-7.

10. Desmeules P, Cousineau J, Allard P. Biological variation of glycated haemoglobin in a paediatric population and its application to calculation of significant change between results. Ann Clin Biochem. 2010;47(1):35-8

11. Ucar F, Erden G, Ginis Z, Ozturk G, Sezer S, Gurler M, et al Estimation of biological variation and reference change value of glycated hemoglobin (HbA1c) when two analytical methods are used. Clin Biochem. 2013;46(15):1548-53.

12. Braga F, Dolci A, Montagnana M, Pagani F, Paleari R, Guidi GC. Revaluation of biological variation of glycated hemoglobin (HbA1c) using an accurately designed protocol and an assay traceable to the IFCC reference system. Clin Chim Acta. 2011;412(15-16):1412-6.

13. Kilpatrick ES, Maylor PW, Keevil BG. Biological variation of glycated haemoglobin. Implications for diabetes screening and monitoring. Diabetes Care 1998;21(2): 261–4.

14. Garde AH, Hansen AM, Skovgaard LT, Christensen J. Seasonal and biological variation of blood concentrations of total cholesterol, dehydroepiandrosterone sulfate, hemoglobin A1c, IgA, prolactin, and free testosterone in healthy women. Clin Chem 2000;46(4): 551–9.

15. Rohlfing C, Wiedmeyer HM, Little R, Grotz V, Tennill A, England J. Biological variation of glycohemoglobin. Clin Chem 2002;48(7):1116–8.

How to cite this article:

Ercan M, Avcı E, Akbulut ED, Oğuz EF, Topçuoğlu C, Yücel Ç, Abuşoğlu S, Turhan T. Biological variation components of glycated hemoglobin HbA1c on tosoh G7 HPLC. J Clin Anal Med 2018;9(6): 469-73.

Table 1. The main characteristics of healthy subjects.

Parameter

All group (n=12)

Female (n=3)

Male (n=9)

Age (year) mean±SD

43.5±9.04

34±6.16

45.77±7.87

BMI(kg/m2) mean±SD

23.63±1.3

22.68±1.68

23.95±1.08

HbA1c (%) mean±SD

(mmol/mol) mean±SD

5.10±0.30

32.33±3.33

4.82±0.23

29.25 ±2.37

5.20±0.25

33.36±2.96

Figure 1. Mean values and absolute range of HbA1c (mmol/mol)

Figure 2. Mean values and absolute range of HbA1c (%)

Table 2. Mean value, estimated average variance components, and indices derived from data on biological variation of HbA1c.

Group

CVA%

CVI%

CVG%

II

RCV%

 

2.81

1.69

7.56

0.22

9.08

Table 3. Present and previous studies on biological variability of HbA1c.

 

Groups specifics

no. of samples

frequency

Mean HbA1c values (%)

Method

CVA (%)

CVI (%)

CVG (%)

RCV (%)

II

Present study

12 (9 M, 3 F, 27–54 years), healthy subjects

2 months, 4 samples

5.10 (32.3 mmol/mol)

HPLC (ion-exchange)

2,81

1,69

7,56

9,08

0,22

Ucar et. al. 2013(11)

29 (9 M, 20 F, 20–45 years) healthy subjects

3 months,

4 samples

-

HPLC (ion-exchange)

1.6

1.17

5.58

5.45

0.35

24 (7 M, 17 F, 20–45 years) healthy subjects

HPLC (boronate-affinity)

3.12

2.15

4.03

10.40

0.93

Braga et. al. 2011(12)

18 (9 M, 9 F, 26–52 years) healthy subjects

2 months, 5 samples

5.47

(36.3 mmol/mol)

Competitive immunoassay

2.4

2.5

7.1

9.5

0.35

Carlsen et al. 2011(9)

15 (6 M, 9 F) healthy subjects

7 days, 10 samples

5.1

HPLC (ion-exchange)

0.5

1.2

5.7

3.6

-

15 (9 M, 6 F) type 1 diabetes (2 patients excluded)

7.0

0.6

1.7

8.2

5

-

Desmeules et al. 201010)

38 (24 M, 14 F) pediatric with cystic fibrosis but without diabetes or impaired glucose tolerance

1 year, 5 samples

-

HPLC (ion-exchange)

1.4

4.8

12.8

-

-

Rohlfing et al. 200215)

45 healthy subjects (only men)

7 days, 12 samples

4.9

HPLC (boronate-affinity)

2.3 (between day)

1.7

4

-

-

1.5

(within day)

Kolatkar et al. 1994(6)

29 insulin-dependent diabetics

3 months, NS

6.5

HPLC (ion-exchange)

1.4

2.4

-

-

-

Table 4. Quality specifications for HbA1c measurement derived from our data on biological variation

Quality level

İmprecision, %

Bias,%

TEa%

Optimal

<0.42

<0.95

<1.64

Desirable

<0.63

<1.91

<2.94

Minimal

<1.26

<2.87

<3.90

Additional Info

  • Recieved: 15.02.2018
  • Accepted: 26.03.2018
  • Published Online: 28.03.2018
  • Printed: 01.11.2018
  • DOI: DOI: 10.4328/JCAM.5761
  • Author: Müjgan Ercan, Esin Avcı, Emiş Deniz Akbulut, Esra Fırat Oğuz, Canan Topçuoğlu, Çiğdem Yücel, Sedat Abuşoğlu, Turan Turhan
  • Identifier: DOI: 10.4328/JCAM.5761
  • Index Page: 469-473
  • How to Cite: Ercan M, Avcı E, Akbulut ED, Oğuz EF, Topçuoğlu C, Yücel Ç, Abuşoğlu S, Turhan T. Biological variation components of glycated hemoglobin HbA1c on tosoh G7 HPLC. J Clin Anal Med 2018;9(6): 469-73.
  • Running Title: Biological variation components of HbA1c
Download attachments:

Article & Author Search JCAM