HbA1c FS*

Diagnostic reagent for quantitative in vitro determination of hemoglobin A1c (HbA1c) in whole blood on photometric
systems

Order Information Reagents

Cat. No. Kit size Components and Concentrations
1 3348 99 10 930 R1 3 x 18 mL + R2 3 x 6 mL R1: Buffer 100 mmol/L
1 4590 99 10 113 1 x 500 mL HbA1c net Hemolyzing Solution FPOX ≥ 0.5 kU/L
1 3350 99 10 044 2 x 0.3 mL TruCal HbA1c net Ethlyene glycol derivative < 10%
R2: Buffer 20 mmol/L
Summary [1,2,3,11,14] Protease ≥ 500 kU/L
Hemoglobin A1c (HbA1c) is a glycated hemoglobin which is formed by the Chromogen ≥ 0.05 mmol/L
non-enzymatic reaction of glucose with native hemoglobin. This process Ethlyene glycol derivative < 10%
runs continuously throughout the circulatory life of erythrocytes (average life
time 100 – 120 days). The rate of glycation is directly proportional to the Storage Instructions and Reagent Stability
concentration of glucose in blood. The blood level of HbA1c represents the The reagents are stable up to the end of the indicated month of expiry, if
average blood glucose level over the last 3 months. Therefore, HbA1c is stored at 2 – 8°C and contamination and evaporation are avoided. Do not
suitable for retrospective long-term monitoring of blood glucose freeze the reagents! Protect reagents from light!
concentration in diabetics. Clinical studies have shown that lowering of
Reagent Preparation
HbA1c level can help to prevent or delay the incidence of late diabetic
complications. Besides, HbA1c testing can be used for diagnosis of diabetes The reagents are ready to use.
mellitus. Bring HbA1c net Hemolyzing Solution to room temperature and homogenize
As the amount of HbA1c also depends on the total quantity of hemoglobin, by repeated inversion. Due to composition of the hemolyzing solution an
the HbA1c value is indicated as ratio of the total hemoglobin concentration. opalescent and slightly turbid appearance remains. Avoid foaming! Do not
shake!
Method Warnings and Precautions
Hemoglobin: Photometric test 1. Reagent 1 contains animal material. Handle the product as potentially
HbA1c: Colorimetric, enzymatic method infectious according to universal precautions and good clinical
laboratory practices.
Principle 2. Hemoglobin and HbA1c values in g/dL determined with DiaSys HbA1c
The concentrations of HbA1c and hemoglobin are determined separately net FS are used to calculate the HbA1c ratio from total hemoglobin
and are used to calculate the HbA1c ratio from total hemoglobin exclusively. exclusively. Individual results for hemoglobin and HbA1c must not be
used for diagnostic purposes.
Hemoglobin measurement
3. Falsely low values (low HbA1c despite high blood glucose) may occur
Whole blood samples are lysed with hemolyzing solution. Hemoglobin is
in people with conditions such as shortened red blood cell survival
released from the erythrocytes. The absorbance of hemoglobin is measured
(e.g. hemolytic diseases) or significant recent blood loss during the
at 570 nm after addition of reagent R1 and is proportional to the total
weeks before (higher fraction of young erythrocytes). Falsely high
hemoglobin concentration in the sample.
values (high HbA1c despite normal blood glucose) have been reported
HbA1c measurement [16] in iron deficiency anemia (high proportion of old erythrocytes). These
After addition of R2, fructosylated dipeptides from the N-terminal part of the circumstances have to be considered in clinical interpretation of
hemoglobin β-chain are released by a protease. Hydrogen peroxide (H2O2) HbA1c values. Care must also be taken in clinical interpretation of
is produced by oxidative cleavage of fructosylated dipeptides by FPOX HbA1c results from patients with hemoglobin variants.
(fructosyl peptide oxidase). The H2O2 generated is determined 4. In very rare cases, samples of patients with gammopathy might give
colorimetrically by reaction with a chromogen in presence of peroxidase at falsified results [15].
660 nm. The absorbance increase is proportional to the HbA1c 5. N-acetylcysteine (NAC), acetaminophen and metamizole medication
concentration. leads to falsely low results in patient samples.
6. Please refer to the safety data sheets and take the necessary
Standardization precautions for use of laboratory reagents. For diagnostic purposes,
The assay is standardized according to IFCC [4] and DCCT/NGSP [7] results should always be assessed with the patient’s medical history,
reference methods. Calculation of patient and control values is possible clinical examinations and other findings.
according to IFCC [mmol/mol] as well as according to DCCT/NGSP [%]. 7. For professional use only!

NGSP and IFCC values show a linear relationship and, therefore, can be Waste Management
calculated from each other using the following equation: Please refer to local legal requirements.
HbA1c (IFCCb) = (HbA1c (NGSPa) – 2.15) / 0.0915 Materials Required but not Provided
HbA1c (NGSPa) = 0.0915 x HbA1c (IFCCb) + 2.15 General laboratory equipment
a: NGSP values in % Specimen
b: IFCC values in mmol/mol
Whole blood collected with EDTA
IFCC: International Federation of Clinical Chemistry [4,5,10] Please collect whole blood by standard venipuncture and fill the blood
DCCT: Diabetes Control and Complications Trial [6] collection tube according to manufacturer specifications.
NGSP: National Glycohemoglobin Standardization Program [7]
Specimen Stability [8]:
HbA1c and Average Glucose Concentrations [11] Whole blood 1 week at 2 – 8°C
Hemolysate 1 hour at 15 – 25°C
Due to a linear correlation between hemoglobin A1c and average glucose
Discard contaminated specimens.
concentrations, HbA1c values can be converted into estimated average
glucose values by means of the following equations:
Standardization according to IFCC (calculated referring to literature
reference [11]):
Average glucose conc. [mg/dL] = 2.63 x HbA1cb + 15.01
Average glucose conc. [mmol/L] = 0.146 x HbA1cb + 0.829
b: HbA1c values in mmol/mol IFCC
Standardization according to NGSP:
Average glucose concentration [mg/dL] = 28.7 x HbA1ca – 46.7
Average glucose concentration [mmol/L] = 1.59 x HbA1ca – 2.59
a: HbA1c-values in % NGSP
No significant differences in the regression equation were observed for
variations in individuals tested regarding sex, presence or absence of
diabetes, type of diabetes, age, race, and ethnicity. Although this equation
can be used for the majority of individuals each laboratory has to verify
whether the regression equations mentioned are applicable for the patient
group to be examined.

HbA1c net FS – Page 1 * Fluid Stable

Sample Preparation Calibrators and Controls
DiaSys HbA1c net Hemolyzing Solution is required for sample preparation. DiaSys TruCal HbA1c net calibrator is recommended for calibration. The
Calibrators, controls and samples have to be hemolyzed before use. assigned values of TruCal HbA1c net have been made traceable to the
Hemolysates have to be processed within 1 hour after production. approved IFCC reference method [4]. DiaSys TruLab HbA1c net controls
Processing in batch mode is recommended. Please refer to subsequent should be assayed for internal quality control. Each laboratory should
pipetting scheme for manual hemolysis: establish corrective action in case of deviations in control recovery.
Preparation
Calibrator Calibrator Cat. No. Kit size
Control Sample
Level 1 Level 2 TruLab HbA1c net Level 1 5 9930 99 10 076 6 x 1 mL
TruCal HbA1c net TruLab HbA1c net Level 2 5 9940 99 10 076 6 x 1 mL
16 µL - - -
Level 1
TruCal HbA1c net
- 50 µL - - Performance Characteristics
Level 2
TruLab HbA1c net Measuring Range
Level 1 and Level 2 - - 50 µL 50 µL The assay has got a measuring range from 20 – 150 mmol/mol according to
/Sample IFCC (4 – 16% according to DCCT/NGSP).
Add The assay is applicable for hemoglobin concentrations in blood from
HbA1c net 6 – 30 g/dL (3.73 – 18.6 mmol/L).
Hemolyzing 1000 µL 1000 µL 1000 µL 1000 µL
solution Specificity/Interferences
Mix and allow standing for 1 minute. Hemolysis is completed after 1 minute. A
slight turbidity remains due to the composition of the hemolyzing solution. According to CLSI protocol EP7-A2, a study on interferences was
conducted.
IFCC
Assay Procedure For each interfering substance three samples with different hemoglobin and
Application sheets for automated systems are available on request. HbA1c values have been tested; a low level sample within a hemoglobin
range of 8 – 10 g/dL and a HbA1c range within 28 – 35 mmol/mol; a medium
Please refer to your distributor.
level sample within a hemoglobin range of 11 – 15 g/dL and a HbA1c range
Basic parameters for Hitachi 917 with TWIN application and within 28 – 35 mmol/mol; a high level sample within a hemoglobin range of
manual calibrator/control/sample hemolyzation 11 – 15 g/dL and a HbA1c range > 60 mmol/mol.
DCCT/NGSP
Hemoglobin determination
For each interfering substance three samples with different hemoglobin and
Wavelength (main/sub) 570/800 nm (bi-chromatic) HbA1c values have been tested; a low level sample within a hemoglobin
Temperature 37°C range of 9 – 10 g/dL and a HbA1c range within 4.7 – 5.4%; a medium level
Measurement TWIN test/3-point sample within a hemoglobin range of 10 – 15 g/dL and a HbA1c range within
4.7 – 5.4%; a high level sample within a hemoglobin range of 10 - 15 g/dL
Sample/Calibrator 30 µL and a HbA1c range > 7.65%.
Reagent 1 180 µL
Reagent 2 60 µL The table below summarizes the results which comply for all tested levels
Addition Reagent 2 Cycle 15 using IFCC as well as DCCT/NGSP standardization.
Absorbance Cycle 15 Interfering substance Interferences < 10% in serum
Calibration linear with hematocrit correction
HbA1c determination Ascorbate up to 50 mg/dL
Bilirubin (conjugated and
Wavelength (main/sub) 660/800 nm (bi-chromatic) up to 10 mg/dL
unconjugated)
Temperature 37°C Glucose up to 1000 mg/dL
Measurement TWIN test/3-point
Hemoglobin, acetylated up to 10 mmol/L
Sample/Calibrator 30 µL Hemoglobin, carbamylated up to 10 mmol/L
Reagent 1 180 µL Lipemia (triglycerides)
up to 400 mg/dL
Reagent 2 60 µL at < 11 g/dL hemoglobin
Addition Reagent 2 Cycle 15 Lipemia (triglycerides)
up to 750 mg/dL
Absorbance 1 Cycle 18 at > 11 g/dL hemoglobin
Absorbance 2 Cycle 34 N-acetylcysteine (NAC) up to 2000 mg/L
Calibration linear Urea up to 300 mg/dL
Uric acid up to 20 mg/dL
Calibration Alcoholism and ingestion of large doses of aspirin may lead to im-
plausible results. For further information on interfering substances refer
The concentrations of HbA1c and hemoglobin in unknown samples are
to Young DS [13].
derived from linear calibration curves.
Each calibration curve is obtained with 2 calibrators at different levels Hemoglobin variants may lead to deviant HbA1c results. The tested
without a zero value. Hemoglobin variants HbS, HbC, HbD, HbE, HbJ, HbG, HbSC, HbSE, HbEE
and HbF showed no significant interference.
Stability of calibration: 6 weeks
Target
Calculation Hemoglobin
Percentage of
Value range
Mean
Hemoglobin Recovery
After entering the calculation formula into the instrument, the calculation of Variant HbA1c
Variant (≤) HbA1c [%]
HbA1c ratio from total hemoglobin is done by the instrument automatically. [% DCCT/NGSP]
Please refer to the instrument manual. AS 40% S 5.2 – 8.8 94.7
AC 36% C 5.0 – 7.4 97.1
Depending on the standardization selected, enter the following formula:
AD 41% D 5.6 – 7.0 93.9
IFCC AE 26% E 5.9 – 7.6 99.1
Values in mmol/mol according to IFCC: AJ 50% J 5.2 – 8.4 100
AG 20% G 6.1 – 6.6 97.4
 HbA1c [g / dL ]  SC 52% S, 44%C 4.5 – 7.0 91.6
HbA1c [mmol / mol ] =   × 1000
 Hb [g / dL ]  SE 65% S, 27% E 7.4 95.4
DCCT/NGSP EE 94% E 5.1 – 8.9 98.0
Values in percent according to DCCT/NGSP: Elevated F 4.6% F 6.5 – 8.1 93.6
Sensitivity/Limit of Detection
 HbA1c [g / dL ]  HbA1c: 0.2 g/dL
HbA1c [%] =  91.5 x  + 2.15
 Hb [g / dL ]  Hemoglobin: 1.5 g/dL

HbA1c net FS – Page 2

Imprecision
Values according to IFCC (Hitachi 917) Manufacturer
Within-run precision Mean SD CV DiaSys Diagnostic Systems GmbH
IVD Alte Strasse 9 65558 Holzheim Germany
n = 20 [mmol/mol] [mmol/mol] [%]
Sample 1 29.5 0.556 1.88
Sample 2 32.9 0.197 0.598
Sample 3 63.5 0.447 0.703

Total precision
Mean SD CV
CLSI
[mmol/mol] [mmol/mol] [%]
n = 80
Sample 1 26.0 1.01 3.88
Sample 2 32.5 1.23 3.78
Sample 3 66.2 1.23 1.86
Method Comparison
A comparison of DiaSys HbA1c net FS (y) to an immunoturbidimetric assay
(x) using 60 samples gave following results (IFCC values):
y = 1.047 x – 0.782 mmol/mol; r = 0.982

A comparison of DiaSys HbA1c net FS (y) to a HPLC assay (x) using 100
samples gave following results (IFCC values):
y = 1.031 x + 0.441 mmol/mol; r = 0.989

Reference Range
Suggested target values for HbA1c [9]:
IFCC NGSP
[mmol/mol] [%]
Non-diabetics 20 – 42 4–6
Target of therapy < 53 <7
Change of therapy > 64 >8
Each laboratory should check if the reference ranges are transferable to its
own patient population and determine own reference ranges if necessary.

HbA1c cut point value for diagnosis of diabetes mellitus [14]:

According to a recommendation of the American Diabetes Association
(ADA): ≥ 6.5% (NGSP) (48 mmol/mol (IFCC))
Patients with HbA1c values in the range of 5.7 - 6.4% HbA1c (NGSP) or
39 - 46 mmol/mol HbA1c (IFCC) may be at high risk of developing diabetes.

Literature
1. Thomas L. Clinical Laboratory Diagnostics. 1st ed. Frankfurt: TH-Books
Verlagsgesellschaft; 1998. p. 142-48.
2. Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER, editors. Tietz
Textbook of Clinical Chemistry. 3rd ed. Philadelphia: W.B. Saunders
Company; 1999. p. 790-6.
3. Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER, Bruns DE,
editors. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics.
4th edition St. Louis Missouri: Elsevier Saunders; 2006; p. 878-884.
4. Jeppsson JO, Kobold U, Barr J, Finke A et al. Approved IFCC reference
method for the measurement of HbA1c in human blood. Clin Chem Lab
Med 2002; 40: 78-89.
5. Hoelzel W, Weykamp C 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): 166-74.
6. The Diabetes Control and Complications Trial Research Group. The
effect of intensive treatment of diabetes in the development and
progression of long-term complications in insulin-dependent diabetes
mellitus. N Engl J Med.1993; 329: 977-86.
7. Little RR, Rohlfing CL, Wiedmeyer HM, Myers GL et al. The National
Glycohemoglobin Standardization Program: A Five-Years Progress
Report. Clin Chem 2001; 47: 1985-92.
8. Data on file at DiaSys Diagnostic Systems GmbH.
9. Pantheghini M, John WG on behalf of the IFCC Scientific Division.
Implementation of haemoglobin A1c results traceable to the IFCC
reference system: the way forward. Clin Chem Lab Med 2007; 45(8):
942-4.
10. Nordin G., Dybkær R. Recommendation for term and measurement unit
for “HbA1c”. Clin Chem Lab Med 2007; 45(8): 1081-2.
11. Sacks DB. Translating Hemoglobin A1c into Average Blood Glucose:
Implications for Clinical Chemistry. Clinical Chemistry 2008; 54: 1756-8.
12. Weykamp C. Carbamylated Hemoglobin Interference in
Glycohemoglobin Assays. Clin Chem 1999; 45: 438-9.
13. Young DS. Effects of Drugs on Clinical Laboratory Tests. 5th ed.
Volume 1 and 2. Washington, DC: The American Association for
Clinical Chemistry Press 2000.
14. Sacks DB, Arnold M, Bakris GL, Bruns DE, AR Horvath et al.
Guidelines and recommendations for laboratory analysis in the
diagnosis and management of diabetes mellitus. Clin Chem 2011;
57(6): e1-e47.
15. Bakker AJ, Mücke M. Gammopathy interference in clinical chemistry
assays: mechanisms, detection and prevention. ClinChemLabMed
2007;45(9):1240–1243.
16. Ferri S, Kim S, Tsugawa W, Sode K. Review of Fructosyl Amino Acid
Oxidase Engineering Research: A Glimpse into the Future of
Hemoglobin A1c Biosensing. Journal of Diabetes Science and
Technology 2009; 3(3): 585-592.

HbA1c net FS – Page 3 844 3348 10 02 00 August 2020/4