HbA1c FS*

Diagnostic reagent for quantitative in vitro determination of hemoglobin A1c (HbA1c) in whole blood on
BioMajesty JCA-BM6010/C

Order Information Storage Instructions and Reagent Stability

Cat. No. 1 3348 99 10 962 The reagents are stable up to the end of the indicated month of expiry, if
R1: 6 x 300 tests stored at 2 – 8°C and contamination and evaporation are avoided. Do not
R2: 6 x 300 tests freeze the reagents! Protect reagents from light!
Cat. No. 1 3348 99 10 964 Warnings and Precautions
R1: 2 x 160 tests 1. Reagent 1 contains animal material. Handle the product as potentially
R2: 2 x 160 tests infectious according to universal precautions and good clinical
laboratory practices.
Method
2. Hemoglobin and HbA1c values in g/dL determined with DiaSys
Hemoglobin: Photometric test HbA1c net FS are used to calculate the HbA1c ratio from total
HbA1c: Colorimetric, enzymatic method hemoglobin exclusively. Individual results for hemoglobin and HbA1c
must not be used for diagnostic purposes.
Principle 3. Falsely low values (low HbA1c despite high blood glucose) may occur
The concentrations of HbA1c and hemoglobin are determined separately in people with conditions such as shortened red blood cell survival
and are used to calculate the HbA1c ratio from total hemoglobin exclusively. (e.g. hemolytic diseases) or significant recent blood loss during the
Hemoglobin measurement weeks before (higher fraction of young erythrocytes). Falsely high
Whole blood samples are lysed with hemolyzing solution. Hemoglobin is values (high HbA1c despite normal blood glucose) have been reported
released from the erythrocytes. The absorbance of hemoglobin is measured in iron deficiency anemia (high proportion of old erythrocytes). These
at 571 nm after addition of reagent R1 and is proportional to the total circumstances have to be considered in clinical interpretation of
hemoglobin concentration in the sample. HbA1c values. Care must also be taken in clinical interpretation of
HbA1c results from patients with hemoglobin variants.
HbA1c measurement [16] 4. In very rare cases, samples of patients with gammopathy might give
After addition of R2, fructosylated dipeptides from the N-terminal part of the falsified results [15].
hemoglobin β-chain are released by a protease. Hydrogen peroxide (H2O2) 5. N-acetylcysteine (NAC), acetaminophen and metamizole medication
is produced by oxidative cleavage of fructosylated dipeptides by FPOX leads to falsely low results in patient samples.
(fructosyl peptide oxidase). The H2O2 generated is determined 6. Please refer to the safety data sheets and take the necessary
colorimetrically by reaction with a chromogen in presence of peroxidase at precautions for the use of laboratory reagents. For diagnostic
658 nm. The absorbance increase is proportional to the HbA1c purposes, results should always be assessed with patient`s medical
concentration. history, clinical examinations and other findings.
7. For professional use only!
Standardization
Waste Management
The assay is standardized according to IFCC [1] and DCCT/NGSP [4]
reference methods. Calculation of patient and control values is possible Please refer to local legal requirements.
according to IFCC [mmol/mol] as well as according to DCCT/NGSP [%]. Reagent Preparation
NGSP and IFCC values show a linear relationship and, therefore, can be The reagents are ready to use. The bottles are placed directly into the
calculated from each other using the following equation: reagent trays.
Homogenize HbA1c net Hemolyzing Solution by repeated inversion. Due to
HbA1c (IFCCb) = (HbA1c (NGSPa) – 2.15) / 0.0915
composition of the hemolyzing solution an opalescent and slightly turbid
HbA1c (NGSPa) = 0.0915 x HbA1c (IFCCb) + 2.15 appearance remains. Avoid foaming! Do not shake!
a: NGSP values in % Specimen
b: IFCC values in mmol/mol
Whole blood collected with EDTA
IFCC: International Federation of Clinical Chemistry [1,2,7]
Please collect whole blood by standard venipuncture and fill the blood
DCCT: Diabetes Control and Complications Trial [3]
collection tube according to manufacturer specifications!
NGSP: National Glycohemoglobin Standardization Program [4]
Specimen Stability [5]:
HbA1c and Average Glucose Concentrations [8] Whole blood 1 week at 2 – 8°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: Sample Preparation:
Standardization according to IFCC (calculated referring to literature For sample preparation the DiaSys HbA1c net Hemolyzing Solution is
reference [8]): required.
Average glucose conc. [mg/dL] = 2.63 x HbA1cb + 15.01 Cat. No. Kit Size
Average glucose conc. [mmol/L] = 0.146 x HbA1cb + 0.829 HbA1c net Hemolyzing Solution 1 4590 99 10 967 4 x 450 tests
b: HbA1c values in mmol/mol IFCC 1 4590 99 10 961 2 x 160 tests
Standardization according to NGSP: The bottles of DiaSys HbA1c net Hemolyzing Solution are placed directly
into the reagent trays.
Average glucose concentration [mg/dL] = 28.7 x HbA1ca – 46.7
Reagent tray position has to be defined as “Diluent position” in application
Average glucose concentration [mmol/L] = 1.59 x HbA1ca – 2.59
for sample, control and calibrator. Position number must be between 1 and
a: HbA1c-values in % NGSP 45 respectively 1 and 50.
No significant differences in the regression equation were observed for Hemolysis is performed on board of the instrument automatically. Whole
variations in individuals tested including sex, presence or absence of blood collection tubes must not be higher than 75 mm; otherwise
diabetes, type of diabetes, age, race, and ethnicity. Although this equation contamination may occur!
can be used for the majority of individuals each laboratory has to verify Processing in batch mode is recommended.
whether the regression equations mentioned are applicable for the patient Calibrators, controls and samples have to be hemolyzed before use. Please
group to be examined. refer to subsequent pipetting scheme for on-board hemolysis:
Reagents Preparation
Calibrator Calibrator
Control Sample
Components and Concentrations Level 1 Level 2
R1: Buffer 100 mmol/L TruCal HbA1c net
1.6 µL - - -
FPOX ≥ 0.5 kU/L Level 1
Ethlyene glycol derivative < 10% TruCal HbA1c net
- 5 µL - -
R2: Buffer 20 mmol/L Level 2
Protease ≥ 500 kU/L TruLab HbA1c net
Chromogen ≥ 0.05 mmol/L Level 1 and Level 2 - - 5 µL 5 µL
Ethlyene glycol derivative < 10% /Sample
Add:
HbA1c net
Hemolyzing 100 µL 100 µL 100 µL 100 µL
solution

Reagent information * fluid stable

Calibration Hemoglobin variants can lead to deviant HbA1c results: The tested
The concentrations of HbA1c and hemoglobin in unknown samples are Hemoglobin variants HbS, HbC, HbD, HbE, HbJ, HbG, HbSC, HbSE, HbEE
derived from linear calibration curves. and HbF showed no significant interference.
Each calibration curve is obtained with 2 calibrators at different levels Target
without a zero value. Percentage of Mean
Hemoglobin Value Range
Hemoglobin recovery
Variant HbA1c
Calculation Variant (≤)
[% DCCT/NGSP]
HbA1c [%]
After entering the calculation formula into the instrument, the calculation of AS 40% S 5.2 – 8.8 94.7
HbA1c ratio from total hemoglobin is done by the instrument automatically.
AC 36% C 5.0 – 7.4 97.1
Please refer to the instrument manual.
AD 41% D 5.6 – 7.0 93.9
Dependent on selected standardization enter the following formula: AE 26% E 5.9 – 7.6 99.1
IFCC AJ 50% J 5.2 – 8.4 100
AG 20% G 6.1 – 6.6 97.4
Values in mmol/mol according to IFCC:
SC 52% S, 44% C 4.5 – 7.0 91.6
 HbA1c [g / dL ]  SE 65% S, 27% E 7.4 95.4
HbA1c [mmol / mol ] =   × 1000 EE 94% E 5.1 – 8.9 98.0
 Hb [g / dL ]  Elevated F 4.6% F 6.5 – 8.1 93.6
DCCT/NGSP
Values in percent according to DCCT/NGSP: Imprecision
Values according to IFCC
 HbA1c [g / dL ]  Within-run precision Mean SD CV
HbA1c [%] =  91.5 x  + 2.15
 Hb [g / dL ]  n = 20 [mmol/mol] [mmol/mol] [%]
Sample 1 32.7 0.309 0.947
Calibrators and Controls Sample 2 33.2 0.207 0.623
Sample 3 63.7 0.308 0.483
For calibration the DiaSys TruCal HbA1c net calibrator is recommended.
The assigned values of TruCal HbA1c net have been made traceable to the
approved IFCC reference method [1]. For internal quality control, DiaSys Total precision
Mean SD CV
TruLab HbA1c net controls should be assayed. Each laboratory should CLSI
[mmol/mol] [mmol/mol] [%]
establish corrective action in case of deviations in control recovery. n = 80
Sample 1 32.1 0.522 1.63
Cat. No. Kit size Sample 2 33.6 0.433 1.29
TruCal HbA1c net 1 3350 99 10 044 2 x 0.3 mL Sample 3 67.6 0.824 1.22
TruLab HbA1c net Level 1 5 9930 99 10 076 6 x 1 mL
TruLab HbA1c net Level 2 5 9940 99 10 076 6 x 1 mL Method comparison (n=100)
Performance Characteristics Test x Competitor enzymatic HbA1c assay
Test y DiaSys HbA1c net FS
Measuring range from 20 – 150 mmol/mol according to IFCC Slope 0.983
(4 - 16% according to DCCT/NGSP). Intercept 0.772 mmol/mol
The assay is applicable for hemoglobin concentrations in blood from
Coefficient of correlation 0.9945
6 - 30 g/dL (3.73 – 18.6 mmol/L).
Limit of detection**:
HbA1c 0.2 g/dL Method comparison (n=100)
Hemoglobin 1.5 g/dL Test x HPLC assay
On-board stability 6 weeks Test y DiaSys HbA1c net FS
Calibration stability 6 weeks Slope 0.996
** lowest measurable concentration which can be distinguished from zero
Intercept -0.0153 mmol/mol
mean + 1.645 x SD (n = 60) of an analyte free specimen Coefficient of correlation 0.9931

Specificity/Interferences Reference Range

According to CLSI protocol EP7-A2 a study on interferences was conducted.
Suggested target values for HbA1c [6]:
IFCC
For each interfering substance three samples with different hemoglobin and IFCC NGSP
HbA1c values have been tested; a low level sample within a hemoglobin [mmol/mol] [%]
range of 8 – 10 g/dL and a HbA1c range within 28 – 35 mmol/mol; a medium Non-diabetics 20 – 42 4–6
level sample within a hemoglobin range of 11 – 15 g/dL and a HbA1c range Target of therapy < 53 <7
within 28 – 35 mmol/mol; a high level sample within a hemoglobin range of Change of therapy > 64 >8
11 – 15 g/dL and a HbA1c range > 60 mmol/mol. Each laboratory should check if the reference ranges are transferable to its
own patient population and determine own reference ranges if necessary.
DCCT/NGSP
For each interfering substance three samples with different hemoglobin and HbA1c cut point value for diagnosis of diabetes mellitus [14]:
HbA1c values have been tested; a low level sample within a hemoglobin
According to a recommendation of the American Diabetes Association
range of 9 – 10 g/dL and a HbA1c range within 4.7 – 5.4%; a medium level
(ADA): ≥ 6.5% (NGSP) (48 mmol/mol (IFCC))
sample within a hemoglobin range of 10 – 15 g/dL and a HbA1c range within
Patients with HbA1c values in the range of 5.7 - 6.4% HbA1c (NGSP) or
4.7 – 5.4%; a high level sample within a hemoglobin range of 10 - 15 g/dL
39 - 46 mmol/mol HbA1c (IFCC) may be at high risk of developing diabetes.
and a HbA1c range > 7.65%.
Literature
The table below summarizes the results which comply for all tested levels
using IFCC as well as DCCT/NGSP standardization. 1. 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
Interfering substance Interferences < 10% in serum
Med 2002; 40: 78-89.
with hematocrit correction
2. Hoelzel W, Weykamp C et al. IFCC Reference System for
Ascorbate up to 50 mg/dL Measurement of Hemoglobin A1c in Human Blood and the National
Bilirubin (conjugated and Standardization Schemes in the United States, Japan, and Sweden: A
up to 10 mg/dL
unconjugated) Method-Comparison Study. Clin Chem 2004; 50 (1): 166-74.
Glucose up to 1000 mg/dL 3. The Diabetes Control and Complications Trial Research Group. The
Hemoglobin, acetylated up to 10 mmol/L effect of intensive treatment of diabetes in the development and
Hemoglobin, carbamylated up to 10 mmol/L progression of long-term complications in insulin-dependent diabetes
Lipemia (triglycerides) up to 1000 mg/dL mellitus. N Engl J Med.1993; 329: 977-86.
N-acetylcysteine (NAC) up to 2000 mg/L 4. Little RR, Rohlfing CL, Wiedmeyer HM, Myers GL et al. The National
Urea up to 300 mg/dL Glycohemoglobin Standardization Program: A Five-Years Progress
Uric acid up to 20 mg/dL Report. Clin Chem 2001; 47: 1985-92.
Alcoholism and ingestion of large doses of aspirin may lead to im- 5. Data on file at DiaSys Diagnostic Systems GmbH.
plausible results. For further information on interfering substances refer 6. Panteghini M, John WG on behalf of the IFCC Scientific Division.
to Young DS [10]. Implementation of haemoglobin A1c results traceable to the IFCC
reference system: the way forward. Clin Chem Lab Med 2007; 45(8):
942-4.
7. Nordin G., Dybkær R. Recommendation for term and measurement unit
for “HbA1c”. Clin Chem Lab Med 2007; 45(8): 1081-2.

Reagent information
8. Sacks DB. Translating Hemoglobin A1c into Average Blood Glucose:
Implications for Clinical Chemistry. Clinical Chemistry 2008; 54: 1756-8.
9. Weykamp C. Carbamylated Hemoglobin Interference in Glyco–
hemoglobin Assays. Clin Chem 1999; 45: 438-9.
10. 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.
11. Thomas L. Clinical Laboratory Diagnostics. 1st ed. Frankfurt: TH-Books
Verlagsgesellschaft; 1998. p. 142-48.
12. 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.
13. 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.
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.
Manufacturer
DiaSys Diagnostic Systems GmbH
IVD Alte Strasse 9 65558 Holzheim Germany

Reagent information
Hemoglobin TWIN test Application for HbA1c net FS
Reagent code 10 334

Application for whole blood samples

This application was set up and evaluated by DiaSys. It is based on the standard equipment at that time
and does not apply to any equipment modifications undertaken by unqualified personnel.

Sub Param. : 2 (Up button)

Analytical Conditions Prozone (MULTI-STD Setting only)
R1 volume 90 Prozone form None
R2e volume 0 Prozone limit 9.999
R2 volume 30 Prozone judge Upper limit
R1 diluent vol 0 Judge limit 9.999
R2e diluent vol 0 M-DET.P.m 0
R2 diluent vol 0 M-DET.P.n 0
Sample vol (S) 15 S-DET.P.p 0
Sample vol (U) 15 S-DET.P.r 0
Reagent 1 mix strong
Reagent 2e mix weak Reac. Type
Reagent 2 mix weak Reac. Type Inc.
Reaction time 10
Reaction Rate Method
Sub-analy. Conditions Cycle 2
Name Hbn Factor 2.0
Digits 2 E2 corre Not do
Unit g/dL Blank (u) 9.999
M-wave. L. 571 Blank (d) -9.999
S-wave. L. 805 Sample (u) 9.999
Analy.mthd. EPA Sample (d) -9.999
Calc.mthd. MSTD
Qualit. judge Not do
Endpoint Method
Re.absorb (u) 9.999
Calculation Method Setting Re.absorb (d) -9.999
M-DET.P.I 0
M-DET.P.m 17
M-DET.P.n 18 Analysis item condition setting (M) [Subwindow button]
Check D.P. 0 Sample Type * Serum Urine
Limit value 0.003 Reac. sample vol. 15 15
Variance 10 Diluent method With dilution With dilution
S-DET.P.p 0 Undil. sample vol. 5 5
S-DET.P.r 0 Diluent volume 100 100
Diluent position ** **
*Has to be defined for controls and Blank as well
**Position number must be between 1 and 45 respectively 1 and 50 (depends on rotor)

Standards setting
FV # BLK H 9.999 STD H 9.999
BLK L -9.999 STD L -9.999

Multi-STD setting (subwindow button)

Formula: Linear correction Blank: Any value Axis conv. : No convert. Points: 2

Reac. smp. Undiluted. Diluent Diluent

FV Dil. method STD-H STD-L
vol. smp. vol. volume position
BLK/0 # 15 With dilution 1.6 100 ** 9.999 -9.999
1 # 15 With dilution 5.0 100 ** 9.999 -9.999
2
3
4
5

#entered by user

For adjustment and maintenance of the sample probe, please contact your local distributor/service technician.

Individual results for hemoglobin and HbA1c in g/dL must not be used for diagnostic purposes.

Application BioMajesty JCA-BM6010/C August 2020/6

HbA1c TWIN test Application for HbA1c net FS
Reagent code 10 334

Application for whole blood samples

This application was set up and evaluated by DiaSys. It is based on the standard equipment at that time
and does not apply to any equipment modifications undertaken by unqualified personnel.

Sub Param. : 1
Analytical Conditions Prozone (MULTI-STD Setting only)
R1 volume 90 Prozone form None
R2e volume 0 Prozone limit 9.999
R2 volume 30 Prozone judge Upper limit
R1 diluent vol 0 Judge limit 9.999
R2e diluent vol 0 M-DET.P.m 0
R2 diluent vol 0 M-DET.P.n 0
Sample vol (S) 15 S-DET.P.p 0
Sample vol (U) 15 S-DET.P.r 0
Reagent 1 mix strong
Reagent 2e mix weak Reac. Type
Reagent 2 mix weak Reac. Type Inc.
Reaction time 10
Reaction Rate Method
Sub-analy. Conditions Cycle 2
Name HbA1cn Factor 2.0
Digits 3 E2 corre Not do
Unit g/dL Blank (u) 9.999
M-wave. L. 658 Blank (d) -9.999
S-wave. L. 805 Sample (u) 9.999
Analy.mthd. EPA Sample (d) -9.999
Calc.mthd. MSTD
Qualit. judge Not do
Endpoint Method
Re.absorb (u) 9.999
Calculation Method Setting Re.absorb (d) -9.999
M-DET.P.I 0
M-DET.P.m 41
M-DET.P.n 42 Analysis test condition setting (M) [Subwindow button]
Check D.P. 0 Sample Type * Serum Urine
Limit value 0.003 Reac. sample vol. 15 15
Variance 10.0 Diluent method With dilution With dilution
S-DET.P.p 22 Undil. sample vol. 5 5
S-DET.P.r 23 Diluent volume 100 100
Diluent position ** **
* Has to be defined for controls and Blank as well
**Position number must be between 1 and 45 respectively 1 and 50 (depends on rotor)

Standards setting
FV # BLK H 9.999 STD H 9.999
BLK L -9.999 STD L -9.999

Multi-STD setting (subwindow button)

Formula: Linear correction Blank: Any value Axis conv. : No convert. Points: 2

Reac. smp. Undiluted. Diluent Diluent

FV Dil. method STD-H STD-L
vol. smp. vol. volume position
BLK/0 # 15 With dilution 1.6 100 ** 9.999 -9.999
1 # 15 With dilution 5.0 100 ** 9.999 -9.999
2
3
4
5

#entered by user

For adjustment and maintenance of the sample probe, please contact your local distributor/service technician.

Individual results for hemoglobin and HbA1c in g/dL must not be used for diagnostic purposes.

Application BioMajesty JCA-BM6010/C August 2020/6