HbA1c FS*

Diagnostic reagent for quantitative in vitro determination of hemoglobin A1c (HbA1c) in whole blood on
Sysmex BX-3010 Chemistry Analyzer

Order Information Warnings and Precautions

Cat. No. Kit size Number of tests 1. Reagent 1 contains animal material. Handle the product as potentially
1 3348 99 10 972 R1 3 x 18.5 mL BX-3010 3 x 100 tests infectious according to universal precautions and good clinical
R2 3 x 8.5 mL BX-3010 3 x 100 tests laboratory practices.
2. Hemoglobin and HbA1c values in g/dL determined with DiaSys HbA1c
Method and Principle net FS are used to calculate the HbA1c ratio from total hemoglobin
Hemoglobin: Photometric test exclusively. Individual results for hemoglobin and HbA1c must not be
HbA1c: Colorimetric, enzymatic method used for diagnostic purposes.
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 (e.g.
and are used to calculate the HbA1c ratio from total hemoglobin exclusively. hemolytic diseases) or significant recent blood loss during the weeks
Hemoglobin measurement before (higher fraction of young erythrocytes). Falsely high values (high
Whole blood samples are lysed with hemolyzing solution. Hemoglobin is HbA1c despite normal blood glucose) have been reported in iron
released from the erythrocytes. The absorbance of hemoglobin is measured deficiency anemia (high proportion of old erythrocytes). These
at 570 nm after addition of reagent R1 and is proportional to the total circumstances have to be considered in clinical interpretation of HbA1c
hemoglobin concentration in the sample. 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 (H 2O2) 5. Please refer to the safety data sheets and take the necessary
is produced by oxidative cleavage of fructosylated dipeptides by FPOX precautions for the use of laboratory reagents. For diagnostic purposes,
(fructosyl peptide oxidase). The H2O2 generated is determined results should always be assessed with patient`s medical history,
colorimetrically by reaction with a chromogen in presence of peroxidase clinical examinations and other findings.
at 660 nm. The absorbance increase is proportional to the HbA1c 6. For professional use only!
concentration.
Waste Management
Standardization Please refer to local legal requirements.
The assay is standardized according to IFCC [1] and DCCT/NGSP [4] Reagent Preparation
reference methods. Calculation of patient and control values is possible
according to IFCC [mmol/mol] as well as according to DCCT/NGSP [%]. The reagents are ready to use. The bottles are placed directly into the
reagent trays.
NGSP and IFCC values show a linear relationship and, therefore, can be Homogenize HbA1c net Hemolyzing Solution by repeated inversion. Due to
calculated from each other using the following equation: composition of the hemolyzing solution an opalescent and slightly turbid
b a
HbA1c (IFCC ) = (HbA1c (NGSP ) – 2.15) / 0.0915 appearance remains. Avoid foaming! Do not shake!
a b
HbA1c (NGSP ) = 0.0915 x HbA1c (IFCC ) + 2.15 Specimen
a: NGSP values in % Whole blood collected with EDTA
b: IFCC values in mmol/mol Please collect whole blood by standard venipuncture and fill the blood
IFCC: International Federation of Clinical Chemistry [1,2,7] collection tube according to manufacturer specifications!
DCCT: Diabetes Control and Complications Trial [3] Stability [5]:
NGSP: National Glycohemoglobin Standardization Program [4] Whole blood 1 week at 2 – 8°C
Hemolysate 1 hour at 2 – 8°C
HbA1c and Average Glucose Concentrations
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: Sample Preparation:
Standardization according to IFCC (calculated referring to literature For sample preparation the DiaSys HbA1c net Hemolyzing Solution is
reference [8]): required.
b
Average glucose conc. [mg/dL] = 2.63 x HbA1c + 15.01 Cat. No. Kit Size
b
Average glucose conc. [mmol/L] = 0.146 x HbA1c + 0.829 HbA1c net Hemolyzing Solution 1 4590 99 10 113 1 x 500 mL
b: HbA1c values in mmol/mol IFCC Calibrators, controls and samples have to be hemolyzed before use.
Hemolysates have to be processed within 1 hour after production.
Standardization according to NGSP: Processing in batch mode is recommended. Please refer to subsequent
a
Average glucose concentration [mg/dL] = 28.7 x HbA1c – 46.7 pipetting scheme for manual hemolysis:
a
Average glucose concentration [mmol/L] = 1.59 x HbA1c – 2.59 Preparation
a: HbA1c-values in % NGSP Calibrator Calibrator
Control Sample
No significant differences in the regression equation were observed for Level 1 Level 2
variations in individuals tested including sex, presence or absence of TruCal HbA1c net
8 µL - - -
diabetes, type of diabetes, age, race, and ethnicity. Although this equation Level 1
can be used for the majority of individuals each laboratory has to verify TruCal HbA1c net
- 25 µL - -
whether the regression equations mentioned are applicable for the patient Level 2
group to be examined. TruLab HbA1c net
Level 1 and Level 2 - - 25 µL 25 µL
Reagents /Sample
Add
Components and Concentrations HbA1c net
R1: Buffer 100 mmol/L Hemolyzing 500 µL 500 µL 500 µL 500 µL
FPOX ≥ 0.5 kU/L solution
Ethlyene glycol derivative < 10% Mix and allow standing for 1 minute. Hemolysis is completed after
R2: Buffer 20 mmol/L 1 minute. A slight turbidity remains due to the composition of the
Protease ≥ 500 kU/L hemolyzing solution.
Chromogen ≥ 0.05 mmol/L
Ethlyene glycol derivative < 10% Calibration
Storage and Reagent Stability The concentrations of HbA1c and hemoglobin in unknown samples are
derived from linear calibration curves.
The reagents are stable up to the end of the indicated month of expiry, if
stored at 2 – 8°C and contamination and evaporation are avoided. Do not Each calibration curve is obtained with 2 calibrators at different levels
without a zero value.
freeze the reagents! Protect reagents from light!

Reagent Information Page 1 of 8

Calculation Hemoglobin variants can lead to deviant HbA1c results: The tested
After entering the calculation formula into the instrument, the calculation of Hemoglobin variants HbS, HbC, HbD, HbE, HbJ, HbG, HbSC, HbSE, HbEE
HbA1c ratio from total hemoglobin is done by the instrument automatically. and HbF showed no significant interference.
Please refer to the instrument manual. Target
Percentage of Mean
Dependent on selected standardization enter the following formula: Hemoglobin Value Range
Hemoglobin recovery
Variant HbA1c
IFCC Variant (≤) HbA1c [%]
[% DCCT/NGSP]
Values in mmol/mol according to IFCC: AS 40% S 5.2 – 8.8 94.7
AC 36% C 5.0 – 7.4 97.1
 HbA1c [g / dL]  AD 41% D 5.6 – 7.0 93.9
HbA1c [mmol/ mol ]     1000
 Hb [g / dL]  AE 26% E 5.9 – 7.6 99.1
DCCT/NGSP AJ 50% J 5.2 – 8.4 100
Values in percent according to DCCT/NGSP: AG 20% G 6.1 – 6.6 97.4
SC 52% S, 44% C 4.5 – 7.0 91.6
 HbA1c [g / dL]  SE 65% S, 27% E 7.4 95.4
HbA1c [%]   91.5 x   2.15
 Hb [g / dL]  EE 94% E 5.1 – 8.9 98.0
Elevated F 4.6% F 6.5 – 8.1 93.6
Calibrators and Controls
DiaSys TruCal HbA1c net calibrator is recommended for calibration. TruCal Precision according to DCCT/NGSP
HbA1c net values have been made traceable to the approved IFCC Within run (n=20) Sample 1 Sample 2 Sample 3
reference method [1]. Use DiaSys TruLab HbA1c net Level 1 and Level 2 for Mean [%] 5.11 5.13 8.19
internal quality control. Each laboratory should establish corrective action in
CV [%] 1.04 1.12 0.70
case of deviations in control recovery.
Between run (n=20) Sample 1 Sample 2 Sample 3
Cat. No. Kit size
Mean [%] 4.75 4.75 8.47
TruCal HbA1c net 1 3350 99 10 044 2 x 0.3 mL
TruLab HbA1c net Level 1 5 9930 99 10 076 6 x 1 mL CV [%] 1.54 1.29 1.37
TruLab HbA1c net Level 2 5 9940 99 10 076 6 x 1 mL
Method comparison (n=100)
Performance Characteristics Test x BioMajesty 6010C / DiaSys HbA1c net FS
Exemplary data mentioned below may slightly differ in case of deviating Test y Sysmex BX-3010 / DiaSys HbA1c net FS
measurement conditions. Slope 1.08
Intercept –0.300 % DCCT
Measuring range from 4 - 16% according to DCCT/NGSP
Coefficient of correlation 0.994
(20 – 150 mmol/mol according to IFCC).
The assay is applicable for hemoglobin concentrations in blood from
6 - 30 g/dL (3.73 – 18.6 mmol/L). Method comparison (n=100)
Limit of detection**: Test x Akray HA–8160 HPLC
HbA1c 0.2 g/dL Test y Sysmex BX-3010 / DiaSys HbA1c net FS
Hemoglobin 1.5 g/dL Slope 1.07
On-board stability 4 weeks Intercept –0.242 % DCCT
Calibration stability 4 weeks Coefficient of correlation 0.990
** lowest measurable concentration which can be distinguished from zero
mean + 1.645 x SD (n = 60) of an analyte free specimen Reference Range
Specificity/Interferences Suggested target values for HbA1c [6]:
According to CLSI protocol EP7-A2 a study on interferences was conducted. IFCC NGSP
IFCC [mmol/mol] [%]
For each interfering substance three samples with different hemoglobin and Non-diabetics 20 – 42 4–6
HbA1c values have been tested; a low level sample within a hemoglobin Target of therapy < 53 <7
range of 7 – 10 g/dL and a HbA1c range within 20.2 – 39.9 mmol/mol and a Change of therapy > 64 >8
high level sample within a hemoglobin range of 10 – 15 g/dL and a HbA1c Each laboratory should check if the reference ranges are transferable to its
range > 59.6 mmol/mol. own patient population and determine own reference ranges if necessary.
DCCT/NGSP
HbA1c cut point value for diagnosis of diabetes mellitus [14]:
For each interfering substance at least two samples with different
hemoglobin and HbA1c values have been tested; a low level sample within According to a recommendation of the American Diabetes Association
a hemoglobin range of 7 – 10 g/dL and a HbA1c range within 4.0 – 5.8% (ADA): ≥ 6.5% (NGSP) (48 mmol/mol (IFCC))
and a high level sample within a hemoglobin range of 10 - 15 g/dL and a Patients with HbA1c values in the range of 5.7 - 6.4% HbA1c (NGSP) or
HbA1c range > 7.6%. 39 - 46 mmol/mol HbA1c (IFCC) may be at high risk of developing diabetes.
The table below summarizes the results which comply for all tested levels Literature
using IFCC as well as DCCT/NGSP standardization.
1. Jeppsson JO, Kobold U, Barr J, Finke A et al. Approved IFCC reference
Interfering substance Interferences < 10% in serum method for the measurement of HbA1c in human blood. Clin Chem Lab
with hematocrit correction Med 2002; 40: 78-89.
Ascorbate up to 50 mg/dL 2. Hoelzel W, Weykamp C et al. IFCC Reference System for
Bilirubin (conjugated and Measurement of Hemoglobin A1c in Human Blood and the National
up to 10 mg/dL
unconjugated) Standardization Schemes in the United States, Japan, and Sweden: A
Glucose up to 1000 mg/dL Method-Comparison Study. Clin Chem 2004; 50 (1): 166-74.
Hemoglobin, acetylated up to 10 mmol/L 3. The Diabetes Control and Complications Trial Research Group. The
Hemoglobin, carbamylated up to 10 mmol/L effect of intensive treatment of diabetes in the development and
Lipemia (triglycerides) progression of long-term complications in insulin-dependent diabetes
up to 750 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 Page 2 of 8

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 Page 3 of 8

HbA1c FS Sysmex BX-3010
TWIN Test – Hemoglobin Application Chemistry Code 100 42

Chemistry Parameters 1 Sysmex BX-3010 Chemistry Analyzer

Analytical Parameters
Method No. 61 Method Name HbA1c Hb Reagent Name Reagent (µL) Water (µL)

Print Name Hb MethodColor R1 HbA1c 150

Sample Type RBC R2 HbA1c 50

Unit g/dL Hemolyzer Disable

Assay Type End Sample Ppt. Wash Disable

Measuring points Start End Stirring Speed R1 Middle R2 Middle

Stirring for hemolysis Fast
Hb 1 23 – 23
Hb 2 Disable – Disable
A1c 1 25 – 26
A1c 2 45 – 46 Normal Range
No. Normal Range Name Min Max
Wave Length 1 Male-G1 * *
Hb Prim. 570 Hb Sec. 800 2 Male-G2 * *
A1c Prim. 660 A1c Sec. 800 3 Male-G3 * *
4 Female-G1 * *

Normal Sample Volume (µL) Hemolyzed Sample (µL) Hemolyzer (µL) Technical Range
Low Normal High (Conc) 0 – 9999
 hemolysis 0.0 < 25.0 < 0.0 (mAbs/10) * – *
Rerun (High/Prozone)
 hemolysis 0.0 < 25.0 < 0.0 Previous Result Comparison (%) * * %
Rerun (Low)
 hemolysis 0.0 < 25.0 < 0.0 Abnormal Range (Conc) * – *

Aspirating position 30 Panic Range (Conc) –

Decimal Point 3 Profile SI Disable

*Entered by user

Chemistry Parameters 2 Sysmex BX-3010 Chemistry Analyzer

Analytical Parameters
Method No. 61 Method Name HbA1c Hb Sample RBC

Limit Checks Blank measurement

 Duplicate Limit 70 mAbs/10 Blank measurement:
Disable reagent blank and C1 blank
 Sensitivity Limit 2200 mAbs/10
Measurement of Reagent Blank during Run:
Linearity Limit % None

(mAbs/10)/min Reagent blank measurement at calibration:

Reagent blank (No sample)
 Prozone Limit %
The number of measurement:
Higher Triplicate

SL1-S 1 – SL1-F 2 Reagent blank limit checks:

 Duplicate Limit 20 mAbs/10
SL2-S 3 – SL2-F 4

Sensitivity mAbs/10 Instrument Factor

 Absorbance Limit a 1.00 b 0.00

Abs. in reaction Increase

Limit 25000 mAbs/10

Application BX-3010 Page 4 of 8

 HbA1c FS Sysmex BX-3010
TWIN Test – Hemoglobin Application Chemistry Code 100 42

Calibration Registration Sysmex BX-3010 Chemistry Analyzer

Analytical Parameters

Reagent Lot No.

Method No. 61 (R1) * Last
(R2) *
Method Name Hb

Sample Type RBC

Replication Duplicate

Check Interval 42

Test without calibration

Disable

Calibration Type Linear

Reagent Lot New Add The calibration curve is lot dependent

Calibrator Name TruCal HbA1c net

Conc. WORK MASTER Calibr. Lot No.  All

C1 Enter conc Level I Automatic entry Automatic entry * Reagent blank mAbs/10 Last
C2 Enter conc Level II Automatic entry Automatic entry *
Automatic entry
C3 * Blank mAbs/10 Last
C4 *
C5 * Calibration Curve Conc.
C6 *
C7 * Absorbance mAbs/10 Recalculation

K Automatic entry
 C1 Blank
 Reagent Blank for C1
*Entered by user

Application BX-3010 Page 5 of 8

 HbA1c FS Sysmex BX-3010
TWIN Test – HbA1c Application Chemistry Code 100 42

Chemistry Parameters 1 Sysmex BX-3010 Chemistry Analyzer

Analytical Parameters
Method No. 60 Method Name HbA1c A1c Reagent Name Reagent (µL) Water (µL)

Print Name A1c MethodColor R1 HbA1c 150

Sample Type RBC R2 HbA1c 50

Unit g/dL Hemolyzer Disable

Assay Type End Sample Ppt. Wash Disable

Measuring points Start End Stirring Speed R1 Middle R2 Middle

Hb 1 23 – 23
Hb 2 Disable – Disable
A1c 1 25 – 26
A1c 2 45 – 46 Normal Range
No. Normal Range Name Min Max
Wave Length 1 Male-G1 * *
Hb Prim. 570 Hb Sec. 800 2 Male-G2 * *
A1c Prim. 660 A1c Sec. 800 3 Male-G3 * *
4 Female-G1 * *

Normal Sample Volume (µL) Hemolyzed Sample (µL) Hemolyzer (µL) Technical Range
Low Normal High (Conc) 0 – 9999
 hemolysis 0.0 < 25.0 < 0.0 (mAbs/10) * – *
Rerun (High/Prozone)
 hemolysis 0.0 < 25.0 < 0.0 Previous Result Comparison (%) * * %
Rerun (Low)
 hemolysis 0.0 < 25.0 < 0.0 Abnormal Range (Conc) * – *

Panic Range (Conc) –

Decimal Point 3 Profile SI Disable

*Entered by user

Chemistry Parameters 2 Sysmex BX-3010 Chemistry Analyzer

Analytical Parameters
Method No. 60 Method Name HbA1c A1c Sample RBC

Limit Checks Blank measurement

 Duplicate Limit 50 mAbs/10 Blank measurement:
Disable reagent blank and C1 blank
 Sensitivity Limit 500 mAbs/10
Measurement of Reagent Blank during Run:
Linearity Limit % None

(mAbs/10)/min Reagent blank measurement at calibration:

Reagent blank (No sample)
 Prozone Limit Higher %
The number of measurement:
Triplicate

SL1-S 1 – SL1-F 2 Reagent blank limit checks:

 Duplicate Limit 20 mAbs/10
SL2-S 3 – SL2-F 4

Sensitivity mAbs/10 Instrument Factor

 Absorbance Limit a 1.00 b 0.00

Abs. in reaction Increase

Limit 25000 mAbs/10

Application BX-3010 Page 6 of 8

 HbA1c FS Sysmex BX-3010
TWIN Test – HbA1c Application Chemistry Code 100 42

Calibration Registration Sysmex BX-3010 Chemistry Analyzer

Analytical Parameters

Reagent Lot No.

Method No. 60 (R1) * Last
(R2) *
Method Name HbA1c

Sample Type RBC

Replication Duplicate

Check Interval 42

Test without calibration

Disable

Calibration Type Linear

Reagent Lot New Add The calibration curve is lot dependent

Calibrator Name TruCal HbA1c net

Conc. WORK MASTER Calibr. Lot No.  All

Enter conc Level I Automatic entry Automatic entry
C1 * Reagent blank mAbs/10 Last
Enter conc Level II Automatic entry Automatic entry
C2 *
Automatic entry
C3 * Blank mAbs/10 Last
C4 *
C5 * Calibration Curve Conc.
C6 *
C7 * Absorbance mAbs/10 Recalculation

K Automatic entry
 C1 Blank
 Reagent Blank for C1

*Entered by user

HbA1c FS TWIN Test – HbA1c Calculation

Calculated Test Sysmex BX-3010 Chemistry Analyzer
Analytical Parameters

Calculated test No. 3003

+ - * / ( )
Calculated Test Name HbA1c2
16 FE 17 GLUHK 18 HDL-C
Print Name HbA1c net 19 LDL-C 20 LDH 21 MG
22 PO3 23 TRIG 24 TP
Sample Type Hemolysate/Hemolysate 25 UREA 26 UA 27 IIBC
e 28 FerrFD 29 HDLpj 30 HCG
Unit mmol/mol 31 LPS 32 CL 33 NA
34 K 35 Glyc 36 CRP
Decimal Point 2 37 CRPU 38 CRPhs 39 PAMY
40 IgG 41 ASO 42 TRF
43 A1cHig 44 Lp(a) 45 MALBs
Expression 46 RF

{60}/{61}*1000

Normal Range
53 16µL 54 8µL
No. Range Name Min Max 55 2µL 56 Olin 57 SPTS
1 Male-G1 0.00 0.00 58 RPTS 59 RPT2S 60 HbA1c
2 Male-G2 0.00 0.00 61 Hb
3 Male-G3 0.00 0.00 1001 ISE-Na 1002 ISE-K 1003 ISE-Cl
4 Female-G1 0.00 0.00 1005 ISE(U)-Na 1005 ISE(U)-K 1007 ISE(U)-Cl
5 Female-G2 0.00 0.00 2001 SI-H 2002 SI-L 2003 SI-I
6 Female-G3 0.00 0.00

Application BX-3010 Page 7 of 8 April 2019/2

 HbA1c FS
Instruction for Entering Calculation Formula into Sysmex BX-3010:

Window A:

2. Choose
„Calculated Tests“ in
„Calculator“

1. Choose preferred
unit mM/M (IFCC) or
% (NGSP).

3. Enter calculation
formula for preferred
unit mM/M (IFCC) or
% (NGSP).

Please note: Hemoglobin and HbA1c values determined with DiaSys HbA1c net FS are
used for calculation of the HbA1c ratio from total hemoglobin exclusively. The individual results
for hemoglobin (HbA1c Hb) and HbA1c (HbA1c A1c) must not be used for diagnostic purposes.

Instruction for entering calculation formular - BX-3010 Page 8 of 8 April 2019/2