HbA1c Capillary

Electrophoresis

24/01/2018
What is
HbA1c?
 Hb A1c: What are we looking for?
Hb A
Hb A0
93-95%
Glycation at the
N-terminal Valin
of the β-globin chain
Hb A1 = GHb
Glycated HbA
5-7%
Hb A1a Hb A1b Hb A1c
0,5% 0,5% 4-6%
Fructose-1,6-diphosphate pyruvate glucose
Hb A2Glucose-6-phosphate

Hb F
+ + +
HbA1c: stable adduct of glucose to the N-terminal amino
group of the β-chain of hemoglobin A0

Yes Hb A1c

Not Hb A1c
 HbA1c and Diabetes
• HbA1c reflects retrospective and cumulative index of
glycemic balance over the previous 120 days before sampling.
• It can be performed at any time of the day because it does
not require a period of fasting for the patient (as the
measurement of fasting plasma glucose level). It is also less
exposed to day-to-day variability
• A HbA1c value of 6.5% is recommended for the diagnosis of
diabetes by American Diabetes Association (ADA).
• Prediabetic patients are defined by a HbA1c value between
5.7% and 6.4%
At the time of DCCT and UKPDS publications (1993-
1998), there was no consistency in the way that HbA1c
were reported: the same person could have different
HbA1c results reported by different laboratories,
depending on the type of analyzer used.

A standardization of reported HbA1c value was necessary

• In 1995, The International Federation of Clinical Chemistry
and Laboratory medecine working group on Hb A1c
standardization was initiated for the development of an
internationally accepted higher order reference method and
reference materials for Hb A 1c measurement

• In 2001, the 2 approved IFCC reference methods, namely

HPLC–mass spectrometry or HPLC–capillary electrophoresis
were approved. This method measures the concentration of
only one molecular species of glycated A1c and give identical
HbA1c results because they use the same primary reference
materials for calibration
 1st step: enzymatic digestion
Staphylococcus aureus Glu-C makes 2 hexapeptides:
Glucose

Val His Leu Thr Pro Glu Glu Lys

N-terminal region of -Globin chain The amount of these 2

HbA1c (m/z= 429.2)

Glu-C peptides can be
determined by LC/MS
or LC/CE

Val His Leu Thr Pro Glu Glu Lys

N-terminal region of -Globin chain

HbA0 (m/z= 348.2)

Glu-C
1. Digestion, Glu-C 2. Separation, LC 3. Detection, MS
1 mg Hb Zorbax SB-CN, [M+2H]+2
pH 4.3, 37 °C, 18 hours 2.1 x 150 mm
50 °C, 300 l/min
 RT: 7.93 - 15.08
10.83 NL:
100 6.00E7

80 [M+2H]2+
60
Native
Hexapeptide
40 m/z 348.2-348.4
Relative Abundance

20

0
11.84 NL:
100 7.75E6

80
[M+2H]2+
60
Glycated
40 Hexapeptide
m/z 429.2-429.4
20

0
8 9 10 11 12 13 14 15
Time (min)

Chromatograms by LC/MS
1. Digestion, 2. Separation, LC 3. Collection, 4. Separation, CE
Glu-C Concentration 5. Detection,
UV 214 nm
 File:FB30027-ce-data.jdx
100
Native
Hexapeptide
Absorbance at 214 nm
(Arbitrary Units)

Glycated
Hexapeptide
Unknown
0
32 34 36 38 40 Time, minutes

CE Electrophoregram
• 1996: In US, The National Glycohemoglobin Standardization
Program (NGSP) is formed. Its purpose is to standardize
HbA1c test to those of the DCCT and UKPDS trials which
established the direct relationships between HbA1C levels
and outcome risks in patients with diabetes.

• Some countries, such as Sweden and Japan, preferred to

continue with their own programs for harmonizing HbA1c
results.
CPRL: Central Primary Reference laboratory
PRLs: Primary Reference Laboratories (3)
CPRL method: DCCT Bio-Rex 70 HPLC reference method
(not suitable for routine analysis)
SRLs: Secondary Reference Laboratories (7)
SRLs: Highly precise commercial methods based on
ion-exchange HPLC, Immunoassay, Boronate – affinity
HLPC or Capillary electrophoresis.

NGSP network laboratories are monitored monthly with 10

pooled frozen whole blood samples spanning the range of
4%–10% Hb A 1c , and all network laboratories are
compared to the CPRL. To pass the NGSP-monitoring
exercise, the estimate of the SD of the difference between
sample replicates (each sample is analyzed on 2 separate
days) must not exceed 0.229% Hb A1c. In addition, the mean of
the differences be tween an individual network laboratory and
the CPRL must not exceed 0.35% Hb A 1c

Comparisons with the IFCC network are performed twice

per year.

NGSP network laboratories assist manufacturers with the

calibration of their methods. A manufacturer is awarded a
Certificate of Traceability if the 95% CI of the differences
between their method and the SRL falls within ± 0.75% Hb A 1c .
 Hb A1c unit
All instruments sold after January ST2011 have to report in both IFCC and
NGSP derived units
International system (IFCC) unit: mmol/mol
Derived NGSP unit: calibrated %

Multiple comparisons between the networks reveal that results obtained

by the IFCC method are highly correlated with NGSP results, but there is a
bias. This bias was translated into “master equations” that describes the
linear relationship between results from the IFCC and NGSP laboratory
networks

* IFCC to NGSP:
NGSP = (IFCC X 0,0915) + 2,15
* NGSP to IFCC
IFCC = (NGSP x 10.93) – 23.50
New capillary technologies for HbA1c analysis

Capillarys Flex Piercing Minicap Flex Piercing

Throughput: 38 samples / hour Throughput: 8 samples / hour
 We apply the following calculation:

%HbA1c =
100 x [HbA1c area / (HbA1c area + HbA0 area)]
% = 0.09148 x mmol/mol + 2.152
IFCC Master Equation
Same as IFCC calculation formula
IFCC and NGSP certificates can
be downloaded from SEBIA
Customer Extranet Website
 SEBIA HbA1c
technique and
technologies
Capillarys 2 Flex Piercing
and Hb A1c

1
• CAPILLARYS 2 Flex Piercing:

• CAPILLARYS Hb A1c kit - PN 2015 (2-8°C):

– Specific buffer (2-8°C): 2 CAPILLARYS vials
– Specific hemolyzing solution (2-30°C) – different from the one used for
CAPILLARYS HEMOGLOBIN(E): 1 CAPILLARYS vial
– Stability on board: 20 days
– Washing solution and dilution segments: same as CAPILLARYS HEMOGLOBIN(E)
• Hb A1c CAPILLARY CALIBRATORS - PN 4755 (-18/-30°C):
– 2 levels (called level 1 and level 2): 1 vial of each
– different from those previously used for Hydragel Hb A1c
– Include barcodes, required for use

• Hb A1c CAPILLARY CONTROLS - PN 4774 or 4768 (2-8°C):

– 2 levels (called level 1 and level 2): 1 vial of each
– different from those previously used for Hydragel Hb A1c
– Include barcodes, required for use for both manual or automatic dilution
– Include colour-coded dilution segments (white for level 1, grey for level 2)
• ACCESSORIES FOR CAPILLARYS Hb A1c:
– Set of conic tubes and caps for controls
(set of 20; PN 9202, set of 500; PN 9205)
– Wedge Adapter (set of 10; PN 9203)
→ for controls, calibrators and samples with volume
lower than 1 mL: minimum volume = 100µL
– Box for freezing and storage of diluted control
segments (set of 2; PN 2082)

• SOFTWARE FOR CAPILLARYS Hb A1c :

– Software 8.50/8.51/8.60/8.61/8.62/8.63/
8.70/ 8.71/ 9.15
 Onboard Hb A1c Hb A1c buffer 8 capped tubes
hemolyzing solution (from 1 to 8)/rack
Use of capped whole blood
samples. Stored at 2-8°C Sample agitation
during 7 days or 72 hours at + piercing + hemolysis
room temperature or
3 months at -70 / -80°C

Migration
à 10.000 volts
Capillary in silicate and
Deuterium thermo-conductive resin
Detector (25µm diameter)
lamp
Direct
Injection < 1nL
quantification
at 415 nm

Cathode - Anode +
 Workload (Phoresis 8.63):
• Start up time: 21 min
• Extinction time: 21min
• Time to get first 8 results: 22 min
• Time for technique change : 23 min (P6->HbA1c or Hb->HbA1c)
• Throughput: 38 samples / hour
Minicap Flex Piercing
and Hb A1c

2
 Special carousel
Use of capped whole blood
samples. Stored at 2-8°C
with tube guides
during 7 days or 18 hours at for hemoglobin
room temperature or analysis
3 months at -70 / -80°C Sample agitation
+ piercing + hemolysis

Migration
until 10.000 volts Capillary in silicate and
thermo-conductive resin
Blue LED (25µm diameter)
Detector
Direct
Injection < 1nL
quantification
at 415 nm

Cathode - Anode +
 HbA1c
• Performances strictly identical to
CAPILLARYS 2 Flex Piercing

• Throughput ≈ 8 samples / hour

• Phoresis CORE 8.62/8.63/9.11
• TRANSFORMATION KITS TO MINICAP Flex Piercing HbA1c
Transformation kit
MINICAP Flex Piercing for HbA1c (PN.1238)

MINICAP (S/N>250) MINICAP Flex Piercing MINICAP Flex Piercing HbA1c

Transformation kit (PN . 1240)

MINICAP → MINICAP Flex Piercing HbA1c
• Kit Minicap HbA1c (PN. 2215)
2 Buffer vials, Hemolysing solution, Wash
solution, dilutions cups
• Hemolyzing solution in position 27 (10ml in tube
16x100 mm for 24 tests, 5 mL in tube 13x75 mm
for 12 tests)
• Same calibrators and controls as for CAPILLARYS
HbA1c
 Workload (Phoresis 8.63):
• Start up time: 17 min
• Extinction time: 34 min
• Time to get first 2 results: 21 min
• Time for technique change : 17 min (P6->HbA1c or Hb->HbA1c)
• Throughput: 8 samples / hour
System calibration

3
• Allows to calibrate the system

• Allows the profile recentering

- The profile is centered using both Hb A0 and Hb A1c peaks
- The positions for centering are:
• Hb A0 : 150
• Hb A1c : 68
• Hb A2: 240
 Calibration in HbA1c method
• HbA1c is a standardized method

• All instruments must be calibrated so that the results obtained on

different instruments, in different laboratories will be as close to each
other as possible

• Calibration is performed before the analysis of controls or samples,

using calibrators with values of HbA1c assigned by the manufacturer

• The software uses the difference between the assigned values of the
calibrators and the values really measured by the system; a calibration
curve is created

• When samples are analyzed, raw measured value is not reported.

Instead, the calibration curve is used to “correct” the value. Only this
calibrated value will be reported
 What is a calibration curve and how is it
used?
Assigned
Calibration
values
curve
of the
calibrators
 

 
Values
measured
by the
instrument
 
 What is a calibration curve and how is it
used?
Calibrated Calibration
(reported) curve
values

This is
the  
value
that will
be
reported Values
measured
by the
instrument

 We apply the following calculation:

%HbA1c =
100 x [HbA1c area / (HbA1c area + HbA0 area)]
% = 0.09148 x mmol/mol + 2.152
IFCC Master Equation
Same as IFCC calculation formula
 Calibrators Controls
2 levels 2 levels
Number
(green & red caps) (white & black caps)
Appearance lyophilisate lyophilisate
Stability (storage) 3 years -30/-18°C 3 years 2-8°C
Given unit Only in mmol/mol mmol/mol and %

Calibrators Controls
-30 to -18°C
max 4 weeks at 2-8°C -30 to +8°C
Lyophilisate transportation
required isotherm boxes for max 4 weeks at 15-30°C
transportation (cool packs)

timestrip with alert if T° >

Temperature sensor during
8°C with unacceptability of none
transportation the calibrators if period >12 h
Up to 12 hours, After 12 hours,
you can use the you cannot use
calibrators the calibrators
 -> Calibrator level 1 (normal HbA1c value)
-> Calibrator level 2 (elevated HbA1c value)
• Reconstitution: with 0.6mL deionized water

Follows this procedure to

ensure correct reconstitution
of calibrator vials

After 30 min incubation,

- Transfer the whole volume in one capped Sebia conical tube for Capillarys 2 Flex
Percing
Use the correct calibrator barcordes according Phoresis version

To use if Phoresis versions 8.71≤ for Capillarys

Flex Piercing and 9.11≤ for Minicap Flex
Piercing

To use if Phoresis versions ≥ 9.15 for Capillarys

Flex Piercing and Minicap Flex Piercing
Use: up to 4 uses of the reconstituted calibrator:
-After each use, freeze the reconstituted calibrator in its conical
capped tube, as soon as possible
-3 defreezing cycles possible : let the reconstituted calibrator
defreeze at 2-8°C before use (swirl the tube before use)
Storage: up to 22 months at -18 to -30°C after reconstitution
 Calibration
on Capillarys 2 Flex Piercing
for Hb A1c analyses

3a
  How to use SEBIA calibrators on Capillarys 2 Flex Piercing:

Calibrator 1 Calibrator 2
First use of Hb A1c program or 3 runs/capillary And 3 runs/capillary
system

-Use rack « F0 », Sebia calibrator barcodes, conic tubes and wedge adapter
Only in mmol/mol

/ 01 IF / xx IF
Phoresis <8.63 Phoresis ≥8.63
Each calibrator is identified by its concentration and its lot number:
changing any character from these will change the calibrator
Calibration protocol at the installation: 3 runs of each calibrator level
within the same day: :
-1st run of the HbA1c calibrator level 1: place the conical tube with calibrator on
rack F0 with green segment, enter calibrator lot number + concentration in
mmol/mol and choose automatic dilution. After analyses, save calibrator
level 1 tube at 2-8°C

-1st run of the HbA1c calibrator level 2: place the conical tube with calibrator
on rack F0 with green segment, enter calibrator lot number + concentration in
mmol/mol and choose automatic dilution After analyses, save calibrator
level 1 tube at 2-8°C After analyses, save calibrator level 2 tube at 2-8°C

Check the optical density, the raw migration and the noise on all
curves for both calibrators
Calibration protocol at the installation: 3 runs of each calibrator level
within the same day:
-1st run of the HbA1c calibrator level 1
-1st run of the HbA1c calibrator level 2
=> Check the optical density, the raw migration and the noise on all
curves for both calibrators

If all previous calibrator results are ok: you can continue the
calibration procedure
- 2nd run of the HbA1c calibrator level 1: place the conical tube with
calibrator on rack F0 with green segment and choose automatic dilution
-3rd run of the HbA1c calibrator level 1: only one possible re-use of the
rejected diluted calibrator segment within 10 min max (reintroduced in
the diluted segment and tube on rack F0 and choose manual dilution)
 Same protocol with HbA1c calibrator level 2
 How to display raw curves
• Go to the folder in which Phoresis is installed (C:/Program files/Phoresis)
and find the file called Phoresys.ini

• Double-click on the file to open it in the

text editor and replace the line:
VIEW_RAW_DATA=0
by VIEW_RAW_DATA=1
(if this line is not present, just insert in
the place indicated on the screenshot)
Recommendations for HbA1c installation

Hb A0 position on the
raw curve between 130-
170 : OK
Recommendations for HbA1c installation

Hb A0 position on the
raw curve outside from
130-170 : Launch an
activation cycle
Hb A1c
Normal profile Other HbA

Hb A1c
Noisy profile Other HbA Check the optical
fibers
• Information a/m for automatic or manual dilutions
on the pattern for calibrators and/or controls for
Hemoglobin, Hba1c, Cord blood, and neonat.
 How are HbA1c calibrators paired?
For example:
Segment 1, Cal 1 3
Segment 2, Cal 1
Re-use Segment 2, Cal 1
1 2 3
Segment 1, Cal 2
Segment 2, Cal 2 2
Re-use Segment 2, Cal 2

How calibrators are paired using calibration procedure at installation

Segment 1, Cal 1 Automatic dilution (A)
Segment 1, Cal 2 Automatic dilution (A)
Segment 2, Cal 1 Automatic dilution (A)
Re-use segment 2, Cal 1 Manual dilution (M)
Segment 2, Cal 2 Automatic dilution (A)
Re-use segment 2, Cal 2 Manual dilution (M)
- The system is calibrated as soon as at least one calibrator of each level
has been analyzed the same day
- After the calibration protocol, the calibration data are memorized and
accessible from the main menu: Capillarys\HbA1c calibration data

Calibration
informations
List of all calibrations, with calibration
ID (refers to work list column)
 Green: both calibrator levels are correctly analyzed on all capillaries
(correct calibration)

Orange: calibration problem on, at least, one of calibrator level for a

capillary (red, yellow, purple, exclamation alarm)

 Red: no capillaries have been calibrated

No color: the calibrator level is not paired with other calibrator level
 Alarm Software criteria What has to be done?

Raw HbA1c values outside

Sebia specifications

Defective profile centering (HbA0 or HbA1c Analyze 1 control on rack "0"

positions outside Sebia specifications) or repeat calibration - if no
valid calibration on some
capillaries, those capillaries
Optical density for calibrator too low are desactivated (no HbA1c
(OD<0.07) results)

Atypical profile Normal peak missing (HbA1c, HbA0, HbA2,

(or in calibration list) other HbA) or presence of supplementary
peak

Always verify that the reconstitution volume, the freezing/thawing cycles

and the mixing procedure for the calibrator are correct before use
Calibrator curve is not used
 Calibrator curve is not used
Level 1 : 4.6-5.7% in Phoresis≥8.60
Level 2 : 10.5-12.8% in Phoresis≥8.60
• Display of warning flags on the mosaic in Phoresis ≥8.60:
• Possibility to cancel one calibration when the
calibration has been erroneous in one or several
capillaries. This is possible only with service
password, in the calibration worklist.
• With the Service password, by clicking on “CT”, the user has
the possibility to visualize the “calibration Informations” table
 Calibration
on Minicap Flex Piercing
for Hb A1c analyses

3b
• Calibrators are reconstituted with 0.6 mL of distilled water
• After reconstitution, divide the volume of calibrator in
2 aliquots (0.4 mL) in conical tubes for control blood
• Storage: 8 hours max at 2-8°C. After use they must be stored
between -18°C and -22°C up to 22 months
• After storage, before use, thaw the reconstituted calibrators
at 2-8°C for at least 45min.
• Homogenize calibrators before each use
• Store the thawed calibrators at 2-8°C for 8hr max
• Do not freeze and thaw the reconstituted calibrators more
than 5 times for the first aliquot after its first use and 6 times
for the second immediately frozen
• 1 set of calibrators allows to perform 12 calibrations
• Place capped conical barcoded calibrator
level 1 in position 28 with a centering ring
• Place HbA1c hemolysing solution (5 mL) in
position 27

• Close the door, the analysis starts

automatically
• Enter in the window which appears: HbA1c
level in mmol/mol, lot number and
expiration date, select the number of
analyses of the calibrator (with the same
reagent cup)
• Remove the tube with calibrator as soon
as the window appears indicating to
remove the tube
• When the analysis of the first calibrator is completed, perform the analysis
of the second calibrator: use the “click to run the control in position 28”
button (the 2 calibrators must be analyzed within the same working day)
• Important: before to force the analysis of the second calibrator, wait that
the Minicap has check the absence of tube in position 1
Minicap : 3 Calibrators 1 and 3 Calibrators 2
Same recommandations to verify:
- Correct optical density (DO>0.07)
- Correct HbA0 position (130-170) on raw curves
- Absence of noisy on both calibrator curves
- 3 correct consecutive calibrator 1 and 2 runs
Cal 1
Cal 1
Cal 1
1 2 3
Cal 2
Cal 2
Cal 2
When is it necessary to perform
a new system calibration
after installation?

3c
 Calibration of Capillarys Flex Piercing for Hb A1c analyses
with both calibrators before any patient analyses
 How to use SEBIA calibrators :

Calibrator 1 Calibrator 2
First use of Hb A1c program or 3 runs/capillary 3 runs/capillary
system
Change of calibrator lot And
numbers
Capillary replacement*
Change of buffer lot number 1 run/capillary And 1 run/capillary

If the warning message 1 run/capillary And 1 run/capillary

‘’analysis of the control not in
conformity ‘’ is diplayed

No need to calibrate after a technique change or after a capiclean cycle

* : If SEBIA criteria are not meet on controls

 Calibration of Minicap Flex Piercing for Hb A1c analyses
with both calibrators before any patient analyses
 How to use SEBIA calibrators :

Calibrator 1 Calibrator 2
First use of Hb A1c program or 3 runs/capillary 3 runs/capillary
system
Change of calibrator lot And
numbers
Capillary replacement
Change of buffer lot number 1 run/capillary And 1 run/capillary
or at least every 2 months
If the warning message 1 run/capillary And 1 run/capillary
‘’analysis of the control not in
conformity ‘’ is diplayed

No need to calibrate after a technique change or after a capiclean cycle

A message that required a validation by the operator, is
displayed before an analysis of calibrator level 1 or 2:

• if the lot number of this calibrator is changed in comparison with

the number previously recorded in Phoresis≥8.60

• if the concentration of this calibrator is changed in comparison

with the concentration previously recorded in Phoresis≥ 8.60

•If both lot number and concentration of this calibrator are changed
in comparison with information previously recorded in Phoresis
≥ 8.60
 Alarm in Phoresis ≥8.60 for a
new calibration after a change
of buffer lot number
 Calibration of capillaries for Hb A1c analyses
after a capillary replacement on Capillarys Flex Piercing

 After the replacement of one or several capillaries and if the lot

number of the calibrators is identical to the one used previously:
1. Perform one calibration (one run of each calibrator level)
2. Then analyze both control levels on the « F0 » rack.
3. Verify that all the obtained values for these controls are
within the customized target ranges established previously for
both control levels.
4. Verify that the bias obtained on every replaced capillary is
≤ 0.2 % for the control level 1 and ≤ 0.3 % for the control
level 2 as compared to the average of the last 3 analyses of
each control on each replaced capillary taken individually.
5. If every points are validated, the system is calibrated for
routine analyses.
 Calibration of capillaries for Hb A1c analyses
after a capillary replacement on Capillarys Flex Piercing
 If the previous requirements are not filled:
1. Perform two calibrations (2 runs of each calibrator level) again
2. Then analyze both control levels on the « F0 » rack again.
3. Verify that all the obtained values for these controls are
within the customized target ranges established previously for
both control levels.
4. Verify that the bias obtained on every replaced capillary is
≤ 0.2 % for the control level 1 and ≤ 0.3 % for the control
level 2 as compared to the average of the last 3 analyses of
each control on each replaced capillary taken individually.
5. If every points are validated, the system is calibrated for
routine analyses.
6. Otherwise, it is necessary to customize the target ranges of
controls according to the protocol described in the package insert.
System validation in HbA1c at
the installation and in routine
using Sebia HbA1c quality
controls

4
 Calibrators Controls
2 levels 2 levels
Number
(green & red caps) (white & black caps)
Appearance lyophilisate lyophilisate
Stability (storage) 3 years -30/-18°C 3 years 2-8°C
Given unit Only in mmol/mol mmol/mol and %

Calibrators Controls
-30 to -18°C
max 4 weeks at 2-8°C -30 to +8°C
Lyophilisate transportation
required isotherm boxes for max 4 weeks at 15-30°C
transportation (cool packs)

timestrip with alert if T° > 8°C

Temperature sensor during
with unacceptability of the none
transportation calibrators if period > 12 h
 Quality Control
on Capillarys 2 Flex Piercing
for Hb A1c analyses

4a
=10 x 2 vials
=1 x 2 vials
Reconstitution: with 0.75mL deionized water
1. Add 0.75mL deionized water
2. Swirl gently the vial to ensure a good homogeneization
3. Allow the vial to stand 30 minutes at 2-8°C
4. Swirl the vial before use and process the control
The reconstituted control must be transferred in a conical
tube before use, with correct barcode !

Use: up to 5 uses of the reconstituted control

+ 4 uses of a single diluted segment : after the 1st control dilution, the segment can be frozen
at -20°C and reused after (3 freezing/defreezing cycles possible).
 Before re-use of a diluted segment, allow to stand at room temperature 20-30 minutes
until complete thawing and homogeneize manually each well before analysis.

NB:
- After each use, freeze the reconstituted control and/or the diluted segment, as soon as possible.
- 30 freezing/defreezing cycles possible: let the reconstituted control defreeze at 2-8°C before use
(swirl the tube before use)
- Reconstitued control stable for 6 months, diluted control segment stable for 1 month
Volume of reconstitution modified for Capillarys 2 Flex Piercing
Reconstitution: with 0.6mL deionized water
1. Add 0.6mL deionized water
2. Swirl gently the vial to ensure a good homogeneization
3. Allow the vial to stand 30 minutes at 2-8°C
4. Swirl the vial before use and process the control
The reconstituted control must be transferred in a conical
tube before use, with correct barcode !

Use: up to 4 uses of the reconstituted control

+ 4 uses of a single diluted segment : after the 1st control dilution, the segment can be frozen
at -20°C and reused after (3 freezing/defreezing cycles possible).
 Before re-use of a diluted segment, allow to stand at room temperature 20-30 minutes
until complete thawing and homogeneize manually each well before analysis.

NB:
- After each use, freeze the reconstituted control and/or the diluted segment, as soon as possible.
- 30 freezing/defreezing cycles possible: let the reconstituted control defreeze at 2-8°C before use
(swirl the tube before use)
- Reconstitued control stable for 6 months, diluted control segment stable for 1 month
Use the correct control barcordes according Phoresis version

To use if Phoresis versions 8.71≤ for Capillarys

Flex Piercing and 9.11≤ for Minicap Flex
Piercing

To use if Phoresis versions ≥ 9.15 for Capillarys

Flex Piercing and Minicap Flex Piercing
 Hb A1c controls analyses on all capillaries for
Capillarys Flex Piercing before any patient analyses

 How to use SEBIA controls:

Control 1 Control 2
After each calibration procedure 1 run/capillary 1 run/capillary
And

After capillaries activation 1 run/capillary 1 run/capillary

Or

After a Capiclean Procedure 1 run/capillary 1 run/capillary

Or

Before any sample analysis 1 run/capillary Or 1 run/capillary

 Hb A1c control analyses on all the capillaries for
capillarys 2 flex piercing during any patient analyses

 How to use SEBIA controls:

 Re-use of thawed diluted HbA1c control
segment analyzed on Capillarys 2 Flex Piercing

When diluted control HbA1c 1 or 2 segment

has been frozen at -20°C and must be
re-used:
• Thaw the segment at room temperature
for 20-30 minutes Put diluted control segment on rack F0
•Mix each well by pipetting up and down
(Do not make any bubbles) Do not forget to put an empty tube
• Do not perform more than with the suitable control HbA1c 1 or 2
3 freezing/defreezing cycles for the same barcode
control segment
 Quality Control
on Minicap Flex Piercing
for Hb A1c analyses

4b
 Hb A1c controls analyses on both capillaries (position 28)
for Minicap Flex Piercing before any patient analyses
 How to use SEBIA controls:

Control 1 Control 2
After each calibration procedure 3 runs/capillary 3 runs/capillary
And

After capillaries activation 1 run/capillary Or 1 run/capillary

After a Capiclean Procedure 1 run/capillary 1 run/capillary

Or

Before any sample analysis 1 run/capillary 1 run/capillary

Or
After a whole carousel of analyses 1 run/capillary 1 run/capillary
Or
• Controls are reconstituted with 0.75 mL of distilled water
• After reconstitution, transfer the whole amount of the
controls, in a single Sebia conical control tube
• Storage: 8 hours max at 2-8°C. After use they must be stored
between -18°C and -30°C
• After storage, before use, thaw the reconstituted controls at 2-
8°C for at least 45 min.
• Homogenize controls before each use
• Store the thawed controls at 2-8°C for 8h max
• Do not freeze and thaw the reconstituted control more than 30
times. However the vial can be analyzed only 19 times in
position 28 (dead volume=100µL)
• Do not leave the reconstituted controls at room temperature
 Verification and alarm on
Minicap/Capillarys Flex Piercing
HbA1c controls

4c
Check your curve aspect and your control values
Check your curve aspect and your control values
OD max to reach using
automatic dilution only
OD max to reach
Check OD max values at the installation
Alarm Software criteria What has to be done?

No HbA1c value calculated (missing HbA1c,

or HbA0, HbA2, other HbA) or presence of
1- Repeat the analysis from the same
a supplementary peak on the profile
vial or dilution segment. Verify the
correct reconstitution, storage and
Defective profile centering (HbA0 or HbA1c
mixing of the control
positions outside Sebia specifications)
2- Reconstitue and reanalyse a new
control vial
Too low optical density for a control (OD
3- Contact your distributor
<0.09 on Capillarys Flex Piercing, 0.07 on
Minicap Flex Piercing)

HbA1c values outside of personalized control Verify the registered values for the
value ranges registered in the QC software control lot number that is on going.
option Analyze the control again

Always verify that the reconstitution volume, the freezing/thawing

cycles and the mixing procedure for the control are correct before use
• Display of warning flags on the mosaic in Phoresis ≥8.60:
 Determination
of customized Control values

4c
It is mandatory to customize HbA1c values and
range for both control levels on Capillarys Flex
Piercing or Minicap Flex Piercing in cases of :

•First installation of HbA1c technique (after first

system calibration)
• Change of calibrator lot numbers
• Use of new control lot number
• Change of one or many capillaries
 Run both control levels on rack F0 to obtain 8 values for each
control using automatic dilution option only

Validation : absence of alarm messages and all HbA1c values

are in the indicated range on Sebia control package insert

Calculate the mean value and CV for these analyses using Phoresis statistic tool

Validation: CV≤2% OR the difference between the minimal value and the maximal
value of HbA1c percentage is ≤0.3 points for control level 1 and ≤0.6 points for
control level 2

Mandatory: Establish your customized HbA1c values in % and mmol/mol for both
control levels for your system
 Run 3 times both control levels on position 28
to obtain 6 values for each control

Validation : absence of alarm messages and all HbA1c values

are in the indicated range on Sebia control package insert

Calculate the mean value and CV for these analyses using Phoresis statistical tool

Validation: CV≤2% OR the difference between the minimal value and the maximal
value of HbA1c percentage is ≤0.3 points for control level 1 and ≤0.6 points for
control level 2

Mandatory: Establish your customized HbA1c values in % and mmol/mol for both
control levels for your system
 Customization of HbA1c controls
ASSIGNATION OF THE HbA1c CONTROLS VALUES: EXAMPLE
Target values given by Sebia (instruction sheet) for controls lots 15124/xx & 16124/xx:
Level 1 = 15124/xx : 5.1 +/- 0.6% Level 2 = 16124/xx : 7.7 +/- 0.7%

Phoresis statistics:

 The average values are in the given ranges for the tested controls
 The CVs are equal to or less than 2.0 % for the A1c percentages values
Assignation of the customized values for this instrument:
- Level 1 : 5.0 +/- 0.3 % or 31 +/- 3 mmol/mol
- Level 2 : 7.5 +/- 0.4 % or 59 +/- 4 mmol/mol
Customization of Capillarys Flex Piercing HbA1c controls
Mandatory: Establish your customized HbA1c values in % and mmol/mol for both
control levels for your system

These customized values must be enter in Phoresis: Quality control -> Setup
control values

Enter both customized

Hb A1c values and ranges in
% and/or mmol/mol
 SEBIA recommendation to customize HbA1c controls for
an instrument follows 6% biais restriction by NGSP
Calculate the mean value and CV on each validated control
HbA1c 1 &2 analyzed (automatic dilution for C2FP only)

Validate that CV≤2% OR the difference between the minimal

value and the maximal value of HbA1c percentage is ≤0.3 points
for control level 1 and ≤0.6 points for control level 2

Apply:
For HbA1c control 1: mean HbA1c%  0.3 & mean HbA1c mmol/mol  3
For HbA1c control 2: mean HbA1c%  0.4 & mean HbA1c mmol/mol  4

This rule follows NGSP biais criteria acceptance: NGSP target  6%

For example : if calculated mean HbA1c% control 2 = 8.9%

=> NGSP biais would be 0.53% (6 x 8.9/100)
SEBIA biais is 0.4% (Sebia biais is more strict)
 CV’s are different
between mmol/mol
and cal%

- Mean values with no decimal for mmol/mol and one decimal for cal%
- Calculated from real values, not from round displayed values
Only for Hb A1c
Westgard’s rules on Levey-Jennings curves
13s 41s

12s 7T

22s 10x

R4s
HbA1c
Samples for HbA1c analysis

5
 Samples for Capillarys/Minicap Flex Piercing Hb A1c
• Only samples collected on K2 EDTA and K3 EDTA tubes (lavender/pink)

• No samples collected on tubes containing NaF

Storage: Whole blood samples stored up to 7 days at

2-8°C or 3 months at -80°C
 Capillarys: hemolysing solution on board
 Minicap: Place a tube with hemolysing solution in position 27 (5 ml in a tube 13
x75mm for 12 analyses, 10ml in a tube 16 x100mm for 24 analyses)
Tube types for Capillarys/Minicap Flex Piercing Hb A1c

- Collection tubes with 13 mm diameter and their corresponding caps

(maximal length of tube with cap : 90 mm, maximal diameter of cap : 17
mm) : BD Vacutainer, Terumo Venosafe 5 mL, Greiner Bio-one Vacuette 1,
2, 3 or 4 mL or Sarstedt S-Monovette 4 mL tubes (13 x 75 mm)

- Collection tubes with 11 mm diameter and their corresponding caps

(maximal length of tube with cap : 90 mm, maximal diameter of cap : 17
mm) : Sarstedt S-Monovette 2,7 mL or Kabe Labortechnik Primavette S
2,6 mL tubes (11 x 66 mm)
 Samples for Capillarys Flex Piercing Hb A1c
Collection tubes with 11 mm diameter and their
corresponding caps (maximal length of tube with cap :
90 mm, maximal diameter of cap : 17 mm) : for
example, Sarstedt S-Monovette 2.7 mL or Kabe
Labortechnik Primavette S 2.6 mL tubes (11 x 66 mm)

Must be analyzed on
this special rack

Reference 1360 :
RACKS CAPILLARYS 2 FLEX PIERCING FOR
TUBES WITH 11 mm
Low volume samples for Capillarys Flex Piercing HbA1c
 Sample analyses:
• Sample volume in primary tubes ≥1ml min : Direct analysis in a
standard rack

• Sample volume in primary tubes 100µl-1ml < : Transfer the sample

volume in a capped conic control tube (ref PN 9202/9205) and analyze
this tube in a standard rack using an wedge adapter (PN 9203)

• Sample volume in primary tubes 100µl< : Mix by pipetting 90µl

hemolyzing solution + 18µl blood in a segment well. Place the segment
on the F0 rack and perform the analysis using ‘’manual dilution’’
Low volume samples for Minicap Flex Piercing HbA1c

For sample volume < 1ml:

 If sample volume between 200 µl and 1ml:

 Transfer 200µl blood (vortexed) in a Sebia conical
control tube (ref 9202/9205), cap the tube
 Stick the “LOW VOLUME AUTO” barcode on the
tube
 Place the tube on the carousel/rack to run the
analysis, close the door, the analysis starts
automatically
Low volume samples for Minicap Flex Piercing HbA1c

If sample volume 200 µl <:

 Vortex the sample and transfer 50 µl in a Sebia conical
control tube (PN. 9202/9205)
 Add 250 µl hemolyzing solution; vortex 5 sec
 Stick the “LOW VOLUME MANUAL” barcode on the
tube
 Place the tube on the carousel/rack to run the
analysis, close the door, the analysis starts
automatically

 Minicap : A tube containing 5ml of hemolysing

solution must still be inserted on the position 27 (5 ml
in a tube 13 x75mm for 12 analyses de 12, 10ml in a
tube 16 x100mm for 24 analyses)
 Indications for low sample volumes
LOW VOLUME AUTO
LOW VOLUME MANUAL
• With Phoresis 8.6.1 release
• Dilution type is displayed in mosaic view
and in “edit curve” window (“a” for automatic,
“m” for manual dilution)
• An individual ID is associated with each label.
This ID can be modified in the worklist
 Whatshall
What do wewemeasure?
measure?
Definition
We apply
of the
theAnalyte
following
according
calculation:
to IFCC:
HbA1c%HbA1c
is biochemically
=
characterised
100 as thearea
x [HbA1c area / (HbA1c stable adduct
+ HbA0 area)]
% = 0.09148 x mmol/mol + 2.152
ofWe
glucose
apply to
thethe N-terminal
calculation amino
formula
IFCC Master Equation
group
as per IFCCofRecommendations
the β-chain of
hemoglobin A0
Precise
HbA1c measurement
 SEBIA follows
the IFCC
standardization
guidelines !
Blue pattern: Normal Profile Orange pattern: Elevated HbA1c Purple pattern: Atypical Profile
 No raw data for Hb A1c

Raw data for other peaks

No possible modification
of profiles and curves

Colour-coded profiles
Colour-coded Hb A1c peak

Hb request

Standardized Hb A1c values and flags

Profile in blue
Profile in orange
Profile in purple
 Degraded Hb

Profile in purple

Correct storage of samples:

7 days at 2-8°C, 3 months at -80°C
Alarm Software criteria What has to be done?

No HbA1c value reported. The HbA1c value In case of profile not recentered (fractions HbA1c,
is not calculated : HbA0, other HbA and HbA2 only visible), repeat the
- if presence of a homozygote variant or analysis
composite heterozygote variants
In case of interference from a heterozygote variant or
- if interference on the HbA1c or HbA0 peak presence of homozygote variant/composite
(shoulder, insufficient baseline return…): heterozygote variants, perform a hemoglobin
suspect a variant with partial separation electrophoresis and a measurement of fructosamines
- if profile not recentered
- if HbA2 peak missing (for Phoresis version
<9.15 only)
- Suspect the presence of a homozygote variant or
composite heterozygote variants, perform a
hemoglobin electrophoresis and a measurement of
Absence of detection for Hb A0 and / or Hb
fructosamines
A1c peaks
- If the profile does not show a variant (s): suspect a
defective profile recentering and repeat the analysis
for confirmation

Repeat the analyse. If the result is confirmed (even if

Optical density too low for a non atypical the alarm is still present), the HbA1c value can be
samples reported
 HbA1c technique: how prevent automatic export of atypical,
pathological and/or low OD samples to LIS/HOST server?

Host 6.0

Use login password: 4644

Select all
2
options
3
 HbA1c technique: how prevent automatic export of atypical,
pathological and/or low OD samples to LIS/HOST server?
OD option only available with Host 6.0

For Capillarys 2 flex piercing : the threshold used by Phoresis for samples is 0.10 => you must enter
the value of 1000

For Minicap flex piercing: the threshold used by Phoresis for samples is 0.07 => you must enter the
value of 700
 Alarm Software criteria What has to be done?
Heterozygote variant (alpha, beta or delta) :
-Verify if the presence of a variant affects the quantification
- Presence of a heterozygote of HbA1c and/or HbA0 peaks. If the HbA1c and/or HbA0
variant (alpha, beta or delta) fractions are not affected, there is no analytical interference
on HbA1c measurement. Report the HbA1c value with a
comment on possible clinical interference of this variant.
Atypical The HbA1c value can be compared with usual decisional
profile threshold

Presence of Hb F (or variant in Hb F position) :

- Presence of Hb F (or variant - The presence of Hb F (or a variant migrating in Hb F
in Hb F position) position) up to 23% does not interfere with the
quantification of HbA1c (above 23% HbF, an alarm message
is displayed (Phoresis 8.63 Patch 5.0)). Report the HbA1c
value with a comment. The HbA1c value can be compared
with usual decisional threshold

In absence of variant on the profile : risk of beta-

thalassemia. Evaluate clinical context and perform a
HbA2 (!) Elevated HbA 2 value >3% hemoglobin electrophoresis to confirm the presence of
beta-thalassemia. Report the HbA1c value with a comment
on possible clinical interference of this variant. The HbA1c
value can be compared with usual decisional threshold
In Phoresis 8.60: (! ) = alarm indicating a HbA2 value >3%. A risk of beta-thalassemia
can be emphasized
 Hb F and HbA1c profile
Curve Indications

Presence of Hb F (or variant in Hb F position) :

- The presence of Hb F (or a variant migrating in Hb F position) until
Atypical profile 23% does not interfere on HbA1c quantification (Above 23% HbF
(Phoresis 8.63 Patch 5.0), an alarm is displayed on the curve and the
worklist

If Hb F>23%*

* On Phoresis 8.63 Patch 4.0≤, this alarm threshold was >15%

Default values in software
• Only for Hb A1c (not for eAG nor other fractions):
- %NGSP: Use « Normal range in % »:
6.0% (IFCC recommendation) by default,
Customizable: change values on all lines
Indicates the upper reference limit, not the cut-off
(! type 6.4% if cut-off is 6.5% !)

- mmol/mol: Use « Normal range in concentration »:

42 mmol/mol (IFCC recommendation) by default,
Customizable : change values on all lines
Indicates the upper reference limit, not the cut-off

Customization: Delete % or concentration

values if only one unit is required by the
customer
 How to set-up displayed values in curves
and worklist?
• By default, Hb A1c values are displayed in % (NGSP),
in mmol/mol (IFCC)
• This must be personnalized by Sebia Service at the
installation step:
– Normal range in % and normal range in concentration
Change the normal values and/or delete one unit for Hb A1c
– Display window Configure using « Display parameters 2/2 »
– Work list by table Use « options » button
– Report Delete one unit for Hb A1c

Whole customization possible for display and report:

Hb A1c results and flags
How to set-up displayed values in curves and worklist?

Customization

eAG (estimated average glucose in mg/dL) is not

displayed automatically anymore. You must select the
option for displaying if necessary
Fractions results:
- Calibrated Hb A1c
- Raw values for the others
customizable

Hb A1c results summary:

Values and flags
customizable
 For personnalization of Standardized Hb A1c results and flags
worklist display and printing

Increased Hb A1c

pathological

atypical

Different flags:
- for all samples, except calibrators: > if Hb A1c value is higher than one cut-off
>> if Hb A1c values are higher than the 2 cut-off
(*) if atypical profile
- for QC controls: +, -, or = according to QC values, no more to cut-off values
HbA1c and Capillary blood sample
 New Sebia automatic HbA1c testing from capillary blood
(PHORESIS 8.63 required)

 The new fully automated technique for the

HbA1c analysis from capillary tubes on
CAPILLARYS 2 Flex Piercing offers full
traceability from sampling to final result.
Results are strictly identical to those
obtained on venous blood.
Sample storage recommendations for capillary blood
samples
Between collection and analysis, the capillary blood samples can be stored in
microtubes with hemolysing solution for :
 8 days maximum at 2-8°C or 15-25°C
 3 days maximum at 30°C
 8 days maximum at -20°C

 Upon receipt at the laboratory, store the samples in the samples in the
device at 2-8°C and analyze them within 8 hours maximum
Capillary blood sample analyses in HbA1c
 The analysis must be performed on whole blood samples that are hemolyzed
within the microtube. Check the feature of the microtube after the receipt by the
laboratory (do not analyze the sample when the microtube has been damaged during
transportation). When a part of the hemolyzed sample is present in the cap of the
microtube, shake it down into the bulk liquid.

 Vortex the capillary blood sample for 5 seconds.

 Put the microtube on his labelled adapter on a specific rack for capillary blood sample
HbA1c analyses (ref 1361). This specific rack has a C barcode.
Utilization of transport tubes with PHORESIS releases < 8.63 (and ≥ 8.61 p4)
• Uncap the tube, let the capillary tube inside the microtube.
• Place a new green dilution segment on the sample rack No. Cxxx, the central pin of the segment must face the operator.
• Take 100 μL of each hemolyzed sample and apply it into wells of the dilution segment
• Cap tightly the tube and let it on the rack. Start the analysis without any delay

Rack Cn
1 2 345678

Utilization of transport tubes with PHORESIS releases ≥ 8.63

• Check each tube is tightly capped.
• Place a new yellow dilution segment on the sample rack No. Cxxx, the central pin of the segment must face the
operator
• Start the analysis without any delay
Rack Cn
 Profiles comparison
(Low HbA1c)

Venous blood Capillary blood

(+4°C for 5 days) (Room T° for 5 days)
The profiles obtained from capillary blood shows an additional peak of degradation due to the
storage conditions at room temperature. The profile is classified as atypical (purple profile), but
the HbA1c value is still accurate
 Profiles comparison
(Medium HbA1c)

Venous blood Capillary blood

(+4°C for 5 days) (Room T° for 5 days)

The profiles obtained from capillary blood shows an additional peak of degradation due to the
storage conditions at room temperature. The profile is classified as atypical (purple profile), but
the HbA1c value is still accurate
 Profiles comparison
(High HbA1c)

Venous blood Capillary blood

(+4°C for 5 days) (Room T° for 5 days)
The profiles obtained from capillary blood shows an additional peak of degradation due to the
storage conditions at room temperature. The profile is classified as atypical (purple profile), but
the HbA1c value is still accurate
 Performances
of the HbA1c technique

7
• High resolution

capillary electrophoresis

• Separation clear-cut and precise

The best equilibrium
• Simple calculation formula in between
direct IFCC formula precision,
• Mean CVs < 1,5% over 8 capillaries robustness
• No interference or altered results for and throughput
common interferents and variants

• Low calibration frequency required

• Automatical OK-calibration / OK-control

• Instruments dedicated