TOSOH G8 Routine Use Training Workbook
TOSOH G8 Routine Use Training Workbook
TOSOH G8 Routine Use Training Workbook
TOSOH G8
Date: 19/07/2021
Document Ref: SUKBMS-24-596
Version: 2.0
Sysmex House, Garamonde Drive, Wymbush, Milton Keynes, MK8 8DF, United Kingdom · Phone +44 333 320 3460 · Fax +44 1908 267 90 1
academy.training@sysmex.co.uk· www.sysmex.co.uk
Contents
Disclaimer .................................................................................... 3
LCD Screen Layout ...................................................................... 8
Main Screen (Page 1) ................................................................ 8
Main Screen (Page 2) .............................................................. 10
High Pressure Liquid Chromatography ....................................... 12
Task 1: Analysis Principles and Parameter Production ............... 13
Maintenance............................................................................... 14
Daily Checks ............................................................................ 14
Pump Clean (Daily Shutdown) ................................................. 14
Replacing the Line Filter .......................................................... 15
Changing the Column .............................................................. 16
Column Calibration ..................................................................... 18
Registering a New Lot of Calibration Material ........................... 18
Preparation of Calibrator Material ............................................. 18
Performing Calibration ............................................................. 19
Quality Control (QC) ................................................................... 21
Registering a New Lot of QC .................................................... 21
Running QC ............................................................................. 22
Running patient samples ............................................................ 24
Sample Requirements.............................................................. 24
System Mode ........................................................................... 24
Sampler Mode.......................................................................... 26
Pre-dilution Mode ..................................................................... 27
Document Ref: SUKBMS-24-596 Version: 2.0 Date: 19/07/2021 Classification: Unrestricted
Page | 1
Chromatogram Interpretation ...................................................... 28
Normal Chromatograms ........................................................... 28
Chromatogram Flagging .......................................................... 30
Checking Patient Results ......................................................... 31
Task 2: Chromatogram Interpretation ....................................... 33
Changing Reagents .................................................................... 38
Dealing with Errors ..................................................................... 39
Troubleshooting.......................................................................... 40
Area Too High/Area High ......................................................... 40
Area Too Low/Area Low ........................................................... 40
Pressure High .......................................................................... 40
Pressure Low (Drain Flush)...................................................... 40
Calib Error................................................................................ 42
Contact Us .................................................................................. 43
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Disclaimer
Please note, the information contained in training resources provided by Sysmex should not be
used as an alternative to your sites Standard Operating Procedure (SOP)/Contract. If you have
any particular questions regarding any site specific use of reagents, consumables and/or
equipment please contact your Management Team.
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TOSOH G8 Overview
The TOSOH G8 is a full automated HPLC analyzer that can be used to perform HbA1C or
haemoglobinopathy analysis (standalone only). The analyzer can fully integrated with Sysmex
XN9000 system.
Parameters HbA1a
HbA1b
HbF
l-A1c
S-A1c
A0
Throughput
Linearity
G8 Variant mode (SA1c area) 700-3000 (Recommended)
500-4000 (Acceptable)
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Analyser Components
1 2
3 7
8
4
9
5
10
1. Sampling unit – houses the needle unit which is responsible for cap piercing and aspiration
of blood samples. The cover to the sampling unit should NOT be removed while the
analyzer is powered ON.
2. Thermal printer – used for printing out chromatograms, error messages and parameter
status.
3. Buffer reagents – There are 3 buffer reagents housed on-board the analyzer, which work
in combination to elute HbA1a, HbA1b, HbF, SA1c, LA1c and any other haemoglobin
variants present.
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4. Drain valve housing – the drain valve (C) is located under the cover and
is responsible for removal of air from the analyzer, when required. The
drain valve should be closed during analysis (as pictured). Also located A
under the cover are the Rotary valve (A) and the Injection valve (B).
B
5. Haemolysis wash – used for the breakdown of red cells and in manual
dilution of samples.
6. Mains power switch – used to turn the mains power ON and OFF.
7. LCD screen – user interface for operation of the TOSOH G8. Contains the
analyzer settings, reagent/consumable information, and version C
information.
8. Control Panel
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10. XN Track/Sampler unit – Samples are placed in racks and are introduced from the XN-
9000 track when the analyzer is in ‘system mode’. If required the analyzer can be placed
into ‘offline mode’ using the [Mode Switch] on the control panel of the conveyer unit (CV-
60). A GREEN mode switch indicator LED indicates ‘system mode’ and an ORANGE LED
indicates ‘offline mode’.
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LCD Screen Layout
Main Screen (Page 1)
1
4 3
6
7
8
3. Pump flow pressure – The pressure should remain initial pressure ±4. The initial pressure
target can be found in the insert included in the column box.
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[BUFF PRIME]: Displayed following power ‘ON’ or following reagent changing. During
BUFF PRIME 5ml of each elution is primed into the flow line.
[PUMP CLEAN]: Displayed following WASH. Plunger seal is automatically washed with
haemolysis wash solution to prevent contamination or buildup of salt precipitate.
6. Calibration Status;
[Filter]: number of injections since filter was replaced. The filter should be replaced
approximately every 400 injections (See page 16)
[Column]: number of injections since column was last replaced. The column is guaranteed
for 3000 injections (See page 17)
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Main Screen (Page 2)
Main screen (page 2) can be accessed by pressing the next button [↓] on main screen (Page 1)
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Reagents On-board the TOSOH G8
The TOSOH G8 has 4 on-board reagents, 3 elution buffers and 1 haemolysis wash, which are
used during analysis. The 3 elution buffers are used in combination during analysis for separation
of the haemoglobin variants and are in colour code bags. The Lot letter of the elution bags should
always match the lot letter of the column in use.
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Analysis Principles and Parameter
Production
The addition of eluents in a specific order results in the elution of haemoglobin fractions with the
weakest bound fractions eluting first.
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Task 1: Analysis Principles and Parameter
Production
Using the information provided annotate the chromatogram below, labeling the axis, peaks and
identifying reagents used:
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Quick Guides
Maintenance
Daily Checks
Before using the analyzer a series of checks should be made;
• Check reagent levels
• Check filter and column injection counts
• Check pressure
• Waste (if required)
In order to shut down the analyzer, ensure that no work is being processed and the
analyzer is in STANDBY. Place the analyzer in to ‘sampler mode’ using the mode
switch on the conveyor unit (See page 8).
To shut down the analyzer press the [POWER] switch on the front of the analyzer. The
following message will appear:
Press [OK]. Once analyzer is fully powered down press the [POWER] switch to start the analyzer
back up. On start up the analyzer will perform a series of background checks including a pump
clean, priming of the buffers and washing of the aspiration line. This is represented by the analyzer
status changing from PUMP CLEAN to BUFF PRIME to WARMING UP. Completion of the startup
procedure takes approx. 9mins. Once WARMING UP is complete the analyzer will go into
STANDBY mode and is then ready for use. Place analyzer back in ‘system mode’ (See page 8).
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Replacing the Line Filter
The line filter should be replaced every 400 injection
or when the pressure is ±4 from the stated target (See
column insert). IMPORTANT: The analyzer will alarm
at 400 injections and will continue to alarm with every
injection over 400. Failure to replace line filter when A
appropriate may result in damage to the column and
analysis errors.
Black ring
Place holder on top of filter and push down. NOTE: if the filter is orientated in the correct direction
it will easily go back into position and the filter will sit proud within the holder. If there is resistance
to the filter, check the filter is orientated in the right position (See picture above). Screw the filter
holder back in place and re fit the filter outlet. Before using the TOSOH G8 perform a pump prime
to check for leaks and ensure pressure is in the correct range.
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To reset the filter count to zero from the main
screen select [MAINTE] then [REAGENT].
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Before using the TOSOH G8 perform a pump prime to check for leaks and ensure pressure is in
the correct range. A pump prime can be performed from the second screen of the main menu by
pressing [PUMP]. To stop the pump press [PUMP] again. Close column oven and column and filter
housing cover.
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Column Calibration
Column calibration should be performed following
• Column replacement
• When there is evidence of QC drift
• Following major analyzer maintenance
Select each calibrant in turn, ‘CAL-L-ID’ and ‘CAL-H ID’ for the low and high
calibrators respectively, and enter the desired 13 digit barcode, for example
Lot No. ACAL01 enter 7 spaces followed by ACAL01 (_,_,_,_,_,_._ACAL01)
The material is lypholized human haemoglobin and one vial should be made up using 4ml of
distilled water. Once reconstituted the material can be aliquoted in to 1ml aliquots and frozen for a
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maximum of 30 days. NOTE: if kept in resealed vial in fridge calibrator is stable for 7days. Do NOT
leave calibrator at room temperature for long periods of time.
Performing Calibration
In order for column calibration to be performed, ensure the analyzer is in STANDBY, the analyzer
is in ‘sampler mode’ and the [BELTLINE] the FIX, CUP and DIL DO NOT have to be highlighted.
Place the rack on the input area on the right side of the conveyor (A) and press [START] button on
TOSOH G8. Once aspiration of calibrators is complete the rack will move to the output area on the
left side of the conveyor (B).
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During analysis, the level 1 calibrator is analyzed
3 times (runs 1 to 3) and level 2 calibrator is
analyzed twice (runs 4 to 5). The first analysis of
calibrator 1 is discarded and the calibration factor
is calculated from the mean %sA1c of analysis 2
and 3 for level 1 and analysis 4 and 5 for level 2.
These values are then compared to those with the
TOSOH G8 settings.
Once analysis is complete, place the analyzer back into ‘whole blood’ mode (See page 25) and
back into ‘system mode’ (See page 24). NOTE: if analyser left with FIX, CUP and DIL highlighted
whole blood samples will not be diluted during analysis this will lead to AREA TOO HIGH and
possible damage to the column.
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Quality Control (QC)
Quality control (QC) should be performed following calibration of the analyzer, troubleshooting or
as part of daily checks/maintenance.
Select each QC in turn, ‘QC-L1-ID’ and ‘QC-H1-ID’ for the low and high QC
respectively, and enter the desired 13 digit barcode, for example Lot No.
AQC01 enter 8 spaces followed by AQC01 (_,_,_,_,_,_._,_AQC01)
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Running QC
In order to perform QC, ensure the analyzer is in STANDBY, the analyzer is in ‘sampler mode’ and
the [BELTLINE] the FIX, CUP and DIL have been highlighted.
Place 1ml of distilled water and 20µl of QC material in appropriately barcoded cups and mix
thoroughly before placing in the rack.
Place the rack on the input area on the right side of the conveyor (A) and press [START] button on
TOSOH G8. Once aspiration is complete the rack will move to the output area on the left side of
the conveyor (B).
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Document Ref: SUKBMS-24-596 Version: 2.0 Date: 19/07/2021 Classification: Unrestricted
Page | 23
Running patient samples
Sample Requirements
HbA1c analysis requires whole blood samples in EDTA tubes with a minimum blood volume of
1ml. Samples should be analyzed as soon as possible after collection. NOTE: Gross lipidaemic
samples my give erroneous results.
System Mode
The location of the feeder section and collection sections can vary due to instrument combination.
A typical example is shown below:
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Load sample racks into the feeder section of the
‘Start Yard’ (ST). Automatic rack recognition will
activate the rack pushers and the rack will be
moved towards the back of the ‘Start Yard’ to be
fed into the BT-40.
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Following sample analysis the completed racks
will be pooled in the stock yard where they can be
removed or I present sent to the tube sorter for
storage.
Sampler Mode
To place the TOSOH G8 in ‘sampler mode’ press the mode switch on the XN-track
conveyor unit control panel. The indicator light will turn ORANGE. NOTE: if running
Calibrators, QC or pre diluted samples the TOSOH G8 will also need to be placed
in Pre-dilution mode (See page 26).
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Pre-dilution Mode
Pre-dilution mode should be used for running Calibration material, QC and any samples less than
1ml. For QC preparation see page 21.
To place the analyzer in pre-diluted mode from the ‘Main menu’ press [MENU] then [BELT LINE].
This will open the ‘Belt line’ screen, select [FIX] and [DIL].
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Chromatogram Interpretation
Normal Chromatograms
1
2
3
4
5 7
6 8
10
11
12
13
14
4. Calibration equation - Displays the calibration equation the HbA1c result is based on.
5. Theoretical plate (TP) - represents the quality of the calibration from the HbA1c peak. This
figure must be greater than 250. A theoretical plate less than 250 indicates old column.
6. Hb fractions – lists the haemoglobin fraction in the order they are eluted within the column.
Weakest fractions appear at the top of the list with the most positive fractions appearing at
the bottom of the list.
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7. Retention time (RT) - shows the retention time for the corresponding haemoglobin
fragment eluted.
• A1A 0.23
• A1B 0.27-0.33
•F 0.39
• lA1c 0.47
• sA1c 0.57 – 0.61
• A0 0.88 – 0.92
NOTE: Retention times should NOT alter between reagent and column lots. RT errors are
usually due to leaks, especially if the RT is late.
8. Area – displays the corresponding area for each haemoglobin fraction detected.
9. Total area (TA) – sum of all the peak areas. Recommended range 700-3000. Acceptable
range is 500-4000. Note: Total areas outside these limits on one sample indicate sample
problem. TA is out of range on multiple samples indicates analyzer problems such as
blockage or dilution issue.
10. HbA1c % - displays HbA1c percentage for that sample. Normal range is 20-42 mmol/mol
(4.0-6.0% ) with a target of <53mmol/mol (<7.0%) for diabetic patient.
11. Hb Fragments – peaks for different haemoglobins eluted with weaker binding to column
resins than HbA1c, typically HbA1a, HbA1b, HbF, and LA1c.
12. sA1c peak – peak for the stable HbA1c (sA1c) fraction.
14. Common Variant field (HbD, S and C) – area where additional strongly bound
haemoglobin variant fragment peaks will appear if present, resulting in an error flag (See
page 29).
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Chromatogram Flagging
Three types of chromatogram flag can be seen
1. HbF flag: generated when HbF is >5%. HbF peak is automatically removed from the
HbA1c peak.
• HbF 5-15% results in a warning
• HbF > 15% no result given
2. Variant flags: indicate the presence of a possible haemoglobin variant. The Hb variant
peak is automatically removed from the HbA1c result. Results can be report with
comment ‘possible haemoglobin variant detected’. IMPORTANT: Do NOT state which
variant is present. These results can NOT be used for diagnosis but can be used for
monitoring.
3. POO peaks: unknown haemoglobin variants. POO peaks which appear after the A0 peak
are ok to report. POO peaks before A0 with total percentage >5% invalidate HbA1c
results and therefore should not be reported. These peaks could be due to other
haemoglobin variant, glycated variant or possible old sample.
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Checking Patient Results
When analyzing chromatograms (See page 27) the following points should be checked.
• Patient information
• The total area (TA) is within the recommended range. Recommended range 700-3000.
Acceptable range is 500-4000. Note: Total areas outside these limits on one sample
indicate sample problem. TA is out of range on multiple samples (>3) indicates analyzer
problems such as blockage or dilution issue.
• The retention times (RT) for each fragment. sHb1c 0.57-0.61, A0 0.88 to 0.92.
• The theoretical plate (TP) value >250. NOTE: As column ages TP will drop.
• All peaks are represented, A1A, A1B, F, LA1C, SA1C and A0.
• Presence of flags HV or PO peaks (See page 29)
• HbA1c is within normal range
• Check for significant other fractions (See page 29)
In order to fully analyze results chromatograms should be studied in conjunction with patients full
blood count. Confirmation of a haemoglobin variant can NOT be made from Hb Variant
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chromatograms. Variant identification can only be done by DNA analysis and any chromatogram
in which such a variant is detected should be discussed with TOSOH.
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Task 2: Chromatogram Interpretation
For each chromatogram below answer the following questions
1. Is the total area within the recommended range?
2. What is the retention time for HbA0 and sHbA1c?
3. What is the TP valve and is this acceptable?
4. Are all peaks present?
5. Are there any other significant fractions present?
6. Can the HbA1c value be reported? Are any comments required?
Chromatogram A
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Chromatogram B
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Chromatogram C
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Chromatogram D
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Chromatogram E
1……………………………………………………………
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3……………………………………………………………
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Changing Reagents
Reagents should be checked as part of daily maintenance and changed as soon as remaining
levels are insufficient for next run. IMPORTANT: there are NO level sensors on the reagent lines
however the TOSOH G8 will alarm when buffer levels reach 3 – 5%. Failure to change reagent
correctly will result in air being drawn into the analyzer.
Before replacing the reagent ensure the analyzer is in ‘STANDBY’ and that the new reagent is the
same lot letter as the column currently in use.
Remove the appropriate reagent container for the analyzer by unhanging reagent from reagent
rack (Elution buffers only) and unscrewing the cap. Place the reagent probe directly into the new
reagent container, ensuring the probe reaches the bottom and rescrew cap tightly. TIP: if changing
more than one reagent at a time ensure the right colour coded line is placed into the appropriate
reagent container. If changing a buffer place buffer back on reagent rack.
IMPORTANT: Do NOT save or pool reagents. Discard any reagent left. Do NOT mix reagent lots.
Once reagent has been replaced it must be registered on TOSOH G8. This can be done by entering
the [MAINTE] screen from the ‘main menu’ screen then pressing [REAGENT].
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Select appropriate reagent or reagents that
have been changed. Press [CHANGE]. The
following message will appear
CHANGE --- EXECUTE?
Press [OK]. The reagents will then be
primed replacing the reagent in the flow
line.
NOTE: approximately 5ml of reagent is
used during a reagent prime.
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Troubleshooting
Area Too High/Area High
AREA TOO HIGH/AREA HIGH will appear as a ‘FLAG MESSAGE’ at the bottom of the result print
out (See page 27). ‘AREA TOO HIGH’ is seen if the total area exceeds 4000 and the ‘AREA HIGH’
message is seen if the total area is between 3000 – 4000. The most common cause of this error is
an inappropriately mixed sample or a high RBC count. Dilute the sample down using haemolysis
wash solution to a 1:400 ratio.
Pressure High
‘PRESSURE HIGH’ error is seen when the pressure exceeds the upper limit of 15MPa and results
in the analysis of samples being stopped and the analyzer going into ‘STANDBY’. The most
common cause is the filter or column replacement period has been exceeded. Alternative if filter
count is less than 400 the filter maybe blocked.
Check filter count and column count on the main screen on the TOSOH G8. Columns are
guaranteed for 3000 injections, however they can be run till between 5000-8000 injections as long
as QC and chromatograms remain okay. The line filter should be replaced every 400 injection or
when the pressure is +4 from the stated target (See column insert). Replace the column or filter as
indicated by the appropriate counter (See page 17 and 16 respectively). If error continues contact
your service provider.
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‘PRESSURE LOW’ error is seen when air is present in the pump check valve. The most common
causes of air entering the system is insufficient reagent(s) or leaks from either filter or column.
Check reagent levels and replace as required (See page 37). Once reagent has been replaced
perform a ‘Drain Flush’ (also known as pump air removal).
Ensure the analyzer is in STAND-BY, form the main screen select [MAINTE] screen and then
[REAGENT] to enter the ‘REAGENT CHANGE’ screen. From here select [D.FLUSH] and the
following message will appear ‘OPEN DRAIN VALVE’. Open drain valve housing (See page 7) and
turn the drain valve anticlockwise 90º degrees as indicated below and press [OK].
Press [OK]. The drain flush procedure takes approximately 7 minutes and the air trap in the pump
will be automatically removed. Once the procedure is complete the following message will appear
‘CLOSE DRAIN VALVE’. Turn the ‘drain valve back clockwise 90º degrees to the closed position
as indicated above and press [OK]. Return to the main screen (Page 2) to perform a ‘PUMP
PRIME’.
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A pump prime can be performed from by pressing
[PUMP]. To stop the pump press [PUMP] again.
Close column and filter housing cover.
Calib Error
‘CALIB ERROR’ occurs when the following conditions are met;
• The difference in sA1c % between the 2nd and 3rd calibration assays is > 0.3%
• The difference in sA1c % between the 4 th and 5th calibration assays is > 0.3%
• The sA1c % result of any of the calibration assays 2, 3, 4 and 5 was ±30% of the assigned
value.
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Contact Us
Mail
Training Academy
Sysmex UK Ltd
Garamonde Drive
Wymbush
Milton Keynes
MK8 8DF
Phone
Product Hotline
For urgent application support 0333 320 3466 (UK)
Service Hotline
For technical support and service team 0333 320 3467 (UK)
Sysmex Academy
https://uk.sysmex-academy.com/
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