Laboratory Procedure Manual

Analyte: Glycohemoglobin (HbA1c)

Matrix: Whole Blood

Method: Tosoh G8 High Performance Liquid

Chromatography (HPLC) Glycohemoglobin
Analyzer

Revised Date: May 5, 2020

As performed by: Diabetes Diagnostic Laboratory

University of Missouri School of Medicine
1 Hospital Dr. Columbia, MO 65212

Contact: Curt Rohlfing, Rhonda Howard, Dr. Randie Little

Important Information for Users

The Diabetes Diagnostic Laboratory periodically refines these laboratory methods. It is
the responsibility of the user to contact the person listed on the title page of each write-
up before using the analytical method to find out whether any changes have been made
and what revisions, if any, have been incorporated.
Glycohemoglobin (HbA1c)
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Public Release Data Set Information

This document details the Lab Protocol for testing the items listed in the following table:

Data File Name Variable Name SAS Label

GHB_K LBXGH Glycohemoglobin (%)

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1. SUMMARY OF TEST PRINCIPLE AND CLINICAL RELEVANCE

The Tosoh Automated Glycohemoglobin Analyzer HLC-723G8 is intended for in

vitro diagnostic use for the quantitative measurement of % hemoglobin A1c
(HbA1c) in whole blood specimens. HbA1c measurements are used in the
clinical management of diabetes to assess glycemic control.1 This test is also
used as an aid in the diagnosis of diabetes identifying patients who may be at
risk for developing diabetes.2

The procedure is specifically designed for the Tosoh Automated

Glycohemoglobin Analyzer HLC-723G8 equipped with appropriate software,
TSKgel G8 HSi Column, Elution Buffers, and Hemolysis & Wash Solution.

The analyzer uses non-porous ion exchange, high performance liquid

chromatography (HPLC) and microcomputer technology to quickly and
accurately measure the HbA1c as a percentage of the total amount of
hemoglobin present in the sample.

Summary and Explanation of the Test

Diabetes causes elevated levels of glucose to circulate in the blood. Maintaining

normal or near normal levels of blood glucose is part of the routine clinical
management of diabetes. Continuous and careful management of blood glucose
levels prevents development of serious long term complications resulting from
vascular impairment such as retinopathy, nephropathy, and neuropathy.

Although a fasting blood glucose measurement gives the clinician information

about the patient’s status over the last twelve hours, the stable HbA1c offers a
more accurate indication of the patient’s long-term diabetic control over the last
two to three months.

Glycohemoglobin is a general term for hemoglobin-glucose complexes in which

glucose is bound to the alpha and beta chains of hemoglobin. The most
quantitatively prevalent complex is called HbA1c, in which glucose binds to the
N-terminus of the beta chain of HbA.

HbA1c is nonenzymatically synthesized in two steps:

The glucose aldehyde group and the free amino group on the valine in the N-
terminus of the hemoglobin beta chain react to form the Schiff base, aldimine
(also known as labile HbA1c or LA1c).

A stable ketoamine form of the hemoglobin complex (SA1c) is then produced by

a reaction known as Amadori rearrangement.

The level of LA1c changes rapidly in response to changes in blood glucose

concentration. However, the level of the SA1c does not fluctuate significantly in

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response to physiological factors. Consequently, the SA1c measurement

provides a better indication of the average glucose level over the previous two to
three months (the average red blood cell life span ~ 120 days).

Formation of Labile and Stable Forms of A1c (LA1c and SA1c)

The Tosoh Automated Glycohemoglobin Analyzer HLC-723G8 can individually

resolve SA1c and LA1c on the chromatogram without manual pretreatment,
allowing accurate measurement of SA1c directly.

The Tosoh Automated Glycohemoglobin Analyzer HLC-723G8 uses non-porous

ion-exchange high performance liquid chromatography (HPLC) for rapid,
accurate and precise separation of the stable form of HbA1c from other
hemoglobin fractions. Analysis is carried out without off-line specimen
pretreatment or interference from Schiff base.

The analyzer dilutes the whole blood specimen with Hemolysis & Wash Solution,
and then injects a small volume of this specimen onto the TSKgel G8 Variant HSi
Column. Specimens may also be diluted offline using the dilution procedure
below. Separation is achieved by utilizing differences in ionic interactions
between the cation exchange group on the column resin surface and the
hemoglobin components. The hemoglobin fractions (designated as A1a, A1b, F,
LA1c+, SA1c, A0, and H-V0, H-V1, H-V2) are subsequently removed from the
column by performing a step-wise elution using the varied salt concentrations in
the Variant Elution Buffers HSi 1, 2, and 3.

The time from injection of the sample to the time the specific peak elutes off the
column is called Retention Time. The Tosoh Automated Glycohemoglobin
Analyzer HLC-723G8 software has been written so that each of the expected
fractions has a window of acceptable retention times. If the designated peak falls
within the expected window, the chromatogram peaks will be properly identified.
When a peak elutes at a retention time not within a specified window, an
unknown peak (P00) results. If more than one peak elutes at times not specified
by the software windows, each is given a sequential P0x title. In order to keep
the peaks within their appropriate windows, it may be necessary to change how
fast or slow the buffers are moving through the system by changing the pump
flow rate.

The separated hemoglobin components pass through the LED photometer flow
cell where the analyzer measures changes in absorbance at 415 nm. The
analyzer integrates and reduces the raw data, and then calculates the relative
percentages of each hemoglobin fraction. The Total Area of the SA1c is divided
by the sum of the total areas of all peaks up to and including the A0 to obtain a
raw SA1c percentage. This uncorrected result is substituted as the “x” value in
the linear regression formula determined during calibration. The analyzer prints
the final numerical results and plots a chromatogram showing changes in

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absorbance versus retention time for each peak fraction. Specimens that show a
deterioration peak, an abnormal looking chromatogram, or a presumptive
hemoglobin variant are reflexed to the boronate affinity method. In addition, G8
results ≤ 10 % HbA1C that have a LA1C ≥ 1/2 SA1C are reflexed to the boronate
affinity HPLC method for verification.

2. SAFETY PRECAUTIONS

1. While working in the lab proper Personal Protective Equipment (PPE) is

enforced. This includes wearing gloves, lab coats, face masks (as applicable),
protective eye wear such as goggles, and closed toe shoes are required for
handling all human blood specimens. Once gloves are removed wash your
hands or use hand sanitizer to ensure your hands are clean before leaving the
lab.
2. Vials containing human blood are only to be opened in a biological safety
cabinet with the sash in the correct position.
3. All plastic tips, sample cups, gloves, etc. that contact blood are considered
contaminated and are to be placed in a biohazard waste container.
4. All hoods, telephones, doorknobs and work surfaces are wiped down with
Oxyvir disinfectant or 10% bleach at least one time during each work shift. Any
area in which blood is spilled is also to be cleaned and disinfected immediately
with Oxyvir disinfectant or 10% bleach. Refer to the Lab Safety Manual for
additional details located in room M764.
5. All healthcare personnel shall routinely use appropriate barrier precautions to
prevent skin and mucous membrane exposure when contact with blood or other
body fluids of any patient is anticipated. All products or objects that come in
contact with human or animal body fluids should be handled, before and after
cleaning, as if capable of transmitting infectious diseases. Wear appropriate
Personal Protective Equipment (PPE), including facial protection, gloves, and
protective clothing. Dispose of all biological samples and diluted specimens in
a biohazard waste container at the end of analysis. Dispose of all liquid
hazardous waste in properly labeled hazardous waste container.

3. COMPUTERIZATION; DATA SYSTEM MANAGEMENT

Data are maintained on a secured Microsoft Access / Microsoft SQL server

client-server system in a 128-bit authenticated Windows domain environment.

1. Laboratory services are requested through the Westat system operations via
an email notification containing a unique manifest list of the samples and
sample analysis type (e.g. GHB), which confirms that specimens have been
shipped to DDL.
2. Each Manifest Form should include and be verified against each sample
received:
a. Patient Sample ID #

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b. Test Name
c. Date Collected
d. Shipment ID #
e. Shipment Date
f. Lab Name
g. Lab ID
h. Survey Year
3. Once specimens are received and verified the corresponding file is imported
electronically into the SQL server database via secure transfer.
4. After analysis the results, date analyzed and tech initials are imported from
the instrument into the SQL server database via secure transfer.
5. Data check sheets are printed out and checked against the instrument
printouts by the supervisor.
6. After results are cleared by the supervisor a results file in the specified
format is exported and uploaded to Westat via secure transfer.

4. SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES;

CRITERIA FOR SPECIMEN REJECTION

1. Patient Preparation: No special conditions such as fasting or special diets

are required.
2. Specimen Type and stability:
a. Collect whole blood specimens in vacuum collection tubes containing
EDTA anticoagulant (purple or pink tops) and mix thoroughly.
b. As per manufacturer instructions; specimens may be stored up to fourteen
days at 2-8°C before analysis. Specimens may be stored up to twenty four
hours at room temperature (10-25ºC) before analysis.1 However, a
stability performed in-house and published through Diabetes Technology
& Therapeutics demonstrated the below WB sample stability 4-7
i. Room Temperature: 6 days
ii. 4°C: 14 days
iii. Frozen (- 20°C);(-70°C): 7 days; 1 year.
c. HbA1c samples are stable at frozen conditions of – 70 °C
for 10 years (refer to the Stability of Haemoglobin A1c
(HbA1c) measurements from frozen whole blood samples
stored for over a decade study; 2005 Diabetes UK, Diabetic Medicine, 22,
1726 – 1730).
d. Liquid Nitrogen for up to 10 years
e. The minimum volume required for analysis directly from collection tubes is
1 mL of whole blood. Whole blood samples as small as 50 µL may be used
when appropriate sample cup and software options are selected.1
f. Specimens collected using the Bio Rad HbA1c Capillary Collection System
are also acceptable for research testing use only.8 Samples prepared
using this procedure are stable for 2 weeks at room temperature or 4
weeks at 2-8 °C or 4 days at 42°C.

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3. Specimens are delivered to the Diabetes Diagnostic Laboratory, Room M764

by overnight courier. Each specimen must arrive in the laboratory with a
unique barcode identification number. Unacceptable specimen criteria:
a. Clotted samples
b. Specimen types and stability not listed above.
c. Unlabeled samples.
4. If an unacceptable specimen is received notify NHANES and Westat, and
add the appropriate comment code in the database.
5. Handling Conditions:
a. Samples are to be kept refrigerated at 2-8oC immediately after collection.
b. Transport under refrigerated conditions.
c. Once received and prepared for analysis, specimens are to be
immediately returned to 2-8oC storage where they are to be kept for one
week before being discarded. If longer term storage is necessary,
specimens may be frozen and stored at -70oC (DO NOT FREEZE
SAMPLES AT -20oC).7

5. PROCEDURES FOR MICROSCOPIC EXAMINATION

Not applicable for this procedure.

6. EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT

PREPARATION, CALIBRATORS (STANDARDS), AND CONTROLS

1. Equipment:
a. Tosoh G8 Glycohemoglobin Analyzer (Tosoh Bioscience, Inc., South San
Francisco,CA).
Parameter Setting
Wavelength Sample: 415nm
Reference: 500nm
Column Temperature 25 °C
Injection Interval 1.6 min
Calibration Two-point
Working Temperature 15°C - 27°C

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b. Hamilton Autodilutor Model Microlab 500 or 600 with 2.5 mL and 25 µL

syringes (Hamilton, Reno, NV)
c. Microsoft Windows Compatible Computer capable of running Microsoft
Internet Explorer, Cerner Pathnet software, and G8 Data Management
Software.
d. G8 Data Management Software (Tosoh Bioscience, Inc., South San
Francisco, CA)
e. Adjustable Variable Volume Pipettes in 0.5-10, 2-20, 20-200, and 100-1000
µL volumes.
2. Equipment Maintenance
a. Tosoh Analyzer System—Routine maintenance
i. Column pre-filters – Replace the filter element if the pressure is
greater than the pressure level that is indicated on the column
inspection report +4 MPa or after 400 injections.1 At least 5 prefilters
should be on hand at all times.
ii. Analytical column—Change after 2500 injections.1 At least two spare
columns are to be kept available at all times.
iii. Record all routine maintenance in the Tosoh G8 Diary.
b. Tosoh Analyzer System—Periodic/Preventative Maintenance
i. Replacing Buffers: Solution of Hemolysis Wash
ii. Confirm that the lot #’s match the corresponding buffers. If the
analyzer is not in STAND-BY mode, press the STOP key and wait
until ‘STAND-BY’ appears on the Status screen.
iii. Remove old buffer bag from analyzer and replace with a new bag.
Be sure that the color of the connection tubing matches the color of
the buffer bag label.
iv. Write the date opened and an expiration date three months into the
future on each bag or bottle as it is opened.
v. From the MAIN screen, select MAINTE, then REAGENT CHANGE.
Once the buffers have been correctly installed, select the appropriate
buffer, and then press CHANGE.
vi. Record new lot information in G8 Data Management software, daily
diary sheet, and buffer record in G8 Diary sheet binder.
c. Removing air from the buffer lines
i. Air can enter the fluid lines if a buffer bag runs dry or after long-term
shutdown. The following procedure removes air from the lines:
ii. From the MAINTE screen, press REAGENT CHANGE key.
iii. Highlight the key(s) for the reagent(s) to be primed.
iv. Press PRIME key. The confirmation message will be displayed. If
everything is ready press the OK key. The reagent(s) in the analyzer
fluidics will automatically be replaced with fresh reagent. The
operation is complete when the “PRIMING…” display disappears.
Approximately 5 mL of each reagent will be consumed when PRIME
is executed.
v. Pump buffers and verify pressure. Repeat if necessary.
d. Removing air from the pump

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i. During pumping, if the pressure does not rise, air may be present on
the outlet side of the pump.
ii. Use the following procedure to remove the air.
iii. Verify analyzer is in STAND-BY mode.
iv. If the analyzer is not in STAND-BY mode, press the STOP key and
wait until ‘STAND-BY’ appears on the Status screen.
v. Press the REAGENT CHANGE key on the MAINTE screen.
vi. Press the DRAIN FLUSH key.
vii. The following message will be displayed requesting that the drain
valve be opened: “Open the door on the left side of the analyzer and
turn the drain valve 90 degrees in the counterclockwise direction to
open the valve.”
viii. Turn the valve ONLY 90 degrees counterclockwise.
ix. Press the OK key.
x. A confirmation message will appear. Ensure the drain valve is open.
Press the OK key again.
xi. Air stuck in the pump will automatically be removed. This procedure
takes approximately 7 minutes to complete and is finished when the
“FLUSHING…” message disappears.
xii. A message will be displayed requesting that the drain valve be
closed. Turn the valve back 90 degrees in the clockwise direction to
securely close it.
xiii. Press the OK key.
xiv. Press the EXIT key to return to the main screen second page. Press
the PUMP key.
xv. If a pressure of within the acceptable range for the filter is displayed
in the HbA1c mode with no pressure fluctuation, air removal is
complete. Press the PUMP key again to stop the pump motor. If the
pressure does not rise 5Mpa or is unstable, stop the pump and
repeat the air removal procedure again.
e. Replacing the Filter Element
i. Replace the filter element after 400 injections or when the pressure
exceeds limits as indicated on the column inspection report +4MPa.
ii. Verify analyzer is in STAND-BY mode.
iii. If the analyzer is not in STAND-BY, press the STOP key and wait
until ‘STAND-BY’ appears on the Status screen.
iv. Open the door below the display.
v. Confirm that the SV1 key is open (O) on the second page of the main
screen.
vi. Remove the filter outlet (peek) tubing from the top of the filter
assembly.
vii. Loosen the top of the filter holder assembly by turning it
counterclockwise. Remove the filter holder by pulling it straight out.
viii. Lightly press the top of the holder to remove the old filter element. If
salt crystals are present in the holder, rinse with distilled or deionized

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water to clean. Position the new element paying attention to how it

is oriented. The gray colored surface is the outlet (up) side.
ix. Firmly tighten the top of the filter holder assembly by hand until no
further tightening is possible.
x. Slide outlet tubing unit it extends ¼ inch past the end of the tubing.
Connect the outlet side tubing.
xi. Press the PUMP key again to start Elution Buffer delivery. Confirm
that the pressure reaches 6 Mpa or more with no leaks from the filter
housing or tubing connections. If a leak is found tighten the
assembly further.
xii. Press the PUMP key to stop the pump.
xiii. Reset filter counter to 0 on the REAGENT CHANGE screen.
xiv. Record change on daily diary sheet.
f. Column Replacement: Replace the column in the following situations.
i. Replace column after 2500 injections.
ii. When the pressure is more than what is indicated on the column
inspection report + 4 MPa and is not reduced by filter replacement.
iii. When peaks on the chromatogram (particular the shaded SA1c
peak) have become broad or broken in two fractions.1
iv. When assay results for quality control samples are consistently out
of assigned ranges even after re-calibration.
v. When the CALIB ERROR persistently occurs.
vi. Please contact Technical Support if the above issues are not
resolved after column replacement.
vii. Replacing the Column: Replace the column if column maintenance
(see above) does not solve the problem and if the column exceeds
2500 injections, according to the following procedure. Verify analyzer
is in STAND-BY mode.
1) If the analyzer is not in STAND-BY mode, press the STOP key
and wait until ‘STAND-BY’ appears on the Status screen.
2) Remove old column.
3) Open the front doors of the analyzer. Release latch and open
the column oven. Unscrew column connections and remove
used column.
4) Confirm that the SV-1 key is open (O) on the MAIN screen,
(second page).
5) Slide the inlet tubing unit it extends ¼ inch past the end of the
fitting. Connect the new column to the pump (right) side only.
Take care that the flow arrow on the column indicates flow
right to left. Press the PUMP key allowing buffer flow into the
column. When the buffer begins to flow from the open end of
the column, press the PUMP key stop the flow.
6) Connect detector tubing to outlet (left) side of column. Slide
the outlet tubing until it protrudes ¼ inch past the end of the
fitting. Insert the outlet tubing into the left side of the column.
Screw the fitting finger tight.

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7) Check for leaks. Press the PUMP key to start the pump and
confirm there is no fluid leakage.
8) Check for fluid leaks at the connections. If leaks occur, tighten
fittings.
9) Verify that pressure stabilizes. The pressure should rise to the
pressure level that is indicated on the column inspection
report + 4 MPa. If leaks occur, tighten fittings.
10) Reference pressure and limits should be recorded onto a label
attached to the instrument. This label should include the data,
reference MPa, low and high MPa limits, and column serial
number.
11) After verifying connections are secure, stop the pump by
pressing the PUMP key.
12) Close column oven.
13) Close front doors of the analyzer.
14) After connecting a new column, reset (zero) to the column
counter in the REAGENT CHANGE screen.
15) Record change on daily diary sheet.
16) Run at least three whole blood samples to prime the new
column. Verify that the retention time for the SA1c peak is
between 0.57 – 0.61 minutes. The ideal the retention time for
SA1c is 0.59 minutes.5
17) If necessary, adjust the flow rate to match the retention time
for the SA1c peak on the reference chromatogram included
with the column.
18) Once the retention time matches within +/- 0.2 min, print off
the chromatogram and submit it along with the included
chromatogram from the manufacturer to the supervisor or
delegee for usage approval.
19) In the event of column lot change, all reagents need to be
replaced to lots corresponding to the new column lot. A
comparison needs to be done between the old and new lots
of columns/reagents (n=40). Comparison must meet these
criteria:
a) XY plot (current lot on x-axis) with linear regression
performed.
b) Slope = 1.0+/- 0.1
c) Intercept = 0.0+/- 0.1
d) R2 > 0.98
e) 95% CI of the differences between x and y within 0 +/-
0.5% HbA1c. Overall mean bias within +/- 0.2%
HbA1c. If any outliers (>1% HbA1c difference between
X-Y) occur, investigate further.
g. Replacing printer paper.
i. Lift the printer cover (upper lid) to the back to open.

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ii. Push the paper holding lever down to the very front and wrap the
remaining paper onto the roll.
iii. Lift the roll up and remove the mandrel.
iv. Insert the mandrel into the new roll with attention to the direction.
v. Return the paper holding lever to the very back and insert the paper
into the printer. Press the feed switch to feed the paper.
vi. Check for twisted paper. If the paper is twisted, push the paper
holder lever to the front, adjust the paper, and return the lever to the
back.
h. Replacing the sampling needle.
i. Replace the needle if it is bent or broken. Although needle
replacement is normally done by field service personnel, the
procedure below may be performed by the operator.
ii. Put on protective clothing (goggles, gloves, etc.) and take care not
to touch the end of the sampling needle during handling.
iii. Press the POWER key to switch off the analyzer.
iv. Use a screwdriver to remove the sampling cover screws.
v. Remove the sampling needle cover.
vi. The sampling needle unit is located behind this cover. Grasp the
upper part of the sampling needle unit by hand and slowly pull the
unit forward as far as possible.
vii. A small volume of reagent may leak during needle replacement.
Place a tissue or plastic pad under the sampling needle tip to absorb
any leakage.
viii. By hand, loosen and remove the tubing connected to the 3-way
block.
ix. Remove the screws on the upper section of the sampling needle. Be
careful not to drop the screws or the holding plate inside the machine
during this operation.
x. Remove the screws that hold the guide through which the tubing
passes.
xi. Slowly lift up the sampling needle to remove it. Place immediately
into a sharps container.
xii. Insert the new sampling needle with the bevel facing forward. The
sampling needle must be positioned with the bevel facing forward or
the needle will not correctly dilute the sample.
xiii. Secure the holding plate with the screws.
xiv. Pass the tubing through the guide, secure with the screw, and
securely connect the tubing to the 3-way block.
xv. Move the sampling unit back and forth and confirm that the tubing
does not catch. If necessary, loosen the screws and change the
guide direction to prevent the tubing from being obstructed. Push
the sampling unit back; close the blue cover by following the above
procedure in reverse. Secure the screws.

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xvi. Turn on the Main Power Switch. Press the POWER key on the
control panel and allow the analyzer to complete the WARMUP
process then to the STAND-BY state.
xvii. Assay 3 whole blood samples to confirm the sample is aspirated
correctly. The Total Area for these samples should be approximately
the same as it was before the sampling needle replacement.
xviii. Adjusting the Flow Rate: The flow factor is generally 1.00 mL/min,
but can be 1.02/1.03 mL/min dependent on instrument factory
setting. The flow factor should only be adjusted +/- 0.05 of the default
factory setting.1
3. Instrument Preventative Maintenance (PM)
a. A monthly PM is performed by a trained member of our DDL staff using the
monthly PM checklist (located on the H:\DDL\D150\Templates drive). The
completed checklist is reviewed by the lab supervisor or delegee for
verification.The Monthly PM is performed using the below steps;
i. With G8 powered on, press the menu button, then utilities, then
password.
ii. Enter “MAINTE” in password screen, then press enter.
iii. Exit back to main screen.
iv. Press Mainte button.
v. Press Sampler Mech button.
vi. Remove the fitting from the top of the filter holder assembly.
vii. Remove the top portion of the filter assembly.
viii. Eject the filter.
ix. Wet a cotton swab with DI water. Clean inside of filter holder
assembly, including the threads. Clean the top portion of the
assembly as well.
x. Reassemble the filter assembly.
xi. Remove the two screws from the top of the blue cover door, and pull
open cover.
xii. Press the “Move Y stat” button. The needle should move to the stat
position.
xiii. Remove the two screws holding down the needle. Remove the small
metal plate under the screws.
xiv. Remove the needle.
xv. Remove the screws from the bottom portion of the needle assembly.
Remove the metal plate beneath these screws.
xvi. Remove the blue o-ring.
xvii. Clean the screws, plates, and o-ring.
xviii. Using an alcohol wipe, clean the needle to remove any dried blood.
Use the corners of the alcohol wipe’s package to clean out the
grooves on two sides of the needle.
xix. Use the alcohol wipe to clean the positioning wheel behind the stat
well.
xx. Replace the o-ring. Place one drop of TriFlow lubricant in the middle
of the o-ring.

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xxi. Replace the metal plate that holds the o-ring in place, and replace
the screws.
xxii. Insert the needle into the top hole, followed by the lower hole. Assure
that the bevel of the needle tip is facing you.
xxiii. While holding needle in place, replace the metal plate at the top, and
screw it into place.
xxiv. Assure needle is secure and press “Move Y Dil” button.
xxv. Use compressed air to blow out dust from interior, metal facing under
blue cover, under the printer cover, and over any vents and fans on
the exterior.
xxvi. Replace blue cover and screw into place.
xxvii. Using Oxivir, clean all exterior surfaces.
xxviii. Remove buffers from chrome holder, remove chrome holder.
xxix. Rinse chrome holder under tap water, dry with paper towel, and
replace holder and buffers.
xxx. Clean sysmex racks with Oxivir, rinse, and replace broken adapter
rings.
b. For every 20K injections, a PM is performed by a Tosoh Service
representative. 10
4. Pipette Preventative Maintenance
a. Hamilton Autodilutor 500/600
i. Verify calibration of the device according to Autodilutor SOP.
ii. Instrument should be cleaned with disinfectant and inspected for
proper functioning daily.
b. Adjustable volume pipettes
i. After each use, the pipette should be wiped with disinfect with soaked
gauze.
ii. Pipettes are calibrated annually by trained field service personnel.
5. Materials:
a. Reagents—Supplied by Tosoh Bioscience (South San Francisco, CA). Part
numbers are subjected to change. Any questions or concerns about the
materials used for this assay please refer to your supervisor/delegee or call
Tosoh Scientific hotline at 1-800-248-6764, customer # 1208. Refer to
Operator’s Manual for additional details relating to the Tosoh G8 HPLC
instrument components and items used for testing.
b. Other Materials
i. Powder free nitrile exam gloves. (Fisher Scientific, Waltham, MA)
ii. Oxivir disinfectant. (AHP Technology, Sturtevant, WI)
iii. Gauze Sponges 4x4 not sterilized (Fisher Scientific, Waltham, MA)
iv. Kim Wipe lintless tissues. (Fisher Scientific, Waltham, MA).
6. Storage Requirements:
a. Unopened Elution Buffer 1, 2, and 3 are stable at room temperature until
the expiration date printed on the label. After opening, Elution Buffers are
stable for three months. Store at 4-30 ºC.

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b. Unopened Hemolysis & Wash Solution is stable until the expiration date
printed on the label. After opening, Hemolysis & Wash Solution is stable
for three months. Store at 4-30ºC.
c. The unopened TSKgel G8 Variant HSi column should be stored at 4-15 °C
in a cool location away from direct sunlight. The column is stable until the
expiration date printed on the label. Replace column after 2500 injections.
d. Reagents must be brought to room temperature prior to use.
e. Reagent labeling: Reagents, calibrators, controls, and solutions should be
traceably identified to indicate the following:
i. Content and concentration
ii. Storage requirements (see above stability requirements for whole
blood).
iii. The below should be followed for working reagents; (open reagents
in use for testing):
1) Preparation date or opened date and the identity of the
preparer.
2) Tech’s initials.
3) Expiration date.
iv. Controls and calibrators obtained from freezer for daily
use should be initialed and dated with the pulled date.
v. Prior to processing controls and calibrators, sample cups
should be labeled (with the name of the control or
calibrator), dated, and initialed (tech responsible for
adding hemolysis wash to the sample cups).
vi. Processed controls and calibrators are immediately
returned to the refrigerator after samples are assayed
from the morning run, and are discarded after daily use.
vii. Buffers and wash should be labeled with the name of the
reagent and reagent source (name of vendor e.g. Tosoh),
preparation date or opened date, expiration date (from
open or preparation date), and tech’s initials.

7. CALIBRATION AND CALIBRATION VERIFICATION PROCEDURES

1. Calibrator Preparation: Donors are recruited and compensated for their

donation of blood. K2EDTA whole blood tubes are pooled together, mixed for
at least 30 min, and aliquoted under refrigerated conditions, see below.
a. Pooled Low Calibrator
i. Single level calibrator prepared from K2EDTA whole blood drawn by
venipuncture from four non-diabetic individuals.
ii. The blood specimens were pooled, dispensed in 30uL aliquots into
400µL microtubes under refrigerated conditions. Batches of these
low calibrators (aliquots) were assigned a lot number, preparation
date and stored at -70°C on the same day. The remaining aliquots

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were assigned the same lot number/date and placed in a cryogenic

(liquid nitrogen) tank at -196°C in freezer boxes in the same day.5-7
iii. Low calibrator HbA1c values were assigned by twenty interassay
determinations along with the previous lot of calibrator. Refer to QC
and Calibrator's binder and the controls and calibrator color key table
(locate at the bench) for the current the controls and calibrator
acceptable ranges.
iv. The acceptable calibrator values used should be within 2 SD of the
assigned value.
b. Pooled High Calibrator
i. Single level high calibrator was prepared from pooled EDTA whole
blood purchased from Aalto Scientific. Refer to Aalto Scientific
product insert for additional details.
ii. The blood was dispensed into 250 µL aliquots under refrigerated
conditions. Batches of these high calibrators (aliquots) were
assigned a lot number, date the high calibrators was made (and
received from Aalto) and stored at -70°C on the same day. The
remaining aliquots were assigned the same lot number/date and
were placed in a cryogenic (liquid nitrogen) tank at -196°C in freezer
boxes in the same day. 5-7
iii. High calibrator HbA1c values were assigned by twenty interassay
determinations along with the previous lot of calibrator. Refer to QC
and Calibrator’s binder and the controls and calibrator color key table
(locate at the bench) for the current the controls and calibrator
acceptable ranges.
iv. The acceptable calibrator values used should be within 2 SD of the
assigned value.
c. Preparation and Stability – Aliquots from Liquid Nitrogen to -70 °C.
i. Removed one box of calibrators from the liquid nitrogen
phase to the vapor nitrogen phase for at least 2 hours.
After 2 hours, transfer box into the -70°C freezer for at
least 2 hours to allow materials to acclimate. If box is
already in the vapor nitrogen phase, move box into the
-70°C freezer and allow materials to acclimate.
ii. After the acclimation period, transfer 50 tubes (while
working in the freezer) into a box labeled the material’s
name, date removed from liquid nitrogen, and with an
expiration date 365 days (one year) into the future.
iii. Return the box with the remaining calibrator aliquots to
the vapor nitrogen phase of the liquid nitrogen.
iv. Low and high calibrator aliquots are pulled from freezer
daily and placed in the refrigerator (2-8 °C) at start of the
day. Prior to use, thawed materials at room temperature,
and mixed by inversion gently.
v. WB calibrations are diluted with hemolysis & wash
solution to obtain a Total Area (TA) in the range of 500-

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4000. Calibrators are discarded after daily use.

2. Calibration Frequency: Calibration is to be performed:
a. Daily prior to the first analytical batch of the day on that instrument.
b. If drift in QC is observed.
c. When controls values are out of range.
d. After a column replacement.
e. After a new reagent lot.
f. After a filter change.
g. After analyzer maintenance.
h. Refer to Operator’s Manual for additional troubleshooting advice.1
3. Calibration Procedure:
a. Verify that there is sufficient volume of Elution Buffers, Hemolysis & Wash
Solution and at least 400 µL of each calibrator in the sample cup.
b. Check analyzer status.
c. If analyzer is in Standby mode, proceed.
d. On Main screen make sure CALIB is reversed highlighted.
e. Place the sample vials in the rack and tubes with low calibrator in position
1 (on the left) and high calibrator next to it in position 2.
i. Press the START key to begin the calibration.
ii. The analyzer measures Calibrator 1 three times and Calibrator 2 two
times for a total of 5 times. The analyzer discards the first
measurement, and uses the remaining four measurements to
calculate the slope and the intercept. Patient sample results following
calibration will be calculated using the new factors.
f. Current calibrator value assignments can be found in the Quality Control
Binder.
4. Calibration Acceptability Criteria—the analyzer has a two-point automatic
calibration function for stable HbA1c (SA1c). When the analyzer processes
calibrators, it calculates the slope and the intercept from a linear regression
equation to determine quantitative results from patient samples and controls.
When the calibration procedure is completed, the analyzer automatically
accepts or rejects the calibration results.
a. Verify the acceptability of the chromatogram. Refer to Operator’s Manual
for additional details. If the calibration is unsuccessful, recalibration is
required. Patient aliquots are not assayed until the calibration results are
acceptable.
b. A Calibration Error message appears and the run aborts if;
i. The two SA1c% results for Calibrator 1 differ by 0.3% or more.
ii. The two SA1c% results for Calibrator 2 differ by 0.3% or more.
iii. Any of the four calibrator results differ from its assigned value by ±
30% or more.

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8. PROCEDURE OPERATING INSTRUCTIONS; CALCULATIONS;

INTERPRETATION OF RESULTS

1. Instrument setup for the Tosoh Analyzer System.

a. Check levels of buffers 1, 2, and 3 and wash solution, making sure there is
sufficient volume to complete the assay. Replace buffer if necessary. All
changes in lot numbers of buffers are to be recorded on the Tosoh Diary
Sheet, and in the Tosoh G8 daily lot info. spreadsheet.
b. Analyzer should be in STANDBY mode prior to beginning analysis.
c. Record # of injections on column. (Listed on MAIN screen)
d. Record # of injections on filter. (Listed on MAIN screen)
e. Check pressure listed on the MAIN screen and record.
f. If pressure is greater than +4 MPa over the reference value listed on the
inspection report, replace the filter.
g. If pressure is less than the reference values, check for leaks and secure
leaking fittings near the filter assembly and column.
h. Check temperatures, including the minimum and maximum, for the room
(M764), the refrigerator in M764, and the ultralow freezer in M764 and
record the values on the weekly environmental conditions checklist. If
values are found to be outside of limits, the supervisor is to be notified and
the course of action documented.
i. Check for air bubbles & leaks:
During warmup, check the tubing connections for leaks, particularly
the filter and column inlet and outlet sections. Tighten connections
if a leak is found. Record check in the Tosoh Diary.
j. Check calibration status.
Verify that the MAIN screen has the CALIB button reverse
highlighted.
k. Check printer paper.
Open the printer lid and check the remaining paper. Be sure that
there is sufficient paper to complete the assay. Replace with a new
roll of paper if needed.
l. Check Flow rate:
From the MAIN screen, press the MENU key, then PARAMETER,
and ▼. Record the flow factor in the Tosoh Diary.
m. Controls at end of run:
Be sure that there are low and high level controls at the end of the
assay.
n. Verify the Tosoh G8 Reporting Software program is open.
2. Sample preparation – For Controls, Calibrators, and specimens requiring pre-
dilution
a. Using a Hamilton Autodilutor, prepare control and calibrator hemolysates
by diluting 10ul of well mixed whole blood with 0.990 mL hemolysis reagent
in the sample vial. Use the following procedure:
i. Wipe outside of tip with gauze wetted with distilled water.

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ii. Insert tip of autodilutor into blood specimen and press the button on
top of the handle to draw 10ul of specimen into the tip.
iii. Insert tip into corresponding 1.5mL sample cup and press button
again to dispense sample and reagent into sample cup.
iv. Wipe outside of tip again with gauze wetted with distilled water.
v. Repeat procedure for all QCs and specimens not suitable for direct
sampling on the instrument from the primary tube.
b. Load calibrators.
Place the sample vials in the rack with PLC in position 1 (on the left) and
PHC in position 2.
c. Load controls: Controls are performed at the beginning of the run, every 50
samples, and at the end of a run.
d. Place controls in the rack with low in position 3 and high in position 4.
e. Place low and high controls in the end positions.
f. Load samples.
i. Mix each sample by gently inverting each capped sample tube.
ii. Place capped sample tubes in the rack in order from left to right. If
you are using barcoded tubes, verify that the labels face the
analyzer.
iii. Position each rack in the rack guide, starting with the right side.
iv. Place a blank rack after the last rack of samples. The blank rack
serves as an end marker.
3. Operation of the Tosoh Analyzer System.
a. Press the POWER button. (Already in standby when performing instrument
setup.)
b. Follow the calibration procedure in the CALIBRATION section of this
manual.
c. Press the START button.
d. Check chromatograms from normal and abnormal QC specimens in
positions 1, 2, 3 and 4.
e. When measurement ends, the analyzer washes the column by pumping
buffer for 15 minutes, and then enters STAND-BY mode.
f. After run is completed, verify and input, when necessary, accession
numbers for each individual specimen in the G8 Reporting Software under
the heading “barcode”.
g. Examine individual chromatograms.
i. Each chromatogram should include six peaks identified as A1A,
A1B, F, LA1C+, SA1C, and A0.
ii. All peaks should be clearly resolved.
iii. The acceptable retention time for SA1c is 0.57 – 0.61.1
iv. The acceptable retention time for A0 is 0.87 – 0.91.1
v. The acceptable range of TOTAL AREA is from 500 to 4000.
However, optimal results are obtained in the TOTAL AREA (TA)
range from 700 to 3000. Do not report results with a TA < 500 and >
4000. Low or high hematocrit samples may display Total Area above
or below the Total Area linear reportable range;

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• Samples that exhibit TA < 500; reassay the specimen using less
Hemolysis & Wash Solution and centrifuge lightly to yield a TA
between 500 – 4000.1
• Samples with TA > 4000; reassay the specimen using more
Hemolysis & Wash Solution.1
vi. Repeat any specimens with %HbA1c values less than 4.0% or
greater than 14.0% for verification.
vii. Any samples which show a deterioration peak (extra peak between
the SA1C and AO peaks usually designated as P00 or P01) are to
be marked “X-PK”. Enter into the Primus data base the specimen ID,
and in the comment section “X-PK”. Also, record the specimen into
the Primus Sample Tracking from Tosoh G8 log and rerun by the
Trinity Premier boronate affinity method. Report the boronate affinity
HbA1c result.
viii. Samples with heterozygous HbD will exhibit an additional peak or
peaks after the A0 peak, and the instrument will designate the main
peak HV-0, and minor peaks Pxx. The chromatogram is labeled with
“HbAD”, and the %HbA1c cannot be reported for these specimens.
Enter into the Primus database the specimen ID, and in the comment
section “Hemoglobin D”. Also, record the specimen into the Primus
Sample Tracking from Tosoh G8 log and rerun by Trinity Premier
boronate affinity method. Report the boronate affinity HbA1c result.
ix. Samples with heterozygous HbS will exhibit an additional peak or
peaks after the A0 peak, and the instrument will designate the main
peak HV-1, and minor peaks Pxx. The chromatogram is labeled with
“HbAS”, and the %HbA1c cannot be reported for these specimens.
Enter into the Primus database the specimen ID, and in the comment
section “Hemoglobin S”. Also, record the specimen into the Primus
Sample Tracking from Tosoh G8 log and rerun by Trinity Premier
boronate affinity method. Report the boronate affinity HbA1c
result.
x. Samples with heterozygous HbC will exhibit an additional peak or
peaks after the A0 peak, and the instrument will designate the main
peak HV-2, and minor peaks Pxx. The chromatogram is labeled with
“HbAC”, and the %HbA1c cannot be reported for these specimens.
Enter into the Primus data base the specimen ID, and in the
comment section “Hemoglobin C”. Also, record the specimen into
the Primus Sample Tracking from Tosoh G8 log and rerun
by Trinity Premier boronate affinity method. Report the boronate
affinity HbA1c result.
xi. Samples with heterozygous HbE will exhibit an additional small
peak between the SA1C and A0 peaks. The instrument will
designate the HbE peak as PHV-E. The chromatogram is labeled
with “HbAE”, and the %HbA1c cannot be reported for these
specimens. Enter into the Primus data base the specimen ID, and

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in the comment section “Hemoglobin E”. Also, record the specimen

into the Primus Sample Tracking from Tosoh G8 log and rerun
by Trinity Premier boronate affinity method. Report the boronate
affinity HbA1c result.
xii. Samples with chromatograms indicating that there is a presumptive
variant peak that is not flagged as HbS, HbC, HbE, and HbD should
be marked as “ABN”.” Enter this sample ID (with the ABN indication)
into the Primus database and record the specimen into the Primus
Sample Tracking from Tosoh G8 log and rerun by Trinity Premier
boronate affinity method. Refer to the supervisor or delegate for if
you have questions interpreting chromatograms.
xiii. Samples with chromatograms showing the presence of both a
variant hemoglobin and HbF > 10 % should not be reported.
xiv. G8 results ≤ 10 % HbA1C that have a LA1C ≥ 1/2 SA1C are reflexed
to the boronate affinity HPLC method for verification due to potential
variants. Enter into the Primus data base the specimen ID, G8 result,
and in the comment section “the LA1c and A1c result”. Also, record
the specimen into the Primus Sample Tracking from Tosoh G8 log
and rerun by Trinity Premier boronate affinity method.
xv. Chromatograms that are not flagged as abnormal, but appear
questionable under the supervisor (or delegate) discretion should be
labeled “primus checks”. Enter into the Primus data base the
specimen ID, G8 result, and in the comment section “Primus Check”.
Also, record the specimen into the Primus Sample Tracking from
Tosoh G8 log and rerun by the rerun by Trinity Premier boronate
affinity method.
xvi. Fetal hemoglobin (HbF) up to 38.4% does not interfere with the
Tosoh G8 HbA1c result as long as there is adequate separation of
the HbF and LA1c peaks. This has been demonstrated based on the
comparison of Tosoh G8 and the IFCC HbA1c Capillary
Electrophoresis Reference Method. b ,l In the event that the LA1c
peak is greater than 10% (an indication of inadequate separation of
HbF from LA1c), the analysis is repeated. If separation is still not
adequate, this is reported to the supervisor and the result is not
reported. The following equation is then used for the calculation of
a reportable result: % SA1c = (((SA1c Area / (A1a Area +A1b Area
+ SA1c Area + A0 Area)) x 100) x Slope + Y Intercept.
xvii. Any abnormal peak pattern consistent with a homozygous variant
hemoglobin (e.g. HBSS) or double heterozygous
variant (e.g. HbSC) should be not be reported. This result is
considered unreportable. The report is noted with the comment, “An
abnormal peak pattern consistent with a homozygous or double
heterozygous variant hemoglobin was detected. This condition is
known to affect the average lifespan of red blood cells, so the
HbA1c test should not be used for this patient. An alternative test to
measure glycemia is fructosamine/glycated serum protein.”

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xviii. Any other abnormal chromatograms should be reported to the

supervisor for further investigation prior to reporting.
h. Result reports are generated by the G8 Reporting Software by highlighting
all the specimens in the run and selecting the “Result Report x 6” function.
A Result List is also generated by again highlighting all of the specimens
from the run and selecting the “Result List – Portrait” function.
4. Reporting Results: Procedure:
a. In G8 Data Management software, select all specimens, calibrators, and
controls for the run, and press the report button.
b. Select “Report x6.” Chromatography report will open in Adobe Reader.
Print.
c. Repeat step 1 above, select Result list (portrait). Result list will open in
Adobe Acrobat Reader. Print to local printer.
d. Review the printed sheets to verify that controls meet acceptance criteria
and that all specimen results requiring further verification have been noted
and had their barcodes appended with the letter V to prevent inadvertent
uploading of these results. Make sure all specimens have barcodes.
e. In the NHANES database, select “Enter Results:, then “A1c Results-Pull”
to display the NHANES samples to be imported from the G8.
f. Select the results to be imported then select “Commit Results”, then
“Close”.
g. Select “Reports”, then “Print Check Sheet” and enter the batch number to
print the check sheet.
h. Submit check sheet(s), chromatography, and result list printouts along
with completed diary sheet to supervisor for verification.
i. Supervisor will verify acceptability of entire run based upon controls, and
acceptability of individual results based upon chromatography.
j. When satisfied, the supervisor will clear the results in the NHANES
database.
5. Panic Results: As this test is utilized strictly as measure of long-term glycemic
control, there are no “panic values” for this test and therefore this section is
not applicable.
6. Reporting Format: Results are expressed on the report as % Hemoglobin A1c
(HbA1c) and are rounded to one decimal place. Results are reported
throughout the entire range of % HbA1c values verified by linearity studies.
Results below 4.0% or above 14.0% are reanalyzed for verification prior to
results being reported. Results outside of current linearity values are reported
as < (low linearity value) or > (high linearity value) as ppropriate. Linearity is
performed every 6 months. The established AMR is 3.1 – 19.5%.

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7. Supervisor Responsibility:
a. The supervisor or delegate ensures quality control passes within the
acceptable ranges prior to releasing patient results.
b. The supervisor or delegate checks every individual chromatogram to
ensure all peaks are resolved and reportable.
c. All chromatograms requiring further evaluation are noted and are not
uploaded into the database.
8. Procedure Notes
a. To avoid an error condition during calibration, be sure to place PLC and
PHC in the first sample rack in positions 1 and 2 respectively.
b. Each reagent lot number supplied by Tosoh is performance matched to
the supplied TSKgel G8 Hsi Columns. Following any announced change
in supplied Tosoh TSKgel G8 HSi Columns, contact Tosoh to determine
suitability of existing reagents.
c. The reagents must be at room temperature prior to use.
d. If the column is not to be used for more than one week, remove it from the
analyzer, seal the ends with the protective plugs and store in cool place at
4-15 ºC. Avoid direct sunlight.
e. The relationship between HbA1c results from NGSP network and the
IFCC network is expressed by using the following equation: NGSP (%) =
0.09148 x IFCC (mmol/mol) + 2.1522
f. Any changes to procedure must be documented. Major changes to the
SOP may include the way a procedure is performed or calculations and
requires the approval of the Medical Director. Minor changes include
typographical errors or other minor corrections that do not change the way
the procedure or calculation is performed and do not require approval of
the laboratory director. Major SOP changes must be reviewed by the Lab
Director prior to SOP update.
g. Any changes to the SOP will be communicated to technical staff via verbal
communication and email notification. Technical staff after reading the
changes made to the SOP will review, sign, and date the SOP.

9. REPORTABLE RANGE OF RESULTS

Results are expressed on the report as % Hemoglobin A1c (HbA1c) and are
rounded to one decimal place. Results are reported throughout the entire
range of % HbA1c values verified by linearity studies. Results below 4.0% or
above 14.0% are reanalyzed for verification prior to results being reported.
Results outside of current linearity values are reported as < (low linearity
value) or > (high linearity value) as appropriate. Linearity is performed every
6 months. The established AMR is 3.1 – 19.5%.

10. QUALITY CONTROL (QC) PROCEDURES

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1. Quality Control
a. Quality Control Preparation: Donors are recruited and compensated for
their donation of blood. Blood products are pooled together, mixed for at
least 30 min, and aliquoted under refrigerated conditions, see below.
i. Pooled Low Control
1) Single level low control was prepared from K2EDTA whole
blood whole blood were drawn from known non-diabetic
individuals (Normal level HbA1c).
2) The blood specimen were pooled, dispensed in 50 μL aliquots
into 400μL microtubes under refrigerated conditions. Batches
of low controls (aliquots) were assigned a lot number,
preparation date (date the controls were collected), and
stored at -70°C on the same day (working stock for daily
usage, no more than 50 tubes). The remaining aliquots were
assigned the same lot number/date and placed in a cryogenic
(liquid nitrogen) tank at -196°C in freezer boxes in the same
day (long term storage).
ii. Pooled High Control
1) The elevated (Abnormal) HbA1c level pooled whole blood
(K2EDTA) controls were purchased from Aalto Scientific.
Refer to Aalto Scientific product insert for additional details.
2) The blood was dispensed into 250 μL aliquots under
refrigerated conditions. Batches of high control, were
assigned a lot number, dated with the day the controls were
collected (prepared), and aliquots were stored at - 70°C on
the same day (working stock for daily usage, no more than 50
tubes). The remaining high control aliquots were assigned the
same lot number/date and were placed in a cryogenic (liquid
nitrogen) tank at -196°C in freezer boxes on the same day
(long term storage).
b. Preparation and stability.
i. Removed one box of calibrators from the liquid nitrogen phase to
the vapor nitrogen phase for at least 2 hours. After 2 hours, transfer
box into the -70°C freezer for at least 2 hours to allow materials to
acclimate. If box is already in the vapor nitrogen phase, move box
into the -70°C freezer and allow materials to acclimate.
ii. After the acclimation period, transfer 50 tubes (while working in the
freezer) into a box labeled the material’s name, date removed from
liquid nitrogen, and with an expiration date 365 days (one year) into
the future.
iii. Return the box with the remaining calibrator aliquots to the vapor
nitrogen phase of the liquid nitrogen.
iv. Low and high control aliquots are pulled from freezer daily and
placed in the refrigerator (2-8 °C) at start of the day. Prior to use,
thawed materials at room temperature, and mixed by inversion
gently.

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v. WB controls are diluted with hemolysis & wash solution to obtain a

Total Area (TA) in the range of 500-4000. Controls are discarded
after daily use.
c. Mean and Ranges
i. Daily means and ranges are calculated from twenty interassay
determinations.
ii. Quality control limits are established by calculating 95% (2sd) and
99% (3sd) confidence limits for both daily means and daily ranges
for each control.
iii. Mean and Range limits for the current controls are posted on each
G8 instruments.
iv. Current Control value assignments and limits can be found in the
Quality Control Binder located in room M767.
d. Tolerance Limits
i. Analytical Batch Quality Control – Daily
1) The system is declared “out of control” if any of the following
conditions occur:
a) The mean from a single run for a single control falls
outside 99% confidence limits (3sd).
b) The means from a single run for both controls fall
outside 95% confidence limits (2sd).
c) The means from eight successive runs for a single
control fall either all above or all below the mean line.
Runs for which the mean falls within 1sd of the
established mean are not counted in this trend.
d) The range from a single run for a single control falls
above 99% confidence limits.
e) The ranges from a single run for both controls fall
above 95% confidence limits.
f) The ranges from eight successive runs for a single
control fall above the mean line.
2) If a run is declared “out of control”, all samples from that runs
are repeated in another run. Additionally, the instrument,
calibration, and controls are investigated to determine the
cause of the problem before further analysis occurs. In the
case of a trend, troubleshoot accordingly by either adjusting
the flow rate and/or performing a recalibration. Results are not
released until the quality control specification are acceptable.
ii. Levey-Jennings Plots – Monthly
1) Mean chart—Plots the mean values for each control in the run
and each compares them to upper and lower two and three
standard deviation limits as well as the mean.
2) Range chart— Plots the range values (maximum value –
minimum value) for each control in each run and compares
them to mean, upper two standard deviation, and upper three
standard deviation limits.

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3) The Laboratory Director or delegee reviews these on a

monthly basis.
e. Routine Quality Control Testing
i. Normal and Abnormal level (elevated) controls are run at the
beginning of a run, every 50 samples, and at the end of the run.
ii. Sample QC—Two percent of HbA1C specimens are randomly
selected and reanalyzed. Duplicate results are hand written on the
laboratory results worklist and reviewed for accuracy by trained
personnel. If the difference in %HbA1c between the duplicate is
greater than 4% (relative) or 0.2% HbA1C (whichever is greater) of
the original HbA1c value, the specimen is reanalyzed and the
chromatograms and instruments from both the original and
duplicate runs are investigated.
f. Inter-instrument QC
i. Comparison between Tosoh G8 instruments—NGSP monitoring
specimens are analyzed each month on each Tosoh G8 HPLC
instrument to validate agreement between instruments. Acceptability
is defined as the estimate of the standard deviation of the difference
in sample replicates that must not exceed 0.229 (99th percentile of
the sampling distribution around a target SD of 0.15).2
ii. Comparison between Tosoh G8 and Trinity ultra2/Premier
instruments:
1) GHBQC procedure is followed for the comparison of results
(n = 30 – 50 per month) between the Tosoh G8 HPLC HbA1c
method and its backup method, Trinity ultra2/Premier HPLC
HbA1c. Criteria for Pass/Fail: Bland/Altman:+/-0.70, Y at
X:+/-0.30, Syx: 0.35
2) Results are reviewed monthly by the Lab Director.
g. Proficiency Testing
i. College of American Pathologists (CAP) survey:
1) Three times a year Diagnostic Diabetes Lab (DDL) will receive
Proficiency Testing (PT) specimens from CAP. The testing
personnel who receives these samples should store and
handle these specimens according to the instructions
provided. CAP PT are tested as patient samples (and named
according to their sample IDs) within the routine lab workload,
results are recorded on the forms included, and then
submitted online by the lab supervisor or delegate.
Interlaboratory communication about proficiency testing
samples or results is NOT allowed until after the close of a PT
event (after the submission deadline has passed).
2) Technologists should rotate testing of proficiency survey
samples (during different PT events), so that the same tech is
not running all the PT surveys. Refer to DDL’s Quality
Management Program for additional details relating to PT
survey handling and reporting.

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3) Due to a recent CMS directive, our institution is only allowed

to order and result one PT survey per analyte; laboratories are
not permitted to test PT samples on multiple instruments.
During a PT event the primary instrument should be utilized
(if this is how testing is routinely performed). Proficiency
Surveys should be rotated among primary instruments during
different PT events.
4) Once the CAP PT results are submitted by the lab supervisor
or delegee, the proficiency samples are placed and
maintained under frozen conditions (-70 °C) until after the
close of that PT event. The PT samples should then be
retested on the other G8 HPLC platforms (that were not used
during the PT event) and the boronate affinity methods
(Premier) after the submission deadline.
5) Results from all DDL HbA1c methods are compared to the
assigned target values obtained from the PT evaluation report
(COM.04250 Comparability of Instruments/Methods) using
the HbA1c CAP Total Allowable Error (pass/fail criteria) of ±
6%. Multiple instrument Comparison PT results from each
survey are entered in the “HPLC Multiple Instrument
Comparison” spreadsheet found on the H drive
(H:\DDL\CAP\Intra-instrument Comparison).
6) The Lab director, manager or delegate will review the results
from this study and approve if the evaluation is satisfactory.
Any discrepant results observed outside of the acceptable
criteria are investigated further.
h. Carryover Studies- Performed following manufacturer’s experimental
design and guidelines;14-15
i. Prepare two specimens, one with a very high %HbA1c and one with
a very low %HbA1c value.
ii. Aliquot these specimen: 11 with low %HbA1c concentration and 10
with high %HbA1c concentration.
iii. No other specimens or test should be assayed on the instrument and
the samples should be assayed using the below carryover sample
order: 3 L (low) / 2 H (high)/ 1 L /2 H/4 L/2 H /1 L/2 H/1 L/2 H/1 L
iv. After analyzing the low and high specimens in the above order, enter
the results in EP Evaluator using the carryover program. Refer to
Teitz (ed). Textbook of Clinical Chemistry , page 421. WB Saunders
Co, Philadelphia, 1986 and the EP Evaluator carryover report
interpretation guide for additional details.14-15
v. The carryover test passes if the results of the High-low sequences
are statistically identical to the results of the low-low sequences
(three times the SD of the low-low result – the SD that would be
expected if no high results were measured). The results of the low-
level sample should not be affected by the high-level sample.

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Carryover studies should be performed roughly every year or after

major instrument maintenance.

11. REMEDIAL ACTION IF CALIBRATION OR QC SYSTEMS FAIL TO MEET

ACCEPTABLE CRITERIA
i. If control results are outside of the acceptable limits, sample results
should not be reported. Samples should be return to optimal
storage conditions until all QC issues are resolved. QC Corrective
actions could include repeating of QC (of the same lot), performing
a recalibration procedure, changing out the appropriate reagents,
retesting patient samples from a previously acceptable run (to
check instrument performance), and performing analytical precision
studies (to test the integrity of the control material in use, etc.).
ii. QC results must be acceptable prior to releasing patient results. If
QC results are still unacceptable after performing corrective
actions, call the appropriate technical vendor hotline for additional
troubleshooting advice and alert the supervisor or delegate
immediately.
iii. All Corrective Action/Preventive Action (CAPA) events concerning
out of range controls or calibrators, or rejected runs that occur at
the time of testing, should be documented (noted on the daily diary
sheet for that particular assay and QC Corrective Action form) and
immediately communicated (verbally and by email) to the Lab
Director or Manager .
iv. If the calibration is unsuccessful, recalibration is required. Patient
aliquots are not assayed until the calibration results are acceptable.
v. QC and calibration results must be considered acceptable prior to
reporting patient samples.

12. LIMITATIONS OF METHOD; INTERFERING SUBSTANCES AND

CONDITIONS

1. Dilution studies demonstrate that the assay is linear from a Total Area of
500-4000.
2. For diagnostic purposes, the results obtained from this assay should be used
in conjunction with other data (for example, signs and symptoms, duration of
diabetes, results of other test, age of patient, clinical impressions, degree of
adherence to therapy, etc.).
3. The life span of red blood cells is shortened in patients with hemolytic
anemias, depending upon the severity of the anemia. As a consequence,
specimens from such patients may exhibit decreased HbA1c levels.1
4. The life span of red blood cells is lengthened in polycythemia or
postsplenectomy patients. Specimens from such patients may exhibit
increased HbA1c levels.1
5. Interference:

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a. The presence of hemoglobin variants (e.g. HbC, HbF >38.4%, HbE, HbD,
HbS, etc.) may interfere with HbA1c results. 17
b. Refer to Operator’s manual Interference section for additional details.

13. REFERENCE RANGES (NORMAL VALUES)

Mean 5.0%
Range 4 - 6 % (equivalent of mean blood glucose of 60 - 120 mg/dL)

The normal range for the HbA1c test was established at the Diabetes Diagnostic
Laboratory in February 2000 based on 181 non-diabetic subjects collected from
the continental United States. Subjects were confirmed to have fasting blood
glucose less than 110 mg/dL (2000 standard for non-diabetic classification).
The mean HbA1c was 5.0%, with a 99% Confidence Interval of 4 to 6%.
Reference Range studies were repeated in 2004, 2009 and 2012 in the same
manner except that subjects were included only if their fasting glucose was less
than 100 mg/dL (Current ADA criteria for non-diabetic classification). In all
studies the original range of 4-6% was confirmed.

Interpretation: The level of HbA1c is an index of average

blood/plasma glucose during the preceding 3-4 months and has
strong predictive value for diabetes complications.
HbA1c ≥ 6.5% is diagnostic for diabetes. HbA1c 5.7-6.4% indicates
increased risk for diabetes Note: Any condition that shortens
erythrocyte survival or decreases means erythrocyte age (e.g.
hemolytic anemia) will falsely lower HbA1c results regardless of
the assay method (ADA Clinical Practice Recommendations).

14. CRITICAL CALL RESULTS (“PANIC VALUES”)

N/A

15. SPECIMEN STORAGE AND HANDLING DURING TESTING

Any specimens not analyzed on the day of arrival in the laboratory are stored in
the refrigerator (2°C - 8°C). Upon completion of analysis, NHANES specimens
are frozen at -70°C and discarded after 1 year.

16. ALTERNATIVE METHODS FOR PERFORMING TEST OR STORING SPECIMENS IF

TEST SYSTEM FAILS

The laboratory has 3 G8 instruments for performing glycohemoglobin (HbA1c). If

none are available for use, the specimens are stored refrigerated at 2 -8°C until
testing can be performed.

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17. TEST RESULT REPORTING SYSTEM; PROTOCOCOL FOR REPORTING CRITICAL

CALLS (IF APPLICABLE)

NHANES data files with results are exported from the NHANES database in the
specified format and uploaded to Westat via secure transfer weekly.

18. TRANSFER OR REFERRAL OF SPECIMENS; PROCEDURES FOR SPECIMEN

ACCOUNTABILITY AND TRACKING

All shipments are recorded on the NHANES Shipping Log upon receipt. Actions
taken during the course of analysis, result reporting, and specimen retention are
also recorded on the log. Specimens are stored frozen at -70oC or colder after
analysis; specimen locations are recorded according to sequential DDL
accession number and box number. After one year specimens may be
discarded.

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19. SUMMARY STATISTICS AND QC GRAPHS

See following pages.

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2019-2020 Summary Statistics and QC Chart

LBXGH (Glycohemoglobin (%))

Start End Standard Coefficient of

Lot N Date Date Mean Deviation Variation
WB27 60 15JAN19 30MAY19 5.23 0.07 1.4
WB30 189 15JAN19 17MAR20 10.27 0.14 1.4
WB29 129 04JUN19 17MAR20 5.06 0.07 1.3

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REFERENCES
(1) Tosoh Automated Glycohemoglobin Analyzer HLC-723G8 Variant
Analysis Mode Operator’s Manual v. 3.0, Tosoh Bioscience, Inc. 2014 and
Training Manual.
(2) National Glycohemoglobin Standardization Program (NGSP) website:
http://www.ngsp.org
(3) Ultra 2 SOP/Premier SOP.
(4) Validation Study Binder for HbA1C.
(5) Rohlfing CL, Hanson S, Tennill AL, Little RR. Effects of whole blood
storage on hemoglobin a1c measurements with five current assay
methods. Diabetes Technol Ther. 2012 Mar;14(3):271-5. doi:
10.1089/dia.2011.0136. Epub 2011 Oct 27
(6) Little RR1, Rohlfing CL, Tennill AL, Connolly S, Hanson S. Effects of
Sample Storage Conditions on Glycated Hemoglobin Measurement:
Evaluation of Five Different High Performance Liquid Chromatography
Methods. Diabetes Technol Ther. 2007 Feb:9(1),36-42.
(7) Stability of whole blood at -70°C for measurement of hemoglobin A1c in
healthy individuals. Clin Chem 2004;50:2460-2461 Jones W, Scott J,
Leary S, Stratton F, Jones R; ALSPAC Study Team.
(8) HCCS Biorad Package Insert.
(9) Tosoh G8 Variant Analysis Mode Chromatogram Interpretative Guide.
(10) Verbal Communication - Tosoh Bioscience Field Service Engineer, Mark
Scheckel Tel: (650)-636-8350 Email: mark.scheckel@tosoh.com
(11) Little RR, Rohlfing CL, Hanson SE, Schmidt RL, Lin CN, Madsen RW,
Roberts WL. “The Effect of Increased Fetal Hemoglobin on 7 Common Hb
A1c Assay Methods.” Clin Chem. 2012 May 1: 58(5), 945.
(12) Shu I, Devaraj S, Hanson SE, Little RR, Wang P. Comparison of
hemoglobin A1c Measurements of samples with elevated fetal hemoglobin
by three commercial assays. Clin Chim Acta 2012: 413, 1712-13.
(13) Proficiency Testing Process (CAP) – Guideline version # 3. Paula Bullock,
approved 5/11/2016.
(14) Trinity Biotech HbA1c Carryover Testing Protocol-Jon Davis R&D
Technical Manager
(15) Tosoh Bioscience Carryover Testing Protocol- Eric Shaw, Clinical Support
Specialist.
(16) Autodilutor Carryover Procedure. Approved by Dr. Hammer on 2/15/2014,
Dr. Lay filed on 3/17/2015, and Dr. Little 6/21/2016.
(17) Rohlfing C, Hanson S, Weykamp C, Siebelder C, Higgins T, Molinaro R,
Yip PM, Little R. Effects of hemoglobin C,D,E and S traits on
measurements of hemoglobin A1c by twelve methods. Clin Chim Acta
2016: 455,80-83.

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