1.0 INTENDED USE
This reagent is intended for the quantitative
determination of Hemoglobin A1c (HbA1c) in human blood. The determination of HbA1c is most commonly
performed for the evaluation of glycemic control in diabetes mellitus. HbA1c
values provide an indication of glucose levels over the preceding 4-8 weeks. A
higher HbA1c value indicates poorer glycemic control. For in vitro diagnostic
use only.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Throughout the circulatory life of the red cell, HbA1c is
formed continuously by the adduction of glucose to the N-terminal of the
hemoglobin beta chain. This process, which is non-enzymatic, reflects the
average exposure of hemoglobin to glucose over an extended period. In a
classical study, Trivelli, et al, (10.1) showed HbA1c in diabetic subjects to
be elevated 2-3 fold over the levels found in normal individuals. Several
investigators have recommended HbA1c serve as an indicator of metabolic control
of the diabetic, since HbA1c levels approach normal levels for diabetics in
metabolic control. (10.2,10.3,10.4) HBA1c
has been defined operationally as the “fast fraction” hemoglobins (HbA1a,
A1b, A1c) that elute first during column chromatography
with cation-exchange resins. The non-glycosylated hemoglobin, which consists of
the bulk of the hemoglobin. Has been designated HbA0. The present
procedure utilizes an antigen and antibody reaction to directly determine the
concentration of HbA1c.
2.2 TEST PRINCIPLE
This method utilizes the interaction of antigen and
antibody to directly determine HbA1c in whole blood. Total hemoglobin and HbA1c
have the same specific absorption rate to latex particles. When mouse antihuman
HbA1c monoclonal antibody is added (R2) latex HbA1c mouse antihuman HbA1c
antibody complex is formed. Agglutination is formed when goat anti-mouse IgG
polyclonal antibody interacts with the monoclonal antibody. The amount of
agglutination is proportional to the amount of HbA1c absorbed on to the surface
of the latex particles. The amount of agglutination is measured as absorbance.
The HbA1c valued is obtained from a calibration curve.
3.0 SPECIMEN COLLECTION AND HANDLING
3.1 PATIENT PREPARATION
No special patient preparation is required. Fasting specimens are not required.
3.2 SPECIMEN COLLECTION.
No special additives or preservatives other than
anticoagulants are required. Collect
venous blood with EDTA using aseptic technique.
All human specimens should be regarded as potentially biohazardous. Therefore, universal precautions should be
used in specimen handling (gloves, lab garments, avoid aerosol production,
etc.).
3.3 SPECIMEN STORAGE
HbA1c in whole blood collected with EDTA is stable for
one week at 2-8°C. (10.5)
Interferences: Bilrubin to 50 mg/dl, ascorbic acid to 50
mg/dl, triglycerides to 2000 mg/dl, carbamylated Hb to 7.5 mmol/L and
acetylated Hb to 5.0 mmol/L do not interfere with this assay. See also the
LIMITATION SECTION (6.2).
4.0 MATERIALS Catalog No 78050 (40 ml)
Reagents necessary for the determination of HbA1c are
included in the kit.
4.1 REAGENT
4.1.1 Reagent 1 (R1): Latex 0.13%, glycine buffer 20 mmol/L.
4.1.2 Reagent 2a (R2a): Glycine buffer 80 mmol/L
4.1.3 Reagent 2b (R2b): Mouse anti-human AbA1c monoclonal
antibody 0.05 mg/ml, goat antimouse IgG polyclonal antibody 0.08 mg/dl,
stabilizers.
4.1.4 Hemolysis Reagent (Lyse):water and stabilizers.
4.2 WARNINGS AND PRECAUTIONS
4.2.1 This reagent
kit is intended for in vitro diagnostic use only.
4.2.2 Reagent is
not for internal or external use in humans or animals.
4.3 REAGENT PREPARATION
R1 and the hemolysis reagent are ready to use as is. R2
is prepared by pouring the entire ciontents of R2b vial into the R2a vial. Mix
gently
4.4 REAGENT STORAGE AND STABILITY
4.4.1 Store reagent
at 2-8°C.
4.4.2 Unopened reagent
is stable until the expiration date.
4.4.3 R1 and R2 are stable for at least one month after opening
when store at 2-8°C.
4.5
REAGENT DETERIORATION
4.5.1 Alterations in the physical appearance of the reagent or values of control materials outside of the acceptable range may be an indication of reagent instability.
4.6 ADDITIONAL MATERIALS REQUIRED
4.6.1 Spectrophotometer
4.6.2 10µl and 1ml
pipettes.
4.6.3 HbA1c
calibrator
4.6.4 HbA1c
controls
5.0 TEST PROCEDURE
The following is a procedure for use on Hitachi 717. Call Biotron for specific instrument applications. (Hitachi 717 is a registered trademark of Nissei Sangyo Co., Ltd, Japan)
5.1 HEMOLYSATE PREPARATION
A hemolysate must be prepared for each sample.
5.1.1 Dispense 1 ml of hemolysis reagent into tubes labeled: Control, Patient, Calibrator, etc. (Plastic or glass tubes of appropriate sizes are acceptable.)
5.1.2 Dispense 10ml of controls or calibrators into the appropriately labeled lyse reagent tube. Mix
5.1.3 Centrifuge whole blood specimens at 2000rpm for 2 minutes and place 10ml of packed cells into the appropriately labeled lyse reagent tube. Mix.
5.1.4 Allow to stand for 5 minutes or until complete lysis is evident. Hemolysates may be stored up to 10 days at 2-8°C
5.2 PROCEDURE CONDITIONS
TEST NAME HbA1c
ASSAY CODE [1-POINT]:[50]-[0]
SAMPLE VOLUME [7]
[3]
R1 VOLUME [240]
[50] [NO]
R2 VOLUME [80]
[20] [NO]
WAVELENGTH [ ] [660]
CALIBRATION [NONLINEAR]
[4] [5]
STD(1) CONC-POS
(saline)
[0.0] [1]
STD(2) CONC-POS
[user def’d] [2]
STD(3) CONC-POS
[user def’d] [3]
STD(4) CONC-POS
[user def’d] [4]
STD(5) CONC-POS
[user def’d] [5]
SD LIMIT [999]
DUPLICATE LIMIT [1000]
SENSITIVTYLIMIT [0]
ABS LIMIT (INC/DEC) [32000]
[INCREASE]
PROZONE LIMIT [-]
[-]
EXPECTED VALUE [-]
[-]
PANIC VALUE [-]
[-]
INSTRUMENT FACTOR [1.0]
5.3 CALCULATION AND RESULTS
Results for the samples are calculated by referencing the absorbance of the sample with the calibration curve.
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.11)
Recommended
values:
Less than
6% for non-diabetic
Less than
7% for glycemic control of a person with diabetes
These values are suggested guidelines. Each laboratory
should establish its own expected values. In using HbA1c to monitor diabetic
patients, results should be interpreted individually. That is, the patient should
be monitored against him or herself. There is a 3-4 week lag time before HbA1c
reflects changes in blood sugar level.
6.2 LIMITATIONS OF PROCEDURE
6.2.1 This assay should not be used for the diagnosis of
diabetes mellitus.
6.2.2 Specimens should always be assayed using a calibration
curve.
6.2.3 It has been reported that results may be inconsistent in
patients who have the following conditions: opiate addiction, lead-poisoning,
uremia (carbamylated Hb), alcoholism, ingest large doses of aspirin.
(10.6,10.7,10.8,10.9)
6.2.4 It has been reported that elevated levels of HbF may lead
to underestimation of HbA1c and, that uremia does not interfere with HbA1c
determination by immunoassay. (10.10)
6.2.5 It has been reported that hemoglobin variants HbS and
HbA2 are not detected by immunoassay, leading to possible inaccurate
determination. Also it has been reported that labile intermediates (Schiff
base) are not detected and therefore do not interfere with HbA1c determination
by immunoassay.
6.2.6 Other very rare variants of hemoglobin (e.g. HbE) have
not been assessed.
7.0 QUALITY CONTROL
Standard practice for quality control should be applied
to this system. Biotron's HbA1c controls can be used to monitor the daily
acceptable variations. These controls
should be assayed daily with the run of patient samples, whenever a new reagent
lot is used, and following any system maintenance.
A satisfactory level of performance is achieved when the
analyte values obtained are within the "acceptable range" established
by the laboratory.
8.0 CALIBRATION PROCEDURES
Use Biotron HbA1c calibrator set . Use saline for 0. The
calibration is stable for 7 days.
The assay is calibrated by referencing the absorbance of
the unknown sample to the absorbance of the calibrators. Calibration is
required with the use of a new lot of reagent, any system maintenance or
whenever indicated by quality control data.
9.0 PERFORMANCE CHARACTERISTICS
9.1 PRECISION
Within-Run:
The intra assay precision was established by assaying bloods with 3 levels of
HbA1c twenty times each.
Mean
(%) SD (%) CV (%)
4.76 0.06 1.26
7.29 0.08 1.10
10.9 0168 1.47
Between
Run: The inter run precision was established by assaying bloods with 3 levels
of HbA1c for ten runs conducted over a
five day period.
Mean
(%) SD (%) CV (%)
4.72 0.06 1.27
7.36 0.08 1.09
11.0 0.17 1.55
9.2 CORRELATION
A study on human specimens between this HbA1c procedure
and a another immunoassay procedure (Roche Diagnostics) yielded a correlation
coefficient of 0.995 and a linear regression equation of
y=1.05x-0.36.(n=45).
9.3 LINEARITY
The HbA1c assay range is 2.0% to 16.0%
9.4 SENSITIVITY
This HbA1c procedure has an approximate 0.073 absorbance
change for 1.0% of HbA1c under the conditions as described in the PROCEDURE
CONDITIONS (5.2) section. Saline showed little or no drift.
10.0 REFERENCES
10.1 Trivelli,
L.A., Ranney, H.M., and Lai, H.T., New Eng. J. Med. 284, 353 (1971).
10.2 Gonen, B. and
Rubenstein, A.H., Diabetologia 15,1 (1978).
10.3 Gabbay, K.H.,
Hasty, K., Breslow, J.L., Ellison, R.C., Bunn, H.F., and Gallop, P.M., J. Clin.
Endocrinol. Metab. 44, 859 (1977).
10.4 Bates, H.M.,
Lab. Manag., Vol. 16 (Jan: 1978).
10.5 Tietz, N.W.
Textbook of Clinical Chemistry, Philadelphia, W.B. Saunders Company p794-795
(1999).
10.6
Corielo, A., et al,
Diabetologia 22, p.379 (1962).
10.7
Goldstein, D.E., et
al, Clin. Chem 32, pp. 364-370 (1986).
10.8
Fluckiger, R. et al,
Med Intelligence 304, pp. 823-827 (1981).
10.9
Nathan, D.M., et al,
Clin Chem 29, pp. 466-467 (1983).
10.10 Engbaek, F., et al, Clin Chem 35, pp 93-97 (1989)
10.11 American Diabetes Association: Clinical Practice
Recommendations (Position Statement.) Diabetes Care 24 (Supp 1): S33-S55
(2001).