1.0 INTENDED USE
This reagent is intended for the quantitative
determination of total Iron and unsaturated Iron binding capacity (UIBC) in
serum. Total Iron binding capacity
(TIBC) is determined as the sum of total Iron and UIBC.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Ferrozine is a sulfonated derivative of diphenyltriazine. It forms a water-soluble magenta complex with
iron (10.1.) Persijn et al, (10.2)
described a method for determining serum iron with ferrozine. This is the basis for the Biotron
procedure. This method avoids protein
precipitation (10.2) and minimizes interference from other trace metals (10.1.)
2.2 TEST PRINCIPLE
At acid pH and in the presence of suitable reducing agent,
transferrin-bound serum iron dissociates to form ferrous ions. These react with ferrozine to produce a
magenta colored complex with an absorption maximum near 560nm. The difference in color intensity at this
wavelength, before and after addition of ferrozine, is proportional to serum
iron concentration.
At alkaline pH, ferrous ions added to serum bind
specifically with transferrin at unsaturated iron-binding sites. Remaining unbound ferrous ions are measured
with the ferrozine reaction. The difference
between the amount of unbound iron and the total amount added to serum is
equivalent to the quantity bound to transferrin. This is the serum UIBC.
3.0 SPECIMEN COLLECTION AND HANDLING
3.1 PATIENT PREPARATION
No
special patient preparation is required.
3.2 SPECIMEN COLLECTION
Collect blood samples using only syringes, test tubes,
etc. that are iron-free. Separate serum
as soon as blood clots. Plasma should
not be used. Although occult hemoglobin
does not interfere with the assay, only clear unhemolyzed serum is suitable for
assay. Each mg of hemoglobin contains
3.4µg iron.
The amount of sample required will depend on the
analyzer used. The amount of serum
required is in the range of 10-500 µl.
Call Biotron's technical service department at 1-800-595 8766 for the
recommended sample volume for your analyzer.
Record the patient's name, date and time of sample
collection and preparation.
3.3 SPECIMEN STORAGE
Serum iron reportedly is stable for at least 4 days
stored at room temperature or 1 week in the refrigerator (10.4.)
It is recommended that testing be done as soon as
possible after sample collection and preparation. If testing cannot occur immediately, store
the sample properly using the guidelines above.
4.0 MATERIALS
(2 X 125 ml)
Reagents necessary for the determination of Iron and
UIBC are included in the kit.
4.1 IRON/UIBC REAGENT
4.1.1 Iron
Buffer Reagent
Hydroxylamine
hydrochloride 1.5%
(w/v)
Acetate buffer, pH 4.5
surfactant
4.1.2 UIBC
Buffer Reagent
Tris
(hydroxymbthyl) aminomethane pH8.1 0.5
mol/L
surfactant
sodium azide as
preservative 0.05%
4.1.3 Iron
Color Reagent
Ferrozine 0.85%
(w/v)
in hydroxylamine hydrochloride solution with stabilizer.
4.1.4 Iron
Standard
Iron 500µg/dl
(89µmol/L)
in hydroxylamine hydrochloride solution.
This standard is traceable to SRM 937.
4.2 WARNINGS AND PRECAUTIONS
For In Vitro Diagnostic Use. Never pipette by mouth. Exercise the normal precautions required for
handling all laboratory reagents.
Iron Buffer Reagent, Iron Color Reagent and Iron Standard
are HARMFUL if inhaled, swallowed or allowed to come in contact with the
skin. The reagents are irritating to
eyes, respiratory system and skin, with possible risk of irreversible effects
and possible mutagen. Target organ(s) are blood and central nervous
system. In case of contact with eyes,
rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing.
UIBC Buffer Reagent contains sodium azide which is toxic
if ingested and which may react with lead and copper plumbing to form highly
explosive metal azides. On disposal
flush with a large volume of water to prevent azide accumulation.
4.3 REAGENT PREPARATION
Reagents
are supplied in liquid form and are ready for use in the assay.
4.4 REAGENT STORAGE AND STABILITY
Store
reagents at room temperature (18-26°C).
Reagents are stable until the expiration date shown on
the respective labels.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer or colorimeter capable of reading absorbance accurately at 560 nm.
4.5.2 1 cm
cuvettes or a flow cell capable of transmitting light at 560 nm.
4.5.3 Test
tubes capable of holding 4 ml.
4.5.4 Pipettes
capable of delivering 0.5, 2.0 and 2.5 ml.
4.5.5 Constant
temperature source which can be adjusted to 37°C.
4.5.6 Timer
for a 10 minute incubation.
4.5.7 Normal
and abnormal controls for quality control.
5.0 TEST PROCEDURE
Application procedures using Biotron Diagnostics
Iron/UIBC reagents are available for most automated instruments. Please contact Biotron Diagnostics Technical
Services Department at 1-800-595 8766
for additional information.
The
following is a general procedure for use on a manual instrument.
5.1 PROCEDURE CONDITIONS
Wavelength 560
nm
Temperature 37°
C
Pathlength 1
cm
Mode End
Point
Reaction time 10
minutes
Sample volume 500
µl
Iron Buffer volume 2.5
ml
UIBC Buffer volume 2.0
ml
Iron Color volume 0.05
ml
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 560 nm may be used. The band width should be 10 nm or less, stray
light 0.5% or less, and the wavelength accuracy within 2 nm.
5.3 CALIBRATION
The procedures are calibrated with the iron standard
provided with the kit.
5.4 PROCEDURE FOR IRON
5.4.1 Label
cuvettes Blank, Standard and Patient.
5.4.2 Add
2.5ml of Iron Buffer reagent to each cuvette.
5.4.3 Add
0.5ml of iron-free water to Blank cuvette.
Add
0.5ml of iron standard to Standard cuvette.
Add
0.5ml of serum to Patient cuvette.
5.4.4 Mix each
cuvette thoroughly.
5.4.5 Read and
record the absorbance of Patient and Standard versus Blank as reference at
560nm. This is the INITIAL A.
5.4.6 Add
0.05ml (50µl) of Iron Color reagent to each cuvette. Mix thoroughly and incubate at 37°C for 10
minutes.
5.4.7 Read and
record the absorbance of Patient and Standard versus Blank as reference at
560nm. This is the FINAL A.
5.5 PROCEDURE FOR UIBC
5.5.1 Label
cuvettes as Blank, Standard and Patient.
5.5.2 Add 2.0
ml of UIBC Buffer reagent to each cuvette.
5.5.3 Add 1.0
ml of iron-free water to Blank cuvette.
Add
0.5 ml of iron-free water and 0.5 ml of Iron Standard to Standard
cuvette.
Add
0.5 ml of serum and 0.5 ml of Iron Standard to Patient cuvette.
5.5.4 Mix each
cuvette thoroughly.
5.5.5 Read and
record the absorbance of Patient and Standard vs Blank as reference at
560nm. This is the INITIAL A.
5.5.6 Add 0.05
ml (50 µl) of Iron Color reagent to each cuvette. Mix thoroughly and incubate at 37°C for 10
minutes.
5.5.7 Read and
record the absorbance of Patient and Standard vs Blank as reference at 560nm. This is the FINAL A.
Note: Occasionally,
the difference between Patient INITIAL A and FINAL A may be very small because
of the high degree of unsaturation of transferrin with iron.
5.6 CALCULATION AND RESULTS FOR TOTAL IRON
FINAL
A - INITIAL A (Patient)
Total
Iron (µg/dl) = -------------------------------------
x 500
FINAL
A - Initial A (Standard)
where
concentration of Iron Standard = 500 µg/dl.
Example:
(0.151
- 0.080)
Total
Iron (µg/dl) = -------------------
x 500 = 99
(0.360 - 0.000)
where: FINAL A Patient = 0.151
FINAL
A Standard = 0.360
INITIAL
A Patient = 0.080
INITIAL
A Standard = 0.000
5.7 CALCULATION AND RESULTS FOR UIBC
FINAL
A - INITIAL A (Patient)
UIBC
(µg/dl) = 500 - -------------------------------------------
X 500
FINAL
A - INITIAL A (Standard)
where
concentration of Iron Standard = 500 µg/dl
Example:
(0.310-0.100)
UIBC
(µg/dl) = 500 -
------------------------- X 500 = 208
(0.360-0.000)
where: FINAL A Patient = 0.310
FINAL
A Standard = 0.360
INITIAL
A Patient = 0.100
INITIAL
A Standard = 0.000
5.8 CALCULATION AND RESULTS FOR TIBC
If the UIBC (unsaturated iron binding capacity) is
known, then TIBC (total iron binding capacity) is calculated:
TIBC
= Total Iron + UIBC
Example:
TIBC (µg/dl) = 99 + 208 = 307
where
iron = 99 and UIBC = 208 µg/dl
5.9 S.I. UNITS
To Convert results into SI units, multiply results in
µg/dl by 0.179. For example, Iron = 100
µg/dl = 100 x 0.179 µmol/L = 17.9 µmol/L.
6.0 INTERPRETATIONS OF RESULTS
6.1 EXPECTED VALUES (10.4)
Iron: 65 ‑ 173 µg/dl (11.6 ‑ 30.9 µmol/L)
TIBC: 224 ‑ 366 µg/dl (40.1 ‑ 65.6 µmol/L)
These values are suggested guidelines.
It is recommended that each laboratory establish the normal range for
the area in which it is located.
6.2 CLINICAL SIGNIFICANCE
Measurements of iron are used in the diagnosis and
treatment of a number of conditions (10.6) such as iron deficiency anemia,
hemochromatosis and chronic liver disease.
In most cases, it is the combination of both serum total iron and iron
binding capacity that is of the most clinical significance.
As with any chemical reaction, the user must be alert to
the possible effect on results caused by unknown interferences from medication
or endogenous substances. All patient
results must be evaluated in light of the total clinical significance of the
patient.
6.3 MEDICAL ALERT VALUES (10.8)
Each laboratory should establish low and high values
beyond which the patient would require immediate attention by a physician. If a "medical alert value" is
reached, always repeat the test to confirm the result and notify a physician if
the result is confirmed.
6.4 LIMITATIONS OF PROCEDURE
Certain drugs and other substances are known to
influence circulating iron levels (10.5.)
7.0 QUALITY CONTROL
Standard practice for quality control should be applied
to this system. Commercially available
lyophilized controls can be used to monitor the daily acceptable
variations. Normal and abnormal controls
should be assayed at the beginning of each run, whenever a new reagent or a
different lot number is being used, and following any system maintenance.
A satisfactory level of performance is achieved when the
analyze values obtained are within the "acceptable range" established
by the laboratory.
8.0 CALIBRATION PROCEDURES
The procedures are calibrated with the iron standard
provided with the kit.
Calibration is required with the use of a new lot of
reagent, any system maintenance or whenever indicated by quality control data.
9.0 TECHNICAL PERFORMANCE
9.1 PRECISION
The estimates of precision shown below were obtained from assays of
human control serum.
Within-Run
In
this study, 20 replicates of 2 control sera were run.
Mean
(µg/dl) SD (µg/dl) CV (%)
Iron 91 ± 1.5 1.7
250 ± 2.8 1.1
UIBC 133 ±
2.5 1.9
263 ± 4.0 1.5
Between-Run
In this study, 5 runs were made, each run consisting of
5 replicates of 3 control sera.
Mean
(µg/dl) SD (µg/dl) CV
(%)
Iron 74 ± 2.2 3.0
91 ± 1.5 1.6
249 ± 2.2 0.9
UIBC 153 ±
2.3 1.5
206 ± 3.3 1.6
9.2 CORRELATION
Iron: A correlation study was done comparing this method
(y) with a comparative method (x). The
samples range between 9 µg/dl and 1000 µg/dl of total iron.
Number Regression Equation Correlation
of
Samples y=Biotron x=Comparative Coefficient
59 y = 0.992 x +
1.25 0.999
UIBC: A correlation study was done comparing this method
(y) with a comparative method (x). The
samples range between 31 µg/dl and 413 µg/dl of UIBC.
Number Regression Equation Correlation
of
Samples y=Biotron x=Comparative Coefficient
40 y = 1.003 x -
0.512 0.994
9.3 LINEARITY
This procedure for total iron is linear to 500 µg/dl. If
serum total iron value is greater than 500 µg/dl, dilute sample with equal
volume of saline, repeat assay and multiply result by 2.
9.4 RECOVERY STUDY
Aliquots of a freshly reconstituted serum control were
diluted with equal volumes of standard containing iron concentrations of 100,
200 and 300 µg/dl. Serum total iron recoveries
were 98.8%, 96.6% and 99.4% respectively.
9.5 TECHNICAL ASSISTANCE
For technical assistance with this reagent performance,
call King at 1-800-262-8655.
10.0 REFERENCES
10.1 Stookey
LL, Ferrozine - A new spectrophotometer reagent for iron. Anal Chem 42: 779,
1970.
10.2 Persijn
JP, Van Der Slik W, Riethorst A: Determination of serum iron and latent
iron-binding capacity (LIBC). Clin Chem Acta 35:91, 1971.
10.3 Pippard
MJ, Stray S: Simple assay for urinary iron after desferrioxamine therapy. Am J. Clin Pathol 77: 324, 1982.
10.4 Clinical
Chemistry Principles and Technics, 2nd ed., RJ Henry DC Cannon, JW Winkelman,
Editors, Harper & Row, Hagerstown (MD), 1974
10.5 Young
DS, Effects on Drugs on Clinical Laboratory Tests - 3rd Edition, AACC Press,
1990.
10.6 Fundamentals
of Clinical Chemistry NW Tietz, Editor, Saunders, Philadelphia, 1976.
10.7 Goodwin
JF, Murphy B. Guillemette M: Direct measurement of serum iron and binding
capacity. Clin Chem 12:47, 1966.
10.8 G.J.
Kost, "Critical Limits for Urgent Clinician Notification at U.S. Medical
Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5.