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www.biomaghreb.com
IVD
FT En 55 Date of updating : 10/ 2020 Version B
IRON
AND TOTAL IRON BINDING CAPACITY
Quantitative determination of iron
and its total binding capacity in human plasma.
A- IRON
CLINICAL SIGNIFICANCE
In humans, 70% of the body’s iron is bound to haemoglobin, the
rest is bound to transport proteins such as ferritin (or transferrin),
or stored in certain tissues such as the liver and bone marrow. Low
serum iron levels may be seen in iron deficiency anemia (martial
deficiency) or in patients with inflammatory anemia.
Conversely, iron overload may occur during hemochromatosis or
acute liver dysfunction.
PRINCIPLE
At pH 4.8 ferric iron (Fe +++) is instantly released from transferrin.
Ascorbic acid reduces it to ferrous iron (Fe++). Ferrozine forms a
soluble colored complex with ferrous iron, measurable from 560
to 580 nm.
The presence of thio-urea eliminates the interference of cuprous
ions.
REAGENT COMPOSITION
IRON FERROZINE
Reagent 1
Guanidine, HCl
Acetate Buffer
4,5 mmol/l
pH 5
Reagent 2 Ascorbic acid 40 mmol/l
Reagent 3 Ferrozine 1 mg/l
Reagent 4 Standard 17,9 µmol/l
SAFETY CAUTIONS
Biomaghreb reagents are intended for use by qualified personnel
for in vitro use (do not pipette by mouth).
• Refer to the current MSDS available on request or on www.bio-
maghreb.com.
• Check the integrity of the reagents before use.
• Disposal of waste: comply with applicable legislation.
For safety reasons, treat any specimen or reagent of biological
origin as potentially infectious. Observe the applicable legislation.
REAGENT PREPARATION
Dissolve the contents of a spatula of ascorbic acid (approximately
250 mg) in 50 ml of Reagent 1 (Reagent A).Add 40 µl ferrozine to
1 ml of Reagent A (Reagent B).
Reagent B is prepared extemporaneously.
SAMPLE COLLECTION AND HANDLING
Serum,plasma heparinazed and non hemolysed
PRESERVATION AND STABILITY
• Before opening: Until the expiry date indicated on the label of
the box at + 4°C.
• After opening : Reagents A and B are stable :
3 days at 20 - 25°C;
2 weeks at 2 - 8°C.
ADDITIONAL EQUIPMENT
• Basic equipment of the medical analysis laboratory ;
• Spectrophotometer or Clinical Biochemistry Analyzer
LIMITS
The use of glass materials requires soaking for several hours in
2N hydrochloric acid and then rinsing carefully with distilled water.
It is therefore preferable to use disposable plastic material. High
doses of anticoagulants (Heparin) can cause disturbances in the
reaction mixture.
QUALITY CONTROL
External quality control program.
It is recommended to control in the following cases:
- At least one test per series.
- Change of reagent bottle.
- After maintenance work on the analyzer.
If a control value is outside the confidence limits, repeat the proce-
dure using the same control.
Use normal and pathological control sera.
CALIBRATION
- Standard (Reagent 3)
The calibration frequency depends on the performance of the ana-
lyzer and the storage conditions of the reagent.
Recalibration is recommended in the following cases:
1. changing the reagent lot ;
2. after maintenance work on the analyzer; and
3. the control values are outside the confidence limits.
LINEARITY
The reaction is linear up to 1000 µg/dl (179.7 µmol/l).Above this
limit, dilute the sample with 9 g/L NaCl solution and repeat the
determination taking the dilution into account in the calculation
of the result. The linearity limit depends on the sample/reagent
volume ratio.
PROCEDURE
-Wavelength: 578 nm (530-590).
Temperature: 20 -25°C.
Tank: 1 cm thick.
Adjust the spectrophotometer zero by :
• Reagent A for Sample Whites ;
• Blank reagent for standard and samples.
Blank
Reagent
Standard Sample
white
Sample
Distilled water 200µl - - - - - -
R4 - - 200µl - - - -
Sample - - - - 200µl 200µl
Reagent A - - - - 1 ml - -
Reagent B 1 ml 1 ml - - 1 ml
Mix and wait 10 minutes
Measuring absorbances at 578 nm
Staining stability: 30 minutes.
CALCULATION
Serum iron =
DO. Sample - DO. White sample
OD Standard
x n
n= 1 mg/l;
n = 17, 9 µmol /l.
REFERENCE VALUES
Men
69 - 158 µg/dl
12.5 - 28.3 µmol/l
Women
59 - 145 µg/dl
10.7 - 26 µmol/l
REFERENCES
Persijn et al Clin. Chem. Acta 35,91 (1971);
Stoockey L. Anal. Chem. 42,779 (1970);
Williams et al. Clin. Chem. 23,237(1977).
B- TIBC
CLINICAL SIGNIFICANCE
Transferrin is an iron transport protein. The amount of iron that can
be bound to transferrin, added to the already bound iron, represents
the total iron binding capacity (TIBC). The ratio of serum iron to
TIBC is the transferrin saturation factor. TIBC increases in the case
of martial deficiency due to inadequate iron intake or secondary to
chronic bleeding. A decrease in TIBC may be associated with an
inflammatory syndrome, hepatocellular insufficiency, or iron over-
load.
PRINCIPLE
The total iron binding capacity is evaluated after saturation of
transferrin with an iron solution and adsorption of the excess on
magnesium hydroxycarbonate. The determination of iron is then
carried out using the iron ferrozine kit.
REAGENT COMPOSITION
Reagent 1
Iron
Saturating Iron Solution
5 mg/l
89,5µmol/l
Reagent 2 Basic magnésium carbonate (adsorbant)
Dosing Spatula
SAFETY CAUTIONS
Refer to the package insert of the reagent used for the iron determination.
REAGENT PREPARATION
The reagents are ready to use and stable until the expiry date in-
dicated on the label.
SAMPLE PREPARATION
Unhemolyzed serum. The sample can be stored for 7 days at
2-8°C or several weeks frozen at -20°C.
PRESERVATION AND STABILITY
Until the expiry date indicated on the box label at +4°C.
ADDITIONAL EQUIPMENT
•
Basic equipment of the medical analysis laboratory ;
•
Spectrophotometer or Clinical Biochemistry Analyzer;
LIMITS
Iron contamination is one of the main causes of error. The use of
plastic and one use pipettes and cuvettes is recommended.
QUALITY CONTROL
- Any control serum titrated for this method ;
- External quality control program.
(Refer to the package insert of the reagent used for the iron de-
termination.)
PROCEDURE
Sample 1 ml
Reagent 1 2 ml
Mix, incubate for 5 minutes, then add:
Reagent 2 about 200 mg
Wait 20 minutes, stirring several times. Centrifuge for 10
minutes at 3000 rpm.
CALCULATION
In the case of iron and total binding capacity determinations, it is
advisable to start with transferrin saturation and then conduct the
serum iron and total binding capacity determinations in parallel.
Take the 1/3 dilution into account for the calculation.
REFERENCE VALUES
•
44,5 - 73,5 µmol/l ;
• 249 - 412 µg/dl.
Each laboratory is recommended to determine its own reference
values for each population concerned.
REFERENCES
TIETZ N.W. Text book of clinical chemistry, 3rd Ed. CA Burtis, E.R.
Ashwood, W.B.Saunders (1999) p. 1701-1703.
IN VITRO DIAGNOSTIC USE
Manufacturer Use by In Vitro Diagnostic Temperature
Limitation
Catalogue number See insert Store away from light Sufcient
for < n > essais
Batch number
