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SUMMARY AND EXPLANATION
Iron in serum exists primarily as a complex with transferrin. Each transferrin molecule binds two atoms of ferric-iron. This mechanism serves for transport of iron from mucosal cells to the body's storage areas such as the liver and bone marrow. Elevated serum iron utilization (lead poisoning), accelerated release of iron from body stores (neocrotic hepatitis), defective iron storage (pernicious anemia) and increased iron absorption (hemochromatosis). Decreased serum iron is found with dietary deficiency, nephrosis, and increased body demand (pregnancy). Clinical interpretation of abnormally low or high iron levels should be made only in conjunction with iron-binding capacity (TIBC) since both values are important for differential diagnosis (1).
Many chromagens have been used in the determination including thiocyanate o-phenanthroline, bathophenanthroline and TPTZ. In 1971, Persijm et al. (1) presented a method using the chromagen ferrozone, described by Stookey. (2) This method did not require protein precipitation and was more sensitive than previous methods. The present procedure is a modification of the Persijn method.
PRINCIPLE
Serum iron: Transferrin-bound iron is released at an acid pH and reduced from ferric to ferrous ions. These ions react with ferrozine to form a violet colored complex which is measured spectrophotometrically at 560 nm. The absorbance measured at this wavelength is proportional to serum iron concentration.
Total Iron-binding Capacity (TIBC): A known amount of ferrous ions are added to serum at an alkaline pH. The ferrous ions bind with transferrin at unsaturated iron-binding sites. The additional unbound ferrous ions are measured using the ferrozine reaction. The difference between the amount of ferrous ions added and the unbound ions measured is the unsaturated iron-binding capacity (UIBC). The TIBC is equal to the serum iron concentration plus the UIBC.
REAGENTS: FOR IN-VITRO DIAGNOSTIC USE
Reagent Set (Cat. No. 3260) provides:
1. IRON BUFFER REAGENT: Hydroxylamine hydrochloride 220mM in acetate buffer, pH 4.5 with surfactant.
2. UIBC BUFFER REAGET: Tris 500 mM, pH 8.1 with surfactant, sodium azide 0.05% (w/v) as preservative.
3. IRON COLOR REAGENT: Ferrozine 16.7mM in hydroxylamine hydrochloride.
4. IRON CALIBRATOR (500 ug/dL): Ferrous chloride in hydroxylamine hydrochloride.
PRECAUTIONS:
1. All reagents are toxic. Do not pipette by mouth. Avoid all contact.
2. UIBC buffer contains Sodium Azide. Dispose with large amount of water to prevent azide reacting with plumbing.
STORAGE AND STABILITY:
Store at 2- 8ºC. Stable until expiration date if sealed tightly.
DETERIORATION:
All reagents should be a clear solution. Turbidity would indicate deterioration.
INSTRUMENTS
Use a spectrophotometer or colorimeter calibrated at 560 nm.
SPECIMEN COLLECTION
PRECAUTIONS:
1. Serum should be separated from the blood clot as soon as possible.
2. Plasma should not be used since anti-coagulants may interfere.
3. Serum iron shows diurnal variation (3). Accordingly, morning specimens are preferred after an overnight fast.
4. Grossly hemolyzed or lipemic samples should not be used.
SAMPLE STORAGE:
Iron appears stable for one week at 2 - 8ºC.
ADDITIVES:
No special additives or preservatives are needed.
INTERFERING SUBSTANCES:
Specimens from patients receiving chelation therapy should not be used. Young et al, (5) have reviewed drug effects on serum iron.
PROCEDURE
MATERIALS PROVIDED:
IRON BUFFER REAGENT (Cat. No. 3261), UIBC BUFFER REAGENT (Cat. No. 3262), IRON COLOR REAGENT (Cat. No. 3263) and IRON CALIBRATOR (Cat. No. 3264).
MATERIALS REQUIRED BUT NOT PROVIDED:
1. 0.05 and 0.50 mL micropipettor
2. 2.0 and 2.5 mL pipettor or dispensor
3. Incubator capable of maintaining 37ºC.
4. Test tubes and rack
REACTION CONDITIONS:
Wavelength 560 nm
Filter Selection 540 - 580 nm
Reaction Type Endpoint
Reaction Time 10 minutes
Temperature 37ºC
Sample Volume 0.5 mL
Iron Buffer 2.5 mL
Iron Color 0.05 mL
UIBC Buffer 2.0 mL
Iron 60 - 150 ug/dL
TIBC 250 - 400 ug/dL
Calibrator Value 500 ug/dL
MANUAL PROCEDURE:
Serum Iron
1. Label test tubes "Blank", "Calibrator", "Control", "Sample",etc.
2. Add 2.5 mL Iron Buffer reagent to all tubes.
3. Add 0.5 mL sample to respective tubes. Mix. Note: Use iron-free water for "Blank".
4. Zero spectrophotometer at 560 nm using the Reagent Blank.
5. Read and record absorbances of all tubes. (A1 reading).
6. Add 0.05 mL iron color reagent to all tubes. Mix.
7. Place all tubes in heating bath at 37º C for 10 minutes.
8. Zero instrument at 560 nm with reagent blank.
9. Read and record absorbances of all tubes. (A2 reading).
CALCULATION OF RESULTS
A = Absorbance
Cal = Calibrator
A2 test - A1 test
----------------------- x Conc. = Total Iron (ug/dL)
A2 Cal - A1 Cal of Cal
Example:
A1 test = 0.08 A2 test = 0.15
A1 Cal = 0.00 A2 Cal = 0.40
Then:
0.15 - 0.08 0.07
----------------- = --------- = 0.175 x 500 = 87.5 ug/dL
0.40 - 0.00 0.40
UIBC (Unsaturated Iron-Binding Capacity)
1. Label test tubes "Blank", "Calibrator", "Control", "Sample",etc.
2. Add 2.0 mL UIBC Buffer reagent to all tubes.
3. To "Blank" add 1.0 mL iron-free water. Mix.
4. To "Calibrator" add 0.5 mL iron-free water plus 0.5 mL calibrator.
5. Mix well.
6. To "Test" add 0.5 mL respective sample plus 0.5 mL iron calibrator.
7. Mix well.
8. Zero spectrophotometer at 560 nm using the Reagent Blank.
9. Read and record absorbances of all tubes. (A1 reading).
10. Add 0.05 mL iron color reagent to all tubes. Mix well.
11. Place all tubes in heating bath at 37º C for 10 minutes.
12. Zero instrument at 560 nm with reagent blank.
13. Read and record absorbances of all tubes. (A2 reading).
CALCULATION OF RESULTS
A = Absorbance
Cal = Calibrator
UIBC (ug/dL) =
A2 test - A1 test
Conc. of Cal. - ------------------------------ x Conc of Cal.
A2 Cal - A1 Cal
Example:
Conc = 500 ug/dL
A1 test = 0.10 A2 tes = 0.20
A1 Cal = 0.00 A2 Cal = 0.40
Then:
( 0.2 - 0.10)
500 - ----------------- x 500 = UIBC (ug/dL)
(0.40 - 0.00)
500 - (0.25 x 500) =
375 ug/dL (UIBC)
TIBC (Total Iron-Binding Capacity)
level + UIBC = TIBC (ug/dL)
Conversion ug/dL x 0.179 = umo/L
STABILITY OF FINAL REACTION:
The test samples should be read within 15 minutes after color development.
CALIBRATION:
It is not necessary to perform a calibration curve with this procedure since the reaction is linear in range of 0-500 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use Iron Calibrator (Cat. No. 3264) which is provided in the reagent set or other suitable calibration material for this purpose. Specimens exceeding 500 mg/dL should be diluted with 0.9% sodium chloride solution and reassayed. Multiply the test result by the dilution factor to obtain the final answer.
QUALITY CONTROL:
The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner as the Unknowns. EAGLE DIAGNOSTICS makes available CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.
LIMITATIONS
1. Copper present in serum can cause interference in the assay by forming a colored complex. Ordinarily this interference is not significant unless the copper level is quite excessive (3).
2. Ingestion of oral contraceptives reportedly elevates serum iron-binding capacity.
EXPECTED VALUES
IRON:
Males 60 - 160 ug/dL (10.7 - 28.6 uM)
Females 40 - 140 ug/dL (7.2 - 25.1 uM)
TIBC:
250 - 400 ug/dL
The ranges represents the 95% confidence intervals from a clinically normal population. It is recommended that each laboratory establish its own range of expected values.
PERFORMANCE CHARACTERISTICS
LINEARITY:
This method is linear to 500 ug/dL.
PRECISION:
Total Iron
MEAN / ST. DEV. / %CV
Within Run
110 / 3.0 / 2.7
184 / 3.1 / 1.7
251 / 3.3 / 1.3
Run to Run
109 / 5.1 / 4.7
177 / 3.8 / 2.1
249 / 4.5 / 1.8
UIBC
Within Run
279 / 3.5 / 1.3
315 / 3.7 / 1.2
392 / 5.8 / 1.5
Run to Run
287 / 16.5 / 5.7
303 / 12.3 / 4.1
390 / 16.3 / 4.2
SPECIFICITY:
A comparison of this SERUM IRON PROCEDURE with another widely used commercial method resulted in a coefficient of correlation of .993 with a regression equation of y = 1.02 x + 7.0. A study performed between this procedure and a similar TIBC procedure resulted in a coefficient of correlation of .976 with a regression equation of y = 0.92 x + 32.5.
SENSITIVITY:
This IRON / TIBC procedure has a sensitivity of 1.5 Ug/dL per 0.001 absorbance unit.
REFERENCES
1. Persijn, J.P.m et al. Clin. Acta 35.91 (1971).
2. Stookey, L.L., Anal. Chem 42:779 (1970).
3. Tietz, N.W., Fundamentals of Clinical Chemistry, Philadelphia, 1982, p. 923-929 (1976).
4. Weissman, N. Pileggi, V.J., in Clnical Chemistry: Principles and Technics, 2nd Ed.., R.J. Henry et al, editors, Hagestown (MD), Harper & Row, pp. 692-693 (1974).
5. Young, D.S., et al Clin. Chem. 21:1D (1975).
6. Henry, J.B., Clinical Diagnosis and Mangement by Laboratory Methods, Philadelphia, W.B. Saunders, p. 1434 (1984).
