IRON PROCEDURE

Intended for the Quantitative Determination of Iron in Serum

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 bodys 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).

Most commercial methods for iron assay employ reagents that form colored complexes with iron. Recently, FERENE has been introduced as a superior iron-complexing agent that offers improved specificity, sensitivity and stability, with greater ease of handling (2, 3). This reagent is particularly well-suited for use on automated instruments (4). The EAGLE IRON PROCEDURE is based on a modified ferene reagent.

PRINCIPLE

Upon addition of serum to IRON COLOR REAGENT, iron disassociates from transferrin and is reduced to the ferrous ion. Ferene present in the IRON COLOR REAGENT combines with the ferrous ion to form a blue complex that is measured at 595 nm. The intensity of blue color is directly proportional to the iron concentration.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 3200 provides:

IRON COLOR REAGENT - (Cat. No. 3201-1)

REACTIVE INGREDIENTS:

1.0 Mm ferene in acetate buffer at pH = 4.5. Surfactant and reducing agent present.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15 - 30ēC. Stable until expiration date if sealed tightly. PROTECT FROM LIGHT.

DETERIORATION:

The reagent should be a clear, blue-yellow solution. Turbidity or a dark color would indicate deterioration. The Reagent Blank in PERFORMANCE OF TEST should have an absorbance less than 0.300 at 595 nm.

IRON BLANK REAGENT - (Cat. No. 3202-1)

REACTIVE INGREDIENTS:

1.7 M acetate buffer at pH = 4.5. Surfactant and reducing agent present.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15 - 30ēC. Stable until expiration date if sealed tightly.

DETERIORATION:

The calibrator should be a clear, colorless solution. Turbidity would indicate deterioration.

IRON STANDARD - (Cat. No. 3203-1)

REACTIVE INGREDIENTS:

500 ug/dL iron (89.5 uM). Reducing agent present.

STORAGE AND STABILITY:

Store at 15 - 30ēC. Stable until expiration date if sealed tightly.

DETERIORATION:

The standard should be a clear, colorless solution. Turbidity or a dark color would indicate deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 595 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. It is recommended that serum separated from the blood clot as soon as possible be used.

2. Plasma should not be used since anti-coagulants may interfere with the test .

3. Serum iron shows diurnal variation (5). 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 - 8ēC.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Specimens from patients receiving chelation therapy should not be used. Young et al, (6) have reviewed drug effects on serum iron.

PROCEDURE

MATERIALS PROVIDED:

IRON COLOR REAGENT (Cat. No. 3201-1), IRON BLANK REAGENT (Cat. No. 3202-1) and IRON STANDARD (Cat. No. 3203-1).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.1 mL micropipettor

2. 1.0 mL pipettor or dispensor

3. Test tubes and rack

REACTION CONDITIONS:

Wavelength 595 nm

Filter Selection nm

Reaction Type Endpoint

Reaction Time 5 minutes

Reaction Temperature 15 - 30° C

Sample Volume 0.1 mL

Reagent Volume 1.0 mL

Total Volume 1.1 mL

Low Normal mg/dL

High Normal mg/dL

Calibrator Value mg/dL

NOTE: A sample volume of 0.1 - 0.25 mL reagent may be used in this method without achange in reaction condition. It is recommended that the highest sample volume feasible be used in order to obtain maximum sensitivity. For greatest accuracy, all samples should have a serum blank as described in PERFORMANCE OF TEST below. Glassware and pipettors should be made iron-free by acid wash.

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

PIPETTING SCHEME

SAMPLE TEST BLANK 1.0 mL Color Reagent

1.0 mL Blank Reagen in each tube in each tube

Reagent Blank 0.1 mL D-H2O 0.1 mL D-H2O

Standard 0.1 mL Standard 0.1 mL Standard

Control 0.1 mL Control 0.1 mL Control

Unknown 0.1 mL Unknown 0.1 mL Unknown

MANUAL PROCEDURE:

1. Dispense 1.0 mL IRON COLOR REAGENT into a series of tubes labeled Reagent Blank, Standard, Control, Unknown 1, etc. These are for the test samples.

2. Dispense 1.0 mL IRON BLANK REAGENT into a second series of tubes labeled Reagent Blank, Standard, Control, Unknown 1, etc. These are for the serum blank.

3. Add 0.1 mL of each sample to its respective COLOR REAGENT and BLANK REAGENT tubes and mix well. Follow the PIPETTING SCHEME shown above.

4. Allow to stand at room temperature for 5 minutes.

5. Zero instrument at 595 nm using the Reagent Blank for the test samples from

STEP 1.

6. Read and record absorbances for Standard, Control and Unknowns from STEP 1. These are the Test Absorbances.

7. Zero instrument at 595 nm using the Reagent Blank for the test samples from

STEP 2.

6. Read and record absorbances for Standard, Control and Unknowns from STEP 1. These are the Blank Absorbances.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 60 minutes after color development.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0 - 120 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use IRON STANDARD (Cat. No. 3203-1) which is provided in the reagent set for this purpose.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for Unknowns. EAGLE DIAGNOSTICS offers 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.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentrations:

EXAMPLE:

A 500 ug/dL (89.5 uM) Standard had Abs. = 0.330 and Serum Blank Abs. = 0.002, while the Unknown Test Abs. = 0.093 and Unknown Serum Blank Abs. = 0.008. The Iron concentration of the Unknown is:

0.093 - 0.008  X 500 ug/dL (or 89.5 uM)

----------------------------------------------------- = 130 ug/dL (or 23.2 uM)

0.330 - 0.002

LIMITATIONS

1. Copper present in serum can cause a positive interference in the assay by forming a colored complex with ferene. Ordinarily this interference is not significant unless serum iron is depressed and the copper level is quite excessive (3).

2. Ingestion of oral contraceptives reportedly elevates serum iron (7).

EXPECTED VALUES

Males 60 - 160 ug/dL (10.7 - 28.6 uM)

Females 43 - 140 ug/dL ( 7.2 - 25.1 uM)

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.

PRECISION:

Normal and abnormal control sera were assayed 20 times each for within run precision and for 10 working days to establish run to run precision.

Within Run

MEAN / ST. DEV. / %CV

Normal 110 / 1.4 / 1.3

Run to Run

Normal 110 / 2.6 / 2.3

Abnormal 227 / 4.9 / 2.2

SPECIFICITY:

A comparison of this IRON PROCEDURE with another widely used commercial method showed a 99% correlation with samples in the normal and abnormal range.

SENSITIVITY:

This procedure has a sensitivity of 1.5 Ug/dL per 0.001 absorbance unit.

REFERENCES

1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd Ed., W.B. Saunders Co., Philadelphia, 1982, p. 921.

2. Higgins, T., Clin. Chem. 27, 1619 (1981).

3. Artiss, J.D., Vinogradov, S. and Zak, B., Clin. Biochem. 14, 311 (1981).

4. David, R.M., and Shjihabi, Z.K., J. Med. Tech. (1985), in press.

5. Statland, B.E., Winkle, P., and Bokelume, H., Clin. Biochem. 9, 26 (1976).

6. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. Vol. 21, 321D (1975).

7. Tatro, D.S., Hosp. Form. Man. 6, 14 (1971).