An Immunoradiometric Procedure

For The Quantitative Analysis of Serum Ferritin.

SUMMARY

The serum ferritin concentration is proportional to the amount of iron in stores in the human body. The measurement of serum ferritin enables the clinician to differentiate between the anemia caused by iron deficiency and other forms of anemia^1,2,3. Serum ferritin concentration is a useful, noninvasive screening test for iron overload, which may allow the detection of idiopathic hemochromatosis in the precirrhotic stages⁴.

TEST PRINCIPLE

This is a one stage, 2-site immunoradiometric (sandwich) assay. Antibody to ferritin is coated on the surface of the plastic beads. Ferritin present in sera or calibrators binds to the antibody coated beads. Radiolabeled antibody in turn binds to the ferritin on the solid phase antibody (forming a "sandwich"). The solid phase is washed and counted in a gamma counter. The amount of radiolabel bound to the solid phase is directly proportional to the concentration of ferritin present in the sera or calibrator solutions.

REAGENTS

Prediluted Ferritin Calibrator Solutions:    6 vials containing 0.3ml of human spleen ferritin diluted to concentrations of 6, 20, 60, 200, 600, and 2000 ng/ml in borate buffer containing bovine serum albumin, rabbit serum, sodium chloride, and inert coloring agents with sodium azide as a preservative.  Store at 2 - 8C.   Do Not Use After Expiration Date.

Note: The ferritin concentration of the base stock of cadmium crystallized human spleen ferritin from which these calibrators were derived was established by a protein determination. As such, these calibrators have more immunologic reactivity than WHO calibrators. Values for the Control Sera and for the Patient Samples will be approximately 25.4% lower than those observed using WHO calibrators.

Solid Phase Antihuman Ferritin:   128 plastic beads (Beads) coated with rabbit antihuman spleen ferritin and stored in a bottle containing borate buffer with bovine serum albumin, rabbit serum, and sodium chloride with sodium azide as a preservative.   Store at 2 - 8C.   Do Not Use After Expiration Date.

Sample Diluting Buffer (Blue Solution):   1 bottle containing 30 ml of borate buffer with bovine serum albumin, rabbit serum, sodium chloride, ethylenediaminetetra-acetate, and inert coloring agents with sodium azide as a preservative.   Store at 2 - 8C.   Do Not Use After Expiration Date on Bottle.

Radiolabeled (¹²⁵I) Antihuman Ferritin (Red Solution):   1 bottle containing 27 ml of radiolabeled (¹²⁵I) rabbit antihuman spleen ferritin dissolved in borate buffer with bovine serum albumin, rabbit serum, sodium chloride, ethylenediaminetetra-acetate, inert coloring agents and sodium azide as a preservative. This kit contains less than 10 uCi (370 kBq) of radioactivity.   Store at 2 - 8C.   Do Not Use After Expiration Date on Bottle.:  

Reaction Trays and Grippers:    4 Reaction Trays containing 32 Reaction Wells each plus 128 bead Grippers.

Graph Paper:    3 sheets of Standard Plot logit-log graph paper.

PRECAUTIONS

WARNING - RADIOACTIVE MATERIAL

1. Do not pipette radioactive material by mouth.

2. Use only in a designated area.

3. Store in the original container in a designated area.

4. Clean up spills with paper towels and dispose of in accordance with approved regulations.

5. Used radioactive solutions and solids should be disposed in accordance with approved regulations (see 10 CRF part 20).

 

WARNING -   The human ferritin in the calibrator solutions has been tested and found to be nonreactive for Hepatitis B Antigen by RIA, and antibody to HTLV III by ELISA. The calibrator solutions as well as the patient specimens and control sera should be considered potentially hazardous and handled in accordance with approved laboratory procedures and regulations.

WARNING -  Reagents in this kit contain sodium azide. Contact with copper or lead drain pipes may result in the formation of explosive azide deposits. It is important during disposal to flush drains with copious amounts of water to prevent azide accumulation. Plumbing that may be contaminated with azides can be flushed with 10 percent sodium hydroxide solution.

 

Other Precautions:

• Avoid splashing or generating aerosols.
• Follow kit recommendations for incubation times and temperatures to avoid possibly erroneous results.
• Microbial contamination of reagents may cause erroneous results.
• Do not use reagents with those from other lots or manufacturers.
• Do not use kit reagents after the expiration date.

 

SAMPLE COLLECTION AND PRESERVATION

Collect 5 ml of venous blood aseptically. Allow the blood to coagulate and separate the serum from the clot by centrifugation. Plasma may also be used for ferritin analysis. Moderate hemolysis will not interfere with the assay. If the assay will be performed within 7 days, store the serum refrigerated. If more than 7 days will elapse before the test is performed, the serum specimen should be frozen. Serum specimens may be stored frozen for 4 months without change in the ferritin content.

MATERIALS REQUIRED BUT NOT PROVIDED

• Deionized or distilled water.
• Precision pipettes capable of delivering volummes of 10 and 200 ul.
• 12 X 75 mm glass or plastic test tubes.
• Gamma counter capable of detecting ¹²⁵I.
• Clinical rotator table or vibrator table

GENERAL INFORMATION ON PROCEDURE

Serum ferritin levels up to 2000 ng/ml may be measured without prediluting serum specimens. If precision is required for higher concentrations, samples may be diluted with sample diluting buffer and reassayed. Precision techniques are necessary for accurate and reproducible results. All solutions should be dispensed directly into the bottom of the reaction tube.

ASSAY PROCEDURE

Determine the number of Solid Phase Antihuman Ferritin Beads needed for the assay and label the Reaction Trays and Grippers accordingly. Allow reagents, sera, and patient samples to reach room temperature before performing the assay.

1.   Beginning with Reaction Well A3 (skip Wells A1 and A2), pipette 10 ul of Prediluted Ferritin Calibrator Solution and sample, in duplicate, into separate Wells. Reaction Wells A1 and A2 measure non specific binding and only contain the Radiolabeled Antihuman Ferritin and Antihuman Ferritin Coated Beads.

2.   Pipette 200 ul of Radiolabeled Antihuman Ferritin (red solution) into each Well and place the tray(s) on a clinical rotator table for 5 minutes at 200 rpm to assure mixing.

3.   Add 1 Bead to each Reaction Well and place the numbered Grippers into the Reaction Wells. Push down firmly to attach the beads.

4.   Incubate on a clinical rotator (200 rpm) or vibrator table for 2 hours at room temperature. (For those requiring a more rapid assay the incubation time can be reduced to 1 hour at room temperature.)

5.   Wash Beads under running deionized water while still attached to Grippers. Shake to remove excess water.

6.   Remove each Bead or Bead plus Gripper to a counting tube.

7.   Count in a gamma counter for 1 minute.

8.   Calculate results. (See CALCULATION section below.)

NOTE:  A rotating table or vibrator table must be used for a 1 or 2 hour incubation period. Extend the incubation period to overnight (14-18 hours) at room temperature if a mixing device is not used. Steps 1 through 8 are otherwise unaltered.

 

SUMMARY OF ASSAY PROCEDURE

Procedure Flow Sheet:

**The 0 ng/ml calibrator consists of the 200 ul radiolabeled antiferritin only and is used to measure nonspecific binding  (NSB).   

After the incubation period, wash each Bead under running deionized water, and shake to remove excess fluid.

Count Beads in gamma counter for 1 minute.

CALCULATIONS

Calculate the mean total count for each calibrator and patient serum. Determine the net count by subtracting the mean count of the 0 calibrator (NSB) from each.

I.   ESTIMATION OF MAXIMUM BINDING (MB)

Maximum Binding (MB) now must be determined. Either of the following techniques are satisfactory:

 METHOD I:     Maximum Binding (MB) can be estimated using the formula:

                     MB = C₂(2C₁C₃ - C₁C₂ -C₂C₃) /[(C₁C₃) -(C₂)²]

               Where:    C₁ = net counts of 20 ng calibrators

C₂ = net counts of 200 ng calibrators

C₃ = net counts of 2000 ng calibrators

 

              Example using Example Data Table:

                    MB = [30493 (2(3736 × 76797)-(3736 × 30493)-(30493 × 76797))] / [(3736 × 76797) -(30493)²]

                    MB = -5.738374 × 10¹³ / -6.429095 × 10⁸ = 89256

METHOD II:   The net counts of the 2000 ng/ml calibrator multiplied by the factor on the quality control sheet enclosed in the kit closely approximates MB.

 

Example:    76797 × 1.15 = 88317

 

II.   CALCULATION OF PERCENT BOUND (PB)

Once (MB) has been derived, the Percent Bound (PB) must be determined for each calibrator and unknown.

PB = 100 x Net counts for each cal. or unknown / MB

 

Example for Normal Serum from Example Data Table:

PB = 100 x 19685/89256 = 22%

 

III.   CONSTRUCTION OF THE CALIBRATION CURVE

Construct a best fit line on the Logit-Log paper supplied with the kit, plotting the Percent Bound of each calibrator versus its ferritin concentration in ng/ml. Read the concentration of each unknown directly from the graph of the calibrator response curve.

IV.   OTHER METHODS OF DATA REDUCTION

Other methods of data reduction may be used to analyze the 2-site IRMA assay of ferritin. These include the plots of:

1. Counts bound (B) versus the log of the ferritin concentration.

2. Counts per minute versus ferritin concentration on a semi-log graph. It has been demonstrated that the data conforms closely to a Scatchard plot.

3. Percent Bound:

Bound / Total × 100 versus the log of the ferritin concentration

4. Automated Methods: Available on most multi-sample gamma counters. Refer to the instrument's operating manual for details.

Alternatively, the serum concentration of ferritin can be calculated from a regression equation. If you desire to use this form of calculation, call Ramco Laboratories at 800-231-6238 or 281-313-1250.

EXAMPLE DATA

(For Demonstration Purposes Only)

COUNT TIME = 1 MINUTE

ESTIMATED MAXIMUM BINDING (MB) = 89256 (88317)

*PERCENT BOUND (PB) was estimated using Method I calculation for MB, the results in parenthesis were estimated using Method II calculation for MB.

**0 ng/ml calibrator measures nonspecific binding (NSB).

Concentrations for normal and iron deficient serum were determined using the Example data Logit-Log Graph.

LIMITATIONS OF THE PROCEDURE

Moderate hemolysis has no effect upon the reproducibility or accuracy of the procedure. No drugs or other administered substances have been found to produce any effect on tests. Anticoagulants have not been shown to influence the test so long as they do not result in dilution of the plasma. Strict adherence to precise laboratory procedure is essential for maximum accuracy of the final results.

EXPECTED VALUES

Normal values are age and sex dependent. Serum ferritin concentrations greater than 300 ng/ml may indicate increased iron stores as seen in idiopathic hemochromatosis.

The serum ferritin concentration reflects the amount of iron in stores. In iron deficiency, the stores are gone and the serum ferritin is very low (less than 20 ng/ml). In other kinds of anemia iron stores are higher than normal and the serum ferritin values are usually over 100 ng/ml. Values between 20 and 100 ng/ml in anemic patients may suggest a combination of iron deficiency with some cause of anemia. Serum ferritin concentrations greater than 300 ng/ml are elevated and may indicate increased iron stores as seen in idiopathic hemochromatosis.

PERFORMANCE CHARACTERISTICS

Intra-Assay Precision:

Inter-Assay Precision:

SENSITIVITY

Sensitivity is defined as the smallest value of ferritin which can be distinguished from the zero standard with a 95% confidence limit (+/- two standard deviations). Using the 10 ul sample size specified in the assay procedures, the smallest concentration of ferritin that can be distinguished from zero is 0.23 ng/ml.

 This Kit Contains Ramco Calibrators

The concentrations of the Ramco Calibrators contained in this kit was established by a protein determination of the base stock of cadmium crystallized human spleen ferritin. As such, these Calibrators have more immunologic reactivity than WHO calibrators. Values for the Control Sera and for the Patient Samples will be 13.0% lower than those observed using WHO Calibrators (3rd International Standards for Ferritin, Recombinant NIBSC code: 94/572; National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK).

To convert the values for the Control Sera and Patient Samples to equivalent WHO values, multiply the values obtained using these Ramco Calibrators by 1.34.

BIBLIOGRAPHY

1. Addison, G.M., Beamish, M.R., Hales, C.N. et al: An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J. Clin. Pathol 25:326, 1972.

2. Jacobs, A., Miller, F., Worwood, M., Beamish, M.R., and Wardrop, C.A.: Ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. British Medical Journal 4:206, 1972.

3. Lipschitz, D.A., Cook, J.D., and Finch, C.A.: A clinical evaluation of serum ferritin as an index of iron stores. N. England J Med. 290:1213, 1974.

4. Halliday, J.W., Cowlishaw, J.L., Russo, A.M., and Powell, L.W.: Serum ferritin in the diagnosis of haemochromotosis. Lancet, 621 September, 1977.

5. Miles, L.E.M., Lipschitz, D.A., Bieber, C.P., and Cook, J.D.: Measurement of serum ferritin by a 2_site immunoradiometric assay. Anal. Chem. 61:209, 1974.

6. Alfrey, C.P.: Serum Ferritin Assay. CRC Critical Reviews in Laboratory Sciences, 179, November 1978.

7. Seiler, M., Alfrey, C., and Whitley, C.: Differentiation of iron deficiency from anemia of chronic disorders: The use of serum ferritin assay. Nuc Compact 9:160, 1978.

8. Li, P.K., Humbert, J.R., and Cheng, C.: Evaluation of commercially obtainable ferritin test kit in relation to the high dose parabolic phenomenon. Clin. Chem. 24:1650, 1978.

Date Issued: February 2, 1987

Revised: August 10, 2001; May 26, 2005;