Unsaturated iron binding capacity

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NameUnsaturated iron binding capacity
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This procedure is valid for the following chemistry analyzers:

  • AU400/AU400e

  • AU640/AU640e

  • AU480

  • AU680

  • AU600

  • AU2700

  • AU5400

  • AU5800

Prepared By

Date Adopted

Supersedes Procedure #

Review Date

Revision Date


Distributed to

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Iron (non-heme) measurements are used in the diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated with widespread deposit in the tissues of two iron-containing pigments, hemosiderin and hemofuscin, and characterized by pigmentation of the skin), and chronic renal disease. Transferrin is the major iron carrying protein in the serum.

Serum iron concentration connotes the Fe (III) bound to serum transferrin and does not include the iron contained in serum as free hemoglobin. Because normally only about one third of the iron binding sites of transferrin are occupied by Fe (III), serum transferrin has considerable reserve Iron Binding Capacity. This is called the serum Unsaturated Iron Binding Capacity (UIBC).1


System reagent for the quantitative determination of Unsaturated Iron Binding Capacity (UIBC) in human serum or plasma on Beckman Coulter AU Clinical Chemistry analyzers.


In the Beckman Coulter AU System UIBC procedure, ferrous-Iron (Fe2+) at alkaline pH, added to serum, binds specifically with transferrin at unsaturated iron binding sites. Remaining unbound ferrous iron reacts with the Nitroso-PSAP [2-Nitroso-5-(N-propyl-N-sulfopropylamino)phenol] to form an intense green complex. The difference between the resulting change in the measured absorbance and the absorbance from the total amount added to serum is equivalent to the quantity bound to transferrin. This is the Unsaturated Iron Binding Capacity (UIBC).


Patient Preparation:

Samples should be taken in the morning from patients in a fasting state, since iron values can decrease by 30% during the course of the day.2

Additional instructions for patient preparation as designated by this laboratory:


Serum or heparinized plasma samples, free from hemolysis, are the recommended specimens. Remove serum from the red cells to minimize hemolysis. Plasma specimens collected with EDTA, oxalate, or citrate are not recommended, since they bind iron, preventing its reaction with the chromogen.2

Additional type conditions as designated by this laboratory:

Handling Conditions:

Serum iron is stable for 7 days at 2 - 8C or 4 days at room temperature (15 - 25C) after the serum is separated from red cells.2

Additional handling conditions as designated by this laboratory:



Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, AU5400 and AU5800 analyzers.


Beckman Coulter AU System Unsaturated Iron Binding Capacity Reagent

Final concentration of reactive ingredients:

Tris buffer pH 8.1 (20ºC)

180 mmol/L


6.9 µmol/L


400 µmol/L

Also contains preservatives.

Reagent storage location in this laboratory:

Test tubes 12 -16 mm in diameter or sample cups (Cat No. AU1063).

Storage location of test tubes or sample cups in this laboratory:

Beckman Coulter Chemistry Calibrator (Cat. No. DR0070)

Storage location of the calibrator in this laboratory:


  1. For in vitro diagnostic use.

  2. Do not ingest. Harmful if swallowed.

  3. R1a contains hydroxylamine, which may cause sensitization by skin contact.

  4. Contains sodium azide as a preservative that may react with lead joints in copper plumbing to form explosive compounds. Even though the reagent contains minute quantities of sodium azide, drains should be well flushed with water when discarding the reagent.

  5. WARNING: POTENTIAL BIOHAZARDOUS MATERIAL. The calibrator is manufactured from human serum. No test method can offer complete assurance that HIV- 1/2, HCV, Hepatitis B, or other infectious agents are absent from biological materials, all calibrator material should be handled at the Biosafety Level 2 as recommended for any infectious human serum or blood specimen in the CDC/National Institutes of Health manual, Biosafety in Microbiological and Biomedical Laboratories, 1993.


1. UIBC R1: Pour an aliquot of R1 buffer into R1a bottle and mix gently by inversion. Pour entire contents of R1a into R1 bottle and mix gently by inversion. Repeat this process once more.

  1. UIBC R2: Pour an aliquot of R2 buffer into R2a bottle and mix gently by inversion. Pour entire contents or R2a into R2 bottle and mix gently by inversion. Repeat this process once more.

Note: It is important that the entire contents of the R2a color reagent be transferred to R2 and thoroughly mixed. Failure to do so may result in high bottle to bottle imprecision of control and patient recovery.

The Beckman Coulter Chemistry Calibrator reconstitution:

  • Remove the vials of calibrator and diluent from storage and let stand at room temperature (15-25C) for 5 minutes.

  • Remove the cap and stopper from the vials of the lyophilized serum and reconstituting diluent.

  • Using a volumetric pipette or a calibrated air-displacement pipettor, add exactly 5.0 mL of reconstituting diluent to DR0070 lyophilized serum vial. DO NOT pour directly from the reconstituting diluent vial.

  • Replace the cap and stopper to the vial of lyophilized serum immediately after adding the diluent

  • Allow the calibrator to stand for 5-10 minutes. Gently swirl the contents until completely dissolved.

Storage Requirements:

1. The unopened reagents are stable until the expiration date printed on the label when stored at 2 - 8°C.

2. Working reagents are stable for 30 days when stored in the refrigerated compartment of the analyzer.

  1. Contamination after opening must be avoided.

  2. Un-reconstituted calibrator and diluent are stable until the expiration date stated on the label when stored at 2 - 8°C.

  3. For Unsaturated Iron Binding Capacity, reconstituted calibrator materials are stable for 7 days from the date of reconstitution when stored at 2 - 8°C. The materials should be capped and stored upright 2 - 8°C when not in use.

Indications of Deterioration:

Discoloration of the reagent, visible signs of microbial growth, turbidity or precipitation in reagent may indicate degradation and warrant discontinuance of use.

Additional storage requirements as designated by this laboratory:


The following data was obtained using this UIBC Reagent on Beckman Coulter AU analyzers according to established procedures. Results obtained at individual facilities may differ.


Estimates of precision, based on CLSI recommendations6, are consistent with typical performance. The within run precision is less than 8%CV and total precision is less than 10%CV. Assays of serum pools and control sera were performed and the data reduced following CLSI guidelines.


Within run


Mean, g/dL




















Method Comparison:7

Patient samples were used to compare this UIBC Reagent. Representative performance data on AU analyzers is shown in the next table.

Y Method

AU640 / AU640e

X Method

Method 2





Correlation Coeff. (r)


No. of Samples (n)


Range (µg/dL)



The typical change in absorbance for 1 µg/dL of UIBC is 0.32 mAbsorbance.


Standard Preparation:

Perform a one-point calibration (AB) using a water blank (blue rack) and the appropriate calibrator in a yellow calibration rack. The frequency of calibration for the Unsaturated Iron Binding Capacity procedure is every 14 days. Calibration of this Unsaturated Iron Binding Capacity procedure is accomplished by use of Beckman Chemistry Calibrator (Cat No. DR0070).

Calibration Procedure:

Recalibration of this test is required when any of these conditions exist:

  1. A reagent lot number has changed or there is an observed shift in control values.

  2. Major preventative maintenance was performed on the analyzer.

3. A critical part was replaced.


During operation of the Beckman Coulter AU analyzer at least two levels of an appropriate quality control material should be tested a minimum of once a day. In addition, controls should be performed after calibration, with each new lot of reagents, and after specific maintenance or troubleshooting steps described in the appropriate AU User’s Guide. Quality control testing should be performed in accordance with regulatory requirements and each laboratory’s standard procedure.

Location of controls used at this laboratory.


A complete list of test parameters and operating procedures can be found in the appropriate User’s Guide and at www.beckmancoulter.com.


For SI units (umol/L), multiply the results by 0.1786.


Reference Ranges:

Adults5: 155 - 355 µg/dL

Expected values may vary with age, sex, diet and geographical location. Each laboratory should determine its own expected values as dictated by good laboratory practice.

Expected reference ranges in this laboratory:

Procedures for Abnormal Results:

Abnormal results are flagged by the listed analyzers according to the normal values entered by the user into the instrument parameters.

Reporting Format:

Results are automatically printed for each sample in mg/dL at 37°C.

Additional reporting information as designated by this laboratory:


The Beckman Coulter AU System Unsaturated Iron Binding Capacity procedure is linear from 55 - 450 µg/dL. Samples exceeding the upper limit of linearity should be diluted and repeated. The sample may be diluted, repeated and multiplied by the dilution factor automatically utilizing the AUTO REPEAT RUN.

Interfering Substances:

Results of studies3 show that the following substances interfere with this UIBC procedure.

The criteria for no significant interference is recovery within 10% of the initial value.


No significant interference up to 40 mg/dL Bilirubin


Interference less than 5% up to 1 mg/dL Copper


No significant interference up to 200 mg/dL Hemolysate


No significant interference up to 1000 mg/dL Intralipid*

* Intralipid, manufactured by KabiVitrium Inc., is a 20% IV fat emulsion used to emulate extremely turbid samples.

The information presented is based on results from Beckman Coulter studies and is current at the date of publication. Beckman Coulter Inc., makes no representation about the completeness or accuracy of results generated by future studies. For further information on interfering substances, refer to Young4 for a compilation of reported interferences with this test.

Laboratory specific procedure notes:


1. Burtis, C.A. and Ashwood, E.R., Tietz Textbook of Clinical Chemistry, Second Edition, W.B. Saunders Co., 1994.

2. Perrotta, G., Iron and Iron-Binding Capacity, In: Pesce, A.J., Kaplan, L.A. editors., Methods in Clinical Chemistry, C.V. Mosby, St. Louis, 1258 - 1261, 1987.

3. CLSI/NCCLS, Interference Testing in Clinical Chemistry, EP7-A2, 2005.

4. Young, D.S., Effects of Drugs on Clinical Laboratory Tests, Fifth Edition, Washington, DC, AACC Press, 2000.

5. Beckman Coulter Inc. data on samples collected from 200 blood donors in North Texas.

6. CLSI/NCCLS Evaluation Protocol EP5 - A2, 2004.

7. Data on file for specific AU analyzers.

© Beckman Coulter, Inc. March 2012 CLSIOSR6x205.02
All printed copies are considered to be copies of the electronic original. Page of

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