Calculations

IRON (TOTAL) A = Absorbance

Precautions
STD = Standard
REAGENT 1. All reagents are toxic. Do not pipette by mouth. Avoid all contact. A2 Test - A1 Test x Conc. = Total Iron (ug/dL)
2. This reagent is for in vitro diagnostic use only. A2 Std - A1 Std of Std.
INTENDED USE
REAGENT STORAGE
For the in vitro quantitative determination of Iron in serum. 0 Example: A1 Test = 0.08 A2 Test = 0.15
1. Store all reagents refrigerated at 2-8 C.
A1 Std = 0.00 A2 Std = 0.40
METHODOLOGY REAGENT DETERIORATION
Iron exists In serum complexed with transferrin, a transport protein. Most 1. All reagents should be clear. Turbidity may indicate contamination Then: 0.15 - 0.08 = 0.07 = 0.175 x 500 = 87.5 ug/dL
early procedures for Iron determination Involved dissociation of the iron and the reagent should not be used. 0.40 - 0.00 0.40
from the Iron- protein complex, precipitation of the proteins, and then
CALIBRATION
measurement of the iron content of the protein free filtrate. SPECIMEN COLLECTION AND STORAGE
The procedure is calibrated with an aqueous iron standard (500ug/dL) or
1. Fresh, unhemolyzed serum Is the specimen of choice.
an appropriate serum calibrator.
Many chromagens have been used in the determination Including 2. Serum should be separated as soon as clot has formed.
QUALITY CONTROL
thiocyanate o-phenanthroline, bathophenanthroline and TPTZ. In 1971, 3. Heparinized plasma may be used but other anticoagulants should
4 The integrity of the reaction should be monitored by use
Persijn et al1 presented a method using the chromagen ferrozine, not be used to avoid possible Iron contamination.
2 of a two level control with known Iron values.
described by Stookey . This method did not require protein precipitation 4. Serum Iron Is reported to be stable for four days at room temperature
EXPECTED VALUES
and was more sensitive than previous methods. The present procedure (15-30°C) and seven days at 2-8°C. Iron (Total) 60 - l50 g/dL
is a modification of the Persijn method. It is strongly recommended that each laboratory
INTERFERENCES Determine the normal range for its particular population.
1. Certain drugs and other substances are known to Influence PERFORMANCE
Principle:
circulating Iron levels. See Young, et al 5. Linearity:
Serum Iron: Transferrin-bound Iron is released at an acid pH and
2. Iron contained In hemoglobin does not react in this method, When run as recommended the assay is linear to
reduced from ferric to ferrous ions. These ions react with ferrozine to 500 ug/dL.
therefore, slight hemolysis will not interfere. However, gross Samples with values above 500 ug/dL must be diluted 1:1 with normal
form a violet colored complex which Is measured spectrophotornetrically
hemolysis (pink or red specimens) will contribute to the absorbance saline, re-assayed and result multiplied by two.
at 560nm. The absorbance measured at this wavelength Is proportional
measured at the wavelength used and should be avoided. 3 Method Comparison:
to serum iron concentration. Studies performed between this procedure and a similar Serum Iron
3. To make tubes, pipettes, etc. Iron free, they must be washed with hot
procedure yielded the following results:
dilute (1:2) hydrochloric or nitric acid, followed by several rinsing with Correlation Coefficient: 0.983
CLINICAL SIGNIFICANCE3
Iron-free deionized or distilled water. Slope: 0.79
In most cases, both serum Iron and TIBC values are necessary for
Intercept: (g/dL) 0.13 (g/dL)
greatest diagnostic significance. Low serum iron values are seen In
ADDITIONAL EQUIPMENT REQUIRED BUT NOT Precision:
chronic blood loss, insufficient intake or absorption of Iron, and increased
PROVIDED Within Run Level 1 Level 2 Level 3
demand on the body stores (e.g. pregnancy). Elevated serum iron values
1. A clinical chemistry analyzer capable maintaining constant Mean (g/dL) 110 184 251
are seen In increased red cell destruction, decreased red cell synthesis, S.D. (g/dL) 3.0 3.1 3.3
temperature (37°C), and measuring absorbance at 560nm.
Increased Iron intake, or increased Iron stores release. C.V. (%) 2.7 1.7 1.3
2. Iron-free deionized water and related equipment, e.g.: pipettes
3. Analyzer specific consumables, e.g.: sample cups Run to Run
Increase in the TIBC may be due to increased production of
4. Control, and Calibrator materials such as those provided by JAS Mean (g/dL) 109 177 249
apotransferrin (e.g. chronic Iron deficiency) or an increased release of S.D. (g/dL) 5.1 3.8 4.5
ferritin, as In hepatocellular necrosis. Diagnostics.
C.V. (%) 4.7 2.1 1.8

ASSAY PROCEDURE REFERENCES

Decreases In the TIBC can occur with cirrhosis and hemachromatosis
due to a deficiency in ferritin, or in nephrosis due to a loss of These instructions are to be used as a general guideline for adapting to
select automated instruments. Refer to your specific JAS instrument 1. Persijn, J.P., et al. Clin Acta 35.91 (1971).
apotransferrin. application instructions available upon request. 2. Stookey,L.L.1Anat.Chem42:779(1970).
3. Tletz, N.W., Fundamentals of CIinical Chemistry
REAGENT COMPOSITION System Parameters Philadelphia, W.B. Saunders, pp. 923-929 (1976).
Iron (Total) 4. Weissman, N., Pileggi V.J., in Clinical Chemistry:
Active Ingredients Concentrations
TEMPERATURE: 37°C Principles and Technics, 2nd Ed.. RJ. Henry et al, editors,
IRON BUFFER REAGENT: Hagerstown (MD), Harper & Row, pp.692-693(1974).
WAVELENGTH: 560 nm
Hydroxylamine hydrochloride 220mM DIRECTION: Increase 5. Young, [Link] al, Clin Chem. 21:ID(1975).
in acetate buffer, SAMPLE / RGT RATIO: 1:5 6. Henry, J.B., Clinical Diagnosis and Management by
e.g. Sample Vol. 0.5 mL (500L) Laboratory Methods, Philadelphia, W.B. Saunders, P. 1434 (1984)
pH 4.6 + 0.1 with surfactant.
IRON COLOR REAGENT: Reagent Vol. 2.5 mL
Chromogen Vol. 0.05 mL (50L) JAS Diagnostics Inc.
Ferrozlne 16.7mM REACTION TIME: 10 Min
In hydroxylamine hydrochloride.
PI: IRO2.04 REV: 10/19/09