UNIT: Heme Pigments (continued)
MLAB 2401 - Clinical Chemistry Lab Manual
– At acid pH, and in the presence of suitable reducing agent, transferrin-bound serumiron dissociates to form ferrous ions. These react with ferrozine to produce a magenta coloredcomplex with an absorption maximum near 560 nm. The difference in color intensity at thiswavelength, before and after addition of ferrozine, is proportional to serum iron concentration.
– At alkaline pH, ferrous ions added to serum bind specifically with transferrin at unsaturatediron-binding sites. Remaining unbound ferrous ions are measured with the ferrozine reaction. Thedifference between the amount of unbound iron and the total amount added to serum is equivalentto the quantity bound to transferrin. This is the UIBC.
– The serum TIBC equals the total iron plus the UIBC.SpecimenBlood should be collected using materials that are iron-free. As soon as blood clots, separate serum. Although occult hemoglobin does not interfere, only clear non-hemolyzed serum (not plasma) issuitable for assay. Each mg of hemoglobin contains 3.4
g iron. Serum iron reportedly is stable for at least four days stored at room temperature or one week in the refrigerator.Procedure ISerum Total Iron1.To test tubes labeled Blank, Standard, Test, and add 2.5 mL Iron Buffer Reagent.2.a.To Blank, add 0.5 mL iron-free water.b.To Standard, add 0.5 mL Iron Standard.c.To Test, add 0.5 mL serum.Mix each test tube thoroughly.3.a.After setting the spectrophotometer wavelength to 560 nm, zero the instrument using theBlank (for double beam spectrophotometers, read Standard and Tests against the Blank).b.Read and record absorbance (A) of Standard and Tests. This is the Initial A.4.To each test tube add 0.05 mL Iron Color Reagent. Mix thoroughly and place in water bath at37°C for 10 minutes.5.Read and record absorbance of Test and Standard vs. Blank as reference at 560 nm. This isthe Final A.6.CalculationsSerum Total Iron (