Chemistry Laboratory 2

Serum Iron Procedure
1. To perform the serum total iron procedure you need five tests tubes (blk, std, L1, L2 and patient) with iron buffer reagent and specified other ingredient. You mix and incubate then record the initial absorbance at 560nm. Next you add iron color reagent, mix and incubate and then record the final absorbance at 560nm. After you have the absorbances and the concentration of the standard, you can calculate the concentration of the controls and the patient’s iron.
UIBC procedure
To perform the serum unsaturated iron-binding capacity (UIBC) procedure need five tests tubes (blk, std, L1, L2 and patient) with TRIS buffer reagent and specified other ingredient. You mix and incubate then record the initial absorbance at 560nm. Next you add iron color reagent, mix and incubate and then record the final absorbance at 560nm. After you have the absorbances and the concentration of the standard, you can calculate the concentration of the controls and the patient’s excess iron. You can also calculate UIBC and then TIBC.
sources of error
iron contamination from water or glassware, incorrect interpretation of low levels as clinically significant when iron levels can fluctuate throughout the day.
The reference values for serum iron (?g/dL)
65-170 for adult males and 50-170 for adult females. The reference range decreases as age decreases, but newborns have the highest reference range at 100-250.
The reference values for serum TIBC (?g/dL)
are 250-450 for adult males and females. The reference range slight decreases as age decreases, but newborns have the lowest reference range at 100-400.
Significance of Controls
The control levels are tested along with the patient in order to insure quality control. They have a known value and reference range, which should be reported in order to release the patient’s results with confidence.
The principle of the procedure for serum total iron
based on the ferrozine complex formation. At acid pH and in the presence of a reducing agent (buffer), transferrin-bound serum iron will dissociate from transferrin and form ferrous iron (Fe2+). Ferrous iron will react with ferrozine to produce magenta colored complex. The difference in color intensity before and after ferrozine is proportional to serum iron concentration using beer’s law.
The principle of the unsaturated iron binding capacity procedure
is that at an alkaline pH, ferrous ions added to serum bind with transferring at its unsaturated iron-binding sites. The remaining unbound iron is measured using ferrozine. The difference between the amount of unbound iron and the total amount added to the serum is equivalent to the quantity bound to transferring (UIBC). The TIBC is the total iron in the sample, plus the UIBC, iron that is bound to transferrin.
To determine concentration of iron: Calculations
First figure out change in absorbance (final-initial)
Then use beer’s law to figure out concentrations

Cu = (Au/As) x Cs, measured in ug/dL

UIBC Calculations
Concentration of Standard – concentration of excess iron
This is a measurement of the quantity of iron bound to transferrin
TIBC calculations
Serum Fe + UIBC
This is a measurement of total iron bound to transferrin and the total iron in the sample
Clinical significance of Serum Iron test and UIBC test
to determine the amount of iron in transport to where it is needed in the body and to diagnose iron deficiencies or iron toxicity
Plasma Protein that transports iron
Transferrin
Two compounds which store iron are
ferritin and hemosiderin
When body iron stores are low, the duodenal mucosa cells contain ____ ferritin and _____transferrin
Less, more
What disease is likely if iron and TIBC are low
Chronic illness possible malignancy
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