Purpose

For the evaluation of body iron status.

 Deficiency
 overload
 Test sample

Either serum or heparinized blood is suitable for

the test.
EDTA, oxalate or citrate anticoagulants are not
recommended for the test.
Avoid haemolysis during the test and separate
from red cells as soon as possible (particularly if
reticulocyte count is high).
Note: stop any iron therapy or iron chelating
therapy at least 12 h before the test.
 Preparation of glassware

 It is essential to avoid contamination by iron.

 If possible, use disposable plastic tubes and
bottles.
 If glassware is to be used:
wash in detergent solution.
Soak in 2 mol/l HCl for 12 h.
Finally, rinse in iron-free water.
 Serum iron

A mixed acid reagent is used to:

1. Release the transferrin-bound iron.
2. Reduce the ferric to ferrous.
3. Precipitate the serum proteins.
 After centrifugation, the ferrous in the
remaining supernatant is colour developed
using chromagen solution (e.g. buffered
bathophenanthroline sulphonate or
ferrozine in in 1.5 mol/l sodium acetate) and
the resulting solution is read
spectrophotometrically.
Normal range:
adults:11-18 µmol/l
higher in neonates
lower in children
 Serum TIBC
Sufficient iron is added to saturate the
serum TIBC, any excess is removed by
adsorption using solid magnesium
carbonate.
After centrifugation the supernatant is
assayed for iron.
Normal range: 47-70 µmol/l.
 %Calculation of transferrin saturation

Transferrin saturation% = serum iron x100%

TIBC
Normal range: 16-60%.
 Serum ferritin

Serum or plasma ferritin is measured by

immunoassays:
1. Non competitive (sandwich) assays:
 RIA
 EIA
2. Competitive assays:
 RIA
Normal range: 15-300 µg/l
Note: ferritin is acute phase reactant.
 Other tests

Serum transferrin
Soluble transferrin receptors (sTfR)
 both they usually measured by immuno-
assays.
 Uses:
This method demonstrates iron granules in
marrow macrophages, erythroblasts (sidroblast)
and in erythrocytes (sidrocytes).
Principle:
Ionized iron react with acid ferrocyanide to give a
blue color.
Test sample:
Air-dried marrow or peripheral blood films.
Equipment:
Coplin jars
 Reagents

Potassium ferrocyanide 20 g/l…..A

HCl 19:64 ml concentrated added to water and
made to 1 litre………………..B
Methyl acohol
Safranin- aqueous 1 g/l.
Standards:
B.M films known to contain iron.
 Method

Fixation: 20 min in methyl alcohol.

Mix equal volume of A & B in the coplin jar
and immerse slides for 10 min at RT.
Wash in running tap water for 20 min.
Rinse thoroughly in D.W.
Counterstain with safranin for 5-10 sec.
Examine using X100
 Result

Iron appear as greenish colour.