1. Determination of Iron, Folic Acid and Vitamin B12

2. Serum Iron Iron is largely mediated by three proteins: transferrin, transferrin receptor and ferritin. Iron is carried in the plasma bound to the protein transferrin. This molecule binds two atoms of F +3 and delivers iron to cells by interaction with the membrane transferrine receptor. Normal range for serum iron 10-30 µmol/l Specimen: sufficient blood to provide 3.5ml serum or heparinized plasma.

3. Principle of serum iron assay Transferrin-Fe + trichloracetic acid Fe+transferrin Fe(III) + thioglycolic Fe(II) Fe(II) +bathophenanthroline Bathophenanthrolin-Fe(II) Colored serum iron (µmol/l)= A test -A blank XCst A st - A blank

4. Total iron-binding capacity 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 called the serum unsaturated iron binding capacity (UIBC) TIBC is a measurement of the maximum concentration of iron that serum proteins, particularly transferrin, can bind. It increased in iron deficiency and decreased in chronic inflammatory disorders or malignancies.

5. Excess iron as ferric chloride is added to serum. any iron which dose not bind to transferrin is removed with excess magnesium carbonate.the iron concentration of the iron- saturated serum is then measured. Normal range for TIBC= 40-75 µmol/l TIBC (µmol/l) =serum iron +UIBC

6. Serum ferritin The quantity of iron in the storage compartment is reflected in plasma ferritin concentration. Serum ferritin is a very sensitive indicator of a iron deficiency. A large number of chronic disease result in increased in serum ferritin concentration. Can measured by radioimmunoassay (RIA),ELISA kits. Normal range=40-340µg/l in males. Normal range=14-150µg/l in females.

7. Folic Acid and Vitamin B12 Two types of methods : microbiological assays and competitive protein binding assay Several commercial kits are available for the competitive protein binding assay (CPB) of vitamin B 12 and folic acid. Most provide for the simultaneous measurement of both substances in same reaction tube by using 125 I- labeled folate and 57 Co-labeled cobalamin.

8. Serum specimen are recommended for this procedure also plasma specimen collected in EDTA may also be used (from fasting individuals). Normal range of serum vitamin B 12 = 160- 925ng/l. Normal range of serum folate 3-15µg/l.In folate deficiency usually <1µg/l. Normal range of red cell folate= 160-640µg/l.

9. Schiling test Principle: the vitamin B 12 absorption test permits differentiation between pernicious anemia and intestinal malabsorption as causes of vitamin B 12 deficiency. Orally administered 57 Co or 58 Co-labeled vitamin B 12 can be measured by determining the radioactivity in faces, urine, or serum(0.5µg). In pernicious anemia <7% is excreted in urine. In normal, excrete in urine 8% or more. Also injected intramuscularly with a “flushing dose” of 1000µg unlabeled vitamin B 12.