Sorry about my blogging absence; it’s my birthday today so things have been a little hectic! This is a little late, but it’s all about our favourite trace element. 

Iron Overview: There’s a lot of little nitty gritty details when dealing with iron, but in as few words as possible: Iron is needed to form hemoglobin, myoglobin, and for certain enzymes to function (eg, cytochromes). Any iron not in use is put into the storage pool. Body iron sits at about 2-4g, most of which is incorporated into hemoglobin. We take in 10-15mg a day, and about 5-10% is actually absorbed, mostly in the duodenum.

Unionized iron (Fe) is an insoluble metal that is neither useful nor healthy. At physiological pH, iron is in its ferric form (Fe3+), but the useful form is Ferrous (Fe2+). Ferrous iron is also toxic. The two ionic forms of iron are always interconverting and generating free radicals so they have to be protein bound:

Transferrin: plasma iron transport protein, though only about 1/3 of the total binding sites are ever occupied. It is apoferritin bound to two ferric irons and a bicarbonate.

Ferritin: The main storage form of iron. Apoferritin and a ferric iron make ferritin, a water soluble protein that is small and diffuse through macrophages and red cell precursors. You can’t see it in stains, but serum ferritin levels are a good indicator of the status of a person’s iron stores.

Hemosiderin: A more concentrated storage form derived from ferritin. This one is nonsoluble and is visualized with the Perl’s Prussian Blue stain (discussed later)

Pappenheimer bodies: Hemosiderin visualized with routine staining. They are precipitated hemosiderin +RNA, and it is the RNA that you’re actually seeing. These siderocytes are considered sideroblasts if the red cells are still nucleated.

Testing Serum Ferritin (Fer): Measures, unsurprisingly, ferritin levels in serum. As a protein, the best way to measure Fer is using immunoassay, whether it is enzyme linked, radioimmunoassay, or radioimmunometric. The test is done using test kits on a big analyzer, but the general gist is the same as all other immunoassays: you have an antibody to the protein and, when it binds with Fer, the resulting reaction can be measured and a quantity can be calculated. I won’t get into the details of it in this post since methods are numerous and also highly varied.

Serum Iron: Serum iron is the Fe3+ that is bound to transferrin and does not include iron that is incorporated into hemoglobin. It is mostly a measure of how much iron tissues have access to.  A serum iron by itself doesn’t tell you very much, and adding a transferrin saturation will provide a lot more clinical information.  The value is highly variable and moves with transferrin levels. In testing, iron freed from transferrin using trichloroacetic acid. The Fe3+ that is released is converted into Fe2+ by acidifying the solution. Fe2+ combines with the chromogen Ferrozine to produce a color, and the intensity of the colour is proportional to the amount of iron.

Total Iron Binding Capacity (TIBC) and Unsaturated Iron Binding Capacity (UIBC): For TIBC, all binding sites on Transferrin are saturated with ammonium ferric citrate, and any excess iron is removed with magnesium carbonate. The iron assay is repeated on the now saturated transferrin.
UIBC is equal to TIBC - Serum iron
Transferrin saturation can also be calculated: Serum Iron divided by TIBC.

Perl’s Prussian Blue: A very pretty stain, usually done on bone marrow smears but can be done on other tissue, particularly liver sections (below), and on peripheral blood when looking for siderocytes. HCl is added to split iron from hemosiderin, allowing the iron to combine with ferrocyanide to create ferri-ferrocyanide (Prussian Blue), which is a bright blue complex. The slide can be counterstained with safranin, which gives you the pinky-red background.

Interpretation: I won’t get into the disease processes and stuff behind all of these results today since this post is already hideously long already and iron is very complex for a trace mineral. But here’s a general table of how to interpret results, bearing in mind that there are always other tests as part of the panel:

As for the Prussian Blue reaction, it is interpreted by the pathologist, but is a semi quantitative interpretation. Generally, in the case of bone marrow, if you can see blue spots macroscopically, it is increased, if you can only see them microscopically, it is normal, and if blue is scant or absent, it is considered decreased.

Marrow stores will be increased in megaloblastic anemia, hemolytic anemias, siderosis, sideroblastic anemia, and anemia of chronic inflammation. On the flipside, stores are decreased in iron deficiency anemia, and polycythemia vera.