Comparison Of Ferritin Levels In Serum And Plasma Biology Essay

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Ferritin is the major iron storage protein in the body, found in all body tissues, but in large amounts in the liver, spleen and bone marrow. The aim of the study was to compare Ferritin results from blood into EDTA and SPS samples, to determine whether either sample bottle could be used to give an accurate Ferritin reading, which would be beneficial to the patient and to the testing laboratory.

For the evaluation of iron stores in serum and plasma ferritin, this study analysedand compareda hundred random blood samples to measure the plasma and serum ferritin levels.

Serum ferritin was measured from clotted samples(SPS) and plasma ferritin was measured from Ethylenediaminetetraacetic acid (EDTA) samples. Measurement was important for the accurate determination of the spread of iron deficiency anaemia combined with systemic diseases, for the changes in plasma and serum ferritin with diet and for the recognition of iron overload and its evaluation.


Ferritin is a protein of a molecular weight (480,000), a spherical apo-protein shell, enclosing a core of hydroxyphosphate with up to 4000 iron atoms(A Jacobs 1975). Human ferritin is made up from 24 subunits, of two immunologically distinct types; H and L. there are multiple gene copies on 12 different chromosomes. The coding loci are located at 11q12-q13 for the heavy chain and 19q13.3-q13.4 for the light chain. An introneless gene on chromosome 5(q23.1), codes for mitochondrial ferritin(A V Hoffbrand 2011).Ferritin that present in serum normally contains a small amount of iron and consist of L subunits(A V Hoffbrand 2011).

Ferritin test is requested to check the iron level in the body, and is usually done along side with iron and total iron binding capacity (TIBC), unbound iron binding capacity (UIBC) or transferrin saturation(AACC 2009).

Ferritin test is requested;

If a full blood count (FBC) indicates low haemoglobin (Hb) or low haematocrit.

When red blood cells (RBCs)are microcytic and hypochromic, due to iron deficiency anaemia.

When there is a suspicion of iron overload as in haemochromatosis (a disease in which too much iron is absorbed from diet)(AACC 2009).

Iron absorption

The amount of iron in the body depends on the blood volume and haemoglobin concentration(A V Hoffbrand 2011). The newborn contains about 80mg/kg at full term. Neonatal iron reserves are utilized for growth as from 6 months to two years old virtually no iron stores are present(A V Hoffbrand 2011). Thereafter iron stores gradually accumulate during childhood to around 5mg/kg. Iron absorption doesn't depend only on the iron in diet but also on bioavailability of that iron as well as the body's need for that iron(GO Walters 1973).

Iron is released from protein complexes by acid and proteolytic enzymes in the stomach and small intestines and haem is liberated from myoglobin and haemoglobin(A Jacobs 1975). Duodenum and less likely jejunum absorb iron by the help of acidic pH, vitamin C and other molecular-weight chelates, therapeutic ferrous iron salts are well absorbed on an empty stomach but when taken with a meal, absorption is reduced as a result of the same log and binding

process that affect non-haem iron dietary like in black tea as it inhibits absorption(A V Hoffbrand 2011).

Iron transport

Only a small proportion of total body iron's daily enters or leaves the body is stored(A V Hoffbrand 2011). The greatest iron mass is found in erythroid cells, which contain about 80% of total body endowment.

Transferrin; plasma transferrin is an 80 KDa glycoprotein with homologus N-terminal and C-terminal iron binding domains(A Jacobs 1975). All circulating plasma iron is bound to transferrin, due to three purposes which are;

It renders iron soluble under physiologic conditions.

It prevents iron-mediated free redical toxicity.

It facilitates transport into cells.

Transferrin is a blood plasma protein that attaches to transferrin receptors found on the blood cells, allowing iron molecules to group together tightly(P Ponka 1998). Transferrin receptor test is given to determine iron imbalance, when a patient is suffering from high or low levels of iron. Transferrin is synthesized in the liver and secreted into plasma, produced locally in the central nervous system (CNS) and testis, as these two sites are inaccessible to proteins in the general circulation (blood; testis, blood; brain barrier)(P Ponka 1998).

Haem synthesis

Haem consists of a protoporphyrin ring with an iron atom at its centre(A V Hoffbrand 2011). The first step of haem synthesis takes place in the mitochondrion, with succinyl CoA and glycine by ALA synthase to form -aminolevulic acid (ALA), under the action of ALAs, with pyridoxal phosphate as a coenzyme, ALAs molecules are transported to the cytosol where a series of reactions produce the ring structure coproporphyrinogen III, ALAs return to the mitochondrion where an addition reaction produces protoporphyrin IX. An enzyme called ferrochelatase inserts iron into the ring structure of protoporphyrin IX to produce haem(A Jacobs 1975). Anaemia along with a number of drugs or toxins as lead, inhibit haem production by interfering with enzymes involved in haem biosynthesis(A Jacobs 1975). After phagocytosis, haem is broken down by haem oxygenase (HMOX1) to release iron. Ferrous iron can then either enter ferritin (where it is oxidized to ferric iron by the ferritin protein) or be released into plasma via ferroprotein 1, where then it binds to transferrin(A Jacobs 1975). Hepcidin controls the release of macrophage iron by reducing iron release especially if there is an inflammation on iron over load. Changes of this macrophage iron release, is thought to account for the diurnal rhythm of serum iron concentration, which was observed to be higher in the morning than in the evening(A V Hoffbrand 2011).

Iron storage

Serum ferritin concentration correlates with iron stores. Normal concentration of serum ferritin range from 15-300g/L and are higher in men in about 30g/L than in women (reference), serum ferritin below 15g/L are virtually specific for storage iron depletion and values above 300g/L do not necessarily indicate iron overload (Haemochromatosis)(A Jacobs 1975). All because ferritin synthesis may be influenced by factors other than iron, as in many inflammatory diseases(A V Hoffbrand 2011). When ferritin synthesis is influenced by other factors than iron, it behaves as an acute-phase reactant, and for this reason, serum ferritin concentrations below 50g/L may be associated with lack of iron storage in patients with anaemia of chronic disease. Also a ferritin value of above 100g/L suggests the presence of storage iron(JD Cook 1974). Bone marrow also stores iron, indications can be observed by bone marrow staining that appears as reticuloendothelial and or erythroblast.

Iron uptake

About 85% of transferrin iron normally enters developing RBC's for incorporation into haemoglobin(A V Hoffbrand 2011). Transferrin-bound iron reflects the expression of transferrin receptors, which are present on high iron requirement cells. A soluble truncated form of the transferrin receptor derived from these cell surfaces is detectable in serum(AACC 2009). The transferrin-receptor complex is taken up by a process of receptor-mediated endougtosis. The iron is released at low pH of the endosome, reduced from Fe to Fe, apotransferrin and receptor are then recycled to the plasma and cell membrane(AACC 2009). Iron released from the endosome is transported into mitochondria by mitoferrin or enters ferritin(AACC 2009). 80- 90% iron taken into developing erythroblasts is converted to haem within one hour. Any iron taken up in excess of the requirement of haem synthesis is incorporated in ferritin. Red cell ferritin content is increased by haemoglobin synthesis impairment, as in thalassaemia or sidroblastic anaemia syndromes. Excess iron can be seen in mature red cell cytoplasm in the form of one or more siderotic granules, which are then removed by the spleen(A V Hoffbrand 2011).

Iron supply to the tissues

Serum iron and the saturation of the total iron-binding capacity of transferrin (TIBC) give a measure of iron supply to the tissues(BJ Fersuson 1992). A rise in TIBC is characteristic of iron deficiency; a reduced serum iron concentration with normal or reduced TIBC is a characteristic response to inflammation, where as a serum transferrin saturation less than 15% is insufficient to support normal erythropoiesis(Reif 1992).

Serum transferrin: as plasma concentration reflects the number of erythroid precursors and iron supply to the bone marrow, in clinical practice, these two factors must be considered in interpreting transferrin receptor levels(A V Hoffbrand 2011).

RBC protoporphyrin: when iron supply is limited, incorporation of iron into haem is restricted, leading to accumulation of the immediate precursor propotoporphyrin IX. This then is lost slowely from circulating red cells. Protoporphyrin levels may increase in patients with sideroblastic anaemias and lead poisoning(A V Hoffbrand 2011).

Hypochromic RBC: as iron supply to erythron diminishes, new RBCs produced are increasingly hypochromic. Values of hypochromic cells of 6% may help in the early identification of impaired iron supply in patients with renal failure who are under a traeatment of recombinant erythropoietin(A V Hoffbrand 2011).

Reticulocyte haemoglobin content (CHr): used in iron screening in dialysis patients particularly, CHr may predict iron treatment in response of anaemia and also appropriate for the assessment of iron-deficient erythropoiesis(A V Hoffbrand 2011).

Iron deficiency anaemia

Is defined as a decrease in total iron content in the body. Iron deficiency anaemia occurs when iron level is sufficient enough to diminish erythropoiesis and develops the anaemia(TL Holyoake 1993), iron deficiency is the most common form of anaemia in the world(AK Leung 2001). RBCs become microcytic and hypochromic and poikilocytosis become more obvious, MCV and MCH are reduced and target cells may be present, platelets are frequently increased, reticulocyte count is low for the degree of anaemia(DJ Talor 2005). Serum TIBC rises and serum iron falls, the number of erythroblasts containing cytoplasmic iron become reduced at an early stage of aneamia development and siderotic granules become entirely absent from these cells, except where very rapid blood loss that mobilizes the iron storage, bone marrow macrophages show a total absence of iron(A V Hoffbrand 2011).

When iron deficiency is chronic and sever, widespread tissue changes may present, including; hair thinning and ridged nails. Part responsibility of tissue change would be the iron dependent enzymes, which may fall and mitochondrial swelling in many different cells occur, this then indicates poor lymphocyte transformation and diminished cell-mediated immunity plus impaired intracellular killing of bacteria by neutrophils(BJ Fersuson 1992).

Causes of iron deficiency

Diet: diet is rarely the major cause of iron deficiency especially in adults, diet may contain insufficient iron content as a result of poverty, religion or vegetarian diet(Clark 2008).

Iron requirement: in infancy, demand for growth may be grater than dietary supply, therefore, iron deficiency is quiet common in infancy. It is also common in adolescence, females and pregnancy(AK Leung 2001). Although iron absorption increases throughout pregnancy, the amount of iron lost with placenta and with bleeding at delivery, may very easily cause iron deficiency, the fetus aquires about 280mg of iron and further 400-500mg is acquired for the temporary expansion of maternal red cell mass, with about 200mg of iron is lost at delivery, this may not be sufficient to meet the resultant net maternal outlay of over 600mg iron(DJ Talor 2005).

Blood loss: is the most common cause of iron deficiency in adults(A V Hoffbrand 2011). The maximum amount of iron absorbed from a normal diet is about 6-8mg a day, a daily loss of blood that equals this amount would be very important. Blood loss would normally be from the genital tract in women (menstrual cycles) or gastrointestinal tract in both sexes(A V Hoffbrand 2011). Most common worldwide blood loss cause is (hookworm)(RJ Stoltzfus 1996), where as in the UK, menorrhagia, haemorrhoids and peptic ulceration are the most common causes of blood loss. Gastric bleeding because of salicylates as (Aspirin) or other steroidal anti-inflammatory drugs or bowel tumors are also common(AF Goddard 2000).

Malabsorption: is also a primary cause of iron deficiency, where dietary iron is poorly absorbed in gluten-induced enteropathy. 50% of patients newly diagnosed coeliac disease have coexistent iron deficiency anaemia(Fleming 1982). Patients often don't show response to oral therapy with inorganic iron. Helicobacter pylori gastritis is another common cause of iron deficiency, H. pylori inhibits gastric hydrochloric acid secretion, interfering with solubilization and absorption of inorganic iron intake and also possible that gastrointestinal blood loss causes iron deficiency(A V Hoffbrand 2011). Iron deficiency also present in congestive heart failure due to malabsorption and iron loss.

Iron overload: excessive iron overload of the parenchymal cells in the liver arises when there is iron absorption, which eventually lead to tissue damage(Fleming 1982). Excessive iron absorption is first taken up by senescent red cells by macrophages and slowly released to transferrin, which then are taken up by parenchymal cells(Fleming 1982). Sever iron overload is defined as an excess of more than 5g is confined to genetic haemochromatoses together with iron-loading anaemia and sub-Saharan dietary of iron overload(Fleming 1982). Iron overload can be related to:

Excess ion absorption due to herediatary haemochromatosis which causes a massive ineffective erythropoietic anaemia.

Increased iron intake in sub-saharan dietary iron overload, in combination with genetic determination of increased absorption(A V Hoffbrand 2011).

Repeated red cell transfusion for congenital anaemic patients, as in beta thalassaemia, sickle cell anaemia and red cell aplasia. Also in acquired refactory anaemia e.g, aplastic anaemia and myelodysplasia.

Genetic haemochromatosis

Classified according to the genetic defect causing iron overload. The vast majority causes are of

Type 1: involving the HFE gene(G Papanikolaou 2003). In northern European populations, about 90% of patients with haemochromatosis are homozygous for the HFE Cys28Tyr mutation (C282Y). In southern Europe only 60% of patients with haemochromatosis are found to be homozygous for C282Y mutation(G Papanikolaou 2003).

Type 2: the haemochromatosis caused by mutation of hemojuvelin or hepcidin genes.

Type 3: mutation is due to transferrin receptor 2.

Type 4: due to mutations of ferroprotein.

Types 1,2 and 3 are autosomal recessive diseases and share common features in hepcidin deficiency and include high transferrin saturation and hepatocyte iron accumulation(A V Hoffbrand 2011). Type 4, is inherited as a dominantcondition and is hetrogenous disease with variable clinical phynotype. In neonatal, haemochromatosis, no mutation is known(A V Hoffbrand 2011). This condition is recognized only at birth but may occur in utero. It is charechterized by heavy parenchymal iron deposition in many organs and causes to antiribonuclear factor antibody at the presence of alloantibody as rhesus incompitability infusion of gamaglobulin in pregnancy(DJ Talor 2005).

Sub-Saharan iron intake

African iron overload (Bantu siderosis) is charechterized by the combination of dietary components and an unknown susceptibility gene(Fleming 1982). It is a cause of hepatic fibrosis and cirrhosis, and associated with diabetes, tuberculosis and infection that is highly prevented in sub-saharan Africa(RJ Stoltzfus 1996). Iron absorption is as in type 4 haemochromatosis, mutation in gene HFE have been excluded but ferroportin gene mutation may play a role(G Papanikolaou 2003).

Other causes of iron overload

Atransferrinanaemia: a rare recessive genetic disorder associated with hypochromic anaemia(A V Hoffbrand 2011).

Acaeruloplasminaemia: a rare recessive disorder where a deficiency of ferroxidase activity as a consequence of mutation in the caeruloplasmin gene exists(A V Hoffbrand 2011).

Hallervorden-spatz syndrome: autosomal recessive neurodegenerative disorder associated with iron accumulation in the brain(A V Hoffbrand 2011).

Neuroferritinopathy: a rare dominantly inherited basal ganglia disease presents with extrapyramidal features similar to those of Huntington or Parkinson diseases and show iron accumulation in the forebrain and cerebellum with the gene (FTL) that codes for ferritin light-chain polypeptide to be venesected to remove iron, may aquire chelation therapy(A V Hoffbrand 2011).

Friedereich ataxia: a neurodegenerative disease charechterized by loss of sensory neurons in the spinal cord and dorsal root ganglia(A V Hoffbrand 2011).

Herediatery hyperferritinaemia: a syndrome charechterized by elevated serum ferritin levels due to heterozygous point mutation in the L-ferritin iron-response element when a monoclonal ferritin is synthesized because of a negative feedback of ferritin syndthesis(A V Hoffbrand 2011).

Iron loading anaemia

In patients with transfusion dependent anaemia, iron removal is essential, patients with thalassaemia major also need iron removal where as in patients with thalassaemia intermedia who are too anemic iron cant be removed(A V Hoffbrand 2011).

Tests for body iron

Serum ferritin: monitors changes in body iron and measures the total body iron.

Liver iron: measured after a liver biopsy or by MRI.

Cardiac iron: iron deposition in myocytes measured by regular monitoring of cardiac iron, cardiac failure is a usual cause of death in transfusional iron overload.

Urine ironexcretion: the test is useful when commencing therapy with DFX or deferiporone, with which iron is highly dosed related, and for monitoring therapy.

Non-transferrin-bound iron: presents in plasma in patients with gross iron overload and 100% saturation of transferrin(AACC 2009).


One hundred subjects were randomly tested for the ferritin levels,in blood plasma (EDTA) and blood serum (clotted) samples.

Ferritin test was performed on both blood samples for each subject. Due to the involvement in other blood testing (FBC), the EDTA samples were tested 24 hours after the serum testing was performed

EDTA blood samples were refrigerated overnight on a temperature of 2-5 degrees Celsius until tests were performed.

Serum and plasma ferritin were measured by the method of Access 2 analyser that uses antigen-antibody reactions to detect a specific antigen or antibody in the sample of body fluid, e.g serum or plasma.

The Access 2 system supports three types of assay calibrations

Quantitive Assay; use a calibration curve to measure the amount of analyte present in a sample.

Semi Quantitive Assay; use a calibration curve to measure the amount of analyte present in a sample.

Qualitative Assay, which use a cutoff value to classify the result of analyte is either reactive or detective.

Depending on the analyte present in a sample, immunoassays use a variety of assay formats to measure this analyte. The most common formats are, sandwich, antibody detection and competitive binding format. Each assay format uses reagent that react with a specific analyte to form immune complexes.

The measurement of Ferritin, the Access anylyser uses the sandwich assay. The sandwich assay (see figure) use coated paramagnetic particles to measure antigen in a sample.

The particles can be either directly or indirectly coated with capture antibody.

A sample is mixed with the particles and an enzyme-labled antibody (conjugate).

The sample analyte (ferritin) and conjugate form immune complexes that bind to the particles.

Magnets separate the particles-bound immune complexes from the unbound components and washing removes the unbound component (see appendix…).

After adding chemiluminescent substrate, the measured RLUs are directly proportional to the amount of antigen in the sample (see appendix … ).

Most tests were completed in 15-20 minutes.

See details of calibration curves and QCs attached.