Introduction

Total iron-binding capacity (TIBC) is an essential test used for the diagnosis of iron deficiency anemias and other disorders of iron metabolism. Iron binding capacity is the capacity of transferrin to bind with iron. Iron binding capacity is of two types, TIBC and unsaturated iron-binding capacity (UIBC). When iron stores are depleted, the transferrin levels increase in the blood. As only one-third of transferrin is saturated with iron, so the transferrin present in serum has an extra binding capacity (67%). This is unsaturated iron-binding capacity. TIBC is the total of serum iron and UIBC. Percentage transferrin saturation is calculated by dividing serum iron by TIBC and multiplying the result by 100.

Etiology and Epidemiology

Anemia is the most common hematological disorder in both adult and pediatric populations.[1][2][3][4] Iron panels are routinely run in patients with anemia. One of the tests included in the iron panel is TIBC. Anemia is classified based on the mean corpuscular volume (MCV) into microcytic, normocytic, or macrocytic anemia. Iron deficiency anemia, which is normocytic in early stages and microcytic in later stages, is the most common type of anemia worldwide. Iron overload conditions can be hereditary or acquired. The most common iron overload disorder is hemochromatosis.[5][6] If left untreated, severe iron overload is potentially fatal, resulting in organ damage. The disrupted homeostasis of iron metabolism results in the deposition of iron in various organs of the body, causing liver cirrhosis, bronze diabetes, neurological deficits, and arrhythmias.[7] The most common cause of death in hemochromatosis is liver cirrhosis and ventricular arrhythmias.[8][9][10] TIBC is used in both iron deficient and overload scenarios to establish the diagnosis.

Pathophysiology

The food we consume has iron in two forms, heme and non-heme. The elemental iron is released from its bound form in the food by the action of hydrochloric acid in the stomach. The ferric form is enzymatically reduced to ferrous form and then absorbed in the gut by the divalent metal ion transporter located on the apical surface of the intestinal epithelium. The heme form of iron is absorbed directly through a heme transporter. The absorbed iron can be stored along with apoferritin, an iron storage protein, in the enterocytes, or can be absorbed into the blood through ferroportin. Ferroportin is a transporter present on the basolateral surface of enterocytes. Before being transported into the blood, the ferrous iron is converted to ferric form by hephaestin. The ferric iron is then picked up by apo-transferrin. Apo-transferrin is an iron-transporting protein present in the blood. It binds to iron and delivers it to various tissues, mainly bone and liver.[11][12] Together, iron and apo-transferrin form transferrin, but occasionally the terms transferrin and apo-transferrin are interchangeably used as the bond formed between them is non-covalent. Most of the iron is incorporated into hemoglobin and myoglobin. It is also used for the synthesis of certain enzymes. Iron is stored in macrophages with storage protein apoferritin. It is lost from the body during menstrual bleeding when epithelial cells of the skin, as well as enterocytes, are shed and during hemorrhage.

The daily requirement of iron in adults is recommended as:

• In men and non-menstruating women 8 mg
• In menstruating women 18 mg
• In pregnant women 27 mg

Generally, the total transferrin in the blood is only 33% saturated. Each molecule of apo-transferrin can bind to a maximum of 2 ferric ions. In iron-deficient states, the total transferrin saturation falls to 16% or less.[13][14]

Specimen Requirements and Procedure

An iron panel is ordered to diagnose disorders of iron metabolism. Tests on the iron panel include; fasting serum iron, TIBC, percentage transferrin saturation, or iron saturation, and serum ferritin. In addition to this, physicians order a complete blood picture and reticulocyte count as well, usually when anemia is suspected.

Specimen

A whole blood sample of the patient is collected as part of an anemia workup and iron panel evaluation. The following steps should be followed by the healthcare worker when drawing a blood sample.

• Explain the procedure to the patient and ask for consent.
• Use a new pair of gloves for each patient.
• Clean the skin over the cubital fossa of the patient with an antiseptic in a spiral fashion, moving from the center to the periphery.
• Tie a tourniquet around the patient’s arm.
• Once the vein becomes prominent, insert the needle.
• After collecting the blood sample, untie the tourniquet, and remove the needle gently.
• Apply a bandaid or cotton piece dipped in antiseptic over the puncture site.
• Correctly label the blood sample before sending it for analysis.

For UIBC, the blood sample should not be hemolyzed. A fasting specimen is recommended. Bilirubin and lipemia offer no interference with UIBC.

For iron, no interferences are offered by lipemia, bilirubin of less than 30 mg/dL, or hemolysis. Serum should be separated within 2 hours after collection. It should be taken care that separated serum should not remain at +15 degrees C to +30 degrees C longer than 8 hours. In case of a delay of more than 8 hours, the serum should be stored at +2 degrees C to +8 degrees C. If the serum sample is to be stored beyond 48 hours, samples should be frozen at –15 degrees C to –20 degrees C. Frozen samples should be thawed only once. Deterioration of samples may occur if they are repeatedly frozen and thawed. It is recommended that samples be drawn in the morning due to diurnal variation. Oral contraceptives can elevate iron or TIBC values. When required, the samples should be stored in borosilicate glass or plastic containers. 

Testing Procedures

A timed-endpoint method is used to measure iron concentration. The steps are as follows:[15]

• The iron (ferric form) bound to transferrin is freed by adding acetic acid.
• Then the iron is reduced to its ferrous form by using hydroxylamine and thioglycolate.
• The iron then forms a complex with FerroZine Iron Reagent.
• The concentration of iron in the sample is measured by the change in the absorbance of light.
• Change in absorbance is tracked at 560 nm wavelength at a fixed-time interval.
• TIBC excess is estimated by adding excess ferric iron to saturate transferrin and the unbound ferric iron is removed.

Interfering Factors

Test results can be influenced by recent blood transfusions, hemolyzed specimens (UIBC measurement), fluoride, oral contraceptives, and chloramphenicol use.[16][17]

Results, Reporting, Critical Findings

Normal reference ranges:

• Iron-binding capacity: 255 mcg/dL to 450 mcg/dL.[18]
• Transferrin-iron saturation percentage: 25% to 35%.[13]
• UIBC - normal values for UIBC may vary among laboratories, but most laboratories define their normal range as 111 mcg/dL to 343 mcg/dL.[18]
• TIBC - normal values for the TIBC test can vary among laboratories. Generally, the normal range is taken as 240 mcg/dL to 450 mcg/dL.[18]

Clinical Significance

Iron binding studies are important for the diagnosis of iron deficiency and iron overload conditions. In iron-deficient conditions, the relative transferrin content compared to iron content increases, and thus the TIBC values are high. The opposite happens in iron overloaded states of the body; the quantity of free transferrin in blood decreases, and consequently, TIBC values are low. Iron binding capacity also decreases in liver diseases, like cirrhosis, as transferrin is synthesized by the liver. TIBC levels may be low in multifactorial anemias or anemias of chronic inflammation. In such cases, additional information regarding a component of iron deficiency can be obtained through the calculation of iron or transferrin saturation. 

Treatment of iron deficiency anemia involves correction of the underlying source of blood loss along with correction of iron deficiency state with iron supplementation. Iron supplementation can be done via oral route or intravenous formulations, depending on the rapidity of iron correction needed. Various intravenous iron formulations are available and are similar in efficacy when compared in studies, with varying adverse effects profiles. 

In iron-deficiency anemia, another interesting finding noted on complete blood count analysis is the reactive elevation of platelets (reactive thrombocytosis). There is evolving literature on the increased risk of thrombosis (more so venous than arterial) associated with elevated platelets and iron deficiency.[19][20] According to a large retrospective analysis by Song et al., the risk of thrombosis is about 15.8% with elevated platelets and iron deficiency compared to 7.8% risk associated with iron deficiency alone and no elevation in platelets. 

In iron-overload states including hereditary hemochromatosis, conditions associated with transfusion dependency seen in myeloid disorders, or thalassemias (which can present in later years with increased ability to absorb and store iron) TIBC levels are low with proportional increases in iron saturation levels. For hereditary hemochromatosis, the initial treatment of choice is therapeutic phlebotomy to keep ferritin levels under 50 to 100 ng/ml, while keeping hemoglobin levels above 11 g/dL. In other cases of iron overload and coexisting anemia, where therapeutic phlebotomies are not safe to perform, iron chelators are employed. Iron chelators are available as oral or parenteral formulations. 

Quality control and Lab Safety

• Healthcare workers handling serum/plasma samples should be vaccinated for hepatitis B as the samples could be positive for hepatitis B. Samples can also be positive for HIV and other blood-borne pathogens. Standard institutional or laboratory protocols must be followed while handling the samples.
• People working in the lab should wear laboratory coats, gloves, and protective eye equipment, as indicated.
• The laboratory should have color-coded bins, and waste items should be discarded properly in sharps or bio-hazardous containers as indicated. 
• The laboratory should be well ventilated.
• Unlabeled serum samples should be rejected.

Enhancing Healthcare Team Outcomes

Evaluating iron-binding capacity, as part of anemia workup requires careful coordination between interprofessional team members at various levels. Key roles are played by physicians who order the test, nurses, or phlebotomists who collect blood samples, pathologists, laboratory assistants, and technicians who carry out the test.