Introduction

Total iron-binding capacity (TIBC) is a crucial laboratory test for diagnosing iron metabolism disorders and inflammatory diseases.[1] Iron-binding capacity is the capacity at which transferrin binds with iron.[2] Transferrin, previously known as siderophilin, is the principal plasma transport protein for ferric iron (Fe³⁺). Transferrin has a molecular weight of 79.6 kDa and comprises 5.5% carbohydrates. Transferrin is a single polypeptide chain with 2 N-linked oligosaccharides and 2 homologous domains, each with a Fe³⁺-binding site.[3] Transferrin is synthesized mainly in the liver and circulates with a half-life of 8 to 10 days. Transferrin reversibly binds 2 ferric ions with high affinity at physiological pH but lower affinity at decreased pH; this permits iron release within intracellular compartments. After cellular delivery of iron through receptor-mediated endocytosis, apotransferrin is recycled back into circulation.[4]

A few clinical indications exist for directly measuring transferrin. However, the indirect laboratory assessment of transferrin concentration may be inferred by TIBC. TIBC may be calculated as total or unsaturated.[5] Depleting bodily iron stores by any mechanism increases circulating levels of transferrin. At optimal health, only one-third of transferrin is saturated with iron, and serum transferrin has an extra binding capacity of 67%, the unsaturated iron-binding capacity (UIBC).[6] TIBC is the total serum iron and UIBC. Percentage transferrin saturation is calculated by dividing serum iron by TIBC and multiplying the result by 100.[7]

Etiology and Epidemiology

Iron studies, which typically include total serum iron level, TIBC, transferrin, and transferrin saturation, are essential for diagnosing patients suspected of iron deficiency, overload, or poisoning. Other laboratory evaluations may be included in iron studies that may be utilized to evaluate specific inflammatory processes.

Anemia is the most common hematological disorder in all age groups, and iron-deficiency anemia is the most commonly encountered anemia worldwide.[8][9] Approximately 30% of the global population is affected by iron-deficiency anemia.[10] Iron studies are routinely performed in patients with anemia, particularly if the anemia is normocytic or microcytic.

The most commonly encountered iron overload disorders are primary and secondary hemochromatosis.[11][12] Hereditary hemochromatosis is one of the most common autosomal recessive disorders among individuals of European ancestry, with a prevalence of 1 in 300 to 500 individuals.[13] Severe iron overload causes iron deposition in most tissues and can be fatal if left untreated.[14][15][16] Other clinical conditions that may result in an increased TIBC include polycythemia vera, late pregnancy, and the use of estrogen-containing medications. TIBC may be decreased in malnutrition, hypoproteinemia, and liver dysfunction. TIBC must be interpreted within the clinical context.[5]

Pathophysiology

Foods contain iron in heme and non-heme forms. Bound elemental iron is released in the stomach through the action of hydrochloric acid. Ferric iron is enzymatically reduced to ferrous iron and is absorbed in the gut by the divalent metal ion transporter located on the apical surface of the intestinal epithelium.[17] Heme iron is absorbed directly through a heme transporter. Absorbed iron is stored with apoferritin within the enterocytes or absorbed into the blood through ferroportin.[18]

Ferroportin is a transporter protein on the basolateral surface of enterocytes and many other cells. Ferrous iron is converted to ferric iron by hephaestin before being transported into the blood. The ferric iron is picked up by apotransferrin, a circulating protein that delivers iron to various tissues, primarily the liver and bone.[19] Technically, apotransferrin carrying 1 or 2 ferric ions is transferrin; these terms are frequently used interchangeably due to the noncovalent bond with ferric irons. The majority of iron is incorporated into hemoglobin or myoglobin; some is used to synthesize certain enzymes.[20] Iron is stored in macrophages with the storage protein apoferritin. In healthy individuals, small amounts of iron are lost through epidermal and enterocytic shedding; small amounts are lost in sweat. Menstruation and other forms of bleeding also cause iron loss.[21]

The recommended daily iron intake for adults is as follows:

• Men and non-menstruating women: 8 mg
• Menstruating women: 18 mg
• Pregnancy: 27 mg

Generally, the total transferrin in the blood is only 33% saturated. The total transferrin saturation falls to 16% or less during iron-deficient states.[22][23]

Specimen Requirements and Procedure

To collect a whole blood sample for iron studies, the following steps are recommended:

• Explain the procedure to the patient and obtain consent to proceed.
• Use a new pair of gloves for each patient.
• Clean the skin over the puncture site with an antiseptic in a spiral manner, moving from the center to the periphery.
• Place a tourniquet above the puncture site.
• When the vein is visibly raised and can be felt upon palpation, proceed to insert the needle.
• After collecting the blood sample, gently untie the tourniquet and remove the needle.
• Apply a bandaid or cotton piece dipped in antiseptic over the puncture site.
• Correctly label the blood sample before sending it for analysis.[24]

Samples for UIBC should not be hemolyzed. A fasting specimen is recommended. Bilirubin and lipemia do not interfere with UIBC.[1]

Lipemia, <30 mg/dL of bilirubin, or hemolysis do not interfere with iron studies. The serum should be separated within 2 hours of collection.[25] Once separated, serum should not remain between 15 °C and 30 °C for more than 8 hours; if a delay of more than 8 hours is anticipated, separated serum should be stored between 2 °C and 8 °C. If the separated serum must be stored for over 48 hours, it should be frozen between −15 °C and −20 °C. Frozen samples undergo only one thawing cycle, as repeated freeze-thaw cycles can cause sample deterioration.[26] Samples should be stored in borosilicate glass or plastic containers.[27] Certain iron studies show diurnal variation of as much as 30%; therefore, morning samples are recommended.[28] Oral contraceptives can elevate iron or TIBC values.

Testing Procedures

A practical and chemical method for determining TIBC in serum or plasma was first reported in 1957, refined in 1978, and revised in 1990.[29] However, in the 1990s, more direct TIBC assays were developed. Most automated chemistry analyzers measure UIBC and calculate TIBC. Transferrin measurements have mostly replaced testing for UIBC and TIBC outside the United States.[30]

In most colorimetric methods, TIBC is measured through a series of steps. First, transferrin is saturated with excess ferric iron. The unbound ferric iron is removed through chelation, typically using magnesium carbonate. The total amount of iron saturating the transferrin is then measured. Non–transferrin-bound iron and ferritin-bound iron can falsely elevate transferrin saturation levels.[31]

Iron concentration is measured through a timed-endpoint method with the following steps:

• Ferric iron bound to transferrin is freed by adding acetic acid.
• The freed ferric ion is then reduced to the ferrous form using hydroxylamine and thioglycolate.
• The resulting ferrous iron complexes with FerroZine™ Iron Reagent.
• The concentration of iron in the sample is measured by the change in the absorbance of light tracked at 560 nm at a fixed-time interval.
• TIBC excess is estimated by adding excess ferric iron to saturate transferrin, and the unbound ferric iron is removed.

An ion-exchange technique may be employed to measure serum iron. In this method, a serum sample is added to an aliquot of ion-exchange resin preloaded with iron. Following a short period of equilibration, the serum sample (transferrin) is saturated with iron; the affinity of iron for the serum transferrin is greater compared to the affinity for the resin. An aliquot of this iron-saturated serum is analyzed using the same method as total iron analysis.

Transferrin iron saturation can be calculated using the total serum iron and TIBC or transferrin measurement. Transferrin concentration can indirectly derive the TIBC and transferrin iron saturation.[32] 

Theoretically derived formulas for these calculations are as follows:

1 mol transferrin is 79,570 Da with the capacity to bind

2 atoms of iron with an atomic mass of 55.84 Da, therefore:

Transferrin (g/L) = 0.007 × TIBC (μg/L), and

TIBC (μg/dL) = transferrin (mg/dL) × [1.41 x TIBC (μmol/L)], or

                    = transferrin (g/L) × 25.2

Transferrin saturation (TSAT %) = [serum Fe (μg/dL) / transferrin (mg/dL)] × 70.9

               = [serum Fe (umol/L) / transferrin (mg/dL)] × 398 [33]

UIBC-based methods generally exhibit a significant negative bias compared to TIBC-based methods.[24] The chemical methods for TIBC require a relatively large sample volume. The TIBC assays are sensitive to contamination of laboratory consumables with Fe and show a large variation; reference intervals differ by as much as 35% among commercial methods.[1]

Interfering Factors

Test results can be influenced by recent blood transfusions, hemolyzed specimens (UIBC measurement), fluoride, oral contraceptives, and chloramphenicol use.[34][35] Response to hemolysis is both heterogeneous and unpredictable, and therefore, no reliable corrective measures are recommended.[36]

Serum from patients with iron (Fe) overload may contain Fe bound loosely to molecules other than transferrin, such as citrate and albumin, and sometimes Fe chelators.[37] Consequently, this may result in the following: (i) TIBC methods generally overestimating the Fe-binding capacity of transferrin and (ii) transferrin saturation calculated from immunochemically measured transferrin measurements exceeding 100%.[38]

Adding excess amounts of Fe(III)-chloride in the TIBC assay can lead to the nonspecific binding of Fe to albumin and other plasma proteins.[39] As a result, an overestimation of TIBC occurs, especially in patients with hyperferritinemia and low transferrin concentrations, as observed in conditions such as liver diseases and nephrotic syndrome. In addition, this may cause a nonlinear relationship between serum transferrin concentration and TIBC.[1]

The intraindividual day-to-day variation of Fe and transferrin saturation is approximately 25% to 30%.[28] In contrast, TIBC (or transferrin values) show only slight day-to-day or diurnal variation, with a variation for TIBC between 4.8% and 8.8% and for transferrin, 3%.[40] Furthermore, variations in results obtained at the same time of the day on sequential days or weeks remain substantially greater compared to analytical variations, even when samples are collected after an overnight fast.[41]

Results, Reporting, and Critical Findings

The following reference ranges for iron metabolism parameters valuable guidance for assessing iron status and diagnosing related disorders.

• Iron-binding capacity: 255 to 450 mcg/dL.
• Transferrin-iron saturation percentage: 25% to 35%.
• UIBC: normal values for UIBC may vary among laboratories, but most laboratories define their normal range as 111 to 343 mcg/dL.
• TIBC: normal values vary among laboratories, generally 240 to 450 mcg/dL.[22][42]

Clinical Significance

Iron studies are important for diagnosing 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.[43] The opposite happens in iron-overloaded states of the body; the quantity of free transferrin in the blood decreases, and consequently, TIBC values are low. Iron binding capacity also decreases in liver diseases, such as cirrhosis, as the liver synthesizes transferrin.[44] 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 by calculating iron or transferrin saturation.[45]

The treatment of iron-deficiency anemia involves addressing the underlying source of blood loss and replenishing the iron-deficiency state with iron supplementation.[46] Iron is supplemented through oral or intravenous formulations, depending on the urgency of iron correction needed. Various intravenous iron formulations are available, all with similar efficacy but differing adverse effect profiles.[47]

In cases of iron-deficiency anemia, another notable finding on complete blood count analysis is the reactive elevation of platelets, known as reactive thrombocytosis. Emerging literature suggests an increased risk of thrombosis, particularly venous thrombosis, associated with elevated platelets and iron deficiency.[48][49] 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 a 7.8% risk associated with iron deficiency alone and no elevation in platelets.[50]

In iron overload states, such as hereditary hemochromatosis, conditions associated with transfusion dependency seen in myeloid disorders, or thalassemias, which can present in later years with an increased ability to absorb and store iron, TIBC levels are low with proportional increases in iron saturation levels.[51] The initial treatment for hereditary hemochromatosis is therapeutic phlebotomy to keep ferritin levels under 50 to 100 ng/mL while keeping hemoglobin levels above 11 g/dL. Iron chelators are employed in other cases of iron overload and coexisting anemia, where therapeutic phlebotomies are unsafe. Iron chelators are available as oral or parenteral formulations.[1]

Quality Control and Lab Safety

Quality control (QC) of the analytical examination process monitors a measurement procedure to verify that this meets performance specifications appropriate for patient care or that an error condition is corrected.[52] For non-waived tests, laboratory regulations require, at the minimum, analysis of at least 2 levels of QC materials every 24 hours. If necessary, laboratories can assay QC samples more frequently to ensure accurate results. QC samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance.[53] To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency, such as once a month, the labs can develop an individualized quality control plan that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.

The design of a QC plan must consider the analytical performance capability of a measurement procedure and the risk of harm to a patient if an erroneous laboratory test result is used for a clinical care decision. An erroneous laboratory test result is a hazardous condition that may or may not cause harm to a patient, depending on the action or inaction a clinician takes based on the erroneous result.[54]

The acceptable range and rules for interpreting QC results are based on the probability of detecting a significant analytical error condition with an acceptably low false alert rate.[55] The desired process control performance characteristics must be established for each measurement before selecting the appropriate QC rules.[56] Westgard multi-rules are typically used to evaluate the QC runs. If a run is declared out of control, the system, including the instrument, standards, controls, and other relevant factors, should be investigated to determine the cause of the problem. Analysis should not proceed until the problem has been thoroughly resolved.[57]

Changing reagent lots can have an unexpected impact on QC results. Careful reagent lot crossover evaluation of QC target values is necessary. Because the matrix-related interaction between a QC material and a reagent can change with a different reagent lot, QC results may not be a reliable indicator of a measurement procedure’s performance for patient samples after a reagent lot change.[58] Using clinical patient samples, the consistency of results between old and new reagent lots is verified because of the unpredictability of a matrix-related bias.[59]

The laboratory must participate in the external QC or proficiency testing program as a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the clinical laboratory improvement amendments regulations.[60] Ensuring the accuracy and reliability of the laboratory concerns other laboratories performing the same or comparable assays. Participation is mandatory and results are monitored by CMS and voluntary accreditation organizations. The proficiency testing plan should be included as an aspect of the quality assessment (QA) plan and the overall quality program of the laboratory.[61]

All specimens, control materials, and calibrator materials should be considered as potentially infectious. Clinicians handling serum or plasma samples should be vaccinated for hepatitis B. The samples could also be positive for HIV and other blood-borne pathogens. The usual precautions required for handling all laboratory reagents should be observed. Disposal of all waste materials should follow local guidelines. Gloves, a lab coat, and safety glasses should be worn when handling human blood specimens. All plastic tips, sample cups, and gloves that come in contact with blood should be placed in a biohazard waste container.[62] All disposable glassware should be discarded into sharps waste containers. All work surfaces should be protected with disposable absorbent bench top paper, which should be discarded into biohazard waste containers weekly or whenever blood contamination occurs. All work surfaces should be wiped weekly.[63]

Enhancing Healthcare Team Outcomes

The assessment of iron-binding capacity within an anemia workup demands a cohesive effort among diverse interprofessional team members, each contributing crucial expertise at various stages of the diagnostic process. Clinicians, serving as the frontline decision-makers, are tasked with ordering the test based on their clinical judgment and suspicion of iron-related disorders. Nurses or phlebotomists, proficient in blood sample collection techniques, ensure the proper procurement of specimens necessary for analysis.

In the laboratory setting, pathologists oversee the overall diagnostic process, providing clinical interpretation and guidance. Laboratory assistants support the logistical aspects of sample handling and processing, ensuring samples are properly labeled and prepared for analysis. Technicians, skilled in conducting the iron-binding capacity test, carry out the analytical procedures with precision and accuracy.

Effective communication and coordination among these team members are essential to ensure seamless workflow and accurate interpretation of results. Collaboration facilitates the timely identification of iron metabolism disorders, enabling appropriate interventions and patient management strategies. By leveraging the expertise of each team member, healthcare providers can optimize patient care and outcomes in the context of anemia evaluation.