Serum Iron Reference Ranges: Ferrozine vs. TVTP Methods
The ferrozine colorimetric method and turbidimetric (TVTP) assays measure serum iron using fundamentally different analytical principles, yielding method-specific reference ranges that are not interchangeable—each laboratory must establish its own reference intervals based on the specific assay platform used.
Why Reference Ranges Differ Between Methods
Analytical Principles Create Method-Specific Results
The ferrozine method measures iron through colorimetric detection after proteins are precipitated with trichloroacetic acid-hydrochloric acid, Fe³⁺ is reduced to Fe²⁺ by sodium ascorbate, and Fe²⁺ reacts with ferrozine to form a lavender complex measured spectrophotometrically at 562 nm 1
Each serum marker has a defined reference range, lower detection limit, and diagnostic cutoff that must be determined in rigorous clinical studies comparing new assays with a criterion standard test 2
Methodologically, assays may be fairly similar from laboratory to laboratory, but there are no internationally standardized reference ranges for all assays 2
Critical Analytical Differences
The ferrozine assay demonstrates linearity to at least 10 mg/L (179 μmol/L) with day-to-day precision (CV) of 2.4% for 1.03 mg/L samples and 1.9% for 2.13 mg/L samples 3
The ferrozine method can detect iron in amounts ranging between 0.2 and 30 nmol with equal detection of ferrous and ferric iron, unaffected by other divalent metal cations 4
Bilirubin and copper do not interfere with ferrozine assays; hemoglobin and gross lipemia interfere only slightly 3
Establishing Laboratory-Specific Reference Ranges
Mandatory Local Validation
Reference ranges must be determined through clinical studies that construct receiver operating characteristic (ROC) curves to determine optimal values based on analysis of sensitivity, specificity, and clinically meaningful likelihood ratios 2
The National Academy of Clinical Biochemistry suggests that reference ranges should set the upper limit of normal at the 99th percentile for the normal healthy population 2
The lower detection limit should be set at the level at which a 10% coefficient of variation is maintained; below this range, results must be considered unreliable 2
Typical Ferrozine Reference Ranges (Not Universal)
Published ferrozine studies report measurements in ranges of 0.338–5.916 μmol/L for non-transferrin-bound iron with within-run coefficients of variation of 0.812–2.37% 5
Standard serum iron measurements using ferrozine show linearity to 72 μmol/L with accuracy of 99.3–101.4% and precision within-run of 0.73% 6
Clinical Implications for Iron Status Assessment
Transferrin Saturation Remains the Key Parameter
Transferrin saturation (TSAT) calculated as (serum iron × 100) ÷ TIBC reflects iron readily available for erythropoiesis and is more clinically meaningful than absolute serum iron values 7
TSAT <16–20% confirms iron deficiency regardless of the assay method used to measure serum iron 7
Serum iron shows diurnal variation, which may affect interpretation if not collected at consistent times 7
Complementary Testing Required
Neither serum iron nor ferritin alone provides a complete picture of iron status—they must be interpreted together 7
Serum ferritin <15 μg/L has 99% specificity for iron deficiency and is the most specific test in the absence of inflammation 2
When ferritin and TSAT results are discordant, the percentage of hypochromic red cells or soluble transferrin receptor may help assess iron status 7
Critical Pitfalls to Avoid
Do not assume reference ranges from one laboratory or assay method apply to another—each facility must validate its own cutoffs based on the specific analytical platform 2
Do not rely on serum iron alone for diagnosis—calculate transferrin saturation and measure ferritin to assess both iron availability and storage 7
Do not ignore the timing of blood collection—serum iron exhibits diurnal variation that can affect results by 30–50% 7
Ferritin is an acute-phase reactant that rises during inflammation, infection, or tissue damage, potentially masking iron deficiency even when serum iron is low 2