Calculated parameters for the diagnosis of Wilson disease

**Background:** Wilson disease (WD) is a rare autosomal recessive disorder caused by ATP7B gene mutations, leading to impaired copper incorporation into caeruloplasmin and toxic free copper accumulation. Diagnosis is challenging because clinical features and biochemical markers are often atypical or within normal limits. Direct free copper measurement is cumbersome and not widely available. Calculated parameters such as non-caeruloplasmin copper (NCC), NCC percentage (NCC%), copper-caeruloplasmin ratio (CCR), and adjusted copper have been proposed but their clinical utility remains uncertain.

**Methods:** This retrospective study recruited 213 patients (median age 35 years, 60.6% male) who presented to University of Malaya Medical Centre between 1 January 2015 and 31 December 2017 with unexplained hepatic, neurologic, or neuropsychiatric presentations and/or were worked up for WD. WD was confirmed in 55 patients (25.8%) and 158 (74.2%) were WD-free. Serum caeruloplasmin was measured by immunoturbidometric assay (Abbott ARCHITECT ci4100) and total copper by ICP-MS. NCC in molar units (NCC1) = total copper (μmol/L) – [47 × caeruloplasmin (g/L)]; NCC in mass units (NCC2) = total copper (mcg/L) – caeruloplasmin-bound copper (mcg/L); NCC% = [NCC (μmol/L)/total copper (μmol/L)] × 100; CCR (μmol/g) = [total copper (μmol/L) × 0.132]/caeruloplasmin (g/L). Adjusted copper was derived using the formula: total copper (μmol/L) – [63.5 × caeruloplasmin (g/L)] + 17.5. A discriminant function was derived: Discriminant function = −3 + [14 × caeruloplasmin (g/L)] + [0.017 × copper (μmol/L)]. A validation cohort of 58 subjects (2018-2019) was used to test the discriminant function.

**Key Results:** Caeruloplasmin, total copper, NCC1, NCC2, NCC%, CCR, and discriminant function medians were significantly lower in WD vs non-WD (all P<0.001 except NCC% and CCR P=0.012). Adjusted copper was not significantly different (P=0.697). Spearman correlation with WD was strongest for discriminant function (−0.731, P<0.001) and caeruloplasmin (−0.731, P<0.001). From ROC analysis, discriminant function and caeruloplasmin shared the highest AUC of 0.982, followed by total copper (0.965). The discriminant function cut-off of <−0.95 yielded specificity 99.4%, sensitivity 98.2%, and overall accuracy 99.1%. Caeruloplasmin <0.14 g/L gave specificity 98.1% and sensitivity 98.2% (accuracy 97.7%), outperforming the <0.20 g/L cut-off (specificity 84.2%, accuracy 87.8%). Total copper <11 μmol/L gave specificity 86.7% and sensitivity 96.4% (accuracy 89.2%). NCC1 at the European cut-off (<1.6 μmol/L) showed 84.2% specificity and 70.9% sensitivity (accuracy 80.3%), while the American cut-off (<3.9 μmol/L) showed 51.3% specificity and 85.5% sensitivity (accuracy 59.6%). NCC2 (<200 mcg/L) showed 51.9% specificity and 85.5% sensitivity (accuracy 61.0%). NCC% (<10%) showed accuracy 75.1%, and CCR (<8 μmol/g) showed accuracy 59.6%. In the validation cohort (n=58; 18 WD, 40 non-WD), both the discriminant function and caeruloplasmin <0.14 g/L achieved 100% classification accuracy, while caeruloplasmin <0.20 g/L achieved 91.4% accuracy.

**Clinical Implications:** The discriminant function combining serum caeruloplasmin and total copper provides excellent diagnostic performance for WD and can be easily incorporated into clinical calculators. A caeruloplasmin cut-off of <0.14 g/L should be considered instead of the traditional <0.20 g/L for improved diagnostic accuracy. Calculated parameters including NCC (both molar and mass units), NCC%, CCR, and adjusted copper are not helpful for diagnosing WD and should not be relied upon clinically. The study is limited by its retrospective design, single-centre setting, small sample size due to disease rarity, and lack of genetic confirmation.