Severe muscle injuries can lead to the release of myoglobin into the bloodstream, subsequently filtering into the urine via the kidneys [1, 2]. Elevated myoglobin levels can be detrimental to the kidneys, causing renal damage or failure. Therefore, urinary myoglobin measurement is crucial; however, existing commercially available kits are designed for serum or plasma [3, 4]. High myoglobin concentrations in serum or plasma can induce a high-dose hook effect [5, 6]. Using a serum or plasma myoglobin assay for urine specimens poses a risk of a hook effect owing to high myoglobin concentrations that may be present in urine from patients with severe muscle injuries [1, 7]. Therefore, the predilution of urine specimens would address high concentrations and potential hook effect but may impact accuracy as diluted specimens might approach the assay sensitivity [4].

Various urinary myoglobin dilutions were evaluated using the Elecsys Myoglobin assay (Cat#. 12178214160; Roche Diagnostics; Indianapolis, IN, USA) on a cobas e602 analyzer, which has an analytical measuring interval (AMI) of 1.18–168.54 nmol/L (21–3,000 ng/mL). Results were reported in ng/mL and converted to the International System of Units based on the molecular weight of myoglobin (17.8 kD) [8]. Residual, de-identified, frozen (−20°C) urine specimens were used (IRB protocol #00007275; informed consent waived). Specimens were pH-adjusted to 8–9 at the time of collection using Na₂CO₃. Specimens were diluted onboard with Elecsys Diluent Universal (Roche Diagnostics), and dilutions of 1:400 were previously investigated in accordance with CLSI guidelines (CLSI EP34) [9].

As a preliminary assessment, urine specimens (N=7; 56.19–1,516.85 nmol/L) were retested without dilution (neat) and at a 1:50 dilution. Of these, one produced a suspected hook effect, where the neat result was 100.00 nmol/L but was 1,496.46 nmol/L at a 1:50 dilution. This suggests that a hook effect in urine may occur at lower analyte concentrations than specified for the serum matrix by the manufacturer, as a hook effect does not occur for serum myoglobin concentrations up to 1,685.39 nmol/L [4]. For all remaining specimens (N=6; 23.76–68.03 nmol/L), the results were elevated by approximately 2.5-fold when using a 1:50 dilution, suggesting that the specimens had concentrations low enough to impede a dilution of that magnitude, potentially affecting measurement accuracy.

To investigate whether a lower dilution factor would allow the detection of a hook effect while providing more accurate results, additional urine specimens (N=35; 56.19–33,539.33 nmol/L) were reanalyzed neat, 1:10, and 1:50 dilutions. Specimens below the AMI (<168.54 nmol/L) were categorized as not exhibiting a hook effect. Specimens that produced results above the AMI after a 1:50 dilution (>8,426.97 nmol/L) were further diluted at 1:400. A hook effect was detected in 30 of the 35 specimens at both the 1:10 and 1:50 dilutions, with several specimens requiring a 1:400 dilution to obtain a result (Table 1).

Further, we performed a recovery experiment by serially diluting a high-concentration urine specimen (48,744.33 nmol/L) with a low-concentration urine specimen (6.18 nmol/L). Expected concentrations were calculated based on the high- and low-concentration specimens used. Each serially diluted specimen was tested neat and at 1:10, 1:50, and 1:400 dilutions, and the results were compared to the expected concentrations. However, the dilutions were only performed when the anticipated concentration was within the AMI of the assay (1.18–168.54 nmol/L). The acceptable limit of recovery (80.4%–119.6%) was based on a previously reported total allowable error for myoglobin in serum [10]. For each of the 1:10, 1:50, and 1:400 dilutions, recoveries exceeded 119.6% when the diluted specimen result (prior to correcting for the dilution factor) was 4.06–37.29 nmol/L (Table 2). The manufacturer recommends that the serum concentration after dilution should exceed 2.81 nmol/L; nevertheless, we observed that a higher concentration may be necessary for diluted urine specimens. Diluted results were within acceptable limits of recovery when the diluted specimen produced results between 35.86–132.19 nmol/L (Table 2). These findings demonstrate that accuracy reduces with decreasing myoglobin concentrations owing to the dilution process. Therefore, a smaller predilution factor (1:10) is recommended with additional dilutions (1:50 and 1:400) as necessary to detect a hook effect while avoiding inaccuracies for low-concentration specimens.

Overall, these findings support a predilution step when analyzing urine specimens using the Roche Myoglobin assay. A hook effect was observed in urine at lower concentrations than the serum hook effect threshold reported in the assay package insert. We conclude that to effectively use a serum myoglobin assay to measure myoglobin in urine, an appropriate predilution factor should be carefully validated to enable the detection of a high-dose hook effect while producing results with good accuracy and clinical acceptability.