Dear Editor,

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).

Table 1 . Detection of a high-dose hook effect using 1:10, 1:50, and 1:400 dilutions

Sample number	Neat (nmol/L)	1:10 dilution (nmol/L)	Hook effect detected (1:10; >168.54 nmol/L)	1:50 dilution (nmol/L)	Hook effect detected (1:50; >168.54 nmol/L)	1:400 dilution (nmol/L)
1	48.89	125.79	No hook effect	154.83	No hook effect
2	41.63	1,049.21	Detected	2,412.81	Detected
3	10.15	274.04	Detected	2,901.46	Detected
4	85.34	74.78	No hook effect	85.45	No hook effect
5	55.85	197.98	Detected	247.36	Detected
6	64.27	>1,685.40	Detected	2,429.38	Detected
7	42.74	>1,685.40	Detected	>8,426.97	Detected	21,166.57
8	25.99	80.62	No hook effect	115.79	No hook effect
9	110.28	1,140.84	Detected	1,316.69	Detected
10	43.51	>1,685.40	Detected	6,093.54	Detected
11	20.08	732.08	Detected	5,770.34	Detected
12	89.61	111.97	No hook effect	110.79	No hook effect
13	20.31	282.58	Detected	530.56	Detected
14	30.52	>1,685.40	Detected	>8,426.97	Detected	31,265.56
15	34.17	459.78	Detected	824.10	Detected
16	28.51	>1,685.40	Detected	>8,426.97	Detected	12,656.46
17	34.17	>1,685.40	Detected	>8,426.97	Detected	15,379.49
18	9.56	901.91	Detected	>8,426.97	Detected	21,466.80
19	28.60	1,326.57	Detected	6,596.07	Detected
20	>168.54	>1,685.40	Detected	3,221.52	Detected
21	31.82	1,133.48	Detected	5,771.01	Detected
22	48.18	952.13	Detected	2,553.48	Detected
23	16.34	384.89	Detected	3,048.03	Detected
24	61.57	1,084.78	Detected	1,499.72	Detected
25	8.31	302.87	Detected	2,155.17	Detected
26	28.19	413.99	Detected	613.09	Detected
27	>168.54	>1,685.40	Detected	2,070.11	Detected
28	7.66	265.34	Detected	390.22	Detected
29	10.99	75.96	No hook effect	152.87	No hook effect
30	63.43	>1,685.40	Detected	6,177.75	Detected
31	96.24	395.45	Detected	431.01	Detected
32	31.87	552.81	Detected	1,724.72	Detected
33	77.53	1,501.52	Detected	2,090.51	Detected
34	108.71	224.44	Detected	259.21	Detected
35	>168.54	1,316.69	Detected	1,617.02	Detected

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.

Table 2 . Evaluation of dilutions using the Roche Myoglobin assay on the cobas e602 analyzer

Expected concentration (nmol/L)	Result of diluted specimen* (nmol/L)	Dilution	Final reported result* (nmol/L)	% Recovery
48,744.33	121.86	1:400	48,744.33	100.0
24,375.28	68.00	1:400	27,201.07	111.6
12,190.73	35.86	1:400	14,342.58	117.7
6,098.48	132.19	1:50	6,609.66	108.4
3,052.36	67.31	1:50	3,365.67	110.3
1,529.27	37.29	1:50	1,864.61	121.9
767.75	84.94	1:10	849.44	110.6
386.97	44.03	1:10	440.34	113.8
196.57	23.51	1:10	235.11	119.6
101.40	12.90	1:10	129.04	127.3
53.82	6.75	1:10	67.53	125.5
30.00	4.06	1:10	40.56	135.2
18.09	Not applicable	No dilution	18.93	104.6
12.13	Not applicable	No dilution	12.87	106.1
9.16	Not applicable	No dilution	9.61	104.9
7.70	Not applicable	No dilution	7.87	102.2
6.97	Not applicable	No dilution	7.08	101.6

*The result of the diluted specimen is calculated by dividing the final reported result divided by the dilution performed (e.g., if the final reported result were 500 nmol/L and the dilution performed was 1:10, the calculation would be 500 nmol/L/10=50 nmol/L).

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.

The authors gratefully acknowledge Taylor M. Snow for specimen acquisition.

Hunsaker JJH performed the experimental work, analyzed the data, and drafted the manuscript. All authors contributed to the study conception and design, were involved in clinical evaluation and result interpretation, and read and approved the final manuscript.

None declared.

This study was supported by the ARUP Institute for Clinical and Experimental Pathology.