1 Department of Medicine, Division of Nephrology and 2 Departments of Pathology, Oncology and Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Correspondence and offprint requests to: Derek M. Fine, MD, Division of Nephrology, Johns Hopkins University School of Medicine, 1830 E. Monument Street, Suite 416, Baltimore, MD 21205, USA. Email: dfine1{at}jhmi.edu
Abstract
Background. In the diagnosis of rhabdomyolysis, the microconcentrator qualitative assay for urine myoglobin (uMb) is often used as a screening tool. The accuracy and clinical utility of this assay in screening patients with rhabdomyolysis have not been examined.
Methods. We conducted a retrospective analysis of the relationship between creatine kinase (CK), serum myoglobin (sMb), the urine qualitative assay for myoglobin and the semi-quantitative assay for urine haem pigments (uH) in patients evaluated for rhabdomyolysis.
Results. There were 673 patients with CK and uMb recorded on the same day. The uMb assay had a sensitivity of only 26.4% [95% confidence interval (CI): 23.129.7%] and specificity of 96.8% (95% CI: 95.598.1%) for the detection of severe rhabdomyolysis, defined as a CK >10 000 U/l. SMb and CK measured simultaneously in 83 patients were highly correlated (R2 = 0.72 for log-transformed values), suggesting that the negative uMb test was not a result of the absence of sMb. In 241 patients who had CK, uMb and uH measured on the same day, the presence of moderate or large uH in the absence of haematuria, indicating presence of myoglobinuria, had a sensitivity of 81% (95% CI: 7686%) for the detection of CK >10 000 U/l vs a sensitivity of 22% (95% CI: 1727%) for the uMb assay.
Conclusions. The microconcentrator-based uMb assay has a poor and clinically inadequate sensitivity in the detection and diagnosis of rhabdomyolysis.
Keywords: creatine kinase; diagnosis; microconcentrator; myoglobin; rhabdomyolysis; urine
Introduction
Tests utilized in the evaluation of rhabdomyolysis include serum myoglobin (sMb), serum creatine kinase (CK), urine myoglobin (uMb) and urine haem pigments (uH) [15]. The test most commonly used in the diagnosis of rhabdomyolysis is serum CK. The CK-MM is the dominant isoform in skeletal muscle and is the most sensitive test to confirm the diagnosis of muscle breakdown [2,3]. Myoglobin, a 16.9 kDa oxygen-binding haemoprotein, is found in all striated muscles and appears in the circulation within a few hours after cardiac or skeletal muscle damage. Testing for sMb is thought to be less sensitive but more specific than serum CK for muscle breakdown, because it may be rapidly eliminated by renal and extra-renal mechanisms [3,6].
The development of acute renal failure in the presence of rhabdomyolysis with significantly elevated CK is well described [3,79]. The pathogenesis of rhabdomyolysis-induced renal failure is multifactorial, including direct tubular toxicity of myoglobin, formation of myoglobin casts within tubules and vasoconstrictive effects of myoglobin [10].
Although the diagnosis of rhabdomyolysis is often suggested by clinical circumstances and is confirmed with serum testing, qualitative assays for urine myoglobin and haem pigments, having excluded haematuria, are frequently employed as screening tools. Identification of myoglobin in the urine by centrifugation through a microconcentrator membrane has been described to qualitatively assess the presence of uMb [11,12]. Using this technique, urine myoglobin (16.9 kDa) and haemoglobin (64 kDa) can be separated by centrifugation through a microconcentrator membrane with a 30 kDa cut-off. This separation allows for individual analysis of the haemoglobin-free urine filtrate for the presence of haem pigment (i.e. myoglobin), using a peroxidase-based dipstick.
In our practice, when evaluating patients with clinically diagnosed rhabdomyolysis, this qualitative assay frequently provided negative results. This observation led us to question the accuracy and the utility of this assay as a screening test. There is literature indicating that this assay may not be accurate in the detection of myoglobinuria [12,13]. In an early study of the microconcentrator membranes, the authors commented in their conclusion that for reasons unknown some devices retained both myoglobin and haemoglobin [12]. Indeed, in a later study, it was observed that the amount of myoglobin recovered in 25 urine samples using this technique varied from <1 to 38% [13].
Early aggressive management of rhabdomyolysis has been shown to limit development of acute renal failure [14,15] and, therefore, prompt and accurate diagnosis is essential. As the use of the uMb microconcentrator qualitative assay to detect myoglobinuria may misdiagnose such patients, we sought to examine the accuracy and clinical utility of this uMb assay in the screening of patients with rhabdomyolysis.
Subjects and methods
We conducted a retrospective analysis of the inter-relationship between serum CK, sMb, the urine qualitative assay for myoglobin and the semi-quantitative assay for haem pigments. With the approval, of The Johns Hopkins Medicine Institutional Review Board we searched the hospital laboratory database for patients evaluated for rhabdomyolysis over a 5 year period between January 1996 and December 2000. Data were extracted in three groups listed below.
Included in group 1 were all patients who had a serum CK and uMb measured on the same day during the above time-period. These patients were selected to explore the relationship of serum CK to the uMb qualitative assay. In this and subsequent groups, a CK level of >10 000 U/l was the primary cut-off used in sensitivity testing. A CK level of >10 000 U/l was chosen as an enzyme elevation at which one is likely to have a high risk of developing acute renal failure [16]. Secondary analysis was performed using different cut-offs to further assess the accuracy of the diagnostic test. The relationship between serum and urine myoglobin was assessed separately.
Group 2 patients, selected to explore the relationship of CK to sMb, had a serum CK and sMb measured on the same blood draw.
Group 3 was structured to assess the relative accuracy of the uMb assay vs presence of uH (in the absence of haematuria) in predicting rhabdomyolysis. This group included those with severe rhabdomyolysis identified as those with peak CK of >10 000 U/l who had serum CK, uH and uMb measured in the same 24 h period.
The presence of uH was assessed using the N-Multistix Reagent Strip (Bayer, Elkhart, IN) on the Clinitek-200+ instrument (Miles, Elkhart, IN) and reported as none, trace, small, moderate or large. This reagent strip detects haem content by a peroxidase reaction and, therefore, does not distinguish myoglobin from haemoglobin.
UMb was reported as negative or positive. The uMb assay was performed in the Johns Hopkins Hospital Department of Pathology laboratory by the following protocol. If the Bililabstix (Bayer) urine dipstick is positive for haem pigments, 7 ml urine is centrifuged between 900 and 1200 g for a minimum of 5 min. If the dipstick is still positive for haem pigments, 2.5 ml supernatant is placed in a Centricon YM-30 microconcentrator and centrifuged between 2500 and 3500 g for 15 min using the Sorvall RT 6000 refrigerator centrifuge (temperature 410°C). The same peroxidase-based Bililabstix dipstick is used on the filtrate portion of the sample and the result reported as negative or positive for myoglobin. All analyses were performed using the Stata 6.0 statistical package (Stata Corp., College Station, TX, USA).
Results
There were 673 patients identified with CK and uMb recorded on the same day (group 1). As shown in Table 1, the uMb assay was insensitive in detecting severe rhabdomyolysis in subjects with a CK >10 000 U/l. This sensitivity of 26.4% [95% confidence interval (CI): 23.129.7%] was minimally improved to 29.7% (95% CI: 26.233.2%) when using an even more severe elevation of CK >30 000 U/l as a cut-off. The sensitivity worsens as the CK cut-off is lowered. It dropped to 21% for a CK cut-off of 5000 U/l and to 13% for a cut-off of 1000 U/l.
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We then set out to determine whether the poor sensitivity of the uMb assay was due to the absence of myoglobin from the urine. Patients were therefore identified who had their CK, uMb and uH measured in the same 24 h period (group 3). Table 2 demonstrates that in the absence of haematuria (hence reflecting presence of myoglobinuria) the uH assay has a greater sensitivity than uMb for the detection of rhabdomyolysis (81vs 22%).
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In the 241 group 3 patients, the likelihood ratios for the detection of CK >10 000 U/l with uH categorized into in four groups: negative/trace, small, moderate or large (Table 3). The data show that uH in the large range is highly predictive of CK >10 000 U/l, while uH in the negative/trace range has a significantly low likelihood for predicting this level of rhabdomyolysis. In the intermediate ranges (small and moderate), there is little predictive value of this assay.
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Our analysis provides the first clinical evidence that the microconcentrator-based uMb assay in the detection of severe rhabdomyolysis, defined by CK >10 000 U/l, has a low and inadequate sensitivity. We have shown that this lack of sensitivity is not due to the absence of sMb, as there is a strong correlation between sMb and serum CK. This is further supported by the poor sensitivity, albeit in a limited sample, in detecting uMb by this assay, even in the presence of confirmed significantly elevated sMb levels. The presence of moderate to large amounts of uH, in the absence of haematuria, which serves as an indirect measure of myoglobinuria, is far more frequently present than a positive uMb test. This suggests that the poor sensitivity of the assay is not likely due to the absence of myoglobin in the urine.
The insensitivity of this assay is probably a consequence of an intrinsic property of the assay. The data indicate that there is myoglobin present in the urine and that it is frequently not detected. We suggest that this is due to its removal by the microconcentrator membrane during the filtration process, despite the expected 30 kDa cut-off. This finding was also reported in a non-clinical setting by Loun et al. [13], whose group analysed ultrafiltration discrepancies in the uMb assay in 25 urine samples in the laboratory. The group found marked differences between myoglobin concentrations pre- and post-ultrafiltration and found that most of their samples had recoveries of myoglobin <5% (range: 138%). Based on their findings they speculated that such discrepancies could be clinically important in the detection of myoglobinuria and questioned the use of the assay. Our findings support their contention that this assay has significant clinical deficiencies and these deficiencies make this assay of little clinical use.
Loun et al. [13] suggested a few possibilities for this discrepancy, such as myoglobin complexing with protein, obstruction of the filter membrane with particulate matter or direct complexing of myoglobin with the membrane filter. They concluded that the discrepancy in the myoglobin level pre- and post-ultracentrifugation was due to non-specific binding of myoglobin to the filter membrane and/or a lack of uniformity to the membrane pores. The initial centrifugation of urine in the assay used in our laboratory should remove any debris which may obstruct the membrane and, therefore, complexing of myoglobin with other proteins is unlikely. We suggest that the discrepancy in the myoglobin level pre- and post-ultracentrifugation is more likely due to myoglobin binding to the membrane.
It is unlikely that TammHorsfall protein binding to myoglobin in the presence of acid urine is responsible for the insensitive assay in our study, as even in those with high urine pH the sensitivity was not improved. Furthermore, Loun et al. [13] obtained the results in their experiments showing poor myoglobin recovery, despite alkalinizing all samples prior to performing the assay.
A possible explanation for the negative dipstick findings could be the rapid clearance of myoglobin from the serum. However, as we have demonstrated, this is unlikely since the serum CK correlated strongly with sMb levels. In addition, the presence of uH by dipstick in the absence of haematuria prior to centrifugation would support the presence of uMb.
The key in the treatment of rhabdomyolysis is early institution of appropriate therapy. Several investigators have demonstrated that victims of crush injuries that resulted in traumatic rhabdomyolysis, who received aggressive fluid repletion early in the course of their evaluation, were less likely to develop renal failure [14,15]. Implicit in the requirement of early treatment is the requirement for early detection. These studies have emphasized the need for an adequate screening test for the detection of rhabdomyolysis and we have, in turn, demonstrated that the microconcentrator uMb assay used by our institution is not sufficiently sensitive as a screening tool.
Though a more sensitive assay, the detection of myoglobinuria by detection of haem in the absence of haematuria is not sufficiently diagnostic with only 81% sensitivity. The lack of adequate sensitivity for the diagnosis of rhabdomyolysis by this assay has been demonstrated previously [3].
There are several potential limitations of this study. First, there could be a discrepancy between the technique or protocol used by the groups that initially described the assay and those utilized by our laboratory. The uMb assay utilized at this institution is consistent with that used in published studies that validated the use of ultrafiltration for detecting myoglobinuria [11,12]. Second, we do not have data on the development of renal failure in the studied population. The absence of uMb by the microconcentrator assay could be explained by the finding that renal clearance of myoglobin may be low in renal failure [17]. However, as we have described above, the presence of uH, as a surrogate marker of uMb in the setting of rhabdomyolysis, indicates that myoglobin is, indeed, present. Third, our study was a retrospective analysis of laboratory data and as such was not designed to address the impact of misdiagnosis by this assay on clinical outcomes. However, based on the severity of rhabdomyolysis not detected by this assay in our study, we believe that the microconcentrator uMb assay is likely to critically delay appropriate therapy, if used as an isolated diagnostic test.
In conclusion, in our analysis of patients who were evaluated for rhabdomyolysis, we have found that utilizing qualitative uMb by the uMb microcentrifugation assay is not sufficiently accurate to be clinically useful. Additionally, we found that the uH assay in the absence of haematuria (as an indicator of myoglobinuria) is a better predictor of high CK and rhabdomyolysis, though not sufficiently diagnostic. The serum CK, which is closely and significantly correlated with sMb, remains the most clinically useful indicator of rhabdomyolysis.
Conflict of interest statement. None declared.
References
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