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Technical Briefs |
Dept. of Pathol., Div. of Clin. Labs., Duke Univ. Med. Center, Durham, NC 27710
a author for
correspondence: fax 919-681-7786, e-mail sedor003{at}mc.duke.edu
Myoglobin is a 17-kDa single-chain hemoprotein found in skeletal and cardiac muscle. This heme protein facilitates the movement of oxygen into cells and provides for local storage of oxygen. Myoglobin is found in the circulation as a result of muscle damage. Several conditions are associated with the release of myoglobin into the circulation, including myocardial infarction, trauma, ischemia, surgery, exercise, rhabdomyolysis, and other myopathy-associated disease states [1–3].
The quantitative measurement of myoglobin in urine is clinically important in diagnosing myoglobinuria, which can subsequently induce acute renal failure, particularly in posttrauma, surgery, and rhabdomyolysis patients. Recent studies have suggested that patients with a urine myoglobin concentration >20 000 µg/L, particularly with a decreased myoglobin clearance rate (<4 mL/min), are at increased risk for decreased renal function (4)(5). Although the mechanism for myoglobin-induced acute renal failure has not yet been elucidated, large amounts of myoglobin present in the tubules may precipitate, particularly under acidic conditions, resulting in increased intratubular pressure and, subsequently, the decreased glomerular filtration rate (6) and (or) free-radical generation from inorganic iron may cause renal damage (7). The identification of the early clinical sequelae of myoglobinuria is important for enabling administration of prophylactic treatment for acute renal failure (8).
Quantitative methodologies, including automated immunoassays, have been advocated and are becoming more widespread for the measurement of urine myoglobin (4)(5)(9) because previous qualitative methods have been shown to be unreliable (9)(10). As with all quantitative assays, it is imperative that appropriate control specimens be analyzed in parallel with all patient specimens. Ideally, such materials should be matrix-matched and treated in a manner identical to that of all patient specimens. The concentration of the material should be at or near those relevant to clinical decision concentrations. In addition, this material should be stable over an extended period of time. To date, a suitable commercial quality-control (QC) material that meets these criteria is not available. The purpose of this study was to evaluate the suitability and long-term stability of an in-house prepared urine pool for use with quantitative urine myoglobin assays.
A pool was prepared from urine obtained from several patients with increased urine myoglobin concentrations. The pool was diluted with buffer [100 mmol/L phosphate buffer, pH 9.0, containing 70 g/L bovine serum albumin (BSA) and 0.1 g/L sodium azide] to a final myoglobin concentration of ~20 000 µg/L. To avoid repetitive freezing/thawing, 100-µL aliquots of the urine myoglobin pool were stored in polyethylene vials at -80 °C. The target concentration of the pool was determined to be 18 800 µg/L as determined by analysis of five aliquots in duplicate over 5 days. A tentative range (mean + 2SD) of 16 200–20 900 µg/L was established with this data and was subsequently used as a target value in assessment of the stability of the pool over the following 6 months. The mean and SD of all QC data were evaluated monthly and at completion of the study and compared with this initial range.
Urine myoglobin concentration was determined by modification of the serum Stratus II immunoassay (Baxter Healthcare Corp.) as previously described (4). As part of our routine workload, the in-house QC material was analyzed in parallel with at least one concentration of a commercial creatine kinase (CK)-MB/myoglobin immunoassay control (Dade International). Both patient urine and urine QC were treated in an identical manner by diluting 1:101 as follows: Twenty microliters of either urine myoglobin QC or patient sample was pipetted into 2 mL of buffer (described above) and vortex-mixed for 30 s before the analysis. This dilution was previously determined to be most effective in detection of clinically relevant urine myoglobin concentrations. Each vial of QC was used once and the remainder discarded. The Dade serum control material was analyzed without dilution.
The values obtained with the in-house urine myoglobin control (n =
199) as well as 194 runs of the commercial serum material over a period
of 179 days are shown in Table 1
. The overall mean + SD of the urine myoglobin QC was 19 200 +
2020 µg/L (CV = 10.5%). There was no difference between the
mean of the initial target determinations and the monthly means
obtained over this time period (data not shown). Similarly, no
difference was observed with the serum control material over this
period.
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Over the 6-month study period a total of 16 outliers were obtained with the in-house QC material and 15 with the Dade material. In 13 of these 16 cases, the results were within allowable limits upon repeat analysis. In many cases, the initial cause for rejection may have been a dilution error by one of 38 technologists who were responsible for specimen analyses. Further corrective action was required in the remaining three cases. In two cases, the outlier was caused by a reagent problem. The final one required recalibration.
The CV of the in-house material was double that of the commercial serum material (10.5% vs 5%). This increased CV may result from the 101-fold dilution before analysis. The fact that the QC was diluted in a manner identical to patient specimens is beneficial in detection of possible dilution errors before analysis by the technologist.
The use of this material offers several advantages for this application. The target concentration of the material is clinically relevant (~20 000 µg/L). Perhaps the greatest advantage lies in the fact that the material is treated identically to patient specimens (as discussed above). This matrix-matched material is useful for detection of errors in dilution and for detection of problems that may occur with the diluent. Secondly, the stability of the material prepared in this manner (at least 6 months) allows for preparation of a large lot, thus obviating the need for weekly preparation of commercially available serum materials, as well as providing consistency for the detection of long-range trends. Finally, the preparation of such an in-house pool has economic advantages.
In summary, we found that urine myoglobin preserved with 70 g/L BSA phosphate buffer, pH 9.0, with 1 g/L sodium azide is stable for at least 6 months when stored at -80 °C. This allows preparation of a QC material suitable for long-term use with newer quantitative urine myoglobin assays.
References
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