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Letters to the Editor |
lmaz
an YücelaMinistry of HealthAnkara Hospital Clinical Biochemistry Laboratory Ankara 06340, Turkey
aAuthor for correspondence. Fax 90-312-3621857; e-mail doyucel{at}yahoo.com.
To the Editor:
The presence of hemolyzed erythrocytes is common in urine and spinal fluid samples, and the released cellular contents, primarily hemoglobin (Hb), may affect protein measurements. Hematuria may be seen with or without the presence of other proteins in urine. Similarly, in hemorrhagic stroke, erythrocytes in the cerebrospinal fluid (CSF) begin to lyse within 2–3 h (1). Hb can be measured by protein assays, but may produce spectral and chemical interferences.
We investigated the effect of hemolysis on Pyrogallol Red (PYR) (2), benzethonium chloride (BTC)(3), and benzalkonium chloride (BC) (4) methods for measurement of protein. We prepared 3 urine and 3 CSF pools with different protein concentrations and added hemolysates at Hb concentrations of 48–3840 mg/L for urine and 186-5580 mg/L for CSF samples as described previously (5). We measured Hb hemolysate concentrations with a Coulter® LH 750 hematology analyzer.
For the PYR method (2), we included sodium dodecyl sulfate at 25 mg/L (6) and measured absorbances with a Shimadzu UV-1208 spectrophotometer. The BC and BTC methods were performed on a Roche Modular P analyzer with bichromatic analysis at 450 and 700 nm and a sample blank (4). In-house reagents were used for all of the methods. The protein concentrations were determined in duplicate by each method on the same day.
Hemolysis affected the PYR and BTC methods at an Hb concentration of 48 mg/L, the lowest concentration tested (see Fig. 1
and Table 1 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue1/), consistent with the report that the PYR method gives increased protein results at Hb concentrations 
16 mg/L (7). These concentrations were considerably lower than the Hb concentrations (
200 mg/L) required for hemolysis to be detected visually (8). PYR was the method most affected by hemolysis in our study, and PYR results were increased 30% even at 48 mg/L Hb. For reasons that remain unclear, BC was not affected by hemolysis at low Hb concentrations (<192 mg/L for urine or <372 mg/L for CSF samples; Fig. 1
), even in the pool with the lowest protein concentration.
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Freedom from an effect of Hb on CSF and urine protein assays may be useful for clinical assays of urine and CSF because the presence or absence of proteins other than Hb provides an indication of the localization and type of the defect in the urinary tract. For example, cystitis, urinary tract neoplasia, and urinary tract stones are associated with hematuria without concomitant proteinuria. Similarly, for CSF protein assays, intracerebral hemorrhage is associated with high erythrocyte count and Hb concentration and a mild increase in CSF protein concentration. In contrast, subdural hemorrhage is associated with parallel increases in protein concentration and erythrocyte count or Hb concentration.
In conclusion, hemolysis interference in urine and CSF is method dependent, and microhemolysis may contribute to unexpected urine and CSF protein results in laboratories using BTC and PYR, whereas Hb does not seem to interfere in the BC method.
References
ilmaz FM, Çelebi N, Yücel D. Automated turbidimetric benzalkonium chloride method for measurement of protein in urine and cerebrospinal fluid. Clin Chem 2004;50:1450-1452.The following articles in journals at HighWire Press have cited this article:
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  E. J Lamb, F. MacKenzie, and P. E Stevens How should proteinuria be detected and measured? Ann Clin Biochem, May 1, 2009; 46(3): 205 - 217. [Abstract] [Full Text] [PDF]
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 J. L. Carter, C. R. V. Tomson, P. E. Stevens, and E. J. Lamb Does urinary tract infection cause proteinuria or microalbuminuria? A systematic review Nephrol. Dial. Transplant., November 1, 2006; 21(11): 3031 - 3037. [Full Text] [PDF]
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, pool 2; 
, pool 3. The dashed line in A is the 10% error limit.