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Technical Briefs |
a author for correspondence: fax 205-975-4468, e-mail hortin{at}wp.path.uab.edu
Dept. of Pathol., Univ. of Alabama at Birmingham, 618 S. 18th St., Birmingham, AL 35233
The reaction of picric acid (2,4,6-trinitrophenol) with creatinine under strongly alkaline conditions, originally described by Jaffe >100 years ago (1), remains the most common means of measuring creatinine in clinical samples. However, picric acid reacts with many compounds besides creatinine to yield colored products (1)(2)(3)(4)(5). Ketones, glucose, proteins, cephalosporins, and other compounds react to yield positive interferences; also, decreases in the color of bilirubin and hemoglobin under the reaction conditions yield negative interferences (2)(3)(4)(5)(6)(7). To counter these interferences, analysts have tried many variations of the Jaffe reaction, including modifications of sample preparation, addition of oxidizers to remove interferents, solvent extraction of interferents, pH adjustment of the reaction, continuous-flow dialysis, and kinetic measurement of the reaction (2)(3)(4)(5). In kinetic methods, the most common approach to decreasing interferences, the timing of the absorbance readings can be selected to minimize the effects of components that react faster or more slowly than creatinine. For most samples, these modifications of the alkaline–picric acid methods have improved the specificity of measurement. Results have become closer to the "true" creatinine values determined with reference methods (2)(3)(4)(5)—improvements in measurement that have led to a downward adjustment of creatinine reference intervals by ~3 mg/L (8)(9) (to express creatinine in µmol/L, multiply mg/L by 8.84).
Despite improvements, substantial problems of interference with creatinine measurements remain (2)(3)(4)(5). Recently, a rate-blanking method has been introduced that compensates for absorbance changes of bilirubin and hemoglobin under the strongly alkaline reaction conditions (6)(7). Although this modification decreases the interference from bilirubin and hemoglobin, we find that it introduces a new interference from lipemic samples.
Introduction of a new rate-blanked compensated creatinine method from Boehringer-Mannheim Corp. (Indianapolis, IN) was considered advantageous in our laboratory because the method decreases negative interference from chromogens such as bilirubin and hemoglobin (6)(7). However, we noted unexpectedly low creatinine values for several patients after introduction of the method. In our clinic laboratory, occasional samples—all lipemic—yielded negative values, and other samples yielded creatinine values substantially below what was clinically expected from the patient's previous values, body mass, and clinical status. Use of some of these measured serum creatinine values to calculate creatinine clearance yielded clearance values of 200 mL/min per 1.73 m2 body surface area or more, which are inconsistent with usual reference values (2)(10). Nonetheless, the manufacturer claims the method is not subject to significant interference from lipemia, as tested by adding a synthetic lipid emulsion to samples to give a final triglyceride concentration of as much as 10 g/L (7). At this concentration, lipid emulsions showed a small negative interference (~1 mg/L decrease in creatinine) for creatinine concentrations near the upper limit of normal (~13 mg/L). Any effect <10% was considered not significant in the evaluation study (7).
To investigate the problem of interference by lipemic samples, we
compared creatinine values obtained with three different methods—the
rate-blanked compensated method from Boehringer-Mannheim, a compensated
kinetic method from Boehringer-Mannheim, and an enzymatic method on the
Vitros 700 analyzer from Johnson and Johnson (Rochester, NY)—for 17
lipemic samples received in our clinic laboratory during 1 week. The
two Jaffe reactions were performed simultaneously with the same lot of
reagents on different channels of the same Hitachi 747 analyzer.
Lipemic samples were identified by the lipemic index reported by the
instrument (an index of changes in absorbance measurements attributable
to light scattering by lipoprotein particles of a specimen diluted in
isotonic saline). Because the lipemic index depends on lipoprotein size
as well as quantity, it does not correlate exactly with triglyceride
concentrations. We also determined triglycerides in some samples as
another measure of lipemia, using the Hitachi 747 and a coupled
enzymatic method from Boehringer-Mannheim without blanking for
glycerol. As listed in Table 1
, the rate-blanked compensated method (Jaffe 1) provided values
that averaged 5 mg/L lower than another compensated method (Jaffe 2)
and 4 mg/L lower than the enzymatic method. For some samples, the
difference between the rate-blanked method and the other comparative
methods was as much as 10 mg/L. However, there was good agreement
between the compensated method and the enzymatic method for the lipemic
samples.
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The magnitude of interference by lipemia in the rate-blanked creatinine method did not correlate closely with the lipemic index or triglyceride concentration. One of the problems with this interference is that it is difficult to identify a cutoff point below which interference from lipemia does not occur. We routinely analyze by an alternative method samples with a lipemic index >65; ~0.5–1% of clinic samples have a lipemic index above this value, so several samples per day require alternative analysis. The frequency of lipemic samples from our clinic is likely to be high relative to that for inpatient settings, because many of our samples are from nonfasting patients. This interference has a large potential clinical impact on the measurement of creatinine in the critical range of 5–15 mg/L, where highly accurate measurements of serum creatinine are needed for calculating creatinine clearance, monitoring renal transplant patients, and detecting early stages of kidney dysfunction from diabetes and other etiologies (2)(10).
Differences between the two Jaffe methods for lipemic samples were not related to differences in calibration. The same calibration material was used, and method comparison studies showed close agreement for nonlipemic samples; analysis of 17 samples with triglyceride <4 g/L (lipemic index range 5–63, mean 20) during the same week as analysis of lipemic samples showed no difference >1 mg/L for comparative analyses by the two methods.
The interference of lipemic samples in the rate-blanked compensated method appeared to relate to nonlinear changes in measured absorbance by lipoproteins during the analysis. After addition of the first reagent containing NaOH, there was a curvilinear increase of absorbance—rapid at first during the period used to rate-blank the absorbance changes in the sample, but slowing before addition of the second reagent (containing picric acid). Thus, the blanking for baseline absorbance changes was excessive. For the rate-blanking to be accurate, the baseline absorbance changes (without picric acid) would need to be linear throughout the entire reaction. Identifying the problem as involving the rate-blanking step explains why a simpler compensated method using the same reagents does not have similar lipemia interference. Ways to avoid the lipemia interference include using Jaffe methods without rate blanking, using enzymatic methods, or storing samples to allow separation of lipoproteins.
Interferences from lipemia are difficult to study, given the lack of a readily available source of lipoproteins for adding to samples. Effects of lipemia are often estimated by using synthetic lipid emulsions, which may or may not accurately replicate the physicochemical properties of actual lipoproteins during analyses. With regard to interference from lipoproteins in a creatinine assay, we found that the effects of lipemia disappeared when samples were stored overnight, even when samples were vigorously vortex-mixed to try to disperse the lipoproteins in the samples. Use of stored samples, as commonly done in many method-evaluation studies, would not detect the interference found in freshly drawn samples. This example illustrates the need to use actual lipemic samples freshly drawn from human subjects when investigating interference from lipemia.
Boeringherr-Manheim Corp., Lab Diagnostics, 9115 Hague Road, P.O. Box 50446, Indianapolis, IN 96250-0446
Hortin and Goolsby have demonstrated not only that the Jaffe creatinine assay remains imperfect (despite all efforts to confer specificity) but also that the interferograph model for assessing interferences has yet another shortcoming. We have previously recognized that the age of the hemolysate can influence hemoglobin interference and that the type of bilirubin can influence the icterus interference. Now, apparently, in addition to the concentration and type of lipids in the sample, even the age of the lipids that give rise to lipemia may also be important in predicting interferences based on turbidity measurements. Clearly, as valuable as the serum indexes are, they are still only indicators of possible interferences, and not quantitative estimates.
Studies are currently under development to determine what, if any, improvement can be made in the current Jaffe creatinine assay that will preserve the improved performance vis-à-vis bilirubin that the rate-blanking introduced, while minimizing this newly recognized influence of endogenous lipemia. We would like to determine more specifically which components of the sample lipids are responsible for the behavior. But we recognize that it is also possible, based on the poor correlation of the bias to the sample lipemia, that the actual interference is due to some other sample constituent, and that the lipemia is simply a marker for this interfering substance. Upon completion of these studies, operating parameters and (or) assay limitation statements will be appropriately amended.
Correction
In the Beckman Conference paper by J.B. Silkworth and J.F. Brown, Jr., entitled "Evaluating the impact of exposure to environmental contaminants on human health," 1996;42:1345–9, insert the underlined words:
p. 1345, line 8 of the abstract: " ... are also produced, often to greater extents, by naturally occurring constituents ... ."
p. 1345, line 7 of the article: " ... (DDT), might have on wildlife and even on human health. The press then popularized the issue and may have been more effective at promoting fear than understanding."
p. 1346, first column, line 9: " ... specific cytoplasmic receptor protein known as the aromatic hydrocarbon receptor."
p. 1348, first column, line 11: " ... effects produced in humans by the synthetic chemicals of this type are no different from those produced—generally to a much greater extent—by long-accepted natural components of the human environment."
On page 1345, second column, end of first paragraph, replace " ... lowest observable effect concentration." with " ... lowest observable effect level."
The final sentence on page 1345 should read: "Some of these congeners are of environmental concern because they tend to bioaccumulate and to produce toxicological effects in heavily dosed test animals 7,9, including immunotoxicity, birth defects ... ."
References
The following articles in journals at HighWire Press have cited this article:
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  G. L. Myers, W. G. Miller, J. Coresh, J. Fleming, N. Greenberg, T. Greene, T. Hostetter, A. S. Levey, M. Panteghini, M. Welch, et al. Recommendations for Improving Serum Creatinine Measurement: A Report from the Laboratory Working Group of the National Kidney Disease Education Program Clin. Chem., January 1, 2006; 52(1): 5 - 18. [Abstract] [Full Text] [PDF]
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J. A. Bornhorst, R. F. Roberts, and W. L. Roberts Assay-Specific Differences in Lipemic Interference in Native and Intralipid-Supplemented Samples Clin. Chem., November 1, 2004; 50(11): 2197 - 2201. [Full Text] [PDF]
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 P J Twomey, A C Don-Wauchope, and D McCullough Unreliability of triglyceride measurement to predict turbidity induced interference J. Clin. Pathol., November 1, 2003; 56(11): 861 - 862. [Abstract] [Full Text] [PDF]
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