Bilirubin on lyophilized specimens.

Q We are unable to obtain consistent and comparable bilirubin levels using lyophilized pediatric bilirubin standards and different instruments and reagent systems. While different systems and reagents give comparable readings for the pediatric controls (lyophilized), the patient values vary by 1 to 4 mg/dl, particularly at levels above 14 mg/dl. This is the case whether or not a blanking method is used. Since other hospitals in our community use several methods, there is incredible inconsistency in the results. How do you suggest we rectify this problem?

A One way I judge the complexity of a test is to see how many papers on it are in my file folder. I have 3.5 inches of papers in the bilirubin file, 2.5 inches for creatinine, and 1 inch for chloride. Although the total bilirubin test has been around for 100 years since Ehrlich described it, and for more than 50 years since Jendrassik published the most commonly used method, it is still tricky and not well characterized.

The total bilirubin test measures bilirubin in many forms including those conjugated to glucuronides and those bound to proteins. The large number of methods--with differences in accelerators, pH conditions, coupling agents, agents for reducing turbidity, time, and temperature conditions--have led to inconsistent results.

Standardization also continues to be a problem. Over 20 years ago, the Bilirubin Standards Subcommittee of the American Academy of Pediatrics proposed a method for making standards in serum. Today, various standards are still used. Blijenberg[1] found that there were differences between the stated values of many of the standards and the values found when they were analyzed by a reference method.

Factors important in the stability of bilirubin reference standards are composition and purity of the bilirubin preparation, pH at which it is maintained, amount and type of protein matrix, and exposure to light during manufacture, storage, and use.

CAP surveys illustrate that there are consistent differences between methods and instruments when the same survey sample is analyzed. For example, the Technicon RA-produces 1000 results that are about 10% lower than those of other systems using the same kind of method. Results obtained with direct spectrophotometric methods are generally about 20% higher than those of other methods. These differences are probably present when human specimens are analyzed as well. Schlebusch[2] reports a positive bias for bilirubinometers using human neonatal specimens.

Factors that affect the measurement of bilirubin and can be a cause for differences between methods are the following: linearity of the method, particularly at the upper end of the testing range; the level at which the test has been standardized; the calibrator used for standardization; sensitivity of the method to hemoglobin or turbidity (lipemia in patients and turbidity in calibrators); and sensitivity to other extraneous materials in the serum, such as pigments, medications, and metabolic products.

It is not surprising that agreement is poor between methods used with this nonspecific test that measures different chemical species. I have reported, for instance, a case in which an unknown inhibitor caused the total bilirubin to be lower than the direct bilirubin. To prevent clinical confusion, every laboratory should place a single method in routine use. If a backup method is needed, its use should be noted along with the result, and the clinician cautioned that a discrepancy may exist between results obtained with the two methods. [1] Blijenberg, B.G.; Roetering, H.A.; and Leijnse, B. Reflections on the standardization of total bilirubin in neonatal serum J. Clin. Chem. Clin Biochem. 25:177 181, 1987. [2] Schlebusch, H.; Axer, K.; Schneider, C.; et al Comparison of five routine methods with the candidate reference method for the determination of bilirubi n in neonatal serum J. Clin. Chem. Clin. Biochem. 28:203 210, 1990.

Daniel M. Baer, MD., professor of clinical pathology, Oregon Health Sciences University, and chief of pathology, Veterans Affairs Medical Center, Portland, Ore.