Basil T. Doumas

Serum and urine albumin: a progress report on their measurement and clinical significance

For about 25 years, bromcresol green and bromcresol purple have been the basis for most of the measurements of serum albumin in the US and perhaps in the world. The longevity of the methods is due to their being simple, sensitive, specific, inexpensive and relatively free from interferences. The lack of change in the serum albumin methodology is balanced by two important developments. First, the recognition of the importance of serum albumin in the maintenance of good health, and the association of decreased concentrations with increased risk of morbidity and mortality. Second, the association of albuminuria with diabetic nephropathy, which without medical intervention could lead to end-stage renal disease. The development of accurate and precise methods for urinary albumin has provided a tool to physicians to extend the length and improve the quality of life of many diabetic individuals.

The measurement of bilirubin fractions in serum

Bilirubin fractions are measured by (1) the direct diazo reaction, (2) high-performance liquid chromatography (HPLC), (3) direct spectrophotometry, and (4) enzymatic methods. HPLC, which effects separation and quantitation of the four bilirubin fractions, is the method of choice, but impractical for routine use. A special application of direct spectrophotometry allows the measurement of unconjugated bilirubin and the sum of bilirubin conjugates. This approach, which provides essentially the same information as HPLC, unfortunately is available only in one clinical analyzer. The direct diazo reaction measures bilirubin conjugates plus delta-bilirubin, albeit not very accurately. Direct diazo methods that measure unconjugated bilirubin as direct could obscure the clinical diagnosis. At acid pH, enzymatic methods measure all direct reacting bilirubins, while at pH 10 only conjugated bilirubins are measured. Because the measurement of conjugated bilirubins is clearly more helpful than that of direct bilirubin in the differential diagnosis of jaundice, direct diazo methods should be replaced by methods specific for bilirubin conjugates.

Relationship between nutrition, weight change, and fluid compartments in preterm infants during the first week of life

This study was conducted to investigate the redistribution of fluid compartments and to examine the factors contributing to the variability of early weight loss in premature infants. Fourteen preterm infants (mean +/- SD: birth weight, 1473 +/- 342 gm; gestational age, 30.7 +/- 2.4 weeks) were studied at 1 and 7 days of age. Total body water was measured by deuterium oxide dilution, extracellular volume by bromide dilution, and intracellular volume by the difference between total body water and extracellular volume. There were significant changes in body fluid distribution per concurrent weight from birth to age 1 week. Extracellular volume decreased by 11%, and intracellular volume increased by 8.5% with no change in total body water. Infants were then grouped according to postnatal weight loss (group 1 (n = 7) > 10% and group 2 (n = 7) < 5% of birth weight). In group 1 there was a significant loss of both weight (mean +/- SD: 15.6% +/- 3.7%) and extracellular volume (15.9% +/- 9% of birth weight), with no change in intracellular volume. In group 2 there was no significant weight loss (1.4% +/- 1.8%), but a significant loss of extracellular volume (13.0% +/- 5.4% of birth weight) and a significant increase in intracellular volume. Other differences between the groups were a lower energy intake in group 1 than in group 2 (mean +/- SD: 177 +/- 46 vs 269 +/- 45 kilojoules/kg per day; p < 0.005) and a higher physiologic stability index in group 1 (p < 0.05). We conclude that significant postnatal weight loss as a result of the contraction of the extracellular compartment occurs only in less stable infants whose energy intake is inadequate. With adequate energy intake, weight loss is minimal because of the expansion of the intracellular compartment, which may be related to the onset of growth.

Laboratory Performance in Neonatal Bilirubin Testing Using Commutable Specimens: A Progress Report on a College of American Pathologists Study

CONTEXT In 2003 the Chemistry Resource Committee of the College of American Pathologists introduced a commutable specimen in the Neonatal Bilirubin Surveys. This specimen was intended to help evaluate all bilirubin methods. OBJECTIVE To evaluate the effect of commutable specimens on the performance of selected clinical analyzers in measuring neonatal bilirubin from 2003 through 2006. DESIGN A human serum-based specimen enriched with unconjugated bilirubin in human serum has been included since 2003 in the Neonatal Bilirubin Surveys. The bilirubin values of these specimens were determined by the reference method and used to evaluate results reported by various chemistry analyzers. RESULTS Coefficients of variation for College of American Pathologists All Data ranged from 4.9% to 6.2% for the Neonatal Bilirubin Survey. However, coefficients of variation for the 4 major instrument groups (Dimension, Olympus, Synchron, and Vitros), which report 65% of all results, varied from 2% to 3%. College of American Pathologists All Data mean bilirubin values were within 0.46 mg/dL (7.8 micromol/L) of the reference method mean in 2003; in subsequent years these differences became larger, peaking at 1.87 mg/dL (32 micromol/L) in 2005. CONCLUSIONS The large systematic error of bilirubin measurements is due primarily to failure of instrument manufacturers to produce reliable bilirubin calibrators. Primary calibrators should consist of human serum enriched with unconjugated bilirubin. Bilirubin values must be assigned by the reference method, the performance and robustness of which are reported in this article. Secondary calibrators distributed to users must be traceable to primary calibrators.

The status of bilirubin measurements in U.S. laboratories: why is accuracy elusive?

In 2003, the Chemistry Resource Committee of the College of American Pathologists introduced a new specimen in the Neonatal Bilirubin Survey. The specimen, consisting of human serum enriched with unconjugated bilirubin and thus resembling a clinical specimen, brought about an improvement in the accuracy of the measurement of bilirubin by laboratories participating in the Neonatal Bilirubin Survey. There was also an improvement in the specificity of methods measuring direct bilirubin. However, persisting inaccuracies and variability in laboratory performance have been traced to calibrators consisting of bovine serum spiked with unconjugated bilirubin and ditaurobilirubin; bovine serum causes underestimation of both bilirubins by 8 major chemical analyzers. To eradicate inaccuracy calibrators and Survey specimens should be made in human instead of bovine serum.

Bilirubin proficiency testing using specimens containing unconjugated bilirubin and human serum: results of a College of American Pathologists study.

CONTEXT Specimens of the College of American Pathologists Neonatal Bilirubin and Chemistry surveys are inadequate for evaluating the performance of clinical laboratories in measuring serum bilirubin because they exhibit strong matrix interference. Recently published data indicate that at least 1 major clinical analyzer provided inaccurate bilirubin values for Neonatal Bilirubin Survey specimens. The composition of the specimens, bovine serum enriched with ditaurobilirubin, was responsible for the erroneous results. OBJECTIVE This article evaluates the performance of major clinical analyzers using a survey specimen free of matrix interference. DESIGN A human serum-based specimen enriched solely with unconjugated bilirubin was included in the 2003 Neonatal Bilirubin and Chemistry surveys. Its bilirubin concentration (19.4 mg/dL [332 micromol/L]) was determined by the reference method for total bilirubin. RESULTS The coefficients of variation for the 4 major clinical analyzers (Dimension, Hitachi, Synchron, and Vitros) ranged from 1.9% to 3.7%. When compared to the bilirubin value measured by the reference method, mean bilirubin values of the 4 major clinical analyzers and College of American Pathologists (CAP) All Data (which refers to the grand mean and overall coefficient of variation of all method principles, all instruments according to CAP terminology) ranged from -3.5% to 5.1%. Direct bilirubin results from most field methods showed good specificity overall. CONCLUSION Human serum-based survey specimens, having their bilirubin concentrations determined by the reference method, should be included as frequently as feasible in the Neonatal Bilirubin Survey. Such specimens may be used by instrument manufacturers as standards for calibrating bilirubin methods and for assigning values to calibrators provided to instrument users. A substantial improvement in bilirubin measurements due to the reduction of systematic error is expected.

The evolution and limitations of accuracy and precision standards

Limits of maximum allowable analytical imprecision have been defined on the basis of normal range (Tonks), composite biological variation (Cotlove), intraindividual biological variation (Aspen Conference, College of American Pathologists), medical significance (Barnett, Skendzel), and a combination of medical significance and biological variation (Fritsche. Klee). Because error limits based on medical significance are less stringent than those based on biological variation, performance goals based on medical significance are more likely to be attained by laboratories than those defined by biological variation. Most clinical scientists realize that the goal to limit the analytical C.V. to one-half or less of the biological C.V. is arbitrary, and for the large majority of laboratory tests of no benefit to the patient, for the major component of total variation is not the analytical imprecision, but the intraindividual variation itself. Furthermore, the purpose of laboratory testing is to help physicians confirm or exclude potential diagnoses and monitoring therapy rather than detecting small deviations from normal values. Small changes in test values are quite often uninterpretable or lead to costly albeit fruitless investigations. In view of diminishing resources for health care we must establish the accuracy and precision required for each test. While improving the accuracy of some tests would be desirable, for most of them further improvement would be irrelevant to patient care because few tests are pathognomonic by themselves and quite often diagnosis is not made on the basis of a single laboratory result. If more accuracy is desirable, it must also be affordable and benefits should outweigh costs.

Interlaboratory comparison of the Doumas bilirubin reference method.

OBJECTIVES To assess the performance of the Doumas bilirubin reference method. DESIGN AND METHODS Ring trails using pooled patient specimens, a calibrator and human sera enriched with unconjugated bilirubin were analyzed in five laboratories using the Doumas bilirubin reference method. RESULTS The coefficient of variation for the linear measurement range between laboratories ranged from 1-3%. CONCLUSIONS The Doumas bilirubin reference method is robust and reproducible. Bilirubin results using this method may be used in the development of more accurate and reliable calibrators.

Laboratory Performance in Albumin and Total Protein Measurement Using a Commutable Specimen: Results of a College of American Pathologists Study

CONTEXT Discrepant results for serum constituents were observed among peer groups in the College of American Pathologists Comprehensive Chemistry Survey. OBJECTIVES To assess the performance of serum albumin and total protein measurement procedures and to evaluate the commutability of the conventional survey specimens. DESIGN A fresh frozen, off-the-clot serum sample was included along with 4 conventional survey specimens. The fresh frozen, off-the-clot serum sample was prepared in a manner expected to confer commutability with native clinical samples. RESULTS For the fresh frozen, off-the-clot serum sample, the mean values for 17 peer-groups were -0.07 to 0.32 g/dL from the bromocresol green albumin designated comparison method, whereas 4 VITROS (Ortho Clinical Diagnostics, Rochester, New York) peer groups differed by -0.29 to -0.37 g/dL (15 of 21 differences [71%] had P < .001). For bromocresol purple albumin methods, the mean differences from the designated comparison method from 8 peer groups were 0.25 to 0.47 g/dL (all had P < .001). For total protein methods, 23 peer group mean values were -0.07 to 0.15 g/dL from the reference measurement procedure (12 of 24 [50%] had P < .001). The Beckman (Fullerton, California) Synchron LX20 had a bias of -0.30 g/dL (P <.001). The commutability of the conventional specimens was acceptable for 23 of 24 bromocresol green method-material combinations (96%) and 13 of 16 bromocresol purple albumin method-material combinations (81%). All (100%) of the 36 method-material combinations had acceptable commutability for total protein. CONCLUSIONS One (2.2%) of the instrument systems (Synchron) using bromocresol green and none (0%) of the instrument systems using bromocresol purple had satisfactory total-error performance for albumin measurement. Differences in results between bromocresol green and bromocresol purple methods precluded using common reference intervals for interpreting results for serum albumin. Eight of 9 instrument systems (86.5%) had satisfactory total-error performance for total protein measurement.

Diabetic Nephropathy: Urinary Albumin or Total Protein?

Excerpt To the Editor:Increased rates of albumin excretion into the urine at rest or after exercise exceeding about 3(μg/min or 45 mg/day are an early manifestation of diabetic nephropathy and may ...