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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 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.

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.

To determine the relationship between BiliChek TcB (Respironics, Marietta GA) and Doumas reference serum or plasma total bilirubin (TSB).Pooled samples with values assigned by the Doumas reference method were used to establish the relationship between a local laboratory and reference Doumas TSB. We then established the relationship between TcB and TSB in the 3 months before and after reassignment of calibrator setpoints undertaken to match the local laboratory to Doumas reference bilirubin values.Before calibrator setpoint reassignment TSB as measured in our laboratory overestimated Doumas reference bilirubin. After calibrator adjustment laboratory TSB was within 1.7-6.8 micromol/L (0.1-0.4 mg/dL) of Doumas reference values. Mean bias between BiliChek TcB and TSB was 42.8+/-22.2 micromol/L (2.5+/-1.3mg/dL) (n=94) before and 49.6+/-22.2 micromol/L (2.9+/-1.3mg/dL) (n=115) after calibration adjustment.BiliChek TcB significantly overestimates TSB as measured by the Doumas reference method.

Background: The analytic performance and accuracy of drug detection below Substance Abuse and Mental Health Services Administration (SAMHSA) cutoffs is not well known. In some patient populations, clinically significant concentrations of abused drugs in urine may not be detected when current SAMHSA cutoffs are used. Our objectives were to define the precision profiles of three immunoassay systems for drugs of abuse and to evaluate the accuracy of testing at concentrations at which the CV was Methods: Drug-free urine was supplemented with analytes to assess the precision in three commercial drugs-of-abuse immunoassay systems below the SAMHSA-dictated cutoffs for amphetamines, opiates, benzoylecgonine, phencyclidine, and cannabinoids. Consecutive urine samples with signals associated with a CV Results: The CV of all immunoassay systems tested remained Conclusion: The precision of three commercial immunoassay systems for drugs-of-abuse screening is adequate to detect drugs below SAMHSA cutoffs. Knowledge of the positive predictive values of screening immunoassays at lower cutoff concentrations could enable efficient use of confirmatory testing resources and improved detection of illicit drug use.

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.

In 2003, the College of American Pathologists (CAP)1 included in the Neonatal Bilirubin Surveys a human serum–based sample enriched solely with unconjugated bilirubin (UBIL). This sample, shipped frozen in a cold pack, resembled closely a clinical sample from a healthy neonate and was commutable, as expected. The mean of all results submitted to CAP (CAP All Data) for bilirubin (199.4 mg/L) was close to that of the reference method (194.4 mg/L)(1). In subsequent years, the bias has increased(2). Most recently (2009, neonatal bilirubin NB-01), the difference between the mean value for CAP All Data and the reference method value was 9 mg/L, and the differences for the Olympus, Ortho Vitros, and Siemens ADVIA assays were closer to 25 mg/L. Because ditaurobilirubin (DTB), which is present in most commercial calibrators, and bovine serum, which is used instead of human serum for preparing calibrators, have been shown to interfere with the measurement of bilirubin(3), we investigated the effects of DTB and bovine serum on the measurement of total bilirubin by diazo methods. UBIL and DTB were purchased from Lee Biosolutions. Human serum pools (nos. 1 and 2) were obtained from volunteers from the laboratory personnel of the Children’s Hospital of Wisconsin. Bovine sera and donor calf serum were purchased from Sigma–Aldrich and MP Biomedicals. Fresh bovine serum was obtained from a local abattoir in Milwaukee, Wisconsin. Stock solutions of UBIL and DTB were prepared in all of the above-described protein matrices. The concentrations of UBIL (210 mg/L) …

A patient suspected of an inborn error of metabolism will commonly have urine organic acid analysis performed as part of their workup. The traditional urine organic acid method involves extraction of the acidic fraction from urine samples using an organic solvent, derivatization of extracted compounds, and identification using gas chromatography-mass spectrometry (GC-MS). Unfortunately, the extraction step results in the loss of many neutral and positively charged compounds, which may be of interest to metabolic physicians and biochemical geneticists. By replacing the traditional extraction step with an enzymatic treatment of the sample with urease, an abundance of organic molecules are available for separation and quantitation by GC-MS. The urease method is a useful adjunct to newborn screening follow-up and it has the additional benefit of being able to identify many classes of biochemical compounds, such as amino acids, acylglycines, neurotransmitters, and carbohydrates. The method below describes the urease treatment, derivatization, and the organic acids, and other biochemical metabolites that can be identified.

Background: The diagnosis and management of hyperbilirubinemia in the newborn requires accurate and precise bilirubin determinations. We evaluated the current status of bilirubin measurements in US laboratories by examining data submitted by laboratories participating in the College of American Pathologists (CAP) Neonatal Bilirubin (NB) and Chemistry (C) Surveys. Methods: We analyzed specimens from the CAP NB and C Surveys by the reference method for total bilirubin in three laboratories. The reference method bilirubin values were compared with bilirubin values reported by Survey participants. Results: The imprecision (CV) for all instruments combined (CAP-All Instruments) ranged from 4.7% to 5.6% at the bilirubin concentrations tested. The CVs of the four most commonly used instruments were smaller, ranging from 1.9% to 4.5%. Differences between bilirubin values by the reference method and mean values from the four most common instruments ranged from −21.6% to 10.9%. When the same specimens from the NB Surveys were used in the C Surveys, the Vitros values were strikingly different from those of the NB Surveys. The use of different methods in the NB and C Surveys coupled with the presence of a nonhuman protein base and ditaurobilirubin (DTB) in the Survey specimens accounted for the discrepant results. Conclusions: The evaluation of accuracy is impossible from the CAP Surveys because the specimens consist of bovine serum containing a mixture of unconjugated bilirubin and DTB. For the evaluation of accuracy, we recommend that Survey specimens consist of human serum enriched with unconjugated bilirubin.

We evaluated hemoglobin (Hb) interference in the total bilirubin (TBIL) and neonatal bilirubin (NBIL) [the sum of unconjugated (Bu) and conjugated (Bc) bilirubins] methods on the Vitros 5,1 FS analyzer and assessed the reliability of the analyzer’s hemolysis and icterus index values by comparing them to measured concentrations of bilirubin and Hb in test specimens. We also compared TBIL with NBIL values in specimens from neonates 1–14 days old and obtained estimates of the extent of hemolysis in these specimens. We prepared a hemolysate from EDTA blood drawn from volunteers. After centrifugation, the cells were washed 5 times with physiologic saline, diluted with deionized water, and stored at −20 °C overnight. After thawing, the hemolysate was centrifuged to remove the stroma. The Hb concentration was measured with the CELL-DYN 4000 analyzer. We prepared a stock solution of Bu (NIST SRM 916) in pooled human serum samples from healthy volunteers as described previously (1); dilutions were made with the same pooled sera. A solution …

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to occur naturally, four have been found in various strains of Salmonella enterica (abequose, tyvelose, paratose, and colitose) and all five, including ascarylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region harboring the abequose biosynthetic genes from Y. pseudotuberculosis serogroup HA, the detailed genetic principles underlying a 3,6-dideoxyhexose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dideoxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (O antigen) cluster of Salmonella spp., we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a detailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In addition, the functional cloning of this region has allowed the expression of Ep (alpha-D-glucose cytidylyltransferase), Eod (CDP-D-glucose 4,6-dehydratase), E1 (CDP-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase), E3 (CDP-6-deoxy-delta 3,4-glucoseen reductase), Eep (CDP-3,6-dideoxy-D-glycero-D- glycero-4-hexulose-5-epimerase), and Ered (CDP-3,6-dideoxy-L-glycero-D-glycero-4-hexulose-4-reductase), facilitating future mechanistic studies of this intriguing biosynthetic pathway.