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Bovine serum-based bilirubin calibrators are inappropriate for some diazo methods.

To the Editor:

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) and DTB (190 mg/L) were the same in all protein matrices. These solutions were analyzed at Children's Hospital of Wisconsin with the reference method for total bilirubin (4) and with 7 clinical analyzers (Table 1), all of which used diazo methods.

Table 1 shows the values for total bilirubin and differences between bilirubin values in human serum pool no. 1 and in bovine sera for each instrument; the values in human serum pool no. 1 were subtracted from the corresponding values in bovine serum. The bilirubin values for bovine sera were much lower than for human sera. For the abattoir serum, UBIL values were suppressed slightly. Suppression of UBIL values was much larger in commercial bovine sera. The suppression could be due to the presence of inhibitors of the coupling reaction in bovine sera or to the addition of inhibitors during the processing of such sera. The variation in suppression could be due to differences in the diazo reagents and accelerators. The widely different bilirubin values obtained with the Roche Modular Analytics system for samples prepared in MP Biomedicals and Sigma-Aldrich bovine sera were confirmed by reanalysis. Human serum pool no. 2 was used to demonstrate the reproducibility of the molar absorptivity of the alkaline azo pigment.

For DTB in human serum, the underestimation of DTB by the Vitros 5,1 FS diazo method (total bilirubin) has already been reported (3). DTB was also underestimated by the ARCHITECT analyzer (Abbott Laboratories). Values from the other analyzers were close to those obtained with the reference method. In fresh bovine serum, DTB values from 5 of the analyzers were close to those of the reference method, confirming previous observations (5). The DTB values were underestimated by the 5,1 FS and ARCHITECT analyzers. In commercial bovine sera, DTB values were suppressed for all clinical analyzers and for the reference method.

BSA has been used for more than 20 years for preparing bilirubin calibrators (2, 4), and we have never encountered inhibition of the coupling reaction. Thus, the suppression of UBIL and DTB values cannot be due to BSA. Because manufacturers of bovine sera have assured us that nothing is added to bovine serum during the processing of bovine blood, we must assume one or more inhibitors may enter the animals' blood through feeding or the leaching of containers used in the process. Azide, which is used as a preservative, inhibits the coupling reaction. The method we found to detect azide was not sensitive enough to detect inhibitory concentrations. Pre screening bovine sera for inhibitors is possible, although not practical. To rule out the possibility that "aging" of bovine serum may cause formation of inhibitors, we prepared solutions of UBIL and DTB in human serum and in "abattoir" bovine serum that had been stored for 10 months at -20[degrees]C and analyzed them by the reference method. The UBIL values in human and bovine serum were 198.5 mg/L and 195.8 mg/L, respectively; the DTB values were 189.3 mg/L and 186.6 mg/L in human and bovine serum, respectively. These data rule out formation of inhibitors during storage.

Recurring problems plaguing bilirubin measurements can be traced, at least in part, to the use of bovine serum instead of human serum for preparing calibrators. The variable and unpredictable underestimation of UBIL and DTB in bovine serum renders the relationship between the actual bilirubin content and assigned values in calibrators unreliable and dependent on the quality and source of the bovine products. In fact, there is no reliable method for accurately measuring the concentration of bilirubin in calibrators and controls consisting of UBIL, DTB, or both in bovine sera from commercial sources. The use of such products unnecessarily compromises the accuracy of bilirubin measurements in neonates. The solution to this problem is simple: Use human instead of bovine serum for preparing bilirubin calibrators.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared. Consultant or Advisory Role: S. Lo, College of American Pathologists Chemistry Resource Committee; B.T. Doumas, Children's Hospital of Wisconsin, Reference Standards Laboratory.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Diane Breitenfeld (Dynacare Laboratories, Milwaukee, WI), Bonnie Lisko (Community Memorial Hospital, Menomonee Falls, WI), Randall Schneider (Waukesha Memorial Hospital, Waukesha, WI), Donald Wiebe (University of Wisconsin Hospitals, Madison, WI), and Steven Zibrat (University of Chicago Hospital, Chicago, IL) for analyzing the samples shown in Table 1.


(1.) Lo SF, Doumas BT, Ashwood ER. Bilirubin proficiency testing using specimens containing unconjugated bilirubin and human serum: results of a College of American Pathologists study. Arch Pathol Lab Med 2004;128:1219-23.

(2.) Lo SF, Jendrzejczak B, Doumas BT. Laboratory performance in neonatal bilirubin testing using commutable specimens: a progress report on a College of American Pathologists study. Arch Pathol Lab Med 2008;132:1781-5.

(3.) Lo SF, Doumas BT, Ashwood ER. Performance of bilirubin determinations in US laboratories--revisited. Clin Chem 2004;50:190-4.

(4.) Doumas BT, Kwok-Cheung PP, Perry BW, Jendrzejczak B, McComb RB, Schaffer R, Hause LL.

Candidate reference method for determination of total bilirubin in serum: development and validation. Clin Chem 1985;31:1779-89.

(5.) Doumas BT, Wu TW, Poon KC, Jendrzejczak B. Chemical nature of a synthetic bilirubin conjugate and its reactivities in the total and direct reactions by the Jendrassik-Grof method. Clin Chem 1985;31:1677-82.

Stanley Lo [2] *

Bernadine Jendrzejczak [2]

Basil T. Doumas [2]

[1] Nonstandard abbreviations: CAP, College of American Pathologists; UBIL, unconjugated bilirubin; CAP All Data, all results submitted to CAP; DTB, ditaurobilirubin.

[2] Department of Pathology Children's Hospital of Wisconsin and Medical College of Wisconsin Milwaukee, WI

* Address correspondence to this author at: Department of Pathology\MS701 Children's Hospital of Wisconsin and Medical College of Wisconsin 9000 West Wisconsin Ave Milwaukee, WI 53201 Fax 414-266-2779 E-mail

Previously published online at DOI: 10.1373/clinchem.2009.138925
Table 1. Values for total bilirubin in human and bovine sera
enriched with UBIL and DTB, as measured with the reference
method and 7 chemistry analyzers. (a)

 CHW (b) Dynacare
 (reference Laboratories
Sample method) (Olympus 5400) (c)

UBIL added
 Human serum pool no. 1 208 220
 Bovine serum (from an 195 (-13) 208 (-12)
 Bovine serum (from MP 177 (-31) 182 (-38)
 Bovine serum (from 179 (-29) 188 (-32)
 Donor calf serum 185 (-23) 198 (-22)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 205 (-3) 220 (0)
DTB added
 Human serum pool no. 1 186 198
 Bovine serum (from an 182 (-4) 190 (-8)
 Bovine serum (from MP 162 (-24) 164 (-34)
 Bovine serum (from 162 (-24) 166 (-32)
 Donor calf serum 164 (-22) 168 (-30)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 183 (-3) 196 (-2)

 (Abbott (Beckman Coulter
Sample ARCHITECT) (c) DxC 600) (d)

UBIL added
 Human serum pool no. 1 207 217
 Bovine serum (from an 162 (-45) 212 (-5)
 Bovine serum (from MP 151 (-56) 193 (-24)
 Bovine serum (from 156 (-51) 193 (-24)
 Donor calf serum 168 (-39) 197 (-20)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 208 (1) 214 (-3)
DTB added
 Human serum pool no. 1 147 192
 Bovine serum (from an 147 (0) 187 (-5)
 Bovine serum (from MP 115 (-32) 164 (-28)
 Bovine serum (from 115 (-32) 165 (-27)
 Donor calf serum 117 (-30) 167 (-25)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 147 (0) 189 (-3)

 UW Hospitals
 (Siemens Dimension) UCH
 (Roche Modular
Sample RxL (d) Vista (c) Analytics) (d)

UBIL added
 Human serum pool no. 1 209 205 238
 Bovine serum (from an 190 (-19) 195 (-10) 233 (-5)
 Bovine serum (from MP 166 (-43) 168 (-37) 162 (-76)
 Bovine serum (from 170 (-39) 173 (-32) 220 (-18)
 Donor calf serum 190 (-19) 192 (-13) 218 (-20)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 211 (2) 210 (5) 237 (-1)
DTB added
 Human serum pool no. 1 187 182 200
 Bovine serum (from an 182 (-5) 185 (3) 191 (-9)
 Bovine serum (from MP 153 (-34) 156 (-26) 163 (-37)
 Bovine serum (from 153 (-34) 152 (-30) 171 (-29)
 Donor calf serum 158 (-29) 160 (-22) 170 (-30)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 182 (-5) 183 (1) 194 (-6)

 CHW (Ortho Vitros 5,1 FS) (c)

Sample TBIL (e) Bu (e) Bc (e)

UBIL added
 Human serum pool no. 1 224 232 7
 Bovine serum (from an 208 (-16) 216 (-16) 3 (-4)
 Bovine serum (from MP 198 (-26) 206 (-26) 7 (0)
 Bovine serum (from 188 (-36) 216 (-16) 9 (2)
 Donor calf serum 201 (-23) 236 (4) 12 (5)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 225 (1) 235 (3) 7 (0)
DTB added
 Human serum pool no. 1 157 22 222
 Bovine serum (from an 159 (2) 17 232
 Bovine serum (from MP 149 (-8) 7 (-15) 214 (-8)
 Bovine serum (from 148 (-9) 8 (-14) 220 (-2)
 Donor calf serum 146 (-11) 8 (-14) 218 (-4)
 (ICN/MP Biomedicals)
 Human serum pool no. 2 158 (1) 18 (-4) 225 (3)

(a) Data are presented as the concentration (in milligrams
per liter), with the difference in concentration in parentheses
(serum sample value minus the value for human
serum pool no. 1).

(b) CHW, Children's Hospital of Wisconsin, Milwaukee, WI; WMH,
Waukesha Memorial Hospital, Waukesha, WI; CMH, Community
Memorial Hospital, Menomonee Falls, WI; UW Hospitals, University
of Wisconsin Hospitals, Madison,
WI; UCH, University of Chicago Hospital, Chicago, IL.

(c) Calibrators prepared with bovine serum.

(d) Calibrators prepared with human serum.

(e) TBIL (total bilirubin), Bu (unconjugated bilirubin), and
Bc (sum of bilirubin mono- and diglucuronide) are symbols used by Ortho
Clinical Diagnostics for tests performed on the Vitros 5,1 FS chemistry
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Title Annotation:Letters to the Editor
Author:Lo, Staney; Jendrzejczak, Bernardine; Doumas, Basil T.
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:May 1, 2010
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