R hle et al.: External quality control for neonatal bilirubin 441

J. Clin. Chem. Clin. Biochem.Vol. 26, 1988, pp. 441 -446© 1988 Walter de Gruyter & Co.

Berlin · New York

External Quality Control in the Determination of Neonatal BilirubinAn Approach to the Improvement of Results

By G. R hle

Institut f r Klinische Biochemie, Universit t Bonn

H. Schlebusch

Universit tsfrauenklinik, Bonn

W. J. Geilenkeuser and R. Kruse

Institut f r Klinische Biochemie, Universit t Bonn

(Received December 22, 1987/March 3, 1988)

Summary: The reliability of bilirubin analyses is especially important in cases of neonatal hyperbilirubinaemia.However, when the means of the results of external quality control surveys and the method-dependent statedvalues for control sera were compared with reference method values, differences of up to 10% were found.Further inaccuracy arose from interlaboratory imprecision, which showed coefficients of variation of at least7%, and from greater or lesser interference from contamination of samples with haemoglobin.

The present work investigates whether the current situation can be improved by available means.

Introduction errors are responsible for interlaboratory varia-_ . . - . . ,. - r tions in the results, even when these laboratoriesOne prerequisite for an unambiguous diagnosis of , ^ Λ, ι ι Μ- ι · . . " j use the same methods,neonatal hyperbihrubmaemia is an accurate deter-mination of the total bilirubin concentration in serum. The diagrams for exchange transfusions (5 — 7) andErrors can lead to therapeutically unsound decisions, phototherapy (8, 9), which are used as aids in estab-which in turn may result in a more or less pronounced lishing a therapeutic scheme, do not take account ofbrain defect (1). Thus, exacting demands are made on method-dependent bilirubin values. A critical situa-the reliability of the clinical^chemical analysis, and so tion therefore arises, in which the unsatisfactory corn-far these demands have only been partially met (2—4): parability of the results from different laboratories

~ . - - .- ~ working with varying methods and equipment can— Current routine methods are not specific. Sources 1 . β , . . , Λ - - ,* mf. r . γ · t_ ι · * lead repeatedly to controversial decisions (10).of interference are inherent in the analysis mate-

rial, especially that of haemoglobin and its deriv- "Total bilirubin" consists of up to 5 fractions in theatives, which affect the principles of analysis in serum, whose relative quantities are so far undefined:different ways and lead to method^related varia^ 4 N . . - 1U . , , u.r u.. " . . " 1) unconjugated, albumin-bound bihrubm,tions in the results. ^ u.v ,. , .,2) bilirubin-monoglucuromde,

— Bilirubin is comparitively unstable. Primary bili- 3) bilirubin-diglucuronide,rubin standards that are easy to use for regular 4) unconjugated bilirubin which is covalent bound tocalibration and applicable for all routine methods albumin ("δ-bilirubin") (11) andare therefore not available. In addition, systematic 5) free, unconjugated bilirubin.

J. Clin. Chem. Clin. Biochem. / Vol. 26,1988 / No. 7

442 R hle et al.: External quality control for neonatal bilirubin

The development of a reference method in its strictestsense (12) is hardly possible for such a group deter-mination.

Downas et al. (13) have, however, described a methodfor the determination of total bilirubin ("candidatereference method") which extensively satisfies the re-quirements of a reference method. This will, in thefollowing, simply be called the reference method, de-spite the reservations inherent here. Furthermore,since 1986, a stable, secondary bilirubin standard de-veloped by Vink (14) has been available, which con-tains bovine albumin, and is suitable only for chem-ical, not for direct photometric, biliruBin determina-tion.

Under the given conditions, an attempt was made totest with optimal reliability the bilirubin concentrationof 3 control specimens used for external quality con-trol surveys as well as of several commercially avail-able control samples, and to compare these resultswith those obtained by routine methods. The resultsfrom external quality control surveys for neonatalbilirubin carried out in cooperation with the DeutscheGesellschaft f r Klinische Chemie served as a criterionfor the performance of routine methods.

Materials and MethodsSample mater ia l

StandardsBilirubin standards 2 and 3 were from the Rijksinstitut voorVolksgesondheit en Milieuhygiene in Bilthoven. the Nether-lands.

Standard 2: Lot no. 850723, assigned value 98.8 μπιοΐ/ΐ.Standard 3: Lot no. 850726, assigned value 290 μπιοΐ/ΐ (15).

Quality control survey samples

Unconjugated bilirubin was dissolved in a solution of albumin,human serum or bovine serum. Identical volumes of each ma-terial were placed in small test tubes and lyophylized. Thematerial had partly been produced in our own laboratory andpartly provided as a finished product by commercial companies.The composition of the material is specified for 6 quality controlsurveys, the results of which will be described in detail:- Samples A and Β of quality control surveys 3/83-3/84 each

contained the same mixture of albumin and bilirubin; spec-imen Β of quality control survey 3/83 was, however, addi-tionally spiked with 310 μηποΐ/ΐ haemoglobin, and specimenΒ of quality control surveys 1/84—3/84 with 93 μιηοΐ/ΐhaemoglobin.

- Specimens 101, 102 and 103, analysed in the quality controlsurveys 1/87, 2/87 and 3/87, consisted of human serum andvaried concentrations of bilirubin.

Other control samplesThe commercially available control samples were provided bythe following companies:

Sta-Bil (human albumin, No BIC-975) Travenol, M nchen.Kontrollogen-Bilirubin (human serum, No 613415) Behring-werke, Marburg.Precibil (human serum No 1-211) Boehringer-Mannheim,Mannheim.Controbil (bovine serum No 705) Merck, Darmstadt.

Reference method values

Time of the determinations: February to April 1987.

The reference method values of specimens 101, 102 and 103had been determined previously under similar, though some-what less elaborate, conditions. The results of this first deter-mination were slightly higher than the second; only the seconddetermination is used here.

Participating laboratories

Six laboratories took part in the present inquiry into referencemethod values. These were headed by W. Appel, Karlsruhe; B.G. Blijenberg, Rotterdam; H. Bret t Schneider, Penzberg; G. Br g-mann, T bingen; H. Schlebusch, Bonn and H. Wisser, Stuttgart.

Determination methodAll quantitative determinations to establish reference methodvalues were carried out according to Doumas et al. (13).

Photometer controlsThe spectrophotometers used were first tested: the wavelengthswith a holmium nitrate solution and the absorption with grayfilters, whose absorption for wave-length 598 nm were measuredby the Physikalisch-Technische Bundesanstalt, Berlin.

Volume controlsEach laboratory controlled its volume measurements accordingto the recommendations issued by the National Committee forClinical Laboratory Standards, USA (NCCLS, Vol. 4 No 6).

Experimental desigtiEach laboratory determined the bilirubin concentration in 2standard samples, 3 quality control survey samples and 4 com-mercial control samples. All determinations were performed induplicate, in 5 series of analyses.

Establishing the reference method valuesAfter eliminating the results from one reference laboratory,which showed the largest scatter, the median of the remaining90 values for one given specimen was fixed as the referencemethod value.

External qua l i ty control surveysQuality control surveys for the determination of neonatal bili-rubin have taken place three times yearly since 1983. Thelaboratories that applied for survey participation are each, withfew exceptions, part of a hospital with facilities for the newborn.For each survey the participants analysed* two samples, A andB. In the first survey of 1983, sixty pairs of analytical resultswere sent for quality control evaluation; in the third survey of1987, this number had increased to 160.

J. Clin. Cheiii. Clin. Biochem. / Vol. 26,1988 / No. 7

R hle et al.: External quality control for neonatal bilirubin 443

Evaluation

After every quality control survey, the results were divided intoseveral subgroups according to the method of analysis. Forevery subgroup, statistical parameters· were calculated: meanvalue, standard deviation and coefficient of variation.

Methods taken into account

Jcndrassik-Grof method (16); 2.5-dichlorophenyldiazonium(DPD)-method (17); direct spectrophotometry without dilution(bilirubinometer). Beginning with quality control survey 2/86,the results of direct speclrometry with dilution (18) were alsoevaluated separately.

Transformation of analysis results with the help of a ctivestandard

The participants' individual results for sample A of the qualitycontrol surveys 1/87 and 3/87 (A;) were multiplied with thequotient from the reference method value for sample B (RMVB)and the individual result for Sample B (B·,).

Αι · RMVB = Αίβ

From the values standardized in this way (Ais), the statisticalparameters — mean value and standard deviation — werecalculated (fig. 1).

Criterion for excluding mean values and standard deviations infigure 1

When a first calculation of mean value and standard deviationgave a single value differing from the mean by plus or minus3 standard deviations, this was eliminated before a second andfinal calculation.

Results and Discussion

Intel-laboratory precision

An important purpose of the quality control surveysis to determine the degree to which the results fromdifferent laboratories differ from one another. Anoften very rough but otherwise very graphic estimateof the extent of scatter in quality control resultsemerges from the standard deviation or the relativestandard deviation. It proved repeatedly to be the

case that interlaboratory precision increased more orless significantly after the start of the quality controlprogramme. This was not the case, however, whenneonatal bilirubin was determined, or, at best, im-provement was only apparent in its initial stage.

In table 1 the mean values of the coefficients ofvariation are given for each of the 6 control samplesthat were analysed within a given year.

According to these, during the last 5 years there wasno convincing improvement in interlaboratory preci-sion for the 3 analysis methods most often used bythe participants. A slight improvement was noticeableonly over the last 2 years when all the results weretaken into account without differentiating the meth-ods. The available data allow the conclusion that thiswas due to a reduction in the differences between themean values of each method collective.

This practically unaltered interlaboratory precisionfound in participants with the same analysis princi-ples, points to the fact that needed improvement canhardly be expected by increased care on the part ofthe analysts. Apparently other measures are neededto improve a precision impaired most likely, and tothe greatest extent, by systematic errors of differingmagnitude.

Accuracy

Table 2 shows that wide differences can exist betweenthe results obtained by reference methods and thoseby routine methods. Not only the mean values of theresults from quality control surveys but also the statedvalues cited by the suppliers of the control sera de-viated up to 10% from those of reference methods.These findings accord with the results of previousstudies (e.g. I.e. (3)). Such a noticeable differenceraises the question of whether the calibration of theusual routine methods might not need to be re-ex-amined and corrected.

Tab. 1. Means of some method-dependent data from three external quality control surveys for bilirubin determinations per year,each including the analyses of two specimens: mean of the number of participants (n), mean of the coefficients of variation(CV), mean of the relative differences (D%) between the methods means and the total means of all values for a givenspecimen.

Year

19831984198519861987

Jendrassik-Grof

n

3040394343

CV(%)

7.39.35.97.26.2

D(%)

5.23.03.81.73.0

2,5-Dichlorophenyldiazonium reagentn

1424344553

CV (%)

10.97.26.66.96.8

D (%)

3.04.72.52.31.5

Direct photometrywithout dilutionn

1725294342

CV (%)

7.99.1

10.07.57.7

D (%)

8.27.78.33.53.3

All methods

CV (%)

9.210:09.57.97.3

J. Clin. Chem. Clin. Biochem. / Vol. 26, 1988 / No. 7

444 R hle et al.: External quality control for neonatal bilirubin

Tab. 2. Results of the determinations of "reference method values" (16% and 84% percentiles in parenthesis) for 9 bilirubinspecimens (2 standards, 3 specimens, 101 — 103, used in external quality control surveys and 4 commercial controlspecimens) and the means of survey results1) and the stated values2) of specimen producers, respectively.

Specimen

Standard 2 (98.8 μιηοΐ/ΐ)Standard 3 (290 μπχοΐ/ΐ)101102103Sta-BilKontrollogen-BilirubinPrecibitControbil

Reference method value

(μηιοΐ/l)

97.9 (96.0- 99.1)286 (280 -289)91.8 (88.5- 93.9)

178 (174 -180)266 (262 -268)316 (311 -321)333 (327 -337)273 (268 -276)303 (297 -309)

Method dependentJendrassik-Grof

(μηιοΐ/ΐ)

96.11)1881)2811)3252)3441)3002)3222)

values2,5-Dichlorophenyl-diazonium method(μπιοΐ/ΐ)

ΙΟΙ1)1951)2881)

3561)3002)31 12)

Direct photometrywithout dilution(μΐηοΐ/ΐ)

1041)201 ')292')

3101)

Possible effects from the use of standards

Independent of the possible necessity for new calibra-tion procedure^ for routine methods, and consideringthe great diagnostic importance of neonatal bilirubindetermination, the calibration of every analytical se-ries should prove more reliable with the help of asupplementary, if only, secondary standard. The ma-terial developed in the Netherlands (14) is availableas a standard for chemical methods.

To what extent this could improve the reliability ofresults is shown in an evaluation made by the controlsurvey 1/87 (fig. 1), in which samples 102 and 103,from the same production supply, were studied. The

first group of columns shows the mean values andstandard deviations arrived at for the first of the twosamples (A). These results would have been muchbetter if the participants had used the second sample(B,) as a standard for the first, which was exactly therequirement we made for our later calculation. Theresulting second group of columns shows a very goodmatch up between the means and the referencemethod values. Likewise, a considerable improvementin interlaboratory precision would have occurred forall methods of analysis. Both accuracy and interlab-oratory precision would, in practice, be feasible ifsystematic intralaboratory errors of varying degreewere eliminated by the use of suitable standards.

330*3* 320| 310^ 300~ 290| 280~ 270

I 260δ 250

0η

Survey 1/87Specimen A

50 60 48 19SD[/jmol/l] 18 18 22 18 8.0 5.5 14 8.2"Standard" without with

Survey 3/87Specimen A

50 41 2115 18 18 17without

10 12 13 Uwith

Fig. 1. Means and standard deviations (SD) of bilirubin determinations in specimen A of the surveys 1/87 and 3/87 andtransiormed j-neans and standard deviations for the same specimen calculated on the basis of the individual results for

s fictive standards with 178 μπιοΐ/ΐ and 333 μηιοί bilirubin respectively.

S 2,5-Dichlorophenyldiazonium methodB Direct spectrophotometry without dilutionH Direct spectrophotometry with dilutionT Standard deviationn = number of results>- reference method value

J. Clin. Chem. Clin. Biochem. / Vol. 26,1988 / No. 7

R hle el al.r External quality control for neonatal bilirubin 445

Again, specimen 103 of the control samples from thequality control survey 3/87 served as sample A,whereas the Kontrollogen-Bilirubin (No. 613415)was sample B (B3). The specimens were from differentsuppliers and therefore had dissimiliar matrices. Thethird column group in figure 1 shows, with no majordifference from the first, the statistical parameters ofthe original results for sample A. The fourth groupshows these same parameters after transformationwith the individual results and with the referencemethod values for sample B3.

Again, with all methods there was a marked, if some-what slighter, improvement in interlaboratory preci-sion because of the transformation. The conversionusing the diazo-methods even matched the referencemethod value almost exactly. But in the case of directphotometry, with and without dilution, the oppositeeffect occurred, i. e. accuracy decreased. This phenom-enon can be partly explained if it is assumed that thehuman serum matrix of sample A contained caroten-oids which could have influenced the photometricdeterminations more than the chemical ones. On theother hand, it is not clear why the mean of thephotometric determinations of sample B3 (Kontrol-logen-Bilirubin) was significantly lower than the ref-erence method value (tab. 2). Control samples forwhich reference method values are available are there-fore not automatically suitable as calibrators for bil-irubin determination.

In connection with this observation it is worth notingthat the specificity of direct photometry is called intoquestion by the varying absorption spectra of theindividual bilirubin fractions (11). This is surely onesource of the criticism that has been levelled at directphotometry, especially when applied without dilutionof the samples (3, 4, 19).

Haemoglobin interference

In four of the quality control surveys from 1983 and1984, two specimens each were studied that wereidentical in matrix and bilirubin concentration. Oneof the two samples, however, was mixed with hae-moglobin to simulate a medium or light haemolysis.These experiments took account of the experience thatin determining neonatal bilirubin in patients' samples,an analysis often must be made with serum or plasmathat is more or less haemolytic.

With one exception all analysis methods gave a me-dian for the sample with haemoglobin that was lowerthan for those without (fig. 2). In the case of routinemethods using the diazo-technique, this differencemust be noted as an effect due to lack of specificityof known origin. Direct photometry without dilution,however, which is supposed to eliminate the interfer-ence from haemoglobin by measurements with 2 dif-ferent wavelengths, does not seem to satisfactorilyfulfil this requirement.

Amount ofhaemoglobinadded [/jmol/l] 310 93 93 93

1.0cσ

i 'S 0.9

ωBilirubin[)umol/'l] 310(approximately)Survey 3/83

180

1/84

400

2/84

300

3/84

Fig. 2. Relative levels of the means of the bilirubin determinations of four quality control surveys in which the specimens Β werespiked with» haemoglobin, compared with the means for the specimens A (1.0) which were identical except for thehaemoglobin contamination.Π Jendrassik-Grof -methodS 2,5-Dichlorophenyldiazonium method• Direct spectrophotometry without dilution

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446 Röhle et al.i External quality control for neonatal bilirubin

Conclusions

Considering the special importance of bilirubin deter-mination for neonatology, the state of analysis todayis unsatisfactory. Substantial improvement couldprobably be brought about by means that are alreadyavailable or could, in all probability, shortly becomeavailable.1. Accuracy and precision of the results would be

decisively improved if their calculation could beexamined with the additional help of secondarystandards. Samples developed as reference materialin the Netherlands (15) are available for use assecondary standards for diazo-methods (cf. Ma-terial and Methods). Despite all reservations aboutusing control samples as calibrators, commercialcontrol series might also serve the same purpose.The prerequisite would be, however, that they aretested for suitability as calibrators for definedmethods and that the reference method values forbilirubin concentrations are available.As a result of being easy to work with, directphotometry is often used and, by a standardizedmethod suggested by Blijenberg et al. (20), couldprove to be a pragmatic step towards more reliableresults.

2. Normal unspecific analysis methods could be re-placed by the specific ones of Donmas et al. if theycould be modified so that smaller sample volumescan be handled. According to the authors' testresults (13), the problem of haemoglobin interfer-ence would thus be solved.Improved analysis could bring about a situationin which a revision of the decision limits given inscientific publications (5 — 7) will need to be dis-cussed.

Acknowledgement

We would like to thankDr. A. B. T. J. Boink, Rijksinstituut voor Volksgezondheid enMilieuhygiene, Bilthoven, for allowing us to use the bilirubinstandards.Prof. Dr. W. Appel, Karlsruhe; Dr. B. G. Blijenberg, Rotterdam;Dr. H. Brettschneider, Penzberg; PD Dr. G. Brügmann, Tübin-gen and Prof. Dr. Dr. H. IVisser, Stuttgart, for their commit-ment to establishing reference method values, a commitmentthat demanded a great deal of work from each of them.Prof. Dr. R. Nink, Physikalisch-Technische Bundesanstalt, Ber^lin, for lending us grey filters for the photometry controls.The companies Travenol, Behringwerke, Boehringer-Mannheirnand E. Merck who made their own manufactured control seriesavailable to us.

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Priv.-Doz. Dr. G. RöhleInstitut f. Klinische BiochemieSigmund-Freud-Str. 25D-5300 Bonn l

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