Different batches of bromocresol green were used in a study of several modifications of the bromocresol green method for serum albumin. No major batch variation was observed. Lyophilized animal sera and a certain batch of lyophilized human sera, however, responded differently from a pool of fresh human sera when the method was modified. These phenomena should be taken into accuont in calibration procedures and in quality assessment.Peer Reviewe

Uldall, A.

Clinical Chemistry and Laboratory Medicine (CCLM)

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Short communication/Kurzmitteilung 305J. Clin. Chem. Clin. Biochem.Vol. 22, 1984, pp. 305-307SHORT COMMUNICATION/KURZMITTEILUNGAnalyte and Matrix Problems in the Calibration and Quality Assessmentof the Bromocresol Green Method for Albumin in SerumBy A. UldallDepartment of Clinical Chemistry, University of Copenhagen, Herlev Hospital, Herlev, Denmark(Received July 18/December 21, 1983)Summary: Different batches of bromocresol green were used in astudy of several modifications of the bromocresol green methodfor serum albumin. No major batch Variation was observed. Lyo-philized animal sera and a certain batch of lyophilized human se-ra, however, responded differently from a pool of fresh humansera when the method was modified. These phenomena should betaken into account in calibration procedures and in quality assess-merit.Analyt- und Matrix-Probleme bei der Kalibrierung und Qualitäts-kontrolle der Bromkresolgrüfc Methode für Albumin im SerumZusammenfassung: Verschiedene Chargen von Bromkresolgrünwurden für eine Untersuchung mehrerer Modifikationen derBromkresolgrün-Methode für Albumin im Serum eingesetzt.Größere Chargenunterschiede wurden nicht beobachtet. Lyophi-lisierte Tierseren und eine bestimmte Charge lyophilisierter Serenvom Menschen reagierten jedoch abweichend von einem Poolfrischer Seren vom Menschen auf die Modifizierung der Methode.Diese Erscheinungen sollten 'bei der Kalibrierung und der Quali-tätskontrolle berücksichtigt werden.IntroductionThe bromocresol green method for serum albumin is still usedextensively in many clijiical chemistry laboratories in spite of con-siderable criticism (1). The methods might wörk properly (2—5),but in external quality assessment the results are often poor (e.g.I.e. (6)).Bromocresol green is marketed äs a pH indicator, and productioncontrol during manufacture can only be expected to cover thistype of use. Therefore it was the aim of the present paper to inves-tigate differences between batches of bromocresol green, and tofind other possible explanations for interlaboratory Variation inthe bromocresol green method for serum albumin.Materials and MethodsBromocresol greenFive batches of bromocfesol green art. no. 8121 from E. Merck(Darmstadt, West Germany) were investigated, three from the la-boratories showing the greatest deviations in the earlier survey(6), a 25 year-old batch and a brand new one.Bromocresol green reagentAppropriate concentrations of bromocresol green were preparedfresh by dissolution of bromocresol green in an aqueous succinicacid/succinate buffer, pH 4.2 (23 °C) (50 mmol succinate and0.75 ml Brij™ 35 in one Ihre).Reference seraThe following reference sera were used:Pool of fresh sera,lyophilized serum from bank blood (D; local production),commercially available lyophilized serum (G; intended for gener-al quality control, "abnormal" level, Dade^, American HospitalSupply Corp., Miami, USA),equine lyophilized serum (H; Nyegaard & Co., Norway),mixed bovine-equine lyophilized serum (I; Nyegaard & Co., Nor-way) anda human albumin solution (Protein Standard, Kabi DiagnosticaAB, Sweden).The materials, identified by letters, were the same äs those used inthe previous investigation (6).ProcedureOne volume of specimen was diluted with 20 volumes of aqueoussodium Chloride (154 mmol in one litre). An aliquot (50 ul) of thisdiluted sample was added to 450 bromocresol green reagentand the absorbance of the mixture was read against water at 630nm between 35s and 1235s after mixing in the GEMSAEC™(Electro-Nucleonics Corp., New Jersey, USA).Albumin was furthermore determined by a bromcresol greenmethod on the SMAC I™ (Technicon Instruments Corp., NewYork, USA); reaction conditions: Specimen dilution l vol + 122vol; preheated at 45 °C in 170 s; bromocresol green 135 / ;succinic acid buffer, pH 4.2; merthiolate; Pegosperse™ 1.5 vol in100 vol; reaction time 17s.J. Clin, Chem. Clin. Biqchem. / Vol. 22, 1984 / No. 4306 Short communication/KurzmitteilungResultsTable l shows the colour yield of different albumin preparationsin comparison with the lyophilized serum from bank blood (D).Thus the data are shown in a way, similar to results obtained in acontinuous flow System using a calibration serum. No major dif-ferences were found between different batches of bromocresolgreen.The behaviour of the fresh serum pool was similar to that of thelocaily produced lyophilized serum from bank blood (D). This isin contrast to the albumin solution, the two animal lyophilizedsera (H and I) and the lyophilized human serum (G). The lattercontained elevated concentrations of many components, e. g. bill·rubins.Using the SMAC I™, the following results were obtained for thealbumin-containing materials shown in table l, in the same des-cending order: 604 μιηοΐ/ΐ, 600 μηιοΐ/l, 455 μιηοΐ/l, 529 μιηοΐ/land 552 umol/1. A value of 685 umol/1 was found for the lyophil-ized serum from bank blood (D) used s calibrator.The bottom of table l records a lack of proportionality betweenabsorbance and concentration of bromocresol green in the purereagents.DiscussionThe big Variation of accuracy often found between laboratoriesusing bromocresol green methods for albumin could not be ex-plained by batch variations of bromocresol green. Furthermore,using high performance liquid chromatography and thin layerchromatography the bromocresol green batches seemed quitepure; "less than 1% of impurities" were fo hd with exception ofthe 25 years old batch "where 2% was found" (K. E. Rasmussen,H. Ravn, personal communication 1981).The present data show th t the type of reference materials con-tribute remarkably to the uiterlaboratory Variation, when meth-ods with different reaction times and different concentrations ofbromocresol green are compared (2, 3); evefi different referencematerials of human origin gave different response. Ageing of rea-gents containing Pegosperse™ have been reported s an addition-al soufce pf error (7) s well s different salt concentration (8).The present fmdings should be taken iiito aecount in selecting cal-ibration materials. Pools of fresh sera should probably be used ssecondary calibration material (9).AcknowledgementsK. E. Rasmussen and H. Ravn, stud. M. Pharm. Sei., are thankedfor the Chromatographie comparison of different batches of bro-mocresol green.Tab. l. The time dependence of the colour yield of albumin preparations at different concentrations of bromocresol green in the re ctipiimixture at 630 nm, read against reagent blank. The absorbance is expressed in percentage of that obtained simultaneously on alyophilized serum from bank blood (D). Each set of data represents mean values obtained on at least three batches of brorho^cresol green (Merck art. no. 8121) from the batch numbers: 625927, 8601737,9622557,9023339 and 1153373. Batch number9622557 showed the most variable results, and examples of these data are given for albumin in parenthesis.Concentration of bromocresol greenReaction timeAlbumin solution(Kabi Diagnostica AB, Sweden)Fresh pool of human seraLyophilized human serum (G)453594(94)857021595(95)8671123594(94)87683592(91)84X)6621589(89)83X)65123588(87)85XX)651353583(81)82X)5521582(80)84X)56123580(77)86X)57Unitμιηοΐ/ΐs%%%(DADE, American hospital Suppliers Corp.Miami)Lyophiiized equine serum (H)(Nyegaard AS, Norway)Lyophilized bovine-equine serum (I)(Nyegaard AS, Norway)69 6871 70 7169 68 7078 78 8065 66 67 %85X) 89X) 88") %Absorbance r nge; lyophilized serum frombank blood (D), read against reagent blankAbsorbance of bromocresol green reagentread against water0.47-0.520.06-0.080.52-0.580.12-0.150.45-0.550.36-0.421.1x) s for all results (3-9) were 1.0 or less if no asterisk is present. One asterisk signifies s between 1.2 and 2.0.**) s for the 9 measurements was 3.6.J. Clin. Chem. Clin. Biochem. / Vol. 22,1984 / No. 4Short communication/Kurzmitteilung 307References1. Duggan, J. & Duggan, P. F. (1982) Clin. Chem. 28, 1407- 6. Uldall, A. (1984) Scand. J. Clin. Lab. Invest. 44, Suppl. 168, in1408. press.2. Robertson, W. S. (1981) Clin. Chem. 27, 144-146. 7. Silsand, T. & Pettersen, J. E. (1981) J. Clin. Chem. Clin. Bio-3. Ingwersen, S. & Raabo, E. (1978) Clin. Chim. Acta 88, 545- ehem. 79, 839-840.550. 8. O'Donnel, N. & Lott, J. A. (1978) Clin. Chem. 24, 1004.4. Walsh, R. L. (1983) Clin. Biochem. 76, 178-181. 9. Keyser, J. W.,Fifield, R. & Watkins, G. L. (1981) Clin. Chem.5. Keller, F., Feldmann, K. & Metz, F. J. (1981) J. Clin. Chem. 27, 736-738.Clin. Biochem. 79, 169—171.Lic. Pharm. Adam UldallDept. of Clinical ChemistryUniversity of CopenhagenHerlev HospitalHerlev Ringvej 75DK-2730 HerlevJ. Clin. Chem. Clin. Biochem. / Vol. 22, 1984 / No. 4

Analyte and Matrix Problems in the Calibration and Quality Assessment of the Bromocresol Green Method for Albumin in Serum

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Analyte and Matrix Problems in the Calibration and Quality Assessment of the Bromocresol Green Method for Albumin in Serum

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