investigation into paediatric bilirubin analyses in ... · investigation into paediatric bilirubin...

J Clin Pathol 1982;35:52-58

Investigation into paediatric bilirubin analyses inAustralia and New ZealandLR WATKINSON, A ST JOHN,* LA PENBERTHY

From the Department of Clinical Biochemistry, Flinders Medical Centre, Bedford Park, South Australia5042 SA

SUMMARY A small regional survey of direct spectrophotometric methods and a larger Australianand New Zealand survey of paediatric bilirubin analyses are described. The overall performance ofboth groups was unsatisfactory with an unacceptable high interlaboratory variation. This inter-laboratory variation was reduced significantly by the use of a spectrophotometric method witha common standard of methyl orange.

The Australian and New Zealand survey also examined the "state of the art" for the measurementof conjugated bilirubin and showed that laboratories could not adequately measure conjugatedbilirubin.

The need for paediatric bilirubin analyses to beperformed with small imprecision and little or noinaccuracy has been discussed by St John andPenberthyl and Blijenberg and Leijnse.2 There isalso a need for interlaboratory agreement of resultsso that the problems of transferring babies to specialcare hospitals are eliminated.

In a previous small regional survey' we showedthat the performance of the group was unsatisfactoryand gave an unacceptably high interlaboratoryvariation. It was suggested that further investigationof this problem particularly in the areas of methodsimplification and the use of common standards mayimprove the performance of these analyses.

Hertz et al3 critically examined a number of directspectrophotometric methods and recommended twomethods as being the most acceptable for measuringtotal plasma bilirubin. We have further investigatedthese methods to determine whether their useimproves the performance of paediatric bilirubinanalyses.

It is axiomatic that if laboratories in an inter-laboratory survey use the same standard then theinterlaboratory variation will be less than whenparticipants use a variety of standards. One of thedifficulties with bilirubin analyses is the choice ofstandard material and we have previously discussed

*Present address: Department of Clinical Biochemistry,Royal Perth Hospital, Perth, Western Australia 6000 WA.

Accepted for publication 4 June 1981

the problems associated with lyophilised sera andpure bilirubin standards.' Jackson4 demonstratedthe use of recrystallised, dried methyl orange inneutral solution as a secondary standard. Such astandard in combination with a direct spectro-photometric method should minimise the problemsof standardisation and so we have investigated thepractical use of this material.

If laboratories were widely to adopt direct readingprocedures such as spectrophotometric methods orsome brands of "bilirubinometers" then this raisesthe problem of assaying conjugated bilirubin(bilirubin esters). Many laboratories maintain the useof diazo coupling techniques because they assay forboth total bilirubin and conjugated bilirubin. Con-sequently the "state of the art" for the analysis ofplasma conjugated bilirubin requires examination.With the above problems to investigate we

conducted:1 a small regional survey to investigate the use of

direct spectrophotometric methods.2 a survey of Australian and New Zealand lab-

oratories where we have examined:(i) the state of the art for this larger group,(ii) the use of direct spectrophotometric methods

by the group,(iii) the use of a methyl orange secondary

standard with the direct spectrophotometricmethod,

(iv) the state of the art for the estimation ofconjugated bilirubin in plasma.

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Investigation into paediatric bilirubin analyses in Australia and New Zealand

Material and methods

AUSTRALIAN AND NEW ZEALAND SURVEY

Two distributions were made to participants in thesurvey of Australian and New Zealand laboratories.The first was in June 1979, when four samples weredistributed to 110 laboratories. Three samples wereplasma from exchange transfusions and the fourthwas a reconstituted lyophilised material, Precibil(Boehringer Mannheim GmbH, Biochemica,Mannheim, West Germany).The second distribution was in November 1979.

Five samples were distributed to 113 laboratories.Two were plasma from exchange transfusions, onewas adult plasma with a high concentration ofconjugated bilirubin and a fourth sample wasreconstituted Precibil from the same batch as theJune survey. The fifth sample was a primary bilirubinstandard prepared from National Bureau ofStandards Pure Bilirubin (Standard ReferenceMaterial 916; Office of Standard Reference Materials,Washington, USA) by dissolving the material in2-0 ml of 0-1 M sodium carbonate and 1-5 ml of0-1 M sodium hydroxide and diluting to 100 ml withpooled sera; as per instructions provided with thismaterial. Borate buffer pH 9 3 (30 5 g/l dipotassiumtetraborate);3 and a secondary standard of methylorange, prepared by dissolving 238-8 mg of dried,recrystallised methyl orange (May and Baker Lab-oratory Chemicals) in distilled water to a finalvolume of one litre, were also distributed.

Transport on both occasions was via expresscourier services. Participants were asked to assaysamples immediately.Table 1 shows the distribution of methods for

Table 1 Methods usedfor bilirubin analyses

Alfethod Total Conjugatedbilirubin bilirubin

Jendrassik and Grofs 25 38Malloy and Evelyn6 20 27Bilirubinometers 27Spectrophotometric 12Others 17 1 1Total 101 76

plasma total bilirubin and conjugated bilirubin.Several different formulae were used by the spectro-photometric group and the "Others" group includeda variety of diazo methods. A variety ofmaterials wereused as standards. Ten different commercial prep-arations and two pools of sera were used for internalquality control purposes by the participants.

REGIONAL SURVEY

"Elevated bilirubin control" (Ortho Diagnostics,

Raritan, New Jersey, USA) was distributed to SouthAustralian laboratories on two occasions. Thematerial was reconstituted in a central laboratoryand received by participating laboratories on themorning of preparation. Borate buffer pH 9*3,a secondary methyl orange standard and a primarybilirubin standard as described above were alsodistributed. The bilirubin concentration in thesamples provided was calculated using the two for-mulae recommended by Hertz et al.3 When a 1/41dilution in borate buffer and a 10 mm path lengthcuvette were used they were:

Formula I(Abs466 x 216 - Abs522 x 27 4) x 41 = ,umol/lFormula II

(Abs463 x 19-9 - Abs559 x 22-8) x 41 = ,umol/l

All specimens were prepared and distributed inliquid form. Care was taken to protect them fromthe light during and after preparation.

Results and discussion

One hundred and thirteen laboratories participatedin the Australian and New Zealand survey. Resultsobtained previously from the South Australianregional group' were unsatisfactory and it wasconsidered necessary to investigate further the stateof the art; this time obtaining information regardingthe methods used, standardisation and qualitycontrol procedures from a larger group. We alsowished to investigate whether our previous sug-gestions of method simplification and the use ofa common standard by all participants would reducethis problem.

Table 2 shows the results obtained for the Precibil

Table 2 Results lreturntedfor Precibil (assigned value327 ,umol/l)

Distribution No of labs Mean SD CV % Outlier

June 1979 100 328 24 4 7-4 1November 1979 93 324 30-8 9-5 3

In all calculations the mean and SD were calculated, outliers(results > 3 SD from the mean) removed and the calculations re-peated until all outliers were eliminated.

lyophilised control. We believed these results wereunacceptable for patient care. The 950% confidenceinterval was 262 to 386 ,smol/l, and the possibility ofreporting one of these two concentrations may leadto quite different clinical actions. This is an exampleof the problems that occur when a baby is transferredfrom one hospital to another. When these resultswere examined more carefully, on the basis of themethod of analysis (Table 3), no one method groupperformed significantly better than any other.

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Watkinson, St John, Penberthy

Table 3 Method comparison of results for the twodistributions ofPrecibil

Method No of labs Mean SD CV % Outliers

Jendrassik and Grof6 25 337 24-9 7-4 026 340 31-2 9-2 1

Malloy and Evelyn" 20 329 21-1 6-4 117 329 32-9 10-0 1

Bilirubinometers 27 319 21-9 6-9 022 311 14-2 4-6 1

Spectrophotometric 12 328 22-0 6-7 010 313 33-9 10-8 0

Others 16 331 29-4 8-8 018 319 38-3 12-0 0

The true test of each laboratory's analyses is itsperformance with neonatal plasma. Table 4 sum-marises the results of these distributions. Once againthe results were unacceptable for patient care. Noindividual method consistently produced the smallestinterlaboratory variation (standard deviation).

Table 4 Results returned for the five neonatal plasmadistributed

Sample No of labs Mean SD CV % Outliers

A 101 109 12-2 11-2 1B 101 200 16-0 8-0 0C 101 288 23-0 8-0 1D 96 156 14-6 9-3 1E 91 232 17-3 7-4 6

The variation found may be due to the inaccuraciesof each participating laboratory and not due tointralaboratory imprecision. However, it is possibleto gain an impression of the intralaboratoryimprecision using the difference between duplicates

formula (SD = Jd-). Table 5 shows the resultsi2nJ

of this approach and whilst these results (on onlyone pair of analyses) appear more clinically accept-able they do not achieve what we believe is the goalfor these analyses, namely a coefficient of variationof <2-5%.

Table 5 Intralaboratory precision from duplicateanalysis ofPrecibil

Method No of labs SD CV %* Outliers

Jendrassik and Grof5 17 15-3 4 7 4Malloy and Evelyn6 13 12-6 3-9 2Bilirubinometers 21 12-4 3-8 2Spectrophotometric 9 21 1 6 5 0Others 11 11-4 3 5 2

*The coefficient of variation was calculated using the assigned valueof 327 romol/l.

STANDARDISATION PROCEDUREMost laboratories were using commercial lyophilisedmaterial as a standard. Eleven laboratories wereusing some form of pure bilirubin standard.However, as a group they did not perform anybetter than those using other forms of standard-isation. Following the previously reported improve-ment in group results when using a commonstandard' we further investigated the use of thisapproach. The commercial lyophilised material(Precibil, stated value 327 pmol/l) and pure bilirubinstandard (NBS material, weighed in value 356,umol/l) distributed to participants were used toinvestigate two possible common standards. Table 6summarises the results returned by participants forthese two sera.When comparing the results of the NBS standard

for each method group both the Jendrassik and Grof5and Malloy and Evelyn6 mean values were sig-nificantly higher (p < 0-01) than 356 kmol/l. How-ever, the Bilirubinometer, "spectrophotometric" and"other methods" group mean values were not sig-nificantly different from the true value. This biasbetween methods poses problems when using NBSmaterial as a common standard. When using thismaterial as a standard, results for neonatal plasmawere recalculated as follows:"Standardised result" =Participant's result for sample alue of standardParticipant's result for standard

Table 6 Summary of results for Precibil and NBS standard

Method Precibil NBS standard

n Meani SD CV % Outliers n Mean SD CV % Outliers

All methods 100 328 24-4 7-4 192 30 95 0 493 324 30-8 9-5 3 92 370 29-5 8-0 4

Jendrassik and Grofs 25 337 24-9 7-4 026 340 31-2 9-2 1 28 384 38-2 9-9 0

Malloy and Evelyn" 20 329 21-1 6-4 1 17 375 30 5 8-1 017 329 32-9 10-0 1I7 35 3- -

Bilirubinometer 27 319 21-9 6-9 0 23 354 22-9 6-5 022 311 14-2 4-6 1

Spectrophotometric 12 328 22-0 6-7 0 10 355 19-8 5 6 010 313 33-9 10-8 010 35 9- 56 0

Others 16 331 29-4 8-8 0 17 369 42-4 11-5 018 319 38-3 12-0 0 17 369 42-4 11-5 0

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Investigationz into paediatric bilirubin analyses in Australia and New Zealand

The interlaboratory variation of these "standardisedresults" for neonatal plasma from the group was

significantly reduced for most methods by using thisapproach (Table 7). However because of the biasfound with the Jendrassik & Grof and Malloy &Evelyn methods "standardisation" of these resultssignificantly reduced the mean values comparedwith the Bilirubinometer and spectrophotometricmethods. The use of this material as a common

standard, although reducing the interlaboratoryvariation requires further investigation with respectto accuracy.When the same approach was used with a com-

mercial lyophilised sera (Precibil) similar resultswere obtained. Whilst the use of Precibil as a

standard did reduce the interlaboratory variation ofthe group's results it was not as consistent as theNBS bilirubin. However, the Precibil control did notintroduce such significant problems with bias as theNBS material.The use of a common standard improved the

performance of paediatric bilirubin analyses for thegroup, however, these results are still not adequatefor patient care and further improvements should besotught.

METHOD SIMPLIFICATION

Hertz et a13 recommended the use of two formulaewhen using spectrophotometric methods. Eightlaboratories in South Australia participated in a

regional survey to examine these formulae and

Table 8 shows the results obtained for two purebilirubin standards and Ortho "elevated bilirubincontrol". Coefficients of variation obtained suggestedthat there may be some potential for the use of theseformulae by all laboratories in one region. Thequality and maintenance of spectrophotometerswould probably be a significant source of variationwith these results. Consequently, provision of a

common standard for this method should reduce thevariation due to spectrophotometers and con-

sequently the use of methyl orange was investigated.The absorption spectrum of methyl orange diluted1/41 in borate buffer is shown in Fig. 1. If methyl

I -- Biirubin-Methyl orange

Absorbance

460 520 560Wavelength (nm)

Fig 1 Absorytion spectrum of Methyl Orange in BorateBuffer

Table 7 Neonatal plasma results using the NBS bilirubin as a common standard

Method Standardisation Sample D Sample E

n Mean SD CV Outliers n Mean SD CV % Outliers

All methods Raw data 96 156 14-6 9 3 1 91 232 17-3 74 6NBS 92 149 10-6 7-1 4 91 221 14-6* 6-6 5

Jendrassik and Grof5 Raw data 28 156 14-4 9-2 0 27 233 14-7 6-3 1NBS 28 145 11-4 7-9 0 27 217 9 4* 4-3 1

Malloy and Evelyn" Raw data 18 157 14 9 9 5 0 18 237 28-4 12 0 0NBS 17 147 5 2* 3-5 0 15 216 6 5* 3 0 2

Bilirubinometer Raw data 23 162 15 3 9 5 0 23 233 15 5 6-6 0NBS 23 160 13-5 8-4 1 23 245 18 2 7-8 0

Sp_ctrophotometric Raw data 10 154 13-2 8 5 0 10 226 21-3 9-4 0NBS 10 154 7-1* 4-6 0 10 226 11 9* 5-3 0

*Significantly reduced dispzrsion (p < 0-05).

Table 8 Summary of the regional survey of direct spectrophotometric methods

Sample No of labs Total no of results Formula used Mean SD CV%

Primary bilirubin standard I 8 8 1 (466/522) 313 18-9 6-011 (463/559) 337 18-4 5-4III (Methyl orange 463/559) 330 12-3 3-7

Primary bilirubin standard 11 8 8 1 (466/522) 323 38-7 12-011 (463/559) 350 17-5 5-0III (Methyl orange 463/559) 350 19-6 5-6

Ortho "elevated bilirubin control" 8 16 1 (466/522) 208 8-0 3 9(two separate analyses) 11 (463/559) 219 10-5 4-8

III (Methyl orange 463/559) 217 6-2 2-9

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orange is to be used as a secondary standard then itmust mimic bilirubin in respect to its absorptionspectrum, having a peak absorbance at approxi-mately 460 nm and negligible absorption at thesecond wavelength where correction is made forhaemoglobin, methaemalbumin, turbidity etc.Methyl orange absorbs significantly at 522 nm butnot at 559 nm. Consequently whilst formula II

(463/559) is suitable as a direct spectrophotometricmethod it is not suitable for use with a secondarymethyl orange standard. Table 8 shows the resultsfrom the regional group using the following formula(III) to determine total bilirubin concentration.Formula III

(Abs463 X 19-9 - Abs559 x 22 8)of Sample

x 370 = ~urol/l

(Abs463 x 19-9 - Abs559 x 22 8)of Methyl Orange Standard

(the methyl orange was assigned a value of370 umol/l).Whilst this approach did not markedly reduce thedispersion of this group the results confirmed the use

of the 463/559 formula and methyl orange as a

secondary standard. This approach was thereforeadopted in the Australian and New Zealand survey.

One hundred and thirteen laboratories in Australiaand New Zealand were supplied with borate buffer,a methyl orange secondary standard and asked toanalyse this standard and the sera distributed usingthe 463/559 nm spectrophotometric method de-scribed previously. Table 9 compares the resultsusing Formula II with those obtained by the group

(All methods) for the same material. The inter-laboratory variation of results from Formula II was

significantly reduced compared to the "All methods"results for the NBS standard, Precibil and one of theneonatal plasma distributed.The mean value for the NBS standard (359 ,umol/l)

is not significantly different from the weighed-in


value (356 ,umol/l). This suggests that Formula II

gives accurate results. However it should be notedthat the "Precibil" value (301 tmol/l) was sig-nificantly (p < 0-01) lower than the stated value of327 ,umol/l; once again demonstrating a bias betweenthe Jendrassik and Grof5 diazo coupling techniqueand the direct spectrophotometric assay.

In an effort to improve these results further,methyl orange was used as a standard as was

previously discussed with the regional survey. Thevalue 388 ,umol/l obtained by the group was assignedto the methyl orange. This resulted in a furthersignificant reduction in the interlaboratory variationwhen compared with the original data from alllaboratories. When examining the absorbancesreported by laboratories it was found that most ofthe outliers obtained were from isolated erroneous

readings. Where a laboratory reported a generalisedbias in the absorbances from all samples the methylorange corrected these results in most cases (54 outof 57). The use of methyl orange therefore appearsto correct for the deficiencies of most spectro-photometers in this survey. Similar improvementsin interlaboratory variation were found when usingeither the NBS bilirubin or "Precibil" as a standardfor the spectrophotometric method. However becauseof the stability of the methyl orange solution, thesimplicity of preparation, and the absence of batchto batch variation we believe that the use of methylorange will reduce standardisation problems. Con-sequently in reporting to participants of this surveywe recommended that regional groups could use

this approach with one central laboratory preparingthe methyl orange secondary standard.

CONJUGATED BILIRUBIN

One of the problems with recommending a spectro-photometric method for total bilirubin is thata laboratory must have an alternate method for theestimation of conjugated bilirubin. Thereforealthough a laboratory may not be entirely satisfied

Table 9 Results from a common spectrophotometric method compared with "All methods" results

Sample Method n Mean SD CV'% Outliers

Methyl orange Formula II 463/559 71 388 16-1 4-2 9NBS standard All methods 92 370 29 5 8-0 4

Formula 11 463/559 76 359 24-6* 6-8 4Methyl orange 463/559 76 364 16-1* 4-4 3

Precibil control All methods 93 324 30-8 9 5 3Formula II 463/559 71 301 20 5t 6 8 6Methyl orange 463/559 72 303 15-6t 5 2 4

Neonatal plasma D All methods 96 156 14-6 9-3 1Formula It 463/559 73 147 11.4* 7-8 7Methyl orange 463/559 72 149 8 0t 5-4 7

Neonatal plasma E All methods 91 232 17-3 7 4 6Formula 11 463/599 76 220 18-5 8 4 4Methyl orange 463/559 73 223 10-0t 4-5 6

Significantly reduced dispersion * p < 0 05. t p < 0-01.

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Investigation into paediatric bilirubin analyses in Australia and New Zealand

Neonatal plasmasample D

Neonatal plasmasample E

20-

151

10

5

0. ..3O 10 20 30 40 500 I/R/g///R/R//.4 c0 10 20 30 40 50Conjugated bilirubin (umol/l)

15

10

5-

Adult plasmahigh conjugatedvalue

0 20 40 60 80 10 120 140

Fig 2 Distribution of Results for Conjugated Bilirubin Estimations

with its analyses for total bilirubin it may continueto use a relatively unsatisfactory method in order tocontinue to provide a conjugated bilirubin deter-mination. Consequently we surveyed the state of theart for these analyses. Fig. 2 shows the distributionsof results obtained with two neonatal plasma andone adult plasma having a high concentration ofconjugated bilirubin.

If this survey reflects the state of the art forconjugaged bilirubin analyses then it is obviousthat laboratories are unable to report accuratelyconjugated bilirubin results. In view of these resultswe question the practice of reporting apparentlyaccurate results when it appears that at best theresults reported are only an approximation to thetrue value. We believe a more responsible approachwould be to rank conjugated bilirubin results asfollows (,umol/l):

<2525-5050-100

100-150150-200

This approach should be adequate for patient careand not lead to overinterpretation of results.Another factor in the use of conjugated bilirubin

results is the frequency with which the laboratory isrequired to perform this assay. Participants in thissurvey varied from "not at all" to "with every totalbilirubin." We believe that a discretionary ratherthan screening approach is necessary particularly as

more blood is required from the patient for eachconjugated bilirubin analysis. Obviously the questionof the frequency of analysis must be discussed withclinical staff but if the frequency of results forconjugated bilirubin estimation is reduced thena simple diazo coupling method could be used.Consideration regarding the most suitable methodfor total bilirubin estimation, where results must beboth precise and accurate, can then include spectro-photometric methods which at present appear toprovide the most acceptable performance.

Conclusions

Our primary aim during these investigations hasbeen to demonstrate the advantages of methodsimplification. We have shown that using thespectrophotometric method in conjunction with amethyl orange standard can produce accurate resultswith reduced imprecision, although improvement inthe latter was not as significant in the Australian andNew Zealand group as within the smaller regionalgroup of laboratories. Objections to using a spectro-photometric method are that it cannot measure therelative amounts of conjugated and unconjugatedbilirubin present in any patient sample but it is ourbelief that precise measurement of conjugatedbilirubin is probably not necessary and in fact oursurvey showed that the reporting of apparentlyprecise results may not be valid with the methodsused at present. We are sure that laboratories can

25

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maintain a method that will give an approximationof the plasma concentration of conjugated bilirubinwhich would be adequate for patient care and there-fore the method used for conjugated bilirubinanalysis should not affect the decision of whichmethod to use for total bilirubin analysis.

We are most grateful for the work by the laboratorieswho participated in the surveys. We would also wishto acknowledge the assistance of the AustralianAssociation of Clinical Biochemists and the RoyalCollege of Pathologists of Australia on whose behalfwe carried out the Australian and New Zealandsurveys.


References

St John A, Penberthy LA. An interlaboratory survey ofpaediatric bilirubin analyses. J Clin Pathol 1979;32:794-7.

2 Blijenberg BG, Leijnse B. A survey report on the deter-mination of total bilirubin in neonatal samples. J ClinChem Clin Biochem 1980;18:27-30.

3 Hertz H, Dybkaer R, Lauritzen M. Direct spectrophoto-metric determination of the concentration of bilirubinsin serum. ScandJ Clin Lab Invest 1974;33:215-30.

4 Jackson SH. A direct-reading bilirubinometer incorporatinghemolysis and turbidity correction. Clin Chemn 1965;2:1051-7.

5 Jendrassik L, Grof P. Vereinfachte photometrische Methodezur Beistimmung des Bilirubins. Biochem Zscht 1938;297:81-9.

6 Malloy HT, Evelyn KA. The determination of bilirubinwith the photoelectric colorimeter. J Bio/ Chem 1937'119:481-90.

Reports and Bulletins prepared by the Association of Clinical BiochemistsThe following reports and bulletins are published by the Association of Clinlical Biochemists. They may be obtainedfrom The Publishing Department, British Medical Journal (ACB Technical Bulletins), BMA House, TavistockSquare, London WCIH 9JR. Overseas readers should remit by British Postal or Money Order.

SCIENTIFIC REVIEWS (price £1E 00/$2.00 each)

1 The asscssment of thyroid function March 1971FV FLYNN and JR HOBBS

2 Renal function tests suitable for clinical practiceJanuary 1972 FL MITCHELL, N VEALL, and RWE WATTS

3 Biochemical tests for the assessment of fetoplacentalfunction May 1975 CE WILDE and RE OAKEY

4 Test of exocrine pancreatic function March 1977AH GOWENLOCK

5 Assay of cholinesterase in clinical chemistry March1979 ELSIE SILK, J KING, and MARY WHITTAKER

TECHNICAL BULLETINS (price £l-00/$2.00 each)

22 Bilirubin standards and the determination of bilirubinby manual and technicon AutoAnalyzer methodsJanuary 1971 BARBARA BILLING, RUTH HASLAM, and N

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23 Interchangeable cells for spectrophotometers andfluorimeters September 1971 Ss BROWN and AH

GOWENLOCK

24 Simple tests to detect poisons March 1972 BW

MEADE et al.25 Blood gas analysers May 1972 K DIXON

26 Kits for enzyme activity determination September1972 SB ROSALKI and D TARLOW

27 Assessment of pumps suitable for incorporation intoexisting continuous flow analytical systems November1972 A FLECK et al.28 Routine clinical measurements of transferrin inhuman serum September 1973 K DIXON

29 Control materials for clinical biochemistry (5thedition) September 1973 JF STEVENS30 Notes on the quality of performance of serumcholesterol assays September 1973 SS BROWN31 Determination of uric acid in blood and in urineJuly 1974 RWE WATTS32 A survey of amino acid analysers readily available inthe United Kingdom September 1974 JE CARLYLEand P PURKISS33 Definitions of some words and terms used in auto-mated analysis November 1974 A FLECK, R ROBINSON,SS BROWN, and JR HOBBS

34 Measurement of albumin in the sera of patientsJanuary 1975 LINDA SLATER, PM CARTER, and JR HOBBS

35 Investigation of the validity of temperature correctionfactors for serum aspartate and alanine transaminasesMarch 1975 SB ROSALKi et al.36 Factors influencing the assay of creatinine November1975 JGII COOK37 A survey of enzyme reaction rate analysers readilyavailable in the United Kingdom July 1977 RASAUNDERS and RF BURNS

38 Transport of specimens for clinical chemistry an-alysis November 1977 P WILDING, JF ZILVA, andCE WILDE

39 A scheme for the evaluation of diagnostic kits May1978 PH LLOYD40 A practical guide to gamma-counting in radio-immunoassay January 1980 CE WILDE and D OTTEWELL

41 The use of biochemical tests in the diagnosis ofdisorders of calcium metabolism July 1980 ANGELAFAIRNEY

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