Linda M. Thienpont, 3 Andre P. Dc Leenheer, Dietmar Stockl,2 and Hans Reinauer2

Transcription

1 CLN. CHEM. 39/6, (1993) Cndidte Reference Methods for Determining Trget Vlues for Cholesterol, Cretinine, Uric Acid, nd Glucose in Externl Qulity Assessment nd nternl Accurcy Control.. Method Trnsfer nd M. Thienpont, 3 Andre P. Dc Leenheer, Dietmr Stockl,2 nd Hns Reinuer2 We describe the testing of trnsferbility of cndidte Reference Methods developed by NSTAND for cholesterol, cretinine, uric cid, nd glucose. The methods re bsed on isotope dilution-gs chromtogrphy-mss spectrometry. The study consisted of two prts: setup of the methods nd self-evlution for rediness in the collborting lbortory, followed by independent mesurements in prllel with NSTAND. Criteri used for judging the trnsferbility nd generl relibility of the cndidte Reference Methods were: the ccurcy nd precision of the collborting lbortory nd the greement between the two lbortories. The ccurcy ws judged from the results on the Stndrd Reference Mteril 99 from the Ntionl nstitute of Stndrds nd Technology. For ll nlytes except glucose the bis from the certified vlue ws <.7%. The men intrlbortory imprecision rnged from.66% to 1.24%. The greement between the results ws tested by n dvnced linerregression nlysis nd Student s t-test. n generl, the results demonstrte tht the cndidte Reference Methods developed by NSTAND cn be successfully trnsferred without loss of their inherent precision nd ccurcy. ndexing Terms: qulity control lborto,y performnce The development nd evlution of cndidte Reference Methods (RMs) for the determintion of trget vlues for cholesterol, cretunine, uric cid, nd glucose in externl qulity ssessment (EQA) nd internl ccurcy control, ccording to the Guidelines of the Germn Medicl Society (1) hve been previously described (2). The performnce requirements of the RMs necessry for these pplictions hve been derived previously from the decision limits for clssifying lbortory s n outlier in the Germn EQA scheme (3). The methods re ll bsed on isotope dilution-gs chromtogrphy-mss spectrometry (D-GC/MS). They hve been evluted by comprison with the certified Stndrd Reference Mteril (SRM) 99 of the Ntionl Lbortori voor Medische Biochemie en voor Klinische Anlyse, University of Ghent, Hrelbekestrt 72, B.9 Gent, Belgium. 2lnstitut f#{252}r Stndrdisierung und Dokumenttion im Mediinichen Lbortonium e.v. (NSTAND e.v.), Johunes Weyer Strsse 1, D-4 D#{252}sseldorf 1, Germny. 3Author for correspondence. 4Nonstndrd bbrevitions: EM, ReferenceMethod;EQA, externl qulity ssessment; D-GC/MS, isotope dilution-gs chromtogrphy-mss spectrometry; SRM, Stndrd Reference Mteril; nd NST, Ntionl nstitute of Stndrds nd Technology. Received September 18, 1992; ccepted Jnury 13, nstitute of Stndrds nd Technology (NST, Githersburg, MD). Here we describe the verifiction nd documenttion of their trnsferbility, which is importnt for the cceptnce of method s RM, ccording to the pproved guidelines of the Ntionl Committee for Clinicl Lbortory Stndrds (4). Trnsferbility of method mens tht it cn be pplied in other lbortories by mens of written specifictions nd procedurl instructions lone, without ny loss of its inherent precision nd ccurcy. For the group who proposed the RMs, the lbortory of the nstitut f#{252}r Stndrdisierung mid Dokumenttion im Mediinichen Lbortorium e.v. (NSTAND e.v., D#{252}sseldorf, Germny), there ws n dditionl driving force for the trnsfer of the developed methods. As one of the officilly recognied Germn reference institutions for the determintion of RM vlues in control mterils for EQA nd internl ccurcy control (1), they felt the need to determine RM vlues by t lest two lbortories, to disclose otherwise possibly undetected smll systemtic errors, nd to mintin or even improve the originl performnce of the cndidte RMs developed t NSTAND. Ordinrily, study for testing interlbortory trnsferbffity of methods is bsed on the vilbility of detiled nlyticl protocol, including specifictions nd instructions on equipment, regents, clibrtion, mesurement, nd clcultion protocol. But becuse the lbortory of the University of Ghent (Belgium) hd mny yers of experience with the use of D-GC/MS s reference methodology for quntifying orgnic serum components (e.g., 5), the protocol for trnsfer of the proposed RMs of NSTAND ws reduced to description of smple pretretment nd fundmentl GC/MS mesurement conditions (type of column, derivtive, nd ml vlues). Regents but not their origin were specified, except for the primry clibrtion mterils used. n other words, it ws left entirely to the responsibility nd experience of the collborting lbortory director to ensure rediness nd optiml opertion of instrumentl equipment nd to perform D-GC/MS mesurements under the conditions of RM. The dvntge of this strtegy of method trnsfer ws tht it becme prt of n dditionl externl evlution protocol of the rebustness of the proposed RMs ginst vritions of equipment, lterntive sources of regent supply, nd differences in mbient operting conditions (4). n tril preceding the trnsferbility study, the lbortory of the University of Ghent performed self-competence evlution bsed on the quntifiction of SRM 99 serum in ddition to four ser previously CLNCAL CHEMSTRY, Vol.39, No.6,

2 mesured t NSTAND. After this preliminry study, the collborting lbortory ws invited to mesure, independently nd in prllel with NSTAND, series of control mterils tht were completely unknown to both lbortories. The results of this trnsferbility study re reported here. Mterils nd Methods Equipment Lbortory A used the equipment nd instrumenttion s described in the ccompnying rticle (2). Lbortory B used combined GC/MS 597B Mss Selective Detector system (Hewlett-Pckrd, Plo Alto, CA) equipped with 25 m x.2 mm (i.d.) methyl silicone chemiclly bonded fused-silic column (Ultr 1; Hewlett-Pckrd) with.33-p.m-thick film. The GC inlet of this system ws splitlsplitless uuector (Hewlett-Pckrd), used in the split mode. The high-performnce liquid-chromtogrphic system (HPLC) in lbortory B ws composed of equipment from Spectr-Physics (Sn Jos#{233}, CA): Model SP 875 pump, Model SP 87 solvent delivery system, nd Model SP 427 integrtor. Monitoring of the effluent ws done with n LC 871 UV-VS detector (Pye Unicm, Cmbridge, UK). For injections, 1-p.L injection loop (Vlco nstruments, Houston, TX) ws used. The HPLC column ws commercilly prepcked reversed-phse C15 column: Econosphere (5 p.m), 15 x.46 cm (i.d.), from Alltech (Deerfield, L), equipped with comptible 1-cm gurd column. For weighing the primry clibrtion mteril, clibrted nlyticl blnce ws used: 1-g Mettler type M5 blnce with redbility of io g (Greifensee, Switerlnd). Volumetric smpling ws performed with clibrted (on 1-g Mettler type B5 blnce with redbility of io g) Hmilton syringe devices (Bendu, Switerlnd). Regents The qulity nd origin of the regents used by lbortory A re described in the compnion rticle (2). Lbortory B lso used nlyticl-grde solvents nd chemicls. The regents for silyltion, i.e., N-(tertbutyldimethylsilyl)-N-methyltrifluorocetinide, N-trimethylsilyl-N-methyltrifluorocetmide, nd Silyl 991 ( mixture of N-trimethylsilyl-N-methyltrifluorocetmide nd trimethylchlorosilne, 99/1 by mss), were from Mcherey & Ngel (DUren, Germny). Pyridine ws purchsed from Pierce (Rockford, L), cetic nhydride from E. Merck GmbH (Drmstdt, Germny), nd hydroxylsrniine hydrochloride from Aldrich (Steinheim, Germny). Clibrtion Mterils Lbortories A nd B used the sme primry clibrtion mterils, i.e., the SRMs from NST, nd internl stndrds s described previously (2). The isotopiclly lbeled compounds were ll purchsed from Cmbridge sotope Lbortories (CL, Woburn, MA). The stock solutions in lbortory B were prepred in the sme solvents s mentioned before (2) for lbortory A. Their pproximte concentrtions were: 11. mmoll (cholesterol),.4 mmol/l (uric cid), 1.6 mmol/l (cretinine), nd 11. mmol/l (glucose). The SRM 99 lyophilied serum from NST ws reconstituted in lbortory B ccording to procedure B of NST. From this procedure, the following certified concentrtions (minol/l) were considered (ltest revision June 1988): cholesterol = 3.66, cretinine =.152, uric cid =.48, glucose = Becuse the revision dted from 1988, the decrese of the glucose concentrtion (-.44 mmol/l per yer) ws tken into ccount, ccording to the dt sheet shipped with the SRM 99. Study Design The trnsferbility study ws performed over 1-yer period with commercil control smples tht were sent to NSTAND for determintion of RM vlues. On three different occsions, the concentrtions of the nlytes investigted hd to be determined in four to six different ser within period of 6 weeks. The totl number of results per nlyte is not lwys equl (see Tble 4), becuse in certin ser, some nlytes were not requested by the mnufcturers. Anlyticl Procedure The nlyticl procedure of lbortory B differed from tht of lbortory A (see 2) concerning the reconstitution, smpling, nd clibrtion protocols. Lbortory B reconstituted lyophilied serum nd performed ll smpling steps on volumetric bsis. The ccurcy nd precision of these steps were ensured by using clibrted positive-displcement pipetting devices from Hmilton, i.e., the Microlb P nd Microlb CR7-5 systems. Clibrtion of ech syringe (5 p.l to 5 ml) ws performed by weighing 12 times the delivered volume of wter t known temperture nd tmospheric ir pressure on the Mettler B5 blnce. Depending on the use of the syringe (t its totl or prtil volume cpcity), clibrtion ws performed for the totl volume, or in ddition for 5% nd 1% of its totl volume cpcity. The mss of the wter ws converted to its corresponding volume by using the dt for clibrtion of glsswre mentioned in the Hndbook of Chemistry nd Physics (6). The inccurcy nd imprecision of ll pipetting devices used for reconstitution nd smpling of serum, clibrtion, nd internl stndrd solutions did not exceed.2%. Attention ws pid to the temperture of the solvents t the moment of use of the clibrted syringes. A mximum devition of 5#{176}C from the temperture t clibrtion ws llowed. For the D-GC/MS mesurements, lbortory B used the brcketing clibrtion technique (7). Three different stndrds ( low-, middle-, nd high-concentrtion stndrd), ech t lest in triplicte, were used. The mounts of nlyte in the stndrds closely encompssed the concentrtion of substnce to be mesured in the serum smples. The rtio of unlbeled to lbeled compound (stndrd/internl stndrd) in the three clibrtion mixtures vried from.8 to 1.2. The middle stndrd hd rtio of the unlbeled to lbeled compo- 12 CUNCAL CHEMSTRY, Vol. 39, No. 6, 1993

3 Tble 1. GC Conditions for Anlyse s n Lbortory B nitil column Retention Het out fter Anlyts Dsrlvtlvs temp., C Rte, C/mm Finl temp., C time, mm nlysis, C Cholesterol TMS (1) Cretinine BDMS 18 (1) (3.2) (2) Uric cid TMS 23 (1) 2 28 (3.5) (3) Glucose NPA 17 (1) 1 23 (2.5) (2.5) TMS, thmethsi1 ether; BDMS, te,t-butyldimethylsilyt ether; NPA, nitrilopentcette. Holding time (minutes) given in prentheses. nent of -1, s did the serum smples. To be ble to nlye different serum smples within the sme clibrtion curve, ech lbortory dpted the serum volumes to contin lwys the sme totl nlyte mount, i.e., tht corresponding to the mount present in 1 L of SRM 99 serum of NST. According to the described procedure, preliminry nd pproximte quntifiction of the concentrtions of the unknown smples ws necessry. GC/MS mesurements of the ser were lso done ccording to the brcketing principle. This mens injection of myimum of three smples between two low nd two middle stndrds or between 2 middle nd 2 high stndrds, nd vice vers. Pek-re rtios of stndrds so mesured hd to gree within 1% for cceptnce, wheres duplictes of the processed smples hd to gree within 2.5%. n cse this criterion ws not met, fourth set of prllels (two smple liquots processed nd mesured on the sme dy) ws processed nd mesured. Clcultion of the concentrtion of the serum smples ws done by liner interpoltion on the dily clibrtion curve, constructed by the method of lest squres (8). For ech dy of mesurement, different clibrtion stock solution ws used. t ws cross-checkedbefore use for cceptble ccurcy ginst the previous one. As requested by the generl study design, ech RM vlue ws obtined from six independent mesurements, performed on three different occsions on two liquots of three different reconstituted ser. n csethe intrlbortory between-dy coefficient of vrition (CV) exceeded 2%, fourth set of mesurements ws done. Specific procedures. For ech nlyte, the protocol for smple pretretment, derivtition, nd GC/MS mesurement (m/ vlues) described by lbortory A ws followed. The specific nlyticl conditions for lbortory B re described in Tbles 1 nd 2. The mounts Tble 2. MS Conditions for Anlyses n Lbortory B m Dwelltim., Multiplier Anlyte Unlbeled Lbeled msb voltge, V Cholesterol Cretinine Uric cid Glucose #{149} Derivtives s n Tble 1. bdwell times were chosen to perform between 15 to 2 mesurement cycles under the peks. used for the MS mesurements were: -13 pmol (5 ng) for cholesterol, -3 pmol (.35 ng) for cretinine, -3 pmol (.5 ng) for uric cid, nd -28 pmol (5 ng) for glucose. Evlution Protocol For cholesterol totl of 17 ser were nlyed in both lbortories, for cretinine 18 ser, for glucose 14 ser, nd for uric cid 18 ser. For ech set of results the individul vlues, the men, the stndrd devition (SD), nd the CV were reported. To compre the men results obtined in the two lbortories, we used n dvnced liner-regression method insted of the lest-squres procedure, becuse there is nlyticl error in the vribles plotted on both the bsciss nd the ordinte. Our method, for which MS DOS computer progrm is vilble on request, is bsed on mximiing the likelihood function of the composed vribles x nd y. As result, estimtes for the slope p nd the intercept nd the covrince mtrix re obtined; from the ltter, the SD for $ nd cn be derived. From these dt, the confidencelimits (P =.95) for slope nd intercept cn be clculted. This confidenceintervl should contin the vlue of 1 for the slope p nd for the intercept, if the results of lbortory A nd B re identicl. More generlly, one cn investigte for different vlues of x whether the clculted + x vlue indeed is identicl to x within the sttisticl error. This is done by clculting the -vlue [ = ( + fix - x) o( + fix - x)], which should hve n bsolute vlue <2 t the 95% confidence level. n principle, in cseof identity of results obtined in two lbortories, this criterion for should be fulfilled for ll vlues of x in the rnge of interest [note tht the confidence intervl for corresponds to the prticulr cse where x =, so tht is reduced to / ()]. We reduced the test for identity between + fix nd x over the whole concentrtion rnge to the clcultion of the -vlues for three representtive concentrtions (low, medium, nd high). n ddition, we constructed for the pired observtions new vribles: result of lbortory B minus result of lbortory A divided by the men of the results. These new vribles not only hve the sme SD, but lso should hve men equl to in the bsence of so-clled lbortory effect. This null hypothesis is investigted with the Student s t-test. CUNCAL CHEMSTRY, Vol. 39, No. 6,

4 Resufts We stress tht ll results obtined during the trnsferbility study re shown in the following tbles nd figures (i.e., no outliers hve been removed). Tble 3 summries the results for the quntifiction of cholesterol, cretinine, uric cid, nd glucose in SRM 99 serum by lbortory B during the trnsferbility study. Tble 4 tbultes for lbortories A nd B the rnge nd men overll imprecision (expressed s CV) obtined for nlysis of ech nlyte in the different ser. Figure 1 shows the percentge devition of the results of lbortory B from those of lbortory A vs the respective men concentrtions clculted from the results of both lbortories. Tble 5 shows for ech nlyte the slope, the intercept, nd their respective 95% confidenceintervls of the regression eqution y = + lix clculted by the method of dvnced liner regression. Tble 6 tbultes the vlues for t three representtive concentrtions nd clculted for testing the identity of + fix nd x over the whole concentrtion rnge. Tble 6 lso lists the tbulted nd clculted t-vlues t the 95% confidence level nd corresponding degrees of freedom. Discussion Accurcy From the dt reported for the internl ccurcy control with the SRM 99 serum of NST (Tble 3), it is shown tht lbortory B met for ll but 1 nlyte (glucose) the predefined criteri of NSTAND of mximum bis of.9% (3). Except for glucose, the men devitions from the certified trget vlue were.6%. For glucose men devition of - 1.3% from the expected trget concentrtion ws observed. This vlue exceeds the mximum expected RMs inccurcy of.7% s outlined in reference 2. Therefore, it ws suspected Tble 4. CVs (%) Obtined n Lbortores A nd B for Ech Anlyte during the Trnsferbility Study Lbortory A Lbo rtory B Anlyte n Rnge Men Rnge Men Cholesterol Cretinine Glucose Uriccid tht in lbortory B problems with the trnsfer of the glucose method rose. This ws supported by occsionlly observed high mesurement imprecisions nd unexplinble devitions from the expected isotope rtios. During the observtion of these problems, lbortory B hd temporry ccess to mss spectrometer connected to Vrin GC equipped with its stndrd split/splitless injector. When the sme smples were mesured with this system, good precision nd ccurcy were obtined. Therefore, we conclude tht conventionl split/splitless injectors my cuse problems when the nitrilopentcette derivtive of glucose is used for the D-GCMS mesurements. As consequence, lbortory B in the future will use progrmmble temperture vporiing injector for the glucose nlyses. mprecision The dt on the intrlbortory CVs (Tble 4) re in generl in ccordnce with the dt reported in Prt (2). They show tht the method trnsfer ws chieved without loss of precision for cholesterol nd uric cid. A smll increse in imprecision ws observed for cretinine nd glucose in lbortory B, but the imprecision never exceeded the limits of mximum of 2% s set by NSTAND (3). Tble 3. Lbortory B Results for SRM 99 Serum during the Trnsferbility Study Dmsrence from Anlyts certified vlue, % Cholesterol Men+.6 Cretinine Men-.1 Uric cid Men -.4 Glucset Men-1.3 #{149} Certifiction vlue bsed on the nlysis of SAM 99 serum ccordingto procedure B. b The devition s clculted on the ssumption tht the glucose concentrtion decreses by.44 mmol/l per yer. 14 CUNCAL CHEMSTRY, Vol. 39, No. 6, 1993 Sttisticl Evlution of the Results A visul inspection of Figure 1 revels tht the percentge devition of the results of lbortory B from CV, those of lbortory A is not concentrtion dependent..7 However, for glucose, unidirectionl devition from.3 the idel ero line is observed. The observed differences.2 rnge from -5.3 to +.4% (men: -1.9%). These dt suest tht lbortory B hs negtive bis in corn- 1. prison with lbortory A for glucose. This is not the 1.8 cse for cholesterol, cretinine, nd uric cid, for which.9 respective differences rnging from -2.2 to + 1.7% 1 3 (men: -.3%), from -3.3 to +2.6% (men: +.4%), nd 9 from -2. to + 1.4% (men: -.2%) re observed. 7 The sttisticl evlution by the bove-described tests led to the following conclusions. The vlues clcu- 1. lted for the slope p ccording to the dvnced liner.9 regression method (Tble 5) do not show sttisticlly 1.2 significnt (P =.5) proportionl difference between the results obtined in both lbortories for ny of the mesured prmeters. ndeed, the confidence intervls clculted for fi ll comprise the vlue of 1. From the confidence limits clculted for (t P =.5), it is reveled tht for glucose the null hypothesis of =

5 CHOLESTEROL CREATNNE = - L. -1 -, 2 C 5. x - Z. 2 f D , URC ACD GLUCOSE 4 - C #{149},.,,.P / #{176} C 2 S #{176} -2.. #{149} S 17 Fg. 1. Devtion of lbortory B from lbortory A in percentge of the men for cholesterol,cretinine, uric cid, nd glucose Tble 5. ntercepts, Slopes, nd Confidence Umlts of the Regression Eqution Clculted by Advnced Uner Regression Men (nd 95% confidence lmts) Cholesterol Cretinine Uric cid Glucose lnterce 3.79 (-81.5,89.1) imol/l -.16 (-1.31,.97) tmolfl -.98 (-4.49, 2.53) mol/l -.14 (-.26, -.2) mmol/l y lbortory B; x = lbortory A. #{149} Sttisticlly sgnificnt (P.5). Stepe.997 (.97, 1.23) 1.4(.995,1.13) 1.(.992, 1.9).998(.982, 1.14) cnnot be ccepted, contrry to the other prmeters. For glucosethe vlue of ero is not comprised in the 95% confidence intervl, which mens tht there exists constnt bis for the results between lbortories A nd B. However, s the outcome of the test on = depends only on identity of the results obtined in two lbortories for concentrtions of x =, it ws interesting to reinvestigte for ll prmeters the outcome of the test fter expnsion over the whole concentrtion rnge (Tble 6). But gin, only for glucose were sttisticlly significnt differences between the two lbortories reveled. The sme observtion ws derived from the Student s t-test (P =.5) s shown in Tble 6. For glucose, it is evident tht the disgreement be- Tble 6. Test for dentity of rv + (ix nd x over the Whole Concentrtion Rnge Conc of Anlyte mmol/l Cholesterol Cretinine Uric cid Glucose Absolute.vluee #{149} Sttisticlly significnt (P =.5) Tbulted #vlues Clculted tween the two lbortories is in ccordnce with the negtive bis of lbortory B compred with the certified vlue of the SRM serum of NST (Tble 3). Though for the other prmeters the sttisticl considertions outlined erlier reveled no significnt differences between the results of lbortory A nd B, devitions up to 3.3%, s in the cse of cretinine, might seem excessive for smples nlyed in replicte with RMs. But for judging the mgnitude of the observed interlbortory CLNCAL CHEMSTRY, Vol. 39, No. 6,

6 devitions, the totl error of the RMs should be tken into ccount. As outlined underaccurcy, the mximum systemtic error in lbortories A nd B ws estimted to be ±.7% (except for glucose with n observed men inccurcy of 1.3%). This mens tht in the worst cse, i.e., lbortory A deviting from the true vlue by -.7% nd lbortory B by +.7%, the discrepncy between both lbortories due to systemtic errors could mount to 1.4%. To this vlue the sttisticl uncertinty of the mesurement results hs to be dded. At the 95% confidence level, this uncertinty is clculted ccording to the t-sttistic s: ± t(5) (CV/V ), in the cse of RM vlue obtined from six determintions (see design of our study). With men CVs of.7%, 1.24%, nd.89% observed for cholesterol, cretinine, nd uric cid (see Tble 4), the vlues for rndom error mount to ±.8%, ± 1.4%, nd ±.9%, respectively. n this wy the mximum devition for cholesterol should be 3.% [2 (.7%) + 2 (.8%)], for cretinine 4.2%, nd for uric cid 3.2%. These vlues were never exceeded in the described trnsfer study, s the mximum observed devition between lbortories B nd A mounted to only 2.2% for cholesterol, 3.3% for cretinine, nd 2.% for uric cid. n summry, the cndidte EMs for cholesterol, cretinine, glucose, nd uric cid developed by NSTAND show goodtrnsferbility. The study demonstrted tht the proposed RMs permit nother lbortory to meet criteri of n intrlbortory CV of less thn 2% nd n inccurcy of less thn.9% (except for glucose).as the collborting lbortory used the pproch of reconstitution nd smpling on volumetric bsis s well s the liner brcketing clibrtion method with stndrds surrounding the concentrtions in the unknown smples, the relibility of the NSTAND pproch of working on weighing bsis nd clibrting with stndrd level of 1 is shown. n ddition, the robustness of the cndidte RMs ginst different sources of regents, different GC/MS pprtus, nd different working conditions is proven (except in the cse of glucose). Lst, different generl pplicbility of the cndidte EMs for relible determintion of trget vlues in control mterils to be used for EQA nd internl ccurcy control is shown. We cknowledge the ssistnce of Prof. Steyert for sttisticl evlution of the results. References 1. RichthnienderBundesrtekmmer ur Qulittsicherung in mediinichen Lbortorien. Dtch Artebl 1988;85A699-A St#{228}ckl D, Reinuer H. Cndidte Reference Methods fordetermining trget vlues for cholesterol, cretinine, uric cid, nd glucose in externl qulity ssessment nd internl ccurcy control 1. Method setup. Clin Chem 1993;39:993-1 (this issue). 3. Stockl D, Reinuer H, Appel W, v Klein.Wisenberg A. Diskussionsvorschlgf#{252}r em einheitliches Referenmethodenkonept uf der Gnmdlge der Richtlinien deebtmdesirteknuner ur Qulittsicherung in mediinischen Lbortorien. Lb Med 1991;15: Ntionl Committee for Clinicl Lbortory Stndrds. Development of ReferenceMethods in clinicl chemistry for the Ntionl Reference System in clinicl chemistry. Approved guidelines. Document NESCL2-A. Villnov, PA. NCCLS, Thienpont L, Sieksmnn L, Lwson A, Colinet E, Do Leenheer A. Development, vlidtion, nd certifiction by isotope dilution gs chromtogrphy-mss spectrometry of lyophilied humn serum reference mterils for cortisol (CRM 192 nd 193) nd progesterone (CRM 347 nd 348). Clin Chem 1991;37: West RC, ed. Hndbook of chemistry nd physic,56th ed. Clevelnd, OH: CRC Press, 1974:D Cohen A, Hert HS, Mndel J, Pule RC, SchfferB, Sniegoski LT, et l. Totl serum cholesterolby isotope dilution/mss spectrometry: cndidte Definitive Method. Clin Chem 198;26: Yp Wi, SchfferB, Hert HS, White E V, Welch MS. On the differencebetween using liner nd non-liner models in brcketing procedures Mss Spectrom in isotope dilution 1983;1: mss spectrometry. Biomed 16 CUNCALCHEMSTRY,Vol. 39, No. 6, 1993