A. Clerico

G. Crepaldi

G. Penno

G. Rigamonti

L. Cruschelli

O. Giampietro

R. Navalesi

CLERICO, ALDO

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Letters to the Editor should betyped double-spaced (including ref-erences) with conventional mar-gins. The overall length is limitedto five manuscript pages, includingnot more than one figure or onetable.UrIne poolFirst phase (1 1 laboratories)1 (low) 552 (medium) 443 (high) 46Second phase (15 laboratories)11 (low) 5612 (medium) 6013 (high) 59Assays,nGreat mean± SDTotal(cvT)aBetween-kIt(cvbk,JbWIthIn-kIt(CV.,,.JcdBetween-lab(CVb.,,,,)WIthIn-lab(CV.,J4.55 ± 2.3218.95 ± 9.138.6 ± 24.351 .048.065.934.6 (46%)g30.4 (40%)g52.3 (63%)g37.5 (54%)”37.1 (60%)h40.2 (37%)”28.2 (57%)29.9 (64%)33.4 (69%)24.7 (43%)22.4 (36%)22.3 (31%)3.42 ± 1 .6620.9 ± 2.952.2 ± 9.448.513.818.19.6 (3.8%)2.9 (3.7%)4.8 (7.2%)47.5 (96%)’13.5 (96%)’17.4 (93%)142.4 (86%)11.5 (72%)14.9 (75%)16.9 (14%)7.1 (28%)8.7 (25%)a CV,? = CVb kit  CVWkn2.b Percent of total variability due to between-kit differences is shown in parentheses: 100 x(CVb kt/CVT)C CVw.kJt2 CVbgab2 + CVW labd Percent of total variability due to within-kit difterences is shown in parentheses: 100 x (CV,, kft/CVT).a Percent of total variability due to between-lab differences is shown in parentheses: 100 x (CVb 2/CV ,,2)f Percent of total variability due to within-lab difterences is shown in parentheses: 100 x(CVW,b2/CVWkft2).a p <o.oi.hp<0005P<O.001.CLINICAL CHEMISTRY, Vol. 38, No. 11, 1992 2333External Quality Control forRadioimmunoassay of UrinaryAlbumin in Diabetic Subjects: AOne-Year ExperienceTo the Editor:In diabetic populations, who havean increased prevalence of hyperten-sion and renal disease, the assay ofurinary albumin excretion (UAE) hasassumed an important role in the pre-vention and monitoring of diabeticnephropathy and in the evaluation ofcardiovascular risk (1-4). Becausescreening for microalbuminuria hasbecome part of routine diabetes careand involves an increasing number ofpatients, a suitable and standardizedmethod should be used for measuringalbumin in urine. To meet theseneeds, one must know and try to mm-imize all analytical sources of varia-tion and error. Although several im-munological procedures (such asenzyme immunoassay, immunotur-bidimetric assay, fluoroimmunoassay,and immunonephelometric assay) havebeen widely used for routine measure-ment of UAE, radioimmunoassay isgenerally considered the most reliablemethod (5, 6). Because external qualityassessment (EQA) programs contrib-ute to improving the analytical perfor-mances of participating laboratoriesthrough the comparison oftheir resultswith those produced by others, our lab-oratory, starting in March 1988, orga-nized a national pilot EQA program forurinary albumin RIA to estimate thevariability among and within laborato-ries in Italy as well as to check thepossibility of improving, by an EQAsurvey, laboratory performance inmeasuring microalbuminuria. Here,we report results from the first year ofthis program.The EQA scheme was based on thedistribution of frozen pooled urinesamples, followed by the computerizedanalysis of the returned results, aspreviously described (7-9). Partici-pant laboratories assayed quality con-trol (QC) samples by their routine pro-cedures, returning results with thename of the method or kit used. Aftercollected results had been processed bycomputer, summaries were preparedand sent back to each participant. TwoEQA cycles have been accomplished(each cycle lasts -6 months). Fifteenlaboratories took part in the EQA pro-gram (11 in the first phase). Only twokits were used by the 15 participantlaboratories: Albumin RIA 100, sup-plied by Pharmacia (Uppsala, Sweden;n = 8; n = 6 in the first phase), andH-Albumin, by Sclavo (Ciisello Bal-samo, Milano, Italy; n = 7; n = 5 in thefirst phase).We prepared 450 QC samples from13 urine pools to be sent to partici-pants in the two cycles. During thefirst one, we mailed out, in five dis-patches, 165 QC specimens to 1 1 lab-oratories (Table 1). All these sampleshad been prepared from three urinepools (pools 1, 2, and 3) with low (con-sensus mean = 5.2 ± 0.95 mgIL), me-dium (23.6 ± 3.3 mgfL), and high (59.3± 8.1 mg/L) albumin concentrations.During the second cycle, we sent 285QC vials derived from 10 urine pools:three pools had been set up by addingto a low-albumin pool weighedamounts of human albumin (pools 4,5, and 6), and four pools had beenprepared by progressive dilution of ahigh-albumin pool (pools 7, 8, 9, and10). These seven pools were preparedto better evaluate the accuracy of par-ticipating laboratories, because the to-tal variability found in the first cyclewas surprisingly large (CVs from 48%to 66%). The last three pools derivedfrom urine samples with low (consen-sus mean = 3.9 ± 1.3 mg/L), medium(19.5 ± 2.9 mgIL), and high (52.9 ± 8.7mg/L) albumin contents (pools ii, 12,Table 1 . One-Factor Analysis of Variance of 1 45 UrInary Albumin Determinations In the First Cycle of the EQASurvey and of 175 Urinary Albumin Determinations In the Second Cyclecv, % Wfthln-kft cv, %2334 CLINICAL CHEMISTRY, Vol. 38, No. 11, 1992and 13; Table 1). All urine samplesused for QC sample preparation werenegative for bacterial growth and con-tamed sodium azide (-2 mL of a 200g/L solution per liter of urine). Pre-pared QC aliquots were frozen at-20 #{176}Cuntil sent to participants,packed in solid CO2 in polystyrenecontainers.All results collected in each EQAcycle were used to compute (7-9) thegreat or general mean (for each pool),the consensus mean (for each pool),laboratory bias, laboratory precision,and total variability or between-labagreement (CV) and components ofvariability. We also calculated theouthier results by two statistical ap-proaches, the target-value method andthe Cochran-Grubbs test (10). For thetarget-value method, throughout eachphase of the survey, we considered asoutliers all the values that were >2SD from the means of the RIA resultsprovided by our laboratory (target vah-ues). During the first cycle ofthe EQAprogram, pools 1, 2, and 3 were tested42, 42, and 28 times, respectively, byour laboratory in different experimen-tal sessions. In the second phase, pools11, 12, and 13 were measured 55, 51,and 47 times, respectively, in differentsessions.The consensus means calculated af-ter deletion of outliers by the Coch-ran-Grubbs test were very similar tothe means obtained after exclusion ofoutliers by the target method, exceptfor QC pool 3. The sequential applica-tion of the Cochran-Grubbs test al-lowed the removal of three haborato-ries in the first phase (one for pool 2and two for pool 3) and none in thesecond phase of EQA. A significantdecrease of variability between thefirst and the second EQA phases wasalso confirmed by the reduction of out-her results as calculated by the target-value method: 62 of 145 results (42%)of the first phase and 46 of 278 results(16.5%; P <0.001) of the second phasewere >2 SD of the target values.The total variability (CVT) of urinepools 1, 2, and 3, sent during the firstphase ofEQA, was very large, rangingfrom 48% to 66%. The within-kit andthe between-kit components of van-ability were quite similar, thus mdi-cating that both the imprecision of thetwo RIA kits and the between-kit dif-ference were important causes of thehigh urinary albumin assay impreci-sion in that EQA phase (Table 1). Infour laboratories, the mean biasshowed a systematic deviation fromthe consensus mean, whereas in twoother laboratories, the confidence him-its of the mean appeared extremelywide. Results from these six laborato-ries accounted for the large number ofoutliers found in the first phase of theEQA program.The analysis ofthe recovery and thedilution tests showed good results for11 of 15 laboratories. Two laboratorieshad poor results for both the recovery(mean of the absolute values of thepercent deviations from the expectedvalues >20%) and the dilution tests(intercept significantly different fromzero). Two other laboratories had poorresults only for the recovery test.These four laboratories accounted for>50% of the outliers. The total van-ability remained very large for pooh 11(48%), but was substantially lower forpools 12 and 13 compared with CVTobserved for similar pools in the firstEQA phase (pools 2 and 3, Table 1).Regardless of the albumin concentra-tion, the between-kit component of thevariability was lower in the second(<10%) than in the first phase (30-50%) of the EQA; no differences wereobserved for results obtained with thetwo kits.Laboratories with both accuracyand precision >25% improved theirperformances substantially during thesecond phase ofthe EQA survey (4/11vs 12/15; chi-square = 5.10;P = 0.024)for assays of urine samples with me-dium and high albumin concentra-tions.A prerequisite for the EQA programis the stability of EQA specimensthroughout the study. We previouslyreported the stability over time ofurine pools at -20 #{176}C(as long as 6months) (11) and after repeated freez-ing and thawing (as many as fivetimes) (12). Stability of EQA sampleswas also confirmed by repeatedly mea-swing them in the reference labora-tory throughout the two phases of thestudy. These data agree with others,indicating that urine samples are rel-atively stable for several months at-20 #{176}C(6).A very high variability (from 40% to60%) emerged from the first phase ofthe EQA program. This finding wassurprising because RJA of urinary al-bumin is generally considered so ro-bust, easy to perform, and reliablethat it is suggested as the standardtechnique for UAE determination (5,6). All participant laboratories mea-sured UAE by only two commercialifiA kits. Conceivably, if laboratoriesusing other RIAs or other methods(such as nephelometnic or turbidimet-nc assay) had participated in the EQAscheme, the between-lab agreementwould have been worse, because sig-nificant biases have been found amongthe different methods currently usedfor UAE (5, 6).The total variability significantlyimproved in the second phase of theEQA survey, especially for albuminconcentrations in the medium andhigh range. A large reduction in thebetween-kit component occurred too.Evidence consistent with ours was re-cently provided by a study (13) de-signed to check the existing variabil-ity in the measurement of albumin inurine among five laboratories usingvarious immunoassays (enzyme im-munoassay, immunoturbidimetnic as-say, fluorescent immunoassay, andzone immunoelectrophoresis assay).Results from this study indicated con-siderable variation both within andamong laboratories; variability wasequally attributable to imprecisionand relative inaccuracy, being partic-ularly high for materials with lowalbumin concentrations (13).In conclusion, our data indicate thaturinary albumin assays need to be ad-equately standardized, and an externalQC of the assay is necessary even forlaboratories that use a common assayprocedure (such as RIA). Actually, theinterlaboratory survey is the only wayto quantitatively evaluate the perfor-mance of an RJA. The improvement inurinary albumin assay in the secondEQA period was particularly relevantbecause it was obtained for those un-nary albumin concentrations generallyconsidered of high clinical useflulnessfor classifying diabetic people accord-ing to well-established risk categoriesfor diabetic nephropathy and early car-diovascular mortality.References1. Viberti GC, Walker JD. Diabeticnephropathy: etiology and prevention. Di-abetes Metab Rev 1988;4:147-62.2. Mogensen CE. Microalbuminunia as apredictor of clinical diabetic nephropathy.Kidney Int 1987;31:673-89.3. Bennett PH. “Microalbuminunia” anddiabetes: a critique-assessment of urinaryalbumin excretion and its role in screeningof diabetic nephropathy. Am J Kidney Dis1989;13:29-34.4. Mogensen CE. Prediction of clinical di-abetic nephropathy in IDDM patients: al-ternatives to microalbuminuria? Diabetes1990;39:761-7.5. Giampietro 0, Chenico A. Microalbumi-nuria in diabetes: which method to employ,which sample to collect. J Nucl Med AlliedSci 1990;34:111-20.6. Rowe N, Dawnay A, Watts GF. Mi-croalbuminunia in diabetes mellitus: re-view and recommendations for the mea-surement of albumin in urine. Ann ClinBiochem 1990;27:297-312.7. Pilo A, Zucchelli M, Piro MA. Externalquality control for T3 and T4 assays inCLINICAL CHEMISTRY, Vol. 38, No. 11, 1992 2335Italy: outline of the scheme and results ofthe first semester experience. J Nucl MedAllied Sci 1981;25:107-18.8. Zucchelhi GC, Piho A, Chiesa MR, PiroMA. Progress report on a national quality-control survey oftriiodothyronine and thy-roxin assay. Chin Chem 1984;30:397-8.9. Piho A, Zucchelli GC, Chiesa MR. Corn-ponents of variance analysis for data col-lected in interlaboratory quality controlstudies ofimmunoassay. J Nucl Med AlliedSci 1985;29:269-73.10. Horwitz W. Protocol for the design,conduct and interpretation of collaborativestudies. Pure Appl Chern 1988;60:855-64.11. Giampietro 0, Clerico A, Cruschelhi L,et al. Microalbuminuria in diabetes melhi-tus: more on urine storage and accuracy ofcolorimetnic assays [Letter]. Chin Chem1989;35:1560-2.12. Gianipietro 0, Miccoli R, Clerico A, et al.Urinary albumin excretion in normal sub-jects and in diabetic patients measured byradioimmunoassay. methodological and chin-ical aspects. Clin Biochem 1988;21:63-8.13. Mueller PW, MacNeil ML, Smith SJ,Miller DT. Interlaboratory comparison ofthe measurement ofalbumin in urine. ChinChern 1991;37:191-5.Giuseppe Penno’Aldo Clerico’Lorella Cruschelli’Giusy Rigamonti2Gaetano Crepaldi3’4Renzo Navalesi’Ottavio Giampietro”51 Cattedra di Malattie del Metab.Istituto di Clin. Medica II and Istitutodi Fisiol. Clin. del CR.R.University of PisaPisa, Italy2 ICI PharinaMilano, Italy3 Istituto di Med. Interna e di Med.Clin.University of Pad uaPadua, Italy4 Study Group Coordinator, Italian Mi-croalbuminuria Study Group.5 Address correspondence to this author, at:Cattedra di Malattie del Metabohismo, Isti-tuto di Chinica Medica H Via Rorna 67,56100 Pisa, Italy.Monitoring NutrItIonal Status InBurn PatientsTo the Editor:Rettmer et al. (1) compared the per-formance of several static measure-ments of serum concentrations withsome functional tests for the nub-i-tional assessment of burn patients.Although their conclusion that func-tional tests generally provide a moreaccurate evaluation of nutritional sta-tus is probably correct, their interpre-tation of their results is questionable.To compare these two categories oftests, the nutritional status of thestudied patients must be known. Theauthors classified all their patients asnutritionally replenished, this judg-ment being based on the estimatedcalories met, the weight change, andthe nitrogen balance. However, thesethree characteristics reflect only theprotein and caloric nutritional status.They do not necessarily indicate thatconcentrations of all trace mineralsand vitamins are normal. Further-more, these measures are also limitedas markers ofprotein-calorie status inburn patients (2, 3). For example, oneof their patients achieved only 46% ofhis estimated calorie needs but wasnonetheless considered to have beenadequately fed. Weight gain is diffi-cult to interpret in burn patients be-cause they can develop considerableedema, which accounts for the greaterpart of the weight gain in the firstweeks after the burn. In calculatingthe nitrogen balance, Rettmer et al.did not account for the losses of proteinin burn wounds, which can amount toseveral grams per day (4).The authors did not mention thecomposition of the diet. There is greatdiversity in the practices of burn cen-ters concerning supplementation withtrace minerals and vitamins (5, 6).Because Rettmer et al. did not evalu-ate the same nutrients with the staticand the functional tests, we cannot besure whether the differences they ob-served only reflect an insufficient sup-plementation of some elements.The choice of some ofthe static testsis also dubious. Even in ambulatorypatients, no one would judge the nub-i-tional status by measuring singly theserum concentration ofcalcium or iron.A low serum concentration of calciummust be interpreted in light of the al-bumin concentration (7). Similarly, theconcentration of transferrin (or thebinding capacity of iron) must beknown before one can interpret a lowserum iron concentration. Also, theunits for transferrin in their Figure 1should read gIL instead of mg/L. If wetake into account the low concentrationof binding proteins, the observed con-centrations of iron and calcium areprobably in the low normal range. Thesame phenomenon could explain the“low” serum copper values obtained (8).The serum concentration of caro-tone can reflect the nutritional statusofvitamin A only ifit is a major sourcein the diet (9). Because their patientswere probably supplemented with thevitamin and not the provitamin (caro-tone), the low concentrations obtainedshould have been expected.Finally, their Figure 4 contains sev-eral errors. The results of the func-tional assays are presented in the formof activity coefficients, which are di-mensionless. The units indicated(prnoh/L) are thus inappropriate. Therange ofthe zinc protoporphyrin/hemeratio cannot be correct because no re-sults below the mean are indicated.Despite the above restrictions, Iagree with Rettmer et al. that there isa great need to develop new nutrition-al-assessment procedures.References1. Rettmer RL, Williamson JC, Labb#{233}RF,Heimbach DM. Laboratory monitoring ofnutritional status in burn patients. ChinChem 1992;38:334-7.2. Pasulka PS, Wachteh TL. Nutritionalconsiderations for the burned patient. SurgChin North Am 1987;67:109-31.3. Jensen TG, Long JM, Dudnick SJ,Johnston DA. Nutritional assessment mdi-cations of postburn complications. J AmDiet Assoc 1985;85:68-72.4. Waxman K, Rebelho T, Pinderski L, etal. Protein loss across burn wounds. JTrauma 1987;27:136-40.5. Shippee RL, Wilson SW, King N. Tracemineral supplementation ofburn patients:a national survey. J Am Diet Assoc 1987;87:300-3.6. King N, Goodwin CW. Use of vitaminsupplements for burned patients: a nationalsurvey. J Am Diet Assoc 1984;84:923-5.7. Gray TA, Paterson CR. The clinicalvalue of ionised calcium assays. Ann ChinBiochem 1988;25:210-9.8. Boosahis MG, McCall JT, Sohem LD, etal. Serum copper and ceruloplasmin levelsand urinary copper excretion in thermalinjury. Am J Chin Nutr 1986;44:899-906.9. Olson JA. Serum levels ofvitamin A andcarotenoids as reflectorsofnutritional status.J Nath Cancer Inst 1984;73:1439-44.Dept. de Biochim.HOpital du Saint -SacrenwntQu#{233}bec,Canada, GiS 4L8J_ MasseThe authors ofthe article referred tocomment:To the Editor:The comments of Masse underscorethe difficulty in determining the nutni-tional status of burn patients (1). Thenutritional support our patients re-ceived from day 1 post-burn consistedof enteral feeding with one of twostandard 1 kcal/L formulas supple-mented by central venous alimenta-tion and a standard multivitaminpackage if needed. By day 5 all pa-tients were meeting 84% of theirestimated requirements. By day 15,

External quality control for radioimmunoassay of urinary albumin in diabetic subjects: a one-year experience

External quality control for radioimmunoassay of urinary albumin in diabetic subjects: a one-year experience

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Archivio della ricerca della Scuola Superiore Sant'Anna