Cecere M

Clerico A

Cruschelli L

Giampietro O

Penno G

Cecere M.

CLERICO, ALDO

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CLINICAL CHEMISTRY, Vol.39, No.3, 1993 53312. Horst RL, Littledike ET, Riley JL, Napoli JL. Quantitation ofvitamin D and its metabolites and their plasma concentrations infive species of animals. Anal Biochem 1981;116:189-203.13. Hollis BW, Frank NE. Solid phase extraction system forvitamin D and its major metabolites in human serum. J Chro-matogr 1985;343:43-9.14. Hollis BW, Napoli JL. Improved radioimmunoassay for vita-miii D and its use in assessing vitamin D status. Clin Chem1985;31:1815-9.15. Hollis BW. Comparison of equilibrium and disequilibriumassay conditions for ergocalciferol, cholecalciferol and their majormetabolites.J Steroid Biochem 198421:81-6.16. Hollis BW, Pittard WB. Evaluation of the total fetomaternalvitamin D relationships at term: evidence forracial differences. JClin Endocrinol Metab 1984;59:652-7.17. Calvo M, EnaJM. Relations between vitamin D and fatty acidCLIN. CHEM. 39/3, 533-536 (1993)binding properties of vitamin D-binding protein. BiochemBiophysRes Commun 1989;163:14-7.18. Williams MH, VanAlstyne EL, Gaibraith RM. Evidence of anovel association of unsaturatedfatty acids with Go (vitaminD-binding protein). Biochem Biophys Res Commun 1988;153:1019-24.19. Bouillon R, Van Herck E, Jam I, Tan BK, Van Baelen H,DeMoorP. Two direct (nonchroniatographic) assays for25-hydroxy-vitamin D. Clin Chem 1984;30:1731-6.20. Chapuy MC,ArlotME, DuBoeuf F, BrunJ, Crouzet B, Arnaud5, et al. Vitamin D3and calcium to prevent hip fractures in elderlywomen. N Engl J Med 1992;327:1637-42.21. Gloth FM, Tobin JD, Sherman SS, Hollis BW. Is the recom-mendeddaily allowance forvitamin D too low for the homeboundelderly? J Am Geriatr Soc 1991;39:137-41.How and How Longto Store Urine SamplesbeforeAlbuminRadioimmunoassay:A Practical ResponseOttavio Giampietro, Giuseppe Penno, Aido Clerico,’ Lorella Cruschelli, and Mauro CecereWe used three study protocols to check the dependenceof albumin stability, measured by an AlA, on differenttemperatures, durations, and materials (i.e., assay tubes)of urine storage. Albumin values obtained for samplesstored in three types of assay tubes (glass, polystyrene,and polypropylene) throughout the 2 months of the first(prospective) protocol were superimposable. The 24-hstorage of six urine samples at room temperature or at4#{176}C,as well as 72-h storage at 4#{176}C,did not affect thealbumin measurement by AlA. After 2 months of storageof these same six urine samples at -20 #{176}C,there was stillno albumin decrease. A significant albumin decreaseoccurred (a mean of -5% per year, throughoutthe rangeof albumin concentrationstested) when samples stored at-20 #{176}Cwere reassayed by AlA after 2 years (secondprotocol, retrospective). Finally, 3 of 21 (14.3%) urinepools stored at -20 #{176}Cfor various periods (4-21 months)showed a significant albumin loss after storage; the timeof storage as well the decrement rates of these 3 poolsdiffered from each other (third protocol, retrospective).Short- and medium-term (2-6 months) freezing of urinesamples at -20 #{176}Cdoes not significantlyaffect the stabil-ity of immunoreactive albumin. For longer preservationperiods, storage of urine samples at -70 #{176}Cmay bepreferable.IndexIng Terms: external quality contml albuminuna diabe-tes mellitus variation, source of sample handllngCattedra di Malattie del Metabolismo, Istituto di ClinicaMedica 2, Via Roma 67, 56100 Pisa, Italy.‘Istituto di Fisiologia Clinica del CNR, Universit#{224}degli Studi diPisa, Pisa, Italy.Received June 29, 1992; accepted September 28, 1992.Measurement of urinary albumin excretion in therange of 20-200 mg/day, so-called microalbuminuria, isused to evaluate kidney involvement in people withdiabetes mellitus (1, 2). Indeed, diabetic renal diseasemay be reversible by tight metabolic control, if detectedearly (1). Accordingly, screening to determine urinaryalbumin excretion has become a frequent part of diabe-tes care (1, 2).Contradictory evidence (2-9) has been reported aboutthe effect of specimen storage conditions on the resultsof these assays. Albumin in urine has been stated to bestable at room temperature for several hours and for afew weeks at 4#{176}Cwithout added preservative, whichimplies that urine samples can be stored for a relativelyshort time (from hours to some days) at 4#{176}Cuntil assay(2). However, epidemiological and (or) multicenter stud-ies require that several samples be collected, stored forlong periods (from several months to some years), andeventually shipped to the laboratory before the assay.Hence, storage conditions such as room temperature or4#{176}Care not suitable; instead, freezing the urine sam-ples seems more appropriate. Some authors claim thatstorage of samples at -20 #{176}Cfor determination of uri-nary albumin excretion by laser immunonephelometry(5) or RIA (7) yields falsely low results, so that freezingurine samples is not suitable. Others (3,8), like us (4,6,9), are of the opinion that freezing urine samples at-20#{176}C(or -70 #{176}C)does not in itself affect the albuminassay, and thus recommend storing specimens at -20 #{176}C(or -70 #{176}C)if the assay cannot be performed within afew days (2-3 days at most).To determine the best way to stabilize the albumin inurine until assay, we stored under different conditionsand for different periods a large number of urine sam-plea and pools treated in various ways.80cv. x6000 20 40 60 80Albumin odd.d, mg/L5working rong.00II>U2010. .MaterIals and Methods100534 CLINICAL CHEMISTRY, Vol.39, No.3, 1993AlA of Urinary AlbuminWe measured urinary albumin with a commercialRIA (Albumin RIA 100; Pharmacia AB, Uppsala, Swe-den) in which purified human serum albumin (HSA) isthe standard, 1I-labeled HSA is the tracer, and theantiserum is specific for HSA. The bound and the freephases were separated by a solid-phase system (anti-rabbit IgG, raised in sheep, bound to Sepharose), andthe standard curve was prepared with solutions contain-ing known amounts of purified HSA. The analyticalcharacteristics of this RIA have been reported in detail(10). The assay was not significantly subject to interfer-ence from bovine serum albumin, transferrin, or humanimmunoglobulins.The RIA was performed according to the manufactur-er’s instructions. In brief, we added to all tubes 50 L ofthe standard solutions or urine samples, 50 pL of thetracer solution (-30 000-40 000 counts/mm of 1I-la-beled HSA), and 2.0 mL of anti-HSA antiserum reagent;mixed; and then incubated the samples for 1 h at roomtemperature (18-22 #{176}C).After centrifugation (1500 x gfor 10 mm, 4#{176}C),the supernates were aspirated anddiscarded, and the radioactivity of the precipitates wascounted in a well-type gamma counter.The mean limit of detection of this RIA was 0.29 (SD0.12) mg/L (n = 40). The mean (n = 40) within-assayimprecision profile and the mean RIA standard curve areshown in Figure 1. The working range [the concentrationrange of albumin that was measured with an imprecision(CV) <10%] was 2-62 mg/L; consequently, the urinesamples with albumin concentrations 60 mg/L werereassayed after dilution. The between-assay variability(CV) for a wide range of albumin concentrations (3.4-60.1 mg/L) observed in 21 urine pools stored at -20 #{176}Cranged between 5.6% and 27.2% (Figure 2), with apattern similar to the within-assay imprecision profile.All sample values were measured and the index ofsensitivity, the precision profile, and quality control forthe RIA were performed according to a previously de-scribed computer program (11, 12). The interpolation ofthe dose-response curve was calculated by a four-pa-rameter logistic function (12).Urine SamplesUrine samples were collected from healthy subjectsand from normo-, micro-, and macroalbuminuric dia-betic patieits. First-morning, 24-h, and overnight col-lections were included in the study. Urinalysis andurine cultures were performed on each specimen. Nopreservative had been added to the urine collections,and the samples were gently mixed, but not centrifuged,before assay.Study Protocols20 40 60 60Albumin added, mg/IFig. 1. Mean imprecision profile (within-assay), working range (rangeof albuminconcentration measured with an imprecIsion <10%), andmean standard curve (insert) for AlA used to measure urinaryalbuminin this studying various albumin concentrations (from 7.2 to 371.0mg/L) were collected in sterile 100-mL urine culturetubes and stored for different times (from 1 day to 2months) at various temperatures [room temperature(18-20#{176}C),4#{176}C,and -20 #{176}C]in several 2-mL aliquotsdispensed into three different types of assay tubes (glass,polystyrene, and polypropylene) until assay. These spec-imens were analyzed for albumin by the RIA in eight30We used more than one protocol to check the depen-dence of albumin stability on various temperatures,lengths, and materials (i.e., assay tubes) of storage.First experimental series. Six urine samples contain-0 10 20 30 40 50 60urinary albumin concentrstlon mg/LFig. 2. Between-assay variabilities of assays of 21 urinepoolswitdifferentalbuminconcentrations242016200 22584706050403020103 7 140 100 200 300 400 500 600II..fI-I #{149} ..U #{149} U #{149}UUN. 42 y-58.9-O.O2thc r.0.43 p.0.005 CV-9.7%21 28 60Time, days of storage at .20#{176}CFig. 3. First experimental series: percent variation(mean± SD) ofalbuminconcentrationin sixurinespecimenstoredat -20 ‘C fors2 monthsindifferentypes of assay tubesU . .0 50 100 150 200 250 300Time, daysFIg.4. Thirdexperimentalseries:changesof albuminconcentrationswith time in thethreeurinepoolsthatshoweda significantalbumindecreaseafterstorageat -20 ‘CCLINICAL CHEMISTRY, Vol.39, No.3, 1993 535separate runs as follows: within 1 h from collection atroom temperature, withdrawing urine (50 L) for theassay directly from the urine culture tubes (controlsample) and from each type of assay tube; after 24 h(stored at room temperature and 4#{176}C);after 72 h (storedat 4 and -20 #{176}C);and after 7, 14, 21, 28, and 60 days(stored at -20 #{176}C).Second experimental series. We assayed in five sepa-rate runs two series of urine samples that had beenstored at -20 #{176}C:the first included 61 specimens thathad been assayed 2 years before by the same RIA(albumin concentrations 2.0-344 mgfL, mean ± SD =30.6 ± 50.7 mgIL); the second included 137 samplesassayed 4-.-5years before (albumin concentrations 0.3-364 mgIL, mean ± SD = 13.1 ± 33.0 mgfL). Most ofthese urine samples had been frozen and thawed morethan once before the last assay.Third experimental series. We repeatedly assayed(from 16 to 82 times in different experimental sessions;total number of assays:692) over various periods (rang-ing between 4 and 21 months) 21 urine pools stored at-20 #{176}Cin 2-mL aliquots until assay.Statistical AnalysisThe statistical analysis was carried out by MacintoshSE and Macintosh ilsi personal computers, with use ofthe Stat View SE+#{176}””’#{176}program (1988 Abacus Con-cepts, Inc.). The results are expressed as mean ± SD.ResultsFirst protocol. No difference [by repeated-measureanalysis of variance (ANOVA), P = 0.8472] was ob-served among results obtained with the three types ofassay tubes throughout the 2 months of the experiment(Figure 3).The 24-h storage of six urinary samples at roomtemperature (for polystyrene tubes, 89.8 ± 121.4 mg/L)and at 4#{176}C(95.9 ± 140.8 mg/L), as well their 72-hstorage at 4#{176}C(114.1 ± 177.4 mg/L) did not affect themeasurement of albumin by RIA (P = 0.4532 byE______________IJo’s.. I0 Po1y.tyrne I .EPOiypropy1enj1’ANOVA), in comparison with the results determinedwithin 1 h after collection (113.1 ± 157.5 mg/L). Storageof these same six urine samples at -20 #{176}Cfor 2 monthsbefore assay produced no significant albumin loss (byANOVA, P = 0.8078) from the initial value (Figure 3).Samples stored at -20 #{176}Cfor 2 years. We found asignificant decrease in albumin when urine samplesstored at -20 #{176}Cwere reassayed by RIA after 2 years.The 61 urine samples frozen for 2 years had a meandecrease in albumin of 13.2%: basal value, 30.6 ± 50.7mg/L; values after 2 years, 26.5 ± 41.2 mg/L (P = 0.0153by paired t-test). The 137 specimens stored at -20 #{176}Cfor4-5 years had a mean albumin decrease of 21.8%: basalvalue, 13.1 ± 33.0 mg/L; value after 4-5 years, 10.2 ±23.6 mg/L (P = 0.0046 by paired t-test). These findingsindicate a mean loss of -5% per year throughout therange of albumin concentrations in urine samples frozenat -20 #{176}C,based on the assumption that the decrease isa linear function of the storage time.Urine pools. Only 3 of 21 (14.3%) pools stored at-20 #{176}Cshowed a statistically significant albumin de-crease during storage. The time of storage and thedecrement rates of these three pools all differed fromeach other (Figure 4).DiscussIonHere we demonstrate that the material of the storageand assay tubes-glass, polystyrene, or polypropylene-520N. 21 y.3.9.0.006x r-0.545 p.0.01 CV- 17.7%0 2.5 50 75 100 125 150 175. UN. 55 y.21.1.0.03X c’.0.701 p.0.0001 CWI0.Oib12536 CLINICAL CHEMISTRY, Vol. 39, No.3, 1993had no effect on urinary albumin measurement by RIA.Moreover, in our hands, albumin in urine was suffi-ciently stable at room temperature for 24 h and at4-8 #{176}Cfor at least 3 days without added preservative,thus confirming previous reports (2).Because storing urine samples at 4#{176}Cfor longer than3 days is impractical, the alternative is to freeze them.To check whether storage at -20 #{176}Caffected albuminassayed by RIA, we analyzed results from a prospectiveprotocol of urine storage and from two retrospectiveseries of urine samples frozen for different long periodsat -20 #{176}C.To minimize the effects of analytical variabil-ity and bias (especially those due to the use of differentreagent lots) during the course of the study, we assayedfrozen specimens in different runs by RIA. Moreover, weassayed both individual urine samples and pools indifferent protocols, because some authors (13, 14) havecriticized the use of only pools in studies on the effectsoftime and temperature on albumin stability in urine.The prospective protocol demonstrated that 2 monthsof storage at -20#{176}Cdid not impair the accuracy of RIAdeterminations of albumin in urine. These results, inconjunction with those previously provided by our groupon the effects of specimen storage at -20 #{176}Cfor 6-7months (6,9), strikingly indicate that albumin in urinesamples is not affected by freezing at -20 #{176}C,at leastnot for short- or medium-term storage (6, 9). Whathappens shortly after that is not known (no data areavailable). However, after 2 (and still more, after 5)years of storage at -20 #{176}C,a significant albumin loss-albeit of negligible clinical relevance-does occur (sec.ond protocol of this study). In fact, albumin from speci-mens frozen long-term at -20 #{176}Cdecreased by -5% peryear throughout the entire range of albumin concentra-tions. This loss is smaller than that previously reportedin other, shorter studies (5, 7, 8, 15). This decrementrate of -5% per year would probably not be detectablein experiments lasting <1 year, because this amount ofvariation is usually less than the between-assay preci-sion of the procedures routinely used to measure uri-nary albumin (16) (Figures 1 and 2). This indirectlyconfirms findings previously reported by us (6,9) and byother laboratories (3, 17).Because storage at -70 #{176}Chas been reported to beadequate to prevent substantial degradation of immuno-reactive albumin in urine samples for at least 160 days,MacNeil et al. (8) recently proposed this alternative.The third protocol of this study indicates that occa-sionally, in individual instances, a more rapid decre-ment rate of urinary albumin may occur (Figure 4),suggesting that an unpredictable rate of degradation ofurinary albumin may be found among somesubjects(8).At present, the reason for this is unknown, so thatanticipating the behavior of the single urine albuminresult in samples stored at -20 #{176}Cis not possible.In conclusion, becauseour findings confirm that freez-ing urine samples does not itself significantly affect thestability of immunoreactive albumin, the question ofhow to treat urine samples before assay needs to beanswered. If albumin cannot be measured within 1 day’sstorage at room temperature or within 2-3 days ofstorage at 4#{176}C,freezing urine samples at -20 #{176}Cfor 4-6months is advisable and should result in no clinicallysignificant albumin loss, regardless of the compositionof the storage tubes. For longer storage, we recommendfreezing the urine samples at -70 #{176}C.References1. Viberti GC, Walker JD. Diabetic nephropathy: etiology andprevention. DiabetesfMetab Rev 1988;4:147-62.2. Rowe DJF, Dawnay A, Watts GF. Microalbuminuria indiabe-tes mellitus: review and recommendations for the measurement ofalbumin in urine. Ann Clin Biochem 1990;27:297-312.3. Silver A, DawnayA, LandonJ, Cattell WR. Immunoassay forlow concentrations of albumin in urine. Clin Chem 1986;32:1303-6.4. Giampietro 0, Miccoli R, Clerico A, et al. Urinary albuminexcretion in normal subjects and in diabetic patients measured bya radioimmunoassay: methodological and clinical aspects. ClinBiochem 198821:63-8.5. Elving LD, Bakkeren JAJM, Jansen MJH, de Kat AngelinoCM, de Nobel E, van Munster PJJ. Screening for microalbumin-uria in patients with diabetes mellitus: frozen storage of urinesamples decreases their albumin content. Clin Chem 1989;35:308-10.6. Giampietro 0, Clerico A, Cruschelli L, Penno G, Navalesi R.Microalbuminuria in diabetes mellitus: moreon urine storageandaccuracy of colorimetric assays [Letter]. Cliii Chem 1989;35:1560-2.7. Osberg I, Chase HP, Garg SK, de Andrea A, Harris S,HamiltonR, Marshall G. Effects of storage time and temperature on mea-surement of small concentrations of albumin in urine. Olin Chem1990;36:1428-30.8. MacNeil MLW, Mueller PW, CaudillSP, Steinberg KK. Con-siderations when measuring urinary albumin: precision, sub-stances that may interfere, and conditionsfor samplestorage.ClinChem 1991;37:2120-3.9. Giaxnpietro 0, Cruschelli L, Penno G, Navalesi R, Clerico A.Moreoneffects of storagetime and temperature on measurementof small concentrations of albumin [Letter]. Clin Chem1991;37:591-2.10. Giampietro 0, ClericoA, Cruschelli L, Miccoli R, Di Palms L,Navalesi R, et al. Measurementof urinary albumin excretion rate(AER) innormal and diabetic subjects:comparison of two recentradioimmunoasaays.J NucI Med Allied Sci 1987;31:321-6.11. Pilo A, Zucchelli GC. Automatic treatment of radioimmuno-assay data: an experimental validation of the results. Olin ChimActa 1975;64:1-4.12. Pilo A, Zucchelli CC, Malvano R, Masini S. Main features ofcomputer algorithms for RIA data reduction. Comparison of somedifferent approaches for the interpolation of dose-response curve. JNucl Med Allied Sci 1982;26:235-48.13 Elving U), Bakkeren JAJM, de Kat Angelino CM. More oneffects of storage time and temperature on measurement of smallconcentrations of albumin [Letter]. Olin Chem 1991;37:592-3.14. Osberg I, Chase PH, Garg SK. More on effects of storage timeand temperature on measurement of small concentrations of albu-min [Letter]. Olin Chem 1991;37:593-4.15. Erman A, Rabinov M, Rosenfeld J. Albumin determination infrozen urine-underestimated results. Clin Chim Acts 1988;174:255-62.16. Giampietro 0, Penno G, Clerico A, Cruschelli L, Lucchetti A,Nannipieri M, et al. Which method for quanti1ring “microalbu-minuria” in diabetics? Acts Diabetol 199228:239-45.17. Torffvit 0, Wieslander J. A simplified enzyme-linked immu-nosorbent assay for urinary albumin. Scand J Olin Lab Invest1986;46:545-8.

How and how long to store urine samples before albumin radioimmunoassay: a practical response

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