A Noninstrumented Quantitative Test System and Its Application for Determining Cholesterol Concentration in Whole Blood

Transcription

1 CLIN. CHEM. 36/9, (1990) A Noninstrumented Quntittive Test System nd Its Appliction for Determining Cholesterol Concentrtion in Whole Blood Michel P. AlIen, Anthony DeLIu, Urs Rmel, Henry Jeong, nd Prithipl Slngh A novel noninstrumented technology hs been developed for quntifying niytes of clinicl interest in biologicl fluids. Appliction of this technology is exemplified by the development of quntittive cholesterol test with performnce equivlent to stte-of-the-rt instrumented methods. The ssy chemistry combines two seprte processes locted in different res of test strip: enzymtic ction on serum cholesterol to produce hydrogen peroxide (5 x 10 mm enzyme regent pd) nd quntifiction of the hydrogen peroxide (5 x 70 mm mesurement region). Color bnds re formed in the mesurement re through the use of redox-coupled indictor system. The height of the color bnd on the strip is directly proportionl to the smple cholesterol concentrtion. A one-step cssette contins ll components necessry to run the test nd includes blood filtrtion nd utomtic smple mesurement, so tht unmesured fingerstick whole-blood specimens cn be nlyzed by the nontechniclly trined user. The test is complete in <15 mm, is red visully like thermometer, nd gives results tht re in excellent correltion with estblished instrumented methods. Self-testing is becoming n importnt prt of helth cre s people re more wre nd involved with the sttus of their own helth (1, 2). Severl qulittive or semi-quntittive self-tests-e.g., for glucose, pregnncy, ovultion, occult blood, nd vrious urine components-hve become estblished s physicins nd ptients relize their potentil for providing improved helth cre. Recently, noninstrumented quntittive test for mesuring theophylline in whole blood ws introduced by Syntex Medicl Dignostics (3).This test, AccuLeveltm is tody the only test of its kind tht quntifies n nlyte without the use of n instrument. Although AccuLevel chieves quntittive results with good ccurcy nd precision, it is not idel for use by nontechniclly trined personnel becuse it requires exct smple mesurement nd hs somewht complex ssy protocol. In certin environments where tests re performed infrequently, or where the stff my not be sufficiently trined in mintining n instrument, noninstrumented technology becomes importnt. In ddition, the cost of n instrument my sometimes be fctor tht reduces the vilbility of needed test. For the brodest ppliction, such technology should be ble to hndle whole blood from fingerstick-idelly, drop of unmesured blood from which one cn obtin quntittive mount of plsm without the use of pipette. Such technology would likely be pplicble to ny biologicl fluid nd thus hve potentil ChemTrk, Inc., 484 Okmed Prkwy, Sunnyvle, CA Received April 30, 1990; ccepted July 5, to be used with most nlytes in the generl chemistry nd immunochemistry res. In ddition, technology tht cn hndle quntittive tests cn lso be pplied to semiquntittive or qulittive tets. We decided to try to ifil the need for user-friendly, noninstrumented, quntittive self-test for mesuring cholesterol. The Expert Pnel on the Detection nd Tretment of High Blood Cholesterol in Adults, Ntionl Cholesterol Eduction Progrm (NCEP), hs estblished guidelines for dults older thn 20 yers to identify risk groups ssocited with vrious concentrtions of blood cholesterol (4): <2000 mg/l (5.17 mmol/l) is desirble cholesterol concentrtion, between 2000 nd 2390 mg/l (6.18 mmoljl) is borderline high, nd >2400 mgfl (6.21 mmol/l) is high. To ssure the qulity of cholesterol testing, the NCEP Lbortory Stndrdiztion Pnel issued guidelines for ccurcy nd precision ssocited with lbortory cholesterol determintions (5). This sme NCEP report recommended tht ll dults should know their cholesterol concentrtion, nd there hs been n effort to identify individuls with high concentrtions of cholesterol nd to initite tretment. Desk-top instrumenttion cpble of performing rpid, on-site cholesterol determintions hs become vilble, mking cholesterol screening more ccessible to the generl public. Although these portble nlyzers hve incresed the number of Americns who re wre of their cholesterol concentrtion, severl limittions ssocited with user competence nd system ccurcy hve been identified (6, 7). We describe here noninstrumented quntittive test system for cholesterol with the following performnce: () n unmesured drop of fingerstick blood is used, (b) the test is complete in <15 min, (c) red out is objective, s in thermometer, (d) precision nd ccurcy re consistent with NCEP guidelines, (e) the test strips cn be stored t room temperture, nd (/) the strips re disposble fter single use. Assy Principle As shown in Figure 1, four pper strip components nd wicking regent comprise the ssy conligurtion. The mesurement region, strip of chromtogrphy pper, contins substrtes for horserdish peroxidse (HRP, EC ) immobilized in uniform coting. The pper strip re djcent to the mesurement region is the enzyme regent pd, which contins cholesterol esterse (EC ) nd cholesterol oxidse (EC ) immobilized Nonstndrd bbrevitions: NCEP, Ntionl Cholesterol Eduction Progrm; CAP, College of Americn Pthologists; HRP, horserdish peroxidse; MBTH, 3-methyl-2-benzothizolinone hydrzone; nd DMA, N-N-dimethylrnuine. CLINICAL CHEMISTRY, Vol. 36. No. 9,

2 R910., En,ym. A..Qnt P,d Smpe R.c.Ing P.d WCkIng Strip.: Sir,,ple WIckrgReigrt. I Sirnpi#{149} A I Wicking B Simpi. Ii.. En,yr,,iiic Cc,wp,iiofl C H,O, Co ::... : Ccl,, Bind Fo,n,.tlon 0 I Bnd Louis, MO. Sodium phosphte monobsic ws from Mllinckrodt, Inc., Pris, KY. Bovine serum lbumin Frction V nd bovine gmm globulins Frction II were purchsed from Miles Lbortories, Elkhrt, IN. Six concentrtions of cholesterol clibrtors were prepred inhouse by supplementing fresh humn serum (cholesterol = 1500 mg/l, 3.88 mmoljl) with humn lipoprotein concentrte to yield finl concentrtions ner 1500 (3.88), 2000 (5.17), 2500 (6.47), 3000 (7.76), 3500 (9.05), nd 4000 mg/l (10.35 mmol/l). Three concentrtions of humn serum reference mteril were purchsed from the College of Americn Pthologists (CAP), Skokie, IL. SdOfl.. vd,og.pdd. Fig. 1. Schemtic representtion of the chromtogrphy pper components, the wicking regent ssocited with the strip ssy, nd the opertion of the ssy: (A) plsm smple ppliction, ( initition of wicking of the buffered HAP solution nd movement of the plsm cholesterol upwrd onto the enzyme regent pd, (C) conversion of cholesterol to hydrogen peroxide with the beginning of color bnd formtion in the mesurement region, (C) the stopping of the wicking once the solution hs reched the top of the mesurement region (the ssy is complete t this point) in dry formultion. The next strip re is the smplereceiving pd, disk of chromtogrphy pper tht holds 5 L of plsm or serum. The re below the smple pd, the wicking strip, trnsports liquid from the re of the wicking regent to the smple pd. The finl component is the wicking regent, buffered protein solution contining HRP. The entire ssy chemistry is performed nd completed during the cpillry migrtion of the wicking regent through the contiguous sections of the ssy strip. The opertion of the test strip is s follows: 5 L of plsm or serum is deposited on the smple pd (Figure IA); wicking is initited by llowing the lower portion of the wicking strip to contct the wicking regent (Figure 1B); the wicking regent, contining HRP, moves the smple into the enzyme pd, where the cholesterol in the plsm is converted to hydrogen peroxide (Figure 1B, C); the hydrogen peroxide then moves with the wicking regent into the mesurement region where, in the presence of HRP, it rects with the immobilized substrte to produce visible color bnd (Figure 1C, D). The ssy is complete when the wicking solution reches the end of the mesurement region, which tkes bout 10 mm. The hydrogen peroxide rects stoichiometriclly with respect to the immobilized substrte in the mesurement re. Becuse the HRP substrte is uniformly distributed in this region, the height of the color bnd is function of the mount of hydrogen peroxide generted in the enzyme regent pd. In this wy, quntittive signl is produced tht cn be visully interpreted by mesuring the height of the color bnd. Mterils nd Methods Cholesterol esterse, cholesterol oxidse, nd HRP were from Boehringer Mnnheim Biochemicls, Indinpolis, IN, nd Toyobo Co., Ltd., Osk, Jpn. o-dinisidine, 3-methyl-2-benzothizolinone hydrzone (MBTH), nd N,N-dimethylniline (DMA) were purchsed from Aldrich Chemicl Co., Milwukee, WI. Sodium cholte, Triton X-100, 4-chloro-1-nphthol, tetrmethylbenzidine, 4-minontipyrine, p-hydroxybenzenesulfonte, phenol, sodium chloride, nd sucrose were from Sigm Chemicl Co., St. Mesurement Region This re of the ssy strip of chromtogrphy pper (5 x 70 mm) contins substrtes to HRP immobilized in uniform coting. We exmined vrious unimoleculr peroxidse substrtes, including tetrmethylbenzidine, o-dinisidine, nd 4-chloro-1-nphthol, s well s coupled systems, e.g., 4-niinontipyrine with p-hydroxybenzenesulfonte, nd MBTH with DMA. In this pper we report our results with the MBTHIDMA substrte system for HRP. Covlent ttchment of the DMA required the synthetic ddition of primry mine to the DMA molecule. The minted substrte molecule ws then coupled to crbonyldiimidzole-ctivted chromtogrphy pper in mnner similr to tht described by Chpmn nd Rtcliffe (8). The MBTH ws subsequently immobilized to the DMA-derivtized chromtogrphy pper by simple dsorption. The mount of HRP substrte immobilized in the mesurement re determines the sensitivity of the ssy. The color bnd height produced from given concentrtion of hydrogen peroxide (or cholesterol) is inversely proportionl to the density of immobilized peroxidse substrte. Higher lodings of substrte give lower migrtion heights. Becuse loding of substrte in this re is predictble, sensitivity cn be djusted to meet the requirements of the prticulr ssy. Enzyme Regent Pd This re of the ssy strip chromtogrphy pper (5 x 10 mm) contins regents in dry formultion, which convert plsm or serum cholesterol to hydrogen peroxide. The pper is prepred by immersing sheet of chromtogrphy pper in phosphte-buffered ph 7.0 solution contining, per liter, 18 ku of cholesterol esterse, 50 ku of cholesterol oxidse, 10 g of sodium cholte, nd 5 g of sucrose. The pper is then dried. Becuse the cholesterol esterse nd cholesterol oxidse re not irreversibly immobilized to the chromtogrphy pper, they move slowly upwrd during wicking. After wicking is complete, these enzymes re spred upwrd from the enzyme regent pd 10 to 15 mm into the lower prt of the mesurement region. Most of these enzymes, however, remin on the enzyme regent pd throughout the wicking process. For the cholesterol enzymes, 1 U of ctivity forms 1 imol of hydrogen peroxide per minute. For this ctivity ssy we used 4-minontipyrine with phenol nd Triton X-100 in 0.10 moljl potssium phosphte buffer, ph 7.0, s doscribed in the Toyobo ctlog (sections C nd COE- 311) CLINICAL CHEMISTRY, Vol. 36. No. 9, 1990

3 Smple Receiving nd Wicking The smple-receiving pd is 5-mm-dimeter disk, the wicking strip is 5 x 22 mm. Both re untreted chromtogrphy pper. The wicking regent contins 5 mg of HRP nd 2 g of bovine gmm globulin per liter of 100 mmol!l phosphte buffer (ph 7.0). To eliminte edge effects commonly ssocited with pper chromtogrphy, we cut the lengthwise edge of the mesurement region with specil die tht produces serrtions. This serrted-edge configurtion results in rockettype color fronts; their convex-rounded leding edge mkes the color bnd height esy to red (Figure 1). This method hs been previously described by Zuk et l. (3). Accu Meter#{174} Cssette System The solid-phse regent chemistry described bove hs been enclosed in cssette (Figure 2), mking it esier for nontechniclly trined individuls to operte the test. This cssette system, the AccuMeter (ptents pending), includes blood seprtion device, smple mesurement system, nd mechnism for relese of wicking regent. Becuse ll the necessry regents, the blood-seprtion system, nd the pipette re self-contined in the cssette, the only dditionl prt needed to conduct the test is finger lnce. The test procedure is very simple: the user lnces finger, pplies drop of blood to the smple ppliction site, nd wits bout 1 mm before pulling the slide to the right. About 10 mm lter, the test-complete window turns green, indicting tht the result cn be red. The height of the color br is red in millimeters nd compred with conversion tble to obtin quntittive result. The entire cholesterol determintion is complete in <15 mm. Figure 3 shows n exploded view of the AccuMeter test system, illustrting the reltive position of internl components. The cssette is composed of three plstic prts: cover, movble slide, nd bse. The ssy strips re positioned, s indicted, with the mesurement region, the enzyme regent pd, nd the wicking strip locted on the right side. The smple pd is positioned in the movble slide. A seled buffer continer on the underside of the cssette cover is directly bove the slide ripper teeth (its position is outlined in Figure 3A). When the blood smple is pplied, the slide is positioned , B Fig. 3. An exploded view of the AccuMeter test system: (A) the reltive position of internl components during smple ppliction; ( the lignment of the smple pd with the ssy strips fter the slide motion such tht the smple pd is ligned with the blood-seprtion device nd the smple well on the cssette cover (Figure 3A). Smple filtrtion proceeds, removing erythrocytes nd depositing the plsm on the smple pd (this tkes bout 1 mm). The cssette slide is then pulled to the right (Figure 3B), which ccomplishes two things simultneously: it ligns the smple pd with the ssy strips, nd it releses the wicking regent by drwing the ripper teeth cross the buffer continer nd rupturing its sel. Smple mesurement is lso complete in tht, t this point, reproducible volume of plsm is delivered into the ssy system regrdless of the volume of the whole-blood smple initilly pplied to the cssette. Smple Appliction Site A 1/ Procedure 1. Lnce Finger 2. Apply Smple 3. Pull Slide 4, Red Result Mesurement Regon M50eble Slide Fig. 2. AccuMeter Cssette System, indicting the loction of vrious res on the cover: (A) n unused test; ( test cssette used ccording to the indicted procedure ( The color bnd mesures 30mm. The control windows re lso colored t this poiflt, the Test Working window being purple nd the Test Complete window being green B S in-house Cholesterol Comprison Assy We use the Boehringer Mnnheim High Performnce ssy s our in-house comprison system for cholesterol. Comprison of this ssy performed in-house (y) with the Abell-Kendll ssy (9) performed in ntionl reference lbortory (x) showed virtully identicl results. The regression line eqution ssocited with this comprison ws y = 0.996x (r = 1, n = 18). Results nd Discussion Smple Preprtion Automtic smple mesurement is significnt feture of the cssette. We exmined the performnce of the smple-mesurement device by chllenging the cssette system with blood volumes from 5 to 50,uL nd then mesuring the weight of the smple-recovery pd. As Figure 4 shows, 20 to 25 L of whole blood is necessry to sturte the metering system; therefter, plsm recovery is constnt. Thus, when whole-blood smple volumes exceed 30 CLINICAL CHEMISTRY, Vol. 36. No. 9,

4 ci E I E 5. 5 Clibrtion Curve Whole Blood Smple > 3OpL Plsm Recovered = 5 pl S.D. = pl C.V. <3% Whole Blood Volume (pl) Fig. 4. Plot of plsm recovery by mss (in milligrms) vs wholeblood smple volume (in microliters) pplied to the cssette Performnce of this device is summrized for whole-blood smples 30 t4. il, the plsm volume obtined is 5 ul (SD rnging from 0.08 to 0.15 FL). Typiclly, the coefficient of vrition (CV) of this smple-mesurement system is 2%. Becuse the smple-mesurement system opertes t volumes >30 L, the blood-seprtion system must lso remin effective under the sme conditions. We evluted the performnce of the blood-seprtion device by chllenging the system with rnge of whole-blood volumes nd then mesuring the hemoglobin concentrtion of the contents of the smple-recovery pd. A properly functioning blood-seprtion system would show only low concentrtions of hemoglobin on the smple pd. Figure 5 compres the performnce of two methods of erythrocyte seprtion: the AccuMeter procedure nd the widely ccepted method of blood seprtion by ifitrtion through glss fibers. In the typicl glss fiber system, 30-FL smples give cceptble performnce with low concentrtions of hemoglobin found on the smple pd; t greter volumes of whole-blood smples, however, hemoglobin recovery increses drmticlly. For exmple, pplying 50 p.l of blood to the glss fiber system results in hemoglobin (49 g/l) ccumulting on the smple pd. This is not cceptble becuse hemoglobin t concentrtions >1.0 g/l interferes with our ssy chemistry. The effectiveness of the blood-seprtion device for the AccuMeter system, on the other hnd, is prcticlly independent of smple volume. This device combines glss fibers with microporous membrne nd hs performnce superior to previously described methods of sttic blood seprtion (10, 11). Applying 50 pl of blood yielded only 0.40 g of hemoglobin per liter on the smple pd (Figure 5). Ech lot of AccuMeter test cssettes is clibrted by using six-concentrtion clibrtor set for serum cholesterol. The six clibrtors re run in replictes of 5 over three consecutive dys, resulting in 15 replictes for ech clibrtor concentrtion. The men of replictes t ech concentrtion is clculted nd the results re plotted s mster curve (Figure 6). By this method of clibrtion we estblished the dynmic rnge of the cholesterol ssy to be from 1500 (3.88) to 4000 mgfl (10.35 mmoijl). Accurte results will be obtined when smple concentrtions re within this rnge. Accurte clibrtion of cholesterol mesurement methods hs historiclly posed significnt chllenge, lrgely becuse of method-specific mtrix effects in vrious cholesterol clibrtion mterils. Processed clibrtion mterils often do not give the sme results s ptients fresh smples, but re either higher or lower thn the true (Abell- Kendll) vlue for fresh smple. Accurcy Accurcy cn be estblished in only one wy (12), n itertive process involving: () direct comprison of the cholesterol ssy in question to the reference Abell-Kendli method by nlyzing ptients fresh smples, (b) clibrtion set-point djustment s indicted by step, nd (c) blind correltion nlysis between the cholesterol ssy nd the reference Abell-Kendll to verify n cceptble bis. We followed this process to clibrte the AccuMeter system nd thereby estblished ccurcy through trcebility to the Ntionl Reference System for Cholesterol. Becuse the Abell-Kendll reference ssy (9,13,14) must be performed in one of the nine lbortories tht re members of the Ntionl Reference Method Lbortory Network (12), we determined ccurcy of the AccuMeter system by correltion to the in-house Boehringer Mnnheim High Performnce reference ssy. Figure 7 shows the vlues obtined for EDTA-treted blood ssyed with the AccuMeter plotted vs plsm vlues obtined with the comprison system. The regression line eqution of results for 74 ptients smples shows slope very close to 1.0 with smll negtive intercept of 13.3 mg/l (0.03 mmol/l) nd correltion coefficient of We lso evluted ccurcy by using n instrument tht ccepts EDTA-treted whole blood smples. For this, we used the Abbott Vision (Abbott Lbortories, N. Chicgo, IL), which hs been demonstrted to give ccurte results Glss Fiber E , Whole Blood Volume (pl) AccuMeter 5 Fig. 5. Plot of hemoglobin concentrtion (gil) recovered on the smple pd vs volume of whole-blood smple pplied The performnce of typicl glss-fiber-type seprtion device is compred with tht of the AccuMeter membrne-glss fiber system 50 Ct Clibrtor Cholesterol Concentrtion (mg/dl) Fig. 6. An AccuMeter mster clibrtion curve (y = x ) Regression prmeters re s follows: slope = (SE ), = (SE ) 1594 CLINICAL CHEMISTRY, Vol. 36. No. 9, 1990

5 ii BMDHP Cholesterol (mg/dl) Fig. 7. Comprison of AccuMeter-determined cholesterol concentrtions in EDTA-treted whole blood vs the Boehnnger Mnnheimdetermined cholesterol concentrtions in plsm (6). Correltion between the two methods ws excellent, s ws the correltion for fingerstick whole blood (Figure 8). Although dditionl clinicl evlutions re needed, especilly in the re of fingerstick determintions, these dt clerly demonstrte the ccurcy of this noninstrumented system. The lck of uncceptble bis lso vlidtes the clibrtion method used for AccuMeter, which is trceble to the Ntionl Reference System for Cholesterol. Precision The precision of the AccuMeter chemistry is primrily result of the coting uniformity of the peroxidse substrte in the mesurement region. The uniformity of this pper is tested by wicking the mesurement region by immersing only the lower 5 rm of the 70-mm strip in 0.5 ml of buffered protein solution contining 5 mg of HRP nd with 2.6 mmol of hydrogen peroxide per liter. The precision of color bnd height produced is n estimte of the substrte uniformity in this strip region, independent of ll other strip res. The SD of migrtion height is 0.5 mm (CV = 2%). y=0.99x+472 r=0.95 The overll AccuMeter precision is influenced by other system components s well, including the utomtic smple-mesurement device nd the uniformity of the enzyme regent pd. It is difficult to estimte the rel contribution to precision from ech system component; however, we hve determined the within-run nd between-run precision for the complete AccuMeter test system. The within-run precision ws determined by ssying EDTA-treted blood in replictes of 20 t four cholesterol concentrtions. As Tble 1 shows, t cholesterol concentr- 400 tions of clinicl interest, the ssy precision rnges from 4%to5%. The totl nlyticl precision ws estimted by running five replictes on ech of nine seprte dys over threemonth period. Becuse whole blood is not stble over the time frme of this study, we used CAP reference serum, reconstituting new bottle of lyophilized reference mteril with 3.0 ml of wter t ech time (Tble 2). One-wy nlysis of vrince ws used to estimte the within-run nd (corrected) between-dy components of the totl vrince (15). These components were then dded together to estimte the totl nlyticl precision. Temperture Men SD CV,% EDTA-treted blood run in replictes of 20 t four cholesterol concentrtions. Effect The effect of mbient temperture on the ssy chemistry ws determined by performing ssys t 4, 18, 23, nd 37 #{176}C with the CAP cholesterol clibrtor (1470 mg/l, 3.8 mmoljl) in replictes of five t ech temperture. There ws no chnge in migrtion height or ssy quntifiction t these tempertures. Tble 1. WIthIn-Run Whole-Blood Preclslon Cholesterol cencn, mg/i Eo.c SC.) 2oo,Af Abbott Vision Cholesterol (mg/dl) y = 1.02x r= 0.98 n = Abbott Vision Cholesterol (mg/dl) Fig. 8. AccuMeter vs Abbott Vision cholesterol concentrtions in (top) EDTA-treted whole blood nd (bottom) fingerstick whole blood Overll Tble 2. Totl Anlyticl Precision Cholesterol concn, mg/l Dy Men SD CV, % CAP reference serum ws nlyzed in replictes of 5 on nine seprte dys over n 85-dy period. Vrince components nlysis ws performed (15) nd totl nlyticl precision ws determined s follows: grnd men of ll results = 2584 mg/l. Within-run vrince = 1139, SD = 106.7, CV = 4.13%. Between-dy (corrected): vrince = (1139/5) = 41.2, SD = 20.3, CV = 0.79%. Totl nlyticl precision vrince = = , SD = 109, CV = 4.2%. CLINICAL CHEMISTRY, Vol. 36. No. 9,

6 Interfering Substnces Tble 3 shows the threshold concentrtion of interfering substnces (the concentrtion t which there is no mesurble effect on ssy quntifiction). Concentrtions greter thn those listed my cuse interference; however, in most cses, the concentrtions listed were the highest tested. Most of the potentil interfering substnces were wter soluble, so tht we could dd incrementl mounts of the mteril to the control serum. The AccuMeter migrtion height for unsupplemented serum ws compred with the migrtion height for the ser supplemented with potentil interferents. To evlute triglyceride nd bilirubin interference, we used lipemic nd icteric clinicl smples. The cholesterol concentrtions of these smples were determined with the Boehringer Mnnheim comprison ssy s well s the AccuMeter. The interference ssocited with bilirubin is uncler becuse the comprison ssy my be subject to interference from this compound t concentrtions >40 mg/l (0.068 mmoljl). The AccuMeter test does not show interference t this concentrtion. Further evlutions by using the Abell-Kendll reference procedure (which is subject to little bilirubin interference) need to be done to determine the ctul mount of interference from this compound. At present, we list 40 mg/l (0.068 mmol/l) s the bilirubin threshold, but experiments with further dditions of bilirubin suggest tht the threshold is well over 100 mg/l (0.17 mmol L). The triglyceride threshold listed in Tble 3, 3600 mgfl, is the highest concentrtion of lipemic smple we tested. Although this concentrtion is cliniclly insignificnt, we do not expect to see interference from lipemic smples. Additionl studies re underwy to evlute potentil interference effects of cholesterol-lowering drugs, common prescription drugs, over-the-counter products, nd metbolites of these compounds. Stbility Stbility hs been evluted for component prts s well s the complete cssettes. All mterils were seled in Substnce Hemoglobin Ascorbte Triglycerides (lipemic smple) Bilirubin (icteric smple) Cretinine Estrdiol Progesterone lodocetic Sodium Nicotinic Oxlic Sodium Uric cid cid citrte cid cid fluoride Tble 3. InterferIng Substnces Reference rnge Threshold concn, x C 8000#{176} C 4200C 7 mg/l The reference rnge is the concentrtion normlly found in serum. #{176}At the threshold concentrtion, no chnge in ssy performnce is seen. All threshold vlues except homoglobin nd scorbte were the highest concentrtions tested. #{176} These re the concentrtions found in blood-collection tubes. Component Mesurement region Enzyme regent pd Wicking regent Complete cssette Tble 4. Assy Stbility Storge condltlonb 8 months, 45#{176}C 6 months, 37#{176}C 6 months, 37#{176}C 1 month, 45#{176}C All ssy components were stored in seled foil pouches with desiccnt. bthe most chllenging condition t which we see no chnge in ssy quntittion. foil pouch with desiccnt nd stored t 45 #{176}C, 37 #{176}C, room temperture, nd 4 #{176}C. Tble 4 is representtive of these dt nd lists the most chllenging stbility condition t which no chnge in ssy quntifiction is seen. All of the stbility studies shown in Tble 4 re ongoing nd the storge times t these ccelerted stbility conditions re expected to be extended further. However, these dt demonstrte stble ssy system. Our gol is to chieve stbility for two yers of storge t room temperture; the dt suggest tht this is very likely. In ddition, in noninstrumented system like the one we describe, it is necessry tht the clibrtion curve remin unchnged throughout the shelf life of the test. To evlute clibrtion curve stbility, we stored pouched test cssettes t 4 #{176}C, room temperture, 37 #{176}C, nd 45 #{176}C, nd t 37 #{176}C with 95% reltive humidity. Periodic evlution of curve response with the three concentrtions of CAP clibrtors showed no shift in curve response for one month under these conditions. These studies re ongoing, nd we expect to estblish curve stbility beyond two yers t room temperture, level of stbility tht llows fctory clibrtion. In summry, this novel noninstrumented technology cn, in one step, seprte plsm from whole blood, mesure smple volume, nd obtin ccurte nd precise quntittive results. Such technology hs potentil ppliction for wide rnge of nlytes in res of generl chemistry nd immunodignostics. The totl cholesterol ssy described here chieved performnce substntilly equivlent to stte-of-the-rt instrumented methods, with excellent stbility nd convenient protocol, mking it idelly suited for on-site consumer use in the physicin s office nd in the home. It should contribute to identifying persons t risk for coronry rtery disese by being vilble to nd usble by brod spectrum of users. The exceptionl performnce of this noninstrumented ssy system is possible through helpful discussions nd meticulous ttention to fine detil from: Sheng Li, Jerry Gin, Pyre Khnn, Bdri Dsu, Sunith Bddm, Dvid Wibbelsmn, Chrles Cypher, Ln-Thnh Huynh, Helen Chw, D. Eline Belrdi, Jonne Miller, John Lnkford, Pul Ptel, Doug Rundle, Jon Blnding, Mur O Brien, Chris Kitzw, nd Edwrd Sssi. We grtefully cknowledge their ssistnce. References 1. Free HM. Self-testing: boom tht won t hurt lbs. MW 1989;21: Free AH, Free HM. Self-testing, n emerging component of clinicl chemistry. Clin Chem 1984;30: Zuk RF, Ginsberg VK, Houts T, et l. Enzyme immunochromtogrphy- quntittive immunossy requiring no instrumenttion. Clin Chem 1985;31: Report of the Ntionl Cholesterol Eduction Progrm Expert Pnel on detection, evlution nd tretment of high blood choles CLINICAL CHEMISTRY, Vol. 36. No. 9, 1990

7 terol in dults. Arch Intern Med 1988;148: Report from the Lbortory Stndrdiztion Pnel of the Ntionl Cholesterol Eduction Progrm. Current sttus of blood cholesterol mesurement in clinicl lbortories in the United Sttes. Clin Chem 1988;34: Nughton MJ, Luepker RV, Stricklnd D. The ccurcy of portble cholesterol nlyzers in public screening progrms. J Am Med Assoc 1990;263: Bchorik PS, Rock R, Cloey T, Trecik E, Becker D, Sigmund W. Cholesterol screening: comprtive evlution of on-site nd lbortory-bsed mesurements. Clin Chem 1990;36: Chpmn RS, Rtcliffe JG. Covlent linkge of ntiser to microprticulte cellulose using 1,1 -crbonyldiimidzole: rpid, prcticl method with potentil use in solid-phse immunossy. Clin Chim Acts 1982;118: Abell LL, Levy BB, Brodie RB, Kendll FE. Simplified method for the estimtion of totl cholesterol in serum nd demonstrtion of its specificity. J Biol Chem 1952;195: Vogel P, Brun HP, Berger D, Werner W. Process nd composition for seprting plsm or serum from whole blood. US ptent no , Hll RR, Woenker WE. Solid-phse cholesterol ssy involving unique whole-blood seprtor. Clin Chem 1987;33: Hssemer N. Helping mnufcturers nd clinicl lbortories verify the ccurcy of cholesterol methods: the Ntionl Reference Method Lbortory network for cholesterol. AACC Lipids Lipoproteins Div Newsl The Fcts of Life 1989;3(4): Cooper GR, Smith SJ, Duncn 1W, et l. Interlbortory testing of the trnsferbility of cndidte Reference Method for totl cholesterol in serum. Clin Chem 1986;32: Duncn 1W, Mther A, Cooper GR. The procedure for the proposed cholesterol Reference Method. Atlnt, GA 30333: Centers for Disese Control, Clinicl Chemistry Division, Center for Environmentl Helth, Ntionl Committee for Clinicl Lbortory Stndrds, proposed guideline EP5-T. User evlution of precision performnce of clinicl chemistry devices. Villnov, PA 19085: NCCLS, 1984; 4:1. CLINICAL CHEMISTRY, Vol. 36. No. 9,