Improvement of creatinine measurement on RA-1000

has b e e n s h o w n that e x c e s s a m o u n t s of h e p a r l n in s y r i n g e s u s e d for b l o o d gas a n a l y s i s can p r o d u c e significant n e g a t i v e r e s u l t s in the d e t e r m i n a t i o n of pCO_ and c a l c u l a t e d bicarbonate. In t~e d a t a p r e s e n t e d In T a b l e s I and II 0.5, l.O, 2.0 and 3.0 mls of v e n o u s b l o o d or q u a l i t y control material (PRIME Fisher Diagnostics - Levels I, II, and Ill) w e r e d r a w n up into s y r i n g e s w i t h o u t heparin, lyophillzed heparin (Autostix) (Msrquest M e d i c a l P r o d u c t s ) and t h o s e c o n t a i n i n g liquid h e p a r l n ( I 0 0 0 USP u n t t s / m l ) w i t h the e x c e s s r e m o v e d (Deseret-Parke Davis). All s p e c i m e n s w e r e a n a l y z e d for pH, p C O 2 , , P O 2 and b i c a r b o n a t e on a C o r n l n g b l o o d gas a n a l y z e r , m o d e l 168. The r e s u l t s of b o t h t h e s e e x p e r i m e n t s can be summarized as f o l l o w s : (a) At least 1 . 0 - 2 . 0 m l s of inaterial m u s t be c o l l e c t e d in o r d e r to m i n i m i z e alterations in p C O 2 . (h) B o t h A u t o s t i x and D e s e r e t p r o d u c t s b e h a v e d s i m i l a r l y w i t h r e s p e c t to heparin. We c o n c l u d e that the use of s y r i n g e s c o n t a i n i n g I y o p h i l i z e d h e p a r i n is a p p r o p r i a t e for the collection of b l o o d for pH m e a s u r e m e n t and b l o o d gas a n a l y s i s . 24 INTERFERENCE OF INDICAN WITH FOUR CO~gdERCIAL PROCEDURES FOR THE MEASUREMENT OF TOTAL BILIRUBIN IN SERUM. R. Peon and I.H. Hinberg, Bureau of Medical Devices, Health and Welfare Canada, Ottawa, Ontario, KIA 0L2. We have s t u d i e d the effect of Indican ( 3 - i n d o x y l sulfate) on 4 commercial procedures for the measurement of total billrubin in serum. We found that total bilirubin measured by the Billrubln A-Gent TM (Abbott Laboratories, Ltd.) 2,4-dichlorophenyl dlazonlum (2,4-DCPD)

procedure

increased

by

50

mg/L

for

each

mmol/L

added

indican. Similarly, total billrubin measured by the Billrubin C-System TM (8oehringer Mannheim Canada, Ltd.) 2,5-dichlorophenyl dlazonium (2,5-DCPD) procedure increased by 33 mg/L per mmol/L Indicen. Indican also interfered with the M i c r o B i l i r n b i n R e a g e n t Set TM (Harleco) M a l l o y Evelyn procedure, but to a much lesser extent. The Jendrassik Bllirubin Reagent System TM (American Monitor Corp.) Jendrsssik-Grof procedure was unaffected by the presence of Indican. The amount of interference with the 2,5-DCPD procedure Increased significantly with color development time and was twice the initial amount after 30 minutes. Concentrations of Indlcan as high as 0.38 mmol/L have been found in sera of patients with renal failure (Ludwig, G.D. et al. (1968) Am. J. Clin. Nutr. 21, 436). Our results show that this concentration of interferent would increase total billrubln values measured by the 2,4-DCFD and 2,5-DCPD procedures by 19 mg/L end 12 mE~L, respectively. Users of these procedures should therefore be s u s p i c i o u s of unexpectedly elevated billrubin values obtained with sera from patients with chronic renal disease.

for Ii months. We used our previously reported kinetic methods with a centrifugal analyser to assay enzyme activities (Moses, G.C. and Henderson A.R. (1983), CZin Ch~mL29, 1159 and C ~ n Biochem 16, AI6). The overall mean values for the 24 individuals ranged from 5.5 - 9.8 U/L and 5.3 - 13.5 U/L for 5' nucleotidase and cholinesterase, respectively. The average within-person and between-person variation werq 0.38 and 2.68 (SD 2) for cholinesterase and 1.41 and 0.96 (SD 2) for 5' nucleotidase, respectively. The overall mean values were higher in males than females (also higher in caucasians than non-caucasians) for both enzymes. However, these differences were not statistically significant (P >0.05). There were no statistically significant differences between mean 5' nucleotidase and cholinesterase activities with respect to subjects' age. However, mean activities of 5' nucleotidase in individuals over 40 years of age were higher than those in individuals ages 25 - 39 years. In conclusion, the relative magnitudes of within-person and between variability thus obtained are useful guidelines in establishing thrEe-individual reference ranges for these two enzymes, which are used in the differential diagnosis of liver disorders. 27 EVALUATION OF AN IMMUNOINHIBITION/II~UNOPRECIPITATION METHOD FOR CK-MB DETERMINATION. A. Sorisk7, D. S. Out, J. T. Hindmarsh, Division of gloche~istry, Ottawa Civic Hospital, Ottawa, Ontario K/Y 4E9. The Roche Isomune-CKTM is a two-step ~---unoinhibition/ immunoprecipitation assay that specifically quantitates CK~MB activity by blanking the activities of CK-MM, CK-BB, atypical CEs, and adenylate kinase. We evaluated the Roche technique adding sodium oxamate to the substrate reagent to prevent false negatives in samples with high levels of pyruvate and LD. This method was compared with CK electrophoresis ( B e c ~ a n Paragon TM agarose gel and fluorescence densitometric measurement). The Roche method was performed on the ABA 200 analyzer at 37°C, with the filter factor adjusted to 3.75. The CK-MB activity was expressed as a percentage of total CK activity, also measured on the ABA 200 analyzer. Preliminary experience showed the technique to have acceptable precision: within run, C.V. 7.3% (n-8); day to day, C.V. 10% (n=6). Correlation of CK-MB activity on 33 samples by the two techniques gave an r value of 0.924, y - 0.795 x + 2.38. Electrophoresis showed one of these samples to have 0% MB and 48.1% BB activity. The Isomune-CK TM quantitated MB as 0.2% activity. The Roche Isomune-CK TM is quick, easy to perform and appears to be specific for CK-MB activity. It has acceptable precision and gives good agreement with CK electrophoresis. However, while electrophoresls provides additional information on the presence or absence of CK-BB or atypical CK bands, the immunoinhibltion/immunoprecipitation method only quantitates MB isoenzyme. 28 EVALUATION OF THE CLINICAL EFFECTIVENESS OF SERUM CK-MB MEASUREMENTS ON THE DUPONT ACA FOR PATIENTS ADMITTED TO A CORONARY C A R E UNIT. M.L. Leroux, J. Rabson, and P.R.E. Desjardins, Dept. of Clinical Chemistry, Health Sciences Centre, Winnipeg, Manitoba R 3 E O Z 3

25 EXPERIENCE WITH THE ROCKET TECHNIQUE FOR SIMULTANEOUS QUANTIFICATION OF APOPROTEINS A 1 AND B. M. T. Meuffels and J. T. Hindmarsh, Division of Biochemistry, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa KIY 4E9. There is some argument that serum apoprotein levels may be superior to serum lipide as predictors of risk for coronary artery disease. As our laboratory is part of a medicalbiochemical group studying patients attending a lipid clinic, we investigated methods for quantifying apoproteins in serum. The method we are currently using is a rocket technique marketed by Sebia (Issy lea Moulineau~, France - distributed by Gelman Corporation) and described by Fruchart et al (Clin. Chem. 28, 59-62, 1982). Because of problems with assay precision and antibody specificity, the Center for Disease Control in Atlanta, Georgia has developed an international quality assurance program for these assays to which we belong. Preliminary data indicates the precision of the assays to be acceptable and after some initial problems the technique is proving to be satisfactory. Advantages of the rocket technique over other procedures such as radial Immunodiffusion include faster turnaround time, lower cost and the ability to measure apoproteins A1 and B simultaneously.

INSTRUMENTATION

26 BIOLOGICAL VARIABILITY OF SERUM 5'NUCLEOTIDASE AND CHOLINESTERASE ACTIVITIES, G.C. Moses, James F. Tuckerman, and A. Ralph Henderson, Dept. Olin. Biochemistry, University Hospital, London, Ontario, N6A 5A5. We measured serum 5' nucleotidase (EC 3.1.3.5) and cholinesterase (EC 3.1.1.8) activities in 24 healthy laboratory workers once a week for 6 weeks and twice monthly

29 IMPROVEMENT OF CREATININE MEASUREMENT ON RA-1000 8. Rossi~nol, D. Rossignol and C. PetltClerc Service of Biochemistry, Hgtel-Dieu de Montreal, 3840, St-Urbain, Montreal, Quebec, H2W IT8 As part of the first order rate routine for creatinine measurement, RA-1000 includes a reagent blank rate correction on every specimen.

CLINICAL BIOCHEMISTRY, VOLUME 17, JUNE 1984

W e evaluated the clinical effectiveness of serum C K - M B measurements in the diagnosis of acute myocardial infarction (AMI) using a Dupont A C A method in patients admitted to our Coronary Care Unit (CCU). The Dupont A C A C K - M B results were compared to serum C K - M B and L D H isoenzyme results using agarose electrophoresis. The tests were performed on seMal blood samples drawn from I00 consecutive patients admitted to the C C U . As a result of the low specificity obtained for C K - M B measurements compared to literature values, the isoenzyme data was re-evaluated using Receiver Operating Charactemstic (ROC) curves. N e w decision levels were established for serum C K - M B and the L D H I/H ratio measured by agarose electrophoresis. These decision levels improved the specificity and efficiency of these tests in establishing the diagnosis of AMI. The R O C curves, confirmed the reference range suggested by Dupont, for serum C K - M B .

203

Unacceptable variability in the reagent blank rates triggered studies of the alkaline picrate stability and of the kinetics of the reaction. As shown previously (Shoucri, R.M. and Pouliot, M. (1977) Clio. Chem. 23, 1527) the rate of formation the creatinine picrate complexe was found to be dependent on picrate concentration. Moreover the rate was found to be non linear (at least, two first order rate components) at 20.8 mmol/l picrate, with most patient serum. Reducing the concentration of picrate to 13.9 mmol/l, resulted in apparent single first order kinetics for most specimens after 30S following serum addition. At this concentration of picrate, the rate was first order with respect to creatinine content of specimen. Regression of paired determinations on SMAC(x) and RA-lO00(y) gave y = 1.0178x + 0.0544, for 20.8 mmol/l picrate and y = 0.977x + 0.114 for 13.9 uunol/l picrate. Overall precision and comparability of RA-IO00 results with those of the SMAC was considerably improved. 30 CREATININE DETERMINATION ON THE COBAS BIO CENTRIFUGAL ANALYSER: TEMPERATURE RELATED WITHIN-RUN DRIFT K.C. Doole~ and M. Mathai, Biochemistry Laboratory, Izaak Walton Killam Hospital for Children and Dept. of Pathology Dalhousie University, Halifax, N.S. B3J 3G9 The Dow Chemical Company application for creatinine determination on the Cohas Bio Centrifugal Analyser was investigated. A consistent positive drift during within-run precision determinations was observed. The Dow procedure uses a kinetic alkaline picrate reaction with lithium picrate and lithium hydroxide as the principal reagent components. The magnitude of the drift was sufficient to contribute 2% to the within run precision values for this method. The drift persisted after major changes in reagent to sample ratio and reaction timing parameters and was also observed when the Dow procedural application for the Cohas was applied to other reagent preparations using either sodium picrate or lithium picrate. It was observed that the magnitude of the drift was proportional to the temperature at which the creatinine determinations were made. It was found that changing the pipetting mode from parallel pipetting (test method 4, with start reagent vol. = 0 and incubation time = 0) to serial pipetting with reagent addition first (test method 4, start reagent vol.=O, incubation time = I0 sec.) eliminated the drift. A revised application of the Dow formulation, using serial pipetting, was then evaluated. The results were as follows: Between Day Precision: N Mean (mmol/L) SD CV (%) 49 81.5 2.9 3.5 34 315 7.85 2.5 Within-Run P r e c i s i o n : 16 80.9 0.86 1.06 Comparison w i t h the SMAC p r o c e d u r e u s i n g r e g r e s s i o n a n a l y s i s : N = 60, Slope = 0 . 9 1 , I n t e r c e p t = 11.26 mmol/L, r = 0.98

31

EVALUATION OF ENZYME MODULE OF BECKMAN'S ASTRA-8 Anand Swaroop, Pathology Department Holy Cross Hospital, Calgary, Alberta T2S IS6

We compared the DuPond ACA method with ASTRA-8 Enzyme method. A linear regression analysis showed: Slope Intercept r n ECF (i/Slope) AST 0.92675 -3.2440 0.98065 85 1.0790 ALT 1.03099 -4.52896 0.99697 85 0.9699 CK 0.97105 -1.66787 0.99844 85 1.0298 LDH 0.86197 5.51 0.99411 85 1.1601 With new ECF patient samples were run in parallel on ASTRA8 and ACA. The linear regression equations were: CK; y = l.OOx + 2.16; r = 1.00 n = 50 LD; y = l.Olx - 3.54; r = 0.999 n = 50 AST; y = 0.97x + 2.22; r = 1.000 n = 50 ALT; y = 1.06x - 4.3; r = 0.981 n = 50 Between run and run to run studies on two controls showed: Between Run Mean CV% Run to Run Mean CV% LDH Control B I 130.1 4.53 132.6 3.77 Control B 2 374.5 1.74 366.5 4.64 ALT Control B I 26.2 8.4 26.9 8.55 Control B 2 69.1 4.05 65,1 5.03 AST Control B I 33.6 8.63 37.5 5.6 Control B 2 89.5 3.69 90.1 3.11 CK Control B I 115.7 3.03 118,2 2.62 Control B 2 225.5 1.95 235.4 2.97 We conclude that the precision and accuracy are within acceptance levels. And using ECF factor, ASTRA-8 can give the approximate numeral answer produced by any other instrument. 32 THE TANDEM SMAC - RA-IO00: AN APPROACH TO OPTIMAL AND COST EFFECTIVE USE OF BIOCHEMICAL ANALYSES C. PetitClere and B. Rossignol Service of Biochemistry,

204

Hgtel-Dieu de Montreal, 3840, St-Urbain, Montreal, Quebec H2W ITS In 1982, Biochemistry Service at HGtel-Dieu de Montreal produced close to I00,000 SMAC profiles equally shared by in patients, out patients and referred cases. Laboratory test prescriptions was channelled through SMAC as the most common tests were run exclusively on this intrument with the exception of the emergency list. Annual total operating cost for SMAC, excluding amortizement approximate $ 220,000. In 1983, stripping of the S ~ C profiles was undertaken with appropriate support to and from medical staff. Phase I included removal of cholesterol and triglycerides followed by Phase II with the removal of AST, ALT, LDH, ALP, GGT, 8ilirubine and S-iron. Based on a previous experience with the abandon of S ~ II profiles in a university hospital, the drop in laboratory tests prescription was expected to be 80% for AST-ALT and 90% for others. After 3 weeks, those figures were obtained, which resulted in an expected annual decrease of SMAC expenses of $ 50,000. It is interesting to note that the prescription of the new SMAC-10 dropped by 45%! The remaining workload: cholesterol-triglycerides, enzymes and bilirubine was well within the capacity of a RA-1000. Taking into account the cost of running the RAlO00, the net annual economy will be $ 25,000 which by itself pays the RA-1000 over 5 years. The RA-IO00 is now use d in tandem with the S ~ C which has allowed us to control the test prescription and adjust for specific needs of attending

33 EVALUATION OF THE HITACHI 705. J. Krahn and A. Hielkema, Dept. of Clinical Biochemistry, St. Boniface General Hospital, Winnipeg, Manitoba R2H 2A6. Although several evaluations on the Hitachi 705 have been reported in the literature (Douville, P., and Forest, J.C., Clln. Chem. 29, 692 1983 and Kineiko, R.W., Foering, D.A., and Morrissey, M., Clin. Chem. 29, 688, 1983) no extensive evaluation has been done on possible interferences due to carry-over of reagents either on the reagent probes or in the reaction cuvettes. The effects of hemolysed, lipemic or jaundiced sera on the tests performed on this instrument have not been previously analyzed. This presentation will address those two general areas. To determine the effect of carry-over of reagents in the reaction cuvettes we filled all the cuvettes with one type of reagent and then ran the other 15 tests. These experiments showed that for most chemistries tested the reagent carry-over in the cuvettes is not a problem. Experiments investigating reagent carry-over on the reagent probes reveal no problems. Interference studies show that hemoglobin interferes with the bilirubin, protein, phosphate and urate methods; lipemia interferes with the bilirubin and phosphate methods; and bilirubin interferes with the protein, cholesterol and phosphate methods. Studies are currently proceeding to determine whether these interferences can be reduced by different wavelength selection in the bichromatic analysis. 3h PERFORMANCE OF HOME-USE BLOOD GLUCOSE MONITORS IN A HOSPITAL SETTING. l.H. Hinberg, R. Pooh and R. Cobbledlck, Bureau of Medical Devices, Fealth and Welfare Canada, Ottawa, Ontario, KIA OL2. Home-use blood glucose analyzers (HBGA) are increasingly being used to monitor blood glucose control in hospitalized patients. We have studied the performance of 5 brands of HBGA in a typical hospital setting, in which blood glucose monitoring is done by more than one nurse, by comparing HBGA results obtained by I0 trained technicians to those obtained with our G l u e o - q u a n t TM ( B o e h r i n g e r M a n n h e i m Canada, Ltd.) hexokinase procedure for whole blood glucose, which has a bias < 0 . 8 % and a %CV < 0 . 5 % . Performance parameters assessed included bias, user-to-user variability and variability o f r e a d i n g s by t h e same u s e r . A l l brands gave clinically unreliable results a t below normal g l u c o s e l e v e l s . At a g l u c o s e c o n c e n t r a t i o n of 55 mg/100 mL, a s y m p t o t i c 95% u p p e r c o n f i d e n c e limits on t h e probabillty t h a t a s i n g l e o b e r v a t l o n by a randomly chosen u s e r would be w i t h i n 20% (AUCL20%) v a r i e d from 0 . 0 3 f o r t h e H y p o c o u n t TM II Model B (Hypoguard Ltd., Suffolk, England) to 0.89 for the Glueometer TM (Ames Division, Miles Laboratories Inc., USA). Although all brands gave AUCL20% values > 0 . 9 at glucose eoncentratioos between 120 and 325 mE/100 mL, the probability of administering a dose of insulin a t least 2 units different from the correct dose varied from 0.13 to 0.33, depending on the brand used. The best results were obtained with G l u c o m e t e r TM and Hypocount TM II Model B and the worst with

C L I N I C A L B I O C H E M I S T R Y , V O L U M E 17, J U N E 1984