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Objective: We investigated the prevalence of asymptomatic pyuria (ASP) in diabetic patients and compared the Sysmex UF-100 with Fuchs-Rosenthal hemacytometer., Design and Methods: ASP prevalence was investigated in 227 diabetic patients. Imprecision, accuracy and correlation of UF-100 with hemacytometer in measuring leukocyte counts were determined., Results: Diabetic women and men had significantly higher ASP prevalence than non-diabetic women (21.4 vs. 8.7%) and men (12.2 vs. 3.4%). Disease duration and HbA(1C) levels were similar in diabetic patients with and without ASP. UF-100 and hemacytometer readings correlated significantly (r=0.88) without a significant bias. Within-run coefficients of variations for UF-100 (8.14, 6.35 and 12.18%) and hemacytometer (5.14, 5.18 and 8.03%) did not differ significantly., Conclusions: Prevalence of ASP is increased in diabetic patients and not affected by duration of disease or control of hyperglycemia. UF-100 seemed to be a reliable, precise and accurate system to determine pyuria.

Objectives: To develop a new, colorimetric and automated method for measuring total oxidation status (TOS)., Design and Methods: The assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. The oxidation reaction of the assay was enhanced and precipitation of proteins was prevented. In addition, autoxidation of ferrous ion present in the reagent was prevented during storage. The method was applied to an automated analyzer, which was calibrated with hydrogen peroxide and the analytical performance characteristics of the assay were determined., Results: There were important correlations with hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide solutions (r=0.99, P<0.001 for all). In addition, the new assay presented a typical sigmoidal reaction pattern in copper-induced lipoprotein autoxidation. The novel assay is linear up to 200 micromol H2O2 Equiv./L and its precision value is lower than 3%. The lower detection limit is 1.13 micromol H2O2 Equiv./L. The reagents are stable for at least 6 months on the automated analyzer. Serum TOS level was significantly higher in patients with osteoarthritis (21.23+/-3.11 micromol H2O2 Equiv./L) than in healthy subjects (14.19+/-3.16 micromol H2O2 Equiv./L, P<0.001) and the results showed a significant negative correlation with total antioxidant capacity (TAC) (r=-0.66 P<0.01)., Conclusions: This easy, stable, reliable, sensitive, inexpensive and fully automated method that is described can be used to measure total oxidant status.

Objective: To measure serum zinc concentration by means of carbonic anhydrase reactivation using an automated analyzer., Methods: The zinc content of carbonic anhydrase (CA), whose cofactor is zinc, was removed by dialysis against pyridine 2 to 6 dicarboxylic acid and a pure apoenzyme was obtained. Serum proteins were precipitated with trichloroacetic acid (TCA) solution and the supernatant fraction of the sample was used to determine the zinc concentration. The negative effects of the precipitant on CA activity in the assay were completely removed, reaction conditions for maximal CA activity were provided and the color of the product was enhanced and stabilized. P-nitrophenyl acetate was used as the substrate and the change of absorbance of p-nitrophenol which was produced was followed at 400 nm. The initial rate of the esterase activity of CA was measured by using an automated analyzer. Analytical performance characteristics of the assay were determined. The zinc concentrations in serum samples of healthy subjects and patients were measured., Results: The enzymatic assay is accurate, sensitive, specific and is not affected by other metals. There was excellent agreement with the results obtained using an atomic absorption spectrophotometer (AAS) (y = 0.98X + 0.18, r = 0.99). Serum zinc concentrations were found to be higher in patients with vivax malaria, and lower in patients with cutaneous leishmaniasis than in healthy subjects., Conclusion: The enzymatic method is suitable for semiautomated measurement of serum zinc concentration.

Objectives: To evaluate a simple assay for macroprolactin for use with the Bayer Immuno 1 analyzer, and to compare the reactivity of macroprolactin in commonly used automated prolactin assays., Methods: Macroprolactin in serum was precipitated in a buffer containing 13.3% polyethylene glycol (PEG) 8000, redissolved, and assayed on the Bayer Immuno 1 for PRL. Presence of macroprolactin was confirmed in some sera by FPLC using a Pharmacia Superose 12 column, followed by prolactin assay of the fractions on the Immuno 1. Sera with and without macroprolactin were then also assayed on the Abbott AxSYM, Bayer Centaur, Beckman Access, and Roche Elecsys., Results: The PEG precipitation assay is simple and reproducible (CVs < 15%), and we established a normal range of < 20% precipitation of total PRL by PEG. The assay correlates well with the amount of macroprolactin separated by FPLC as a peak with a MW of approximately 180 kDa. Macroprolactin showed the following cross-reactivities in commonly used PRL assays: Roche Elecsys > Bayer Immuno 1 > Abbott AxSYM > Bayer Centaur > Beckman Access, with the Centaur showing more variability than other assays., Conclusion: Macroprolactin can be easily quantitated using the Immuno 1 PRL assay after PEG precipitation. It cross-reacts to different degrees with common prolactin assays, and is a major source of variability between them.

Objectives: Several pediatric advisory groups have recommended selective screening for dyslipidemia in children. Low-density lipoprotein cholesterol (LDL-C) is measured clinically with the Friedewald calculation in fasting samples. Nonfasting measurement of LDL-C would be clinically useful in children., Design and Methods: In the present study, we examine the performance of two surfactant-based direct LDL-C assays in paired samples, fasting and nonfasting, from 100 children., Results: LDL-C in the fasting state was significantly lower with the Friedewald estimation: 2.43 +/- 0. 61 mmol/L, N-geneous (Genzyme Corp.) direct LDL-C: 2.30 +/- 0.59 mmol/L, and Roche (Roche Diagnostics) direct LDL-C: 2.32 +/- 0.57 mmol/L than with ultracentrifugation-dextran-sulfate-Mg(2+) precipitation (UC-DS): 2.47 +/- 0.64 mmol/L. Moreover, there was increased negative bias using nonfasting samples with N-geneous: 2. 25 +/- 0.56 mmol/L and Roche: 2.26 +/- 0.56 mmol/L compared with fasting UC-DS. Correlation with US-DS was highest for Friedewald (r = 0.974) and fasting N-geneous (r = 0.973), and lowest with nonfasting N-geneous (r = 0.849) and Roche in fasting (r = 0.891) and nonfasting samples (r = 0.747). The sensitivity at LDL-C concentration of 2.85 mmol/L for the two direct methods when either fasting or nonfasting samples were used, was lower than that obtained with Friedewald., Conclusion: Overall, these direct LDL-C assays demonstrated limited utility in screening children but may be useful in the management of lipid disorders.

Objectives: We evaluated the analytical performance of the Elecsys 2010 immunoanalyzer (Boehringer Mannheim Canada), which is based on a new detection technology, electrochemical luminescence., Design and Methods: We used six representative assays from the initial launch menu of the instrument: thyroid stimulating hormone (TSH), free thyroxine (FT4), troponin T, human chorionic gonadotropin (hCG), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA). Within-run and between-day imprecision were evaluated using pools of human specimens at low, mid and high concentrations. Linearity was evaluated by diluting specimens with high analyte concentrations with specimens that had a low level of this analyte. Carry over and hook effect were investigated using specimens with high concentrations of hCG. Functional sensitivity was studied by running low TSH specimens over 21 daily runs, and by comparing the scatterplot of FT4 as a function of TSH. Over 100 specimens distributed across the analytical range were analyzed with two comparison methods: ES 300 (Boehringer Mannheim Canada) and AxSYM (Abbott Laboratories)., Results: Within-run and between-day imprecisions were less than 4% and 10%, respectively, for most assays. All assays were linear over the whole analytical range. Carry over was minimal (< 0.0002%). A hook effect was present for hCG levels greater than 560,000 U/L. The functional sensitivity of the TSH assay was lower than 0.02 mlU/L. Correlation coefficients were all > 0.94. Small proportional errors were observed in comparison studies for the CEA and PSA assays., Conclusions: The Elecsys 2010 system was shown to have an acceptable analytical performance for the rapid analysis of a wide variety of analytes. The hook effect observed with hCG assay would imply that the laboratory informs the clinicians of the possibility of falsely low values in trophoblastic diseases or that all specimens with values greater than 3000 U/L are reassayed after dilution.

Monitoring parent drug cyclosporine (CsA) concentrations in whole blood has been facilitated by the introduction of automated nonisotopic immunoassays [fluorescence polarization monoclonal whole blood assay (FPIA), EMIT Cyclosporine Assay]. The latter assay currently has a defined application only for Cobas Mira Chemistry Systems. The purpose of our work was to develop an application for this assay on the Technicon AXON. Instrument settings were optimized to arrive at the following assay performance characteristics. Limit of sensitivity was 50 micrograms/L. Interassay coefficients of variation (CV) were 11.2% (n = 16; mean = 81 micrograms/L) and 9.4% (n = 16; mean = 418 micrograms/L). Recoveries of 102, 112, and 117% were obtained by spiking aliquots of 10 whole blood patient pools of known CsA concentrations with 50, 100, and 200 micrograms/L CsA, respectively. Serial dilutions of two patient specimens demonstrated a linear relationship between expected and actual CsA concentrations (r = 0.996, 0.998; regression lines; y = 0.989x + 11.7; y = 0.979x + 9.5). Carryover and interference (lipemia) were not evident. Instrument calibration stability is at least 1 month. Comparison with CsA concentrations analyzed in renal transplant patients by the FPIA assay produced a linear regression equation of EMIT = 1.113x - 44.5, r = 0.968, Sy/x = 20.8, n = 32. Comparison with high-performance liquid chromatography (HPLC)-derived values in the same patient population produced a linear regression equation of EMIT = 1.114x - 16.4, r = 0.970, Sy/x = 20.2. FPIA-derived CsA concentrations averaged 14.2% more than those obtained with the EMIT method with the latter averaging 1.3% more than HPLC values.(ABSTRACT TRUNCATED AT 250 WORDS)

A semi-automated enzymatic colorimetric method for determining Cu-Zn superoxide dismutase (SOD) in erythrocytes using an FP9 parallel analyzer is described. The method is based on the reaction described by McCord and Fridovich (xanthine/xanthine oxidase, ferricytochrome C), which permits kinetic measurements to be determined colorimetrically for the assay of enzyme activity. The coefficients of variation for within- and between-run analyses were less than 6%. The limit of linearity is 3.5-times the mean value of the normal range while recovery of added SOD ranged from 91 to 120%. The method, which is both reliable and simple, allows rapid, simultaneous measurements of multiple samples.

We have modified a manual assay method for the determination of serum 5'-nucleotidase so that the reaction product, phosphate, is assayed colorimetrically using a continuous flow system. The contribution of non-specific phosphatase enzymes is assessed in the presence of nickel ions which specifically inhibit 5'-nucleotidase. The detergent sodium lauryl sulfate is used to eliminate the need for deproteinization in the phosphate assay. We have measured 5'-nucleotidase activity in the sera of 123 breast cancer patients and correlated our data with the patients' clinical status. Receiver operating characteristic curve analysis showed the most advantageous cut-off point for indication of secondary spread to be 10 U/L. Using this cut-off point, 14% of patients who were clinically free of disease and 35% of patients with disease clinically present had elevated 5'-nucleotidase activity.

A procedure to measure total and direct bilirubin using the Abbott Bichromatic Analyser is described. The method is an adaptation of a sodium dodecyl sulfate method. Reaction products are measured at an acid pH in a buffered matrix using a 600-650 filter wheel. The method has very little spectral interference from either hemolysis or lipemia and correlates well with the manual Jendrassik-Grof method.

A rapid, semi-automated method for determining hexoses which are bound to components of whole blood; e.g., hemoglobin, is described. The method is an improvement on similar lengthy manual methods (1,2) in which 5-hydroxymethylfurfural is produced and measured. Heating of diluted blood samples at 15 lb./sq.in. above atmospheric pressure in a pressure cooker for 20 minutes is shown to give the same results as those obtained with other methods in which 2-5 hours heating at atmospheric pressure is specified (1,2,3). The trichloracetic acid precipitation step needed in the manual methods is avoided by continuous flow dialysis on a Technicom Auto Analyzer II system. Various experimental factors have been studied and comparisons made with a commonly-used ion-exchange resin column technique. Two different methods of calibration, one using pure 5-hydroxymethylfurfural standards, and the other using patients' samples with pre-determined, known values as standards, are presented.

1. A method for the analysis of salicylate in serum is described utilizing an American Monitor KDA. The method correlated well with a manual Trinder method. 2. The within-run coefficient of variation at a salicylate level of 4.4 mg/dl was 2.9%, 0.71% at a level of 16.5 mg/dl and 0.71% at a level of 62.6% mg/dl. 3. The day-to-day coefficient of variation was found to be 8.1% at a salicylate level of 13.1 and 1.3% at a level of 39.0 mg/dl.

Urinary orotic acid is believed to be a valuable probe for early diagnosis of inborn errors of metabolism leading to hyperammonemia and increased pyrimidine synthesis. For the purpose of our urinary mass screening programme, we developed an automated colorimetric method which is reliable in the range of 1 to 50 micrograms/ml orotic acid and allows analyses at a rate of 160 samples per hour. Preliminary results are presented which illustrate that various disorders can be recognized by measuring orotic acid in urine.

An automated enzymatic method is described for the determination of Cu,Zn-superoxide dismutase (SOD) in plasma or erythrocytes using the xanthine-xanthine oxidase and cytochrome C coupled assay. This method was adapted to an Abbott ABA-200 discrete analyzer. Coefficients of variation for within-run and day-to-day analyses were less than 5%. Only 2.5 muL of serum or erythrocyte extract is required so a capillary tube sample of blood (70 muL) is sufficient for the assay. Recovery of added SOD ranged from 92 to 101%. The method reported here is practical for use in a clinical chemistry laboratory for monitoring changes in this enzyme, which is a sensitive early indicator of alterations in copper status.

We describe a fast and efficient method for the isolation of drugs from blood serum by use of the DU PONT PREP I Automated Sample Processor and octadecyl bonded phase columns. Drugs are selectively adsorbed onto C18 bonded phase and eluted with methanol. The recovered residue is essentially free of sample matrix chromogens and shows very low background on thin layer chromatography. It is also suitable for analysis by gas liquid chromatography and high pressure liquid chromatography. Recovery of drugs added to serum is greater than 90 per cent for most drugs studied which include weakly acidic, neutral and alkaline drugs. The method offers tremendous savings to the user in time and dollars, since it is fully automated from the extraction step to the drying of the extract, and the columns are reusable at least 10 times. Up to 12 samples can be processed simultaneously in less than 60 minutes.

Rational antihyperlipemic therapy based on the use of nicotinic acid and its derivatives and/or combinations demands a rapid and reliable method for monitoring nicotinic acid blood levels. To this end, an automated colorimetric method for the analysis of nicotinic acid in serum has been elaborated. For serum from rats, dogs and man given p.o. nicotinic acid or 3-pyridine methanol, the method is greater than 90% specific for nicotinic acid. The limit detection for nicotinic acid is 2 microgram/ml based on 0.15 ml of serum or 0.3 microgram/ml based on 1.5 ml of serum.

We describe a "one-minute" direct bilirubin procedure using the ABA-100. The procedure correlates well with the manual Jendrassik-Grof method and gives good precision at a relatively low direct bilirubin level. In comparison with certain other Abbott ABA-100 direct bilirubin methods, the proposed procedure appears to be the least affected by the presence of high unconjugated bilirubin levels.

A semi automated procedure for the determination of 3-methoxy-4-hydroxy-mandelic acid (HMMA) in urine has been evaluated. This procedure offers advantages in simplicity, speed and precision over existing manual techniques which utilize spectrophotometric fluorimetric and gas liquid chromatographic methods. The urine is extracted with ethyl acetate from a highly acidic sample, purified by extraction into acetate buffer and the diazo colour reaction developed. All stages of the procedure are carried out using the Brown and semi-automatic extractor equipped with partition tube. The within and between assay coefficient of variation obtained on pooled freeze-dried urine samples was 6.7% and 7.7% respectively.

A study into the substitution of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate for phenol in the indicator reaction for an automated glucose procedure is presented. Apart from having physical properties that are more conducive to easy handling than phenol, the former material affords considerably more sensitivity than does the latter. Results obtained with either of these glucose oxidase-coupled systems demonstrate good correlation not only with each other but also with the glucose oxidase procedure of the Beckman Astra 8.

1. A new, quick, sensitive and specific assay for the quantitative determination of proline is presented together with its automatization. The use of glacial acetic acid-formaldehyde as the solvent for ninhydrin lends a series of advantages to the reaction. 2. A serial automated procedure for the simultaneous detection of proline, hydroxyproline and hydroxylysine in one single sample is pesented. The advantage of the triple assay lies in the use of one single sample instead of three previously aliquoted fractions in three individual automated assays. The triple assay is as specific, reproducible and sensitive as the individual assays. 3. A quick, manual assay for hydroxylysine is presented. This method represents a further simplifiction of previous ones by the same author and adds the advantage of rapidity to their sensitivity and specificity.

A simple, sensitive, and precise continuous-flow (Technicon Auto Analyser II) method using enzymic reagents to measure high-density lipoprotein (HDL) cholesterol in the supernatant of serum treated with heparin-manganese reagent is described. 1. The sensitivity of the system was improved by increasing the flow rates of both sample and re-sample manifold lines to 0.23 ml/min. This allowed a recorder full-scale deflection equivalent to 840 mg cholesterol per litre, and permitted a precise determination of cholesterol in the 200-600 mg/l range. 2. Interaction between Mn++ and the enyzmic reagents caused false elevations of the peak plateau on the chart readings, especially when 2M MnCl2 was used. Addition of disodium ethylene-diaminetetra acetate (8 mmol/L) to the enzyme reagent eliminated the spike-like interference in the recorder tracings, and permitted more accurate chart readings. 3. The heparin-Mn++ reagents contributed to the measured HDL-cholesterol. Use of a saline blank improved precision. 4. The inter-assay variation was determined by analysing (n = 26) 2 quality control serum pools: X1 = 362 +/- 11.5 mg/l (CV = 3.18%) and X2 = 252 +/- 14.4 mg/l (CV = 5.74%).

Three quantitative assays for detection of proteins are reported. One is an adaptation to the Auto-Analyzer of the widely used method of Lowry et al. In another procedure, the product of the reaction of proteins with a modified biuret reacts with the phosphomolybdic--phosphotungstic reagent of Folin and Ciocalteu. The third method, performed on hydrolyzed sample, is based on the measurement of proline (Pro) and the conversion of the Pro content into protein. The universal presence of proline in serum proteins suggested this assay. The values for total serum proteins as assayed by the three procedures are similar. The second and the third assay for proteins, described in this paper, can also be performed manually. Studies on interfering substances and their elimination as well as on the sensitivity of the assays are reported.

1. The EMIT-AED procedures for phenobarbital and diphenylhydantoin have been adapted for use on the ABA-100. 2. A basic language computer program has been developed for data reduction and quality control. 3. Within-run and run-to-run coefficients of variations were, in general, less than 8% across the concentration range tested. 4. Performance data on agreement of duplicates and recovery from "spiked" samples were aceptable. 5. The automated procedure provides a two-thirds savings in reagents.

We describe an automated, kinetic nephelometric method for fibronectin on the Multistat III F/LS Centrifugal Analyzer (Instrumentation Laboratory Inc., Lexington, MA 02173). Antiserum is diluted with polyethylene glycol. Calibrators and samples are prediluted with potassium phosphate buffer (10 mmol/L, pH 7.0) containing 8.5 g of NaCl per litre. Intensity (I) is read at 5 and 180 s and the resulting delta I plotted against concentration. A non-linear least squares curve fitting and interpolation of results is carried out automatically. Three controls, with values of between 190-370 gave coefficients of variation between 4 and 7 percent, and the sensitivity of the method is to 25 mg/L.

An automated turbidimetric procedure for determining serum concentrations of apolipoprotein A1 (apo A1) is described. This procedure allows for rapid, precise and inexpensive assay of apo A1 in large numbers of serum samples.

Some analytical parameters have been investigated for a recently described stabilized liquid coenzyme technology in which water-free NAD is dissolved in 1,2 propanediol. Correlation for 108 specimens assayed for AST, ALT and LD with a reference method in which glycol-based NAD was absent was greater than or equal to 0.998 with near identical reproducibility over a period of at least 107 days. Mean recovery of exogenous serum enzymes in this linear kinetic assay is 103%. With the option of mixing only the volume of reagent needed for the enzymatic assay, waste can be eliminated as compared to more costly preparations stabilized by lyophilization. Hazards from an impure water supply are avoided since no reconstituting volume is required.

1. An automated method is described for the assay of pregnancy serum cystine aminopeptidase (Oxytocinase, E.C. 3.4.1.10) using continuous flow analysis. S-Genzyl-L-cysteine-p-nitroanilide, the most highly specific substrate so far available for oxytocinase is employed in this assay. Comparison with the manual method from which it has been developed, gave a correlation coefficient of 0.98. Reaction of oxytocinase on the substrate liberated p-nitroaniline, the absorption of which is measured at 410nm. 2. Blank values are eliminated by the incorporation of a dialyzer in the manifold design and thereby effectively increasing the rate of assay sample to 40 per hour. The lag phase (a) of the continuous flow analysis is 0.17 min while the exponential phase (b) is 0.31 min. Using a 2:1 sample to wash ration the carry-over is 3% at a 95% of steady states.

1. Reference values for the plasma creatinine were established using the alkaline picrate method with the Greiner Selective Analyzer GSA II in relation to the Cr 51-EDTA Clearance. Individuals with normal GFR between 93 to 159 ml/min/1.73 m2 had creatinine values in men (n = 65) from 53.7 to 119.5 mumol/l (0.61 to 1.35 mg/100 ml) and in women (n = 59) from 37.7 to 107 mumol/l (0.42 to 121 mg/100 ml). 2. The creatine determinations with the GSA II were compared to those on the Technicon Analyzer, the Beckman Creatinine Analyzer, the Gemsaec-Fast Analyzer and to the enzymatic creatinine method. A good correlation (r = 0.9780-0.984) was observed. 3. With the GSA II and the enzymatic method, bilirubin showed a minor interference which was more marked with the Beckman analyzer.

A colorimetric method is described for determining total serum iron and unsaturated iron binding capacity (UIBC) of serum using Technicon AutoAnalyzer II equipment. The determination of UIBC has been fully automated by means of a simple modification to the Sampler II module, whereby 2 double probes permit the simultaneous aspiration of a sample and of a measured volume of the iron saturating solution. No manifold changes are needed when changing from serum iron to UIBC.

In this paper we report a method to enable the Beckman BUN analyser to be used for urine urea nitrogen determinations. Satisfactory urine samples could be obtained by diluting the urine with 10 volumes of 0.85% saline. We also report our experience with the use of reagent prepared in house and with a modified pipette, to reduce the cost of using the instrument.