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An assay for fondaparinux (Arixtra) is described based on a modified commercial assay for heparin. The assay is automated on a Dade Behring BCS XP (Siemens Healthcare Diagnostics, Deerfield, IL) and uses the inhibition of activated factor X to quantitate the drug. The assay was unaffected by platelet contamination or the presence of warfarin. The assay was affected by the antithrombin level, and the value obtained in the assay decreased significantly when the antithrombin level was less than 60%. The assay is, however, not specific for fondaparinux. Specimens containing unfractionated or low-molecular-weight heparin will yield results by this assay that will not be an accurate estimation of concentration.

Serum prostate-specific antigen (PSA) assays differ in calibration and response to different PSA forms. We examined intermethod differences in total PSA (tPSA) and free PSA (fPSA) measurements. We tested 157 samples with tPSA concentrations of 2 to 10 ng/mL (2-10 microg/L) using 6 PSA/fPSA method pairs and 1 tPSA method: ADVIA Centaur (complexed and total; Siemens Diagnostics, Tarrytown, NY), ARCHITECT i 2000(SR) (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), IMMULITE 2000 (Siemens Diagnostics), Modular E170 (Roche Diagnostics, Indianapolis, IN), UniCel DxI 800 (Beckman Coulter, Brea, CA), and VITROS ECi (tPSA only; Ortho-Clinical Diagnostics, Raritan, NJ). Regression analysis was performed for PSA, fPSA, and percentage of fPSA with the ARCHITECT i 2000(SR) comparison method. Differences between test and comparison methods were estimated at 2.5, 4.0, and 10.0 ng/mL (2.5, 4.0, and 10.0 microg/L) for tPSA and 15%, 20%, and 25% for percentage of fPSA. Relative differences were more than 10% at 4.0 ng/mL (4.0 microg/L) tPSA for the Centaur, IMMULITE, ECi, and DxI methods. At 20% fPSA, the relative difference was more than 10% for all methods except the AxSYM. Additional harmonization is needed for tPSA and fPSA methods.

Cancer antigen 125 (CA 125) is a high-molecular-mass glycoprotein that is used as a tumor marker to monitor disease progression and response to therapy and in early detection of recurrence after treatment for ovarian cancer. The Access 2 (Beckman Coulter, Brea, CA), ADVIA Centaur (Bayer Diagnostics, Tarrytown, NY), ARCHITECT i2000 (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), Elecsys 2010 (Roche Diagnostics, Indianapolis, IN), IMMULITE 2000 (Diagnostic Products, Los Angeles, CA), and VITROS ECi (Ortho Clinical Diagnostics, Raritan, NJ) assays for CA 125 were evaluated for detection limit, dilution linearity, imprecision, correlation, and reference intervals. The maximum average deviation from target recoveries for dilution linearity studies ranged from 3.7% for the ADVIA Centaur to 18.2% for the IMMULITE 2000. Imprecision studies yielded total coefficients of variation of 2.0% to 8.3% at CA 125 concentrations of 35 and 114 U/mL (35 and 114 kU/L). Method comparison studies revealed good agreement with the VITROS ECi comparison method, with slopes ranging between 0.88 to 1.19 and correlation coefficients of more than 0.95. All methods show acceptable performance characteristics and generally compare well. However, for some samples, substantial differences exist between methods, necessitating parallel testing when introducing a new method.

This is a report of 3 cases of body fluid containing numerous cholesterol crystals that caused falsely elevated cell counts on an automated cell counter. Two of the cases were pleural effusion fluid from patients with long-standing rheumatoid arthritis. Fluid in the third case was from upper extremity cystic lesions of a patient with squamous cell carcinoma of the head and neck. Microscopic examination revealed abundant cholesterol crystals in all fluid samples. In all 3 cases, initially, the automated cell counter reported very elevated WBC and RBC counts that were much higher than those from the manual count. This interference by cholesterol in the automated cell counter is discussed. In addition, possible pathophysiology of cholesterol formation in the body fluid is discussed and chylous and pseudochylous (chyliform) effusions are reviewed. Finally, the use of automated instruments in the evaluation of body fluid is reviewed.

Schistocytes are circulating RBC fragments. The morphologic identification of schistocytes is difficult because the shapes to which they correspond are still under discussion. Automated hematology systems permit the possibility of direct measurement of RBC fragments. We compared schistocyte counts performed by different biologists and technicians with the automated counts by the ADVIA 120 (Bayer Health Care, Tarrytown, NY). The agreement between the ADVIA 120 and the average of the observers gives a correlation coefficient of 0.7274 (95% confidence interval, 0.6285-0.8019). The ADVIA 120 has a tendency to overestimate the count (average, +0.445%). No false-negative case was recorded. The maximum sensitivity (detection of 100% of samples with schistocytes) of the analyzer was determined at a threshold value of 0.25%, but the specificity was low (20%). Therefore, a blood smear examination remains necessary to confirm schistocyte presence. However, the clinical features correlated particularly with negative automated RBC fragments, and the high negative predictive value of RBC fragments ruled out thrombotic events (macroangiopathies or microangiopathies).

Background: Commercially available testosterone immunoassays give divergent results, especially at the low concentrations seen in women. We compared immunoassays and a nonimmunochemical method that could quantify low testosterone concentrations., Methods: We measured serum testosterone in 50 men, 55 women, and 11 children with use of eight nonisotopic immunoassays, two isotopic immunoassays, and isotope-dilution gas chromatography-mass spectrometry (ID/GC-MS)., Results: Compared with ID/GC-MS, 7 of the 10 immunoassays tested overestimated testosterone concentrations in samples from women; mean immunoassay results were 46% above those obtained by ID/GC-MS. The immunoassays underestimated testosterone concentrations in samples from men, giving mean results 12% below those obtained by ID/GC-MS. In women, at concentrations of 0.6-7.2 nmol/L, 3 of the 10 immunoassays gave positive mean differences >2.0 nmol/L (range, -0.7 to 3.3 nmol/L) compared with ID/GC-MS; in men at concentrations of 8.2-58 nmol/L, 3 of the 10 immunoassays tested gave mean differences >4.0 nmol/L (range, -4.8 to 2.6 nmol/L)., Conclusion: None of the immunoassays tested was sufficiently reliable for the investigation of sera from children and women, in whom very low (0.17 nmol/L) and low (<1.7 nmol/L) testosterone concentrations are expected.

We studied the usefulness of an automated screening instrument for processing Papanicolaou (Pap) smears to determine whether it could speed human examination by recording the time to screen 1,007 cervical Pap smears with an AutoPap primary screening instrument with location-guided screening (LGS) software and by conventional microscopic examination. We also assessed the accuracy of the methods to determine preparation adequacy, and we compared the diagnosis by each method. The AutoPap with LGS satisfactorily determined the adequacy of Pap smears and identified the marked abnormal cells for human examination. An accurate diagnosis was possible when only the marked cells were examined, and this method reduced the screening time to less than half that required for conventional screening. With low-grade squamous intraepithelial and more severe lesions as a threshold, there were 37 cases in the conventional group and 29 cases in the LGS group. With atypical squamous cells of undetermined significance as the threshold, there were 111 cases in the LGS group and 93 cases in the conventional group. The AutoPap with LGS can significantly speed the examination of Pap smears without lowering the detection rate of clinically important lesions, thus helping alleviate the cytotechnologist shortage.

Clinical laboratory automation has developed over the past decade as one means of consolidating testing, reducing costs, and improving the effectiveness of laboratory testing. Most of the developments have been aimed at core clinical laboratory operations, and have primarily addressed preanalytical and analytical processing of traditional specimens arriving in blood collection or similar aliquot tubes. Much less attention has been given to specialized applications such as processing specimens for urine toxicology, and only recently have vendors attacked the problems associated with sorting and maintaining the laboratory's inventory of specimens. This report highlights selected developments in these areas, describes one approach to cost-effective custom platform development, and discusses the advantages and pitfalls to solving problems with laboratory automation.

Use of immunoassays and other ligand-binding assays in clinical diagnosis has increased dramatically during the last several years. Despite impressive technical advances, "mass production" of these assays in a routine laboratory still presents many difficulties. This review of ligand-binding assay technology highlights some recent developments, emphasizing challenges and possible solutions for cost-effective patient care.

Cyclosporine is a widely used and potent immunosuppressant drug with a narrow therapeutic index. Therefore, cyclosporine concentrations should be monitored closely. Various automated immunologic methods for cyclosporine whole blood determinations are available. Two new methods, fluorescence polarization immunoassay (FPIA) for the AxSYM by Abbott Laboratories, Chicago, IL, and the cloned enzyme donor immunoassay (CEDIA) by Boehringer Mannheim, Mannheim, Germany, have been introduced. In addition, Dade Behring improved its enzyme multiplied immunoassay (EMIT) assay. The present study evaluated all 3 new methods in comparison with high-performance liquid chromatography (HPLC) and the FPIA for the TDx analyzer. We measured whole blood cyclosporine concentrations of 179 samples obtained from 127 patients after kidney, bone marrow, heart-lung, and liver transplantation. All 4 automated immunologic methods can be used for routine measurement of cyclosporine whole blood concentrations. Disadvantages, such as higher cross-reactivity (Abbott TDx, CEDIA) or a limited linearity range (EMIT), are accompanied by advantages, such as a high precision (Abbott TDx) or an easy sample handling procedure (CEDIA). Information presented in this article should help to find the most adequate cyclosporine method for each medical laboratory.

We evaluated the Sysmex UF-100 urine flow cytometer (TOA Medical Electronics, Kobe, Japan) with 269 uncentrifuged urine specimens by comparing it with Sternheimer staining and particle counting in 1-microL disposable chambers with both brightfield and phase-contrast microscopy (the reference method). Results of routine test strip analysis, sediment microscopy (182 specimens), and bacterial culture (204 specimens) were also available. Detection of urinary WBCs and RBCs was highly reliable with the UF-100 compared with manual chamber counting (r = .98 and .88, respectively). Identification of bacteria was equal to that with visual microscopy of uncentrifuged specimens; sensitivity was 55%, and specificity 90%, compared with bacterial cultures at a cutoff of > 10(3) colony-forming units per milliliter. Renal damage was difficult to evaluate even with manual methods because of the low counts of renal tubular cells and casts; with standard manual Sternheimer-stained sediment analysis, sensitivity was 65% to 69% and specificity 66% to 91%, compared with the uncentrifuged chamber method at a cutoff of 3 and 10 particles per microliter, respectively. Renal damage was demonstrated with the UF-100 with a sensitivity of 26% to 69% and specificity 92% to 94%, compared with chamber counts. Automated urinalysis with the UF-100 urine flow cytometer offers considerable savings in time and labor. When high sensitivity is needed, visual microscopic review should be performed to detect renal disease.

A method is described for automated measurement of serum ceruloplasmin ferroxidase activity. In this method, Fe2+ ions are used as the substrate. In addition, a new calibration system without ceruloplasmin is also presented. Optimum assay reaction conditions were determined. Maximal catalytic activity was obtained at 0.45 mol/L acetate buffer, pH 5.8. The reagents and calibrator are stable for at least 6 months. Significant correlations between serum ferroxidase and p-phenylenediamine oxidase activities (r = 0.96; P <0.0001) and copper concentration (r = 0.93; P <0.0001) were found. The range for serum ceruloplasmin ferroxidase activity in healthy persons was 198-1107 U/L, and in patients with bronchial asthma it was 601-1912 U/L.

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine--cysteine, homocysteine, cysteinylglycine, and glutathione--as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97-107%, with little difference between plasma and urine samples. The detection limit was approximately 50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 micromol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.

A fully automated solid-phase extraction gas chromatographic/mass spectrometric (SPE/GC/MS) method was developed for determination of pesticides in wine. All steps from aspiration of infiltrated wine to printout of the integrated chromatogram were performed without human interaction. A dedicated robot performed addition of internal standard, application of wine onto the SPE cartridge, elution of analytes, drying and concentrating of eluate, and passing of concentrate to the GC sampler. All steps were performed in standard liquid chromatography/GC vials, using a minimum of organic solvent. The method permits determination of 21 different pesticides. Individual detection limits were 0.005-0.01 mg/L. The regression coefficients relating to linearity were > 0.99; only 4,4-dichloro-benzphenone and dicofol showed lower coefficients. The recoveries for 17 pesticides ranged from 80 to 115%.

Thiocyanate found in serum ordinarily is the metabolite of cyanide that is inhaled with tobacco smoke and ingested with cyanogenic foods. We investigated the effect of the thiocyanate ion (SCN-) on the ionized magnesium (iMg) and ionized calcium (iCa) results determined with the AVL and Nova magnesium and calcium ion-selective electrodes (ISEs). We analyzed saline and pooled serum with added SCN-, and serum from apparently healthy nonsmokers (n = 20) and smokers (n = 20). The mean (and range) of the measured serum SCN- concentration was 0.019 (0.008-0.046) mmol/L for nonsmokers and 0.077 (0.020-0.138) mmol/L for smokers. Only the Nova iMg results decreased with increasing SCN- concentration, and the change was dependent on the baseline iMg concentration. In the absence of Mg, SCN- decreased the voltage response of the Nova Mg ISE to calcium ions. At apparently normal serum iMg and iCa concentrations, the interference by SCN- appeared to be equimolar (iMg = -1.04 x SCN- + 0.52). Thus, the serum SCN- commonly found in smokers causes a significant (P < 0.0001) decrease in the Nova iMg results.

The interferences of o-raffinose cross-linked hemoglobin (Hemolink) were examined and compared in two serum creatinine methods on the Hitachi 717 [Boehringer Mannheim (BMC) and Synermed] and in an enzymatic creatinine method on the Vitros 750 (Johnson & Johnson). Interference was considered significant when the change in creatinine concentration from the control exceeded the 95% confidence limits of each method. Significant interference was observed for the BMC/Hitachi 717 method with Hemolink > or = 5 g/L. No interference was observed for the Synermed/Hitachi 717 assay with Hemolink up to 30 g/L in normal samples and up to 50 g/L in samples with increased creatinine. No interference was observed for the Vitros 750 assay with Hemolink up to 50 g/L at both normal and increased concentrations of creatinine. Although the BMC/Hitachi 717 method was considered unacceptable, the Synermed/Hitachi 717 and the Vitros 750 methods allow accurate quantification of serum creatinine in the presence of Hemolink.

Performance characteristics of the Abbott nonpretreatment AxSYM Digoxin II assay were evaluated for quantification of digoxin at four independent sites. Correlation of digoxin measurements with the Abbott pretreatment AxSYM, Baxter Stratus II, Abbott TDx/ TDxFLx II, Abbott IMx, Emit 2000, and Beckman Synchron CX digoxin assays showed acceptable agreement, as indicated by: slope values > 0.84, r > 0.90, y-intercepts for all comparisons at or below the assay detection limit, and Sy/x ranging between 7.5% and 15.4% of the average digoxin value. Susceptibility to interference from digoxin-like immunoreactive factors (DLIFs) was examined in 233 samples from renal patients, liver disease patients, cord blood, and third-trimester pregnancies; the AxSYM Digoxin II assay demonstrated the least DLIFs interference. DLIF susceptibility for four of the methods was significantly greater (P < 0.05) than in the AxSYM Digoxin II assay; susceptibilities of the Stratus II and Emit 2000 methods were similar to the AxSYM Digoxin II assay.

We evaluated a new homogeneous assay for the measurement of HDL-cholesterol (HDL-C) in six European laboratories. The assay includes two reagents and is applicable to most autoanalyzers, which allows full automation. The total CVs of the new method ranged between 1.3% and 6.7%. Thereby determined HDL-C values were in good agreement with those obtained by precipitation with phosphotungstic acid/MgCl2 or by a combination of ultracentrifugation and precipitation (0.956 < r < 0.994). The assay was linear up to at least 1500 mg/L HDL-C. Hemoglobin did not interfere, whereas icteric samples with bilirubin > 100 mg/L showed discrepancies between the homogeneous and the precipitation assay. Lipemia up to total triglyceride concentrations of 8000 mg/L did not interfere with the homogeneous HDL-C assay. The homogeneous HDL-C assay was easy to handle and produced similar results in all laboratories participating in this study. This method will significantly facilitate the screening of individuals at increased risk for cardiovascular disease.

We evaluated three newly introduced systems for automated determinations of hemoglobin (Hb) A1c, which allow the processing of large amounts of samples in a routine clinical laboratory. We compared these methods--the Variant HPLC, the Hi-Auto A1c analyzer system, and the Roche immunoassay--with the Diamat HPLC system. All showed good precision and good concordance with the Diamat HPLC. The reference range for Hb A1c has to be determined by the laboratory for each assay system. Interference study showed no statistically significant influence of anemia, polycythemia, rheumatoid factor, or chronic hemodialysis, although individual Hb A1c values can be influenced by polycythemia (when measured with the Hi-Auto A1c analyzer) and by chronic hemodialysis (when measured with the Variant HPLC). HPLC was not suitable for measuring Hb A1c in the examined cases of hemoglobin variants; assaying fructosamine seems to be better for monitoring these patients.

Constructs consisting of a channel, a membrane, and an absorber are designed for autonomously carrying out various liquid-handling functions of analytical tests. These so-called fluid elements can be used to set up various circuits for conducting several kinds of analytical tests. To demonstrate the feasibility of this concept, we constructed such a circuit and used it to perform, with two handling steps, an ELISA of hepatitis B surface antigen. The detection limit of the assay was comparable with those of state-of-the-art ELISAs for screening blood, and results could be obtained within a total test time of 20 min. We anticipate that this concept of automation may also serve as a basis for new, highly simplified immunoanalyzers.

We performed an immunoassay evaluation for various analytes on a fully automated random-access analyzer, the Technicon Immuno 1 system from Bayer Corp. This system involves latex agglutination, magnetic separation sandwich, and magnetic separation competitive immunoassay configurations. The evaluated analytes were thyrotropin (TSH), triiodothyronine, thyroxine, free thyroxine, follitropin, lutropin, prolactin, beta subunit of human chorionic gonadotropin, cortisol, ferritin, alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen. We tested the assay precision, linearity, and correlation with comparison methods for these analytes. The functional sensitivity of the TSH assay and the sample-to-sample carryover were also studied. Excellent results were obtained for within-run and between-day precision studies, with most assays showing within-run CVs <4% and between-day CVs <6%. The linearity for all assays was acceptable and the correlation between Immuno 1 assays and comparison methods showed satisfactory results. The functional sensitivity of the TSH assay was estimated at 0.04 mU/L. No sample-to-sample carryover was detected.

Achieving high quality and high productivity with automated testing processes will require process control systems that are optimized for the necessary error detection, minimum false rejection, and maximum run length. This study investigates whether run length could be monitored by average of normals (AON) algorithms that truncate the patient test distribution and estimate the average of a suitable number of patient results. The design of AON algorithms for individual analytes is facilitated by computer-simulated power curves that consider the ratio of the population biological variation (Spop) to the test method variation (Smeas), represent a range of Spop/Smeas ratios from 2 to 15, and include numbers of patient test results from 10 to 600. The potential applications of AON algorithms are assessed for 38 tests whose quality requirements represent the total error criteria from the Ontario Medical Association Laboratory Proficiency Testing Program, Spop/Smeas ratios from 0 to 32, critical systematic shifts from 0.02 to 10.85 Smeas, and test workloads representative of a regional reference laboratory. Approximately half of these tests provide high potential for applying AON algorithms to monitor run length.

This study aimed to simplify the spectrophotometric fluorescein method for measuring plasma bromide, improve its reproducibility, and automate it. After major modifications of the method, we obtained an essentially linear calibration curve for plasma concentrations of bromide between 0 and 5.0 mmol/L. The intraassay CV for measuring bromide in the supernatants of deproteinized plasma samples (initial plasma concentrations 2.5-5.0 mmol/L) was as low as 0.5-1.0% (n = 9). When all the procedures were incorporated, including deproteinization and dilution of plasma, the intraassay CV was 2% at 2.5 mmol/L (n = 5) and 1% at 5.0 mmol/L (n = 5). The interassay CV for measuring bromide in plasma supernatants (initial plasma bromide concentration > 2.5 mmol/L) was 1%. Analytical recovery of bromide added to plasma was 99.1% +/- 2.5%. The new method is simpler and more reproducible than other spectrophotometric methods. Conditions for its automation are described.

The catalytic concentration of pleural adenosine deaminase (ADA) and the ratio of pleural lysozyme (PL) to serum lysozyme (SL) were measured in consecutive patients (49 tuberculous and 179 nontuberculous) with two automated procedures in a Hitachi 717 analyzer. Using sensitivity and specificity curves, we established cutoff values at 33 U/L for ADA and 1.7 for the PL/SL ratio. The sensitivity of ADA activities for tuberculous effusion was 90%, specificity 85%. Combining ADA with the PL/SL ratio enhanced specificity to 99%. However, high values for ADA and lysozyme ratios are not, alone or in combination, sensitive or specific enough to replace pleural biopsy or culture of pleural fluid for the diagnosis of tuberculous empyema.

We describe an automated enzymatic procedure for quantifying creatine in erythrocytes. In this assay, after sample clean-up, creatine kinase (CK; EC 2.7.3.2) and pyruvate kinase (EC 2.1.7.40) are used as auxiliary enzymes; lactate dehydrogenase (EC 1.1.1.27) is used in the indicator reaction. CK is also used as the starting reagent. Data obtained with the present method (y) correlated as follows with those from the comparison method (x) for creatine measurement in erythrocytes (colorimetric, diacetyl-alpha-naphthol): y = 1.017x - 27.5 (S(y/x) = 11.9, r = 0.997). Precision data were obtained by analyzing three different samples of erythrocytes nine times per day for 25 days with both methods. The total CVs determined by our method and the comparison method were respectively 3.0-4.8% (measured creatine range, 171-463 micromol/L) and 5.1-7.1% (creatine, 185-485 micromol/L). We established the reference interval (mean +/- 2SD) for creatine in erythrocytes from healthy individuals as 194-538 micromol/L for men (n = 71) and 241-677 micromol/L for women (n = 100). Using erythrocytes from patients with various hematological abnormalities and chronic renal failure, who were being treated with continuous ambulatory peritoneal dialysis or hemodialysis, we performed a clinical evaluation and obtained results in agreement with existing data on biochemical markers for the mean age of erythrocytes.

A cold vapor atomic absorption technique for blood or urine mercury analysis that uses persulfate oxidation to prepare samples for total mercury analysis and acid permanganate oxidation to prepare samples for inorganic mercury analysis is described. The linearity of the procedures ranged from 0.5 to 25 micrograms/L. Precision ranged from 20% at 1 microgram/L to 7% at 20 micrograms/L. Documentation of accuracy is based on analysis of samples prepared by an international proficiency survey program. The development of a two-step digestion procedure followed by automated flow-injection mercury analysis was a necessary precursor to the assessment of inorganic and alkylmercury exposure in a large unexposed human population. Application of this technique to 902 blood and 902 urine samples collected from a normal human population who had no extraordinary mercury exposure generated mean plus two standard deviation skewed confidence-limit ranges of results as follows: blood total mercury, 0-8.4 micrograms/L; blood inorganic mercury, 0-1.7 micrograms/L; blood organic mercury, 0-7.5 micrograms/L; urine total mercury, 0-9.9 micrograms/L; urine inorganic mercury, 0-8.6 micrograms/L; and urine organic mercury, 0-1.8 micrograms/L.