[Current issues in measurement and reporting of urinary albumin excretion]. / Données actuelles sur le dosage de l'excrétion urinaire de l'albumine.

Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the erro-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences has been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no referance materials for either analyte in urine. The recommanded reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethicity) nor the continuous increase in risk related to albumin excretion. Clinical needs have been identified for standardization of (a) urine collection methodes, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

Peak selection from MALDI-TOF mass spectra using ant colony optimization.

MOTIVATION: Due to the large number of peaks in mass spectra of low-molecular-weight (LMW) enriched sera, a systematic method is needed to select a parsimonious set of peaks to facilitate biomarker identification. We present computational methods for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectral data preprocessing and peak selection. In particular, we propose a novel method that combines ant colony optimization (ACO) with support vector machines (SVM) to select a small set of useful peaks. RESULTS: The proposed hybrid ACO-SVM algorithm selected a panel of eight peaks out of 228 candidate peaks from MALDI-TOF spectra of LMW enriched sera. An SVM classifier built with these peaks achieved 94% sensitivity and 100% specificity in distinguishing hepatocellular carcinoma from cirrhosis in a blind validation set of 69 samples. Area under the receiver operating characteristic (ROC) curve was 0.996. The classification capability of these peaks is compared with those selected by the SVM-recursive feature elimination method. AVAILABILITY: Supplementary material and MATLAB scripts to implement the methods described in this article are available at http://microarray.georgetown.edu/web/files/bioinf.htm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effect of substrate size on immunoinhibition of amylase activity.

Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.

Macromolecular chromogenic substrates for measuring proteinase activity.

BACKGROUND: Proteinase activities are often measured using chromogenic substrates that are much smaller than physiological substrates. METHODS: The hydrodynamic size of macromolecular substrates (macrosubstrates) prepared by linking small chromogenic substrates to polyethylene glycol was determined by gel filtration. Efficiency of macrosubstrate cleavage by proteinases and alpha(2)-macroglobulin-proteinase complexes was monitored spectrophotometrically. RESULTS: Macrosubstrates had hydrodynamic radii of approximately 20 A, similar to proteins with a molecular weight of 18,000. Different macrosubstrates served as efficient substrates for chymotrypsin, trypsin, and thrombin. Linking small substrates to a polymer variably affected substrate efficiency, with the impact on activity ranging from a 60-fold decrease to a 30-fold increase. Proteinases complexed with alpha(2)-macroglobulin had approximately 10-fold lower activity vs macrosubstrates than small substrates. CONCLUSIONS: Macrosubstrates are efficient substrates that allow decreased measurement of sterically hindered proteinase molecules such as alpha(2)-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.

Detection of indinavir crystals in urine: dependence on method of analysis.

OBJECTIVES: To determine the frequency of crystalluria in patients treated with the human immunodeficiency virus protease inhibitor indinavir and to compare methods of detecting crystalluria. METHODS: A total of 308 freshly voided urine specimens from 168 patients treated with indinavir were evaluated by manual microscopy of sediment and microscopy with an automated workstation and by dipstick analysis. RESULTS: Crystals were detected in 22%, 31%, or 32% of specimens using, respectively, an automated workstation, manual microscopy, or both methods. Proteinuria or hemoglobinuria occurred significantly more often in specimens with (28%) than without (18%) crystals. Frequency of crystalluria was unrelated to specific gravity, but it increased at higher pH. Crystals were detected in 21% of specimens with pH less than 6 and 42% of specimens with pH of 6 or higher. CONCLUSIONS: Crystalluria occurs in more than 30% of urine specimens from patients treated with indinavir, but detection rates vary substantially with method of analysis. Manual microscopy detected crystalluria 41% more often than did an automated workstation.

Interferences with semen detection by an immunoassay for a seminal vesicle-specific antigen.

A commercial enzyme-linked immunosorbent assay (ELISA), the SEMA assay, for a seminal vesicle-specific antigen (SVSA) provides highly sensitive detection of semen. Here we show marked interference of proteins such as albumin, serum proteins, or mucin with the assay. This would substantially decrease the sensitivity for detecting semen mixed with other biological fluids such as blood or vaginal secretions.

Detection of azide in forensic samples by capillary electrophoresis.

Azide salts are highly toxic compounds that have been difficult to detect in forensic samples. Here, anion analysis by capillary electrophoresis with indirect spectrophotometric detection was applied to detect azide in forensic specimens from two suicide victims. Gastric specimens from the victims were shown to have high azide concentrations; azide represented one of the major anionic components and no corresponding component occurred in normal gastric juice. Samples of blood and bile had low concentrations of azide near the limits of detection. The method described for azide analysis used simple steps for sample preparation and analysis time was less than 10 min per sample. It offers a simple and reliable method for detecting azide in biological fluids.

Progress toward preparation of universal donor red cells.

Coupling of an inert polymer to the surface of red cells was examined as a potential means of covering blood group antigens and producing cells that could serve as universal donor cells for transfusion. Effective blockade of red cell antigens was achieved with N-hydroxysuccinimide-activated esters of polyethylene glycol. It was possible to block all antigens tested, but lower concentrations of reactants were required to block peptide-defined antigens than carbohydrate-defined antigens. Red cells remained intact after modification but were significantly damaged. Our results demonstrate the feasibility of antigenic blockade of red cells, but there is a need to reduce damage during coupling reactions to produce viable red cells.

Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers.

Compact analyzers suited to near-patient testing estimate hematocrit by measuring the conductivity of undiluted blood. We evaluated the accuracy of hematocrit determination of one such analyzer (Instrumentation Laboratory BGE analyzer) against an automated cell counter (EPC) and packed cell volume (PCV) microhematocrit. When specimens (n = 34) from outpatient and ward patients were analyzed with all three methods, the BGE analyzer correlated well with both EPC and PCV hematocrit determinations (BGE = 1.00 PCV + 0.3%, S(y)/x = 2.0%), suggesting that all three methods are similar in performance for most patients. However, a patient with increased plasma osmolality showed significant decreases in BGE and PCV hematocrits relative to the EPC method. The differences in hematocrit measurements could be reproduced by adding solutes to blood in vitro or by modifying the plasma osmolality of rats in vivo. Samples from patients undergoing cardiac surgery, whose blood had large changes in protein concentration, showed discrepancies between hematocrits by conductivity and other methods; similar effects could be produced by changes in protein concentration or in vitro addition of polyethylene glycol. We conclude that conductivity measurements provide accurate hematocrit results for physiologically normal subjects but not for some intensive-care and surgical patients.