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Biomedical subjects

R Daigneault

Publications and source records attributed to R Daigneault.

At least 19 recordsLinked to original sources
PubMed1991

High-resolution two-dimensional protein electrophoresis of pathological plasma/serum.

The potential usefulness of an optimalized high-resolution two-dimensional gel electrophoresis (2-DGE) protocol was studied by comparative analysis of plasma/serum obtained from apparently healthy individuals and from patients with a few selected known diseases. Despite their apparent complexity, patient electrophoretograms revealed readily detectable modifications of the 'reference' protein profile for those selected diseases (listed below). Abnormal profiles were characterized by presence or absence of particular spots, by reduction or enlargement of spot size, or by alterations of spot microheterogeneity. Combinations of several modifications enabled different 'disease-associated spot pattern' to be distinguished on the protein maps of patients with: monoclonal gammopathies, hypogammaglobulinemia, hepatic failure, chronic renal failure and hemolytic anemia. This study demonstrates that identification of plasma/serum protein alterations by 2-DGE allows a few selected diseases to be diagnosed solely on the basis of protein map modifications.

Agammaglobulinemia↗
PubMed1986

Evaluation of the Sysmex CC-800. An automated eight-parameter hematology instrument.

An evaluation of the Sysmex CC-800 hematology analyzer (TOA Medical Electronics, Kobe, Japan, and distributed by American Scientific Products, Chicago, IL) was performed at University Hospital at Boston University Medical Center to assess the analytic performance and ease of use of the instrument. The Sysmex CC-800 is the first self-contained, fully automated, eight-parameter hematology analyzer. It can handle as many as 100 whole blood specimens without constant operator intervention. Stat and predilute modes are also available. The optional PDA-410 (particle distribution analyzer) was not evaluated in this study. Precision, linearity, carryover, and reproducibility of values over time in the automode were well within the manufacturer's specifications. The correlation study was performed with the existing Coulter S-Plus (Coulter Electronics, Hialeah, FL). The Sysmex CC-800 allowed us to expand the linear range without dilution for white blood cells to 160 X 10(3)/microL, hemoglobin to 26 g/dL, and platelets to 2,000 X 10(3)/microL. The samples on the automode exposed to normal laboratory atmosphere for up to 120 minutes showed no significant difference from baseline. The two instruments correlated well (r greater than 0.99). The authors concluded that the Sysmex CC-800 would be a reliable and time saving instrument in their laboratory.

Automation↗
PubMed1981

Limitations of conventional laser nephelometry for the measurement of beta 2-microglobulin, lysozyme, alpha 1 fetoprotein and myoglobin in serum and urine.

We have tested some assay procedures for the measurement of beta 2-microglobulin, lysozyme, alpha 1-fetoprotein and myoglobin in serum and/or urine with the use of a manual Behring laser nephelometer. The assay working ranges were: beta 2-microglobulin: 0.0038-0.038 g/L; lysozyme: 0.005-0.325 g/L. We have studied the effect of different antiserum dilution ratios and of different concentrations of polyethylene glycol 6000 on the calibration curves. The best standard curves were obtained with the use of the following antiserum dilutions: anti-beta 2-microglobulin: 1:3 with saline, 40 g/L PEG; anti-lysozyme: 1:5 with saline, 40 g/L PEG; anti alpha 1-fetoprotein: concentrated; anti-myoglobin: concentrated with added 40 g/L PEG. In the case of beta 2-microglobulin and lysozyme, laser nephelometry, could be a fast and simple procedure if a 10 times increase in sensitivity can be achieved. For the measurement of alpha 1-fetoprotein and myoglobin, the sensitivity of laser nephelometry was disappointing when compared to those reported for radioimmunoassay and enzyme immunoassay.

Beta-Globulins↗
PubMed1981

Basic characteristics and evaluation of a partially automated Behring laser nephelometer for the measurement of IgG, IgA, IgM and C3c in serum.

We have evaluated a partially automated Behring laser nephelometer for the measurement of IgG, IgA, IgM and C3c in serum. The system consisted of a manual Behring laser nephelometer, an automatic cuvette carrier and a Hewlett-Packard 9815 A calculator/printer. The system could process 240 preincubated samples per h when the interval between each voltage reading was set at 15 s. Day-to-day precision was near 6%. We obtained the worst precision for the determination of IgG which requires the smallest volume of diluted sample (10 microliters). The Frigen treatment used to clarify turbid sera seems to decrease IgG and increase C3c concentrations. The addition of polyethylene glycol 6000 at a concentrations of 40 micro/L in the reaction mixture did not improve the assay ranges. Comparison studies with radial immunodiffusion for the four proteins and with the IgM - BMC Immunological Turbidity Test using either least-squares or Deming's regressions gave very good correlation figures, except for C3c and for some IgM paraproteins. We could decrease the cost per test by re-using the plastic cuvettes. The utilization of the calculator-printer greatly simplified data handling but the automatic carrier was not considered a real asset without complete automation.

Autoanalysis↗
PubMed1980

A study of the enzymatic inactivation of chloramphenicol by highly purified chloramphenicol acetyltransferase.

We report the purification of chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) by a two-step procecdure involving chromatography on a Sepharose 4B-reduced chloramphenicol matrix and DEAE-Sephadex A-50. This procedure resulted in a 120-fold purification with 50% recovery of the enzyme. Only one band of enzyme activity was present after electrophoresis on polyacrylamide gel. The enzyme is active over a broad pH range, maximal activity being observed near pH 7.6. Both chloramphenicol 1-acetate of chloramphenicol 3-acetate were found to be very stable in Tris-maleate buffer at pH 6.09 with negligible interconversion. The incubation at pH 6.0 of chloramphenicol 1-acetate with the purified chloramphenicol acetyltransferase yielded chloramphenicol 1,3-diacetate. These data indicate that the enzyme acetylates specifically at the 3-hydroxy position and the diacetylation is possible only because of non-enzymatic interconversion of chloramphenicol 3-acetate to chloramphenicol 1-acetate at higher pH values.

Acetylation↗
PubMed1980

In search of optimum conditions for the measurement of creatine kinase activity: a critical review of nineteen formulations.

We compare the reagent composition recommended by six different groups including three European societies for the determination of creatine kinase activity in serum using the coupled hexokinase/glucose-6-phosphate dehydrogenase (EC 2.7.1.1/1.1.1.49) reactions. Even though discrepancies exist between these methods, there are, nevertheless, major areas of consensus which permit a reasonable extrapolation of an approximate composition for optimum response. We then ascertain how reagents used in thirteen commercial kits differ from these approximated optimum conditions. Except for four companies, all the reagent compositions differ remarkably from the conditions recommended by the six groups.

Adenosine Diphosphate↗
PubMed1979

Pitfalls to avoid when comparing CK-MB fractionation by antibody inhibition reaction and DEAE-Sephadex column chromatography.

1. We compare the Roche ion-exchange column chromatography and the Merck antibody inhibition reaction for CK-MB fractionation in 51 sera. Measurements of total CK and CK-MB activities must be done under the same conditions for each method in order to correlate the results. 2. A decisional value must be used for the interpretation of CK-MB results. We have used 10% for the inhibition assay and 3% for the chromatography procedure. The use of a percentage should be preferred to use of CK-MB activity alone. 3. When the % of CK-MB was established for the 51 patients only 4 results disagreed between the two methods. Three of these could be explained by a lack of sensitivity of the column chromatography procedure. 4. The antibody assay produces reliable results. Since CK-BB and CK-MB are simultaneously measured, the method is therefore prone to interference by CK-BB when present in serum. The assay is greatly affected by the presence of adenylate kinase in serum. It is not necessary to run a serum blank with this procedure when the serum in pre-incubated for 7 minutes with the reagents. 5. The Roche method also produces reliable results but offers less sensitivity when total CK remains in the normal range. The procedure is much less affected by the presence of adenylate kinase.

Chromatography, Ion Exchange↗
PubMed1979

Aminoglycoside antibiotic measurement by bioluminescence, with use of plasmid-coded enzymes.

We describe a bioluminescent assay for gentamicin in serum that is applicable to the measurement of other aminoglycosides as well. The assay is based on the measurement of residual ATP with the luciferase reaction after incubation of the antibiotic with a plasmid-coded enzyme. Two aminoglycoside-inactivating enzymes were used: an adenylytransferase and an acetyltransferase coupled to S-acetyl coenzymeA synthetase. We investigated the latter system further because of the good stability of the acetyltransferase, its recovery in high yield from bacteria, and its more favorable ATP/gentamicin mass ratio. Serum ATPases were inactivated at 60 degrees C for 20 min. The operating range of the assay was 0-15 mg of gentamicin per liter. The precision (CV) was 10.1% at a concentration of 2 mg/L and 1.1% at 10 mg/L. The method correlated well with a radio-enzymatic assay for mock unknown sera (r = 0.981). The results were available within 2 h.

Acetate-CoA Ligase↗
PubMed1978

Evaluation of a Behring laser-nephelometer prototype in the measurement of IgG, IgA and IgM.

A helium-neon laser nephelometer prototype manufactured by Hoechst Behring Institut was evaluated and used in our laboratory during a two-month period for the measurement of IgG, IgA and IgM. Two methods of dilution were usedto prepare standard curves. The best results were obtained when the patient serum was diluted one hundred fold (10-100 microliter) and incubated for 15 min at room temperature in a disposable plastic cuvette with the specific antiserum diluted five fold (100-300 microliter). The intensity of the light scattered by the antigen-antibody complexes was then immediately read on the digital voltmeter. The working ranges were 186-5944 mg/dl for IgG, 33-1066 mg/dl for IgA and 17-545 mg/dl for IgM. Standard curves obtained for the three immunoglobulins on six different days could be superimposed perfectly demonstrating an excellen reproducibility. The coefficient of variation for IgG (n = 8) was 4.1% at 5800 mg/dl. The results (n = 37) produced by the laser nephelometer (y) correlated very well with those obtained by radial immunodiffusion (x) for: IgG y = 8.07 + 1.00 X, r = 0.997, IgA y = - 6.51 + 1.05 X, r = 0.996, IgM y = 3.35 + 0.98 X, r = 0.998.

Evaluation Studies as Topic↗
PubMed1977

Gentamicin pharmacokinetics during hemodialysis in patients suffering from chronic renal failure.

Because the elimination of gentamicin, a potent aminoglycoside antibiotic, is dependent almost entirely on renal excretion, renal functional impairment drastically changes the pharmacokinetics of this drug. As a first step in the study of the effects of renal insufficiency and the anephric state on the pharmacokinetic parameters of gentamicin, serum and urine levels of this drug were studied after a single intravenous bolus dose during hemodialysis in patients suffering from chronic renal failure. The data were fitted to the two-compartment open model and the appropriate kinetic parameters were calculated with the COMPT computer program modified by Pfeffer. The rate constant of metabolism was estimated from plasma and dialysis rate constants of elimination. The rate of renal excretion was shown to be very weak in patients who were not anuric. The use of the mathematical equations of the two-compartment open model demonstrated that, after a single dose of gentamicin, the percentage of decrease of serum concentration with time does not represent, because of tissue binding retention, the percentage of drug eliminated from the body. It was shown that pharmacokinetic parameters represent a useful tool in the optimization of gentamicin therapy.

Adult↗
PubMed1976

An enzymatic assay for chloramphenicol with partially purified chloramphenicol acetyltransferase.

In an enzymatic assay for chloramphenicol, chloramphenicol acetyltransferase partially purified by affinity chromatography was used; [3H]acetyl coenzyme A served as a substrate. The purified enzyme was sensitive to p-hydroxymercuribenzoate but insensitive to 5,5'-dithio-bis-2-nitrobenzoic acid. The Michaelis-Menten constant was 10.5 muM. The operating range of the enzymatic assay was 0-30 mug/ml. The coefficient of variation was 1.9% at a concentration of 10 mug/ml. The method correlated well with a microbiological agar-paper disk method for mock unknown sera (r = 0.997) and serum or urine specimens (r = 0.993). The enzymatic assay was unaffected by the presence of any of 12 other antibiotics in tested serum.

Acetyltransferases↗
PubMed1974

[Alpha-1 antitrypsin deficiency: correlation between biochemical tests and phenotypes in one family].

Phenotypes (Pi) for serum alpha-1 antitrypsin (AAT) were determined in a family of 19 members spanning three generations and presenting a deficiency in this protein. The standard Fagerhol crossed immunoelectrophoresis procedure was used for this purpose. The individual phenotypes consisted of seven MM, three ZZ, five MZ, one SZ and three MS. AAT levels were obtained by radial immunodiffusion, trypsininhibitory capacity measurements and from cellulose acetate electrophoresis. Good correlation was found between the phenotype and the three biochemical methods for demonstrating the normal phenotype MM and the severe deficiency state ZZ. Some discrepancies were observed for the heterozygotes. It is concluded that these various assays are inaccurate for the interpretation of intermediate AAT concentrations. Since Pi typing is not suitable for use on a routine basis, it is suggested that this analysis should be performed by a specialized laboratory if intermediate deficiency is suspected.

Adolescent↗