[The dam. Obsolete method, unappreciated method or method of the future].
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Dental Cavity Preparation↗
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[Selection of measuring method--continuous method or equilibrium method (author's transl)].
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Enzymes↗
[Surgical treatment of scoliosis using the Harrington method. i. Comparison with other methods. II. Some complications of the Harrington method].
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Adolescent↗
A critical comparison of the freehand razor-blade dissection method according to Wilson with an in situ sectioning method for rat fetuses.
Two methods of dissecting rat fetuses are compared: the generally well-known freehand razor-blade dissection method according to Wilson and an in situ sectioning method (ISM). For this purpose the substance EGYT 1978 was used, which induces--inter alia--cardiovascular anomalies. Thirty-nine gravid rats were given either the test drug (EGYT 1978, 400 mg/kg body weight) or the vehicle orally from the 6th to the 15th day of gestation. The fetuses were assigned randomly to either dissection group. Both methods of examination revealed approximately the same number of anomalies per organ system. A comparison of the types of anomaly found showed, however, relevant differences in the nature of the changes revealed by the two methods. The in situ sectioning method was clearly superior to the freehand razor-blade dissection method for the detection of discrete anomalies of the cardiovascular system. We therefore recommend the in situ sectioning method as the method of choice for examining small fetuses from teratological experiments.
Animals↗
Comparison between the fluorometric HPLC method and the conventional method for determining urinary delta-aminolevulinic acid and coproporphyrin as indices of lead exposure.
Fluorometric methods for determining delta-aminolevulinic acid (ALA) and coproporphyrin (CP) in urine have been recently developed by using high-performance liquid chromatography (HPLC). In the present study, urinary ALA and CP in lead-exposed workers were determined with these fluorometric HPLC methods and the conventional methods, and the results obtained were compared. In lead workers with a urinary ALA greater than or equal to 5 mg/l, the values obtained with the fluorometric HPLC method corresponded well with those measured with the conventional colorimetric method. In contrast, in lead workers with ALA less than 5 mg/l, ALA values obtained with the fluorometric HPLC method were lower than those measured with the conventional method, suggesting the possibility of matrix interference in urine. The urinary CP values obtained with the conventional method of Rimington (1971) were higher than those measured with the fluorometric HPLC method, though the correlation was good.
Adult↗
Electron microscopical demonstration of horseradish peroxidase by use of tetramethylbenzidine as chromogen and sodium tungstate as stabilizer (TMB-ST method): a tracing method with high sensitivity and well preserved ultrastructural tissue.
Until now methods using tetramethylbenzidine (TMB) for electron microscopy (TMB-EM methods) are all unable to provide a maximum demonstration of transported horseradish peroxidase (HRP) while maintaining good ultrastructural tissue preservation. In order to solve this problem, we have attempted to adapt a newly developed, highly sensitive TMB method using sodium tungstate (ST) as the stabilizer (TMB-ST method) for HRP electron microscopic retrograde and anterograde fiber tracing. The present study shows that the TMB-ST method combined with diaminobenzidine-cobalt (DAB-Co) is more sensitive than existing TMB-EM methods and that ultrastructural details are well preserved with this combined method. The resultant reaction product complex after osmication is stable and is observed as characteristic crystal-like structures which are extremely electron dense and often aggregated into clumps. In contrast, the TMB-ST method without the DAB-Co step frequently produces a moderate electron-dense reaction product. Therefore, we recommend the TMB-ST method combined with DAB-Co for HRP electron microscopy.
3,3'-Diaminobenzidine↗
Drug susceptibility testing in tuberculosis: a comparison of the proportion methods using Lowenstein-Jensen, Middlebrook 7H10 and 7H11 agar media and a radiometric method.
In the present investigation, the conventional drug susceptibility testing procedures using the 1% proportion method on Löwenstein-Jensen (LJ) and Middlebrook 7H10/7H11 agar media were compared with a recent radiometric method. The latter using "Bactec 460-TB" apparatus and a special broth medium (Becton and Dickinson Diagnostic Instrument Systems, Towson, MD, USA) gave drug susceptibility results within a week as compared to 4-5 weeks with the LJ method, and 3-4 weeks using 7H10/7H11 agar media. This report describes the critical concentrations retained for each of the methods and the methodology used. Also, the compatibility of the results obtained with the radiometric method with those obtained with conventional 7H11 agar method was perfect. In this report, we justify the choice of the 7H11 agar method instead of the LJ method as our conventional drug susceptibility procedure, and suggest also that radiometric drug susceptibility determination by Bactec 460-TB apparatus may be the method of choice for routine, rapid drug susceptibility determinations.
Colony Count, Microbial↗
Preparation of human frozen-thawed seminal specimens using the SpermPrep filtration method: improvements over the conventional swim-up method.
OBJECTIVE: To examine the qualitative and quantitative characteristics of frozen-thawed spermatozoa recovered through the SpermPrep (ZBL, Inc., Lexington, KY) filtration method or the swim-up technique for the purpose of intrauterine insemination (IUI) or other techniques for assisted reproduction. DESIGN, SETTING, PATIENTS: Thirty pairs of frozen specimens purchased from three commercial semen suppliers were used in this study. Each pair consisted of two aliquots from the same semen specimen. MAIN OUTCOME MEASURES: Spermatozoa recovered via the SpermPrep filtration and swim-up processes were evaluated for sperm numbers recovered, sperm motility, grade of sperm motility, percentage of morphologically normal spermatozoa, the response of spermatozoa to a hypo-osmotic environment (hypo-osmotic swelling test), and the amount of debris present. RESULTS: Application of the SpermPrep filtration method resulted in recovery of significantly greater numbers of spermatozoa (P less than 0.01) than were recovered with the swim-up method (31.1 +/- 3.2 x 10(6) versus 10.2 +/- 1.8 x 10(6) spermatozoa, respectively). This represents a mean recovery of approximately one half (49%) of all spermatozoa applied to the filter, whereas for the swim-up method, it was only 15%. The overall quality of recovered spermatozoa was virtually identical between the two methods (P greater than 0.05). The percent motile sperm improved by a mean of 18% to 20%, the grade of motility improved by a mean of 0.4 points (scale 0 to 4), the percent of morphologically normal spermatozoa increased by a mean of approximately 10%, the percent of spermatozoa reactive to a hypo-osmotic medium test increased by a mean of approximately 9%, and the debris score decreased by a mean of 0.2 to 0.3 points (scale 0 to 4). Most importantly, the mean total number of motile, morphologically normal spermatozoa after filtration through the SpermPrep column was 20.2 +/- 1.1 x 10(6), representing a mean recovery of 73% of the normal spermatozoa originally applied to the column. This was 316% greater than the yield obtained with the swim-up method (6.4 +/- 0.8 x 10(6)), which was significantly greater (P less than 0.01) than that recovered via the swim-up method. Also, the time required to harvest sperm through SpermPrep filtration was 20 to 25 minutes versus 80 minutes required for the swim-up method (P less than 0.05). CONCLUSION: Considering that the effectiveness of frozen-thawed semen is already limited when compared with fresh semen, SpermPrep filtration is the method of choice over the swim-up technique of sperm selection because the former provides significantly greater numbers of high quality sperm. It should be considered as an adjunct in semen preparation for IUI or other forms of assisted reproduction.
Evaluation Studies as Topic↗
A new scanning method for thallium-201 myocardial SPECT: semidecubital position method.
The authors have developed a new scanning method for Tl-201 myocardial SPECT using a rotating gamma camera with 180-degree data collection. This requires a new type of imaging table on which the patient is placed in a 50-degree right semidecubital position to allow close proximity to the camera-collimator system. Using this method, attenuation by the imaging table is completely avoidable because the projected views in the left oblique directions are not obstructed by the table. In addition, the radius of camera rotation is about 25% shorter than in the conventional method. Phantom and 16 clinical studies were performed to compare the performance of conventional and semidecubital position methods. In phantom studies, tomographic images produced using the new method showed better lesion contrast and more counts in the posteroinferior wall of the heart than those produced using the conventional method. Clinically, 12 of the 16 studies showed more counts in the posteroinferior wall of the heart with the semidecubital position method than with the conventional method. The semidecubital position method is useful for Tl-201 myocardial SPECT, especially for evaluation of posteroinferior wall hypoperfusion.
Gamma Cameras↗
Automated amidolytic method for evaluating the activated partial thromboplastin time compared with a conventional coagulation method.
We report studies of the validity and clinical application of a new amidolytic method for evaluating the activated partial thromboplastin time (APTT) compared with a conventional clotting method. The results with the two methods were well correlated for normal plasma and plasma from hemophilia patients. Congenital deficiencies of of the intrinsic coagulation pathway other than hypo- and dysfibrinogenemia detected by the amidolytic method agreed with those detected by the clotting APTT. The results with the two methods for plasma from patients under heparin treatment were statistically different, suggesting a lesser sensitivity of the amidolytic method to heparinization. The use of the amidolytic APTT reagent in combination with factor VIII and IX deficient plasma allowed the measurement of both factors. The results obtained with normal and hemophilic plasma were in agreement with those obtained with the one-stage clotting method in all except two occasions. Even though the amidolytic method demonstrated the presence of the lupus anticoagulant in the majority of tested samples of known lupus subjects, it was less sensitive to the abnormality than the clotting method.
Adult↗
Candidate reference method for determining creatinine in serum: method development and interlaboratory validation.
We describe a "high-performance" liquid chromatographic (HPLC) method for accurately determining creatinine in serum. After prechromatographic precipitation of protein, we performed isocratic ion-exchange chromatography with ultraviolet detection (234 nm). Analytical results showed linearity up to 1770 mumol/L, a detection limit of 22 mumol/L, an average analytical recovery of 101%, and a CV ranging from 3% to 11%. We used certified human serum (National Institute of Standards and Technology), and additional lyophilized serum pools also assayed by definitive isotope-dilution mass spectrometry, to validate the accuracy of the HPLC method. In addition, the isocratic HPLC results showed close agreement with those obtained with a step-gradient HPLC method. We also compared the isocratic HPLC method with alkaline picrate and enzymatic methods. Our findings with samples from nonuremic, uremic, and diabetic ketoacidotic patients confirmed the positive bias previously reported with the alkaline picrate method. Interlaboratory transferability of the method was demonstrated with various commercial instruments and analytical columns. We evaluated column stability and possible interference from endogenous or exogenous compounds. On the basis of our analytical findings, we recommend the isocratic HPLC method as a candidate Reference Method for determining creatinine in serum.
Chemistry, Clinical↗
Comparison of three liquid chromatographic methods with FDA optimized Monier-Williams method for determination of total sulfite in foods.
Three liquid chromatographic (LC) methods employing amperometric detection were compared with the collaboratively studied FDA optimized Monier-Williams distillation method for the determination of total sulfite in 5 food types. The foods included lemon juice, white wine, instant mashed potatoes, golden raisins, and onion flakes. Two of the LC methods (one employing headspace sampling and the other direct injection) used ion-exchange chromatography with a basic mobile phase (pH about 10.8) and a glassy carbon electrode; the third (employing direct injection) used ion-exclusion chromatography with an acidic mobile phase (pH about 2) and a platinum electrode. All 4 methods produced similar results for the wine, lemon juice, and raisins. Results were different for instant mashed potatoes and onion flakes. The headspace-LC method and direct ion-exclusion LC method, both of which employed an alkaline sample extraction, yielded significantly higher values for sulfite in instant potatoes than did the other 2 methods. A large interfering peak with both direct LC methods prevented quantitation of sulfite in the onion flakes. All methods can detect sulfite as low as about 1 microgram/g in 4 of 5 food types examined.
Chromatography, Gel↗
[Use of quantitative methods in cineangiocardiography: a comparison of 9 methods of motion analysis for the detection of regional function in patients with coronary heart disease].
In order to evaluate the usefulness of various quantitative methods for the assessment of regional left ventricular function, a total of 9 methods (3 hemiaxial models, 1 area model and 5 radial-axes models) were applied to the radiopaque ventriculograms of 16 patients with anterior wall infarction and 13 patients with posterior wall infarction. A series of 17 patients without clinical or angiographic evidence of heart disease were used as control group. While the hemiaxial methods were consistently found to be useful and reproducible, the area method did not provide significant additional information. The diagnostic accuracy was highest with the use of 2 radial-axes methods. The sensitivity and specificity data were 90% to 100% and 70% to 100%, respectively. The remaining 3 radial-axes methods were found to be of limited value since regional wall motion abnormalities were projected to the contralateral wall. Because of this methodological shortcoming these techniques may falsely indicate asynergy in areas with normal contraction patterns. This was reflected by high sensitivity but low specificity values. A feature of the latter 3 methods was the use of the gravity center of the end-systolic frame as the reference point. The position of this reference point is decisively influenced by the extent and localization of asynergy. These findings suggest that methods using the gravity center of the end-systolic frame should not be employed in the evaluation of regional left ventricular function. In general, some quantitative methods for assessing regional left ventricular function require a considerable expense for technical apparatus which is not always justified by the extent to which additional information is gained.
Angiocardiography↗
Simple, rapid cleanup method for analysis of aflatoxins and comparison with various methods.
A method is described for simple and rapid determination of aflatoxins in corn, buckwheat, peanuts, and cheese. Aflatoxins were extracted with chloroform-water and were purified by a Florisil column chromatographic procedure. Column eluates were concentrated and spotted on a high performance thin layer chromatographic (HPTLC) plate, which was then developed in chloroform-acetone (9 + 1) and/or ether-methanol-water (94 + 4.5 + 1.5) or chloroform-isopropanol-acetone (85 + 5 + 10). Each aflatoxin was quantitated by densitometry. The minimum detectable aflatoxin concentrations (micrograms/kg) in various test materials were 0.2, B1; 0.1, B2; 0.2, G1; 0.1, G2; and 0.1, M1. Recoveries of the aflatoxins added to corn, peanut, and cheese samples at 10-30 micrograms/kg were greater than 69% (aflatoxin G2) and averaged 91%, B1; 89%, B2; 91%, G1; 78%, G2; and 92%, M1. The simple method described was compared with the AOAC CB method, AOAC BF method, and AOAC milk and cheese method. These methods were applied to corn, peanut, and cheese composites spiked with known amounts of aflatoxins, and to naturally contaminated buckwheat and cheese. Recoveries were much lower for the BF method compared with our simple method and the CB method.
Aflatoxins↗
A randomized and blinded comparison of three bleeding time techniques: the Ivy method, and the Simplate II method in two directions.
We compared the Ivy bleeding time method and two alternatives of the Simplate II method (incisions in horizontal and vertical direction) with each other, with regard to the sensitivity, the specificity, the costs and the burden for the patient. In the aspirin study an aspirin-induced bleeding defect was used. Seventy-two healthy volunteers were randomized to receive either 500 mg acetylsalicylic acid (ASA) or a placebo. Double blinding was maintained throughout the study. In the anticoagulation study 62 patients participated, who received oral anticoagulants (OAC) for various reasons. All participants received two bleeding time methods. The burden for the participants of each method was screened by a small standard questionnaire. The differences in sensitivity and specificity between the three methods proved minimal. The Ivy method was more often preferred by the participants than the Simplate methods. Since a choice on the basis of sensitivity and specificity appears not possible, we prefer the Ivy method because of lower costs and less burden.
Adult↗
Validation of a new closed circuit indirect calorimetry method compared with the open Douglas bag method.
New equipment designed for the routine measurement of oxygen uptake (VO2) using a closed circuit method has been validated by comparing it with a standard Douglas bag method. The equipment (The Caloric Measurement Unit, CMU) has been tested in 10 critically ill patients during mechanical ventilation (MV) and in 10 spontaneously breathing healthy subjects. Determinations of VO2 and of the resting energy expenditure (REE) were measured in duplicate with the standard method and once with the CMU. Six additional patients receiving MV were studied with the CMU to evaluate the reproducibility and the effect of FIO2 = 1 vs FIO2 = 0.43 on VO2 measurements. Considering the whole group of 10 patients and 10 subjects, the mean difference of VO2 between both methods was -2 +/- 21 ml/min (95% confidence interval, -11.8 to 7.8 ml/min, p = 0.6) standard deviation. Both methods had a similar reproducibility and the mean difference of VO2 measured at the two different FIO2 with the CMU was -3.2 +/- 11 ml/min (95% confidence interval, -14.7 to 8.4 ml/min, p = 0.5). No statistically significant difference was found between derived REE values obtained from either method. These data show a good correlation between the two methods suggesting that CMU may be used in place of the standard method with the same accuracy in measurement of VO2 even at FIO2 = 1.
Adult↗
Newly Developed Structure-Based Methods Do Not Outperform Standard Sequence-Based Methods for Large-Scale Phylogenomics.
Recent developments in protein structure prediction have allowed the use of this previously limited source of information at genome-wide scales. It has been proposed that the use of structural information may offer advantages over sequences in phylogenetic reconstruction, due to their slower rate of evolution and direct correlation to function. Here, we examined how recently developed methods for structure-based homology search and tree reconstruction compare with current state-of-the-art sequence-based methods in reconstructing genome-wide collections of gene phylogenies (i.e. phylomes). While structure-based methods can be useful in specific scenarios, we found that their current performance does not justify using the newly developed structure-based methods as a default choice in large-scale phylogenetic studies. On the one hand, the best performing sequence-based tree reconstruction methods still outperform structure-based methods for this task. On the other hand, structure-based homology detection methods provide larger lists of candidate homologs, as previously reported. However, this comes at the expense of missing hits identified by sequence-based methods, as well as providing sets of homolog candidates with higher fractions of false positives. These insights help to guide the use of structural data in comparative genomics and highlight the need to continue improving structure-based approaches. Our pipeline is fully reproducible and has been implemented in a Snakemake workflow. This will facilitate a continuous assessment of future improvements of structure-based tools in the AlphaFold era.
Phylogeny↗
HLA-DQB1 allele typing by a new PCR-RFLP method: correlation with a PCR-SSO method.
We have developed a new PCR-RFLP method for HLA-DQB1 typing. This method was easy to follow, requiring only one DQB1 generic amplification and 5 endonucleases to assign 14 out of 15 HLA-DQB1 alleles. In addition, we determined that by using one generic amplification and two enzymes (Sau96 I and Hae III) it was possible to type the generic specificities: DQw2, DQw4, DQw5, DQw6, and DQw7, DQw8-9, providing a practical alternative for serological HLA-DQ generic typing. We also performed a side-by-side correlation with a PCR-SSO typing method and found an almost 100% concordance between the methods. The limitations of these methods were: 1) the PCR-RFLP method did not allow the differentiation between the HLA-DQB1*0602 and *0603 alleles; 2) the PCR-SSO method gave crosshybridization signals in the detection of *0302 or *0303 alleles. Our results suggested that both methods, PCR-RFLP and PCR-SSO, are useful alternatives for HLA-DQB1 typing.
Alleles↗