An evaluation of the British Pharmacopoeial assay of heparin: a comparison with other methods.

The potencies of six commercially manufactured heparins have been measured by the British Pharmacopoeial (BP) assay and activated partial thromboplastin time (APTT), protamine sulphate, and anti-Xa assays. The APTT/BP potency ratios were found to vary with the preparation but this was not dependent on the tissue source of heparin. For mucosal heparins, the anti-Xa/BP potency ratios were close to unity, but for heparin of lung origin the anti-Xa potency was approximately one-quarter of the BP potency. Four heparin fractions prepared by column gel chromatography of a commercial heparin were similarly examined by all four assays, and there was a wide divergence between the BP potency estimates and those obtained with the other methods. The degree of divergence was found to depend on the molecular size of the fraction.     

Two rapid methods for determination of serum theophylline concentrations: a comparison

Two methods for determining serum theophylline concentrations were compared against a standard laboratory assay. Each method was evaluated for ease of use, accuracy, and precision. Although both methods were found to be easy to use, there were major differences between the assays in both accuracy and precision.     

A sensitive fluorimetric assay for serum angiotensin-converting enzyme.

A simple, rapid, highly sensitive and reproducible assay for angiotensin-converting enzyme in untreated serum is described. It is based on the conversion of the substrate analog, hippuryl-L-histidyl-L-leucine (5 mM in 0.1 M K phosphate, pH 8.3-0.3 M NaCl) to hippurate and L-histidyl-L-leucine, which is quantified spectrofluorimetrically (lamdba excitation = 360 nm; lamdba fluorescence = 500 nm) by formation of a fluorescent adduct with ophthaldialdehyde. The chloride requirement and inhibition and activation patterns correspond to those for angiotensin-converting enzyme. The Km for hippuryl-L-histidyl-L-leucine was 1.33 mM. The mean value of serum angiotensin-converting enzyme for 58 normal human subjects (mean age, 32 years; range 19-57) was 32.2 +/- 1.30 (SE), with a standard deviation of 9.87 nmol/min/ml serum. The assay is useful for the diagnosis and possible management of sarcoidosis and may have other applications in the future.     

Sensitive fluorimetric assay for serum angiotensin-converting enzyme with the natural substrate angiotensin I.

A simple, rapid, highly sensitive and reproducible fluorimetric assay for angiotensin-converting enzyme in untreated serum using the natural substrate based on a similar assay with hippuryl-L-histidine-L-leucine is described. Angiotensin I (0.2 mM in 0.1 M potassium phosphate-30 mM NaCl, pH 7.5; 37 C) is converted to angiotensin II and L-histidyl-L-leucine, which is quantified fluorimetrically (excitation = 360 nm; fluorescence, 500 nm) by formation of a fluorescent adduct with O-phthaldialdehyde. L-Histidyl-L-leucine peptidase was also monitored in order to correct for significant activity, which was observed only once, in less than 1% sera. The mean value of serum angiotensin-converting enzyme in sera from 45 normal subjects was 4.69+/-0.194 (SEM)+/-1.30 (SD) nmol/min/ml serum, compared with 31.7+/-1.53 (SEM)+/-10.3(SD) with the substrate analog hippuryl-L-histidine-L-leucine. There was a high degree of correlation between the velocity of cleavage of angiotensin I and hippuryl-L-histidyl-L-leucine (r - 0.903 to 0.993). The assay of serum angiotensin-converting enzyme is of use in the diagnosis and possible management of sarcoidosis and Gaucher's disease, and may have other applications.     

Improved auxanographic method for yeast assimilations: a comparison with other approaches.

An improved pour-plate auxanographic method has been developed for determining the assimilation of 14 different carbohydrates by medically important yeasts. Reduction of a dye incorporated into the agar has been correlated with the growth and carbohydrate assimilation of the yeasts, allowing the speciation of many yeasts within 24 to 48 h. This technique has been found to compare more than favorably with existing yeast assimilation techniques, in terms of rapid identification, total cost, and technician time in preparing and inoculating the plates. The dye pour-plate auxanographic technique provides an easier-to-interpret, rapid, and reproducible method of mycological identification for small clinical laboratories.     

Comparative analysis of two rapid automated methods and a semiautomated version of the urease method for determining aminoglycoside concentrations in serum.

The Syva EMIT Autolab 6000 and the Abbott TDX automated methods for measuring serum aminoglycoside concentrations were compared with each other and with a new semiautomated version of the urease technique. Each was evaluated for accuracy, reproducibility, cost, and ease of use. All three methods gave satisfactory results, although the Abbott TDX was the most accurate. Both automated methods gave results within minutes, whereas the new urease method, which involved more initial manipulation, took 1.25 h to complete (mainly incubation time). There was no difference in accuracy of performance between experienced and inexperienced operators. The new urease method was much cheaper in terms of initial capital expenditure and in cost per test and needed no provision of manufacturer's back-up maintenance services. This could prove invaluable in situations where financial resources are at a premium. Otherwise, the Abbott TDX is currently our method of choice, and reasons for its preference to the EMIT system are listed.     

A new method of allergen standardization. Passive cutaneous anaphylaxis inhibition in a mouse-to-rat system in comparison with other methods.

Various methods have been employed for standardization of potency of allergen extracts. We used passive cutaneous anaphylaxis (PCA) inhibition in a mouse-to-rat system as a new means of standardization. Several allergen extracts, including mite, short ragweed, house dust, Aspergillus, Candida, and Japanese cedar, were examined. Mouse IgE antibodies were produced after two or three injections of antigen and alum. The potency measured by PCA inhibition in the mouse-to-rat system was compared with that of protein nitrogen (PN) content, end point of prick test in humans, and 50% radioallergosorbent test (RAST) inhibition. Our studies suggest that the mouse-to-rat system could be used to determine allergenic potency and to standardize allergen extracts.     

Evaluation of dot enzyme-linked immunosorbent assay for mucocutaneous leishmaniasis and comparison with microplate enzyme immunoassay.

A dot enzyme-linked immunosorbent assay (dot ELISA) was evaluated and compared with a standard microplate ELISA (immunoglobulin G [IgG] ELISA) for the serological diagnosis of mucocutaneous leishmaniasis. The two assays were used to test 113 serum specimens from the following groups: normal individuals and patients with deep mycoses, toxoplasmosis, mucocutaneous leishmaniasis, visceral leishmaniasis, Chagas' disease, malaria, and schistosomiasis. Both tests exhibited cross-reactivity when testing specimens from cases of visceral leishmaniasis and Chagas' disease. The dot ELISA proved to be economical with respect to use of reagents and was easy to perform. Interpretation could easily be made by visual inspection of reaction endpoints in the nitrocellulose disks, obviating the need for spectrophotometric readings. There were no significant differences in sensitivity between the dot ELISA and the IgG ELISA at a cutoff level either of 20 or 40. However, its most remarkable feature was the high specificity compared with that of the IgG ELISA. Because of its ease of performance and high sensitivity and specificity, the dot ELISA should be an excellent test to be executed in the field during seroepidemiological surveys.     

Use of an improved E. coli method for the measurement of cobalamin in serum: comparison with the E. gracilis assay results

Owing to the higher serum cobalamin results that are obtained by R-binder radioisotopic dilution assay compared to microbiological assays (E. gracilis and L. leichmannii) it was suggested that serum contained a cobamide(s) that could not be detected by the more specific microbiological assays and that a much less specific test organism, which responds to most naturally occurring cobamides, such as the cobamide-dependent E. coli mutant, might respond to these cobamide(s) in serum. In an attempt to investigate this possibility an improved and simplified E. coli assay for the measurement of cobamide in serum was developed. The method is described, and the results obtained in normal subjects, in patients with megaloblastic anemia, and in anaemic pregnant women not suffering from megaloblastic anaemia are reported and compared with E. gracilis assay results.     

Resistotyping of Proteus mirabilis and a comparison with other methods of typing

Resistotyping of P. mirabilis using 10 compounds is reported. The method was tested for reproducibility and specificity and results were compared with those obtained by serological, bacteriophage, and proticine typing methods and the Dienes test. The possible relationship between resistance to the chemicals used in the test and antibiotics was also studied.The method was found to be simple, reproducible, show good specificity, and compare favourably with other typing methods. No linkage between resistance to the chemicals and antibiotic resistance was detected.     

Assessment of immune response to meningococcal disease: comparison of a whole-blood assay and the serum bactericidal assay.

A whole-blood assay (WBA), which assesses the complete bactericidal activity of blood, was compared with the serum bactericidal assay (SBA), which measures antibody and complement mediated cell lysis. Twenty children infected with serogroup B strains and 25 infected with serogroup C strains were studied 8-12 weeks after disease, and 29 healthy children were used as controls. The infecting strain (convalescent children only) and two reference strains, MC58 (B:15:P1.7, 16) and NCTC 8554 (C:NT:P1.5) were used. In children previously infected with a serogroup B strain, bactericidal activity was detected in 95% and 85% to their infecting strain by the WBA (>50% killing) and the SBA (s), respectively. Bactericidal activity to the reference serogroup B and C strain was detected by WBA in 70 and 75% of children, respectively, and the SBA in 45% and 20%. In contrast bactericidal activity was detected to both serogroup C strains in >80% of children previously infected with a serogroup C strain using either assay and in 48% (WBA) and 20% (SBA) to the reference serogroup B strain. Levels of bactericidal activity were detectable in fewer control children. Children convalescing from meningococcal disease develop an immune response to their infecting strain, detectable by both the WBA and SBA, which is independent of age. However, the WBA appears to be a more sensitive measure of bactericidal activity to heterologous strains than the SBA.     

The assay of canine serum haptoglobin and its diagnostic implications. A comparison of two assay techniques

A Sephadex gel filtration technique was compared with a radial immunodiffusion (RID) method for the assay of serum haptoglobin in the dog. Administration for a haemolytic agent (acetylphenylhydrazine) to dogs resulted in reduced serum haptoglobin but without a significant change in red cell indices. It is suggested that the assay of serum haptoglobin in the dog may be of value in detecting low grade haemolysis when other criteria fail. After standardising the assay using the gel filtration technique, a number of samples can be screened simultaneously using RID thus enabling routine clinical laboratory monitoring for haemolysis. The clinical value of serum haptoglobin assay in the diagnosis of canine haemolytic anaemia is discussed.     

Interlaboratory comparison of serum vitamin B12 assay.

The results have been compared of microbiological and radioisotope dilution (RID) assay of serum vitamin B12 by participants in national interlaboratory trials in Britain. There was wide variation between the individual participants, especially marked in the L. leichmannii microbiological assay and in the RID methods, whereas excellent correlation, reproducibility, and recovery were obtained in reference laboratories by microbiological assay with both E. gracilis and L. leichmannii. In general, RID gave higher results than microbiological assay. The need for suitable reference sera is emphasised.     

Variation in serum electrolytes and enzyme concentrations in patients with sickle cell disease.

AIM--To assess levels of some biochemical variables in sickle cell disease patients from eastern Saudi Arabia during steady state and in crises states, with a view to comparing biochemical and clinical manifestations of the disease with those in other geographical locations. METHODS--Serum calcium, uric acid, total bilirubin, lactate dehydrogenase, hydroxybutyrate dehydrogenase, and haemoglobin were measured in 110 sickle cell patients when in steady state. The same variables were measured on 30 of the patients when they went into crisis. RESULTS--Serum calcium tended to be lower in sickle cell patients than in healthy controls, while uric acid tended to be in the high normal range. Crises did not make any difference to serum calcium but they increased the uric acid level significantly. All the other variables measured were significantly abnormal and more so during crises. CONCLUSIONS--Although the abnormal levels obtained for these biochemical variables in patients with sickle cell disease from eastern Saudi Arabia were similar to those from other geographical locations, there were noticeable differences in the severity of the abnormalities, which probably explains the differences in the clinical manifestations of the disease between geographical locations. Values of some of these variables could be adapted for use to monitor crises.     

Rapid bioluminescent assay for determining netilmicm and tobramycin concentrations in serum

A rapid bioluminescent assay for determining netilmicin and tobramycin concentrations in serum based on the dose-dependent effect of these agents on the accumulation of extracellular ATP inEscherichia coli LU 14 cultures is presented. This strain ofEscherichia coli is unaffected by antibiotics used in combination with aminoglycosides and it lacks significant ATP-ase activity, which is a prerequisite for extracellular ATP accumulation. ATP was quantified by the firefly bioluminescence system. The accuracy of the bioluminescent assay expressed as mean coefficient of variation over the therapeutic range was 3.2 %; corresponding figures for EMIT and an agar disk diffusion assay were 4.2 % and 4.8 % respectively. All methods used correlated well (r=0.935?0.986) when they were evaluated on clinical serum specimens. The bioluminescent assay requires 25μl serum and results are available within 75 min.     

Comparison of different methods for serum folate assay]

Folates were determined in 148 patient sera, using four different methods: a microbiological assay (Reference Method), two radioassays (Magic B12 FOL [NB] and SimulTRAC SNB no boil kits) and a non isotopic competition method (Magic Lite kit). Folate mean varied according to the methods 10.0 nmol.l-1 (reference method), 12.2 nmol.l-1 (Magic Lite), 8.7 nmol.l-1 (Magic B12 FOL [NB]) and 10.8 nmol.l-1 (SimulTRAC SNB NO boil). Poor correlations were also noted (0.83 < r < 0.95, figures 1 and 2). Differences between methods were explained according to the literature.