Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

In the UK, under present Whitley Council Regulations for payment for out-of-hours pathology services, there is a complex relationship between the number of requests received, the time taken to analyse each request, and the number of calls for which payment may be claimed for work done. At a fixed average analysis time, the rate of increase of remuneration slows down as workload increases until at higher workloads remuneration falls. The introduction of methods with a shorter average analysis time to improve the clinical service increases remuneration disproportionately. We suggest that a fixed sessional payment would be a better way of funding the service.

The relationship between plasma HDL-cholesterol subfractions (HDL2 and HDL3), measured using a differential precipitation method, and serum triglycerides, was studied in 402 healthy Caucasion males attending a health screening centre in London. Mean values for HDL2 and HDL3 were 0·42± 0·24 mmol/L and 0·81±0·15 mmol/L respectively. HDL2 was found to show a stronger negative correlation with serum triglyceride and a stronger positive correlation with total HDL than HDL3. HDL2 also showed a stronger correlation with age than either total HDL or HDL3. Mean levels of HDL2 were 20% higher in subjects over 55 years of age compared with those who were less than 55 years of age. Reference values of HDL2 and HDL3 are presented for different age ranges.

A method is described for the identification of apolipoprotein E phenotypes. Lipoproteins were precipitated by magnesium chloride/phosphotungstate reagent and isoelectric focusing performed on flat-bed agarose. Sensitivity was enhanced by blotting with nitrocellulose prior to staining. The system described requires no ultracentrifugation step and allows up to 20 samples to be processed in each run. Agarose has some advantages over the more commonly used polyacrylamide gels for isoelectric focusing.

Plasma tocopherol was measured in 85 alcoholic patients and 40 control subjects from a local factory. Cholesterol, triglycerides and phospholipids were measured individually and summed to give an estimate of total serum lipids. Plasma tocopherol concentrations of the alchoholics were significantly lower than those of the controls and showed wide variation from marked deficiency to the upper limit of the normal range. Using regression analysis, 1·11 μmol tocopherol/mmol total lipids were calculated as the threshold of deficiency equivalent to 0·8 mg tocopherol/g total lipid established by Horwitt et al. ¹ The sensitivity and specificity of other tocopherol:lipid ratios for identifying vitamin E deficiency was compared with the tocopherol:total lipid ratio. Thresholds of deficiency for the different tocopherol:lipid ratios were calculated. The tocopherol:cholesterol+triglyceride ratio was found to be almost as powerful in identifying vitamin E deficiency as the tocopherol:total lipid ratio (sensitivity 95%, specificity 99%). Of the tocopherol:individual lipid ratios, the tocopherol:cholesterol ratio gave the best results (sensitivity 86%, specificity 94%).

The activity of δ-aminolaevulinic acid dehydratase (ALA-D; porphobilinogen synthase) was measured in whole blood from a group of workers with acute exposure to lead and with low blood lead levels, a group of workers with chronic lead exposure and high blood lead levels, and a group of people without undue environmental lead exposure. The activity of ALA-D was reduced significantly at low blood lead levels only if undue exposure to lead had occurred, and was thus a reflection of low level lead poisoning. In chronic lead exposure the enzyme was not invariably reactivated fully with dithiothreitol, indicating more severe enzyme poisoning. The one lead worker with symptomatic lead poisoning had the most marked enzyme suppression. Measurement of both ALA-D activity and blood lead levels was more useful than the measurement of blood lead levels alone in the diagnosis and assessment of the severity of lead poisoning.

Simple methods for the detection of hypoxanthine-guanine phosphoribosyltransferase and/or adenine phosphoribosyltransferase deficiencies using dried filter paper blood spots were studied. Enzyme activities in the eluate from dried filter paper blood spots stored for 4 weeks at room conditions were shown to be quite stable. Autoradiographs prepared from dried filter paper blood spots and DE-81 papers soaked with enzyme reaction mixtures containing ¹⁴C-hypoxanthine and/or ¹⁴C-adenine showed sharp radioactive spots in normal subjects. No activity was evident in the cases of the Lesch-Nyhan syndrome and/or adenine phosphoribosyltransferase deficiency. The methods seem to be suitable for screening.

The relationship between serum calcium and albumin was studied using a data base containing biochemical results on approximately 687 000 samples. To exclude patients with established bone, liver and kidney disease, calcium and albumin values had to be accompanied by additional results falling within the laboratory reference range for urea, phosphate, globulin, alkaline phosphatase and aspartate transaminase activities. The regression coefficients of calcium on albumin changed significantly following a change of instrumentation, even though the analytical principles of the methods remained the same. In addition, using standard laboratory methods, the relationship between calcium and albumin deviated from linearity at albumin concentrations less than 30 g/L. Care is needed in the selection and clinical application of factors for the adjustment of serum calcium for changes in the concentration of circulating albumin. Using a large data base, an albumin-related reference range for serum calcium may be calculated, eliminating the need for adjusting calcium values.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30°C and correlate well with the manual spectrophotometric method of Ellman¹ for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

Bloodspot 17α-hydroxyprogesterone, plasma cortisol, plasma sodium and urinary 17α-hydroxyprogesterone, cortisol, sodium and creatinine levels were determined in 24 term and 32 pre-term infants on the third, eighth and fourteenth days of life. Pre-term infants, whether ‘well’ or ‘sick’, had significantly raised bloodspot 17α-hydroxyprogesterone levels (up to 158 nmol/L) compared with those found in term infants (up to 18·8 nmol/L). Urinary 17α-hydroxyprogesterone/creatinine ratios were also higher in pre-term infants. Plasma cortisol results showed similar ranges for term and pre-term infants, and bloodspot 17α-hydroxyprogesterone/plasma cortisol ratios for day 3 specimens correlated with the degree of prematurity. These results may be due either to immature enzyme systems in the pre-term baby or to an excess of related steroids cross-reacting in the 17α-hydroxyprogesterone assay.

We propose the use of two distinct upper limits of normal of 20 nmol/L (term infants) and 200 nmol/L (pre-term infants), for the interpretation of bloodspot 17α-hydroxyprogesterone levels at the end of the first week of life.

A sensitive HPLC method that allows the simultaneous determination of imipramine, amitriptyline and their desmethyl metabolites, using a 10 cm straight silica cartridge column with a mobile phase consisting of 0·004% perchloric acid in methanol is presented. Detection of the eluent is at 210 nm. The drugs are back-extracted from serum, giving both a good recovery and sensitivity of at least 5 μg/L.

This method has proved to be extremely robust and reliable, with coefficients of variation around 3% being reported.

A simple method is described for adjusting the mobile phase composition to obtain optimum separation of commonly prescribed antiepileptic drugs by high-performance liquid chromatography. The drugs are separated, in less than 6 min, on a column of Hypersil ODS 5 μm (100 mmx5 mm i.d.) with a high mobile phase flow rate. The final mobile phase composition is a mixture of methanol, acetonitrile, tetrahydrofuran and water, adjusted to give the best separation with our laboratory conditions. The method is described and evidence of precision and accuracy presented. The improved separation resolves the major metabolites of carbamazepine which have been ignored in most methods for antiepileptic drugs. The advantages of mobile phase optimisation to improve the separation of antiepileptic drugs are discussed.

Plasma and buffy layer vitamin C concentrations have been measured in 19 patients before and following major surgery, and falls of 36% and 43% respectively were recorded on the first post-operative day. However, the change in the buffy layer could not be accounted for by a change in the concentration of vitamin C in platelets, mononuclear cells or polymorphonuclear leucocytes, the cell types which form the buffy layer. The buffy layer change could be explained by a post-surgical increase in the number of polymorphonuclear leucocytes which, when the buffy layer vitamin C is expressed per 10⁸ white cells, dilutes the platelet and mononuclear cell contribution to the buffy layer vitamin C concentration. The fall in buffy layer vitamin C does not, therefore, reflect any increased demand for white cell vitamin C in the immediate post-operative period, although the decrease in plasma vitamin C could imply some increased utilisation or redistribution of the non-cellular compartment.

A new immunoblotting method is described for the detection of Bence Jones proteinuria by the routine laboratory. Unconcentrated urine specimens are subjected to electrophoresis on agarose gels. Separated proteins are transferred to a nitrocellulose membrane and the immunoglobulins located and identified by horseradish-peroxidase double-antibody staining. The new method has been compared with that used routinely, and an improved rate of detection of both Bence Jones protein and intact urinary monoclonal immunoglobulin has been obtained. Among urine specimens received for routine testing for Bence Jones protein from 83 patients, 64 monoclonal components were found by the new technique compared with 45 by the method used routinely. Other advantages of the new procedure include: no need to concentrate urine specimens before electrophoresis; unlike immunofixation, the proteins may be detected successfully over a wide concentration range without using several specimen or antibody dilutions; and interpretation is easier.

The design and results of a UK external quality assessment scheme for six enzymes are described, from 21 surveys over a period of 7 years. Improvements in interlaboratory agreement and the adoption of reliable methods are documented. The potential of enzyme calibration materials in further improving numerical concordance between laboratories using different assay conditions, including temperature, is demonstrated and discussed.

Modern automatic analysers are making increasing use of microprocessor technology, which is difficult for the user, or even computer scientists, to evaluate. This paper offers some practical advice for the prospective purchaser on how to assess the capabilities and limitations of the computer hardware and software provided in such instruments, and considers some of the problems of interfacing an analyser to a laboratory computer system.

In the biochemical evaluation of hirsutism, 50% or less of patients have an elevated total serum testosterone. Recent work has suggested that measuring salivary testosterone or a derived serum ‘free testosterone index’ may be of use in the evaluation of hyperandrogenism. We have measured serum total, derived serum free indices and salivary concentrations of testosterone and 5α-dihydro-testosterone in an unselected group of hirsute patients in order to assess their value in the routine evaluation of hirsutism. The assays were performed using a novel oxidation procedure to overcome the need for chromatographic separation. The ‘free testosterone index’ gave the best discrimination. Salivary androgen concentrations were comparatively poor and cannot be recommended for routine use.

A rapid, single-reagent, non-separation, non-isotopic immunoassay was developed for determining levels of the nicotine metabolite, cotinine, in urine.

The single reagent was prepared by pre-mixing an appropriate dilution of sheep anti-cotinine serum with a fluorescein-labelled cotinine tracer.

All normal reliability criteria were satisfied. The assay was found to be specific for cotinine and there was no cross reactivity with other available nicotine metabolites and structurally-related compounds. The results obtained correlated closely with those of an established radioimmunoassay. The assay was well-suited to application in the discrimination of active smokers from non-smokers (and passive smokers).

Free phenytoin has been determined using micro-scale ultracentrifugation followed by analysis by EMIT. The effect of temperature on the determined free fraction was investigated and the ultracentrifugation procedure validated against ultrafiltration. Ultracentrifugation gave free fractions which were on average 16% lower than those obtained using ultrafiltration, but correlation was good, as was the correlation with measurements of total phenytoin (r=0·90). Micro-scale ultracentrifugation is a simple procedure which can be of great utility in the measurement and investigation of free drug levels.