Annals of Clinical Biochemistry

Published by SAGE Publications
Online ISSN: 1758-1001
Print ISSN: 0004-5632
Publications
The commonly accepted method of analysing data from method comparison studies is regression analysis, a method which has limitations. This study illustrates the use of a graphical presentation of data, the difference plot, which can be used as an alternative to least squares regression analysis. The data from comparison studies performed on five methods were analysed both by Deming's regression analysis, with calculation of the correlation coefficient, and by the difference plot. The results show that in most cases much more relevant information was obtained from the difference plot.
 
The short synacthen test (SST) is the gold standard investigation for the evaluation of adrenal insufficiency and is also frequently used for the evaluation of the hypothalamic-pituitary-adrenal (HPA) axis. The 0900-h serum cortisol concentration has also been evaluated as an indication of cortisol reserve, and a result > 450 nmol/L is highly suggestive of a normal serum cortisol response to the insulin tolerance test, while no patient with a 0900-h serum cortisol < 100 nmol/L had a sufficient response. The aim of this study was to determine if the number of inappropriate SSTs could be reduced if a 0900-h serum total cortisol was done prior to the dynamic function test. Two hundred and ten SSTs were performed at 0900 h and the response at 30 min evaluated. Of the 210 SST, 151 (71%) demonstrated a maximum response at 30 min of serum cortisol > 550 nmol/L. All the patients with a 0900-h serum cortisol > 500 nmol/L had an adequate response ( > 550 nmol/L), while no patient with a 0900-h serum cortisol of < 100 nmol/L had an adequate SST response. Twenty one per cent of patients were shown to have had an unnecessary invasive procedure. We conclude that the SST is of little added value in patients with a 0900-h serum cortisol of less than 100 nmol/L or more than 500 nmol/L and it should be included in the appropriate protocols for endocrine investigation.
 
Ethylene glycol in plasma, urine or dialysis fluid is analysed as the phenylboronate derivative by mixing with acetonitrile/acidified 2,2-dimethoxypropane containing phenylboronic acid. After centrifugation, a portion of the supernatant is analysed directly by gas-liquid chromatography using a 3% OV-101 column at 150 degrees C and flame-ionisation detection. Propane-1,3-diol is used as a reactive internal standard. The limit of accurate measurement is at least 0.1 g/L and the linear range extends up to 5.0 g/L. No sources of interference have been identified.
 
We evaluated a novel assay for the measurement of 1,25 dihydroxyvitamin D (1,25 (OH)2D). Immunoextraction of 1,25(OH)2D is performed using a mini column containing a solid-phase monoclonal antibody followed by radioimmunoassay (RIA) using an 125I-labelled 1,25(OH)2D derivative tracer and Sac-cell separation. The mean recovery of 1,25(OH)2D3 was 101%, linearity was excellent, inter- and intra-assay coefficients of variation were 9, 8 and 13% and 11, 10 and 14% at low, medium and high concentrations of 1,25 (OH)2D3, respectively. The cross-reactivity of vitamin D metabolites was < 0.0015% for 25-hydroxyvitamin D3, 24, 25 dihydroxyvitamin D3 and dihydrotachysterol and 0.54% for 1 alpha calcidol. 1,25 dihydroxyvitamin D2 cross-reactivity was 79%. The detection limit of the assay was 5 pmol/L. Comparison with a commercial radio receptor assay (RRA) and an in-house RIA gave regression equations of y = 0.94x + 11.8 (r = 0.98) and y = 0.91x-1.7 (r = 0.95), respectively, with no major discrepancies between the methods in all patient groups studied. Plasma concentrations of 1,25(OH)2D obtained with the assay were as follows: normal, unsupplemented subjects: mean 88, range 48-155 pmol/L, n = 68, patients with chronic renal failure: mean 11, range 3-36 pmol/L, n = 27, primary hyperparathyroidism: mean 198, range 130-299 pmol/L, n = 23, Paget's disease: mean 92, range 42-149 pmol/L, n = 24, osteomalacia: mean 43, range 27-61 pmol/L, n = 9. A minimum sample volume of 300 microL is required, the hands-on time is significantly less than other commercial assays and the measuring procedure is gamma counting rather than scintillation counting. The assay offers several advantages over previous methods and should allow more laboratories to offer measurement of 1,25(OH)2D as part of their repertoire.
 
The seasonal variation of serum 25-hydroxy vitamin D3 and 1,25-dihydroxy-vitamin D has been investigated. Blood was taken from 27 healthy volunteers, aged 21-44 years old at 3 monthly intervals over a period of 1 year. A scrolling monthly programme with 12 quarterly (3 month) time periods was developed. A summer associated increase in 25-hydroxy vitamin D3 was significantly correlated with but lagged behind by 2 months, the increase in recorded sunlight hours. However, four individuals showed no seasonal rise but maintained constant concentrations throughout the year within the established reference range. Serum 1,25-dihydroxy vitamin D showed marked intra-individual variability with no seasonal pattern although the highest concentration (180 pmol/L) was observed in the winter and no concentration greater than 108 pmol/L in the summer.
 
We measured serum and urinary 1,5-anhydro-D-glucitol (1,5-AG) during a glucose tolerance test (GTT) in patients with chronic renal failure (CRF) and compared the fractional excretion of 1,5-AG (FEAG) with that of diabetes mellitus (DM) patients and healthy controls. The mean serum 1,5-AG in CRF patients [60 +/- 23(SE) mumol/L] was significantly lower than in controls (155 +/- 7 mumol/L) in spite of a normal glycaemia. The levels in the CRF group were similar to those in the DM group. During GTT, the blood glucose profile in the CRF group was not significantly different from that of the control group, and urinary glucose excretion was negligible. However, FEAG was significantly higher in CRF patients than in controls. These data suggest that serum 1,5-AG in patients with CRF decreases due to a decrease in 1,5-AG reabsorption, independently of glucose excretion, and that serum and/or urinary 1,5-AG can be a useful marker for renal tubular dysfunction because the 1,5-AG reabsorption system is more vulnerable than the glucose reabsorption system.
 
Correlation between HbA 1C and serum 1,5-AG (logarithmically converted). The figure indicates correlation between estimated HbA 1C and serum 1,5-AG in the CLD patients (W) and between measured HbA 1C and serum 1,5-AG in the control subjects ( †).1,5-AG, serum 1,5-anhydroglucitol; CLD, chronic liver disease; HbA 1C glycated haemoglobin 
Serum 1,5-anhydroglucitol (1,5-AG) is a known marker reflecting recent glycaemic control. In this study, we examined serum 1,5-AG levels in chronic liver disease (CLD) patients with and without diabetes mellitus. Eighty patients with CLD were compared with 667 subjects without CLD. Glycaemic control of the CLD patients was evaluated by estimated glycated haemoglobin (HbA(1C)) calculated using the equation by Rohlfing et al. from mean plasma glucose because CLD patients have apparently low HbA(1C). When the study participants were divided into subgroups stratified by HbA(1C) levels, the CLD patients whose estimated HbA(1C) levels were less than 7.0% showed significantly lower 1,5-AG than their counterparts of the control subjects. Stepwise multivariable analysis revealed that estimated HbA(1C) was the significant explanatory variable for 1,5-AG in the CLD patients. However, in the CLD patients with estimated HbA(1C) less than 5.8%, only hepaplastin test was the significant explanatory variable for 1,5-AG. Serum 1,5-AG levels are low irrespective of plasma glucose levels in the CLD patients with and without diabetes. The CLD patients who had low serum 1,5-AG levels were associated with deteriorated liver function.
 
The specific binding of 1,9-dimethylmethylene blue (DMB) with glycosaminoglycans (GAGs) was studied including molecular analysis of the GAG species using mass spectrometry. The dye solution was unstable under any storage conditions. Inorganic analysis showed that the purity of the dye was variable from different sources. False negative results could be obtained when using impure dye. Binding of the dye with GAG resulted in the formation of a complex with an absorption maximum at 525 nm. The absorbance of the complex was linearly correlated with GAG concentration up to 150 mg/L. The specific molar extinction coefficients of individual GAG molecules were calculated in relation to the molar absorbance of the GAG-dye complex and the carbon and nitrogen contents of the GAGs. The results indicated that binding of dye occurred at both the ionized sulphate and carboxyl groups on the GAG molecules. Improvements of the DMB-binding method for the measurement of GAGs are suggested.
 
A high-performance liquid chromatographic method was developed for the measurement of carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZE) in human breast milk and plasma. The method involves rapid C18 solid-phase extraction of CBZ and CBZE. Chromatographic separation was achieved with a reversed-phase C8 column using a mobile phase of potassium dihydrogenphosphate (pH 2.5) and acetonitrile (67:33 v/v), with ultraviolet detection at 254 nm. 2-Methyl CBZ was used as the internal standard. Determination of both CBZ and CBZE was possible in the range of 0.01-6.0 mg/L and 0.02-6.0 mg/L in milk and plasma, respectively. The recoveries of CBZ and CBZE added to the milk and plasma were 90.6-98.0% and 88.9-104.0%, respectively, with coefficients of variation less than 8.3% and 10.5%, respectively. The method has been used for drug level monitoring in milk and plasma samples obtained from CBZ-treated patients. The mean (SD) levels for CBZ in milk and plasma samples were 3.50 (0.4) mg/L and 6.18 (2.9) mg/L, and for CBZE were 1.28 (0.3) mg/L and 1.85 (1.0) mg/L, respectively. The mean (SD) milk/plasma ratios of CBZ and CBZE were 0.64 (0.2) and 0.79 (0.3), respectively. The milk/plasma ratio of CBZE was slightly higher than that of CBZ.
 
Stable lyophilized ethylenediaminetetra-acetic acid (EDTA)-blood haemolysates were applied in an external quality assurance programme (SKZL, The Netherlands) for glycohaemoglobin assays in 101 laboratories using 12 methods. The mean intralaboratory day-to-day coefficient of variation (CV), calculated from the assay of 12 unidentified pairs over a period of 1 year, was 5.2% (range: 0.2-28.7). Forty-seven per cent of laboratories did not meet the criterion of CV < 5%, whereas 68% did not meet the clinically more desirable 3.3-3.6%. Linearity, as derived from the analysis of five combinations of two haemolysates with low and high glycohaemoglobin percentages over 6 months, was excellent (mean correlation coefficient 0.9953; range: 0.9188-0.9999). Analysis of two samples with high and low glycohaemoglobin percentages gave mean interlaboratory coefficients of variation of 10% for one method performed by several laboratories and 22% for all methods performed by all laboratories. It is concluded that the majority of laboratories do not meet the clinically desirable intralaboratory precision and that an unacceptably high interlaboratory precision exists.
 
N-terminal pro-B type natriuretic peptide (NTpBNP) is a potential marker of cardiac failure. The Roche Elecsys(trade mark) 1010 and 2010 assays for NTpBNP were evaluated for precision, sample stability, and correlation between sample types and with other natriuretic peptides. Samples from 290 individuals aged 45-89 years with no cardiovascular risk factors, renal failure, electrocardiogram changes, evidence of structural abnormalities, or wall motion abnormalities on echocardiography and with an ejection fraction >50% were used to provide reference NTpBNP ranges. The intra-assay imprecision was <10% across the analytical range and >3% at all concentrations analysed <30 ng/L. Inter-assay imprecision was 5.3-6.7% on the Elecsys 1010 and 4.4-5.0% on the Elecsys 2010, in the range 380-13000 ng/L. There was no statistically significant change in NTpBNP following storage in whole-blood samples at room temperature for 24 h before centrifugation; serum samples at room temperature for 7 days, at 4 degrees C for up to 11 days on clot-activation gel or 22 days separated from the gel. NTpBNP concentrations were stable throughout five freeze-thaw cycles. There was a close correlation between NTpBNP concentrations in matched serum, EDTA plasma and lithium-heparin plasma samples. NTpBNP and BNP were more closely associated than were N-terminal proatrial natriuretic peptide and NTpBNP. This association was stronger at lower concentrations. NTpBNP concentrations increased with age, with values higher in women than men. NTpBNP is a stable molecule that can be measured easily and precisely using the Roche Elecsys 1010 or 2010 immunoassay analysers.
 
The analytical performance of a new, whole blood glucose and lactate electrode system (EML 105 analyser. Radiometer Medical A/S. Copenhagen, Denmark) was evaluated. Between-day coefficients of variation were < or = 1.9% and < or = 3.1% for glucose and lactate, respectively. Recoveries of glucose were 100 +/- 10% using either aqueous or protein-based standards. Recoveries of lactate depended on the matrix, being underestimated in aqueous standards (approximately -10%) and 95-100% in standards containing 40 g/L albumin at lactate concentrations of 15 and 30 mmol/L. However, recoveries were high (up to 180%) at low lactate concentrations in protein-based standards. Carry-over, investigated according to National Clinical Chemistry Laboratory Standards EP10-T2, was negligible (alpha = 0.01). Glucose and lactate biosensors equipped with new membranes were linear up to 60 and 30 mmol/L, respectively. However, linearity fell upon daily use with increasing membrane lifetime. We conclude that the Radiometer metabolite biosensor results are reproducible and do not suffer from specimen-related carry-over. However, lactate recovery depends on the protein content and the lactate concentration.
 
Familial defective apolipoprotein B-100 (FDB) is commonly attributable to mutation of glutamine to arginine in codon 3500 of the apolipoprotein B (APOB) gene. APOB, the protein component of low-density lipoprotein (LDL) acts as the ligand for the LDL receptor (LDLR), mediating the clearance of LDL from plasma. This mutation causes hypercholesterolaemia and consequent coronary artery disease phenotypically similar to familial hypercholesterolaemia (FH) attributable to LDLR gene defects. APOB gene mutation R3500Q is prevalent in Western Europe, attributable to a single founder mutation, and occurring at up to one in 1000 in the general population and 1%-2% in apparent FH collections. Many studies applying direct assay of patient groups for R3500Q have been undertaken, but there are indications that other ligand defects exist, R3531C and R3500W having been described. Different regions or countries may display different mutational spectra which can be instructive for research and useful for establishing genetic diagnostic assays. Codon region 3456-3553 of the APOB gene contains the mutations so far identified, and therefore is a strong candidate for a functional role in receptor binding. We have applied denaturing gradient gel electrophoresis (DGGE), a sensitive de novo mutation scanning technique, to this region in 106 apparent FH index cases from the South of England.
 
11 beta-hydroxysteroid dehydrogenase (11 beta HSD) has both dehydrogenase (11 beta DH) and reductase (11 beta R) activities, which catalyse the interconversion of cortisol and cortisone, and prednisolone and prednisone. This enzyme confers specificity on the mineralocorticoid receptor by local oxidation of cortisol to cortisone. Using radiolabelled cortisol 11 beta HSD activity has been shown to be lower in some cases of essential hypertension. This study investigated a novel approach to estimating 11 beta HSD activity in vivo. Plasma steroid kinetics were investigated following oral hydrocortisone (a substrate for 11 beta DH) and prednisone (a substrate for 11 beta R) in five normotensive volunteers after dexamethasone suppression of endogenous steroid production. This approach was evaluated by inducing partial deficiency of 11 beta HSD in the volunteers who took liquorice (to inhibit 11 beta DH) and then carbenoxolone (to inhibit both 11 beta DH and 11 beta R). The ratio of cortisol to prednisolone (formed from prednisone) provided a measure of the activity of both 11 beta DH and 11 beta R. At 75 min after the steroid bolus the ratio increased from 1.1 (0.6-1.3) (median, range) under control conditions to 1.2 (0.8-1.7) after liquorice (P = 0.01, n = 5), and 2.0 (1.3-5.9) after carbenoxolone (P = 0.02, n = 5). It may therefore be applied to the measurement of 11 beta HSD activity in vivo in large numbers of hypertensive patients without the use of radioisotopes.
 
The excretion of urinary free 11-hydroxycorticosteroids and total oestrogens was studied daily in morning urine specimens throughout the menstrual cycle of six normal women (age range 18-24 years). The follicular phase of the cycle was characterised by apparently random fluctuations in the excretion of urinary free corticosteroids. However, after the mid-cycle oestrogen peak there occurred a significant drop in corticosteroid excretion, which then rose to a peak eight to 10 days after ovulation and was synchronous with the second oestrogen peak during the luteal phase.
 
Graphs illustrating relationship between serum cortisol and 11DOC with metyrapone dose. (a) Individual value plot of difference between serum cortisol measurements (immunoassay-LC-MS/MS) with metyrapone dose and (b) serum 11DOC concentration and metyrapone dose. Horizontal lines represent mean value. (c) Scatter graph showing positive linear association between delta cortisol and serum 11DOC concentration. Pearson's correlation coefficient ¼ 0.47. LC-MS/MS, liquid chromatography-tandem mass spectrometry; 11DOC, 11-deoxycortisol 
The accurate measurement of cortisol by immunoassay is compromised by the potential for cross-reactivity of reagent antibodies with structurally related steroids present in serum. This susceptibility is potentiated when normal steroid metabolism is altered pharmaceutically by antisteroidogenic drugs utilized in the management of Cushing's syndrome to moderate cortisol production. The clinical implications of falsely elevated cortisol results include over-treatment and unrecognized hypoadrenalism. To investigate the effect of the 11β-hydroxylase inhibitor metyrapone on serum cortisol assay, a comparison of measurement by immunoassay versus liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted. Cortisol was measured in serum from three patient groups: (1) patients receiving metyrapone (n = 112 samples from 10 patients); (2) control group of patients diagnosed with Cushing's syndrome currently receiving no treatment (n = 31); and (3) control group of patients with no adrenal pathology and not receiving medication known to interfere in cortisol immunoassay (n = 67). Bland-Altman plots showed agreement between methods for the control group (bias = 1.1% [-4.3 nmol/L]) and Cushing's control group (bias = 1.3% [-3.7 nmol/L]). This was in stark contrast to the metyrapone therapy group (bias = 23% [59 nmol/L]). The difference between LC-MS/MS versus immunoassay in the metyrapone therapy group positively correlated with metyrapone dose and serum 11-deoxycortisol concentration (Pearson's correlation coefficient r = 0.47, 95% CI 0.32-0.61; P < 0.0001). These data show that liability of immunoassay measurement of serum cortisol to interference in patients receiving metyrapone may lead to erroneous clinical decisions concerning dose titration.
 
Urinary free 11-hydroxycorticosteroid/creatinine ratios were determined in early morning urine samples from 113 females (age range 20-45 years) and 65 males (age range 22-45 years). Basal values in normal subjects fluctuated between 5 and 55 mumol/mol creatinine. In four patients in whom Cushing's syndrome was diagnosed, urinary free 11-hydroxycorticosteroid/creatinine ratios were greater than 85 mumol/mol creatinine. Administration of dexamethasone (0.5 mg/q.i.d.) to nine normal laboratory staff for two days resulted in a suppression of the urinary free 11-hydroxycorticosteroid/creatinine ratio to less than 50% of the mean basal value in all cases. This degree of suppression did not occur in two cases of Cushing's syndrome due to adrenal tumours.
 
A model consisting of overlapping Gaussian distributions of disease and reference values has been used to calculate the effects of analytical imprecision on the proportions of patients wrongly classified by a test. Using published data the model has been applied to the urinary excretion of 11-hydroxycorticosteroids in the diagnosis of Cushing's syndrome. At the lowest levels of imprecision encountered in hospital laboratories, doubling the imprecision increased false negatives (missed diagnoses) from 3.6% to 4.1% and false positives from 9.4% to 11.2%. Although improved imprecision may thus produce only marginal improvements of misclassification, there is no level of imprecision below which further improvement does not improve misclassification. It is suggested that the concept of an 'acceptable level of imprecision' should be replaced by the concept of costing improvements of imprecision, equating the benefits in terms of patient classification with cost in terms of additional resources.
 
An enhanced immunochemiluminometric assay for serum TSH ('Amerlite', Amersham, Bucks, UK) was studied in 1127 patients in routine clinical practice to assess its value as a first-line test of thyroid status. Good correlation with clinical thyroid status was found in the untreated euthyroid patients, in the untreated hyperthyroid and hypothyroid patients, in pregnancy and in the sick euthyroid. However, a large proportion of clinically euthyroid patients with nodular goitre, as well as those treated by thyroidectomy, radioiodine or antithyroid drugs and those on replacement l-thyroxine showed TSH values outside the reference range. Therefore, additional tests are likely to be needed frequently in these categories.
 
The CA-125 antigen is expressed on a glyco­ protein produced by the cells derived from the coelomic epithelium and can be detected in the pleura, pericardium, peritoneum and the Mill­ lerian duct derivatives.' Serum CA-125 concen­ tration is elevated in the vast majority of patients with ovarian cancer and has proved to be a very useful tumour marker in these patients. How­ ever, serum CA-125 concentrations may also be elevated in a large number of other malignant diseases such as colorectal, breast, lung and pancreatic cancer. In addition, elevated serum CA-125 concentrations may be found in other conditions, including pleural effusions and ascites irrespective of the cause. In recent years elevated serum CA-125 has also been demonstrated in patients with non-Hodgkin's Iymphoma.v? In the cases reported, no immuno­ reactivity for CA-125 on the lymphoma cells could be demonstrated and it was proposed that the elevated CA-125 concentration was a reactive phenomenon.t-' However, it was shown recently that lymphoma cells might secrete CA-125 under specific conditions.!" We report a case which provides further evidence that high serum CA-125 concentrations in patients with malignant lymphoma may be derived from the tumour cells.
 
Comparisons of Pls extraction efficiencies from human serum in different extraction methods 
Serum plasmalogens (Pls) have gained interest in several clinical symptoms such as metabolic syndrome/atherosclerosis or Alzheimer's disease possibly because of their antioxidant properties. We have developed a highly sensitive and simple method to determine plasmenylcholine (PlsCho; choline plasmalogen) and plasmenylethanolamine (PlsEtn; ethanolamine plasmalogen) separately, using a radioactive iodine and high-performance liquid chromatography ((125)I-HPLC method). The present study reports the improvement and validation of (125)I-HPLC method by introducing a quantitative standard (QS) and online detection with a flow γ-counter. 1-Alkenyl 2,3-cyclic glycerophosphate was prepared as QS from l-α-lyso plasmenylcholine by enzymatic treatment with phospholipase D. Online detection with a flow γ-counter was investigated to be available to quantify Pls. The method validation was carried out in terms of selectivity, sensitivity, linearity, precision, accuracy and recovery. Linearity was established over the concentration range 5-300 μmol/L for Pls and QS with regression coefficients >0.99. The accuracy and reliability were satisfactory. The method has been applied to the determination of human serum Pls from healthy subjects and the elderly with dementia or artery stenoses. The improved (125)I-HPLC method is useful as an autoanalytical system for a routine diagnostic test of human serum Pls.
 
A simple and inexpensive procedure for the preparation of high specific activity 125I radioiodinated triiodothyronine and thyroxine is described. The specific activities achieved are approximately 350 Ci/g for 125I-T4, and either 220 Ci/g or 500 Ci/g for 125I-T3 depending on the reagents employed. The high specific activity 125I-T4 and 125I-T3 are suitable for the routine radioimmunoassay of serum total T3 and T4. When properly stored they have a shelf life of at least seven weeks. © 1976, Association for Clinical Biochemistry. All rights reserved.
 
An assay using an antiserum raised against a dexamethasone 21-hemisuccinate conjugate and the heterologous radioligand dexamethasone 21-(carboxymethyl) ether was developed, validated, and used to study the pharmacokinetics of this steroid for 12 h following administration to patients with congenital adrenal hyperplasia. Coupling the antiserum to magnetizable cellulose allowed rapid separation of bound/free steroid. A C-21 rather than a C-3 antiserum was used to minimize interference with a main metabolite, 6 beta-hydroxydexamethasone. Close correspondence of assay (0.35 nmol/L) and curve (0.25 nmol/L) sensitivities suggests that interference by matrix effects is minimal. This was confirmed by good agreement in data from the in-house assay and that of a reference procedure. Good precision was demonstrated by the precision profile and Shewhart chart quality control data. The latter also demonstrated the assay was robust and reliable in routine practice.
 
A new kit (Thyrolute, Ames) for the combined determination of serum total thyroxine (T-4) and sequential free thyroxine index (F.T.I.) using Sephadex G-25 and ¹²⁵ I-thyroxine was evaluated in 136 patients and normal subjects. The T-4 determination was virtually identical to that used in the Ames Tetralute kit and had a similar accuracy and precision. The sequential F.T.I. was compared with a two-stage F.T.I. The two F.T.I.s showed highly significant correlations in the various groups of patients except euthyroid women with raised thyroxine-binding globulin (TBG) (pregnant or oral contraceptive). The overlap found for the sequential F.T.I. between euthyroid, hypothyroid, and thyrotoxic patients was slightly inferior (9%) to that found with the two-stage F.T.I. (6%), but its diagnostic success rate was higher than that of the serum T-4 determination alone. Serial observations of serum T-4 and sequential F.T.I. were also made on eight patients receiving carbimazole therapy for hyperthyroidism. The sequential F.T.I. showed complete parallelism with serum T-4 regardless of thyroid status, so that it was of no practical value in these patients. It was concluded that the sequential F.T.I kit would be of most value in the smaller hospital laboratory lacking facilities for the radioimmunoassay of thyroid hormones and thyroid stimulating hormone.
 
A radioimmunoassay for testosterone in male plasma utilising a gamma-emitting radioligand and a solid-phase antiserum is described. The radioligand is testosterone-3-(O-carboxymethyl)-oxime coupled to ¹²⁵ I-iodohistamine, and the solid-phase antiserum is prepared by coupling antitestosterone-3-bovine serum albumin to cyanogen bromide activated cellulose. The new procedure retains much of the specificity associated with a published, specific radioimmunoassay using an antiserum raised against testosterone-11α-BSA and a tritium radioligand and incorporating a dextran-coated charcoal separation procedure; values obtained by the two procedures are in excellent agreement (r = 0·98, n = 20). The combination of an ¹²⁵ I-radioligand and a solid-phase separation technique greatly increases sample throughput and has the further advantage of reduced running costs and a greater potential for automation. The method gives satisfactory levels of sensitivity, precision, and accuracy.
 
Commercial RIA kits for the assay of serum total oestriol using 125I label are used widely in monitoring fetal well-being in pregnancy; they are, however, very expensive for large-scale routine use. An 'in-house' assay using commercially available [125I]oestriol label and antiserum produced by the S.E. and S.W. Thames Regional Antibody Production Unit has been developed. Technical aspects of the method are described. The assay is compared with two commercial kits and correlates very well with one of them. The assay gives good precision and can be used with substantial savings in cost.
 
Solid-phase first antibody (SP), liquid-phase double-antibody (DA), and pre-precipitated double-antibody (PPT) separation methods have been compared in a radioimmunoassay (RIA) for cortisol in unextracted serum. Both double-antibody methods gave values for pools close to target values assigned by gas chromatography-mass spectrometry (GCMS) whereas the SP assay had a significant negative bias, p less than 0.001 (mean biases: SP -8%, DA -2%, PPT -3%). The SP and DA assays gave average values on patients' samples 12% and 4% lower than values by PPT. These differences could be attributed to different recoveries in the three methods. Between-assay precision of the DA and PPT systems was better than SP (mean CV:SP 11%, DA 7%, PPT 5%). This allowed sensitivity in the PPT assay comparable to that of SP and DA to be achieved, despite the less steep slope of the calibration curve. The DA and PPT assays have several practical advantages over SP. In addition, the PPT assay requires only a 1-hour incubation. It is concluded that the PPT separation system is the method of choice in terms of precision and practical convenience.
 
A radioimmunoassay for plasma cortisol featuring the gamma-emitting radioligand 125I-iodohistamine, coupled to cortisol-3-(O-carboxymethyl)-oxime, is described. The new procedure retains much of the specificity associated with the use of anti-cortisol-3-BSA sera with tritium-labelled radioligands, and has the further advantages that running costs are lower and there is a greater potential for automation. Cortisol values obtained by this procedure agree well with those obtained by a published specific radioimmunoassay using the tritiated cortisol radioligand. Specificity of the procedure was checked by comparing values obtained with and without thin-layer chromatography purication: correlation was excellent (r = 0.96). Satisfactory levels of sensitivity, precision, and accuracy were obtained.
 
Background: Decreased serum concentrations of vitamin B12 are associated with Alzheimer's type dementia. The transcobalamin II gene (TCN2) 776C → G polymorphism affects transcobalamin II function as a carrier of vitamin B12 and might modify its availability. The association of the TCN2 776C → G polymorphism with Alzheimer's type dementia is unclear and was investigated in the present study. Methods: Case-control study including 27 individuals diagnosed with Alzheimer's type dementia and 28 healthy controls. Serum concentrations of vitamin B12, homocysteine and other analytes were determined and the presence of TCN2 776C → G and 5, 10-methylenetetrahydrofolate reductase 1298A → C polymorphisms genotypes was ascertained by polymerase chain reaction-restriction fragment length polymorphism. Results: Serum concentrations of vitamin B12 were lower while those of homocysteine were higher in patients than in controls (P < 0.05). The frequency of individuals carrying at least one 5, 10-methylenetetrahydrofolate reductase 1298C allele was higher (59% versus 32%) while frequency of individuals harbouring at least one TCN2 776G allele was lower (58% versus 86%) in patients than in controls (P < 0.05). Univariate logistic regression showed negative association of TCN2 776CG genotype with Alzheimer's type dementia (OR = 0.17 versus CC genotype, P < 0.02). Multivariate logistic regression identified TCN2 776C → G polymorphism as independent predictor of Alzheimer's type dementia together with higher concentrations of homocysteine, cholesterol and uric acid and lower concentrations of oestradiol. Association of TCN2 776C → G polymorphism with Alzheimer's type dementia was observed for individuals carrying the 5,10-methylenetetrahydrofolate reductase 1298AA genotype but not the AC or CC genotypes, indicating interaction between the two polymorphisms. Conclusions: The TCN2 776C → G polymorphism is negatively associated with Alzheimer's type dementia, suggesting a protective role against the disease in subjects with the 5, 10-methylenetetrahydrofolate reductase 1298AA genotype.
 
The clinical requirements of the users of assay results must be at the centre of assay development. We aimed to develop a single liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for drugs of abuse in urine that would meet the needs of our service users and replace the multiple screening and confirmatory techniques previously in use. After discussion with our users, it was decided that 13 drugs and metabolites should be measured in our panel: morphine, codeine, norcodeine, dihydrocodeine, 6-monoacetylmorphine, acetyl codeine, methadone and its metabolite, buprenorphine and its metabolite, amphetamine, benzoylecgonine and cotinine. Urine samples were prepared by the addition of internal standard, enzymatic hydrolysis and solid-phase extraction. Chromatography conditions were optimized so that the analytes were separated within a run time of 6 min. Optimal parent to daughter m/z ion transitions were chosen for all drugs and daughter ion ratios were used. The LC-MS/MS assay was successfully validated with acceptable precision and lower limits of quantification for all drugs. No matrix effects were seen. The results produced by the LC-MS/MS assay compared well with the previous combination of techniques in use. We have developed and validated a fit-for-purpose LC-MS/MS assay for 13 drugs of abuse in urine that obviates the need for multiple screening and confirmatory analytical techniques.
 
We describe a simple enzyme-linked immunosorbent assay for the measurement of immunoglobulin G (IgG) subclasses in serum. The microtitre plate is coated directly with diluted samples, standards or controls, blocked with phosphate-buffered saline containing 1% Tween to prevent non-specific protein binding to the plate, and each subclass assigned with specific mouse monoclonal antibody. The specific antibody is detected with alkaline-phosphatase linked anti-globulin and the colour development of the paranitrophenyl phosphate indicator reagent is measured at 405 nm until the highest concentration of standard gives an absorbance in excess of 1.4. Coefficients of variation measured at three concentration levels were less than 7% for within-assay variation and less than 10% for between-assay variation. Regression analysis of total IgG with the sum of the measured IgG subclasses gave a correlation coefficient of 0.932 (P < 0.001). The assay has been used to establish age-related reference ranges for serum IgG subclasses; these are found to be slightly different from those established for radioimmunodiffusion methods. Our study does not confirm the previously reported absence of IgG4 in normal individuals.
 
An evaluation has been carried out of 13 commercial testosterone kits. The within- and between-assay imprecision was found to be unsatisfactory for some kits. Sensitivity and linearity were acceptable for all the kits but several showed concentration-dependent biases to the ALTM or GCMS values. The occurrence of anomalous results and changing performance characteristics of some of the direct kits is also presented.
 
Serum concentrations of the thyroid hormone binding proteins, thyroxine binding globulin, prealbumin, and albumin were determined in 30 thyrotoxic patients before and after 131I treatment. Each patient was placed into one of three groups according to response to treatment. The serum concentration of all three proteins rose significantly in 10 patients who became euthyroid, and a greater increase was seen in 10 patients who developed hypothyroidism. There was no significant change in thyroid hormone binding protein concentrations in 10 subjects who remained hyperthyroid. Changes in the concentration of thyroid hormone binding proteins should be borne in mind when total thyroid hormone concentrations are used to monitor the progress of patients receiving treatment for hyperthyroidism.
 
Concentrations of 14 commonly-requested plasma hormones were measured in octuplicate in each of six subjects to determine their stability when unseparated from red cells for periods up to 1 week. Most of the analytes were stable when stored in this way and although statistically significant changes were recorded, in the great majority of cases the changes seen would have no bearing on the clinical interpretation of the result. In the light of these findings, we would confidently report results of analyses for these hormones in plasma that had remained in contact with red cells at ambient temperature for long periods of time.
 
A 14-year-old boy who presented with debilitating lethargy was shown to have an elevated serum ferritin of 572 microg/L and a C282Y homozygous HFE genotype. Liver iron concentration was measured non-invasively by magnetic resonance imaging, which revealed a liver iron concentration of 59 micromol/g dry weight (children's reference range < 14). The early phenotypic expression was further investigated by screening genomic DNA for the presence of co-inherited mutations in genes responsible for non-HFE haemochromatosis. Coding regions and splice sites in genes encoding hepcidin and haemojuvelin were sequenced and previously described mutations in ferroportin 1 and transferrin receptor 2 genes were screened. Although no mutations were found, the most likely cause for the early expression is the presence of novel mutations or gene(s).
 
We describe a method for obtaining the specific activity of 14C in urea, essential in the measurement of the synthesis rate of a plasma protein in vivo, which is simpler than the original procedure. The principle is the measurement of 14CO2 and NH4+ separately, after incubation with urease. A simple alteration gives samples of 13CO2 for mass spectrometry. The 'recoveries' of 14C and 13C in urea were invariably between 90 and 96% and the CV was 3%.
 
The ¹⁴ C-triolein breath test, a recognised index of fat absorption, and the p-aminobenzoic acid (PABA) test, a ‘tubeless’ test of exocrine pancreatic function, have both been widely used in the diagnosis of malabsorption and exocrine pancreatic insufficiency. This study evaluates the potential of a combination of both tests in the investigation of fat absorption and exocrine pancreatic function. Combination of the tests has become technically feasible because of the introduction of high pressure liquid chromatography as the preferred method of analysis for PABA, and use of p-aminosalicylic acid (PAS) as the marker for PABA absorption and metabolism We studied 25 healthy subjects, 11 patients with exocrine pancreatic disease and 12 patients with gastrointestinal disease. The combined test identified subjects with reduced fat absorption and distinguished subjects with exocrine pancreatic insufficiency from those with an intestinal cause of fat malabsorption. The test could be completed in 7 h and had high patient acceptibility. These findings suggest that the combined ¹⁴ C-triolein breath test and PABA test can be used as a non-invasive, 1-day investigation of fat absorption and exocrine pancreatic function.
 
A simple gas chromatographic technique for the measurement of 16α-hydroxydehydroepiandrosterone sulphate in urine from pregnant women is described. An assessment was made of the effectiveness of the measurement of this oestriol precursor for the antenatal diagnosis of placental steroid sulphatase deficiency. Twenty-two patients whose pregnancies were complicated by subnormal oestrogen excretion for gestation were studied. In nine of these, where placental steroid sulphatase activity was found subsequently in vitro to be normal, the excretion of 16α-hydroxydehydroepiandrosterone sulphate was <27 μmol/24 h. In the remaining 13 patients, in whom postnatal in vitro assay demonstrated absence of placental steroid sulphatase activity, urinary excretion of 16α-hydroxydehydroepiandrosterone sulphate was 59–360 μmol/24 h. The excretion of this metabolite was below the limit of detection (20 μmol/24 h) in 30 uncomplicated pregnancies. It is concluded that urinary excretion of 16α-hydroxydehydroepiandrosterone sulphate >50 μmol/day or a ratio of urinary 16α-hydroxydehydroepiandrosterone sulphate to urinary oestrogen >2·0 correctly identifies, before delivery, those pregnancies in which fetus and placenta are deficient in steroid sulphatase activity.
 
Specimens taken into Becton Dickinson lithium heparin Vacutainers with plasma separator were compared with specimens taken into ordinary Becton Dickinson lithium heparin Vacutainers. After overnight storage, results for sodium, potassium, phosphate, albumin, lactate dehydrogenase, alkaline phosphatase, and urate concentrations showed statistical differences. Only the difference observed in urate concentration is likely to affect clinical interpretation.
 
Protein 1 (P1)/Clara cell 16 kDa protein (CC16, previously named CC10), a potentially immunosuppressive protein secreted by non-ciliated cells of the tracheobronchial epithelium, has been found to be a new useful lung-specific biomarker in several pathological lung conditions. Particularly, urinary P1 (uP1) may reflect the altered lung functions in pneumoconiosis. We investigated the relationship between uP1 values and lung functions in 31 non-smoking pneumoconiotic males (mean age 73 years) with a history of dust exposure work in shipbuilding. The protein was measured using an originally prepared enzyme-linked immunosorbent assay system. The forced expiratory volume in 1 s % (FEV(1.0)%) and % vital capacity (%VC) were tested with a spirometer. The mean values of uP1 were 4.62 +/- 4.82 (mean +/- standard deviation) ng/mol creatinine. A univariable correlation test showed a significant positive correlation between uP1 and %VC (r = 0.356, P = 0.049). Also, a multiple regression analysis, when adjusted for age, disease duration, FEV(1.0)% and %VC, showed a significant correlation of uP1 with %VC (beta = 0.467, P = 0.030). The results suggest that a decreased uP1, corroborated by a decreased %VC, may be the result of damage to secretory cells. Measurement of uP1 may become a possible index of fibrotic changes in pneumoconiosis.
 
Intentional iron overdose appears to be an increasingly common form of attempted suicide. We present a case of iron overdose in a 16-year-old girl who was found unconscious in her bed and brought to our emergency department. The most remarkable diagnostic findings were the patient's comatose condition, divergent eye position and positive Babinski foot pad reflexes. Laboratory tests showed hyperglycaemia and mild metabolic acidosis. A computed tomography scan of the cerebrum showed no signs of intracerebral haemorrhage or elevated intracerebral pressure. Toxicology screening showed no use of acetaminophen, ethanol or drugs of abuse. The patient was stabilized and monitored on the intensive care ward. When she woke up, she confessed to having taken Fero-Gradumet(®). Retrospectively analysed, the serum iron concentration in the first blood sample (seven hours after ingestion) was 62 μmol/L which corresponds with moderate iron intoxication. The patient received whole bowel irrigation with 2 L polyethyleneglycol solution and de-ironing treatment with intravenous deferoxamine 20 mg/kg in eight hours. She was discharged from the hospital after three days in a good clinical condition. Retrospectively, serum hepcidin concentrations were determined and evaluated in conjunction with serum iron concentrations and the installed treatment. Before medical de-ironing interventions were started, we saw that the serum iron concentration in our patient was already declining. At the same time, we observed a sharp increase in the serum hepcidin concentration. After normalization of serum iron concentrations, hepcidin normalized as well.
 
The Dade Behring Syva(R) EMIT (enzyme multiplied immunoassay technique) method for the measurement of tacrolimus in whole blood was evaluated against the Abbott IMx(R) microparticle enzyme immunoassay (MEIA) method. EMIT measures tacrolimus colorimetrically, whereas MEIA measures the analyte using fluorimetry. Both methods incorporate a protein precipitation step prior to measurement. Whole blood specimens were treated by two types of precipitation technique followed by analysis for tacrolimus by either MEIA or EMIT on the Bayer Advia 1650. Linearity and precision were assessed and correlation analysis performed to evaluate the EMIT assay on the Bayer Advia 1650. The EMIT tacrolimus assay was linear over the concentration range 0.0-22.0 micro g/L; the limit of detection was 1.2 micro g/L. Correlation between the Syva EMIT and IMx tacrolimus assays was excellent (r = 0.959) and no significant bias existed between the two methods (mean difference, delta = 0.221 micro g/L). Calibration data for the EMIT assay was stable for a period of 24-48 h on the Advia between runs. The Syva EMIT assay for the measurement of tacrolimus in whole blood is suited for daily routine use on the Bayer Advia 1650.
 
A robust assay for routine measurement of blood-spot 17 alpha-hydroxyprogesterone (17-OHP) concentrations has been developed using a magnetizable, solid-phase antiserum and an 125I-radioligand. The working range of this assay (13.5-500 nmol/L) is well suited for the initial diagnosis of congenital adrenal hyperplasia (CAH) and for monitoring replacement therapy in CAH patients. Data derived from multiple blood-spot samples, collected on two consecutive days, provide 17-OHP profiles. These profiles have been used to construct a chart allowing a rapid visual assessment of the efficacy of replacement therapy in CAH patients. Measurement of 17-OHP in the blood-spots of overtreated patients and accurate determination of normal range values in healthy infants relied on development of a sensitive assay (range 1.7-34 nmol/L). In the blood-spots of normal male (n = 50) and female (n = 50) infants collected 5-7 days after birth, 17-OHP concentrations were 7.62 +/- 2.55 nmol/L and 7.32 +/- 2.87 nmol/L respectively. Retrospective measurement of this steroid in samples from known CAH patients (n = 4), which had values ranging from 224 to 2145 nmol/L, support a role for measurement of blood-spot 17-OHP in high-risk screening programmes.
 
Top-cited authors
Julian H Barth
  • The London Clinic
Brian G Keevil
  • University Hospital Of South Manchester NHS Foundation Trust
Mario Plebani
  • University of Padova
Chris M Florkowski
  • Canterbury Health Laboratories
Laura Jane Owen
  • Bangor University