Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

Lipoprotein(a) [Lp(a)] was first identified by Kare Berg in 1963 as an LDL variant. Subsequent studies showed it to be a family of lipoproteins whose wide density range overlaps those of low and high density lipoproteins. Although clinical interest in this particle was limited for many years, it was stimulated again in the late 1970s with the discovery that high concentrations of Lp(a) may be associated with coronary heart disease (CHD). Numerous follow-up studies confirmed that Lp(a) is an independent risk factor for CHD and that raised values are associated with the severity of CHD and with an increased risk of future cardiac events. The relative risk of myocardial infarction has been reported to be 1·75-fold higher when Lp(a) levels are raised above 300 mg/L. Clinical interest in Lp(a) has stimulated the development of commercial methods for its determination. Currently, over 15 methods are available, varying in principle, antibody specificity, standardization and method of detection. This personal view considers the impact that different methodological approaches have had on the interpretation of Lp(a) levels in clinical studies and the initiatives now being taken to standardize the measurement of this heterogeneous particle.

This review will primarily address methodological aspects of homocysteine measurement, highlighting changes since the subject was comprehensively reviewed in 1993, and will bring together practical information of relevance to laboratory planning or to providing an analytical service including homocysteine measurement. Because the physiological state of the patient during blood collection is often neglected, and because it has become apparent that the bloodsampling procedure may contribute considerably more variation to measurement of homocysteine than the total variation of the analytical procedures, the review will pay particular attention to the critical roles of controlled blood collection and sample processing.

The soluble adhesion molecules P-selectin (sP-selectin) and intercellular adhesion molecule-1 (sICAM-1) are derived from platelets and endothelial cells. Circulating concentrations of these soluble adhesion molecules are raised in patients with atherosclerosis and following percutaneous transluminal coronary angioplasty (PTCA). We have investigated the effects of vitamin E supplements (800 IU/day) on circulating plasma ICAM-1 and P-selectin levels pre- and post-PTCA. Patients, randomized to group, were pre-treated with vitamin E or placebo (soybean oil) for 1 month before routine PTCA. Plasma sICAM-1 and sP-selectin were measured by enzyme-linked immunosorbent assay on blood taken immediately pre- and post-PTCA. Total protein and α-tocopherol were measured on the same samples. Plasma α-tocopherol concentrations increased in patients receiving vitamin E: 19·1 (1·5) [mean (standard error of the mean, SEM)] mg/mL post-PTCA versus 13·9 (0·6) mg/mL pre-PTCA (n=23; P<0·01). Plasma sP-selectin and sICAM-1 levels were not significantly increased following PTCA in the vitamin E group. Pre-angioplasty mean (SEM) plasma sP-selectin concentration in the vitamin E group was 8·83 (0·97) ng/mg protein; the corresponding mean post-angioplasty value was 9·34 (0·89) ng/mg protein (P=0·85). The mean (SEM) pre-angioplasty sICAM-1 concentration in this group was 2·18 (0·24) ng/mg protein, and was 2·20 (0·23) ng/ mg protein following angioplasty (P=0·84). In the placebo group (n=24) there was a significant increase in mean (SEM) sP-selectin concentration following angioplasty, from 7·48 (0·73) to 9·70 (0·78) ng/mg protein (P<0·05). The change (mean, SEM) in plasma sP-selectin concentration following angioplasty was significantly greater for the placebo group [2·22 (0·50) ng/mg protein] than for the group receiving vitamin E [0·50 (0·50) ng/mg protein] (P<0·02). This difference remained significant (P<0·05) even after adjustment for pre-angioplasty P-selectin concentrations. Mean (SEM) plasma sICAM-1 concentrations remained unchanged following angioplasty [preangioplasty: 2·16 (0·20) ng/mg protein; post-angioplasty: 1·97 (0·13) ng/mg protein]. Vitamin E may therefore limit platelet or endothelial activation during PTCA.

Diabetic patients have higher cardiovascular risk than non-diabetic subjects, and fibrinogen has been reported to be one of the independent predictors for diabetic vascular complications. We examined 101 subjects (24 men, 77 women) who were newly diagnosed with type 2 diabetes. Thirty non-diabetic subjects were recruited as controls. The mean (standard deviation, SD) baseline fibrinogen concentrations in men and women were 3·87 (1·17) and 3·42 (1·00) g/L, respectively (not significantly different). Of these 101 subjects, 70 were treated with diet alone and 31 were treated with oral agents. After a mean (SD) follow-up period of 2·38 (0·63) years, there was an 18·5% increase in the fibrinogen concentration from 3·53 (1·06) g/L at baseline to 3·97 (1·07) g/L at follow-up (P<0·001). The normal subjects had a mean (SD) follow-up period of 1·17 (0·38) years, and showed no significant change in their fibrinogen concentrations from 3·49 (0·90) g/L at baseline to 3·15 (0·61) g/L at follow-up. In the 101 diabetic subjects, only changes in plasma triglyceride correlated with the changes in fibrinogen concentration. Using multivariate analysis with age, sex, duration of diabetes, baseline and changes in body mass index, blood pressure, glycaemic and lipid parameters as independent variables (R ²=0·106, F=6·840, P=0·011), triglyceride (β=0·282, P=0·011) was identified as the only independent variable that predicted the changes in the fibrinogen concentrations. Improved glycaemic control was not accompanied by a reduction in plasma fibrinogen concentration.

We investigated the use of soluble transferrin receptor (sTfR), together with more traditional measurements such as iron, transferrin and ferritin, to assess iron status in 70 adult cystic fibrosis patients. sTfR concentration was measured using particle-enhanced immunonephelometry. We found that 69% of subjects as determined by transferrin saturation had iron deficiency, but only 29% as determined by sTfR and 11% as determined by ferritin. We found a significant correlation between C-reactive protein (CRP) and both ferritin (r=0·38, P=0·0001) and transferrin saturation (r = -0·54, P<0·0001). In addition, because the CRP concentration was elevated in 64% of subjects, we feel that the transferrin saturation was overestimating, and the ferritin underestimating, iron deficiency in these patients. The sTfR concentration, on the other hand, is unaffected by the acutephase response and was therefore thought to be the most useful test for detecting iron deficiency in this group of patients.

Smoking is a major risk factor for periodontal disease, causing bone and tooth loss. Effective management of many oral conditions requires accurate information about smoking. We describe a disposable, 10-min, near-patient saliva test to monitor cigarette smoking. A plastic device contains the dried reagents to measure nicotine and its metabolites by a colorimetric assay. The device can be used to give a semi-quantitative assessment of tobacco consumption by observing a colour change and comparing it to a reference chart. Alternatively, the test can be quantified by measuring the light absorbance with a colorimeter and determining the concentration of nicotine metabolites by reference to a cotinine standard. This method correlates with reported daily cigarette consumption (r=0·62, P< 0·0001) and nicotine yield of cigarettes smoked (r=0·35, P<0·01) and compares favourably with cotinine, as measured independently by radioimmunoassay (r=0·57, P<0·005). The method provides a simple-to-use, inexpensive way of monitoring tobacco consumption in dental surgeries.

Blood and other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), so that isolation of DNA is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances and makes DNA isolation from blood unnecessary for PCR. When this reagent was included in the PCR mixture, DNA fragments of the-globin gene could be efficiently amplified directly from human blood samples treated with various anticoagulants or PCR-inhibitory substances. We confirmed the usefulness of this cocktail by examining a large number of blood samples with various PCR primer sets. In addition to fresh blood, this method enabled PCR amplification from blood samples stored at 4 C, -20 C or -80 C for a minimum of 1 year.

We describe a 6-year-old boy with histologically confirmed pancreatoblastoma and a markedly elevated serum α-fetoprotein (AFP) concentration. Due to local tumour invasion, cytotoxic chemotherapy was given to debulk the tumour before attempting surgical resection. Serial serum AFP concentrations were measured on this patient. During chemotherapy there was a > 95% fall in total AFP. Tumour-specific variants of AFP, detected by isoelectric focusing, also disappeared during chemotherapy but recurred when chemotherapy was withdrawn. It is suggested that although there was no change in overall size of the tumour, as assessed by various imaging techniques, the changes in serological markers may indicate that the treatment did in fact cause considerable tumour necrosis. AFP and its variants may be useful markers of tumour response in patients with pancreatoblastoma. The expression of AFP and its variants in pancreatoblastoma may be related to the embryonic origin of the pancreas.

Lower reference limits for vitamin B₁₂ are often defined solely in relation to haematological criteria. This may be misleading and there is evidence indicating that biochemical anomalies should also be considered. In 50 patients we measured creatine, as the major product of B₁₂-mediated remethylation, to see if this helps the definition of the B₁₂ reference interval and to investigate the possible effect of low B₁₂ on essential transmethylation. Vitamin B₁₂ values were grouped into six fractiles covering the range 50-500 ng/L; the corresponding creatine results were assessed by analysis of variance giving F=0·94 and a significance of 0·466. Although no correlation between B₁₂ and creatine was found, and therefore no obvious effect on transmethylation of low B₁₂ concentration, this must be interpreted with respect to methionine availability. Other factors indicate that biochemical perturbations should be taken into account when defining vitamin B₁₂ reference intervals.

A method for the simultaneous analysis of urine for the major drugs of abuse is described. The analytical procedure uses solid-phase extraction (SPE), gas chromatography-mass spectrometry (GC-MS) and a semi-automated identification process. It allows simultaneous extraction, derivatization and analysis of acidic, neutral and basic drugs from urine. Urine samples were subjected to enzymatic hydrolysis followed by SPE using Bakerbond narc-2 columns. The eluant was selectively derivatized with N-methyl-bis-trifluoroacetamide (MBTFA) and N-methyl-N-trimethylsilyltrifluoroacetamide + 1% trimethylchlorosilane. Analysis was performed using a GC-MS system operating in full scan mode. A simple macro programme was written to enhance the mass spectra identification capabilities of the MS software by producing extracted ion chromatograms (EIC) for the drugs of interest. Once a suspect compound was indicated by EIC, the mass spectrum of the compound was searched manually against reference libraries for positive identification and the retention time checked against that of the standard. This procedure has increased both the amount and the reliability of information given to clinicians without increasing the cost per sample. The system has been in routine operation for 24 months, processing up to 40 urine samples per day, with a usual turn-around time of 48 h.

We tried to establish the reference values of plasma lipoprotein (a) [Lp(a)] concentration in phenotype groups of apoliprotein(a) [apo A] classified by a new criterion. Lp(a) concentration was determined by latex agglutination immunoassay, and apo A was analysed by electrophoresis in sodium dodecyl sulphate-polyacrylamide gel and a Western blotting technique. According to the relative mobility to the apo B-100 band, apo A was classified into 11 isoforms, i.e. F, B, and S1-S9, and the phenotype was defined by their apparent combination. The frequency ratio of single-band versus double-band was approximately 2:1. In 382 cases of single-band, the most frequent phenotype was S5 (24·3%), followed by S4 (17·3% ), S6 (15·4% ) and S3 (14·4%). In 181 cases of double-band, S5/S6 phenotype was observed most frequently (12·2%), followed by S4/S5 (10·5%) and S3/S6 (7·2%). The reference value was determined between antilogs of the mean ± 1·96 standard deviation by logarithmic transformation of all observed values for individual phenotype cases. These results suggest that the reference values shown to be variable with apo A phenotypes should be useful for evaluating Lp(a) values in diagnosis of atherosclerosis.

We have developed a novel analytical method for serum lipoproteins using a commercially available capillary electrophoresis apparatus, BioFocus 3000 (Bio-Rad Laboratories Co Ltd, USA). The analytical principle is isotachophoresis (ITP), using a carrier ampholyte, BioLyte 7/9, as a spacer ion. The method allows a much higher resolution of lipoproteins than of amino acid mixtures. Serum lipoproteins are normally separated into 13-15 peaks, including some shoulder peaks. The reproducibility of repeated analysis within a day was relatively good with the coefficient of variation within the range 0·9-1·1%. VLDL, LDL and HDL prepared by discontinuous density ultracentrifugation could be further separated by capillary ITP. This high-resolving ability of our method enabled detection of small amounts of abnormal lipoprotein species. For example, small dense LDL, which is thought to be an atherogenic lipoprotein, could be detected within the LDL group peak. Moreover, an abnormal HDL, apolipoprotein E-rich HDL, was also detected by a single analysis. These findings suggest that our capillary ITP method is a useful means for detailed analysis of lipoproteins and thus for clinical diagnosis of hyperlipoproteinaemic subjects.

We describe a radioimmunoassay (RIA) for total leptin and a gel filtration procedure for the separation of free and bound leptin in human serum. The RIA, based on a locally prepared antibody, has a minimum detection limit of 0·9 ng/mL, a working range (CV< 10%) of 2·5-50 ng/mL, inter-assay precision of 10·2, 7·2 and 8·9%CV at 7·9, 15·4 and 30·0 ng/mL, respectively, 94% recovery of exogenous leptin (range 81·1-120·6%), exhibited parallelism and demonstrated no significant cross-reactivity or interferences. A difference plot of results from this method and those from a commercially available kit (Linco Research) demonstrated satisfactory agreement up to concentrations of 50 ng/mL total leptin, with no significant bias. A gender-dependent correlation was obtained between body mass index (BMI) and total leptin (r=0·91, P<0·001, n=75 for men; r=0·79, P<0·001, n=72 for women), with women having higher leptin concentrations than men for any given BMI. Gel filtration studies (inter-assay precision: 4·7%CV, n=18) demonstrated that a variable fraction (between 10% and 40%) of total leptin in serum was bound with high affinity (K _eq=1·0-1·45x10⁹ L/mol) to a non-albumin, non-lipid macromolecule. Binding affinities were found to be similar irrespective of gender or fat mass. A significant positive correlation between free or bound leptin concentrations and BMI was obtained for both men and women (r=0·87-0·94); free and bound leptin concentrations were also significantly higher in women ( P < 0·01) than in men for any given BMI, and higher in obese (P<0·01) than in lean individuals. We conclude that leptin 'resistance' associated with obesity cannot be accounted for by reduced free leptin concentrations in serum and that the methods described are suitable for the investigation of total, free and bound leptin for both clinical and research purposes.

The diagnosis of hypoglycaemia depends upon the demonstration of a low blood glucose concentration during a spontaneous symptomatic episode. Glucose monitoring devices may misdiagnose many healthy individuals with nonspecific symptoms as having hypoglycaemia. We present three illustrative cases of 'glucose meter non-hypoglycaemia'.