Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

Calpain and caspase are families of cysteine proteases that have important roles in the initiation, regulation and execution of cell death. The function of both groups of proteases in the progression of apoptotic and necrotic pathways is presented here in the context of a concise overview of regulated cell death. Many of the morphological differences between apoptotic and necrotic processes are thought to be as a consequence of the action of cysteine proteases. Recent studies suggest that caspase and calpain cascades are tightly interrelated and an appreciation of how these proteases cross-talk should enable a greater understanding of how the boundaries between apoptotic and necrotic cell death have become blurred. Furthermore, an assessment of the contribution that caspase and calpain make to human physiology and pathology is provided, with a description of how these proteases can be detected and quantified. Lastly, an evaluation is made of how caspase and calpain activation might be exploited diagnostically.

The measurement of B-type natriuretic peptides in plasma is under intense promotion as a method of screening for heart failure. This article provides a historical context for this contention, and attempts to highlight what practical problems may be encountered in establishing a screening service from a clinical biochemistry standpoint. B-type natriuretic peptide measurements may also prove, in future, to have a significant role in the objective monitoring of treatment for heart failure, and to be a valuable prognostic indicator in patients suffering from acute coronary syndrome.

Hepatic fibrosis is an important consequence of inflammatory disorders affecting the liver, and ultimately progresses to cirrhosis. Here we explore methods for the detection and monitoring of hepatic fibrosis, particularly in hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease and during methotrexate therapy, in all of which progressive fibrosis can develop over a number of years in a minority of patients.

Liver biopsy currently remains the gold standard to assess fibrosis. However, it has several limitations, including manpower issues, cost, risk of patient injury, including mortality and morbidity, observer variability and sampling variation. Several non-invasive diagnostic tests for fibrosis and cirrhosis have therefore been evaluated. The usefulness of a laboratory test for screening for a pathological abnormality such as fibrosis is critically dependent on the prevalence of the pathology in the population under investigation. When the prevalence is expected to be low, screening tests should have a high negative predictive value so that large numbers of patients can be spared the next diagnostic step, namely liver biopsy. For the moment, clinical chemistry laboratories should offer the aspartate aminotransferase alanine aminotransferase ratio, AST/platelet ratio and the Rosenberg fibrosis index as part of their routine service for monitoring the development of hepatic fibrosis.

Background: Abnormal thyroid function tests (serum thyrotropin [TSH], free thyroxine [T₄] and free triiodothyronine [T₃]) are frequently seen in hospitalized patients. Assessment of thyroid function in these patients is difficult. It has been suggested that acutely ill patients may be hypothyroid at the tissue level. Erythrocyte zinc (EZn) has been shown to be increased in hypothyroidism. The aim of this study was to examine EZn as an index of thyroid status of hospital patients.

Methods: In order to assess the thyroid status at tissue level, we measured EZn in 26 healthy subjects, 39 critically ill patients and 19 hospitalized geriatric patients. EZn was measured in young cells, as the effect of illness is likely to be seen in the newly formed cells.

Result: TSH and free T₃ were lower in critically ill patients and serum free T₃ was lower in geriatric patients. EZn in young cells was higher in both patient groups (by 13% and 23%, respectively). EZn in old cells was also higher in the geriatric group.

Conclusion: We conclude that EZn is higher in hospitalized patients, suggesting that these patients may be hypothyroid at the tissue level.

Background: The outcome of parathyroid surgery is often not clear for at least 24 h after the operation. A frozen section does not always distinguish between an adenoma and hyperplasia. Minimally invasive surgical techniques are being refined, so the need for perioperative assurance about the completeness of surgery has increased. The value of intraoperative parathyroid hormone (PTH) measurements in 26 surgical cases undergoing parathyroidectomy has been evaluated.

Methods: Twenty-one patients were diagnosed as having primary hyperparathyroidism, including two patients with multiple endocrine neoplasia type I (MEN I). Five patients had tertiary hyperparathyroidism, including one patient with X-linked hypophosphataemia and four with renal hyperparathyroidism (RHPT).

Blood samples were taken at the onset of surgery, at the time of tumour resection and at 5-min intervals following removal of the tumour. PTH was measured using a PTH Turbo assay on the DPC Immulite analyser.

Results: Current practice suggests that the PTH concentration should fall to less than 50% of the pre-incision value or to less than 50% of the level at the time of tumour resection (time equals zero). PTH levels were therefore monitored at 5-min intervals following removal of the tumour. In most of the case studies PTH followed the suggested pattern, but not when further exploration was warranted to determine if another adenoma was present. In some cases the PTH levels fell by the appropriate margin to deem the procedure a success but at 10 min post-gland excision the PTH began to rise again. Further exploration was required to confirm the continued source of PTH.

Conclusion: We recommend that intraoperative PTH measurements continue until at least 15 min post-gland removal in cases of suspected single-gland disease. A decline in PTH concentration to at least 50% of the pre-incision or time of gland resection levels should be observed. If the PTH remains elevated or rises again after an appropriate decrease in levels, then multigland disease or ectopic sources should be considered.

Caution is recommended in interpreting intraoperative PTH measurements to ensure complete success of the surgical procedure.

Background: Elevated plasma total homocysteine (tHcy) predisposes to vascular disease and results from interactions between genetic and nutritional factors. MTHFR C₆₇₇T increases tHcy in association with low folate. CBS 844ins68 lowers tHcy and negates the raising effect of MTHFR C₆₇₇T in healthy subjects, but it is unclear if this is the case in subjects at high risk of vascular disease. This study examines the effect on plasma tHcy of interactions between these polymorphisms in an at-risk group.

Methods: Blood samples were collected from 376 subjects at increased risk of coronary artery disease. Plasma tHcy and vitamin B₆ were measured by HPLC and red cell folate and serum vitamin B₁₂ were measured by immuno-luminometric assay. MTHFR C₆₇₇T and CBS 844ins68 status was established by standard PCR techniques.

Results: MTHFR TT predisposed to hyperhomocysteinaemia; this was increased in the presence of low folate (P<0.05) and vitamin B₁₂ (P<0.01). An inverse relationship was found between tHcy and folate (r= -0.42, P<0.0001), vitamin B₁₂ (r= -0.26, P<0.0005) and vitamin B₆ (r= -0.25, P<0.01). There was no interaction between plasma tHcy, vitamins or MTHFR C₆₇₇T and CBS 844ins68.

Discussion: In this population at high risk of coronary artery disease, plasma tHcy was determined by vitamin status. This was exacerbated by the MTHFR C₆₇₇T mutation. CBS 844ins68 did not influence tHcy and did not negate the tHcy-raising effect of MTHFR C₆₇₇T.

Background: Increasing activity in cholesterol lowering is placing increasing demands on lipid clinics to be able to cope with the increase in demand. A combination of interventions has been used to improve laboratory testing, focus interpretative results and provide educational and advisory facilities for general practitioners in order to increase shared care of many potential clinic patients.

Methods: Retrospective study of clinic waiting times, results in patients managed on a shared care basis, and overall clinical activity over a three-and-a-half-year intervention period between March 2001 and August 2004 in a single-consultant, single-centre secondary care clinic serving three primary care trusts, covering a population of approximately 270,000 people. The interventions involved a change to the laboratory request form, outreach educational meetings and promotion of use of 'written advice only' for certain patients as an alternative to direct clinic referral. The main outcome measures were percentages of patients followed up in primary care, change in cholesterol and triglyceride results after advice, and clinic waiting time.

Results: A total of 520 patients were referred over three and a half years, either to be seen or for written advice only. About 291 of these were handled by advisory letters. In all, 90% of these patients were already receiving or had received lipid-lowering therapy at the time of referral. 98% of patients were followed up by their general practice post-advice. Cholesterol and triglyceride concentrations fell by 23% and 39%, respectively, post-advice. Waiting times fell from a peak of 35 weeks before the interventions to an average of three weeks after. This fall has been maintained over the three-and-a-half years the intervention has run.

Conclusions: The interventions have resulted in a large fall in clinic waiting times, improvement in lipid results, and high rate of general practice follow-up, all of which have been sustained in the long term.

Background: Dehydroepiandrosterone sulphate (DHEAS) is a steroid that is increasingly being recognized as a potential drug of abuse in many countries. This is due to its reputation as a hormone that may be able to retard the ageing process.

The measurement of DHEAS is useful in the diagnosis of medical conditions such as congenital adrenal hyperplasia and polycystic ovary syndrome. Thus, a liquid chromatography-tandem mass spectrometry method has been developed to determine DHEAS concentrations in human serum.

Method: The chromatography was performed using a Waters^TM 2795 Alliance HT LC system coupled to a Mercury Fusion-RP column fitted with a SecurityGuard^TM column.

Results: DHEAS and the internal standard, deuterated DHEAS, both had a retention time of 1.5 min. The transition determined by the Micromass Quattro^TM tandem mass spectrometer for DHEAS was m/z 367.3>96.7 and for the internal standard m/z 369.3>96.6.

The method was linear up to 20 µmol/L; the lower limit of detection and the lower limit of quantitation were both 1 µmol/L. The intra- and interassay imprecision were <11% over a concentration range of 1-18 µmol/L for the in-house quality control and <12% for the intra- and interassay imprecision for the Bio-Rad Lyphocheck QC.

Conclusion: The measurement of DHEAS by liquid chromatography-tandem mass spectrometry is robust and has a simple sample preparation procedure with a rapid cycle time of only 4 min.

Background: Ascorbic acid can interfere with methodologies involving redox reactions, while comprehensive studies on main chemistry analysers have not been reported. We therefore attempted to determine the interference of ascorbic acid with analytes on the Beckman Synchron LX20^®.

Methods: Various concentrations of ascorbic acid were added to serum, and the serum analytes were measured on the LX20.

Results: With a serum ascorbic acid concentration of 12.0 mmol/L, the values for sodium, potassium, calcium and creatinine increased by 43%, 58%, 103% and 26%, respectively (P<0.01). With a serum ascorbic acid concentration of 12.0 mmol/L, the values for chloride, total bilirubin and uric acid decreased by 33%, 62% and 83%, respectively (P<0.01), and were undetectable for total cholesterol, triglyceride, ammonia and lactate. There was no definite influence of ascorbic acid on analytical values for total CO₂, urea, glucose, phosphate, total protein, albumin, amylase, creatine kinase, creatine kinase-MB, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total iron, unbound iron-binding capacity or magnesium.

Conclusions: Ascorbic acid causes a false increase in sodium, potassium, calcium and creatinine results and a false decrease in chloride, total bilirubin, uric acid, total cholesterol, triglyceride, ammonia and lactate results.