Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

The Joint British Societies' recommendations for coronary heart disease (CHD) prevention have been updated in the light of recent developments. Key features of the new guidelines include focusing on cardiovascular disease (CVD) prevention rather than CHD and adopting strategies that target high-risk groups, with equal priority being given to all those with established CVD or diabetes mellitus. Subjects without CVD but with a risk factor profile resulting in an absolute risk of developing CVD -20% over 10 years should be included in these strategies and CVD prevention should also be applied to subjects with particularly unfavourable single risk factors including familial dyslipidaemias, such as familial hypercholesterolaemia, hypercholesterolaemia when the total cholesterol:HDL cholesterol ratio is -7.0, hypertension when the blood pressure (BP) -160 mmHg systolic or -100 mmHg diastolic or lesser degrees of hypertension when there is end-organ damage. The optimal target for total cholesterol is <4.0 mmol/L and for LDL cholesterol <2.0 mmol/L, or a reduction of 25% in total cholesterol and a 30% reduction in LDL cholesterol, whichever achieves the lower absolute level. The optimal BP target is <140 mmHg systolic and <85 mmHg diastolic pressure with lower targets in subjects with CVD, diabetes or chronic renal failure. If these conditions are present, treatment should aim to achieve <130 mmHg systolic and <80 mmHg diastolic pressures.

Gilbert's syndrome (GS) is a benign and inherited state characterized by mild, lifelong, unconjugated hyperbilirubinaemia in the absence of haemolysis or evidence of liver disease. Its molecular basis, mutations in the TATA box upstream of the uridine diphosphoglucose glucuronyltransferase gene, leads to impaired bilirubin glucuronidation. This synopsis outlines the pathophysiology and investigation appropriate for this innocent anomaly.

Ocular light plays a key role in human physiology by transmitting time of day information. The production of the pineal gland hormone melatonin is under the control of the light-dark cycle. Its profile of secretion defines biological night and it has been called the 'darkness hormone'. Light mediates a number of non-visual responses, such as phase shifting the internal circadian clock, increasing alertness, heart rate and pupil constriction. Both exogenous melatonin and light, if appropriately timed, can phase shift the human circadian system. These 'chronobiotic' effects of light and melatonin have been used successfully to alleviate and correct circadian rhythm disorders, such as those experienced following travel across time zones, in night shift work and in circadian sleep disorders. The effectiveness of melatonin and light are currently being optimized in terms of time of administration, light intensity, duration and wavelength, and melatonin dose and formulation. The aim of this review is not to replicate information that has been reported in a number of reviews of the human circadian timing system and the role of melatonin and light, but rather to extract findings relevant to the field of clinical biochemistry.

Background: We have developed a new thiopurine S-methyltransferase (TPMT) phenotyping method that measures TPMT activity in whole blood. To evaluate this assay, we compared it with conventional TPMT phenotyping, which uses a red blood cell (RBC) lysate and genotyping for analysis of common TPMT mutations.

Methods: Whole-blood and RBC lysates were prepared from 402 patients' samples received for routine analysis. The TPMT activity of lysates was determined using 6-thioguanine as substrate with high-performance liquid chromatographic (HPLC) analysis and fluorimetric detection. DNA was extracted from buffy coats using phenol-chloroform extraction. A multiplex amplification refractory mutation system (ARMS) strategy was used to screen for the common TPMT mutations TPMT*2 and TPMT*3 (TPMT*3A, TPMT*3C and TPMT*3D).

Results: TPMT activities were higher in the whole-blood (mean TPMT activity 51 nmol 6-MTG/gHb/h) compared with the RBC lysate (37 nmol 6-MTG/gHb/h). Overall, concordance with TPMT genotypic analysis was 97% for both the new whole-blood and standard RBC lysate methods. Between low TPMT activity and heterozygotes, both phenotypic methods gave a concordance of 79%.

Conclusion: Using multiplex ARMS testing for TPMT*2 and 3*C mutations to define the cut-off between low and normal TPMT activity, we have demonstrated that the new whole-blood TPMT phenotyping method performs as well as the conventional RBC lysate assay. This new method overcomes the need to prepare a RBC lysate, a process which is time consuming and increases analytical variation. The resulting assay is better suited to a regional or national TPMT phenotyping service.

Background: Reflective testing incorporates the clinical judgement, knowledge and experience of an individual biochemist to request additional tests appropriate to the clinical scenario. Despite being vigorously debated within the biochemistry profession, little is known about how the clinicians directly involved in patient care feel about it. We have conducted a survey to elicit our service users' opinion of reflective testing.

Methods: Ten clinical scenarios, each involving the possible addition of a specific test, were circulated to 520 hospital doctors and 152 general practitioners. The four response options were to add further tests, phone and discuss the case, add a comment to the original results or do nothing.

Results: A total of 216 (32%) responses were received. Overall, the majority were in favour of 'adding on' free triiodothyronine (86%), γ-glutamyltransferase (78%), lipid profile (59%), thyroid peroxidase antibodies (63%), pituitary hormones (58%), troponin (55%) and serum electrophoresis (68%) in the given scenarios. However, only 30% would like a pregnancy test added and only 45% a prostate-specific antigen added without prior consultation. The response differed according to grade and specialty.

Conclusions: Reflective testing is generally welcomed by our service users, provided the nature and implications of the specific test is considered in addition to the full clinical scenario.

Introduction: Reflective testing refers to the practice of adding on tests by laboratory staff. Little is known about what patients think of this practice.

Methods: We surveyed patients attending a general practice surgery and patients attending hospital outpatient clinics. We sought their views about the practice of adding on tests and about the information they received from requesting clinicians about their investigations.

Results: In both groups of patients, large majorities favoured an approach in which relevant additional tests are performed without consulting the requesting clinician or patient first. Most patients also felt that the requesting clinicians had provided a satisfactory explanation about what tests were to be performed and why.

Conclusion: Most patients are content to let NHS professionals add on relevant tests if this is felt to be in their interest.

Background: Discretionary laboratory requesting of serum protein electrophoresis (SPE) is widespread throughout the UK despite limited evidence to support clinical usefulness. We aimed to examine the clinical usefulness of discretionary SPE requesting on serum samples with globulins > 45 g/L by documenting the number of discretionary tests performed and the number of paraproteins identified in an audit period, by determining if such findings led to further investigation/specialty referral, and by identifying the final diagnosis and subsequent management of these patients.

Methods: A retrospective one-year audit from July 2002 to June 2003 was carried out using information from biochemistry databases and review of medical records.

Results: Of 1332 discretionary SPEs performed in one year, 72 paraproteins were identified (5.4%). Further clinical information could be obtained in 66 cases. Appropriate follow-up occurred in 84% of cases. The most common diagnosis was monoclonal gammopathy of undetermined significance (56%). Eighteen percent of patients with an identified paraprotein were diagnosed with B-cell malignancies that warranted treatment. A further 6% progressed to requiring treatment during follow-up.

Conclusion: Using screening criteria of serum globulins > 45 g/L, one in 20 discretionary laboratory requests resulted in a clinically relevant finding. These results suggest such requesting is worthwhile and should aid future debate on the appropriateness of this practice.

Background: We evaluated the diagnostic discrimination of a new micro-flow cell device (Nanoduct^®) which measures sweat conductivity in situ at a regional referral centre for cystic fibrosis (CF).

Methods: The device was evaluated in comparison to the measurement of sweat chloride with the established quantitative pilocarpine iontophoresis test (QPIT) and extended in a number of patients to conductivity measurements in liquid sweat collected with the Macroduct^® system. Sweat testing was conducted simultaneously on patients referred for diagnostic sweat testing, on patients known to have CF and on adult volunteers. The intra-individual variability, the failure rate and diagnostic accuracy were determined.

Results: A total of 110 tests were performed on 100 individuals, 36 of whom had classical CF and six of whom had non-classical CF. The Nanoduct system produced a false negative result in one quarter of the patients with classical CF. Moreover, conductivity was negatively biased compared with chloride in this group. Repeat testing of the false negatives using a new batch of sensors and/or measuring conductivity in liquid sweat collected with the Macroduct device gave accurate diagnostic discrimination indicating that the original sensors were faulty. Photographic examination confirmed that a batch of sensors were defective.

Conclusions: Our experience suggests that the prototype microflow cell conductometric device cannot be used for the diagnosis of CF due to the high false negative rate. As a consequence of this study, the manufacturers have implemented a pre-testing system to quality control the sensors prior to issue.

Background: In the context of post-transplant immunosuppression, cyclosporine A (CSA) is dose adjusted in accordance with whole blood drug monitoring. While currently available immunoassay systems primarily target the parent drug, cross-reactivity results in the detection of the major circulating CSA metabolites, though their contribution to both immunosuppression and toxicity remain unclear. This study examines the relationship of CSA metabolites to hepatic and renal dysfunction and the incidence of graft-versus-host disease (GvHD) through parallel assaying of parent drug and drug/metabolites expressed as a metabolite ratio (Cp:mR).

Method: Sequential pre-treatment (trough) whole blood samples (n = 527) were collected from 31 allo-stem cell transplantation (SCT) recipients. Both parent drug and drug/metabolite levels were determined using the Abbott fluorescence polarization immunoassay.

Results: The average mean Cp:mR was significantly higher in patients with hepatic (P = 0.004) and renal dysfunction (P = 0.004) than in those without. Significantly higher Cp:mR were also found in patients with grades II-IV GvHD (P = 0.001) than were observed in patients who did not experience significant GvHD. When measured prospectively, an increasing Cp:mR predated the rise in serum creatinine concentration by a median of two weeks.

Conclusions: This study demonstrates a clinically useful CSA metabolite ratio that shows association with hepatic and renal dysfunction and with GvHD. The measure can be used to predict those patients on CSA therapy who are likely to develop renal dysfunction.

Background: Serum testosterone remains the most important investigation in the diagnosis of androgen deficiency in men. Most of the circulating testosterone is bound to albumin and sex hormone-binding globulin (SHBG), whereas free testosterone accounts for approximately 2% of total testosterone. Because direct measurement of free testosterone is impractical in routine practice, several equations are used to provide clinically useful estimates of free testosterone concentration. This study aimed to (1) obtain locally derived reference limits for total testosterone and calculated free testosterone (CFT) concentrations, and (2) critically evaluate the equations commonly used to estimate free testosterone.

Methods: Serum total testosterone, SHBG and albumin were assayed in morning blood samples obtained from 126 healthy men (aged 20-45 years) known to have normal semen analysis. CFT concentrations calculated using four published methods (i.e. the Sodergard, Nanjee-Wheeler, Vermeulen and Ly-Handelsman equations) were compared with one another and the free androgen index.

Results: Reference intervals for total testosterone and CFT by the Vermeulen equation were 9.4-31.0 nmol/L and 0.245-0.785 nmol/L (2.5-97.5 percentile), respectively. CFT values varied considerably with the four equations examined. Mean biases ranged from 5.8 to 56.0%; the Nanjee-Wheeler and Ly-Handelsman equations yielded positive and negative biases, respectively, against the other equations. Free androgen index was shown to correlate poorly with CFT (r ² = 0.21-0.46) and over-estimate the CFT at low SHBG concentrations.

Conclusions: We have used various equations to derive reference ranges for CFT in healthy men aged 20-45 years. We suggest that CFT be incorporated into the investigation regimen for suspected hypogonadism when total testosterone results are equivocal.

Background: The purpose of this study was to determine a cut-off above which a result is considered abnormal for 'spot' calcium-to-creatinine ratio (Ca/Cr) in urine in the paediatric age group. While the cut-offs are not well established, there are many published reports of urine Ca/Cr excretion ratio in the literature. Rather than undertaking a difficult, time-consuming, expensive, and ultimately redundant study, an analysis of these published works was undertaken to determine urine Ca/Cr cut-offs for the laboratory.

Methods: Fourteen studies reporting urine Ca/Cr ratio available in the literature were reviewed. Each study was abstracted for several characteristics. The data were plotted and the line of best fit describing the data was determined. Using the function describing the line, cut-offs by pertinent age were recalculated. These cut-offs were then compared to the original data and to in-house data.

Results: The function describing the line of best fit was y = -0.3175 ln(x) +1.46 (y = Ca/Cr in mmol/mmol and x = age in years), r ² = 0.85. After consultation with the paediatric nephrologists and comparison with our in-house data, cut-offs for age groups of <1, 1-<2, 2-<5, 5-<10, and 10-18 years in mmol/mmol of 1.50, 1.25, 1.00, 0.70, and 0.60, respectively, were derived.

Conclusions Using data from 14 published studies, suitable spot urine Ca/Cr cut-offs from birth to 18 years of age were determined.

Background: We compared a widely used immunoassay method for urine free corticoids with a high-performance liquid chromatography (HPLC) method for the investigation of Cushing's syndrome.

Methods: Urine free corticoid analysis using an Immulite:2000 automated immunoassay analyser and urine free cortisol analysis by a specific HPLC method on 39 patients with an elevated baseline immunoassay 24 h urine free corticoid result who were referred from 2000 to 2004.

Results: Reference ranges for urine free corticoids by immunoassay and for urine free cortisol by HPLC were <900 and <170 nmol/day, respectively. Of the 39 patients with an elevated immunoassay urine free corticoid result, Cushing's syndrome was surgically proven in 22 patients, giving a positive predictive value of 56%. Cushing's syndrome was excluded in 12 of the remaining 17 patients by applying the specific HPLC method and at least one of the following: a clinical evaluation which was not suggestive or adequate suppression of plasma cortisol following dexamethasone. Urine free cortisol analysis by HPLC gave a positive predictive value of 81% for Cushing's syndrome.

Conclusions: Although immunoassay has a low specificity for urine cortisol, it is a widely available test which serves as a valid initial investigation of Cushing's syndrome.

Self-monitoring of blood glucose (SMBG) is a cornerstone of diabetes care. However, the effectiveness of any glucose-monitoring programme depends on the ability to integrate SMBG into a program of self-care and therapeutic decision-making. Because the accuracy of SMBG is instrument dependent, we analysed 45 heparinized whole blood specimens using four marketed portable glucose meters to evaluate whether their precision and accuracy would be efficient and safe for clinical use. All measurements were standardized and performed by a single expert health-care professional at the same clinical chemistry laboratory. Results were compared with those obtained on the same plasma samples by the hexokinase method on a secondary reference analyser and further analysed according to the error tolerance criteria and the current American Diabetes Association (ADA), Clinical Laboratory Improvement Amendments (CLIA), and National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The within-run imprecision ranged from 2.2% to 3.2%. Passing and Bablok regression analysis yielded slope values from 0.93 to 1.07 and correlation coefficients between 0.994 and 0.998. When compared with the secondary reference analyser, mean variations were between -4.9% and 14.1%, fulfilling in three out of four cases the 5.5% current desirable analytical quality specifications for total error. Nevertheless, when considering the two standard deviations level of this bias, several results exceeded this limit. Although three out of four devices tested achieved or came closer to the NCCLS C30-A2, CLIA and error tolerance targets, none of them met the current analytical ADA thresholds. Despite the acceptable analytical performances, we demonstrated that standardization and harmonization of results in SMBG have not been fully achieved.

The differential diagnosis of abdominal pain with associated hyponatraemia should include acute intermittent porphyria. Development of hyperthyroidism in a patient with latent porphyria may precipitate an acute attack and increase disease severity. Treatment of hyperthyroidism may prevent recurrent episodes.

Context: Ten percent of serum total cortisol (TC) is unbound; the remainder is bound to cortisol-binding globulin (CBG) and, to a lesser extent, albumin. CBG concentrations can drop significantly, particularly in critical illness, resulting in a low TC although the free, active, cortisol may be normal or increased. In the context of a low CBG, the diagnosis of pituitary-adrenal insufficiency with measurements of TC is difficult.

Objective: To remind clinicians of the difficulty in interpreting TC when the CBG is low, the circumstances when CBG concentrations may decrease and that measurement of CBG and calculation of the free cortisol index can help in the assessment of pituitary-adrenal reserve.

Case: We present two patients at risk of primary and secondary adrenal insufficiency with a poor response to 250 μg Synacthen. In both cases we confirmed low CBG concentrations but a normal free cortisol index (FCI), confirming normal pituitary-adrenal reserve.

Intervention: In case one, we have been able to avoid long-term steroid replacement therapy. We continue to reduce the steroid dose in case 2 but have been limited by the need for high-dose steroid treatment for exacerbations of the patient's airways disease.

Conclusion: The use of TC in the assessment of the hypothalamic-pituitary-adrenal (HPA) axis may give rise to misleading results if the CBG concentration is low. The FCI may be a better marker of pituitary-adrenal reserve in these subjects. Clinicians should be cautious when interpreting abnormal cortisol results and we emphasize the importance of good clinical assessment.