Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

Most hospital laboratories estimate the concentration of total circulating testosterone using a non-extraction method on an automated multi-channel immunoassay analyser supplied by a small number of multi-national diagnostic companies. Although these platforms offer advantages of quick turnaround times, small volume sampling and random access analysis, proficiency testing schemes suggest the quality of results produced remains similar to that of the early manual radioimmunoassay. An estimate of the bioavailable, non-sex hormone binding globulin (SHBG) bound fraction of circulating testosterone, be that the free or the free plus albumin-bound, may be a better index of gonadal status than total testosterone alone, especially when a borderline hypogonadal level of total testosterone is found, and may avoid misclassification of hypogonadal or eugonadal men. Free or bioavailable testosterone may be calculated or measured. The free androgen index may not give a true reflection of androgen status in men. In the interpretation of serum testosterone concentrations with results >40 nmol/L, the possibility of exogenous administration or abuse needs to be considered. The marked diurnal rhythm in total testosterone should also be taken into account. There may be a diminution of testosterone secretion with advancing age, but the great majority of older men have a circulating total testosterone concentration well within the accepted reference intervals established for younger men. As testosterone concentration may fluctuate markedly both seasonally and from day to day, it may be judicious to measure levels on more than one occasion.

Provided that estimates of serum testosterone are unequivocally eugonadal (12.5-40 nmol/L) or hypogonadal (<7.0 nmol/L), results produced by routine automated immunoassays will in all probability give a satisfactory assessment of androgen status in men.

Routine biochemical assessment of gonadal function in men should include measurement of early morning luteininzing hormone, follicle stimulating hormone, prolactin and SHBG together with total testosterone, and if necessary some estimate of bioactive testosterone.

Clinical Guideline CG3 from the National Institute for Health and Clinical Excellence (NICE) makes recommendations on appropriate clinical practice in preoperative testing for elective surgery. Unfortunately, there is minimal evidence on which the guidelines could be based and therefore they were constructed on the basis of professional opinion. This resulted in the construction of a decision matrix of Byzantine complexity built on foundations of sand: surgical risk is estimated using an unvalidated ad hoc risk estimation method; anaesthetic risk is estimated using the American Society of Anesthesiologists (ASA) risk method that has been demonstrated to be incapable of generating consistent risk assessments. The resultant matrix may be suitable for use as a template for future research, but is extremely complex and inadequately rigorous for routine use.

The publication of guidelines for the investigation of unilateral pleural effusion in adults by the British Thoracic Society has focused attention on this subject which, although comprising only a small proportion of laboratory workload, is a fairly common clinical problem. We critically reviewed the guidance applicable to clinical biochemistry laboratories and found a number of deficiencies. In particular, the need for anaerobic sample collection for pH measurement and preservation of samples for glucose assay is not mentioned and health and safety issues related to the handling of potentially infected fluids are also not considered. There are discrepancies between recommendations in the text and in the accompanying diagnostic algorithm, which require clarification. Measurement of total protein is an essential first step in the analysis of pleural fluid and will usually distinguish transudates from exudates. Measurement of lactate dehydrogenase activity is only required when total protein results are equivocal. There are practical difficulties with measurement of fluid pH as recommended in the guidelines and there is little evidence that such measurements are valuable. Similarly, there is little evidence to support the recommendation for measurement of complement in suspected rheumatoid effusions, and the recommendation for amylase isoenzyme studies if acute pancreatitis is a possibility is not practical. The different nature of pleural fluid demands a good understanding of the handling of these samples, the limitations of the analytical methods and the subsequent result interpretation by laboratory staff. We propose a modified diagnostic algorithm reflecting our criticisms of the original. Dialogue between the laboratory and local clinicians, possibly with the production of local guidelines, informed by these recommendations, should help optimize diagnostic management of patients with pleural effusion.

Background: Clinical demand for quick, cheap, precise and accurate 25-hydroxyvitamin D (25(OH)D) results has led to the development of a variety of assay methods. Lack of standardization of these methods has resulted in intermethod disagreement and challenged whether current assays recognize 25(OH)D₂ and 25(OH)D₃ equally.

Methods: We studied 172 patient samples from hip fracture cases using DiaSorin (DS) and IDS radioimmunoassays and the Nichols Advantage-automated protein binding assay (NA-CLPBA) in comparison to high-performance liquid chromatography (HPLC). 52 patient samples were analysed before and after three months treatment with 1000 IU of daily ergocalciferol (vitamin D₂).

Results: Linear regression analysis in pre-treatment samples demonstrated a positive Y-intercept for each immunoassay compared with HPLC, and a slope that varied from 0.64 (IDS) to 0.97 (DS, NA-CLPBA). Bland Altman analysis demonstrated that all the three assays had a proportional positive bias relative to HPLC at values from 20 to 50 nmol/L. Regression analysis of post-treatment samples demonstrated a slope that was not significantly different from zero for the IDS and NA-CLPBA and 0.2 for the DS method, with a positive intercept for all assays of between 8 and 22, indicating less than 50% of 25(OH)D₂ measured by HPLC was detected.

Conclusions: These results demonstrate the need for assay-specific decision limits for 25(OH)D₃ in order to define appropriate thresholds for treatment institution. Treatment with vitamin D₂ may not be accurately monitored with any of the three commercial assays studied. Clinicians and biochemists who continue to use 25(OH)D assays need to be urgently informed of these issues.

Introduction: Lithium-heparin plasma is the most commonly used sample type in many hospitals, but it has been suggested that it is not suitable for protein electrophoresis due to the presence of fibrinogen, which can potentially mask a paraprotein band or be misconstrued as one. Here we aimed to demonstrate that lithium-heparin plasma samples could be used for protein electrophoresis and paraprotein typing without or with ethanol treatment to remove the fibrinogen.

Method: A lithium-heparin sample from a patient with IgGλ, IgGκ, IgAλ and IgAκ myeloma, a non-specific polyclonal increase and a serum control were treated with ethanol prior to protein electrophoresis. Immunofixation electrophoresis was undertaken to investigate the effect of ethanol treatment on immunoglobulin and light chains. Nephelometry was undertaken to investigate whether ethanol treatment affected the quantification of IgG levels. Densitometric evaluation of proteins after electrophoresis was used to study whether ethanol treatment affected other serum proteins. An audit was also undertaken to ascertain the magnitude of the potential interference from the fibrinogen band in heparinized samples.

Results and conclusions: Ethanol treatment significantly but incompletely removed the fibrinogen in lithium-heparin plasma samples and did not affect the integrity of any of the proteins investigated. Even without ethanol treatment, lithium-heparin plasma can be used for protein electrophoresis and paraprotein identification as the instances of interference between fibrinogen and paraproteins was low (2.3%). In rare cases where there is uncertainty or ambiguity, immuno-fixation electrophoresis is recommended. This report has implications in terms of reducing costs and turn-around time as it prevents the need for requesting another serum sample from patients. This may be one step towards a universal sample for all tests.

Background: The UK Prostate Cancer Risk Management Programme has recommended that all assays for prostate-specific antigen (PSA) should be both equimolar in their response to free and complexed PSA and calibrated to the World Health Organization (WHO) First International Standard for PSA (90:10). To determine which assays currently being used by diagnostic laboratories in England fulfil these criteria, a PSA recovery experiment was performed.

Methods: In all, 15 samples containing varying mixtures and concentrations of the WHO International Standards for PSA and free PSA were sent to 223 laboratories in England who participate in the UK National External Quality Assessment Service (UK NEQAS) scheme for analysis. Analytical platforms were assigned to one of 11 methods, and the results were converted into recovery percentages and analysed by means of a linear random intercept model.

Results: No method was both unbiased and equimolar; estimates of bias ranged from -5% to 22% and estimates of non-equimolarity (change in bias per percent increase in free PSA) ranged from -0.18% to 0.28%.

Conclusions: Analytical methods currently in use for PSA in England have inter-method differences; some methods have estimated biases over 10% which can result in unacceptable clinical performance characteristics. Methods for PSA should be equimolar and calibrated to the international standard to minimize the likelihood of clinical errors.

Background: The cardiac troponins have been shown to be sensitive and specific biochemical markers of myocardial infarction and highly prognostic for future adverse events in patients with acute coronary syndromes. There have been reports suggesting that haemolysis causes a negative interference in the cardiac troponin T (cTnT) assay but the mechanism(s) involved remain unknown. Here we show the effects of haemolysis and haemoglobin per se on the cTnT assay.

Methods: The effect of haemolysis was studied by the addition of prepared haemolysate to serum samples with known and clinically relevant cTnT levels. The effect of haemoglobin was studied by the addition of haemoglobin of increasing concentrations and noting its effect on the level of cTnT measured. The effect of putative proteases was determined indirectly by incubating samples with spiked cTnT with various protease inhibitors and observing the changes in the measured cTnT levels.

Results: The results show that both haemolysis, which is the release of haemoglobin and corpuscular contents, and haemoglobin itself negatively interfere in the cTnT assay in a concentration-dependent manner, although the former had a greater magnitude of effect. On haemolysis, indirect evidence suggests that proteases are released which degrade the cTnT in serum, thus causing the decreased levels detected. Pepstatin A, a reversible inhibitor of aspartic proteinases, effectively inhibited the loss of cTnT in serum at 37°C and pH 7.4 over a 48-h period. We found that at a haemoglobin level of 0.75 g/L, cTnT declined by more than 10% of the initial concentration, suggesting that falsely decreased levels due to haemolysis may significantly affect the clinical utility of the assay.

Conclusions: Haemolysis, haemoglobin per se and possibly proteolysis play a role in the negative interference in cTnT assays. Measures to reduce this interference must be implemented.

Background: Macroprolactin is a high molecular mass complex of prolactin that may be the cause of elevated serum prolactin as determined by immunoassay. The composition of macroprolactin and its reactivity in immunoassays are variable but the complex has minimal bioactivity in vivo. Hyperprolactinaemia due to unrecognized macroprolactinaemia can lead to misdiagnosis and mistreatment.

Methods: Serum from a patient with an unusual pattern of immunoreactivity was distributed to three users of each of the most popular immunoassays represented in the UK National External Quality Assessment Scheme (NEQAS) for prolactin. Clinical details were provided and participating centres were invited to investigate the prolactin content of the specimen according to their local protocol, and to comment on their results. The nature of the macroprolactin in the specimen was investigated in detail by gel filtration chromatography of the native serum and of the serum after adsorption of IgG with protein A, and by affinity chromatography with concanavalin A.

Results: Gel filtration studies revealed two peaks of macroprolactin in this serum. These macroprolactins were shown to be different in their IgG content and degree of glycosylation. All eight immunoassays reacted strongly with the macroprolactin present. The majority (78%) of centres that interpreted their results either demonstrated the presence of macroprolactin in the specimen, or suggested it as a likely cause of the hyperprolactinaemia. However, two centres inappropriately excluded macroprolactinaemia as the cause of the elevated prolactin, and a further two did not consider it at all. Data from previous UK NEQAS distributions (between 1996 and 2003) of macroprolactin containing sera are presented which suggest that the frequency of recognition of macroprolactin as a possible cause of hyperprolactinaemia has increased over time.

Conclusions: Very high molecular mass forms of prolactin and the presence of multiple molecular mass forms, as detected in the case presented here, are uncommon. Also, the pattern of immunoreactivity reported in this specimen was unusual as most macroprolactins studied previously react less strongly in, for example, the Bayer ADVIA Centaur assay compared to the Roche E170 assay. Both peaks of macroprolactin in this serum reacted in all assays tested. This case highlights the variable nature and immunoreactive behaviour of macroprolactin species.

Background: Cerebrospinal fluid (CSF) amino acid analysis is fundamental to the investigation of several inherited metabolic diseases, particularly those presenting with unexplained seizures. CSF glycine measurement is often crucial to the diagnosis of glycine encephalopathy (GE), low CSF serine concentrations are characteristic of 3-phosphoglycerate dehydrogenase deficiency (3-PGDD) and the presence of sulphocysteine is pathognomonic of sulphite oxidase deficiency (SOD), and a vital clue to molybdenum cofactor deficiency (MCD). Limited information is available in the literature on reference values of amino acids in CSF during infancy and CSF samples from healthy individuals are not easily obtained.

Methods: In order to establish paediatric CSF amino acid reference ranges, we performed a retrospective analysis of all quantitative CSF amino acid data collected in our laboratory over a five-year period. Amino acid analysis was performed using ion-exchange chromatography on a Biochrom-20 amino acid analyser with ninhydrin detection. CSF samples were collected from infants undergoing investigation for unexplained seizures.

Results: About 18 of the 95 samples received were excluded from the reference data-set; one was from a patient in whom a diagnosis of GE was confirmed by enzyme analysis, one was from a patient with CSF sulphocysteine of 19 μmol/L in whom a diagnosis of SOD was confirmed by enzyme analysis; the remaining 16 were clearly bloodstained (n = 4) or xanthochromic (n = 12). Frequency of distribution analysis revealed that concentration values for each amino acid demonstrated a right-skewed distribution which was not normalized by log transformation. Data were therefore analysed using non-parametric descriptive statistics and reference ranges were defined by the 2.5th and 97.5th centile limits.

Conclusions: Our reference data were derived from 77 CSF samples taken from 77 infants. Median CSF glycine concentration was 9 μmol/L with a reference range of 3-19 μmol/L. For serine, the median CSF concentration was 52 μmol/L with a reference range of 25-105 μmol/L. Sulphocysteine was not normally present in detectable quantities (<1 μmol/L).

Background: The recommendations of the UK National Screening Committee (NSC) are based on the findings of the Serum, Urine and Ultrasound Screening Study (SURUSS). Our study sought to establish if the SURUSS findings, in terms of detection rates (DR) and false-positive rates (FPR) for various second-trimester marker combinations, could be replicated in a local setting.

Methods: We investigated the effects of adding inhibin-A and unconjugated oestriol to our existing double test protocol. This retrospective study examined 1000 control pregnancies and 128 affected pregnancies.

Results: The inhibin-A method was associated with considerable assay drift and very marked within-batch imprecision (intrabatch percent coefficient of variation [CV] = 17%). At a cut-off of 1 in 250, the quadruple test showed a DR of 72%, the triple test 70% and the double test 63%. There were no significant differences between the FPRs for any of the combinations, which were all between 6.6% and 7.0% for a 1 in 250 cut-off.

Conclusions: In our view, the current inhibin-A assay is unacceptable as a screening marker due to poor assay performance. We have reviewed the NSC benchmark programme outcomes, and would suggest that the 2005 target of a DR of at least 60% with an FPR of less than 5% is achievable using triple testing in the second trimester in conjunction with universal scan dating. Our results suggest that the April 2007 target of a DR of 75% with an FPR of less than 3% is unachievable using current second-trimester maternal serum screening.

A case is described of a patient with a ganglioneuroblastoma, initially located in the right adrenal, which produced an excess of dopamine (7646 and 7959 nmol/24 h), approximately two and a half times the upper limit of the normal daily urine output. The urinary excretion of noradrenaline, adrenaline and methylated derivatives was always within the normal reference ranges. The patient was generally well, with normal blood pressure and only mild flushes. Two years after surgical resection, recurrence was indicated by an increase in urinary dopamine (8507 nmol/24 h); it was located in the tumour bed and left side of the neck by CT and ¹²³I MIBG scans. The patient was treated with a high dose of ¹³¹I MIBG, with subsequent reduction in dopamine production. This was repeated on four other occasions, the latest being in January 2005. The output of dopamine was thus used as a marker of tumour diagnosis and progression and it is recommended that the assay of dopamine be included in the screening of catecholamine-secreting tumours to avoid possible misdiagnosis.

Thyroid function tests are the most commonly requested endocrine investigations in both primary and secondary care. Attention to detail is vital, as the appropriate interpretation may point to conditions other than thyroid disease itself. We describe two cases of hypopituitarism masquerading as borderline thyroid function tests.

The porphyrias are a group of inborn or acquired disorders of haem synthesis that can result in neurovisceral or dermatological symptoms. Diagnosis is usually made using a combination of clinical presentation and biochemical parameters. This case report describes a 25-year-old woman clinically presenting with a rash and then found to have elevated porphobilinogen concentrations in her urine. The initial presumptive diagnosis of variegate porphyria was not supported by analysis of her plasma, urine and faeces, which suggested a combination of acute intermittent porphyria and porphyria cutanea tarda. Sequencing of the hydroxymethylbilane synthase and uroporphyrinogen decarboxylase genes confirmed the relatively rare diagnosis of dual porphyria, and revealed a novel uroporphyrinogen decarboxylase mutation.