Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

Pregnancy-associated plasma protein A (PAPPA) is an emerging biomarker used in various medical fields but has yet to be evaluated in critical care medicine. This study evaluates the value of PAPPA as a biomarker in predicting myocardial dysfunction and 28-day mortality in patients with severe sepsis.

Serum concentrations of PAPPA and traditional cardiac biomarkers including cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP) were measured on admission in 118 severely septic patients. The value of PAPPA for the diagnosis of sepsis-related myocardial dysfunction (SRMD) and for the prediction of 28-day mortality was subsequently evaluated using statistical methods.

PAPPA was elevated (≥4.5 ng/mL) in 76 (64.4%) patients; patients with elevated PAPPA were more likely to have SRMD (76.3% vs. 38.1%, P < 0.001). By comparison of the area under the curve (AUC) in receiver operating characteristics analysis, PAPPA had comparable value (AUC 0.661, P = 0.003) to conventional biomarkers (BNP: AUC 0.699, P < 0.001; cTnI: AUC 0.647, P = 0.007) in the diagnosis of SRMD and offered superior value (AUC 0.771, P < 0.001) over them (all AUC<0.7, P > 0.05) in the prediction of 28-day death. Increased PAPPA (≥5.4 ng/mL) was associated with lower 28-day survival (χ² = 19.78, P < 0.001) and independently predicted 28-day mortality in septic patients.

Serum PAPPA concentration frequently increases in patients with severe sepsis and appears to be associated with SRMD. PAPPA can be used as a novel biomarker for the diagnosis of SRMD and the prediction of outcomes in critically ill patients.

Current sampling and transport conditions of samples in general practice can result in pseudohyperkalaemia. This study was undertaken to determine, in a general practice setting, whether there is any difference in haemolysis obtained when using BD Vacutainer® Rapid Serum Tubes (BD RST) compared with using BD Vacutainer® SST™ II Advance Blood Collection Tubes (BD SSTII).

Blood was collected from 353 patients requiring blood sampling who were attending 31 general practitioner practices in Belgium. For each patient, two BD SSTII tubes and two BD RST tubes were drawn in a randomized order. One of each pair of tubes was inverted five times, the other was not. Serum potassium concentration, serum LDH activity and haemolysis index were measured in each sample.

There was no significant difference in measured potassium concentration according to tube type (P = 0.16). Measured LDH activities were 1.7% higher in serum collected into BD SSTII tubes compared to BD RST tubes (P = 0.02). When comparing serum from unmixed BD RST with BD SSTII tubes, there was a slight reduction in the haemolysis index but no significant difference in measured potassium concentration or LDH activity. Risk of hyperkalaemia was 4.8 times higher in serum from tubes that were incompletely filled compared to those that were filled with the correct amount of blood.

Both types of blood tubes are suitable for the measurement of serum potassium and LDH in patients from general practice. Tube inversion does not improve the accuracy of either serum potassium or LDH measurement. Blood tubes should be filled to the level recommended by the manufacturer to avoid artefactual increases in measured serum potassium concentration and LDH activity.

Medication-related interferences with measurements of catecholamines and their metabolites represent important causes of false-positive results during diagnosis of phaeochromocytomas and paragangliomas (PPGLs). Such interferences are less troublesome with measurements by liquid chromatography with tandem mass-spectrometry (LC-MS/MS) than by other methods, but can still present problems for some drugs. Levodopa, the precursor for dopamine used in the treatment of Parkinson’s disease, represents one potentially interfering medication.

Plasma and urine samples, obtained from 20 Parkinsonian patients receiving levodopa, were analysed for concentrations of catecholamines and their O-methylated metabolites by LC-MS/MS. Results were compared with those from a group of 120 age-matched subjects and 18 patients with PPGLs.

Plasma and urinary free and deconjugated (free + conjugated) methoxytyramine, as well as urinary dopamine, showed 22- to 148-fold higher (P < 0.0001) concentrations in patients receiving levodopa than in the reference group. In contrast, plasma normetanephrine, urinary noradrenaline and urinary free and deconjugated normetanephrine concentrations were unaffected. Plasma free metanephrine, urinary adrenaline and urinary free and deconjugated metanephrine all showed higher (P < 0.05) concentrations in Parkinsonian patients than the reference group, but this was only a problem for adrenaline. Similar to normetanephrine, plasma and urinary metanephrine remained below the 97.5 percentiles of the reference group in almost all Parkinsonian patients.

These data establish that although levodopa treatment confounds identification of PPGLs that produce dopamine, the therapy is not a problem for use of LC-MS/MS measurements of plasma and urinary normetanephrine and metanephrine to diagnose more commonly encountered PPGLs that produce noradrenaline or adrenaline.

To assess glucose meter performance in a diabetes antenatal clinic, focussing on clinical and pre-analytical factors that might impact on the quantification of meter accuracy and precision.

The Freestyle Lite and the Performa glucose meters were assessed by trained researchers. Finger stick glucose was measured and compared with plasma venous glucose, obtained from a concomitantly collected antecubital fossa sample. Venous plasma was separated on-site then sent to the laboratory for measurement of glucose using the hexokinase method (comparative method). Additional data collected included: (i) timing of and also (ii) quantity of last carbohydrate intake; (iii) time periods between collection, preparation and analysis of the venous sample; (iv) the haemolysis index of the plasma sample and (v) haematocrit.

There were 104 participants. Both meters fulfilled ISO 15197 standards, with 99% and 97% of Freestyle Lite and Performa results, respectively, falling within acceptable limits for this standard. Both meters showed minor proportional bias, reading low at higher glucose values. Consensus error grid analysis showed 100% of results from the Freestyle Lite and 99% from the Performa falling within Zone A, thus the observed differences in measured capillary and venous plasma glucose were sufficiently minor that they would have little effect on clinical action. No association was observed between [capillary-plasma] glucose difference and the five variables outlined above.

The two glucose meters tested showed a reassuringly acceptable level of performance, when assessed by a research team in the setting of a diabetes antenatal clinic.

Combined lipid and glucose point-of-care testing (POCT) devices could facilitate widespread population screening for cardiovascular disease (CVD) and diabetes as part of the NHS Vascular Risk Assessment and Management Program (NHS Health Checks). An evaluation of the Cholestech LDX and CardioChek PA POCT analysers was performed in collaboration with the Wolverhampton City Primary Care Trust (PCT).

Intra-/inter-batch imprecision, between-analyser variation and the effect of haematocrit and ascorbic acid assay interference were investigated. Accuracy of the POCT capillary whole blood total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C) and glucose measurements was estimated by comparison with those from the laboratory analysis of paired venous samples. POCT usability and clinical governance were also assessed.

The LDX exhibited lower intra- and inter-batch imprecision and external quality assessment (EQA) scheme between-analyser variation for the measurement of TC, HDL-C and glucose when compared to the CardioChek. Ascorbic acid negatively interfered in all three assays on both POCT analysers and results reported by the CardioChek were influenced by the specimen haematocrit. The LDX displayed closer agreement with the laboratory methods for the measurement of TC and HDL-C but both the LDX and the CardioChek displayed positive bias for the measurement of glucose.

POCT has clear advantages for delivering NHS Health Checks over the laboratory-based approach although device performance does differ. Users should also be aware of the potential clinical governance and interference issues associated with these devices.

The association of plasma cardiovascular risk markers and metabolic syndrome (MetS) with non-alcoholic fatty liver disease (NAFLD) has not been well defined.

Japanese men (n = 809) had standard anthropometric measurements done, and had their liver fat quantitated by ultrasound. Three groups were identified: (1) normal controls without significant disease, (2) preliminary-metabolic syndrome (pre-MetS) cases and (3) MetS cases. Plasma adiponectin, high sensitivity-C reactive protein (hs-CRP), HOMA-IR, lipids, lipoproteins and liver enzymes were evaluated among the three groups.

The prevalence of fatty liver was 13% in controls, 39% in pre-MetS and 62% in MetS. Plasma adiponectin and high density lipoprotein cholesterol (HDL-C) were significantly decreased, and HOMA-IR, hs-CRP, TG, remnant lipoproteins (RLPs) and small dense-LDL-C (sd LDL-C) were significantly increased in subjects with fatty liver compared to those without fatty liver. Multivariate analyses of serum parameters associated with fatty liver revealed that adiponectin and hs-CRP were more strongly associated with the presence of fatty liver than waist circumference. However, HOMA-IR, HDL-C, TG, RLP-C, RLP-TG and sd LDL-C were more strongly associated with waist circumference than with fatty liver. Factor analysis revealed that adiponectin and HDL-C were linked to liver enzymes, lipoproteins and HOMA-IR associated with fatty liver, but not with waist circumference.

Adiponectin was found to be a more specific diagnostic marker for the presence of fatty liver regardless of MetS status, and was inversely correlated with liver enzyme concentrations. However, RLPs were found to be more specifically associated with the presence of MetS.

Glomerular filtration rate (GFR) and mortality is more accurately determined by gold standard measures than serum creatinine-based estimates in cirrhosis. No formal validation of any gold standard method has been reported.

An isocratic methanol-based method incorporating the reference standard iohexol-related compound C was developed and validated in 12 patients with cirrhosis by simultaneously determining GFR using iohexol and chromium-51 labelled ethylenediamine tetraacetic acid (⁵¹Cr-EDTA) clearance. Iohexol pharmacokinetics was also studied with the collection of blood and ascitic fluid at intervals following an iohexol bolus.

Triplicate assays produced a linear calibration curve (R²= 0.99, N = 5) over an iohexol concentration range of 23.6–755 µg/L. Mean (range) extraction recovery of iohexol from serum was greater than 95% (94–97%), with an intra-day coefficient of variation less than 3%. Twelve patients with cirrhosis with mean Child-Pugh score of 9 displayed a mean difference (bias) −1.3 mL/min/1.73 m² (−18 to + 16) comparing iohexol with ⁵¹Cr-EDTA. Iohexol equilibrated between blood and ascitic compartments after 4 h.

A simple, cheap, and accurate isocratic, methanol-based method for the determination of iohexol concentrations is described, validated according to Food and Drug Administration guidance. Iohexol demonstrated comparable performance with ⁵¹Cr-EDTA in determining GFR. Delayed equilibrium of iohexol between blood and ascitic compartments suggests sampling beyond 4 h would improve accuracy of GFR determinations in patients with cirrhosis.

Kisspeptin is a hypothalamic neuropeptide playing a physiological role in human reproduction. Genetic over-activation of kisspeptin causes precocious puberty in children. Concentrations of circulating kisspeptin are low in adults. The concentrations of plasma kisspeptin in boys and girls have not been studied previously.

Blood was obtained from 51 children and 63 adults. Plasma samples were analysed using radioimmunoassay. Children were aged 2–18 years, and attending hospital for a medically requested blood test unrelated to reproductive development. Data on pubertal status were not collected due to ethical reasons.

Mean plasma kisspeptin was significantly higher in children when compared with adults (mean plasma kisspeptin in pmol/L: 12.3 ± 0.9, adults; 40.9 ± 3.3, children, P < 0.001 vs. adults). Overall mean concentrations of plasma kisspeptin were not significantly different between sexes (mean plasma kisspeptin in pmol/L: 39.5 ± 3.2, boys; 44.3 ± 6.3, girls, P = 0.48). In both sexes, concentrations of plasma kisspeptin increased with age to peak concentrations between 9 and 12 years of age, before decreasing beyond 12 years of age to adulthood. Plasma kisspeptin concentrations were highly significantly elevated in both girls and boys aged 9–12 when compared with adults (mean plasma kisspeptin in pmol/L: 59.5 ± 18.3, girls, P < 0.01 vs. adult women; 43.8 ± 6.2, boys, P < 0.001 vs. adult men).

We report that circulating kisspeptin is elevated in both boys and girls when compared with adults. Furthermore both boys and girls may have distinct, age-dependent concentrations of circulating kisspeptin. Further studies may determine if plasma kisspeptin could be used as a clinically useful biochemical marker of reproductive development in children.

Therapeutic drug monitoring of ciclosporin A (CsA) and tacrolimus is traditionally performed using venous whole blood sampling. A number of reports have described development of ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for the quantitation of CsA and tacrolimus from dried blood spots (DBS), which may offer a convenient alternative. As yet, no such reports have validated this methodology using fingerprick capillary DBS samples collected from transplant patients.

Capillary fingerprick DBS were collected from heart and lung transplant patients in a specialist cardiothoracic transplant centre. We utilized our previously published method for the extraction and simultaneous quantitation of CsA and tacrolimus from DBS using UPLC-MS/MS. Drug concentrations measured from DBS were compared to concentrations measured in venous whole blood by our routine clinical UPLC-MS/MS assay.

In total, 91 heart or lung transplant patients were enrolled onto the study; 46 patients were on CsA therapy and 45 on tacrolimus therapy. Passing–Bablock analysis demonstrated excellent agreement between capillary fingerprick DBS samples and venous whole blood samples. There was a mean positive bias of 2.6 µg/L (95% confidence interval (CI) −2.2 to 7.5 µg/L) for CsA (n = 45) and mean negative bias of −0.7 µg/L (95% CI −1.1 to −0.3 µg/L) for tacrolimus (n = 42).

We demonstrate utility of DBS for serial monitoring of CsA and tacrolimus using UPLC-MS/MS in heart and lung transplant patients. This may offer significant advantages for these patients including the ability to take capillary DBS samples in the community prior to clinic visits.

The present study was aimed at determining the effect of the tube type used for primary sample collection and the manner of transport prior to assessment (either manual or by pneumatic tube) on the degree of pseudohyperkalaemia in leukaemic patients.

Blood from six leukaemic patients was collected into seven primary sample tubes (Monovette®, Sarstedt): sample A, heparinized blood gas syringe (potassium reference value); sample B, plasma Li-heparin without separator gel; sample C, plasma Li-heparin with separator gel; and sample D, serum with separator gel. The primary sample tubes designated B, C and D were transported to the laboratory manually. Duplicates of the same sample tubes, B^PT, C^PT and D^PT, were sent to the laboratory by pneumatic tube.

In patients with chronic lymphocytic leukaemia (CLL), there was no increase in the concentration of potassium in samples B, C and D when compared to the reference value. Transport of the samples by pneumatic tube led to a pronounced increase in potassium concentration in samples B^PT and C^PT, whereas there was no increase in sample D^PT when compared to the reference value. In the patient with chronic myeloid leukaemia (CML), an increase in potassium concentration occurred in sample D and in samples B^PT, C^PT and D^PT. A similar finding was observed in the patient with acute lymphocytic leukaemia (ALL), furthermore with an extremely high concentration of potassium in samples C and C^PT.

Manual transport of non-coagulable blood (plasma Li-heparin without separator gel) to the laboratory results in the least possible artificial increase in potassium concentration in the sample.