Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

We evaluated the performance of ultra-performance liquid chromatography-tandem mass spectrometry to measure serum 3-epi-25-hydroxyvitamin D₃, 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ concentrations in 519 infant, paediatric and adolescent serum samples in Korea.

We used a Kinetex XB-C18 column and isocratic methanol/water (77.5/22.5, v/v) with 0.025% (v/v) high-performance liquid chromatography solvent additive flowing at 0.25 mL/min, yielding an 11 min/sample run time. A TQD triple quadrupole mass spectrometer in electrospray ionization positive ion mode with multiple reaction monitoring transition via an MSMS vitamin D kit was used to evaluate precision, carryover, ion suppression and linearity. Samples were prepared using the 4-phenyl-1,2,4-triazoline-3,5-dione derivatization method.

Intra- and inter-run precisions were 1.23–13.28% and 1.02–10.08%, respectively. Group carryovers were −0.27% and 0.10%, respectively. There was no ion suppression. The calibration curve showed good linearity from calibrator Level 1 (11.75 nmol/L) to 6 (375 nmol/L) with R²> 0.9999. The 3-epi-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃ peaks were clearly separated in the extracted ion chromatogram. Infant serum samples 3-epi-25-hydroxyvitamin D₃ concentrations were significantly higher than paediatric and adolescent concentrations.

The ultra-performance liquid chromatography-tandem mass spectrometry assay performed acceptably, clearly separating 3-epi-25-hydroxyvitamin D₃ from 25-hydroxyvitamin D₃. High 3-epi-25-hydroxyvitamin D₃ concentrations were observed in infant but not in paediatric and adolescent serum samples.

Inflammation and hypercholesterolaemia contribute to atherosclerotic changes which can start in childhood. Children with hyperlipidaemias are at high risk for early coronary atherosclerosis. This study evaluates the relationship between lipoprotein-associated phospholipase A₂ (Lp-PLA₂), carotid intima-media thickness (CIMT) and flow-mediated dilatation in hypercholesterolaemic dyslipidaemic children.

We performed a case–control study consisting of 43 cases, aged 2 to 17 years, and 24 age-matched controls. Fasting blood samples were obtained from both groups for the measurement of a lipid profile (total cholesterol, LDL-C, HDL-C and triglycerides) and Lp-PLA₂ in mass units. The latter was determined with a turbidimetric immunoassay method (PlacTest, DiaDexus Inc.) applied to an automated analyser. CIMT and flow-mediated dilatation measurements were undertaken by a paediatric cardiologist, using high-resolution B-mode ultrasonography.

Total cholesterol, LDL-C and Lp-PLA₂ concentrations were significantly higher in the cases than in the controls (p < 0.001 for all three parameters). While CIMT values were also significantly higher in the patients compared to the controls (P = 0.001), flow-mediated dilatation values were significantly lower (P = 0.001). We found positive correlations between Lp-PLA₂ and total cholesterol (r = 0.41, P = 0.001), Lp-PLA₂ and LDL-C (r = 0.36, P = 0.004), Lp-PLA₂ and CIMT (r = 0.44, P = 0.019) and LDL-C and CIMT (r = 0.41, P = 0.032); there were negative correlations between Lp-PLA₂ and flow-mediated dilatation (r = −0.15, P = 0.045), total cholesterol and flow-mediated dilatation (r = −0.45, P = 0.017), LDL-C and flow-mediated dilatation (r = −0.51, P = 0.006) and CIMT and flow-mediated dilatation (r = −0.45, P = 0.016).

Lp-PLA₂ concentrations are significantly elevated in hypercholesterolaemic dyslipidaemic children. Given the association of Lp-PLA₂ with markers of atherosclerosis (total cholesterol, LDL-C, CIMT and flow-mediated dilatation), the finding of increased concentrations of Lp-PLA₂ could be used to identify early atherosclerotic changes in hypercholesterolaemic dyslipidaemic children and may inform their clinical management.

Human chorionic gonadotrophin is a marker of early pregnancy. This study sought to determine the possibility of being able to distinguish between healthy and failing pregnancies by utilizing patient-associated risk factors and daily urinary human chorionic gonadotrophin concentrations.

Data were from a study that collected daily early morning urine samples from women trying to conceive (n = 1505); 250 of whom became pregnant. Data from 129 women who became pregnant (including 44 miscarriages) were included in these analyses. A longitudinal model was used to profile human chorionic gonadotrophin, a Cox proportional hazards model to assess demographic/menstrual history data on the time to failed pregnancy, and a two-stage model to combine these two models.

The profile for log human chorionic gonadotrophin concentrations in women suffering miscarriage differs to that of viable pregnancies; rate of human chorionic gonadotrophin rise is slower in those suffering a biochemical loss (loss before six weeks, recognized by a rise and fall of human chorionic gonadotrophin) and tends to plateau at a lower log human chorionic gonadotrophin in women suffering an early miscarriage (loss six weeks or later), compared with viable pregnancies. Maternal age, longest cycle length and time from luteinizing hormone surge to human chorionic gonadotrophin reaching 25 mIU/mL were found to be significantly associated with miscarriage risk. The two-stage model found that for an increase of one day in the time from luteinizing hormone surge to human chorionic gonadotrophin reaching 25 mIU/mL, there is a 30% increase in miscarriage risk (hazard ratio: 1.30; 95% confidence interval: 1.04, 1.62).

Rise of human chorionic gonadotrophin in early pregnancy could be useful to predict pregnancy viability. Daily tracking of urinary human chorionic gonadotrophin may enable early identification of some pregnancies at risk of miscarriage.

The clinical utility of serum thyroglobulin in the follow-up of patients with differentiated thyroid carcinoma may be compromised by the presence of endogenous antithyroglobulin antibodies. To prevent interference by antithyroglobulin antibodies several groups have developed real-time PCR-based assays for quantification of blood thyroglobulin mRNA levels. For accurate quantification of thyroglobulin mRNA in blood preanalytical factors must be recognized and controlled. In this study, we evaluate the effect of different blood RNA stabilizing systems – the Tempus Blood RNA system and the PAXgene Blood RNA system – and storage time on RNA yield and quality, and thyroglobulin mRNA stability.

Blood samples from 11 patients previously treated for differentiated thyroid carcinoma were collected in K₂-EDTA, Tempus and PAXgene tubes and maintained at room temperature. RNA was isolated following storage for 0 and 72 h, and RNA yield, integrity and purity was determined. Thyroglobulin, GAPDH and ACTB mRNA levels were quantified by semi-quantitative real-time PCR.

The RNA yield was significantly higher for blood collected in Tempus tubes compared with PAXgene tubes following storage for 72 h at room temperature (P = 0.0011). High-quality RNA could be extracted from blood collected in PAXgene and Tempus tubes. Blood collected in K₂-EDTA tubes, but not in PAXgene and Tempus tubes, showed significant changes in thyroglobulin mRNA levels following storage for 72 h at room temperature (P = 0.0263).

Stabilization of blood in PAXgene and Tempus tubes enables storage at room temperature for up to 72 h, without compromising thyroglobulin mRNA levels.

Dietary habits are associated with obesity which is a risk factor for coronary heart disease. The objective is to estimate the change of lipoprotein(a) and other lipoprotein classes by calorie restriction with obesity index and Framingham risk score.

Sixty females (56 ± 9 years) were recruited. Their caloric intakes were reduced during the six-month period, and the calorie from fat was not more than 30%. Lipoprotein profiles were estimated at baseline and after the six-month period of calorie restriction. Cholesterol levels in six lipoprotein classes (HDL, LDL, IDL, VLDL, chylomicron and lipoprotein(a)) were analysed by anion-exchange liquid chromatography. The other tests were analysed by general methods. Additionally, Framingham risk score for predicting 10-year coronary heart disease risk was calculated.

Body mass index, waist circumference, insulin resistance, Framingham risk score, total cholesterol, LDL-cholesterol and IDL-cholesterol were significantly decreased by the calorie restriction, and the protein and cholesterol levels of lipoprotein(a) were significantly increased. The change of body mass index was significantly correlated with those of TC, VLDL-cholesterol and chylomicron-cholesterol, and that of waist circumference was significantly correlated with that of chylomicron-cholesterol. The change of Framingham risk score was significantly correlated with the change of IDL-C.

Obesity indexes and Framingham risk score were reduced by the dietary modification. Lipoprotein profile was improved with the reduction of obesity indexes, but lipoprotein(a) was increased. The changes of obesity indexes and Framingham risk score were related with those of triglyceride-rich lipoproteins, e.g. IDL, VLDL and CM.

Antiphospholipid syndrome is an autoimmune disease characterized by thrombosis, fetal losses and thrombocytopenia associated to antiphospholipid antibodies. They are directed to phospholipids, such as cardiolipins (anticardiolipin) and lupus anticoagulant or to complexes formed by phospholipids and protein cofactors, such as β2 glycoprotein 1 (a-β2GP1) and annexin V (a-annexin V). These auto-antibodies may be considered as a family of antibodies involved in thrombotic events and antiphospholipid activity. On the other hand, some proangiogenic factors are involved in the normal development of placental vasculature, such as the vascular endothelial growth factor. Overexpression of vascular endothelial growth factor receptor in its soluble form (sVEGFR-1) has been associated to a higher antiangiogenic activity. Our aim was to analyse the association between anticardiolipin, lupus anticoagulant, a-β2GP1, a-annexin V and sVEGFR-1 with recurrent miscarriage before week 10 of gestation in females with antiphospholipid syndrome.

We studied 24 females (primary or secondary antiphospholipid syndrome), who were divided into two groups: females with recurrent miscarriage before week 10 of gestation (M; n = 12) and females with no history of fetal loss (NM; n = 12). Anticardiolipin, a-β2GP1, a-annexin V and sVEGF-R1 concentrations were assessed by ELISA, while lupus anticoagulant was assessed by screening and confirmatory tests.

A significant association was observed between the number of positive biomarkers and the belonging group (P < 0.05). Besides, a positive result for lupus anticoagulant and a-β2GP1 was found to be significantly associated to the M group (P < 0.05).

Lupus anticoagulant and a-β2GP1 may be implicated in pregnancies complicated by recurrent miscarriage in females with antiphospholipid syndrome.

Compelling evidence indicate that traumatic brain injury is highly related to accelerated bone fracture repair, but the underlying mechanism still remains elusive. Fracture repair process relies greatly on the formation of new blood vessels in fracture site, and angiogenic factors have been confirmed to be essential for the initiation and maintenance of the fracture healing. Hypoxia-inducible factor-1α was demonstrated to be a critical regulator of angiogenic–osteogenic coupling during bone development and regeneration. The aim of the present study was to investigate the local and circulating concentrations of hypoxia-inducible factor-1α in patients with long-bone fractures and concomitant traumatic brain injury and to determine the potential role of hypoxia-inducible factor-1α in fracture healing.

Twenty-five patients with a long-bone fracture and concomitant traumatic brain injury (FT group) and 33 without a brain injury (Fr group) were enrolled in this study. Healthy subjects donated serum samples as control. Serum samples were collected over a period of six months, following a standardized time schedule. Hypoxia-inducible factor-1α concentrations were measured in fracture haematoma and serum of patients in both groups using enzyme-linked immunosorbent assay.

Patients in FT group had a short time to union. Serum hypoxia-inducible factor-1α concentrations elevated in the early healing period and reached the maximum level during intramembranous bone formation phase in both groups. Thereafter, it decreased continuously and approached to the minimum levels until the end of the observation period. Serum hypoxia-inducible factor-1α concentrations in both groups were significantly higher compared with controls and hypoxia-inducible factor-1α concentrations in both serum and fracture haematoma were higher in FT group than that in Fr group. Fracture haematoma contained significantly higher hypoxia-inducible factor-1α concentrations compared with hypoxia-inducible factor-1α concentrations in serum. Serum hypoxia-inducible factor-1α concentrations had a positive correlation with hypoxia-inducible factor-1α concentrations in fracture haematoma in patients with fractures.

These findings suggest the local and systemic involvement of hypoxia-inducible factor-1α in fracture healing and the accelerated fracture repair in patients with traumatic brain injury might be associated with elevated hypoxia-inducible factor-1α concentrations in fracture haematoma and serum.

Centrifugation of blood samples is an essential preanalytical step in the clinical biochemistry laboratory. Centrifugation settings are often altered to optimize sample flow and turnaround time. Few studies have addressed the effect of altering centrifugation settings on analytical quality, and almost all studies have been done using collection tubes with gel separator.

In this study, we compared a centrifugation time of 5 min at 3000 × g to a standard protocol of 10 min at 2200 × g. Nine selected general chemistry and immunochemistry analytes and interference indices were studied in lithium heparin plasma tubes and serum tubes without gel separator. Results were evaluated using mean bias, difference plots and coefficient of variation, compared with maximum allowable bias and coefficient of variation used in laboratory routine quality control.

For all analytes except lactate dehydrogenase, the results were within the predefined acceptance criteria, indicating that the analytical quality was not compromised. Lactate dehydrogenase showed higher values after centrifugation for 5 min at 3000 × g, mean bias was 6.3 ± 2.2% and the coefficient of variation was 5%.

We found that a centrifugation protocol of 5 min at 3000 × g can be used for the general chemistry and immunochemistry analytes studied, with the possible exception of lactate dehydrogenase, which requires further assessment.

Tandem mass spectrometry (MS/MS) has recently become an alternative method for the newborn screening of sickle cell disorders (SCD), as it is able to detect haemoglobin (Hb) peptides following digestion of bloodspots with trypsin. Using the SpOtOn Diagnostics Reagent Kit, we previously developed a screening protocol to detect only the disease states of SCD, using action values based on the ratio between the variant Hb peptide to wild-type peptide abundances for the HbS, C, D^Punjab, O^Arab, E and Lepore peptides.

Action values using the ratios between the wild type HbA (ßT1-3) peptides and the foetal Hb (γT2) peptide were developed to identify bloodspot samples from premature and transfused infants. An evaluation was undertaken to assess the transferability of the action values onto an additional MS/MS instrument. We report here our experience using this MS/MS protocol.

During a three-year period, we screened 100,456 babies and identified 10 SCD cases (1 HbS/HPFH, 5 HbS/S and 4 HbS/C) and a case of HbE/ß-thalassaemia that was identified as a by-product. The Hb variant to wild-type peptide ratio action values were transferable to a second MS/MS instrument. Our protocol prevented the identification of an estimated 810 carrier infants. Gestational age-related action values for HbA to HbF peptide ratios were required to minimize the number of samples referred for second-line testing to exclude ß-thalassaemia.

MS/MS is a robust alternative screening technology for SCD; in addition, it also optimizes the use of equipment and expertise that currently exist in newborn screening laboratories.

Thyroid-stimulating hormone (TSH) is used as the first-line test of thyroid function. Siemens Healthcare Diagnostics recommend that Siemens Centaur reagents must be protected from light in the assay information and on reagent packaging. We have compared the effect of light exposure on results using Siemens TSH-3Ultra and follicle-stimulating hormone reagents. The thyroid-stimulating hormone reagent includes fluoroscein thiocyanate whereas the follicle-stimulating hormone reagent does not.

Three levels of quality controls were analysed using SiemensTSH-3Ultra and follicle-stimulating hormone reagent packs that had been kept protected from light or exposed to light at 6-h intervals for 48 h and then at 96 h.

Thyroid-stimulating hormone results were significantly lower after exposure of TSH-3Ultra reagent packs to light. Results were >15% lower at all three levels of quality control following 18 h of light exposure and continued to decrease until 96 h. There was no significant difference in follicle-stimulating hormone results whether reagents had been exposed to or protected from light.

Thyroid-stimulating hormone results but not follicle-stimulating hormone results are lowered after exposure of reagent packs to light. Laboratories must ensure that TSH-3Ultra reagents are not exposed to light and analyse quality control samples on every reagent pack to check that there has not been light exposure prior to delivery. The labelling on TSH-3Ultra reagent packs should reflect the significant effect of light exposure compared with the follicle-stimulating hormone reagent. We propose that the effect of light exposure on binding of fluoroscein thiocyanate to the solid phase antibody causes the falsely low results.