Stability of serum placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1).

Placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1) are biomarkers used for diagnosis and risk estimation of preeclampsia. Stability in room temperature (RT) may affect the usefulness of these analyses, as shipping at ambient temperature is the most practical and suitable way to ship samples. To date, scientific studies of such stability are lacking. We aimed to assess the stability of PlGF and sFlt-1 at RT in serum from pregnant women. In addition, a smaller study of stability at 4 °C was performed. Serum was collected from 69 pregnant women and stored at RT or at 4 °C for up to 192 h. Analytes were considered stable if the mean percent change ± 90 confidence interval of the mean was within the baseline concentration ± allowable bias. Allowable bias was calculated from data on biological variation. In addition, an instability equation was calculated to assess loss of stability, in line with recent European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) recommendations. The mean percent change was <3.5% for PlGF, <1% for sFlt-1 and <4.5% for sFlt-1/PlGF ratio up to 192 h. PlGF was considered stable for 168 h, and sFlt-1 and sFlt-1/PlGF ratios were considered stable for 192 h at RT. At 4 °C, PlGF was considered stable for 120 h, sFlt-1 for 168 h and sFlt-1/PlGF ratio for 120 h. Both PlGF and sFlt-1 as well as sFlt-1/PlGF ratio show sufficient stability (minimum 168 h) for samples to be shipped at RT.

Testing the limits: the diagnostic accuracy of reference change values.

Reference change values (RCVs) are used by the physician to judge whether a change in analyte concentration from one sample to the next may represent a clinically significant change. Published RCVs are usually given as fixed percentages of the analyte concentration in the first sample. The accuracy of published RCVs is not well known. We obtained public-use data from the US National Health and Nutrition Examination Survey (NHANES) 2001-2002 to study the distribution of changes in the concentration of eight commonly used analytes. Specimens were obtained on two occasions 7-47 days apart from 279 to 411 individuals with an analyte concentration within the reference interval in both samples. The analytes were albumin, calcium, cholesterol, phosphate, potassium, sodium, hemoglobin and thrombocytes. For each analyte, normal within-subject biological coefficient of variation from the EFLM Working Group on Biological Variation and the NHANES analytical coefficient of variation were used to calculate the 5 and 95 percentile RCVs. These RCVs were calculated as fixed percentages of the analyte concentrations in the first sample and compared to the empirical 5 and 95 percentiles. The sensitivity of the RCVs in detecting changes outside the empirical percentiles ranged from 0.35 for sodium to 0.80 for albumin. The specificity of the RCVs in detecting changes inside the empirical percentiles ranged from 0.85 for potassium to 0.97 for thrombocytes. Calculating RCVs as fixed percentages of the analyte concentration in the first sample lessened the diagnostic accuracy. RCVs given as a function of the first result would perform better.

The effects of dry ice exposure on plasma pH and coagulation analyses.

BACKGROUND: We recently observed that exposure to dry ice lowered sample pH and increased clotting times in lupus anticoagulant analyses, and that such changes could be prevented by placing samples at -80°C after dry ice exposure. In the current study, we sought to evaluate the effects of dry ice exposure on pH and various commonly used coagulation analyses. METHODS: Citrated plasma from 30 healthy blood donors was allocated to four preanalytical regimes: (1) immediate analysis of fresh plasma or (2) storage at -20°C; (3) storage at -20°C followed by dry ice exposure for 24 h or (4) storage at -20°C followed by dry ice exposure for 24 h and storage at -80°C for 24 h before analysis. Analyses of pH, prothrombin time international normalized ratio (PT-INR), activated partial thromboplastin time (APTT), antithrombin, fibrinogen, protein C and protein S was performed. RESULTS: Samples exposed to dry ice had significantly lower pH, prolonged clotting times in PT-INR, APTT and fibrinogen analyses as well as lower levels of protein C, than samples not exposed to dry ice. These changes in coagulation analyses were not present if samples were stored at -80°C for 24 h after dry ice exposure. Antithrombin and protein S were not significantly affected by dry ice exposure. CONCLUSIONS: Dry ice exposure lowered sample pH and affected various coagulation analyses. These effects were avoided by storing samples at -80°C for 24 h after dry ice exposure.

Paediatric reference intervals - an update. / Pediatriske referanseområder ­ en oppdatering.

Children are not small adults, including when it comes to reference intervals. As reference intervals can vary considerably with age, sex and stage of development, it is important to use the correct reference intervals and decision limits in order to ensure optimal diagnostics. Norway's national user manual in clinical chemistry has now been updated to include paediatric reference intervals.

Dry ice exposure of plasma samples influences pH and lupus anticoagulant analysis.

BACKGROUND: Tests for lupus anticoagulant (LA), including silica clotting time (SCT) and diluted Russel's viper venom time (dRVVT) are used to diagnose antiphospholipid syndrome. Due to sample instability, it is recommended that samples are frozen if analysis is postponed >4 h. Shipping on dry ice is common practice to keep samples frozen during transport. Recent data suggest that exposure to dry ice may affect sample pH and results in subsequent analyses. We aimed to determine the effect of dry ice on pH and LA analysis. METHODS: Citrated plasma from eight healthy volunteers was allocated to three preanalytical regimes: 1) storage at -20 °C; 2) dry ice exposure followed by storage at -20 °C; or 3) dry ice exposure followed by storage at -80 °C. RESULTS: Samples stored at -20 °C after dry ice exposure had significantly lower median pH (1.2 units, p=0.01) and prolonged clotting time ratios (up to 55% for dRVVT tests, p<0.02) in LA analysis, compared to samples not exposed to dry ice. This resulted in poor test specificity (25%). Similar changes were not observed in samples placed at -80 °C after dry ice exposure. CONCLUSIONS: Dry ice may affect sample pH and increase the fraction of false positive LA results. This preanalytical factor should be taken into account by laboratories receiving frozen samples for these tests.

Whole-genome microarray and targeted analysis of angiogenesis-regulating gene expression (ENG, FLT1, VEGF, PlGF) in placentas from pre-eclamptic and small-for-gestational-age pregnancies.

OBJECTIVE: To compare the placental pathology associated with pre-eclampsia (PE) and/or fetal growth restriction, the transcriptomes of placental tissues from PE and small-for-gestational-age (SGA) pregnancies were explored. In addition, a targeted analysis of angiogenesis-regulating gene expression was performed. METHODS: Whole-genome microarray analysis was performed on placental tissue from gestational age-matched PE (n = 10), SGA (n = 8) and PE + SGA (n = 10) pregnancies. The expression of genes regulating angiogenesis (endoglin (ENG), fms-related tyrosine kinase 1 (FLT1), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)) was analyzed by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Microarray analysis did not reveal any significant differences between groups. However, an increased expression of ENG and FLT1 was detected by qRT-PCR in the PE + SGA group. CONCLUSIONS: The placental transcriptome did not differ between groups, although an increased anti-angiogenic gene expression in PE + SGA was observed with qRT-PCR analysis. Based on this, we conclude that although microarray technology may represent a powerful tool in generating new hypothesis in complex fields, it may not be sensitive enough to detect subtle changes in gene expression.

Should total calcium be adjusted for albumin? A retrospective observational study of laboratory data from central Norway.

OBJECTIVES: Albumin-adjusted total calcium is often used as a surrogate marker for free calcium to evaluate hypocalcaemia or hypercalcaemia. Many adjustment formulas based on simple linear regression models have been published, and continue to be used in spite of questionable diagnostic accuracy. In the hope of finding a more pure albumin effect on total calcium, we used multiple linear regression models to adjust for other relevant variables. The regression coefficients of albumin were used to construct local adjustment formulas, and we tested whether the diagnostic accuracy was improved compared with previously published formulas and unadjusted calcium. DESIGN: A retrospective hospital laboratory data study. DATA SOURCES: The local hospital laboratory data system. SETTING: Norway, 2006-2015. PARTICIPANTS: 6549 patients above 2 years of age, where free calcium standardised at pH 7.40, total calcium, creatinine, albumin and phosphate had been analysed in a single blood draw, including hospitalised patients and patients from outpatient clinics and general practice. MAIN OUTCOME MEASURES: Diagnostic accuracy by Harrell's c and receiver operating characteristic curve analysis, using free calcium standardised at pH 7.40 as a gold standard, in subgroups with estimated glomerular filtration rate (eGFR) ≥60 or <60 mL/min/1.73 m2. RESULTS: In the subgroup with eGFR <60 mL/min/1.73 m2, the Harrell's c of unadjusted total calcium (0.801) was significantly larger than those of the local formulas (0.790, p=0.002) and the best formula taken from literature (0.791, p=0.004). In the subgroup with eGFR ≥60 mL/min/1.73 m2, no significant differences were found between these three formulas. CONCLUSIONS: Our study shows that the diagnostic accuracy of unadjusted total calcium is superior to several commonly used adjustment formulas, and we suggest that the use of such formulas should be abandoned in clinical practice. If the clinician does not trust total calcium to reflect the calcium status of the patient, free calcium should be measured.