COMPARISON OF PROTHROMBIN TIME AND ACTIVATED PARTIAL THROMBOPLASTIN TIME IN PRIMARY TUBES AND SAMPLE ALIQUOTS AFTER 4 HOURS DURATION

Background: Coagulation tests are widely applied in clinical practice, among which PT and aPTT are the most commonly done. Among many preanalytical conditions –time, temperature and storage conditions are few of the factors affecting the results of samples. This study aims to compare PT and aPTT values in primary tubes and sample aliquots after 4 hours duration with the initial value. Materials & Methods : An observational study was done at Central Diagnostic Laboratory, Gandhi Hospital, Secunderabad, T.S. Samples were collected in citrated tubes, centrifuged, PT and aPTT were processed with plasma of primary tubes. Sufcient quantity of plasma was aliquoted immediately from primary tubes and kept at room temperature for 4 hours after which the plasma of primary tubes and aliquots were re-analyzed separately and values were compared. Results: Pearson correlation was performed to show the relation. PT and aPTT values obtained in the primary tube and aliquot were correlating with the initial value (PT-r =0.833,0.831 respectively) and (aPTT-r =0.97,0.87 respectively) and also with each other (PT-r =0.99) and (aPTT-r =0.90) respectively. There is no statistical signicant difference in PT of primary tube and aliquots when compared to initial PT (p>0.01). Incase of APTT there is statistically signicant difference in primary tube and aliquot when compared to initial value (p<0.01) where as there is no signicant difference of APTT values in aliquot and primary tube after 4 hours (p>0.01). Conclusion: The present study shows no difference of PT values in aliquots and primary tube when compared to initial value, whereas APTT values differ from initial values in samples stored for more than 4 hours in primary tube and aliquot.

Biobanking as a necessary tool for research in the field of personalized medicine in the scientific medical center

The National Medical Research Center for Preventive Medicine of Russia (NMRCPM) conducts epidemiological and clinical research for the development of personalized medicine. This is why NMRCPM has faced the problem of how to standardize preanalytical conditions for all biospecimens from various scientific projects and of how to provide long-term responsible standardized regulated safe storage of blood and its derivatives. This article describes various aspects of establishing a biobank in a large medical center dedicated to integrating the biomarkers research activities of different departments. To date, >205,000 serum/plasma/whole blood specimens have been stored. Collaboration with >25 scientific projects as well as the biobank’s own research project has been organized. The availability of this biobank became a platform for the establishment of the Personalized Medicine Center in NMRCPM.

Evaluation of Preanalytical Conditions and Implementation of Quality Control Steps for Reliable Gene Expression and DNA Methylation Analyses in Liquid Biopsies

BACKGROUND Liquid biopsy provides important information for the prognosis and treatment of cancer patients. In this study, we evaluated the effects of preanalytical conditions on gene expression and DNA methylation analyses in liquid biopsies. METHODS We tested the stability of circulating tumor cell (CTC) messenger RNA by spiking MCF-7 cells in healthy donor peripheral blood (PB) drawn into 6 collection-tube types with various storage conditions. CTCs were enriched based on epithelial cell adhesion molecule positivity, and RNA was isolated followed by cDNA synthesis. Gene expression was quantified using RT-quantitative PCR for CK19 and B2M. We evaluated the stability of DNA methylation in plasma under different storage conditions by spiking DNA isolated from MCF-7 cells in healthy donor plasma. Two commercially available sodium bisulfite (SB)-conversion kits were compared, in combination with whole genome amplification (WGA), to evaluate the stability of SB-converted DNA. SB-converted DNA samples were analyzed by real-time methylation-specific PCR (MSP) for ACTB, SOX17, and BRMS1. Quality control was assessed using Levey–Jennings graphs. RESULTS RNA-based analysis in CTCs is severely impeded by the preservatives used in many PB collection tubes (except for EDTA), as well as by time to analysis. Plasma and SB-converted DNA samples are stable and can be used safely for MSP when kept at −80 °C. Downstream WGA of SB-converted DNA compensated for the limited amount of available sample in liquid biopsies. CONCLUSIONS Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic.

Short preheating at 41°C leads to a red blood cells count comparable to that in RET channel of Sysmex analysers in samples showing cold agglutination

AimsThe presence of cold agglutinin in blood samples can cause a spontaneous agglutination of red blood cells (RBCs) when low temperature occurs. This phenomenon causes a spurious lowering of RBC count on the automated haematological analysers that are detected by incongruous values (≥370 g/L) of the mean cellular haemoglobi concentration (MCHC). A preheating at 37°C can remove the RBC agglutination generally resulting in a reliable count. It has been reported that the same result can be reached by using the optical reticulocyte (RET) channel of Sysmex analysers where the RBC count is not influenced by the presence of cold agglutinin. This study aims to evaluate these data in a larger population, with regard to environmental conditions on Sysmex analysers. We have also evaluated the influence of different thermal pretreatments on the RBC count.MethodsThis study was performed on 96 remnants of peripheral blood samples (48 with MCHC in normal range and 48 with MCHC>370 g/L) which have been analysed in different preanalytical conditions on the Sysmex analysers.ResultsA preheating of samples at 41°C for 1 min leads to a reversibility of the cold agglutination comparable to the one observed in the RET channel and yields better results compared with 37°C for 2 hours.ConclusionsNone of described procedures assure the complete cold agglutination reversibility in every case. Consequently, since the haematological analysers not yet provide reliable parameters to confirm the complete resolution of agglutination, further verification of RBC count accuracy needs to be performed.

Oxidation of PTH: in vivo feature or effect of preanalytical conditions?

Background: Posttranslational oxidation of parathyroid hormone (PTH) modifies its biological activity. Measurement of non-oxidized PTH (n-oxPTH) could be an improvement in assessing PTH status, as intact PTH may rather reflect oxidative stress. However, it is debated whether oxidation of PTH occurs in vivo, or whether it is mainly an in vitro artifact. The aim of this study was to investigate the influence of different preanalytical conditions on the oxidation of PTH within a wide range of plasma PTH concentrations and oxidation propensity. Methods: n-oxPTH was separated from its oxidized form using an affinity column capturing the oxidized PTH. n-oxPTH was measured in eluate using commercially available PTH assays. The study included ethylenediaminetetraacetic acid plasma samples from 17 patients undergoing hemodialysis and 32 healthy subjects. We determined effects of storage temperature, time until centrifugation and freeze-thaw cycles. PTH and n-oxPTH concentrations were measured in each sample using six different immunoassays. Results: n-oxPTH concentrations remained unchanged up to 180 min until centrifugation, two freeze-thaw cycles or after storage at −20°C or −80°C up to 79 days. Various methods for n-oxPTH and PTH measurements yielded highly comparable results, apart from standardization differences between various PTH and n-oxPTH assays. Conclusions: n-oxPTH concentrations were stable under our study conditions, indicating negligible ex vivo oxidation of PTH. In addition, PTH immunoassays have a different sensitivity for n-oxPTH than for total PTH. For this reason, the n-oxPTH/total PTH ratio cannot be used in absence of a n-oxPTH standard. Clinical implications of determining n-oxPTH require additional study.