Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles

Background: Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA. Methods: 3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n=12) and without (n=35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-) automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n=72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed. Results: RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 mu g, p < 0.001) followed by the choice of blood collection tube. (Semi-) automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed. Conclusion: We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles

Assay validity of point-of-care platelet function tests in thrombocytopenic blood samples

Point-of-care (POC) platelet function tests are faster and easier to perform than in-depth assessment by flow cytometry. At low platelet counts, however, POC tests are prone to assess platelet function incorrectly. Lower limits of platelet count required to obtain valid test results were defined and a testing method to facilitate comparability between different tests was established. We assessed platelet function in whole blood samples of healthy volunteers at decreasing platelet counts (> 100, 80-100, 50-80, 30-50 and < 30 x109/L) using two POC tests: impedance aggregometry and in-vitro bleeding time. Flow cytometry served as the gold standard. The number of platelets needed to reach 50% of the maximum function (ED50) and the lower reference limit (EDref) were calculated to define limits of test validity. The minimal platelet count required for reliable test results was 100 x109/L for impedance aggregometry and in-vitro bleeding time but only 30 x109/L for flow cytometry. Comparison of ED50 and EDref showed significantly lower values for flow cytometry than either POC test (P value < 0.05) but no difference between POC tests nor between the used platelet agonists within a test method. Calculating the ED50 and EDref provides an effective way to compare values from different platelet function assays. Flow cytometry enables correct platelet function testing as long as platelet count is > 30 x109/L whereas impedance aggregometry and in-vitro bleeding time are inconsistent unless platelet count is > 100 x109/L

Digital competence in laboratory medicine

Objectives: Even though most physicians and professionals in laboratory medicine have received basic training in statistics, experience shows that a general understanding of data analysis is not yet available on a broad scale. Therefore, data literacy, data-driven decision making, and computational thinking should be implemented in future educational training. To evaluate the state of digital competence among young scientists (YS) in laboratory medicine, we launched a worldwide online survey. Methods: A global online survey was conducted from 25/05/2022 to 26/06/2022 and was disseminated to YS who are listed in three large networks: YS of the DGKL, the EFLM Task Group-YS, and IFCC Task Force-YS and its corresponding members, covering a base of 53 countries. Results: A total of 119 young scientists from 40 countries participated in this survey. 80% did not learn digital skills in their academic education but 96% felt they needed to. Digital literacy was associated with terms such as programming, artificial intelligence and machine learning, statistics, communication, Big Data and data analytics. Conclusions: The results of our survey show that more knowledge and training in the area of digital skills is not just necessary, but also wanted by young scientists. A varied learning environment consisting of tutorial articles, videos, exercises, technical articles, collection of helpful links, online meetings and in person bootcamps is crucial to meet the challenges of an international project with different languages, health systems and time zones

Thrombomodulin in patients with mild to moderate bleeding tendency.

INTRODUCTION: A massive increase of soluble thrombomodulin (sTM) due to variants in the thrombomodulin gene (THBD) has recently been identified as a novel bleeding disorder. AIM: To investigate sTM levels and underlying genetic variants as a cause for haemostatic impairment and bleeding in a large number of patients with a mild to moderate bleeding disorder (MBD), including patients with bleeding of unknown cause (BUC). PATIENTS AND METHODS: In 507 MBD patients, sTM levels, thrombin generation and plasma clot formation were measured and compared to 90 age- and sex-matched healthy controls. In patients, genetic analysis of the THBD gene was performed. RESULTS: No difference in sTM levels between patients and controls was found overall (median ([IQR] 5.0 [3.8-6.3] vs. 5.1 [3.7-6.4] ng/ml, p = .762), and according to specific diagnoses of MBD or BUC, and high sTM levels (≥95th percentile of healthy controls) were not overrepresented in patients. Soluble TM levels had no impact on bleeding severity or global tests of haemostasis, including thrombin generation or plasma clot formation. In the THBD gene, no known pathogenic or novel disease-causing variants affecting sTM plasma levels were identified in our patient cohort. CONCLUSION: TM-associated coagulopathy appears to be rare, as it was not identified in our large cohort of patients with MBD. Soluble TM did not arise as a risk factor for bleeding or altered haemostasis in these patients

Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

<div><p>Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene <i>36B4</i> using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.</p></div

Comparison of TL and SCG quantification using single-plex assays and a multiplex assay.

<p>For evaluation of telomeres, qPCRs were performed as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143889#pone.0143889.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143889#pone.0143889.ref031" target="_blank">31</a>] with adjustments as described in Material and Methods. Dilutions of a control pool sample were used as standard curve. Standard curves for telomere <b>(A)</b> or single copy gene 36B4 <b>(B)</b> qPCR assays using the single-plex (green) or multiplex (grey) assay. Correlation between the multiplex and the single-plex assays for TL <b>(C)</b> and SCG <b>(D)</b>. <b>(E)</b> Correlation of TL/SCG ratio in 192 DNA samples.</p

Effect of freezing and degradation on DNA isolation efficiency.

<p>Amounts of DNA isolated from 7 ml whole blood aliquots from 8 pools (M/F; n = 4/4) depending on preanalytical conditions. Actinomycin D was used for apoptosis-induced sample degradation. <b>(A)</b> Non-frozen, non-degraded samples. <b>(B)</b> Frozen, non-degraded samples. <b>(C)</b> Non-frozen, degraded samples. <b>(D)</b> frozen, degraded samples.</p