Whole transcriptome analysis of platelet concentrates during storage.

BACKGROUND: Platelets are anucleated blood cells and contain various RNA species. We investigated the changes in the whole transcriptome expression profile of platelet concentrates (PC) during storage to explore biological functions and biomarkers in platelet storage damage. MATERIALS AND METHODS: Platelets were collected by apheresis from eight healthy blood donors and stored from day 0 to day 4. Platelet phenotyping and function analysis were used to detect platelet activity during storage. RNA-sequencing was used to detect changes in expression of mRNA, lncRNA and circRNA in the PC during storage. Gene ontology and KEGG analyses were applied to predict the functional distribution of differential expression of mRNA. Gene set enrichment analysis was used to analyze the differential levels of gene pathways. Finally, polymerase chain reaction (PCR) analysis was performed to verify the expression of three mRNA (POLE2, DCUN1D4, DAD1). RESULTS: In total, 10,767 mRNA, 2,923 lncRNA and 68,550 circRNA were detected in the PC by RNA-sequencing. The expression levels of 222 mRNA changed significantly from day 0 to day 4 of storage: 58 increased continuously and 145 decreased continuously. Differentially expressed mRNA may be involved in physiological processes such as platelet activation, platelet aggregation, endocytosis, and apoptosis. Expression levels of 1,413 lncRNA were obvious. The levels of 42 species increased and the levels of 28 species decreased. The expression levels of 198 species of circRNA changed significantly, with those of 13 species changing continuously. The differential levels of expression of DAD1, DCUN1D4 and POLE1 mRNA, shown by RNA sequencing, were validated by PCR assay. DISCUSSION: Changes in mRNA, lncRNA and circRNA during platelet storage may be closely related to platelet apoptosis and physiological functions in the platelet storage lesion. The expression levels of DAD1, DCUN1D4 and POLE1 could be biomarkers to monitor platelet status in PC bags.

Molecular biology analysis of ABO blood group variants caused by natural chimaerism.

BACKGROUND AND OBJECTIVES: The chimaerism phenomenon constitutes a significant mechanism underlying ABO phenotype discrepancies; however, its detection has technical challenges. In the current study, we explored different techniques to establish the chimaeric status of ABO blood types. MATERIALS AND METHODS: Fifteen individuals with possible chimaeric ABO blood type, as suggested by standard tube or column agglutination method and RBC adsorption-elution test, were enrolled in the study. The red blood cells from 11 investigated subjects showed mix-field agglutination with anti-A or anti-B in blood typing; weak A or B antigens on the other four individuals' RBCs were detected by adsorption-elution tests. The genetic study was conducted with PCR-SSP genotype, DNA sequencing of the ABO gene, STR analysis and ddPCR. RESULTS: The genetic chimaeric status was confirmed in four (27%) individuals by SSP test alone. The ABO gene sequencing identified an additional ABO allele and enabled chimaerism detection in 10 (67%) subjects. The STR analyses established the chimaerism status in 13 (87%) individuals. In the two cases where neither of the tests mentioned above had positive findings, the ddPCR was adopted, and microchimaerism, with an extremely low degree of chimaerism (0.77% and 0.12%), was revealed. The ddPCR revealed the unequal haplotypes (29.5% B vs. 70.5% O) in one subject and distinguished this B/O-O/O chimaera from certain B subgroups (B/O genotype without any mutation) like B3 . CONCLUSION: The ABO blood type chimaerism can be genetically established by comprehensive molecular methods, including PCR-SSP/DNA sequencing, STR and ddPCR, which is particularly sensitive for the detection of microchimaerism.

2-Mercaptoethanol (2-ME)-based IATs or Polybrene method mitigates the interference of daratumumab on blood compatibility tests.

OBJECTIVES: Treating red blood cells (RBCs) with dithiothreitol (DTT) is a wildly-recommended to overcome the interference of the daratumumab (DARA) with blood compatibility testing. Nevertheless, DTT can be hard to obtain in the clinical laboratory, while its use in routine practice may be time-consuming. In the following study, we explored the feasibility of using a commercial 2-mercaptoethanol (2-ME) working solution or the time-saving Polybrene method to mitigate DARA interference. METHODS: Antibody screening and cross-matching were performed using 2-ME or DTT-based indirect antiglobulin tests (IATs) and Polybrene method (with human IgG anti-E same IATs titer as DARA as positive control) on 37 samples. Most clinically important blood group antigens on RBCs were detected after treatment with 2-ME or DTT. RESULTS: Treating RBCs with 2-ME eliminates the DARA interference with the antibody screening or cross-matching; yet, K antigen is denatured during treatment. DARA does not interfere with antibody screening and cross-matching via Polybrene method, while 2+ agglutinations of anti-E antibody with the same titer (IATs method) as DARA could be observed in the positive controls via this method. CONCLUSION: 2-ME-based IATs or Polybrene method could replace DTT-based IATs to mitigate DARA interference.