Emergency Supply Policy of Cryopreserved RBC and PLT: The Czech Republic Concept.

Supply of blood for urgent substitution is a strategic logistical problem for the military medical services across the world. The limited shelf life of blood- derived bioproductsin the liquid state and the need for special transport and use conditions, apart from donor and donations availability are among the causes for concern. To solve these problems many national health-care authorities implemented the national emergency blood crisis policy, to get a large amount of blood at any time at any place in the case of disaster, terrorist attack or war. The civil therapeutic problems in immunohematolgy cases can also be solved by stocks of fresh and cryopreserved homologous or autologous blood for patients with rare RBCs antigens or HLA / HPA platelet refractoriness with no chance to use common blood. The short shelf life of fresh platelets limits their efficient inventory management and availability during a massive transfusion protocol. Building an inventory of frozen blood components can mitigate the risk of insufficient availability. Since the beginning of the century in the Czech Republic, used, like other countries, the use of of cryopreserved blood-derived bioproducts has become the current method used to overcome the shortages of a timely supply. The Military University Hospital, Prague, and its bank of cryopreserved blood have been operating under this policy since 2006. There is currently a stock of frozen RBCs for military reserve, for a national blood crisis and, also, a stock of rare RBC units. For crisis management there are also stored, frozen PLTs, which are used in the treatment of heavily bleeding polytrauma patients. Both the containment and research development mitigation policy programs are in place for civil / military emergency situations. Even pathogen reduced frozen PLTs and frozen RBCs were successfully investigated for clinical use if demands arose. Currently, it is possible to meet operational demand while reducing the number of resupply transports and loss of products due to expiration. A lesson has been learned from the current containment, reseach and mitigation programs of efficient blood supply management with cryopreserved blood and blood derived bioproducts.

Optimizing the supply of whole blood-derived bioproducts through the combined implementation of cryopreservation and pathogen reduction technologies and practices: An overview.

The essential historical knowledge and expertise developed over the past 5-6 decades on the safety / efficacy of conventional blood components therapy by blood transfusion establishments have guided the development of validated methods which have ensure optimal safety margins for frozen blood and its bioproducts with or even without pathogen reduction. Newer generations of pathogen reduced frozen red blood cell, plasma and platelet products and the standardised and safer pooling of human platelet lysate are now become available for potential clinical use. These types of whole blood-derived bioproducts not only reduce the risk of transmission of range of pathogenic blood-borne pathogen. As cryopreservation can be combined with PRT without significantly compromising in vitro quality characteristics or physiological capabilities, it allows us to maximize the available inventory of these blood products in both civil and military trauma settings. The main objective of this overview is to update readers and scientific / medical communities of the various building blocks needed to optimally grantee the pathogen safety of whole blood-derived bioproducts, with minimal untoward events to the recipients. While this is an emerging area, we are seeing the numerous potential opportunities that cryopreservation and pathogen inactivation can have on the transfused patient outcomes. This manuscript is informed by recent publications on this topic.

DGTI Register of Rare Donors.

For patients with antibodies against the most common blood groups a rapid and efficient supply of compatible erythrocyte concentrates is self-evident. But typically we have to make the greatest effort providing blood for these patients, which have made antibodies against common blood groups. There are however patients with antibodies against rare blood group antigens that need special blood. The supply of such blood can be very difficult and mostly time-consuming. For this reason we set up a database of blood donors with rare blood groups. Since 2005 the BTS SRC Berne Ltd. has run this database on behalf of the Swiss BTS SRC. After a reorganization and extension of the database, conducted during 2011/2012, the data file was renamed 'DGTI Register of Rare Donors' and is now run under the patronage of the German Society for Transfusion Medicine and Immunohematology (DGTI).

Comparative study of automated cryopreservation of red blood cells.

BACKGROUND: Strategic blood reserves are an important component in meeting blood needs and this can be accomplished through the establishment of a frozen blood program. METHODS: One hundred units of packed RBC were glycerolized using the Haemonetics ACP 215 automated cell processor and placed in a -86 °C deep freezer for freezing and storage. Product weight, hematocrit, RBC count, WBC count and hemoglobin were recorded prior to freezing. Twenty five bags were thawed and deglycerolized after every three months starting at one year from the date of first glycerolization In addition to the earlier parameters the bags were assessed for supernatant osmolality, pH, supernatant hemoglobin, ATP levels and supernatant potassium and from these red cell recovery, percentage hemolysis, supernatant glycerol and red cell viability were estimated. All tests were repeated at the end of 7 and 14 days. RESULTS: The mean red cell recovery was found to be 86.12% on Day 0 and 84% on Day 14. All the bags showed residual glycerol and pH within the acceptable limits upto Day 14. Percentage hemolysis, Mean ATP levels and mean supernatant potassium levels were within acceptable limits upto Day 14. All the units were sterile upto Day 14. CONCLUSION: The data in this study showed that the red cells which were glycerolized using the automated platform ACP 215, frozen at -80 °C for more than a year and deglycerolized again using the ACP 215 had excellent viability while being stored at 4 °C during the 14 days of post-thaw storage.

Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugs.

Genotoxicity is an important information that should be included in human biomonitoring programmes. However, the usually applied cytogenetic assays are laborious and time-consuming, reason why it is critical to develop rapid and economic new methods. The aim of this study was to evaluate if the molecular profile of frozen whole blood, acquired by Fourier Transform Infrared (FTIR) spectroscopy, allows to assess genotoxicity in occupational exposure to antineoplastic drugs, as obtained by the cytokinesis-block micronucleus assay. For that purpose, 92 samples of peripheral blood were studied: 46 samples from hospital professionals occupationally exposed to antineoplastic drugs and 46 samples from workers in academia without exposure (controls). It was first evaluated the metabolome from frozen whole blood by methanol precipitation of macromolecules as haemoglobin, followed by centrifugation. The metabolome molecular profile resulted in 3 ratios of spectral bands, significantly different between the exposed and non-exposed group (p < 0.01) and a spectral principal component-linear discriminant analysis (PCA-LDA) model enabling to predict genotoxicity from exposure with 73 % accuracy. After optimization of the dilution degree and solution used, it was possible to obtain a higher number of significant ratios of spectral bands, i.e., 10 ratios significantly different (p < 0.001), highlighting the high sensitivity and specificity of the method. Indeed, the PCA-LDA model, based on the molecular profile of whole blood, enabled to predict genotoxicity from the exposure with an accuracy, sensitivity, and specificity of 92 %, 93 % and 91 %, respectively. All these parameters were achieved based on 1 µL of frozen whole blood, in a high-throughput mode, i.e., based on the simultaneous analysis of 92 samples, in a simple and economic mode. In summary, it can be conclude that this method presents a very promising potential for high-dimension screening of exposure to genotoxic substances.

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples.

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes. Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments. Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry.

Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell-surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL-2, INF-γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL-2 and INF-γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4+ cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL-2 and 21% for and INF-γ. In CD8+ cells from activated PBMCs, the concordance was 15% for IL-2 and 32% for INF-γ. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen PBMCs Alternate Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen blood.

Comparison of three different kits for extraction of high-quality RNA from frozen blood.

Extraction of high-quality RNA is a crucial step in gene expression profiling. To achieve optimal RNA extraction from frozen blood, the performance of three RNA extraction kits- TRI reagent, PAXgene blood RNA system (PAXgene) and NucleoSpin RNA blood kit (NucleoSpin)- was evaluated. Fifteen blood specimens collected in tubes containing potassium ethylenediaminetetraacetic acid (EDTA) and stored at -80°C for approximately 5 years were randomly selected. The yield and purity of RNA, RIN (RNA integrity number) values and cycle threshold (Ct) values were assessed. Mean RNA yields with TRI reagent, PAXgene and NucleoSpin were 15.6 ± 8.7 µg/ml, 3.1 ± 1.7 µg/ml and 9.0 ± 5.5 µg/ml, respectively. Mean A260/280 ratios of RNA for the three kits were 1.7 ± 0.1, 2.0 ± 0.1, and 2.0 ± 0.0, and mean RIN values recorded as 3.2 ± 0.8, 6.0 ± 1.1, and 6.4 ± 0.9, respectively. The Ct values of housekeeping genes, 18S rRNA, ß-actin, RPLP0 and HPRT1, were as follows: TRI reagent (19.2 ± 1.6, 30.6 ± 1.8, 29.9 ± 1.4 and 36.3 ± 1.3), PAXgene 16.6 ± 1.4, 26.4 ± 1.3, 28.2 ± 1.8 and 33.8 ± 1.1), and NucleoSpin (16.3 ± 1.5, 27.2 ± 1.3, 27.0 ± 1.6 and 32.9 ± 1.6). RNA yield using TRI reagent was 1.7 times higher than that with NucleoSpin and 5 times higher than that with PAXgene. However, the purity and integrity of TRI-extracted RNA was lower than that extracted with PAXgene and NucleoSpin. Moreover, the Ct values of housekeeping genes after extraction with TRI reagent were approximately 1.7-3.8 times higher than those obtained with PAXgene and NucleoSpin. The PAXgene and NucleoSpin kits produced similar results in terms of RNA purity and integrity and subsequent gene amplification. However, RNA yields from NucleoSpin were 2.9-fold higher, compared to PAXgene. Based on these findings, we conclude that NucleoSpin is the most effective kit for extraction of abundant and high-quality RNA from frozen blood.

Requirement of Establishment of Frozen Blood Storage System for Management of Rare Blood Supply and Strategic National Stockpile / 대한수혈학회지

The blood supply can become disrupted in situations of increased demand during unexpected national catastrophes and when a patient needs a rare blood transfusion, which depends on the blood inventory in peacetime. Cryopreservation of blood, which can be stored up to 10 years, represents a possible solution to this problem by avoiding storage lesions. This review describes frozen red cell technologies, quality control issues related to post-thaw red blood cells, and preconditions and practical considerations for implementation of a frozen blood banking system in Korea.