Bacterial contamination of blood components and the prevention of transfusion-associated bacterial infection still remains a major challenge in transfusion medicine. Over the past few decades, a significant reduction in the transmission of viral infections has been achieved due to the introduction of mandatory virus screening. Platelet concentrates (PCs) represent one of the highest risks for bacterial infection. This is due to the required storage conditions for PCs in gas-permeable containers at room temperature with constant agitation, which support bacterial proliferation from low contamination levels to high titers. In contrast to virus screening, since 1997 in Germany bacterial testing of PCs is only performed as a routine quality control or, since 2008, to prolong the shelf life to 5 days. In general, bacterial screening of PCs by cultivation methods is implemented by the various blood services. Although these culturing systems will remain the gold standard, the significance of rapid methods for screening for bacterial contamination has increased over the last few years. These new methods provide powerful tools for increasing the bacterial safety of blood components. This article summarizes the course of policies and provisions introduced to increase bacterial safety of blood components in Germany. Furthermore, we give an overview of the different diagnostic methods for bacterial screening of PCs and their current applicability in routine screening processes.
Bacterial detection methods; Platelet concentrates; Screening strategies; Culture methods; Rapid methods; National guidelines Germany
Hepatitis E virus (HEV) has been recognized since 2004 as a transfusion-transmissible infectious agent, and recent epidemiological data suggest that it may pose a safety threat to the blood supply. It has recently become obvious that hepatitis E is endemic in industrialized countries, and that more infections are autochthonous than travel-associated. Epidemiological and phylogenetic analysis suggests that HEV infection has to be considered as a zoonosis and that viral transmission from animals (pigs, wild animals) occurs through food or direct contact. The seroprevalence and incidence of HEV in the general population and blood donors in European countries indicate an underestimated risk for transfusion transmissions. Recently reported cases of transfusion transmission of HEV infection, and detection of viremic, asymptomatic blood donors in nucleic acid amplification technique screening programs give an indication of the importance of this virus. Diagnostic assays for detection of anti-HEV antibodies, HEV antigens and RNA are discussed. Recent studies support the idea that active immunization can prevent hepatitis E, highlighting the need for vaccination programs. Here we review current knowledge of HEV and its epidemiology, blood transmission and prevention of this disease with emphasis on blood supply.
Hepatitis E virus; Transfusion-transmitted HEV infection; Seroprevalence; Incidence; Transfusion transmission; HEV blood donor screening; HEV RNA
Parvovirus B19 (B19V) is a transfusion-transmissible virus. To obtain data about the prevalence, incidence, the course of B19V infection in blood donors and whether B19V might impair their blood counts, samples from blood donors with B19V infection were investigated.
Blood donations were screened for B19V DNA using the Cobas TaqScreen DPX Test® in mini-pools. B19V DNA concentration, anti-B19V IgG antibody titer and blood counts were determined in positive donors.
157/23,889 (0.66%) donors provided 347 B19V DNA-positive samples. Prevalence of B19V infection was 0.45%, incidence 0.20%. B19V DNA concentrations were predominantly low; only in 8 samples were viral loads of ≥105 IU B19V DNA/ml plasma detectable. Besides a slight decrease in hemoglobin, hematocrit, mean corpuscular volume, mean cellular hemoglobin and mean hemoglobin concentration, no major differences in blood counts occurred in B19V DNA-positive samples. In samples with a low B19V DNA concentration, anti-B19V IgG titers were rather high. 98 donors provided at least 1 B19V DNA-positive follow-up sample, indicating a prolonged viremia.
B19V infection induced no major impairment in the blood counts. In donors with low-level viremia, infectivity through their donations is probably reduced by high antibody titers. Low-level viremia is prolonged, probably exceeding 1 year in many cases.
Parvovirus B19; B19V infection; Blood donor; B19V DNA
During the last few decades, blood safety efforts were mainly focused on preventing viral infections. However, humanity's increased mobility and improved migration pathways necessitate a global perspective regarding other transfusion-transmitted pathogens. This review focuses on the general infection risk of blood components for malaria, dengue virus, Trypanosoma cruzi (Chagas disease) and Babesia spp. Approximately 250 million people become infected by Plasmodium spp. per year. Dengue virus affects more than 50 million people annually in more than 100 countries; clinically, it can cause serious diseases, such as dengue haemorrhagic fever and dengue shock syndrome. Chagas disease, which is caused by Trypanosoma cruzi, mainly occurs in South America and infects approximately 10 million people annually. Babesia spp. is a parasitic infection that infects red blood cells; although many infections are asymptomatic, severe clinical disease has been reported, especially in the elderly. Screening assays are available for all considered pathogens but make screening strategies more complex and more expensive. A general pathogen inactivation for all blood components (whole blood) promises to be a long-term, sustainable solution for both known and unknown pathogens. Transfusion medicine therefore eagerly awaits such a system.
Blood safety; Malaria; Dengue virus; Chagas disease; Babesia
Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat).
Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems.
Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening.
HIV differs from other blood-borne viruses with regard to its fast evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1 variants.
HIV-1 nucleic acid amplification technique; Blood safety; HIV-1 variant
Traditionally, leukoreduction and selection of blood products from seronegative donors have been used as alternative strategies to reduce the risk of transfusion-transmitted cytomegalovirus infections (TT-CMV) in atrisk patients. After the introduction of universal leukoreduction for red blood cell and platelet concentrates in Germany, a controversy evolved as to whether the additional selection of blood products from seronegative donors would reduce or even increase the risk of TT-CMV. This review summarizes the current knowledge about CMV infections in blood donors and the implications of this information on the effect of potential transfusion strategies. Even though there are conflicting data about the incidence of TT-CMV remaining after the introduction of leukodepletion, it has been clearly shown that both prevalence and concentration of CMV DNA in peripheral blood are highest in newly seropositive donors. Therefore, avoidance of blood products from these donors is the most important goal of any transfusion strategy. This goal can be reached by: i) selection of blood products from seronegative donors, ii) provision of CMV DNA-negative blood products, or iii) provision of blood from long-term seropositive donors. In cases of suspected TT-CMV, all implicated donors should be investigated carefully to gather further knowledge on which donors confer the lowest risk for TT-CMV.
Human cytomegalovirus; CMV; Blood donor; Transfusion-associated infections; Transfusion
Contamination of cell culture and biological material by mollicute species is an important safety issue and requires testing. We have developed a singletube real-time polymerase chain reaction (PCR) assay for rapid detection of Mollicutes species stipulated by the European Pharmacopeia.
Primers and TaqMan probes (FAM-labeled) were deduced from 16S rDNA sequence alignment of 18 mollicutes species. A synthetic internal control (IC) DNA and an IC-specific TaqMan probe (VIC-labeled) were included. The analytical sensitivity of the assay was determined on DNA dilutions from 12 mollicute strains. Specificity was proven by the use of DNA from other bacteria.
Analytical sensitivities of the PCR assay were in the range of 405-2,431 genomes/ml for 11 of the 12 tested mollicute DNA samples. The lowest sensitivity was found for Ureaplasma urealyticum (19,239 genomes/ml). Negative results for DNA samples from 3 different ubiquitous bacteria demonstrated the specificity of the PCR assay for Mollicutes. Direct testing of cell culture supernatants spiked with Mycoplasma orale revealed similar sensitivity compared to isolated DNA.
Our single-tube real-time PCR assay with internal reaction control enables rapid and specific detection of mollicute contaminants. The test protocol is suitable for routine quality control of cell therapeutics.
Cell culture; Cell therapeutics; Mycoplasma; Mollicutes; Quality control
The need for an alternative to fetal bovine serum (FBS) is known to scientists and users involved in cell therapy or advanced therapy medicinal products. Human serum (huS) and platelet lysate (hPL) can be used as alternatives resulting in similar or even superior results concerning cell expansion.
We developed protocols for the production of huS and two types of hPL and tested them in the expansion of human fibroblasts and adipose tissue-derived stem cells (ASC). Quality control included cell counts (platelets, red and white blood cells), sterility testing, pH levels, total protein concentrations and growth factor levels. ASC and fibroblasts were expanded for three passages in media supplemented with FBS, huS or hPL and evaluated microscopically. Proliferation in terms of population doubling times (PDT) was determined. In case of ASC, differentiation was performed as well.
All three alternatives demonstrated shorter PDT for fibroblasts and ASC compared to FBS. Furthermore, ASC maintained their differentiation potential.
We conclude that hPL and huS can be used as alternatives to FBS for the cultivation and expansion of cells intended for human use.
Regenerative medicine; Human platelet lysate; Human serum; Fetal bovine serum; Human adipose tissue-derived stem cells
Development of cell therapy and advanced therapy medicinal products depends on in vitro expansion of human cells in fetal bovine serum (FBS) supplemented media. Human-derived supplements, such as human serum (huS) and human platelet lysate (hPL), represent suitable alternatives to FBS. Various studies demonstrated that the use of these human alternatives result in comparable or even improved proliferation and expansion ratios.
Within this study three human supplement alternatives, huS, hPLP (plasma containing hPL) and hPLN (plasma replaced by saline), were compared by 2D gel electrophoresis, an important tool in proteomic analysis. 2D gel electrophoresis allows the determination of the protein number and the detection of protein changes (decreasing/increasing concentration).
Results and Conclusion
The comparison of huS, hPLP, and hPLN gels resulted in clearly visible differences in protein pattern, protein number and concentration, particularly when comparing huS with hPL and hPLP with hPLN.
Cell therapy; Human platelet lysate; Human serum; Fetal calf serum; Proteomics
The broaden application of adoptive T-cell transfer has been constrained by the technical abilities to isolate and expand antigen-specific T cells potent to selectively kill tumor cells. With the recent progress in the design and manufacturing of cellular products, T cells used in the treatment of malignant diseases may be regarded as anticancer biopharmaceuticals. Genetical manipulation of T cells has given T cells desired specificity but also enable to tailor their activation and proliferation potential. Here, we summarize the recent developments in genetic engineering of T-cell-based biopharmaceuticals, covering criteria for their clinical application in regard to safety and efficacy.
Adoptive cell transfer; Chimeric antigen receptor; CAR; T-cell receptor; TCR; Clinical trial
Increasing scientific knowledge and technical innovations in the areas of cell biology, biotechnology and medicine resulted in the development of promising therapeutic approaches for the prevention and treatment of human diseases. Advanced therapy medicinal products (ATMPs) reflect a complex and innovative class of biopharmaceuticals as these products are highly research-driven, characterised by innovative manufacturing processes and heterogeneous with regard to their origin, type and complexity. This class of ATMP integrates gene therapy medicinal products, somatic cell therapy medicinal products and tissue engineering products and are often individualized and patient-specific products. Multiple challenges arise from the nature of ATMPs, which are often developed by micro, small and medium sized enterprises, university and academia, for whom regulatory experiences are limited and regulatory requirements are challenging. Regulatory guidance such as the reflection paper on classification of ATMPs and guidelines highlighting product-specific issues support academic research groups and pharmaceutical companies to foster the development of safe and effective ATMPs. This review provides an overview on the European regulatory aspects of ATMPs and highlights specific regulatory tools such as the ATMP classification procedure, a discussion on the hospital exemption for selected ATMPs as well as borderline issues towards transplants/transfusion products.
ATMP; European Community; Medicinal product regulatory issues
The use of platelet-rich plasma (PRP) in regenerative approaches in cartilage repair is becoming more common. Information about PRP composition and its content of putative bioactive chondrogenic growth factors (GF) that may support cartilage regeneration is scarce.
GF composition of a pool of 6 PRP preparations was determined using Protein Antibody Membrane Arrays covering 507 GF, signaling molecules, and receptors. To verify the chondrogenic GF variability in PRP, Growth Factor Antibody Membrane Arrays covering 26 GF were applied to 6 individual PRP preparations. Selected GF involved in chondrogenic differentiation were quantified by Enzyme-Linked Immunosorbent Assay (ELISA).
417 out of 507 possible detectable proteins were present in the PRP pool, including 76 GF. Quantification of selected chondrogenic GF by ELISA showed an average of 0.31 ng/ml bone morphogenetic protein-2, 0.50 ng/ml connective tissue growth factor, 0.76 ng/ml fibroblast growth factor-2, and 0.59 ng/ml transforming growth factor-β3.
PRP as a therapeutic option in regenerative cartilage repair strategies is a powerful tool for the local application of chondrogenic GF to the site of injury. Chondrogenic GF are present in PRP and may support cartilage repair by inducing cell differentiation and cartilage matrix formation.
Platelet-rich plasma; Cartilage regeneration; Chondrogenesis; Growth factors; Apheresis
Skeletal muscle trauma leads to severe functional deficits, which cannot be addressed by current treatment options. Previous investigation could show the efficacy of a local transplantation (TX) of mesenchymal stroma cells (MSCs) for the therapy of muscle injury. Underlying mechanisms remain to be elucidated. The aim of the present work was to characterize the fiber composition changes following MSC-TX after open crush injury.
20 male SD rats received an open crush trauma of the left soleus muscle. 2.5 × 106 autologous MSCs were transplanted into the crushed soleus muscle of 10 animals 7 days after trauma (group 1, n = 10). Control animals received an injection of saline solution (group 2, n = 10). Histologic analysis of fibrosis, fiber type composition, and muscle force measurements were performed 28 days after trauma.
MSC-TX improved muscle force significantly (fast-twitch, treated: 0.76 (0.51–1.15), untreated: 0.45 (0.32–0.73); p = 0.01). Tetanic stimulation resulted in a significant increase of force development (treated: 0.63 (0.4–1.21), untreated: 0.34 (0.16–0.48); p = 0.04). Histological analyses showed no differences in the amount of fibrotic tissue (treated vs. untreated, p = 0.42). A shift towards fastMHC-positive fibers was observed following MSC-TX (treated vs. untreated; p = 0.01 (mm2) or 0.007 (%)).
This study demonstrated an effect of locally administered MSCs in the treatment of skeletal muscle injuries on a structural level. For the first time a fiber type shift towards fastMHC following MSC-TX after crush injury could be demonstrated and related to MSC-TX. These results might open the discussion of an alternative mode of action of MSCs in tissue regeneration.
Muscle trauma; Regeneration; Stem cells; Fiber type; Fast myosin heavy chain; Slow myosin heavy chain; Shift; Tissue engineering
Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation.
Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1–5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells.
In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups.
These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells.
Osteoblast-like cells; Osteoclast-like cells; Co-culture; Mechanical stimulation; Differentiation
Discrepant results in antigen and reverse ABO blood typing are often caused by a variant ABO gene. Molecular analysis can help to characterize such variants. Here, we describe the identification of a novel ABO gene variant in a patient with aberrant ABO phenotype and discrepant genotyping results.
A patient with discrepant results in automated forward and reverse ABO phenotyping was further investigated by serological (gel and tube technique) and molecular (commercial and inhouse PCR-SSP, DNA sequencing) methods. A PCR-SSP system was established to screen the novel mutation in 1,820 blood donors.
Standard serological tests confirmed blood group O, however, only anti-B isoagglutinins were present. A monoclonal anti-AB antibody detected very weak agglutination in gel technique. Standard ABO genotyping using PCR-SSP led to discrepant results (O1/O1 or O1/A) depending on the test system used. ABO exon re-sequencing identified a novel missense mutation in exon 6 at position 248A>G (Asp83Gly) in the binding region of PCR-SSP primers for the detection of 261G alleles. Blood donors with regular ABO blood groups were all negative for the 248G allele designated Aw34.
The novel ABO gene variant Aw34 is associated with very weak A antigen expression and absent anti-A isoagglutinins. The mutation is located in exon 6 close to the O1-specific 261G deletion in the binding region of PCR-SSP primers. Presumably, depending on the primer concentration used in commercial ABO genotyping kits, the mutation could lead to a false-negative reaction.
ABO phenotyping and genotyping; ABO sequencing ABO gene variant; PCR-SSP
Infectious disease marker testing is obligatory for the release of human tissue for transplantation. Most CE-marked tests are not validated for postmortem blood. In a previous study we have validated the testing for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc. Here, we present the validation of testing for antibodies against T. pallidum, which is the last marker obligatory for tissue release for transplantation.
17 samples of postmortem sera and 10 samples of both pre-und postmortem sera were obtained from cornea donors and tested for anti-T.
pallidum on the Siemens-BEP-III-System. These sera were spiked with anti-T.
pallidum-positive standard sera in concentrations which give low- and high-positive results at the respective dilution.
Two of the unspiked postmortem sera were false-positive most likely due to intense hemolysis (free hemoglobin > 50 mg/dl). Of the 25 negative postmortem sera, none of the spiked samples was false-negative after 0, 24 and 60 h.
There is no indication that postmortem samples give false-negative or false-positive results with the test system and test kits used in cases of low hemolysis. The procedure described might serve as a model for validating other test kits on postmortem samples.
Infectious disease serology; Postmortem blood; Blood testing; Tissue donation; Treponema pallidum
Transfusion of ex vivo expanded megakaryocytes (MKs) has been proposed to sustain platelet recovery after cord blood (CB) hematopoietic stem cell transplantation. In this study, we investigated the effects of heparin on ex vivo colony forming unit-megakaryocytes (CFU-MKs) and MKs expansion from CB CD34+ cells.
CB CD34+ cells were stimulated by a combination of thrombopoietin (TPO), stem cell factor (SCF), Flt3-Ligand (FL), IL-6, and IL-11 supplemented with autologous serum and heparin during 14 days. Expanded cells were analyzed by flow cytometry and cultured in a CFU-MK assay.
Compared to control cultures, the 5-factor combination with heparin induced significantly (p ≤ 0.05) higher numbers of: CFU-MKs and CD41+ cells on days 7 and 14; CD41+ cells displaying hyperploidy levels (≥8N) on day 14; platelets on day 14. The culture-derived platelets were activated upon collagen stimulation.
Heparin can significantly enhance the stimulating effects of a combination of TPO, SCF, FL, IL-6, and IL-11 supplemented with autologous serum on CFU-MK, MK, and platelet production from CB CD34+ cells. This expansion system could represent a promising method to generate CFU-MKs and MKs cells for transfusion to sustain platelet reconstitution following CB transplantation.
Megakaryocytopoiesis; Glycosaminoglycan; Expansion; Cord blood; CD34+ cells
Thrombocytopenia is a common hematologic disorder. Stimulation of thrombopoiesis may reduce the risk for thrombocytopenia-induced bleeding, prevent severe thrombocytopenia, and reduce the need for platelet transfusion. The key cytokine is thrombopoietin (TPO). It regulates proliferation and maturation of megakaryocytes as well as platelet production. TPO is synthesized in the liver. Development of TPO from the laboratory into a therapeutic tool has turned out to be an unexpected challenge. Clinical trials on first-generation thrombopoietic growth factors were stopped in 2001. At present, second-generation thrombopoiesis-stimulating agents have only been approved as orphan drugs for third-line therapy of patients with chronic immune thrombocytopenia. Larger groups in need are patients with myelodysplastic syndrome, chemotherapy-induced thrombocytopenia, other forms of hereditary and acquired bone marrow failure, hepatitis C infections, or liver cirrhosis.
Eltrombopag; Romiplostim; ITP; Cirrhosis; Myelodysplastic syndrome
Scientific data regarding effects of platelet transfusion on platelet count in dengue-related thrombocytopenia is scanty.
A single center, randomized non-blinded trial was conducted on adult patients with dengue fever and platelet counts less than 30,000/μl. Patients were randomized to treatment and control group. Treatment group received single donor platelets. Patients with post-transfusion platelet increment (PPI) ≥10,000/μl and/or corrected count increment (CCI) ≥5,000/μl 1 h post-transfusion were considered responders. Primary outcome was platelet count increments at 24 and 72 h.
87 patients were enrolled, and 43 (48.2%) received platelet transfusion. Mean PPI and CCI at 1 h post-transfusion in the treatment group were 18,800/μl and 7,000/μl respectively. 22 (53.6%) patients in the treatment group were non-responders. Mean platelet increments at 24 and 72 h were higher in the treatment group as compared to the control group. Responders showed significantly higher increments when compared to non-responders and the control group at 24 h (p = 0.004 and p ≤ 0.001, respectively) and 72 h (p = 0.001 and p ≤ 0.001, respectively). Significant differences were found between non-responders and the control group at 24 h (p ≤ 0.001), but not at 72 h (p = 0.104). Patients with lower baseline platelet count were more likely to be non-responders. Platelet transfusion neither prevented development of severe bleeding nor shortened time to cessation of bleeding. Three severe transfusion reactions and two deaths occurred in treatment group.
In this trial, almost half the patients showed no response to a high-dose platelet transfusion. Platelet transfusion did not prevent development of severe bleeding or shorten time to cessation of bleeding and was associated with significant side effects. Therefore, platelet transfusion should not be routinely done in the management of dengue fever.
Dengue fever; Dengue hemorrhagic fever; Single donor apheresis platelets; Platelet transfusion; Corrected count increment; CCI; Transfusion reaction