Selective Detection of Erythrocytes with QCMs-ABO Blood Group Typing.

Blood transfusion, as well as organ transplantation, is only possible after prior blood group (BG) typing and crossmatching. The most important blood group system is that of Landsteiner's ABO classification based on antigen presence on the erythrocyte surfaces. A mass sensitive QCM (quartz crystal microbalance) sensor for BG typing has been developed by utilizing molecular imprinting technology. Polyvinylpyrrolidone (crosslinked with N,N-methylenebisacrylamide) is a favorable coating that was imprinted with erythrocytes of different blood groups. In total, 10 MHz quartz sheets with two resonators, one for MIP (molecularly imprinted polymer) and the other for NIP (non-imprinted polymer) were fabricated and later used for mass-sensitive measurements. The structure of erythrocyte imprints resembles a donut, as identified by AFM (atomic force microscope). All the erythrocytes of the ABO system were chosen as templates and the responses to these selective coatings were evaluated against all blood groups. Each blood group can be characterized by the pattern of responses in an unambiguous way. The results for blood group O are remarkable given that all types of erythrocytes give nearly the same result. This can be easily understood as blood group O does not possess neither antigen A nor antigen B. The responses can be roughly related to the number of respective antigens on the erythrocyte surface. The imprints generate hollows, which are used for reversible recognition of the erythrocytes. This procedure is based on molecular recognition (based on supramolecular strategies), which results from size, shape and enthalpic interactions between host and guest molecules.

Why Does the Precautionary Principle Suffice for Blood Regulation?

Traditional approaches to blood regulation emphasise the precautionary principle and pursue zero-risk for viral transmission; these traditional approaches have usually followed tragedy, such as the HIV and hepatitis C infections that followed the use of factor VIII concentrates. However, a much more haphazard haemovigilance system operates for general adverse events. Such imprecise assessment of hazards prevents sound benefit-risk assessment, and for blood products this is further confounded by the fact that their efficacy has attracted little systematic study. The ongoing COVID-19 pandemic has now prompted the proposal of a convalescent plasma (CP) blood product. Clearly, mere freedom from infectious agents will not suffice in assessing CP, and an objective measure of efficacy, so as to permit formal benefit-risk analysis, is essential. This is both a scientific and an ethical demand, as has been the case for other experimental COVID-19 treatments. With special reference to COVID-19 CP, the well-recognized adverse events of transfusion-associated lung injury (TRALI) and transfusion-associated circulatory overload (TACO) will be important. Furthermore, not only efficacy but also product quality attributes (e.g., antibody titre) will have to be defined. Both of these are outside the traditional regulatory philosophy for blood products and are needed to truly assess the benefit-risk of this putative therapeutic product.

Detection of hepatitis C virus and parvovirus B19 in human plasma pools by nucleic-acid amplification techniques - Trends in results of EDQM proficiency testing studies from 2004 to 2018.

The European Directorate for the Quality of Medicines & HealthCare (EDQM) has run proficiency testing schemes on the detection of viral contaminants in human plasma pools by nucleic-acid amplification techniques since 1999 for hepatitis C virus and since 2004 for parvovirus B19. A retrospective analysis was performed to assess their impact and identify trends and progress in the results obtained by participating laboratories over a 15-year span, from 2004 to 2018. The results demonstrate that overall performance improved over that time, especially among the regular participants. Participation in these proficiency testing schemes is therefore recommended for all interested control laboratories. This analysis also shows that hepatitis C virus detection now seems well established compared to that of parvovirus B19, which still appears more challenging.

Contamination of therapeutic human immunoglobulin preparations with apolipoprotein H (ß2-glycoprotein I).

Polyclonal immunoglobulin (Ig) concentrates are important biological medicinal products and the assurance of their quality and safety is crucial. In our present approach we used proteomic methods to check the purity of commercial Ig products of different origin. The experimental setup included nonreducing 2DE or DIGE combined with MALDI-TOF and the thrombin generation assay, a routine safety test for pharmaceutical Ig preparations, and was complemented by a specific immunoassay. 2DE patterns displayed contaminations with trace amounts of human apolipoprotein H (Apo-H), transferrin, albumin, and its fragments. In contrast to the latter, Apo-H is a protein that is active in the coagulation cascade, and thus a potential involvement in thromboembolic events in vivo cannot be excluded. It was found by 2DE and MALDI-TOF to be a contaminant of several Ig preparations. Spiking experiments of Ig preparations with pure Apo-H demonstrated an Apo-H concentration dependent increase in thrombin generation assay values. Traces of Apo-H are possibly also contributing to unwanted side effects, as already known for factor XIa. The significance of Apo-H contaminations for these side effects might be verified by detailed analyses of pharmacovigilance data.

Assessment of immunoglobulin concentrates on thrombogenic activity by thrombin generation assay, prekallikrein activator assay, and size-exclusion chromatography.

BACKGROUND: Immunoglobulin G (IgG) concentrates have recently been found to be contaminated with procoagulant impurities causing thromboembolic events (TEEs) in vivo. In this study the question was raised whether a thrombin generation assay (TGA) will be able to characterize IgG samples from the Austrian market with regard to their thrombogenic potential. STUDY DESIGN AND METHODS: A total of 44 IgG concentrates have been assayed by TGA employing pooled normal plasma and Factor (F)XI-deficient plasma (FXIdp). Furthermore, the prekallikrein activator assay including determination of blank values, size-exclusion chromatography, and further test systems required for batch release testing of IgG concentrates according to the European Pharmacopeia (Pharm. Eur.) were carried out. RESULTS: All samples complied with acceptance criteria stated in the Plarm. Eur. and/or prescribed by the marketing approval. One intravenous immunoglobulin (IVIG) involved in TEEs exceeded a threshold level of 350 nmol peak thrombin, which was not exceeded after change of manufacture and by all the other IVIGs tested. Two hyperimmune globulins revealed elevated peak thrombin levels of up to 810 nmol in FXI and up to 285 nmol in FXIdp. CONCLUSION: The study indicates that the TGA is able to reliably predict procoagulant activities probably associated with the presence of FXIa and potential thrombogenicity. Comparison of thrombin generation with product-specific acceptance criteria as well as variables from other test systems as amidolytic activity and molecular size can help to monitor IgG quality and manufacturing changes with regard to thrombogenicity.

Synthetic receptors for selectively detecting erythrocyte ABO subgroups.

Surface imprinting techniques with erythrocytes as templates yield polymer coatings with selective recognition sites towards red blood cells. The resulting cavities in the respective surface exhibit selectivity between blood subgroups as shown by Quartz Crystal Microbalance (QCM) measurements. Mass sensitive effects in the kilohertz range could be observed for concentrations down to 0.5 x 10(8) cells/mL. Frequency response as well as recovery of the sensor took place within a few minutes, indicating that no covalent binding is involved. Linear concentration dependence over a defined region provides ideal conditions for cross selectivity measurements. A1 imprinted sensor coatings resulted in an effect of 40 kHz when exposed to the template blood group, while A2 erythrocytes yielded just 11% of that value on the same layer. Furthermore, A2 imprinted coatings incorporated only one third the amount of A1 erythrocytes as compared to A2 ones. Therefore, imprinted materials depict the entire cell surface and utilize it for recognition, whereas natural antibodies bind on the defined antigen position and thus usually cannot distinguish between cells carrying different amounts of them.

Pollen-imprinted polyurethanes for QCM allergen sensors.

Molecularly imprinted polymers for detecting plant pollen were designed as artificial recognition materials for quartz crystal microbalances in the gaseous phase. Imprints of birch (diameter, 25 mum) and nettle (diameter, 15 mum) pollen can be generated by polydimethylsiloxane stamping technique as proven by atomic force microscopy. If pollen grains are able to access the cavities and thus are incorporated, the resulting sensors display Sauerbrey-like negative frequency shifts. Non-Sauerbrey behaviour can be observed as soon as pollen is prevented from entering the selective hollows: this results in grain mobility on the electrode surface leading to frequency increases. Access to the cavities is determined by the diameter ratio between pollen grains and imprints as can be revealed during cross-selectivity measurements of nettle and birch pollen imprinted layers. When the amount of pollen grains on the electrode surface exceeds the number of available imprints, the excess particles move freely, resulting in positive, non-Sauerbrey frequency shifts.

Sensors for bioanalytes by imprinting--polymers mimicking both biological receptors and the corresponding bioparticles.

Structuring of thin polymer layers by soft lithography with template bioparticles results in the formation of selective surface cavities, leading to highly effective sensor systems when combined with mass-sensitive transducers, especially QCM. These sensors allow selective differentiation of various stages of development of yeast cells. In order to achieve a higher degree of standardisation, we fabricated plastic yeast cells and utilised them for the stamp imprinting procedures. These sensitive layers are capable of the differentiation between Saccharomyces cerevisiae and Saccharomyces bayanus. Aside from achieving the same sensitivity compared to the polymers that were structured using native cells, we realised further enhancement of selectivity exceeding a factor of three regarding the two cell strains. These ideas could also be transferred to develop a recognition system for the more flexible erythrocytes and therefore MIP-layers of polyvinylpyrrolidone were combined with QCMs. These devices provide sensor-based ABO blood group typing. Additionally, the differentiation of the subgroups A(1) and A(2) is shown with the generated MIP-layers that are decorated by high selectivity, namely the threefold frequency effect for the imprinted template, and negligible unspecific effects. Application of soft lithographic methods furthermore allows the design of artificial erythrocytes. These "plastic" blood cells possess an increased robustness compared to the native cells, thus opening up multiple novel strategies of surface patterning.

Capillary isoelectric focusing hyphenated to single- and multistage matrix-assisted laser desorption/ionization-mass spectrometry using automated sheath-flow-assisted sample deposition.

In this paper CIEF combined with MALDI-MS is described using a sheath-liquid-assisted automatic sample deposition from the separation capillary onto a MALDI target. Sample/matrix preparation techniques on the target resembling the dried droplet and the thin layer methods were evaluated in the context of the automatic spotting. Volatile buffers were used as IEF catholyte solutions. Test samples consisting of tryptic peptides, glycopeptides, and phosphopeptides of well-known proteins showed that CIEF-MALDI-MS can be used as effective preseparation method prior to MS, allowing to obtain the amino acid sequence coverage of proteins similar to that achieved with CZE-MALDI-MS and CZE-ESI-MS. Particularly, completeness and reliability of glycopeptide analysis is much enhanced by the preseparation. The effect is less pronounced but still significantly found with phosphopeptides present in the test protein. Finally, a test sample of five standard proteins demonstrates the suitability of this technique also for the treatment of intact proteins. This technique has potential to emerge as a faster method analogous and complementary to 2-DE and to IPG-IEF-MALDI-MS demonstrated before by the group of Loo [1].