In vitro assays and clinical trials in red blood cell aging: Lost in translation.

The age of erythrocyte concentrates (EC) in transfusion medicine and the adverse outcomes when transfusing long-term-stored EC are highly controversial issues. Whereas the definition of a short-term-stored EC or a long-term-stored EC is unclear in clinical trials, data based on in vitro storage assays can help defining a limit in addition of the expiration date. The present review merges together these data in order to highlight an EC age cut-off and points out potential misleading consideration. The analysis of in vitro data highlights the presence of reversible and irreversible storage lesions and demonstrates that red blood cells (RBC) exhibit two limits during storage: one around 2 weeks and another one around 4 weeks of storage. Of particular importance, the first lesions to appear, i.e. the reversible ones, are per se reversible once transfused, whereas the irreversible lesions are not. In clinical trials, the EC age cut-off for short-term storage is in general fewer than 14 days (11 ± 4 days) and more disperse for long-term-stored EC (17 ± 13 days), regardless the clinical outcomes. Taking together, EC age cut-off in clinical trials does not totally fall into line of in vitro aging data, whereas it is the key criteria in clinical studies. Long-term-stored EC considered in clinical trials are not probably old enough to answer the question: "Does transfusion of long-term-stored EC (older than 4 weeks) result in worse clinical outcomes?" Depending on ethical concerns and clinical practices, older EC than currently assayed in clinical trials should have to be considered. These two worlds trying to understand the aging of erythrocytes and the impact on patients do not seem to speak the same language.

Undercoverage and Nonresponse in a List-sampled Telephone Election Survey

For landline telephone surveys in particular, undercoverage has been a growing problem. However, research regarding the relative contributions of socio-demographic bias and other composition effects is scarce. We propose to address this issue by analyzing an election survey which used a sample from a register-based sampling frame containing basic socio-demographic information and to which telephone numbers were subsequently matched. With respect to socio-demographic representation of the final sample, we find that difficult to match groups are also difficult to contact, while those who cooperate tend to have different characteristics. We find bias due to undercoverage to be of greater magnitude than noncontact bias, while noncooperation falls between the two. As for substantive variables, both additional efforts to match missing telephone numbers and the construction of better weights are successful in closing the gap between survey estimates of voting behavior and true values from the election results.

Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.

Microparticles from stored red blood cells induce thrombin generation

Background: Microparticles are small phospholipid vesicles of <1 lm shed in blood flow by various cell types including red blood cells. Erythrocyte-derived microparticles (EMPs) accumulate in erythrocyte concentrates (ECs) during their storage time. EMPs are considered as part of storage lesion and as their exact role is not elucidated, they could be involved in these clinical outcomes. Aims: The aim of this study is to evaluate the impact and implication of EMPs isolate from ECs on coagulation. Methods: EMPs were first isolated from erythrocyte concentrates by centrifugation and counted by flow cytometry. Using a calibrated automated thrombogram, EMPs were then added to different type of plasmas in order to evaluate the potential of thrombin generation. Results: We demonstrate that EMPs isolated from ECs are capable to accelerate and amplify thrombin generation in presence of a low exogenous tissue factor concentration, thanks to their negatively charged membrane necessary for the assembly of coagulation complexes. Interestingly, in the absence of exogenous tissue factor, EMPs are also able to trigger thrombin generation. In addition, thrombin generation induced by EMPs is not affected by the presence of anti-TF antibodies. Finally, thrombin generation induced by EMPs is not affected by using plasma samples deficient in factor VII, XI or XII. However, thrombin generation is reduced in plasma deficient in factor VIII or IX and is completely abolished in plasma deficient in factor X, V or II. No thrombin generation was observed in plasma samples without EMPs. Summary/conclusion: Several studies have shown a link between storage time of blood products and post transfusion complications. We provide evidence that EMPs accumulated during storage of erythrocyte concentrates were not only able to accelerate and support thrombin generation in plasma in presence of a low exogenous tissue-factor concentration, but also to trigger thrombin generation in absence of exogenous TF. The impact of those transfused EMs is unknown on recipients, nevertheless it could be hypothesized that under certain circumstances, transfused EMPs could be involved in thrombin generation and could be linked to adverse clinical outcome. Further work is needed to determine whether procoagulant EMPs transfused with erythrocyte concentrate may account for some of the complications occurring after red blood cell transfusion, and more particularly after transfusion of ''older''stored blood, rich in EMPs.